Cqu 7854+SOURCE1+SOURCE1.3

CHARACTERISATION AND POTENTIAL USES
OF MICROBIALLY ENHANCED COMPOST

EXTRACTS FOR PLANT GROWTH
PhD THESIS
Submitted in Partial Fulfillment of the

Requirements for the Degree of
Doctor of Philosophy
CENTRE FOR PLANT AND WATER SCIENCE
KARUNA SHRESTHA
MARCH, 2011
Faculty of Sciences, Engineering and Health

CQUniversity, Rockhampton
Australia
ABSTRACT
Microbially enhanced compost extracts are increasingly being used to
improve soil and crop ‗health‘. In this thesis, studies were made both on the process
of preparing compost extracts and on their possible roles in agriculture. Compost
extraction and microbial enhancement processes were characterised for a system
based on open air incubation using chemical, biochemical and molecular techniques,
with variation in additives, dilution, aeration, compost age, compost source including
vermicasts (earthworm casts based on rumen compost) and commercially available
inocula (Living SoilTM and Nutri-Life 4/20TM). The nutritional and microbial
properties of the compost extracts could be improved or manipulated as desired by
supplying additives or aeration in the extract during incubation. Improvement in
community level functional diversity was noted in vermicast leached extract
compared to the original rumen compost, vermicasts and rumen compost extract. The
Biolog and DGGE tools were useful in the overall characterisation of the microbial
assemblages of these liquid samples. The commercial products were comparable in
terms of microbial diversity to compost extracts based on fresh rumen content
compost.
The following hypotheses for the action of compost extracts in improving
crop ‗health‘ and, therefore, their roles in agriculture were tested: (a) direct
improvement of plant nutrition via the nutrient content of the compost extracts; (b)
improvement of plant nutrition through an indirect action, via microbial action in
mineralising of organic or solubilising of inorganic components of the soil; (c)
acceleration of litter decomposition; (d) bio-protection against fungal pathogens
(namely, Fusarium oxysporum, Fusarium solani, and Rhizoctonia solani); and (e)
protection to root growth against high levels of aluminium (Al) ions, via chelation of
Al ions by humic acids or uptake by microbes. The noted increase in tomato and
sorghum plant growth associated with a compost extract treatment (from composted
rumen waste) was attributed to nutrient addition inherent in the treatment rather than
solubilisation of minerals and/or mineralisation of organic matter. Litter
decomposition rates (sugarcane trash mat), as indicated by cumulative respiration
rate, were increased when microbially enhanced compost extract produced from a
mixture of composted rumen waste and sugarcane stubble, was applied. However,
there was no difference between extracts made from composts of different ages. In
vitro studies showed a distinct effect of compost extracts in suppressing fungal wilt
diseases caused by three fungal pathogens (namely, Fusarium oxysporum, F. solani,
Rhizoctonia solani), however, the disease suppressive effect of compost extracts was
not consistent for the three tested pathogens in hydroponic tomato trials. Aluminium
toxicity on maize seedling root growth was alleviated by compost extract application.
A field study indicated that compost extracts enhanced the soil bacterial community
diversity compared to the conventional treatment. It is likely that compost extracts
will find an expanded role in broadscale agriculture and a role as a liquid organic
fertiliser in intensive horticulture.
i
ACKNOWLEDGEMENTS
First and foremost, I would like to thank my principal supervisor, Professor

David J. Midmore, for his keen interest, valuable comments and guidance throughout
my PhD studies.
I wish to express my sincere gratitude and appreciation to Professor Kerry
B. Walsh for his critical comments, valuable suggestions, and guidance. I am very
thankful to my associate supervisor, Associate Professor Keith M. Harrower, who
not only encouraged me to go into microbiology but also taught me the skills
personally in this totally new field to me. Likewise, my sincere thanks go to my
associate supervisor Associate Professor Nanjappa Ashwath for his helpful
discussions and suggestions.
My special thanks go to Rob Lang from Broadmeadows Pty Ltd for
continuously supplying the rumen compost, David Wyatt from Vermicrobe
International Pty LtdTM for donation of vermiculture facilities, William (Brock)
McDonald for the maintenance of the Vermicrobe system and also Professor Andrew
S. Ball and Dr. Eric M. Adetutu for providing the technical and laboratory support at
the Faculty of Science and Engineering of Flinders University, Adelaide. I would
like to acknowledge Ray O‘Grady of O‘Grady Rural (NSW) and Graeme Sait from
NutriTech Solutions (NTS) Pty Ltd (Qld) for supplying their valuable products for
research use. I am also grateful to BSES Ltd, Bundaberg, Qld for supplying the
sugarcane trash material, John Eigens of Pacific Seeds Company for providing maize
seeds for my research and Tom Nicholas for allowing me to accompany him during a
field survey in central and southern Qld and northern NSW to review the current
practices and production of compost extracts in 2008. Moreover, I am highly obliged
to Scott Stevens for providing soil samples from the trial site located in Baralaba on
the central Queensland highlands and treated with different field management
practices including the application of compost extracts.
I express my gratefulness to various reviewers of my thesis for their useful
comments, constructive criticism and invaluable suggestions. I would also like to
thank the anonymous reviewers and editors for their valuable comments on the
revised version of Chapter 2, 3, 4 which have been published in ―Bioresource
Technology‖ and Chapter 7 which has been accepted in ―Biological Control‖.
ii
I would like to extend my sincere gratitude to the Endeavour Postgraduate
Program (EPA) for providing me a scholarship to carry out my PhD studies at
CQUniversity, Australia and I am also grateful to various Endeavour case managers
for resolving the official as well as personal matters throughout my study period. I
am indebted to CQUniversity for their financial support to undertake research studies
during my PhD course.
My sincere acknowledgement goes to Centre for Plant and Water Science
(CPWS) staffs, members, colleagues and friends for their cooperation and support. I
would personally like to thank Dr. Surya P. Bhattarai and Dr. Phul P. Subedi for their
invaluable advice and encouragement. Dr. Alan Keen and Associate Professor Steve
Mckillup from CQUniversity and Christina Playford from DPI are greatly
acknowledged for providing statistical support during the early phase of my studies.
Linda Ahern, indeed, deserves appreciation for her supportive attitude and readiness
to help in any circumstances. Many thanks to Rob Lowry, Jeffrey Conaghan and
Graham Fox for providing technical assistance while undertaking my research.
My husband deserves a special thanks for his endless support, guidance and
assistance in each and every step of my career. I appreciate his patience and deep
understanding throughout my study period. Most importantly, I am thankful to him
for his supervisory role in order to complete my studies.
Last but not least, I would like to extend an earnest thanks to my family,
especially to my parents for their inspiration, love and great support to achieve my
goals; without their encouragement and blessings my journey wouldn‘t have been
successful.
iii
DECLARATION
I hereby declare that this thesis is my original research work and it has not
been submitted anywhere for any award. Contributions of others, if involved, are
either referenced or acknowledged.
I authorise CQUniversity to reproduce this thesis in whole or in part for
purposes of research.
……………………………
Karuna Shrestha
iv
Table of Contents
ABSTRACT ................................................................................................................................i
ACKNOWLEDGEMENTS ........................................................................................................... ii
DECLARATION.........................................................................................................................iv
Chapter 1: A literature review on the use and efficacy of compost extracts in crop production ... 1
1. Overview. ........................................................................................................... 1
2. Soil ‘Health’ ......................................................................................................... 3
3. Organic inputs into agricultural systems ................................................................... 6
4. Composting ......................................................................................................... 8
5. Compost extract ................................................................................................. 12
5.1. Definition of compost extracts ............................................................................................. 12
5.2. Production methods of compost extract .............................................................................. 13
5.3. Defining compost extract quality .......................................................................................... 20
6. Characterising microbial diversity ................................................................. ……..21
7. Current use of compost extract in central and southern Qld and northern NSW .............. 29
8. Compost extract application and claimed benefits ..................................................... 33
8.1. Direct nutrient addition ........................................................................................................ 36
8.2. Soil inorganic fraction solubilisation ..................................................................................... 37
8.3. Soil organic matter decomposition ....................................................................................... 38
8.4. Leaf litter and stubble decay ................................................................................................. 39
8.5. Free living N2 fixation ............................................................................................................ 40
8.6. Plant disease suppression ..................................................................................................... 41
8.7. Acquired systemic resistance ................................................................................................ 47
8.8. Bioremediation...................................................................................................................... 48
9. Summary and hypotheses ..................................................................................... 50
10. Conclusion ......................................................................................................... 53
Chapter 2: Microbial enhancement of compost extracts based on cattle rumen content compost
– characterisation of a system ................................................................................................ 54
Abstract ..................................................................................................................... 54
1. Introduction ....................................................................................................... 55
2. Materials and methods ........................................................................................ 58
2.1. Experiments and treatments ................................................................................................ 58
2.2. Source compost .................................................................................................................... 59
2.3. Physico-chemical analyses .................................................................................................... 60
2.4. Microbial analyses ................................................................................................................ 61
2.5. Statistical analysis ................................................................................................................. 61
3. Results and discussion .......................................................................................... 62
3.1. General trends ...................................................................................................................... 62
3.2. Experiment 1: Comparison of compost extracts with different additives (control, kelp,
molasses and kelp+molasses) ............................................................................................... 63
3.3. Experiment 2: Comparison of compost extracts with different dilution levels .................... 68
3.4. Experiment 3: Comparison of aerated and non-aerated compost extracts with and without
bags ....................................................................................................................................... 72
4. Conclusion ......................................................................................................... 76
Chapter 3: Changes in microbial and nutrient composition associated with rumen

content compost incubation .............................................................................................. 78
Abstract ..................................................................................................................... 78
1. Introduction ....................................................................................................... 79
v
2. Materials and methods ........................................................................................ 81
2.1. Rumen compost .................................................................................................................... 81
2.2. Vermicomposting process, vermicasts and vermicast leachate ........................................... 81
2.3. Physico-chemical analyses .................................................................................................... 82
2.4. Preparation of compost extracts and sampling regimes ...................................................... 82
2.5. Microbial determinations ..................................................................................................... 83
2.6. Microbial biomass carbon analysis ....................................................................................... 84
2.7. Community level physiological profiles of bacteria and fungi .............................................. 84
2.8. PCR and DGGE ....................................................................................................................... 85
2.9. Statistical analysis................................................................................................................. 86
3. Results and discussion .......................................................................................... 86
3.1. Physico-chemical analysis ..................................................................................................... 86
3.2. Microbial and biochemical assays......................................................................................... 87
3.3. Characterisation of compost extracts ................................................................................... 87
3.4. Community level physiological profiles of bacteria and fungi .............................................. 91
3.5. PCR-DGGE analysis ................................................................................................................ 95
4. Conclusions ........................................................................................................ 97
Chapter 4: Comparison of microbially enhanced compost extracts produced from

composted rumen content material and from commercially available inocula ........... 98
Abstract ..................................................................................................................... 98
1. Introduction ....................................................................................................... 99
2. Materials and methods ...................................................................................... 101
2.1. Source compost and commercial preparations .................................................................. 101
2.2. Preparation of compost extracts and commercial extracts ................................................ 102
2.3. Physico-chemical analyses .................................................................................................. 103
2.4. Biochemical and microbial analyses ................................................................................... 103
2.5. Community level physiological profiles .............................................................................. 104
2.6. Microscopic analyses .......................................................................................................... 104
2.7. Genetic diversity of microbial communities of compost extracts ...................................... 104
2.8. Statistical analysis ............................................................................................................... 105
3. Results and discussion ........................................................................................ 105
3.1. Culture characterisation ..................................................................................................... 105
3.2. Microbial and biochemical analyses ................................................................................... 109
3.3. Community level physiological profiles of bacteria and fungi ............................................ 111
3.4. Microscopic analyses .......................................................................................................... 117
3.5. PCR and DGGE ..................................................................................................................... 118
4. Conclusions ...................................................................................................... 122
Chapter 5: Microbially enhanced compost extract: does it increase solubilisation of

minerals and mineralisation of organic matter and thus improve plant nutrition?.. 123
Abstract ................................................................................................................... 123
1. Introduction ..................................................................................................... 124
2.1. Compost and compost extract ............................................................................................ 127
2.2. Experiment 1 ....................................................................................................................... 128
2.3. Experiment 2 ....................................................................................................................... 129
2.4. Experiment 3 ....................................................................................................................... 131
2.5. Statistical analyses ............................................................................................................. 132
3. Results ............................................................................................................ 133
3.1. Characterisation of compost extract .................................................................................. 133
3.2. Experiment 1: Tomato growth in sand-compost mix ......................................................... 134
3.2.1. Plant biomass .................................................................................................................. 134
3.2.2. Tomato leaf chlorophyll concentration ........................................................................... 135
vi
15
3.2.3. δ N of fertilisers (compost and compost extracts) and plants grown in compost-sand
mix ................................................................................................................................... 136
3.3. Experiment 2: Tomato growth on soil ................................................................................ 137
3.3.1. Soil ................................................................................................................................... 137
3.3.2. Tomato growth, yield and chlorophyll concentration ..................................................... 138
15
3.3.3. δ N of fertilisers and plants grown on soil ..................................................................... 139
3.3.4. Microbial enumeration of aerated and non-aerated compost extracts ......................... 141
3.3.5. Soil microbial populations ............................................................................................... 141
3.3.6. Soil respiration and temperature .................................................................................... 142
3.3.7. Mass balance of nutrients ............................................................................................... 142
3.4. Experiment 3: Sorghum crop rotation ................................................................................ 143
3.4.1. Plant growth, shoot biomass, chlorophyll concentration and light interception ............ 143
4. Discussion ........................................................................................................ 145
4.1. Effects of treatments on growth and nutrition of tomato plants ....................................... 145
4.2. Effects of treatments on tomato leaf chlorophyll concentration ....................................... 147
4.3. Influence of soil types on crop growth ............................................................................... 148
4.4. Nutritional and microbial supply from compost extracts ................................................... 149
4.5. Effects of treatments on soil microbial properties............................................................. 150
5. Conclusions ...................................................................................................... 151
Chapter 6: Biodegradation of sugarcane trash through the use of enhanced compost

extracts.............................................................................................................................. 152
Abstract ................................................................................................................... 152
1. Introduction ..................................................................................................... 153
2.1. Soil, plant and compost material ........................................................................................ 156
2.2. Biodegradation of trash mat ............................................................................................... 156
2.3. Analytical methods ............................................................................................................. 158
2.3.1. Physico-chemical analyses .............................................................................................. 158
2.3.2. Microbial analyses ........................................................................................................... 158
2.4. Statistical analyses .............................................................................................................. 159
3.1. Physico-chemical, biochemical and microbial analyses of different aged composts ......... 159
3.2. Characterising the incubation process using different aged compost extracts as inoculum161
3.3. Comparison of chemical, microbial and biochemical characteristics of different aged and
sugarcane stubble amended compost (24 h incubated) extracts ....................................... 164
3.4. Effects of compost extract treatments on sugarcane trash degradation ........................... 167
3.5. Weight loss of trash and cumulative respiration rate ......................................................... 170
4. Conclusions ...................................................................................................... 174
Chapter 7: Suppressing fungal wilt diseases of tomato caused by Fusarium

oxysporum, F. solani and Rhizoctonia solani using compost extracts in vitro and in
hydroponics culture ......................................................................................................... 175
Abstract ................................................................................................................... 175
1. Introduction ..................................................................................................... 176
2.1. In vitro hyphal growth trials ................................................................................................ 180
2.2. Hydroponics trials ............................................................................................................... 182
3.1. In vitro trials ........................................................................................................................ 185
3.1.1. Mycelial growth of fungi .................................................................................................. 185
3.1.2. Spore count ..................................................................................................................... 188
3.1.3. Total fungal biomass........................................................................................................ 190
3.2. Hydroponics trials ............................................................................................................... 192
vii
4. Conclusions ...................................................................................................... 199
Chapter 8: The effect of compost extract on root elongation of maize (Zea mays L.)
in the presence of aluminium .......................................................................................... 200
Abstract ................................................................................................................... 200
1. Introduction ..................................................................................................... 201
2.1. Rumen compost extract and vermicast leachate ............................................................... 205
2.2. Chemical and microbial analyses of compost extracts ....................................................... 205
2.3. Plant material and experimental set up ............................................................................. 206
2.4. Statistical analysis ............................................................................................................... 207
3.1. Effect of Al on root growth of maize seedlings - Preliminary trial ...................................... 208
3.2. Composition of compost extract ........................................................................................ 209
3.3. Experiment 1 ....................................................................................................................... 210
3.4. Experiment 2 ....................................................................................................................... 212
4. Conclusion ....................................................................................................... 214
Chapter 9: Enhancing microbial activity of a field soil with long-term use of liquid
biofertilisers...................................................................................................................... 215
Abstract ......................................................................................................................................... 215
1. Introduction ........................................................................................................................ 216
2. Materials and methods ....................................................................................................... 219
2.1. Study site and treatments................................................................................................... 219
2.2. Soil sampling ....................................................................................................................... 222
2.3. Soil characterisation ........................................................................................................... 223
3. Results ................................................................................................................................. 225
3.1. Physico-chemical, microbial and biochemical analyses ...................................................... 225
3.2. Community level physiological profiles of bacteria and fungi ............................................ 229
3.3. PCR-DGGE analysis for bacteria and fungi .......................................................................... 233
4. Discussion ........................................................................................................................... 237
4.1. Seasonal variation ............................................................................................................... 237
4.2. Comparison of brigalow and cultivated soils ...................................................................... 238
4.3. Effects of management practices on soil properties .......................................................... 240
5. Conclusions ......................................................................................................................... 241
Chapter 10 ........................................................................................................................ 243

Summary and future directions ..................................................................................... 243
References ........................................................................................................................ 250
viii
List of Tables
Chapter 1
TM
Table 1. 1. Carbon substrates of Ecoplate and FF microplate (Biolog ), respectively divided
into different substrate guilds according to Preston-Mafham et al. (2002) ................................ 25
Table 1. 2. List of farmers [permissions granted to use their data] from Qld and NSW .............. 30
Table 1. 3. List of companies [permissions granted to use their data] visited with Tom Nicholas
or contacted that were involved in producing compost extract .................................................. 32
Table 1. 4. Summary of reports on proven disease suppression following application of
compost extracts (CEs) produced from known input materials................................................... 44
Chapter 2
Table 2. 1. Comparison of physico-chemical and microbial characteristics of compost extracts
incubated with different additives ............................................................................................... 67
incubated with different dilutions ............................................................................................... 69
incubated for 24 h in presence and absence of aeration ............................................................. 75
Chapter 3
Table 3. 1. Initial physico-chemical, microbial and biochemical characteristics of rumen
compost and vermicast (n = 3). .................................................................................................... 87
Table 3. 2. Comparison of chemical, biochemical and microbial characteristics of rumen
compost extract (RCE), vermicast leached extract (VLE), vermicast extract (VCE) ...................... 91
Table 3. 3. Shannon weaver Diversity Index (H’), Equitability Index (J) and number of bands (S)
for bacteria and fungi present in rumen compost (RC), vermicast (VC), rumen compost extract
(RCE) and vermicast leached extract (VLE) .................................................................................. 96
Chapter 4
Table 4.1. Comparison of physico-chemical and microbial characteristics of different liquid
cultures sampled at 24 h and at 48 h of incubation. .................................................................. 106
Table 4.2. Sample analysis data of nine month old rumen compost extract (9MCE) and LS
TM
(Living Soil ) .............................................................................................................................. 117
Table 4.3. Diversity and equitability of microbial communities in different liquid cultures...... 120
Chapter 5
Table 5. 1. Nutrient (macro and micro) concentrations for aerated and non-aerated compost
extract samples analysed by Wesfarmers CSBP Limited, Australia............................................ 133
Table 5. 2. Characterisation of vertisol and ferrosol used in the greenhouse trial ................... 138
Table 5. 3. Enumeration of bacteria and fungi populations in representative samples of ACE
and NCE. The compost extracts were produced at the ratio of 1:10 w/v .................................. 141
Table 5. 4. Effect of compost extracts on microbial (bacterial and fungal) populations (cfu Log
-1
10 mL ) in two soil types sampled at 36 days after transplanting ............................................. 142
Table 5. 5. Mass balance of N in compost extract and tomato crop grown in vertisol. ........... 143
Chapter 6
Table 6. 1. Physico-chemical, biochemical and microbial comparisons of different aged, and
sugarcane stubble amended, composts .................................................................................... 161
Table 6. 2. Comparison of chemical, biochemical and microbial characteristics of different
aged and sugarcane stubble amended compost extracts incubated for 24 h with 1% kelp and
0.5% molasses as amendments ................................................................................................. 165
Table 6. 3. Chemical, biochemical and microbial characteristics of soil samples (collected after
168 days of incubation). ............................................................................................................. 169
ix
Chapter 7
Table 7. 1. Fusarium infection in roots based on rating scale of 0 (pathogen absent) to 1
(pathogen present) on eight tomato plant roots in each hydroponics container ..................... 194
Chapter 8
Table 8. 1. Comparison of physico-chemical and microbial characteristics of sterilised and non-
sterilised rumen compost extract and vermicast leachate ........................................................ 209
Chapter 9
Table 9. 1. Details of treatments and fertility programs of a long-term field trial (commenced
in 2007) conducted in Baralaba, Qld .......................................................................................... 221
Table 9. 2. Variation in the physico-chemical, microbial and biochemical characteristics of soil
samples collected from plots treated with different management practices at the Baralaba trial
site in two different seasons ...................................................................................................... 228
Table 9. 3. Shannon-Weaver Diversity Index (H’), Equitability Index (J) and number of bands (S)
for bacteria and fungi present in soil samples collected in the dry-winter season from plots
treated with different management practices ........................................................................... 236
x
List of Figures
Chapter 1
Figure 1. 1. Enzymatic conversion of FDA to fluorescein (Source: Adam & Duncan, 2001) ........ 22
Figure 1. 2. Flow diagram showing the different steps in the analysis of microbial community
structure by PCR-DGGE ................................................................................................................ 28
Chapter 2
-1
Figure 2. 1. Comparison of pH, electrical conductivity (dS m ), fluctuations in temperatures
(˚C) and dissolved oxygen (% of saturation) over time (readings taken at every 3 h intervals
until 24 h and final readings taken at 48 h) in rumen compost extracts with different additives65
Figure 2. 2. Total microbial activity over time (readings taken at 12, 24 and 48 h) as estimated
by the FDA hydrolysis method in compost extracts with different additives .............................. 67
-1
Figure 2. 3. Time course of solution pH, electrical conductivity (dS m ), temperatures (˚C) and
dissolved oxygen (% of saturation); readings taken at every 3 h intervals until 24 h and final
readings taken at 48 h in rumen compost extracts with different dilutions. ............................... 70
Figure 2. 4. Comparison of total microbial activity at different times (readings taken at 12, 24
and 48 h) following the FDA hydrolysis method in compost extracts with different dilutions .... 71
-1
(˚C) and dissolved oxygen (% of saturation) over time ................................................................ 73
Figure 2. 6. Comparison of total microbial activity over time (readings taken at 12, 24 and 48 h)
following the FDA hydrolysis method in compost extracts in presence and absence of aeration
with and without the use of bags. ............................................................................................... 75
Chapter 3
-1
Figure 3. 1. Comparison of pH, EC (dS m ), fluctuations in temperatures (°C) and dissolved
oxygen (%) per unit time (readings taken at every 3 h intervals until 24 h after commencement
of compost extract incubation ..................................................................................................... 89
Figure 3. 2. Comparison of total microbial activity per unit time (readings taken at 12, 24 and
48 h) following the FDA hydrolysis method ................................................................................. 91
Figure 3. 3. Kinetics of average well colour development (AWCD) and optical densities of (A)
bacterial (570 nm) and (B) fungal (490 nm) communities. .......................................................... 93
Figure 3. 4. Principal component analysis of optical densities produced by microbial activities
of rumen compost (RC), vermicast (VC), rumen compost extract (RCE) and vermicast leached
extract (VLE) ................................................................................................................................. 94
Figure 3. 5. UPGMA (Unweighted Pair Group Method with Arithmetic mean algorithm)
dendrogram generated from responses to 16S rDNA and Internal Transcribed Spacer region
based Denaturing Gradient Gel Electrophoresis (DGGE) analyses .............................................. 96
Chapter 4
-1
Figure 4.1. Comparison of (A) pH, (B) electrical conductivity (dS m ), (C) temperatures (˚C) and
(D) dissolved oxygen (ppm) over time (readings taken at every 3 h intervals until 24 h and final
readings taken at 48 h) in different cultures .............................................................................. 107
Figure 4.2. Comparison of total microbial activity at different times (readings taken at 12, 24
and 48 h) following the FDA hydrolysis method in different cultures ....................................... 110
Figure 4.3. Kinetics of average well colour development (AWCD) and optical densities of (A)
bacterial (570 nm) and (B) fungal (490 nm) communities, respectively, from different compost
extracts and microbial preparations. ......................................................................................... 112
Figure 4.4. Average carbon substrate utilisation from different substrates by samples from
three month old rumen compost extract (3MCE), nine month old rumen compost extract
TM
(9MCE), nine month old rumen compost extract plus Nutri-Life 4/20 inoculum (9MTCE),
TM TM
Living soil (LS) and Nutri-Life 4/20 (NL-4/20) ...................................................................... 114
xi
Figure 4.5. Principal component analysis (PCA) of optical densities produced by microbial
activities of five different cultures consuming 32 (Biolog Ecoplate) and 96 (Biolog FF
microplate) different food sources including the control .......................................................... 116
Figure 4.6. UPGMA (Unweighted Pair Group Method with Arithmetic mean algorithm)
dendrogram for (A) bacteria and (B) fungi ................................................................................. 119
Chapter 5
Figure 5. 1. Effect of compost extracts on (A) total plant biomass (g d wt) and (B) shoot: root
biomass of tomato plants in Experiment 1 grown in a thoroughly washed compost-sand mix at
1: 5 ratio in two replicate trials .................................................................................................. 135
Figure 5. 2. Effect of compost extracts in Experiment 1 on chlorophyll concentration in leaves
in (A) trial I, and (B) trial II .......................................................................................................... 136
15
Figure 5. 3. δ N values of fertilisers applied including the rumen compost and of the tomato
plants grown in compost-sand mix (1: 5) under growth chamber conditions ........................... 137
Figure 5. 4. Data from Experiment 2 on (A) total above-ground biomass (g d wt per plant) of
tomato subject to four treatments after four months of planting on two soil types, (B) fruit
yield of tomato (g d wt per plant), (C) leaf chlorophyll concentration (SPAD unit) ................... 139
15
Figure 5. 5. δ N values of tomato plants grown in ferrosol conducted in a greenhouse
(Experiment 2)............................................................................................................................ 140
Figure 5. 6. Data on sorghum from Experiment 3 on (A) above-ground dry weight biomass per
plant, (B) chlorophyll concentrations of leaves and (C) percent light interception ................... 144
Chapter 6
-1
(˚C) and dissolved oxygen (% of saturation) over time (readings taken at every 3 h intervals
until 24 h and final readings taken at 48 h) ............................................................................... 163
Figure 6. 2. Comparison of total microbial activity at different times (readings taken at 12, 24
and 48 h) following the FDA hydrolysis method in different aged (one, three, six and ten
month old) and sugarcane stubble amended compost extracts ............................................... 166
Figure 6. 3. On the left (A) showing sugarcane trash treated with water control and on the
right (B) showing the same trash treated with compost extract ............................................... 171
Figure 6. 4. (A) Time course of respiration rates which was assessed as accumulation of CO 2
over a 24 h period and (B) comparison of cumulative respiration rates of sugarcane trash
materials lying on top of the ferrosol soil over six month (28 weeks) ....................................... 173
Chapter 7
-1
Figure 7. 1. Effect of compost extracts in trial 1 on mycelial growth rates (mm d ) of Fusarium
oxysporum, f. sp. lycopersici six days after inoculation.............................................................. 186
-1
Figure 7. 2. Effect of compost extracts in trial 2 on mycelial growth rates (mm d ) of Fusarium
oxysporum, f. sp. lycopersici, F. solani DAR 66102, and Rhizoctonia solani DAR 61830 over (A)
three and (B) six days after inoculation .................................................................................... 187
Figure 7. 3. Effect of compost extracts in trial 1 on sporulation (number of spores per plate) of
Fusarium oxysporum, f. sp. lycopersici six days after inoculation .............................................. 188
Fusarium oxysporum, f. sp. lycopersici and F. solani DAR 66102 ............................................... 189
Figure 7. 5. Effect of compost extracts in trial 1 on total biomass (d wt) of Fusarium oxysporum,
f. sp. lycopersici six days after inoculation ................................................................................. 190
f. sp. lycopersici (black bar), F. solani DAR 66102 (light grey bar), and Rhizoctonia solani DAR
61830 (dark grey bar), six days after inoculation ....................................................................... 192
Figure 7. 7. (A) A graph showing an effect of application of compost extracts in hydroponics
trial 3 on two month old tomato plants against the severity of fungal wilt caused by F.
oxysporum f. sp. lycopersici grown in the greenhouse (B) pictures of pathogen inoculated
control plants ............................................................................................................................. 193
xii
Figure 7. 8. Effect of application of compost extracts in hydroponics trial 4 on two month old
tomato plants against the severity of fungal wilt diseases caused by (A) F. oxysporum, (B) F.
solani and (C) R. solani in greenhouse assays ............................................................................ 196
Figure 7. 9. Effect of application of compost extracts in hydroponics trial 4 on the leaf
chlorophyll concentration of two month old tomato plants grown in the greenhouse and
inoculated with three different fungal pathogens ..................................................................... 198
Chapter 8
Figure 8. 1. Relative root extension (% of control, where control plants exhibited a root
extension of 14 cm) of maize (Zea mays L. cv. Hycorn 675 IT) .................................................. 208
Figure 8. 2. Experiment 1: Root elongation rate of maize (Zea mays L. cv. Hycorn 675 IT)
seedlings (n = 4) following growth for 96 h on a solution culture with or without 200 µM of Al
at pH 4.5, with treatments of non-sterilised and sterilised rumen compost extracts and
vermicast leachate (1:500 dilution) ........................................................................................... 210
seedlings (n = 4) measured 96 h after imbibition in solution culture in the presence or absence
of 200 µL Al (solution pH 4.5) ..................................................................................................... 213
Chapter 9
Figure 9. 1. Kinetics of average well colour development (AWCD) of (A) bacterial (570 nm) and
(B) fungal (490 nm) communities originating from soil samples collected from the trial site
during the dry-winter season at Baralaba .................................................................................. 230
Figure 9. 2. Average carbon substrate utilisation for different substrate categories in terms of
(A) 32 (Biolog Ecoplate) and (B) 96 (Biolog FF microplate) different food sources ................... 231
Figure 9. 3. Principal components analysis of optical densities produced by microbial activities
of soil samples collected from the trial site at Baralaba ............................................................ 232
Figure 9. 4. Unweighted Pair Group Method with Arithmetic mean algorithm dendrogram
constructed from the DGGE profiles of (A) bacterial 16S rDNA and (B) fungal ITS genes
amplified from duplicate DNA samples of soils ......................................................................... 234
xiii
Chapter 1: Literature review
Chapter 1
A literature review on the use and efficacy of
compost extracts in crop production
1. Overview
The large increase in world agricultural productivity during the 1960s and
70s (the ‗green revolution‘) was supported by improved genetics and by
increased chemical inputs (inorganic fertilisers, herbicides and pesticides).
However, there is a current trend to minimise (or exclude, in the case of ‗organic‘
production systems) chemical inputs, on the basis of reducing harm to the
environment and, in some cases, to reduce input costs. A key element of this
trend is a focus on soil ‗health‘, with attention to biological components (e.g. ‗soil
food web‘) as well as physico-chemical attributes (e.g. soil aeration, cation
exchange capacity) (Elfstrand et al., 2007; Supanjani et al., 2006).
A wide array of formulations and products are now marketed in
developed countries in support of this focus on soil ‗health‘. Typically, these
products are biological amendments that are applied with the aim of restoring and
regenerating soil microbial communities, with typical claims including action as a
bio-fertiliser, a bio-decomposer and/or a disease suppressor (Antonio et al., 2008;
1
Elliott & Lynch, 1994; Girvan et al., 2004; Grayston et al., 2004; Hall et al.,
2006; Liu et al., 2007). However, documentation of the efficacy, and more
specifically the mechanism of action, of these products is typically very poor.
One class of such products are ‗compost extracts‘, which are commonly
known as ‗compost tea‘. Production of ‗compost extract‘ has progressed from a
simple, anaerobic extraction process to an ‗aerobic bio-fermenter‘ approach,
expanding from small, simple on-farm activity (cow manure soaking in a 44
gallon drum) to large, relatively elaborated activity (e.g. specialised incubating
and aeration apparatus, addition of microbial food sources, enhancement with
specific microbial strains, centralised production with distribution to farms, and
sophisticated distribution/application systems on farm). The term ‗microbially
enhanced extract‘ is thus technically more correct, and the term ‗compost extract‘
is thus simplistic. However, for convenience, the term ‗compost extract‘ (CE)
will be used in this thesis.
Compost is bulky and hence is costly to transport. Use in broad-acre
production is, therefore, challenging and often replaced by ‗green manuring‘ (i.e.
growth of a crop to be left in the field to decompose). Compost extract can be
seen as a value-add to compost. As a liquid, it is easily applied. Further, compost
can only be applied to the soil, while compost extract can be applied to the soil,
foliage and plant remains, either by drenching or by spraying (Litterick et al.,
2004; Scheuerell & Mahaffee, 2004; Weltzein, 1992; Yohalem et al., 1994).
Compost extracts are gaining popularity, particularly, among those who
are seeking substitutes to agrochemicals (Bess, 2000; Touart, 2000). Organic
farmers are paying more attention to compost extract because of its claimed
disease suppressive properties and the lack of standard disease management tools
2
in the organic sector (Scheuerell & Mahaffee, 2002; Siddiqui et al., 2009).
‗Mainstream‘ growers are also looking for alternative approaches to the use of
petrochemical-derived fertiliser products, as prices soar due to increasing global
demand and limited supply. As an example, the US Department of Agriculture
reported a 65% increase in price of synthetic fertiliser, such as nitrate, potassium
and phosphate over 12 months from date to date (Etter, 2008).
However, there is relatively little verification of these claims
within the scientific literature (as reviewed later in this thesis). This situation is
likely due, in large part, to the variation in the product (effectively each compost
extract is a unique biological community), to the scope of the claims (from
disease suppression to soil phosphate solubilisation), and to the time frame that
may be required (multiple years).
The aim of this review is to summarise reports on the efficacy and mode
of action of compost extracts in agriculture.
2. Soil ‘Health’
Soil health has been defined as the capability of soil to act as a living
system, sustaining life (plants, animals including microorganisms) and
maintaining air and water quality (Doran & Zeiss, 2000). Ask a farmer to define a
‗healthy soil‘, and she/he will speak of the feel and smell of the soil.
Plants are autotrophic, that is, they require only sunlight, CO2, water and
the essential mineral elements. In hydroponic systems these mineral requirements
are typically completely met by supplying them in a soluble inorganic form. In
soil, there is a reserve pool of these minerals in both inorganic and organic
3
insoluble forms. Soil biota are essential to functions of mineralisation of organic
matter and solubilisation of inorganic minerals.
Plants have evolved functional linkages with certain groups of soil biota.
For example, the mycorrhizal symbiosis that exists between plants and certain
ascomycete and basidiomycete fungi is well established to improve plant access
to soil nutrients, particularly P (Finlay & Rosling, 2006; Nehls, 2008). The
functional linkage between some plant species and certain N2 fixing bacteria is
also well established. This linkage involves a tight symbiosis in some cases, for
example, legumes with rhizobia, casuarina with frankia, cycads and azolla with
blue green algae, and a looser commensal relationship in other cases (e.g.
enhanced populations of free living N2 fixers, such as Azotobacter and
Azospirillum are examples of microbiota found in the rhizosphere or phylloplane)
(Kahindi et al., 1997; Unkovich et al., 2008).
Other soil microbiota are not directly associated with plants, but
nevertheless contribute to processes that favour plant growth. Examples of
organisms that contribute to mineral solubilisation in the soil include the
phosphate solubilising bacteria (Ekin, 2010; Rodríguez & Fraga, 1999).
Examples of organisms that contribute to organic matter breakdown, and thus
nutrient availability through mineralisation include bacteria (e.g. Nitrococcus,
Nitrobacter), fungi (e.g. Rhizopus, Fusarium spp.), flagellates, amoebae and
nematodes (Bloem et al., 1994; Bourne et al., 2008). Other microbiota are
considered beneficial through an antagonistic effect on the growth of pathogenic
organisms (e.g. Dubeikovsky et al., 1993; Hoitink et al., 1997; Siddiqui et al.,
2008a).
4
Thus, soil biota are important to ecosystem function, and to that subset of
ecosystems, agricultural systems. One cause of decreased crop yields over time,
that requires increasingly greater chemical inputs, may be farming practices that
have led to a decrease in soil biota. For example, it is concluded that ‗long fallow
disorder‘, wherein a poor crop yield is obtained after a long fallow period, is
linked to a loss of soil biota (Thompson, 1996; Thompson, 1987). Heavy use of
inorganic fertilisers and synthetic biocides are believed to result in reduced
populations of beneficial soil microorganisms, for example, by creating excess
salt and nitrates in the soil (Abbott & Murphy, 2003; Lampkin, 1990). This loss
of soil microbiota is considered to result in a greater dependency on the chemical
inputs.
However, while loss of microbial diversity, is generally associated with a
decline in soil ‗health‘ (Swift et al., 1996), this is not always so. Further, it is
necessary to separate ‗cause‘ and ‗effect‘ between levels of soil microbiota
(density and diversity) and soil fertility/plant growth.
For example, no difference was reported in soil microbial biomass and
microbial diversity between soils differing in intensity of use (e.g. Jesus et al.,
2009; Wardle et al., 1999). Jesus et al. (2009) analysed soil samples (0-20 cm
depth) collected from sites cultivated with crops (maize, banana, cassava,
pineapple and sugarcane), pasture, agroforesty (peach palm, cupuassu, pineapple,
banana and coffee), young and old secondary forest (aged 5 and 5-30 years old,
respectively) and compared with the primary forest, all of which were highland
forest originally. They found that the bacterial diversity, assessed by terminal
restriction fragment length polymorphism (T-RLFP) followed by cloning and
sequencing, did not reduce when forest was converted to pasture and crops, or
5
when cleared land was returned to forest. In fact, they found more diverse
communities in pasture compared to the primary forest. Wardle et al. (1999)
investigated three levels of agricultural intensification, namely, (i) least intensive
cultivation (i.e. hand-hoeing), (ii) repeated cultivation (i.e. cultivating between
the maize rows for weed control) and (iii) herbicide application (rimsulfuron
followed by primisulfuron) and mulching (with 10 cm thick layer of non-treated
pine sawdust) in two sites. They did not find much variation in microbial biomass
and activity between treatments, although mulching was noted to improve soil C
compared to other treatments in both sites.
A recent interesting development of a formal assessment system for soil
‗quality‘ has been the Grains Research and Development Corporation (GRDC)
supported development of a website (www.soilquality.org.au). It allows
comparison of input data of physical (e.g. bulk density), chemical (e.g. lime
benefit), and biological (e.g. organic matter biomass, yield potential) properties of
a given region. While currently established for Western Australia, the intent is to
extend this service to the cropping lands of other Australian states. The characters
used to define soil biology are total organic C, labile C, microbial biomass C, soil
microbial activity (as indexed by soil respiration), soil N supply, presence of
pathogenic fungi Rhizoctonia or take-all, and nematode populations.
3. Organic inputs into agricultural systems
During the past century, the invention of synthetic fertilisers, herbicides
and pesticides has revolutionised the farming industry. However, indiscriminate
use of ‗modern‘ management practices has had negative consequences on the
ecology of agriculture (Danielle & Rai, 2006).
6
Soil organic amendments within cultivated agricultural ecosystems have a
strong influence on the overall structure and function of soil microbial
communities (Buckley & Schmidt, 2001; Steenwerth et al., 2003), resulting in a
higher mineralisation and solubilisation capacity than for a soil to which mineral
fertiliser only is applied (Gerhardt, 1997; Liang et al., 2005; Marcote et al., 2001;
Melero et al., 2006; Pascual et al., 1998; Rao & Pathak, 1996). However, the
influence of these inputs depend largely upon type and quantity of amendment
(Barzengar et al., 2002; Nelson & Oades, 1998). For example, while the likely
impact of large quantities of green manure or compost (representing both a
microbial food and inoculum source) to a soil on soil microbiota levels and
diversity is obvious, the impact of a small volume of a microbial culture over a
large area of land is less obvious.
Organic farming involves crop production without using synthetic
chemicals or most inorganic fertilisers (Winter & Davis, 2006). Proponents of
organic farming believe that organic farming is environmentally benign and
organic products have increased nutrient content and can improve human health
(Bourn & Prescott, 2002). Organic farming is highly reliant on the biological
fertility of the soil as a determinant of soil physical and chemical fertility (Abbott
& Murphy, 2003; Bulluck et al., 2002; Ferreras et al., 2006). The crux of this
farming practice is thus ―soil health‖, and a major theme of organic agriculture is
to build a diverse microbial community in the expectation of maximising nutrient
use efficiency in the soil (Giller et al., 1997; Janvier et al., 2007; Moeskops et al.,
2010). Management practices, such as addition of organic matter, maintaining
plant diversity, reduced tillage and adding soil amendments may benefit soil
biology and are commonly employed to improve soil health (Berner et al., 2008;
7
Cotxarrera et al., 2002; Erhart et al., 1999; Lee & Pankhurst, 1992; Pe´rez-
Piqueres et al., 2006; Rincon et al., 2006). These practices enhance nutrient
availability, soil physical structure and soil microflora (Canullo et al., 1992;
Midmore, 2006; van Elsas et al., 2002). The use of compost extract is another
potential management tool.
4. Composting
Compost extract quality is dependent on the quality of compost used
(Carpenter, 2005). Composting can be viewed as an organic waste reduction
process (Eiland et al., 2001), but its greater value lies in the use of compost as a
soil conditioner containing soluble mineral nutrients, organic matter, including
humic acids, and beneficial microorganisms (Leu, 2006). Compost quality is
typically determined by the absence of viable weed seeds and disease-causing
organisms (Jones & Wyatt, 2005), by its nutrient content (e.g. soluble and total
N) and by its physical structure. The Australian standard for compost quality lists
the following specifications: pH 5.0-7.5; EC 0-1 dS m-1; organic matter content ≥
25% dry matter; total N ≥ 0.8% dry matter; total P ≤ 0.1% dry matter, if for P
sensitive plants, and soluble P ≤ 5 mg L-1, if for P tolerant plants and C:N ≤ 22
(AS4454, 1999). While microbial diversity and total microbial load is a quality
factor, it is not assessed.
Composting is a process of usually aerobic decomposition of organic
matter by microbiota, producing a stable organic end product, with carbon-
dioxide, water and heat as by-products (Rynk et al., 1992). This process is an
extension of that occurring in natural ecosystems (e.g. forest floor leaf litter
decomposition). The major factors that influence compost quality include
8
materials added (C/N ratio, particle size, fibre and lignin content), and
temperature, aeration, pH, and moisture (Avnimelech et al., 2004). The main
biota in compost include bacteria, fungi, protozoa, nematodes and rotifers
(Ingham, 2003).
As a generalisation, fungi will dominate, relative to bacteria, in compost
containing a relatively high proportion of cellulose and lignin (Pe´rez et al., 2002;
Tuomela et al., 2000). Typical bacterial genera present in compost include
Azotobacter, Nitrobacter, Azospirillum, Pseudomonas, Proteobacteria,
Actinobacteria and so on (Tang et al., 2007). Typical fungal genera present in
compost include Fusarium and Trichoderma.
The windrow or pile system is the most common form of composting. The
recommended size for manure based windrow is 1.5 m in height and 2.5 m wide
at the base (Rynk et al., 1992). Composting typically involves three different
phases, namely, (i) mesophilic, (ii) thermophilic and (iii) maturation, the first
being a moderate temperature phase (0-45˚C) that lasts for a few days, the second
represents a high temperature phase (50-60˚C or above) that persists for a few
days to several months, and the last, a cooling phase (to ambient temperature)
that lasts for several months (Rynk et al., 1992). During the second phase, when
temperatures are typically at or over 55˚C, most weed seed and human and plant
pathogenic microorganisms are destroyed (Grundy et al., 1998; Suarez-Estrella et
al., 2003). For good compost production, the temperature should be maintained at
57-68˚C, and always below 70˚C, during this phase (Rynk et al., 1992). High
temperatures, greater than 70˚C result in a decline in microbial activity (OSUE,
1999).
9
Aeration is typically achieved by structural means (e.g. inclusion of
coarse organic materials into the pile to provide aeration pathways) and by
practices, such as frequent turning of the pile (Tiquia, 1996). Compost
temperature, an index of aerobic decomposition, is monitored to determine the
frequency of aeration (Michel et al., 1996a; Tiquia, 1996). Turning frequency
also depends on decomposition rate, porosity and moisture of the composted
materials (Tiquia et al., 1996), and the size of the pile (Michel et al., 1996b).
During the thermophilic phase, daily turnings may be required for easily
decomposable mixes high in nitrogen, while the frequency of turning decreases,
for example, to weekly or fortnightly turnings as the pile matures (Misra et al.,
2003; Rynk et al., 1992). The windrow pile is usually turned if temperatures fall
below 48˚C so as to stimulate aerobic decomposition (Misra et al., 2003). If
compost dominated by fungal growth is desired, less turning is required than for
bacterial compost as fungal growth is inhibited by frequent turning (Rynk et al.,
1992).
Aerobic composting occurs at a broad range of pH (5.5-9.0), however the
preferred range for pH is 6.5-8.0 (Rynk et al., 1992). The ideal bulk C:N ratio for
active composting is 25:1 to 30:1; with ranges as low as 20:1 acceptable (Rynk et
al., 1992). If this ratio is above 40:1, a longer composting period is required, and
there is the risk that, on application, the compost will immobilise soil N. If the
ratio is too low, nitrogen will be lost in the form of ammonia, resulting in
unpleasant odours.
Anaerobic composting, e.g. Japanese composting system is an efficient
way to decompose organic wastes using microbial inoculants, such as species of
lactic acid bacteria, photosynthetic bacteria, yeasts, and Actinomycetes,
10
producing ―bokashi‖ as an end product (Xu, 2000). This composting makes use
of anaerobic (oxygen free) process that relies on fermented enzymes for enhanced
decomposition of organic materials in one or two weeks. It produces no odours,
enhances crop growth and restores the microbial activity and fertility in heavily
depleted or nutrient poor soils.
Generally, the moisture content in composting materials should be
maintained between 40-65% (Misra et al., 2003). Moisture is necessary not only
for supporting microbial activity and metabolism but also for nutrient transport
(Shi et al., 1999). A moisture level below 45% results in slowed microbial
activity whereas at moisture levels above 65% pore spaces are occupied with
water making the process anaerobic (Rynk et al., 1992). Post production, compost
should not be over dried (i.e. less than 45% moisture) because microbial activity
will be lost, dust will be generated while handling, and it will be difficult to re-
wet the compost (Moon, 1997).
Inadequate protection and improper storage (e.g. open air storage) of
compost can subsequently result in pathogen contamination (Deportes et al.,
1995). Various groups of pathogens may exist in composted raw materials of
various origins (e.g. manure, sewage sludge, green waste and municipal solid
wastes). Such pathogens are mainly of faecal origin (Deportes et al., 1998), and
the group of pathogens such as E. coli, Clostridium botulinum, Salmonella,
Bacillus cereus, and Classical Swine Fever Virus are mainly responsible for
spreading human and animal diseases (Jones & Martin, 2003).
Effectively any organic material can be composted, although ‗home‘
producers generally avoid animal flesh and fats due to the potential to attract
other animals, for fly strike and for development of off-odours. The key to
11
consistent compost quality is consistent inputs. Given compost is a relatively low
value and bulky material, production from locally available materials is essential
to minimise transportation costs. Typical materials are plant-based (leaves, bark,
crop residues, grass clippings, paper wastes and food processing wastes) and
animal based (cattle manure, horse manure, poultry manure, fish processing
wastes, sewage sludge, slaughterhouse wastes).
Raw input material is typically screened to remove unwanted material,
and shredded to reduce particle size. The final compost is also often screened, to
remove clumps of compost, stones and uncomposted materials (Rynk et al.,
1992).
Unlike a chemical fertiliser, compost is not static for it contains live
microbes (bacteria, fungi, nematodes, protozoa). Thus, physical and chemical
properties of compost change continuously. Generally, plant-based composts are
ready in two to six months. Well aged compost will have microbial populations in
a plateau growth phase, and thus in a relatively static phase (Reeve et al., 2010).
Commercial sale of compost (e.g. bagged compost in retail stores) should be well
aged compost.
5. Compost extract
5.1. Definition of compost extracts
‗Compost extract‘ and ‗compost tea‘ are colloquial and ill-defined terms,
although there have been attempts to better define the term (e.g. Ingham, 1999b).
The dark brown coloured solution that leaches from a pile of compost may be
defined as compost leachate, while the solution obtained after soaking a compost
(either as a slurry or contained in a sack, the latter to remove particulates that
12
would foul irrigation or misting systems if to be used in that manner) in water,
typically for 2 to 21 days (e.g. Weltzein, 1989), may be defined as compost
extract (Diver, 2002). A water-based preparation of compost, incubated with
various microbial food sources (such as molasses, fish and kelp hydrolysate), and
in some cases inoculated with specific microbiota, is best defined as a
‗microbially enhanced compost extract‘.
Compost extracts contain living biota, insoluble organic matter, soluble
nutrients (such as ammonium and phosphate) and organic compounds, such as
proteins, amino acids, carbohydrates, sugars, plant growth regulatory compounds,
siderophores, tannins, phenols and humic acids (Antonio et al., 2008; Dianez et
al., 2006; Ingham, 2000). These components can stimulate plant growth and
suppress soil-borne pathogens (Ingham, 2005; Joshi et al., 2009; Litterick et al.,
2004; Scheuerell & Mahaffee, 2004).
5.2. Production methods of compost extract
Principal production variables include choices of inoculum (compost
feedstock, age), inoculum:water ratio, water quality, added nutrients, incubation
time, solution oxygen level, temperature and pH (Alms, 2002).
Self-evidently, the microbial population of a compost extract will be
dependent on that of the inoculant compost. This consideration extends to the age
of the compost.
Compost extract can be produced by direct addition of a source of
inoculum, typically compost, to water amended with microbial food sources.
However, the resultant product will require filtering to remove particulate matter
to prevent clogging of emitters or spray nozzles (Weltzien, 1991). This issue can
13
be avoided by containing the compost in a permeable bag (e.g. made of muslin,
cotton cloth, nylon stockings or similar) (Ingham, 2005). Following incubation,
the solid materials of the remaining compost can be returned to the composting
pile (Campbell, 2006) or used as a soil amendment.
The compost to water ratio (w/w) utilised in published scientific studies
involving compost extract ranges from 1:1 (Zhang et al., 1998) to 1:50 (Weltzien,
1990). Many studies have followed the method developed by Weltzien‘s
laboratory, employing 1:3 to 1:10 ratios (Haggag & Saber, 2007; Scheuerell &
Mahaffee, 2004; Welke, 2000). Given the high concentration of inoculum to
water, this method may be described more in terms of microbial extraction rather
than microbial population growth. Commercial production of compost extract
may involve considerably lower compost to water ratios (e.g. SmartBugs Pty Ltd,
Lismore, Australia) recommends use of their LivingSoilTM inoculum at a rate of
10 g/L or 1:100). If the compost:water ratio is too low, there is a risk that
contaminating organisms may overwhelm the culture. There is little published
work on the effect of dilution ratio on plant health. For example, in a container
medium study, Scheuerell and Mahaffee (2004) observed significantly reduced
suppression of seedling damping-off disease caused by Pythium ultimum at
higher dilutions (1:9, 1: 4, 1: 1) compared to the neat compost extract, i.e. at 1: 0
dilution. The effect of dilution ratio on the community of extracted and cultured
microorganisms should be studied further.
Typical additions made with the intent of acting as microbial food source
include a carbohydrate source (e.g. molasses) and a N source (e.g. baker‘s yeast,
sea kelp or fish hydrolysate) (e.g. Ingham, 1998). These materials are typically
added during incubation (to support microbial population growth in the culture).
14
Similar additions may be made just before application to the crop, or as a
concomitant application to the crop, to support microbial growth in the crop
environment (Scheuerell & Mahaffee, 2002). Molasses is commonly used in
Queensland and northern NSW where this carbohydrate source is widely
available and rather economical. Generally, molasses is added at a concentration
of 0.5 to 2% to a compost extract batch. Elsewhere, other sugar sources may be
used, for example, Sackenheim et al. (1994) reported use of 3% sucrose (in both
aerated and non-aerated compost extracts).
Further, additional inorganic and organic additives, such as humic acid
and rock dust, may be included with the aim of enhancing microbial growth. For
example, addition of rock dust and humic acid to aerated compost extract has
been reported to enhance the effectiveness of the compost extract in managing
plant disease caused by Botrytis cinerea (Scheuerell & Mahaffee, 2006).
A typical guide to the quality of water required for compost extract
production is for water fit to drink, with rain water commonly used. Use of
surface waters (e.g. dam water) risks biological contamination of the compost
extract. Use of sterilised surface water or town water supply risks chemical
contamination of the compost extract. Where chlorine has been used for
sterilisation, vigorous stirring for about half an hour is recommended to de-gas
chlorine from water (Ingham, 2003).
The pH of the culture solution will also affect the growth and diversity of
microorganisms, with bacterial population growth favoured at neutral pH while
fungal (including yeasts) growth is favoured under acidic and alkaline conditions
(Ingham, 2003). In a large scale food composting, microbial activity as indicated
by the carbon-di-oxide production was inhibited by low pH below 6 during the
15
early phase of composting (Sundberg et al., 2004). Rousk et al. (2010) found that
pH has a significant influence on the phospholipid fatty acid compostion of
microbial community as the community changed along the pH gradient of soil,
showing a correlation of 0.97.
The microbial population composition of compost extract can also be
expected to be affected by the incubation temperature. Scientific publications on
the impact of variable temperature on microbial population and composition of
soil as well as on composting process are numerous (Feng & Simpson, 2009;
Herrmann & Shann, 1997; Holtan-Hartwig et al., 2002; Tang et al., 2007; Uvarov
et al., 2006). For example, Tang et al. (2007) found that the microbial community
pattern (DGGE) during the thermophilic and mesophilic composting processes
changed at different incubation temperatures of 60˚C and 30˚C. The authors
found a relatively lower population and diversity of microorganism, higher
biomass of Proteobacteria and lower biomass of Actinobacteria in the mesophilic
compared to the thermophilic compost.
Parallel studies on the effect of temperature on the microbiology of
compost extracts should be undertaken.
Compost extract can be produced under aerobic or anaerobic conditions,
with obvious consequences to the microbial community supported. The
anaerobic, or fermentation, method involves incubation of compost in water with
or without amendment and without extra supply of aeration (Diver, 2002),
producing non-aerated compost extract. Even without enclosure of the container,
anaerobic conditions will prevail through most of the vessel given the slow rate
of diffusion of oxygen through water. Fermentation for up to several weeks, with
occasional stirring, is practiced. Non-aerated compost extract is typically
16
produced using longer fermentation times than that used for incubation of aerated
compost extract, to allow for greater extraction (separation of substances from the
solid matrix to liquid) of nutrients and beneficial microbes.
This production of non-aerated compost extract is simpler than that of
aerated compost extract (avoiding the need to maintain aerobic conditions); but
the reduced oxygen condition will lead to nutrient losses, particularly through
volatilisation of N following denitrification. Further, the community of obligate
and facultative anaerobes produced in such a culture may have little relevance to
that of the aerobic soil. Also, phytotoxic materials, such as organic acids, phenols
and ammonia may be produced by the anaerobic microorganisms (Campbell,
2006; Ingham, 2005).
To produce aerated compost extract, the extract is aerated to maintain a
level of dissolved oxygen not less than 6 ppm (Ingham, 2005). The duration of
incubation is usually 2-3 days. Ingham (2000) reported that the optimum
incubation time should be characterised in terms of when the maximum active
microbial biomass is achieved, and indicated that this commonly occurs in 18-24
hours with commercial extract-making equipment and procedures.
As a batch culture, respiratory demand is not constant over the incubation
period. At peak respiratory demand, there is a risk of decreased oxygen levels in
the solution. Some operators (O‘Grady, 2008; pers. comm.) suggest that short
periods (presumably a few hours) of dissolved oxygen less than 6 ppm are not
detrimental to the development of the microbial community.
Aerobic incubators have become commercially available in recent years.
The basic components include the container (typically 60 to 5,000 L), fitted with
a drainage point that can be connected to a dispensing pump, an aeration system
17
serviced by an air blower, and an optional compost basket (Diver, 2002). Earlier
designs involved circulation of the extraction solution, with cascade systems
aiming to provide solution aeration, or circulation over the compost with the aim
of improving extraction. However, at present, improved designs to meet the high
oxygen demand of the culture are available in the market (e.g.
www.smartbugs.com.au, www.soilsoup.com, www.greenorganics.biz,
www.trustnature.com.au, www.composttea.com, www.cleanairgardening.com,
www.jollyfarmer.com). In general, current systems employ a much higher rate of
gas flow to achieve aeration, and this process serves the secondary purpose of
vigorously mixing the solution.
The rate of oxygen diffusion through water is 10,000 times less than that
through air. Efficient aeration systems will therefore maximise the surface area of
the gas-solution interface. This requirement drives the use of aeration systems
that produce smaller bubbles and that maximise the residency time of the bubble
in the solution column. Aquarium stones, as recommended, for example, by
Diver (2002), are typically inadequate to maintain the required rate of
oxygenation. Various aeration mechanisms have been recommended, for
example, vortex-mixing high pressure nozzle systems for entrainment of air into
the water stream (Ingham, 1999a), membrane diffusers (smartbugs.com.au) or
micro incubators fitted with a pump and hose (www.asapsupplier.com) to infuse
air into the compost extracts.
The air pumps required for these systems are characterised by a low head,
high volume specification. Few compost extract incubators would involve a
solution depth of more than 2 m (static pressure of 0.2 atm), and generally less
than 1 m. However, the aeration (bubbling) system may have a high resistance to
18
gas movement, necessitating use of a pump capable of delivering a higher
pressure.
In practice, the aerator is stopped for about half an hour to allow insoluble
materials to settle prior to using the extract (Campbell, 2006). At this point the
batch culture may enter a plateau phase in population growth. Population growth,
however, can be restarted by addition of food sources.
In the plateau phase, the culture maintains an (albeit lower) respiration
rate. Storage of this material without aeration therefore risks development of
anaerobic conditions and change in the species composition of the microbial
culture. It is therefore generally recommended that the compost extract be applied
as soon as possible after production, and if stored it should be in a well-ventilated
tank with adequate agitation (Bess, 2000). However, some users (e.g. O‘Grady,
2009; pers. comm.) suggest that amending the cultures to low or high pH might
cause the microbiota to enter a dormant phase, which may allow for storage or
long distance transport of the material. Soil Foodweb Institute
(www.soilfoodweb.com.au) suggests that such ‗put-to-sleep‘ extracts can also be
produced by prolonging incubation time or reducing the activity of microbiota
with some chemicals, however there are chances of losing around 50% of the
microbial diversity in this process.
Cleaning of incubators after incubation of compost extracts is important,
as residue left inside the unit may generate anaerobic conditions and favour
contaminating organisms such as Escherichia coli, Pseudomonas spp. (Alms,
2002). Hypochlorite, hydrogen peroxide and detergents can be used effectively to
clean the inside of the incubator (Ryan, 2003).
19
5.3 Defining compost extract quality
As noted previously, compost extract quality is dependent on the nature
and content of inoculum (compost) used (Carpenter, 2005), and also to conditions
prevailing during the incubation process. Unfortunately, there can be
considerable batch-to-batch inconsistency in compost extract composition
(Campbell, 2006; Ingham, 1999a). In general, a compost extract that has high
level of plant available nutrients and high species richness, evenness and total cell
number of beneficial microorganisms is desired, that is, when used to amend soil,
the compost extract should have organisms that play key functions in the soil.
While there is no officially recognised standard on what constitutes a
good quality compost extract, either in Australia or elsewhere, ‗private label‘
quality standards do exist.
In Australia, the Soil Foodweb Institute (Lismore, NSW;
www.soilfoodweb.com.au) offers a service in (biological) quality assessment of
compost extract and soils using both qualitative and quantitative methods.
Ingham (2005) has reported that compost extract can be qualitatively assessed in
terms of microbiota diversity and density by microscopic examination. A whole
mount preparation of compost extract is examined at 400X magnification, and
biota in up to ten fields of view assessed. The compost extract is assessed as poor
if no or very few microbes are present, good if more than 500 bacteria and at least
one fungal hypha are present in five fields, and very good if innumerable bacteria
and at least one fungal hypha are present in first five fields. If innumerable
bacteria and at least one fungal hypha, of rich species diversity, and high numbers
of protozoa (flagellates, amoeba and ciliates) and nematodes are present, the
compost extract is assessed as excellent. Soil Foodweb Institute (SFI) also offers
20
a quantitative assessment service. Viable cell counts are presumably established
using visualisation by epi-flourescence microscopy, with fluorescein diacetate
staining (e.g. Ingham, 2004). Quantitative assessment requires the assessment of
a known volume of sample and the bacterial and fungal biomass is presumably
estimated based on a calculation involving cell numbers and dimensions. SFI
measures a value of plant available N, presumably from the microbial biomass
figures. SFI also measures nematode density and species diversity to genus level,
as this is considered to index soil ecosystem health.
Quantitative analysis of soil microbiota following the application of
compost extracts at field level to index soil health is crucial to demonstrate any
effect of compost extract use.
6. Characterising microbial diversity
A range of techniques are available for the study of microbial diversity in
environmental samples. Perhaps the most commonly used of these techniques is
simple microscopic examination; the plate count method and the fluorescein
diacetate hydrolysis method are also in use at present. However, these traditional
methodologies may assess only a small fraction of the total microbial diversity
(Scheuerell, 2004). Such limitations of culture-based techniques underestimate
the total representation of microbiota because the microorganisms may remain in
dormant but viable states in the environmental samples but usually fail to grow
on Petri plates. Complex inter-dependencies between microorganisms in the
environmental samples cannot be generated in the laboratory. DNA based
methodologies, as discussed later, may offer potential for greater insight into the
microbial community (diversity) of compost extract.
21
The plate count method is a standard method for microbial enumeration,
generally expressed in colony forming units (cfu). This method can either be
spread plate or pour plate or by placing a membrane filter, after filtering the
tested material, on the plate surface. The preferred method is to serially dilute the
sample and to place the aliquot (usually 1 mL) onto the surface of the plates or to
pour the aliquot to the agar media to determine the cfu mL-1 (g) of the initial
sample.
The fluorescein diacetate (FDA) hydrolysis method is a simple and widely
used spectrophotometric method in which the enzymatic activity of
microorganisms is measured. This approach provides an estimate of total
microbial activity (Adam & Duncan, 2001; Green et al., 2006; Schnurer &
Rosswall, 1982). The assay is sensitive to the activity of several ubiquitous
enzymes, such as lipase, esterase and protease, and thus is considered as non-
specific. Activity of these enzymes results in the hydrolysis of FDA releasing
fluorescein (Fig. 1.1) which can be measured spectrophotometrically.
Figure 1. 1. Enzymatic conversion of FDA to fluorescein (Source: Adam & Duncan, 2001)
This technique has been widely used with environmental samples, for
example, soil (Schnurer & Rosswall, 1982); activated sludge (Fontvieille et al.,
1992); river sediments (Marmonier et al., 1995); and in compost (Ntougias et al.,
2006). This technique should therefore be applicable to the estimation of
22
microbial activity of compost extract although there are other techniques
available for the measurement of enzymatic (e.g. dehydrogenase, urease,
phosphatase) and biochemical (e.g. ergosterol) activities of various environmental
samples.
Techniques based on microbial culture on a range of food sources have
also received great attention in recent years to characterise microbial community
diversities from a range of ecological samples. BiologTM Inc, USA offers a wide
range of single C sources supplied in 96 well plates, and based on their utilisation
pattern, microbial functional diversity is estimated (Preston-Mafham et al., 2002).
To measure the community level physiological profile (CLPP), which is
the metabolic fingerprinting of the microbial community, Biolog Ecoplate and FF
microplate are common in use. The Biolog Ecoplate has 96 wells consisting of 31
single C sources and the control, replicated three times, in addition to a
tetrazolium dye. The Biolog Ecoplate is specially designed for measuring
functional diversity of the bacterial community. The 31 C sources are divided
into six groups of carbohydrates (7), carboxylic acid (9), polymers (4), amino
acids (6), amines (2) and miscellaneous substrates (3). The utilisation of any C
source by the bacterial community results in the respiration dependent reduction
of tetrazolium dye and purple colour formation that can then be quantified and
monitored over time.
Similarly, the FF microplate is designed for measuring fungal functional
diversity utilising 95 single C sources, which were divided into six groups of
carbohydrates (44), carboxylic acid (17), polymers (5), amino acids (13), amines
(6) miscellaneous substrates and (10) the control (without any C sources). Here,
iodonitrotetrazolium violet is used as a redox dye to colorimetrically indicate the
23
mitochondrial activity during the oxidation of C sources producing reddish
orange colour (Insam, 1997). The carbon substrates found in the Ecoplate and FF
microplate from BiologTM, respectively, divided into different substrate guilds
according to Preston-Mafham et al. (2002), are presented in Table 1.1.
24
Table 1. 1. Carbon substrates of Ecoplate and FF microplate (BiologTM), respectively divided into different substrate guilds according to Preston-Mafham et al. (2002).
Carbohydrates Carboxylic acids Polymers Amino acids Amines/amides Miscellaneous

-Methyl-DGlucoside D-Galactonic Acid ã-Lactone Tween 40 L-Arginine Phenylethylamine Pyruvic Acid Methyl Ester
D-Xylose Dgalacturonic Acid Tween 80 L-Asparagine Putrescine Glucose-1-Phosphate
i-Erythritol 2-Hydroxy Benzoic Acid  -Cyclodextrin L Phenylalanine D,L--Glycerol Phosphate
D-Mannitol 4-Hydroxy Benzoic Acid Glycogen L-Serine
N-Acetyl-DGlucosamine -Hydroxybutyric Acid L-Threonine
D-Cellobiose D Glucosaminic Acid Glycyl-Lglutamic Acid
 -D-Lactose Itaconic Acid
 -Ketobutyric Acid
D-Malic Acid
N-Acetyl-DGalactosamine D-Mannose D-Galacturonic Acid Tween 80 -Amino-butyric Acid D-Glucosamine Amygdalin
N-Acetyl-DGlucosamine D-Melezitose D-Gluconic Acid  -Cyclodextrin L-Alanine Glucuronamide Glucose-1-Phosphate
N-Acetyl-DMannosamine D-Melibiose D-Glucuronic Acid -Cyclodextrin L-Alanyl-Glycine Succinamic Acid Glycerol
Adonitol  -Methyl-DGalactoside 2-Keto-D-Gluconic Acid Dextrin L-Asparagine Alaninamide Salicin
D-Arabinose -Methyl-DGalactoside Fumaric Acid Glycogen L-Aspartic Acid 2-Amino Ethanol Bromosuccinic Acid
L-Arabinose  -Methyl-DGlucoside -Hydroxy-butyric Acid L-Glutamic Acid Putrescine D-Lactic Acid Methyl Ester
D-Arabitol -Methyl-DGlucoside -Hydroxy-butyric Acid Glycyl-L-Glutamic Acid Succinic Acid Mono-Methyl Ester
Arbutin Palatinose p-Hydroxyphenylacetic Acid L-Ornithine Adenosine
D-Cellobiose D-Psicose  -Keto-glutaric Acid L-Phenylalanine Uridine
i-Erythritol D-Raffinose L-Lactic Acid L-Proline Adenosine-5'-Monophosphate
D-Fructose L-Rhamnose D-Malic Acid L-Pyroglutamic Acid
L-Fucose D-Ribose L-Malic Acid L-Serine
D-Galactose Sedoheptulosan Quinic Acid L-Threonine
Gentiobiose D-Sorbitol D-Saccharic Acid
-D-Glucose L-Sorbose Sebacic Acid
m-Inositol Stachyose Succinic Acid
-D-Lactose Sucrose N-Acetly-Lglutamic Acid
Lactulose D-Tagatose
Maltitol D-Trehalose
Maltose Turanose
Maltotriose Xylitol
D-Mannitol D-Xylose
25
Serially diluted environmental samples are incubated to each well of the
Biolog Ecoplates and FF microplates for a few days and optical densities are
measured at 24 h intervals to obtain the kinetics of average well colour
development (AWCD) of bacterial and fungal communities. The AWCD and
optical density (OD) are calculated for all different C substrates utilised in
Ecoplate and FF microplates before statistically analysing the data (Garland,
1997).
DNA-based methods have enormous potential to underpin knowledge of
structural and functional characteristics of microbial communities. Such methods
have been employed widely within the field of microbial ecology to analyse
diversity and composition of microbe communities. Most of these methods rely
upon the use of polymerase chain reaction (PCR) using group specific primers
associated with selected ribosomal DNA (rDNA) regions followed by digestion
with endonuclease restriction enzymes and sequencing of selected ribosomal
DNA (rDNA) regions (Dees & Ghiorse, 2001; Ntougias et al., 2004). These steps
are followed in amplified ribosomal DNA restriction analysis (ARDRA)
(Koschinsky et al., 1999), a commonly used fingerprinting method, which allows
grouping of the organisms under study by simple restriction endonuclease
digestion of PCR products.
Denaturing gradient gel electrophoresis (DGGE) (Muyzer et al., 1993) or
temperature gradient gel electrophoresis (TGGE) (Muyzer, 1999) are also used in
this context. The following steps are as for ARDRA, with cloning and sequencing
allowing phylogenetic analysis. The phylogenetic affiliation of the predominant
community members, as represented in the DGGE band pattern, can be inferred
by comparative analysis of sequences from excised and re-amplified DNA
26
fragments (lanes 2-7 as shown in Figure 1.2) and sequences stored in nucleotide
databases (Muyzer, 1999).
To investigate the genetic diversity of the microbial populations (at
species level) in the environmental samples, DGGE followed by cloning,
sequencing and phylogenetic analysis of 16S rDNA is the most commonly used
approach (Fig. 1.2). DGGE has been used mostly in sediments and soils for
complexity and community change studies (Muyzer & Smalla, 1998; Postma et
al., 2008). DGGE has generally been used to analyse the genetic diversity of
bacterial populations in environmental samples (Muyzer et al., 1993), but the
technique has also been applied to fungal communities (Anderson, 2003; Jany &
Barbier, 2008). While there are some studies on microbial diversity in compost
using DGGE (Adams & Frostick, 2009), investigation of diversity in compost
extracts, and the impact of extracts on soil microbial diversity, is warranted.
27
Figure 1. 2. Flow diagram showing the different steps in the analysis of microbial community
structure by PCR-DGGE. DNA is extracted from an environmental sample and used as a template
to amplify (PCR) the 16s rRNA encoding genes of microorganisms (e.g. bacteria). Thereafter, the
PCR products are separated by denaturing gradient gel electrophoresis (DGGE), which is
followed by cloning, sequencing and phylogenetic analysis of 16S rDNA.
For DGGE analysis, total DNA extraction is followed by polymerase
chain reactions (PCR) using universal primers and a thermocycler (Girvan et al.,
2003). The PCR-amplicons are subjected to agarose gel electrophoresis and
visualised after staining under UV irradiation. Following electrophoresis, the
DGGE gels are silver stained (Girvan et al., 2003) and scanned using advanced
analysis package. The UPGMA (Unweighted Pair Group Method with Arithmetic
mean algorithm) dendrogram is generated and the digitised image is analysed to
express the relatedness of microbial communities as similarity clusters. The
Shannon-Weaver diversity index, H‘ (Shannon & Weaver, 1963) and Equitability
index, J (Begon et al., 1990) are calculated from the number and intensities of
bands present in each lane with the following equations: H‘ = -∑(piLNpi), where,
pi = ni/N, ni is the intensity of the band i, N is the total intensity of all bands in
28
the lane and LNpi is the natural log of pi; J = H‘/(LNni), where H‘ is the
Shannon-Weaver diversity index and LNni is the natural log of the total number
of species in a lane. The Shannon-Weaver diversity index is thus a measure of
biodiversity which takes account of the number and evenness of species in the
community profile, while the Equitability index rates only the overall evenness of
band intensities and the S value the number of bands per sample (Dilly et al.,
2004; Krebs, 1999). The DGGE generated bands can be excised from the gels,
sequenced, and tested up to species level to determine their phylogenetic
associations with the database (e.g. www.ncbi.nlm.nih.gov) of GeneBank (Jing et
al., 2010).
7. Current use of compost extract in central and southern Qld
and northern NSW
The use of compost extract is accepted within the organic farming
community, and, in something of a social phenomenon, is gaining acceptance
amongst conventional broadacre farmers and some graziers in central and
southern Qld and in northern NSW. This trend is prompted by farmer interest in
soil health and by the rising price of fertilisers. Users report that they can cut
costs without compromising yield. It appears that a dramatic rise in the rate of
uptake of this technique will occur over the next few years.
Users of compost extract in central Queensland and northern NSW
include certified organic horticultural small crop producers on the Capricorn
coast (generally small scale), broadacre wheat/sorghum/chickpea and cotton
producers of the central highlands, and graziers (Table 1.2).
29
Table 1. 2. List of farmers [permissions granted to use their data] from Qld and NSW visited with Tom Nicholas who are using compost extracts in different crops, and their
claimed benefits (2008).
Area of Rate of Method of Frequency of

Farmer/Contact Crops production application Dilution application application/year Claimed benefits
Lex Webb, Baralaba, QLD Wheat in rotation to 2500 ha Compost extract - Foliar spray and - Improves growth and yield
Tel: 0428581162 sorghum / mungbean @ 30 L/ ha liquid injection
E-mail: belvedere@bluemax.com.au CalSap @ 3L/ha
Paul Murphy, Capella, QLD Spelt wheat and 900 ha Compost extract 20% water addtion Seed priming Once Improves root growth, soil carbon
Tel: 049816767 / 0428816768 bread wheat @ 8 L /ton of seeds to the concentrate and microbial population
E-mail: murphyfarming@bigpond.com CalSap @ 2L/ha compost extract
from CQ Compost
Bryson Taylor, Clermont, QLD Sorghum, chickpea, 2200 ha Compost extract Concentrate compost Liquid injection Once -
Tel: 0749835365 / 0427125022 wheat @ 30L/ha extract obtained
E-mail: kilmacolm@bigpond.com from CQ Compost
Dave Daniels, Clermont, QLD Wheat and chickpea 2830 ha Compost extract Concentrate compost Seed priming Once Improves soil biology, release of
Tel: 0749835272 @ 30 L/ha extract obtained locked-up nutrients, improves soil
E-mail: daniels.travellon@bigpond.com UAN @ 20L/ha from CQ Compost structure, increases soil carbon,
CalSap @ 3L/ha better crop
Tom Nicholas, Clermont, QLD Fallow/field covered 2000 ha Compost extract - Liquid injection Once Better root system, grain quality
Tel: 0749835252 / 0428835463 with sorghum stubble @27 L/ha in seed furrow and better growth of plants
E-mail: tgnicholas@gmail.com CalSap @ 3L/ha
Stuart Larson, Mallanganee, NSW Cereals, soybean, 2040 ha Compost extract - Foliar spray Once Improved soil moisture
Tel: 61266645145 / 0427645171 barley, pasture @ 190 L/ha and organic matter,
E-mail: stuartlarsson@maraseeds.com.au cheap source of C,
cost effective
Geoff Brown, Spring Ridge, NSW Wheat, barley 1080 ha Compost extract 1:50 (w/v) Foliar spray Twice or Insect and disease control,
Tel: 0267473848 / 0428456088 @ 20-40 L/ha more low input cost, improvement
E-mail: goodgerwirri@activ8.bnet.au Molasses, fish emulsion, in growth and yield of crops,
liquid kelp and humic no residual chemicals
acid @ 2 L each/ha
Colin Seis, Winona, Gulgong, NSW Oats, cereal rye 840 ha Compost extract 1:500 (w/v) Liquid injection Once Yield comparable to chemical
Tel: 0263759256 @ 50 L/ha fertiliser, no incidence of
E-mail: colin@winona.net.au Molasses, fish emulsion, disease recorded in the last ten
liquid kelp and humic years, cost effective
acid @ 2 L each/ha
30
The increase in use of compost extracts is being supported by a range of
commercial suppliers of either soil biological amendments including compost
extracts, compost extract incubating tank designers and compost extract and soil
biological quality assessment services (Table 1.3). For example, CQ Compost Pty
Ltd (Emerald) is a major supplier of compost extracts and relevant amendments,
primarily to broadacre cotton/wheat/sorghum/chickpea growers. Compost
extracts are distributed in large volume tanks (1000 L) to farmers. BioNutrient
Solutions Pty Ltd, Moree, NSW was a previous producer of compost extract, but
ceased production because of difficulty in managing the liquid extracts (B.
Davidson, 2008; pers. comm.).
31
Table 1. 3. List of companies [permissions granted to use their data] visited with Tom Nicholas or contacted that were involved in producing compost extract and relevant
organic and biological products and/or providing services and their claims (2008).
Company/Contact Products/Services Claims
CQ Compost, Emerald, QLD Compost extract from compost made from Improves soil health
Tel: 0749876511 / 0427876643 wormcast
Earthlife Pty Ltd., Toowoomba, QLD Several agricultural products for garden, Increases soil organic matter, and soil carbon,
Tel: 07 4633 2219 horticulture and broadacre improves crop quality
E-mail: enquiries@earthlife.com.au
Web: http://www.earthlife.com.au
McLeod's Agriculture, Toowoomba, QLD Organic soil conditioner, zeolite, soil microbes, Improves soil aggregation, organic matter,
Tel: 1800 062616, 07 46993360/0419669115 liquid fertilisers nutrition, cut costs, adds biology in soils
Fax: 0746993359
E-mail: mcleodsagriculture@yahoo.com.au
Web: http://www.mcleodsorganicfertiliser.com/
Grazing BestPrac, Yeppoon, QLD Education and extension service aimed at Best- Encourages regenerative grazing practices to
Tel: 07 4938 3919 Practice for grazing - primary services include improve soil carbon and to improve soil health
E-mail: mick@grazingbestprac.com.au farmer training workshops and consulting by microbial management, produces compost
Website: www.grazingbestprac.com.au focussed on grazing management and strategies extracts and molasses extracts, develops
to develop healthy soils sustainable strategies and increases profits
BioNutrient Solutions Pty Ltd., Moree, NSW Bionutrient solutions, fertilisers, seed treatments, Improves soil aggregation, nutrition, organic
Tel: 0732711058 liquid humate and many other products matter, cut costs, adds biology in soils
E-mail: bart@bionutrient.com.au
Web: http://www.bionutrient.com.au
Soil Food Web Institute, Lismore, NSW Quantitative assessment for soil, compost and Suppresses foliar and root diseases, improves
Tel: 0266225150 compost tea (extracts) measuring biomass by plant nutrition, health, and crop yield
E-mail: contact@soilfoodweb.com.au direct count methods: bacteria, fungi, protozoa,
Web: www.soilfoodweb.com.au nematodes, mycorrhizal root colonization,
consultancy service
O'Grady Rural, South Lismore NSW Regenerative agricultural consultancy/education, Encourages regenerative agricultural practices to
Tel: 02 66216088 / 0429200492 produces and markets microbial nutrients and improve soil carbon and improve soil health
Email: rogrady@bigpond.net.au microbial fermentation equipment (e.g. compost
Web: www.smartbugs.com.au tea brewers), biochar and pandachar, conducts
farm-ready workshops to assist farmers achieve
these outcomes
32
8. Compost extract application and claimed benefits
Users and producers of compost extract have claimed benefits (e.g. Alms,
2002; Elad et al., 1996) in terms of enrichment of soil nutrient level and
enhancement of populations of specific soil microorganisms. Microbial
enhancement is claimed to provide nutrient turnover in the soil, suppress
diseases, remediate soil contaminants, increase soil C levels particularly through
accumulation of humates, improve soil physical structure, improve water holding
capacity and to increase anion/cation exchange capacity (Adams, 1990; Bess,
2000; Cook & Baker, 1983; Griffiths, 1965; Scheuerell & Mahaffee, 2002;
Touart, 2000).
Compost extract is commonly applied to the soil with the aim of
enhancing soil health. Application methods include soil drenching, targeting the
root zone (rhizosphere) of the plants, for example, using planters modified to
deliver a stream of compost extract into the planting furrow, and via irrigation
systems to established plants (Sebti, 2005). Soil application may be undertaken
with a view to suppress disease and/or to improve plant nutrition (Litterick et al.,
2004). Soil application of compost extract (10 L tree-1) plus four foliar
application of yeast (1%) and humic acid (0.5%) is reported to increase fruit yield
and quality with improvement in nutrition status of olive trees compared to the
control treatment (Fayed, 2010b).
Direct application of compost extract to plant foliage is also reported
(Elad & Steinberg, 1994; Ryan et al., 2005; Segarra et al., 2009; Weltzien, 1991).
Spraying is done with a broad range of sprayers, however, mist spraying is the
preferred method (Grobe, 2003). Foliar application is intended to alter the
composition of the microbial population on the leaf surface through nutrient
33
supplementation (Fayed, 2010a) and direct addition of microbes, and is generally
undertaken with a view to suppress disease (Ingham, 1999a). Application under
moist conditions has obvious relevance to microbial survival. Also, it is
recommended that compost extract be applied either in the morning or evening on
sunny days because the microorganisms are sensitive to UV light (Ingham, 2005).
The rate of compost extract application varies significantly according to
intended aim and mode of application (Ingham, 2005). For foliar application,
reported application ranges from 20-50 litres per hectare per application
(sufficient to wet both surfaces of all leaves). This further depends on the canopy
area of crops (Campbell, 2006). Four to six foliar applications may be made to a
crop during its growth period, however, frequency may increase with disease
infestation. S. Stevens (2008; pers. comm.) suggests that soil application of
between 150-200 litres per hectare per application is typical but lesser amounts
are used when compost extract is applied within the seed furrow. However, it is
recommended to apply sufficient volume of compost extract so that it reaches the
entire root zone (Scheuerell, 2003). Variables in application procedure include
the application equipment, dilution ratio, and use of spray adjuvant and the
concomitant application of a microbial food source.
The fundamental aim of compost extract incubation is to alter microbial
populations towards a more plant-beneficial community (Hoitink & Boehm,
1999a). The working principle of compost extract varies from that of the
synthetic chemicals in the way that compost extract adds microorganisms to the
soil and ‗brings the soil back to life‘ (Martens, 2001). There are also a range of
other biological amendments (e.g. direct compost addition, incorporation of green
34
manure crops) used with the aim of restoring and regenerating soil microbial
communities (Larkin, 2008).
Examples of specific claims include use for fertility management
(Scheuerell & Mahaffee, 2002) as compost extract is considered to add nutrients
(Reeve et al., 2010), to improve soil structure by building soil aggregates and to
increase water holding capacity of soils (Ingham, 2005). There is also a growing
body of published work on the efficacy of compost extract in plant disease
suppression (Brinton, 1995; Kai et al., 1990; Koné et al., 2010; Siddiqui et al.,
2008a; Zmora-Nahum et al., 2008).
The hypothesised benefits claimed by proponents of compost extract can
be categorised as follows:
 direct nutrient addition
 soil inorganic fraction solubilisation
 soil organic matter turnover (mineralisation)
 leaf litter and stubble decay
 increased free living N2 fixation
 suppression of plant diseases
 acquired systemic resistance
 bioremediation
 improved soil structure
 improved water holding capacity and
 improved soil C content.
35
8.1 Direct nutrient addition
Compost extract contains a range of soluble elements essential to plant
growth and yield enhancement (Fayed, 2010a; Ryan, 2003; Sebti, 2005; Siddiqui
et al., 2008b). This source will be significant to plant growth when compost
extract is applied at high rates, but is generally insignificant when compost
extract is applied in broadacre applications. When used in broadacre applications,
compost extract is generally used in ‗spot‘ applications, for example, when
applied with seed in the plant furrow. As such, it may have localised nutritive
value (e.g. supporting early seedling establishment).
Some examples of direct influence of compost extracts have been
demonstrated in some studies. For instance, the growth and performance of pak
choi plants were improved when compost extracts were applied as a soil drench
or foliar spray (Pant et al., 2009). The authors reported an improvement in an
uptake of both macro and micro nutrients (e.g. total N, P, K, Ca, Mg and S) as
well as greater total carotenoid content. Likewise, Sebti (2005) demonstrated
increased growth, yield and biomass of a tomato crop compared to the water
control and found that growth effects of compost extracts were comparable to
those of compost and organic fertiliser (Guanito) when applied through foliage or
through fertigation. Soil and foliar applications of compost extracts on
pomegranate trees at the rate of 5 L per tree combined with an antioxidant spray
of ascorbic acid and citric acid mix produced the highest yield with better fruit
quality as indexed by greater vitamin C, total sugar, total soluble solid and total
anthocyanin compared to that of other treatments.
36
8.2 Soil inorganic fraction solubilisation
A range of plant essential elements are present in insoluble inorganic
forms in the soil. Soil microbiota may assist in accessing this resource, effecting a
transfer from the insoluble to the soluble. Perhaps the best example of this
function is for phosphorus (P). Relatively large amounts of P can be locked up in
insoluble forms in the soil (e.g. calcium phosphate) limiting plant growth
(Raghothama, 1999). Indeed much applied (inorganic or organic) P is ‗lost‘ to
insoluble forms. Phosphate solubilising bacteria, such as Pseudomonas, Bacillus
and Rhizobium spp. and fungi, especially mycorrhizal fungi, can mobilise this
source (Rodríguez & Fraga, 1999). For instance, rhizosphere microorganisms
associated with phosphate solubilisation have been reported in the mangroves of
a semiarid coastal lagoon (Vásquez et al., 2000). Furthermore, the reported
growth stimulating action of several isolates of Trichoderma on beans has been
attributed to their roles in producing auxins, siderophores or by phosphate
solubilisation in the rhizosphere (Hoyos-Carvajal et al., 2009). The possible
mechanisms for converting phosphate into a soluble form involve acid
production, redox activity or chelation by metabolites released into the
rhizosphere (Altomare et al., 1999).
Similarly, strains of fluorescent Pseudomonas promoted growth of
various crops, such as rice, maize and sorghum, and demonstrated their potential
to be used as bioinoculants or biofertilisers (Suresh et al., 2010). These strains
were demonstrated to possess antifungal properties and to produce siderophores,
hydrogen cyanide, proteases and plant growth hormones, such as indole acetic
acid solubilise phosphate. These strains of varying potential for crop benefits are
likely to be present in compost extracts produced from various sources. Janzen et
37
al. (1995) reported that addition of a sterile extract of compost to a phosphate
limiting bacterial growth medium (at 10 ug C mL-1), sourced from
phosphogypsum, which is the residue of rock phosphate obtained during the
manufacture of phosphate fertiliser, increased microbial-P in a culture of
Azospirillum brasilense.
8.3 Soil organic matter decomposition
Soil microbiota play a central role in decomposition of organic residue in
soils, and the rate of this turnover can be increased by microbial enhancement
using compost extract (Ingham, 2005; Ryan, 2003). Some of the fungal species
known to aid in degradation of organic matter, and prevalent in most soils, are
Trichoderma spp. (Haaba et al., 1990) and cellulose digesting brown rot fungi,
such as Coniophora prasinoides, Coniophora puteana (Highley, 1980) and
Cellulomonas spp. (Lines-Kelly, 2004). Naidu et al. (2010) reported the presence
of several advantageous bacteria, such as lactic acid bacteria (production of lactic
acid), Bacillus spp. (biocontrol of disease and N2 fixation), and fungal species,
such as Penicillium spp. (food and drug production), Trichoderma spp. (disease
suppression and leaf liter decay), Aspergillus spp. (medical and commercial
production), yeast (nutritional and health benefits), Streptomyces spp. (disease
suppression through antibiotic production) in compost extracts. These beneficial
microorganisms are generally prevalent in composts and are likely to be water
extracted, with microbial multiplication possible with or without microbial
additives and/or aeration.
The microbial decomposition of soil organic matter can yield humates and
other organic fractions that improve soil physical structure and soil cation/anion
38
exchange capacity (Sparling, 1997). Further, the decomposition-mineralisation
process will make nutrients available to plants (Anderson, 2003; Bourne et al.,
2008).
8.4 Leaf litter and stubble decay
In agricultural production, nutrients are exported from the farm with soil
lost through erosion and in harvest products. For the latter, a wheat yield of 5t ha-
1
year-1 and 1% N will remove 50 kg N ha-1year-1 in grain. Nutrients, such as N,
get lost through gaseous emissions (e.g. nitrous oxide via nitrification and
denitrification), ammonia volatilisation and nitrate ions via leaching.
Soil microbial enhancement can promote the tapping of plant essential
elements from inorganic and organic pools within the soil. However, this equates
to ‗soil mining‘, and such pools will eventually be exhausted unless replenished
by inorganic or organic sources. Admittedly, however, the total reserve in the soil
can be very large for some elements (e.g. phosphorus, potassium, iron), capable
of supporting plant growth for many decades.
The issue of leaf litter and stubble decay is an extension of the soil
organic matter turnover, exacerbated by the dry conditions that often prevail
above-ground. Under dry conditions above-ground dead plant material is
essentially preserved. This is a particular challenge to CQ graziers who would
prefer standing grass biomass converted to soil organic matter rather than lost by
burning (O‘Grady & Alexander, 2008), but also applies to farming practices
involving no or low tillage, and elimination of fire (e.g. of cane harvest trash in
sugar cane production) (Hall et al., 2006). Loss of soil organic matter results in
the loss of microbial biomass, activity and diversity, contributing to soil
39
degradation in sugarcane burning systems prior to harvest (Dominy & Haynes,
2002; Dominy et al., 2002). Graham and Haynes (2005) found lower community
diversity in soils under the pre-harvest burning system compared to that covered
with sugarcane stubble following the green cane harvest. The decline in loss of
microbial diversity can, however, be curtailed either by ceasing burning or by
retaining the trash in the soil (Graham et al., 2002). Microbial diversity could also
be enhanced by applying microbially enhanced compost extracts in the soil over
harvest residues with the aim of accelerating the rate of residue decomposition
(Hall et al., 2006).
8.5 Free living N2 fixation
For N there is an additional potential benefit – that of N2 fixation by
certain bacteria. Rhizobium and Bradyrhizobium, the microsymbionts of the
legume N2 fixing symbiosis, are well known microbial enhancement products
adopted across conventional, broad scale agriculture (Kahindi et al., 1997).
However, there are also a range of free living N2 fixing bacteria (including
Azotobacter, Azospirillum, Nitrobacter) that occur in the soil (and potentially also
stems and leaves) and that can actively fix N2, albeit typically at lower rates per
unit area than symbiotic fixation (Döbereiner & Pedrosa, 1987; Franche et al.,
2009; Kahindi et al., 1997) given access to a carbohydrate source and (generally)
micro aerophilic conditions. Such conditions may prevail in the rhizosphere.
Janzen et al. (1995) reported that pure isolates of N2 fixing Azospirillum,
amended with compost extract in a soil microcosm fixed more N2 than isolates
without compost extract. This suggests that compost extract stimulates microbiota
involved in nutrient cycling.
40
While less productive than a legume in terms of N2 fixation, free living N2
fixers have been reported to be responsible for significant rates of N2 fixation
(e.g. 75 kg N ha-1 year-1) (Jones & Bangs, 1985; Vadakattu & Paterson, 2006).
Some microbial enhancement products contain free living N2 fixing
bacteria. For example, the compost extract inoculant ‗Living Soil‘ (from
SmartBugs, Lismore, NSW) carries a claim of containing diverse species of N2
fixing microorganisms (e.g. Azotobacter spp., Azospirillum brasilense and
Bacillus spp.). This area has not received scientific attention, insomuch as there
are few reports in the literature of the efficacy of compost extract application in
enhancing soil N2 fixation (Janzen et al., 1995).
8.6 Plant disease suppression
Certain soil microbes have the ability to suppress numerous plant diseases
(Adams, 1990). Weltzien (1991) reviewed the experimental evidence related to
the suppressive effects of a variety of water-based compost preparations. It was
concluded that watery fermented extracts of different composts can suppress,
both in vivo and in vitro, a wide range of plant-pathogenic fungi. Several leaf
diseases, such as grey mold, apple scab, powdery mildew, downy mildew have
been controlled by compost extracts, prepared from different organic sources, and
as effectively as the conventional fungicides (Cronin et al., 1996; Hoitink et al.,
1997; Ketterer et al., 1992; Litterick et al., 2004; McQuilken et al., 1994;
Weltzein & Ketterer, 1986). Drench application of compost extracts has also been
reported to control soil-borne diseases (Litterick et al., 2004; Scheuerell &
Mahaffee, 2004).
41
The beneficial microorganisms in compost extract may act upon
pathogenic ones through: (i) direct competition (for the pathogen‘s resources,
such as nutrients, oxygen, space and water), (ii) antibiosis (inhibition or
destruction of the pathogen by metabolic products of the antagonist), or (iii)
parasitism (of the pathogen by the antagonist) (Brinton, 1995; Scheuerell and
Mahaffee, 2002). Management of the soil microbial population, including
addition of soil amendments, is a promising strategy for developing natural
inhibition of soil-borne diseases, thus improving crop production (Mazzola,
2004).
For example, incidence of Verticillium wilt and common scab of potato
has been reported to be reduced by the application of organic amendments
(Bailey & Lazarovits, 2003), and enhancement of growth and yield of okra has
been demonstrated for crops treated with compost extract enriched with
Trichoderma spp., on the premise that Trichoderma spp. are strongly antagonist
to other fungi (Siddiqui et al., 2008b). Similarly, Scheuerell and Mahaffee (2004)
reported that compost extract could be used as a drench in a soil-less container
medium to control damping-off in cucumber (cv. Marketmore 76) seedlings
caused by Pythium ultimum. An aerated compost extract amended with kelp and
humic acid was reported to be more effective than a non-aerated compost extract.
Also, Hibar et al. (2006) reported that in vitro application of compost extract
inhibited mycelium growth of Fusarium oxysporum f. sp. radicis-lycopersici and
also showed improvement in root and vegetative growth of an infected tomato
crop. Non-aerated compost extract has also been recommended in the context of
disease suppression (Weltzien, 1990). Minimum effective fermentation periods of
as short as one day (Urban & Trankner, 1993) have been reported for this
42
application, although Scheuerell and Mahaffee (2006) reported that 14 day old
non-aerated compost extract was more effective than 7 day old non-aerated
compost extract in suppressing Botrytis cinerea.
A summary of reports available in the scientific literature on disease
suppression by compost extract application is presented in Table 1.4.
43
Table 1. 4. Summary of reports on proven disease suppression following application of compost extracts (CEs) produced from known input materials
Author Pathogen Crop Dilution ratio / CEs Rate of Compost Methodology Comments
(Disease) application (Country)
Larkin (2008) Rhizoctonia solani Potato - 100 mL/kg Vermicompost containing USA Applied to soil
(Stem canker) composted horse manure,
coffee grounds, pepper, straw, clay
containing nutrient additives,
bluegreen algae, kelp, sugar and
yeast
Al-Mughrabi et al. Streptomyces scabies Potato - 140 L/ha - Field plot Applied to soil
(2008) (Common scab) (ACE) Canada
Haggag and Saber Alternaria solani Tomato 1:5 v/v 10 mL/pot Rice ash, bean straw and vegetative Greenhouse Applied to foliage
M.S.M.(2007) (Early blight) Onion (ACE and NCE) food waste, chicken manure Egypt
Alternaria porri
(Purple blight)
Welke (2000) Botrytis cinerea Strawberries 1:8 and 1:4 v/v 1.3 L/m2 Cattle compost and chicken Field plot Applied to soil
(Fruit rot) Broccoli (ACE and NCE) manure compost Canada
Rhizoctonia solani
(head rot)
El-Masry et al. Pythium debaryanum - 1:2 w/v - Leafy fruit compost, crop compost, In situ, in vitro Applied to Petri plates
(2002)a (Dieback) (CE) garden compost Egypt and culture broth
Fusarium oxysporum
f.sp. lycopersici
(Fusarium wilt)
Sclerotium bataticola
(Charcoal rot)
Utkhede and Koch Clavibacter michiganensis Tomato 1:21 L w/v - Kelp In vitro and greenhouse Applied to foliage on
(2004) (Bacterial canker) (CE) Canada pinched cotyledons
Scheuerell and Pythium ultimum Cucumber 1:1 and 1:4 v/v - Yard trimming, vermicompost extract Hydroponics Applied to soilless container
Mahaffee (2004)b (Damping off) (ACE) compost USA medium drench
Olanya and Larkin Phytophthora infestans Potato - - - Laboratory and Applied to Petri plates
(2006)c (Late blight) (ACE) growth chamber and potato foliage
USA
Dianez et al. 9 fungal pathogens 1:3 w/v 5, 10 and Grape marc (grapevine waste) compost In vitro Applied to Petri plates
-
(2006)d ( ACE) 15 % v/v Spain
a
in situ and in vitro experiments are done applying various concentrations.
b
compost extract was diluted by mixing compost in the ratio of 1:1, 1:4 and 1:9 with tap water and applied to the seeded pots.
c
effect of compost extract was found to be very feeble here (only 0-15% disease suppression) against Phytopthora infestans.
d
The pathogens used were: Rhizoctonia solani, Fusarium oxysporum (4 strains), Verticillium dahliae, Pythium aphanidermatum, Phytopthora parasitica and Verticillium fungicola
44
Table 1.4. continued…..

Author Pathogen Crop Dilution ratio / CEs Rate of Compost Methodology Comments
(Disease) application (Country)
Kone et al. (2010) Alternaria solani Tomato 1:5 v/v 15% v/v Composts from sheep manure, In vitro, greehouse Applied to Petri plates
(Early blight) (NCE) spray until runoff chicken manure, bovine manure, Canada and tomato foliage
Botrytis cinerea shrimp powder or seaweed
(Gray mold)
Phytophthora infestans
(Late blight)
Oidium neolycopersici
(Powdery mildew)
Siddiqui et al. (2009) Choanephora cucurbitarum Okra 1:5 w/v 0.1 mL /Petri plate Rice straw, empty fruit bunch In vitro and in vivo Applied to Petri plates
(Wet rot) (ACE) 1:1 CE to of oil palm composts Malaysia and sterilised PDA
sterilised PDA
Zmora-Nahum et al. (2008) Sclerotium rolfsii - 1:2 w/w 4.5 mL/Petri plate Compost mix of municipal In vitro Applied to Petri plates
(Southern blight) (Centrifused and filtered) sewage sludge and green Israel
waste (1:1 v/v) compost
Joshi et al. (2009) Phaeoisariopsis griseola French bean 1:5 v/v 100%, 1000 L/ha Composts of poultry manure, Field experiment Applied to foliage of
(Angular leaf spot) (NCE) farmyard manure, vermicompost, India French bean
spent mushroom compost,
Lantana camara and Urtica spp.
PDA - Potato dextrose agar.
45
Scheuerell (2003) reported that there was no difference between use of
aerated or non- aerated compost extract in control of powdery mildew on field-
grown roses, in control of grey mold on greenhouse grown geraniums, or in
control of damping-off of basil seedlings indicating the lack of difference in
microbial density and diversity between those compost extracts.
Larkin (2008) evaluated aerated compost extract in the field and
greenhouse alone and in combination with different rotations for its efficacy to
enhance the soil microbial community and to suppress soil-borne diseases of
potato. In the barley/ryegrass rotation, he found that soil application of aerated
compost extract combined with a beneficial microorganism mix of Trichoderma
griseoviridis, Bacillus spp., Trichoderma harzianum and organic nutrients (humic
acids, kelp and yeast) reduced diseases, such as stem canker, black scurf and
common scab separately by 18-33% on tubers and increased yield by 20-23% but
not in other rotations. These results indicated that soil amendments generally add
or enhance microbiota in the soil and the strong effect of crop characteristics to
shape the microbial community is possibly the reason for developing disease
suppressive soils.
Various parameters of the compost extract production process were tested
for suppression of grey mold (Botrytis cinerea) on geranium (Scheuerell &
Mahaffee, 2006) and it was shown that compost extract produced with continuous
aeration did not suppress grey mold disease whereas the non-aerated compost
extract did. Although not consistent, when the aerated compost extract with
continuous aeration was mixed with kelp extract, rock dust and humic acid, it
reduced the disease significantly. The reason for this was unclear because
46
suppression of grey mold was not matched by an increase in the number of
cultivable bacteria in the extract.
Organic compost extracts prepared from pig, horse and cow manure were
tested against fusarium wilt of sweet pepper (Fusarium oxysporum f. sp.
vasinfectum) and all the treatments were effective in suppressing this pathogen by
up to 88.5% (Ma et al., 2001b). Compost water extract (non-aerated) prepared
from cattle manure successfully reduced tomato leaf grey mold (B. cinerea) as
opposed to water control in a greenhouse assay, however the best suppression
was achieved with the chemical fungicide, vinclozolin (Elad & Steinberg, 1994).
8.7 Acquired systemic resistance
A good example of acquired systemic resistance due to the non-
pathogenic colonisation has been given by a team of researchers at the University
of Delaware, U.S. (Bryant, 2008). They found that when the plant leaf is attacked
by a pathogen, the plant recognises the attack and sends a signal to the root
system. The root responds by secreting organic acids into the rhizosphere which
attract beneficial bacteria to help protect the plant by inducing systemic resistance
against the disease causing pathogens. They reported that the leaves of the plants
(the flowering plant, Arabidopsis thaliana) infected with a pathogenic bacterium
(Pseudomonas synngae) but without root inoculation of the beneficial microbe
(Bacillus subtilis) showed chlorosis and other disease symptoms, however the
same disease-infected plants but with root inoculation (B. subtilis) remained
healthy.
Several greenhouse and field studies have shown plant induced systemic
resistance against a variety of pathogens when cucumber seeds were treated with
47
plant growth promoting rhizobacteria (Liu et al., 1995; Raupach & Kloepper,
1997; Raupach et al., 1996). Wei et al. (1991) also reported induced resistance
response against Colletotrichum orbiculare in the leaves of cucumber when
rhizosphere bacteria were applied to their seeds. Such growth promoting
rhizobacteria have demonstrated a great potential in biological control of several
diseases (Kloepper et al., 1996). Similarly, Pseudomonas spp. was reported to
induce systemic resistance in carnation to fungal wilt caused by F. oxysporum
and accumulated increased concentration of phytoalexins in the carnation stem
(van Peer et al., 1991).
Biocontrol of plant diseases is a complex system and its understanding
and exploitation requires a deep insight of several sciences, including microbial
ecology, system analysis, epidemiology, biochemistry and molecular
biotechnology (Elad et al., 1996).
8.8 Bioremediation
Bioremediation is the term given based on the activity of introduced
living organisms to the restoration of an area contaminated with, typically, a
chemical pollutant. Various techniques have been developed in the past to
rehabilitate polluted soils (Alexander, 1994; Romantschuk et al., 2000; Skladany
& Metting, 1992). The chemical pollutant may be a metal (e.g. arsenic associated
with old cattle dips; or aluminium ion in acid soils), a hydrocarbon (e.g. a diesel
oil spill), or other classes of organic compounds (e.g. herbicide residues), or
chemical compounds (e.g. cyanide from ore processing and manufacturing
processes). A variety of microbial consortia that are capable of degrading
environmental pollutants (e.g. bacteria, fungi, grazing protozoa) can be involved
48
in the bioremediation process (Watanabe, 2001). The process of bioremediation
may involve a single organism, or may involve a succession of communities of
organisms. Organic wastes, such as petroleum can be effectively biodegraded
using indigenous organisms in the presence of suitable nutrients and an oxygen
supply (Allard & Neilson, 1997). Microbial degradation of cyanide has been
demonstrated and species known to act in cyanide degradation included
Pseudomonas fluorescens, Trichoderma koningii, Fusarium oxysporum, Bacillus
stearothermophilus, Staphylococcus seiuri, and so on (e.g. Dursun & Aksu, 1999;
Ezzi & Lynch, 2002; Gurbuz et al., 2009).
Bioremediation of chemical pollutants in soil is seen as a cost-effective
and environmentally acceptable approach (Buswell & Eriksson, 1994). The
effectiveness of a soil in bioremediation will be partly physio-chemical (retention
of the material within the soil, catalytic conversion to a non-toxic form) and
partly biological. Microbially enhanced compost extract (‗MECE‘ or ‗CE‘) may
increase relevant soil microbiota and may contribute chemical components
relevant to this issue.
While no peer-reviewed journal article was found on the topic of the
efficacy of compost extract in bioremediation, there is published work on the
remediation of polluted soil using compost (e.g. Diatloff et al., 1998; Dumestre et
al., 1999). The use of compost extract to remediate contaminated soil is a logical
extension.
A potential area for use of compost extract could be biostabilisation or
detoxification of the toxic trivalent aluminium ion. Aluminium toxicity arises
from the high solubilisation of aluminium with decreases in soil pH, a serious
factor causing yield reduction in many acidic soils (Wright, 1989). Previous
49
research has shown that humic and fulvic acids can complex aluminium, reducing
trivalent monomeric aluminium in nutrient solutions, with a resultant increase in
the root length of corn (Diatloff et al., 1998). Compost extracts should contain
humic and fulvic acids, and so should be able to biostabilise aluminium in the
rhizosphere.
9. Summary and hypotheses
Given this interest, the field claims for the benefit of compost extract, and
the need for a theoretical basis for such claims, it is timely for further scientific
investigation of this product and practices involved in its use. While not a
‗complete solution‘, it is likely that this technology may be a useful additional
tool to support ‗biological farming‘ and sustainable agriculture.
Farmer interest in, and uptake of, compost extract use is increasing. This
change is underpinned by interest in sustainable farming practices (building soil
‗health‘) and in lowering the cost of inputs (particularly driven by rising fertiliser
prices). There is a diverse array of potential benefits claimed for the use of
compost extract. While most of these benefits can be generally understood in
terms of mechanism of action in the context of current soil, plant and microbial
science, some claims are less explicable. As an example of the latter, how can
compost extract enhance microbiota generally while inhibiting some microbiota
specifically (i.e. plant pathogens)? Likewise, what is the mechanism behind the
claim that compost extract can cause the plant to develop systemic resistance (i.e.
that compost extract applied to plant roots can result in increased plant resistance
to leaf pathogens)?
50
While mechanisms for compost extract action can be hypothesised, there
is, unfortunately, a paucity of consideration of this topic in the scientific
literature. For example, Carpenter (2005) notes the need to explore for a ―base
theory‖ about compost extract, in terms of type of microbial community,
classification of beneficial microbes, different conditions for incubation and
subsequent effects on microbes. Research to date has largely focused on the areas
of plant nutrition and disease suppression, but this has generally been at an
empirical field level with little consideration of the underlying mechanism of
action.
There is a very limited published work on the characterisation of compost
extract. This is unfortunate, given that compost extract may be a highly variable
product. This variation may be a major factor in reports of inconsistency in plant
response to compost extract application, with reports ranging from highly
beneficial to complete failure, especially from a disease suppression point of view
(Bess, 2000; Larkin, 2008; Siddiqui et al., 2008a).
Based on the literature review, the following hypotheses could be
addressed in understanding the possible benefits of compost extract to plants:
(a) Application of compost extract to the soil can directly improve plant
nutrition through nutrients derived from the inoculant compost and other
amendments added to the compost extract.
(b) Application of compost extract to the soil and leaf surfaces at low rates (e.g.
20-50 L ha-1) can result in a persistent change in soil and phylloplane
microbial populations.
(c) Application of compost extract to the soil can indirectly improve plant
nutrition through mineralisation of the soil organic fraction. This benefit may
51
especially apply to P nutrition, through P-solubilising bacteria and increased
mycorrhizal density.
(d) Above-ground application of compost extract can accelerate the degradation
rate of leaf litter and standing stubble.
(e) Application of compost extract to the soil can increase N2 fixation by free
living soil bacteria.
(f) Application of compost extract to the soil and to above-ground biomass can
result in suppression of root and foliar plant diseases, respectively, through
competition, antibiosis or parasitism, by compost extract microbiota, of the
pathogen.
(g) Application of compost extract to one part of the plant can result in induction
of systemic resistance throughout the plant.
(h) Application of compost extract to the soil can result in bioremediation of
toxic organic and inorganic species present in the soil. For example, compost
extract may allow increased root growth in the presence of Al+++ ions via
chelation of these ions by humic acids or sequestration by micobiota.
(i) Application of compost extract to the soil can result in improved soil C
content through addition of organic matter via partial decomposition of plant
residues.
(j) Application of compost extract to the soil can result in improved soil
structure through direct addition of humates to the soil and through addition
of organic matter via partial decomposition of plant residues.
(k) Application of compost extract to the soil can result in improved water
holding capacity through improvement in soil structure and through addition
of organic matter via partial decomposition of plant residues.
52
(l) Long-term application of compost extracts to the soil can alter soil
microbiota at the field level and influence soil fertility and the possibility of
building a disease suppressive soil.
10. Conclusion
Given the growing interest in using compost extract, as a sustainable
management practice, with the consideration of its huge potential benefits in crop
production including commercial cultivation, and the lack of scientific research
on its possible roles in agriculture, it is timely to consider and evaluate the
potential effects of compost extracts on crop production. Attention should also be
given to the employment of various practices including microbiological and
molecular methods to be used for the monitoring of microbial populations in the
trials. The findings of this research may provide an insight to all users/producers
of microbially enhanced compost extracts including small and large scale farmers,
and researchers.
To achieve this, a number of experiments were conducted on production
and characterisation of compost and of compost extracts. Based on the literature
review, the potential contribution of compost extract to soil ‗health‘ may be
through, although not limited to: (a) soil mineral solubilisation, (b) organic matter
turnover (mineralisation), (c) aluminium chelation, and (d) disease suppression.
These claims were experimentally tested and explanations for the mode of each
effect have been sought.
53
Chapter 2: Compost based extract - characterisation
Chapter 2
Microbial enhancement of compost extracts based
on cattle rumen content compost – characterisation
of a system
A version of this chapter has been published in Bioresource Technology by Karuna Shrestha,
Pramod Shrestha, Kerry B. Walsh, Keith M. Harrower and David J. Midmore
Abstract
Microbially enhanced compost extracts (‗compost tea‘) are being used in
commercial agriculture as a source of nutrients and for their perceived benefit to
soil microbiology, including plant disease suppression. Rumen content material is
a waste of cattle abattoirs, which can be value-added by conversion to compost
and ‗compost tea‘. A system for compost extraction and microbial enhancement
was characterised. Molasses amendment increased bacterial count 10-fold, while
amendment based on molasses and ‗fish and kelp hydrolysate‘ increased fungal
count 10-fold. Compost extract incubated at 1:10 (w/v) dilution showed the
highest microbial load, activity and humic/fulvic acid content compared to other
dilutions. Aeration increased the extraction efficiency of soluble metabolites, and
microbial growth rate, as did extraction of compost without the use of a
54
constraining bag. A protocol of 1:10 dilution and aerated incubation with kelp
and molasses amendments is recommended to optimise microbial load and
fungal-to-bacterial ratio for this inoculum source.
1. Introduction
Composting can be viewed as an organic waste reduction process (Eiland
et al., 2001), but its greater value lies in the use of compost as a soil conditioner
(Leu, 2006). For large scale composting, point sources of organic material of
uniform quality are required. The two abattoirs in Rockhampton (Qld, Australia)
slaughter in total approximately 1700 head a day, producing 140 cubic meters of
rumen content per day (R. Lang, 2007; pers. comm.). Rumen content material is
composed of partly digested plant material. This waste has been historically
disposed across neighbouring grazing properties, but in recent years it has been
composted and sold. A windrow composting process is undertaken over 9
months, with windrows turned mechanically at monthly intervals in order to
provide aeration and improve homogeneity.
A water-based preparation of compost, incubated with various microbial
food sources and in some cases inoculated with specific microbiota, is defined as
a ‗microbially enhanced compost extract‘ or ‗compost extract‘ in short (and
popularly termed as ‗compost tea‘). Compost extracts are gaining popularity,
particularly, amongst those who are seeking substitutes to agrochemicals (Bess,
2000). ‗Mainstream‘ growers are also looking to reduce petrochemical-derived
products such as fertiliser, as prices soar.
Compost extract contains a high population of microbiota, e.g.
Rhizobacteria, Trichoderma, and Pseudomonas spp., which may enhance growth
55
and yield of crops (Sylvia, 2004). These microbiota produce plant growth
hormones and chemical compounds (e.g. siderophores, tannins, phenols) which
are antagonistic to various soil pathogens (Antonio et al., 2008). Other microbiota
may benefit plants through mechanisms such as nitrogen fixation and phosphate
solubilisation (Dubeikovsky et al., 1993). The use of compost extract is also
claimed to increase soil C levels, improve soil structure, nutrient cycling and
water holding capacity, and suppress plant diseases (Ha et al., 2008). However, to
achieve these benefits, several variables have to be considered to produce
compost extracts of desired quality. These include microbial food sources,
compost to water ratio, levels of aeration, compost quality, compost age, duration
of incubation, and the quality of water used. There is also a need for consistent
compost quality, which depends on consistency of inputs and methods used to
produce compost.
Given that the claims for the benefits of compost extracts are varied (from
disease suppression, and improved soil nutrient cycling to a simple direct plant
nutrition effect), the definition of compost extract ‗quality‘ is open ended. For
this study, we assume that the process goal is to maximise nutrient extraction
from compost, and to maximise microbial growth, particularly fungal growth.
Sugar or molasses, kelp extract, fish emulsion and rock dust are
commonly used as cost effective substrates usually available in bulk (Ingham,
2005). Addition of such microbial food sources to the compost extract will
increase microbial population growth during the incubation period (Naidu et al.,
2010). The additives used will affect the C-N balance, the form of C
(carbohydrate), and the form of the N source in growth media and this in turn will
affect the species composition (e.g. fungal: bacterial ratio) of the culture. Pant et
56
al. (2009) indicates that further work is required to relate the impact of such
additives on extract quality (in terms of minerals and microbiota).
Compost to water ratio is an important factor influencing the nutritional
and microbial status of the final product (Weltzien, 1990). Researchers have
employed a wide range of dilution ratios ranging from as low as 1:1 (Zhang et al.,
1998) to as high as 1:50 (Weltzien, 1990). Although dilution ratios of 1:3 to 1:10
are common (Scheuerell and Mahaffee, 2004), ratios of up to 1:1000 are currently
practiced in agriculture and horticulture (D. Daniels, 2008 pers. comm.). The
dilution ratios will obviously impact the nutrient concentration and microbial load
of the final product. There is also increased risk of biological contamination in
heavily diluted extracts.
Compost extract can be produced under aerobic or anaerobic conditions,
with obvious consequences to the microbial community supported. Past research
has mostly focussed on the production and application of non-aerated compost
extracts (Elad & Steinberg, 1994), but increasing attention is being given to
aerobically produced compost extracts, particularly for the control of a variety of
plant diseases (e.g. Siddiqui et al., 2009).
Filtering of compost extracts is required to prevent clogging of emitters or
spray nozzles (Weltzien, 1991). To avoid separate filtration steps, many users
incubate compost contained in a permeable bag (e.g. made up of muslin)
(Ingham, 2005). This practice can be expected to reduce diffusion between the
compost and the bulk solution, and so reduce the rate of microbial and nutrient
extraction from the compost. Whether this practice impacts on species
composition of the extract is not known.
57
The production and use of compost extracts for agronomic and
horticultural purposes appears to be gaining in popularity. For example, in the
central Queensland, compost extracts are used in horticultural, broadacre grain
and grazing enterprises, either produced on-farm or supplied by contractors. A
few scientific studies have been undertaken that have evaluated compost extracts
in terms of nutrients and microbiota (Naidu et al., 2010; Pant et al., 2009;
Scheuerell, 2004; Scheuerell & Mahaffee, 2006). Further studies are essential to
understand the impact of specific incubation conditions on the microbial
outcomes. In this study, the impacts of four major production variables, namely,
additives, dilution rate, level of aeration and use of bags during the incubation of
compost extract are investigated.
2. Materials and methods
2.1. Experiments and treatments
The experiments were carried out indoors at ambient temperatures
between a range of 22 to 32˚C. Compost extractions were undertaken with
variation in (i) additives (experiment 1), (ii) compost: water ratio (experiment 2)
and (iii) aeration and use of a bag to contain the compost (experiment 3). Each
experiment consisted of four treatments with three replications, utilising 12
buckets of 60 L size. A completely randomised design was used for subsequent
experiments. The default extract condition consisted of 3 kg (f wt) compost
contained in a cotton (28 g m-2) bag, placed in 30 L of town water (i.e., a 1:10
ratio) and continuously aerated at the rate of 36 L min-1 through air stones located
at the base of the bucket, as per Shrestha et al. (2011a). The water was aerated
58
vigorously for 30 minutes to remove residual chlorine prior to initiation of
treatment.
The first experiment involved aerated incubation of compost extracts with
two different additives, namely, ‗fish and kelp hydrolysate‘ (kelp) from Searle
Pty Ltd, Australia, molasses (molasses), ‗fish and kelp hydrolysate‘ + molasses
combination (kelp + molasses) and a no additive treatment (control). ‗Fish and
kelp hydrolysate‘ and/or molasses were added at the rate of 1% and/or 0.5%
(v/v), respectively, except in the control. ‗Fish & kelp hydrolysate‘ consists of
78% fish hydrolysate, 20% kelp, 2% stabiliser and 4% fish oil on w/w basis and
contains 10.3% N, 2.5% P, 2.5% K, 7% Ca, 0.2% Mg, 0.5% S, 690 mg kg-1 Fe, 5
mg kg-1 Cu, 14 mg kg-1 Mn, 32 mg kg-1 Zn, 14 mg kg-1 B, and 1 mg kg-1 Mo in
organic form. Molasses is a by-product of cane processing (simple sugars).
The second experiment consisted of compost extracts incubated in
different dilution ratios (non-diluted, 1:10, 1:100, and 1:1000 compost:water on a
w/v basis) with all incubations involving kelp and molasses additions under
aerated conditions.
The third experiment consisted of aerated (ACE) and non-aerated
compost extract (NCE) treatments incubated without or with (ACEb and NCEb)
the use of cotton bags to retain the compost during incubation. Here also, kelp
and molasses were added to each treatment immediately after addition of
compost.
2.2. Source compost
Compost based on cattle rumen content was obtained from a commercial
composting operation (Broadmeadows Pty Ltd). In the Rockhampton area, most
59
animals are fed by grazing (rather than feedlot). All three experiments utilised
freshly collected rumen compost (nine month old) as a basic source material to
produce compost extracts. As there can be considerable batch-to-batch
inconsistency in compost composition (Campbell, 2006), a single batch of
compost was used to produce the extract in each experiment.
2.3. Physico-chemical analyses
In all treatments, the extract pH, electrical conductivity (EC), temperature
and dissolved oxygen, as well as ambient temperature were determined at 3 h
intervals over a 24 h period and a final reading was taken at 48 h. The pH and EC
were measured using a TPS labCHEM Cond/pH meter while dissolved oxygen
and temperature of the extracts were measured using a TPS WP 82 Dissolved
Oxygen Meter (EnviroEquip, Australia).
Samples of compost extracts were collected after 24 h of incubation and
concentrations of inorganic soluble ions (NO3−-N, NH4+-N, PO4−-P and K+-K)
were determined in triplicate using colorimetric test strips (RQflex plus).
Humic and fulvic acids were determined from samples collected at 24 h
using a UV-Vis spectrophotometer following a slightly modified method by
Yamada et al. (1998). Briefly, 2 mL of centrifuged and filtered (0.45 µm
membrane filter) sample of compost extract was added to 8 mL of reverse
osmosis (RO) water followed by addition of 10 M hydrochloric acid (500 L). The
precipitated humic acid was separated from the supernatant solution by
centrifugation at 7000 rpm for 72 minutes at 4˚C. The supernatant solution was
neutralized by adding 10 M sodium hydroxide and the absorbance was measured
at 350 nm using UV spectrophotometer to index fulvic acid level. The
60
precipitated humic acid was dissolved with 1M sodium hydroxide followed by
neutralization with 1 M hydrochloric acid. The absorbance was then measured at
350 nm to index humic acid level. The humic and fulvic acid concentrations of
the samples were then calculated by reference to humic (Sigma Aldrich,
Switzerland) and fulvic acid (International Humic Substances Society, USA)
standards, respectively.
2.4. Microbial analyses
Compost extract samples were collected in triplicate after 24 h of
incubation and cultivable bacterial and fungal populations of serially diluted
samples were estimated by the pour plate technique using plate count agar for
bacteria and half strength potato dextrose agar with chloramphenical (100 µg mL-
1
) for fungi (Harrower, 2006). Bacterial plates were incubated at 37˚C for 1-2
days while fungal plates were incubated for 3-5 days at 25˚C.
Total microbial activity of the samples collected after 12, 24 and 48 h of
incubation were determined using the fluorescein diacetate (FDA) hydrolysis
method given by Adam and Duncan (2001).
2.5 Statistical analysis
Statistical analysis of non-transformed data was undertaken by two-way
ANOVA using the statistical package (XLSTAT, 2010) using GLM model.
Differences between means were separated by Tukey‘s test (p< 0.05). Data in
figures is presented as a mean with associated standard error, with indication of
significant differences between means for a given time of treatment.
61
3. Results and discussion
3.1. General trends
The three experiments consisted of a common treatment with 1:10 w/v
compost contained within a muslin bag in an aerated solution containing
molasses and kelp as additives. Similar ammonium, phosphate and potassium
levels were recorded for this treatment, indicative of a consistent compost source,
although the first experiment produced an extract relatively richer in humic acid,
and the third experiment produced an extract lower in microbial population,
although with a similar bacterial to fungal ratio (Tables 1-3). While these
differences could be due to changes in growing conditions, it is most likely they
represent changes in compost source, reinforcing the need to use compost
produced under consistent conditions.
Adegunloye et al. (2007) reported that most of the microorganisms in
compost best survived under neutral pH, and presumably this is also true for
compost extract. On this basis, a pH of 7 is ideal for compost extracts. Increasing
the EC of an extract is indicative of increased extraction of anions and cations,
which should increase the benefit of the extract as a plant fertiliser; however, an
EC above 5 dS m-1 is likely to decrease microbial survival (Okur, 2002) and is
thus an upper quality limit for compost extracts. In general, the pH of the
compost extract increased during incubation to around 6, and EC increased to 4
dS m-1. The increase in pH is likely due to an overall uptake of more anions than
cations by the microbiota (as commonly documented in agricultural systems, e.g.
Yan et al., 1996) while the increase in EC presumably represents leaching and
breakdown of the organic matter of the compost.
62
Solution temperature and oxygen levels are indices of microbial activity
(e.g. as reported by Garcia et al., 1991 for compost). Tiquia et al. (1996)
correlated temperature with dehydrogenase activity, ATP content and oxygen
consumption rate during composting of spent litter from pig pens at differing
moisture levels and reported that these parameters are indicative of microbial
activity. During incubation, temperature increased above ambient and solution
oxygen levels declined dramatically around 9 h after start of incubation. The
latter decrease indicates that the aeration method failed to keep pace with the
oxygen demand of the culture, despite these liquid cultures being aerated
continuously at the rate of 36 L air min-1 per vessel (30 L). Oxygen depletion to
levels below 6 ppm for extended periods may alter microbial composition of the
culture (Ingham, 2005). This depletion could be addressed by either improving
the aeration system or by decreasing microbial activity (e.g. less microbial food
per bucket, or gradual addition of microbial food during incubation).
3.2. Experiment 1: Comparison of compost extracts with different
additives (control, kelp, molasses and kelp+molasses)
The pH and EC of a 1:5 dilution (with distilled water) of molasses and
kelp was 5.5 and 4.3, and 2.3 and 1.0 dS m-1, respectively. The additives lowered
the pH of compost extracts, and increased extract EC. Significantly higher pH
values (Fig. 2.1A) were recorded in the control (no additives), while the kelp +
molasses treatment showed the highest EC, followed by molasses treatment (Fig.
2.1B). Naidu et al. (2010) have reported that fortification with additives such as
kelp, peptone, humic acid and yeast extracts increased the pH, while brown sugar
and corn meal decreased the pH of compost extracts. They found that EC values
63
of compost extracts were also increased with all the additives tested compared to
the no-additive control. Scheuerell and Mahaffee (2004) also found an increase in
pH values from 7.4 in the non-additive control to between 7.9 and 8.6 in compost
extracts produced with addition of a bacterial nutrient solution obtained from Soil
Soup Inc. WA, USA, and a fungal medium based on soluble seaweed powder,
liquid humic acids and rock dust, adapted from Ingham and Alms (1999). In
summary, in the non-buffered solution of compost extract, pH of an additive is a
major determinant of the pH of a final extract.
64
Figure 2. 1. Comparison of pH, electrical conductivity (dS m-1), fluctuations in temperatures (˚C)
and dissolved oxygen (% of saturation) over time (readings taken at every 3 h intervals until 24 h
and final readings taken at 48 h) in rumen compost extracts with different additives. Control -
compost extract with no additives, Kelp - compost extract with ‗fish and kelp hydrolysate‘,
Molasses - compost extract with molasses, Kelp+Molasses - compost extract with ‗fish and kelp
hydrolysate‘ and molasses. Values are mean ± SE (n = 3).
65
The difference between EC values at the start of incubation must
represent difference in EC of additives. The rise in EC with incubation time must
represent extraction of ions from the compost. The rapid rise within 3 h, followed
by a plateau, indicates that mixing conditions were adequate to ensure solution
diffusion through the compost bag.
The inorganic phosphate and potassium concentrations did not differ
among the treatments; however, the concentrations of ammonium in the molasses
and kelp + molasses treatments were lower than those of the control (Table 2.1).
In contrast, Pant et al. (2009) found higher levels of nitrate, ammonium and
potassium after adding kelp extract and humic acid to their original liquid
preparation. Similarly, Naidu et al. (2010) reported significantly higher
concentrations of nitrogen, phosphorus and potassium along with iron, calcium
and manganese in microbially enriched compost extracts compared to the no
additive control. Specifically, the authors used yeast extract and humic acid as the
source of nutrients. In our study, the increased microbial activity associated with
the addition of molasses and kelp presumably resulted in mineralisation of some
organic forms into mineral forms in the solution. The lower ammonium level in
extracts amended with additives could be due to increased volatilization (for pH
below 6) or to uptake by the increased microbiota present in these treatments.
Although no difference in humic acid was noted, fulvic acid level was
significantly higher in the kelp treatment compared to the control treatment (0.8
vs. 1.23 µg g-1 d wt), which suggests that ‗fish and kelp hydrolysate‘ was a
significant source of fulvic acid (Table 2.1).
66

incubated with different additives and sampled after 24 h of incubation.
Parameter Control Kelp Molasses Kelp+Molasses

+ -1
NH4 -N (µg g d wt) 1.32 ± 0.04b 1.19 ± 0.03ab 1.06 ± 0.07a 1.09 ± 0.04a
- -1
PO43 -P (µg g d wt) 32.83 ± 2.27a 30.44 ± 2.14a 29.35 ± 0.75a 32.39 ± 0.43a
+ -1
K -K (µg g d wt)-1 0.55 ± 0.00a 0.59 ± 0.02a 0.61 ± 0.03a 0.69 ± 0.06a
Humic acid (µg g d wt) 2.33 ± 0.17a 2.08 ± 0.46a 2.17 ± 0.03a 1.44 ± 0.30a
-1
Fulvic acid (µg g d wt) 0.82 ± 0.08a 1.23 ± 0.07b 0.96 ± 0.07ab 0.98 ± 0.07ab
Bacterial population (cfu Log10) @ 24h 10.76 ± 0.07b 7.78 ± 0.02a 11.49 ± 0.05c 10.64 ± 0.07b
Fungal population (cfu Log10) @ 24h 5.88 ± 0.02a 6.62 ± 0.02c 6.14 ± 0.05b 6.78 ± 0.01d
d wt Dry weight. Control - compost extract with no additives, Kelp - compost extract with 'fish and kelp hydrolysate',
Molasses - compost extract with molasses, Kelp+Molasses - compost extract with 'fish and kelp hydrolysate' and molasses.
Within rows, means (n = 3) with the same letter are not significantly different according to Tukey's test (p< 0.05).
The molasses treatment showed the highest temperature followed by kelp
+ molasses treatment (Fig. 2.1C), while these two treatments also demonstrated
the quickest decrease in the level of dissolved oxygen (Fig. 2.1D), presumably
reflecting microbial activity. The level of dissolved oxygen in each treatment
(Fig. 2.1D) was consistent with the activity of microorganisms at 48 h of
incubation (as assessed using FDA, Fig. 2.2) and plate counts of culturable
microorganisms (Table 2.1).
Figure 2. 2. Total microbial activity over time (readings taken at 12, 24 and 48 h) as estimated by
the FDA hydrolysis method in compost extracts with different additives. Control - compost
extract with no additives, Kelp - compost extract with ‗fish and kelp hydrolysate‘, Molasses -
compost extract with molasses, Kelp+Molasses - compost extract with ‗fish and kelp hydrolysate‘
and molasses. Within a time, treatment means with the same letter do not differ significantly and
values are mean ± SE (n = 3).
67
Bacterial and fungal populations as indicated by the plate counts were in
the order molasses > kelp + molasses = control > kelp and kelp + molasses > kelp
> molasses > control, respectively. In agreement with the bacterial plate counts,
the lowest microbial activity was observed in the kelp treatment as also
demonstrated by FDA hydrolysis (Fig. 2.2), although the ranking of other
treatments (kelp + molasses = molasses = control > kelp) opposed to the plate
counts (Table 2.1). The difference between the order of results of the fungal plate
count method and the FDA hydrolysis method agrees with the finding by Garcia-
Gomez et al. (2003), and presumably reflects the difference between a measure of
bacterial activity (FDA method), as opposed to a measure of number of
propagules (plate counts).
Different microorganisms have specific substrate requirements, therefore,
it is expected that the types of organic compounds added to a culture will
influence microbial diversity. The highest bacterial population was achieved with
molasses amendment, while the highest fungal to bacterial ratio was achieved
with both kelp and molasses amendment (Table 2.1). The reason for this could be
due to the nature of the C substrate in each of these additives (molasses should be
simple sugar, while kelp should have included cellulose and lignified plant
tissues). Of the additives tested, the use of both molasses and kelp is
recommended.
3.3. Experiment 2: Comparison of compost extracts with different
dilution levels
Increasing the ratio of compost to water resulted in significantly higher
values of pH and EC. EC plateaued after 3-6 h of incubation even in the 1:10
68
ratio treatment, indicating that a reasonable diffusion of solutes was occurring
through the compost into the bulk solution, despite its containment in a bag.
Compost extract produced using a ratio of 1:10 was also higher in concentrations
of ammonium, humic acid and fulvic acid compared to those of other extracts
(Table 2.2).

incubated with different dilutions and sampled at 24 h of incubation.
Parameter Non-diluted 1:10 1:100 1:1000

+ -1 0.18 ± 0.07a 1.29 ± 0.10b 0.28 ± 0.03a
NH4 -N (µg g d wt) 0.16 ± 0.04a
- -1 37.61 ± 3.20a 33.05 ± 1.52a 39.35 ± 1.15a 40.00 ± 2.27a
PO43 -P (µg g d wt)
+ -1
K -K (µg g d wt) 0.53 ± 0.03a 0.73 ± 0.02b 0.69 ± 0.06ab 0.63 ± 0.02ab
-1
Humic acid (µg g d wt) 0.12 ± 0.02a 0.60 ± 0.20b 0.08 ± 0.01a 0.09 ± 0.01a
-1
Fulvic acid (µg g d wt) 0.71 ± 0.03a 1.80 ± 0.10b 0.78 ± 0.07a 0.62 ± 0.07a
Bacterial population (cfu Log10) @ 24h 9.24 ± 0.04a 11.79 ± 0.01d 10.96 ± 0.04c 9.39 ± 0.03b
Fungal population (cfu Log10) @ 24h 7.84 ± 0.04a 8.40 ± 0.07b 8.28 ± 0.05b 8.21 ± 0.01b
d wt Dry weight. Non-diluted - compost extract with no compost inoculum but with 'fish and kelp hydrolysate'
and molasses, 1:10 - compost extract at the ratio of 1 part compost to 10 parts water (w/v), 1:100 - compost
extract at the ratio of 1 part compost to 100 parts water (w/v), 1:1000 - compost extract at the ratio of 1 part
compost to 1000 parts water (w/v). Within rows, means (n = 3) with the same letter are not significantly
different according to Tukey's test (p< 0.05).
Solution temperatures of the four compost dilution treatments did not
differ initially; however, after 12-15 h of incubation, temperatures of three levels
of dilution were significantly higher than that of the control treatment (Fig. 2.3C).
Dissolved oxygen decreased rapidly (to approx less than 10%) between 6-12 h
after culture initiation in the 1:10 treatment, and 9-15 h in 1:100 and over 15-21 h
in 1:1000, indicative of the level of microbial activity during incubation (Fig.
2.3D). Indeed, the compost acted as a bacterial inoculum, with higher bacterial
plate counts related to increasing compost ratios (Table 2.2). The compost was
also a source of fungal inoculum; however, the culturable fungal load in the 48 h
incubated extracts was not proportional to the compost:water ratio (Table 2.2).
69
A 9
8
7
6
5
pH
4
3 Non-diluted
1:10
2 1:100
1 1:1000
0
0 3 6 9 12 15 18 21 24 48
5.0
4.5
4.0
3.5
EC (dS m-1)
3.0
2.5
2.0
1.5 Non-diluted
1:10
1.0 1:100
0.5 1:1000
0.0
0 3 6 9 12 15 18 21 24 48
C 30
29
28
Temperature (°C)
27
26
25
Non-diluted
24 1:10
1:100
23 1:1000
22
0 3 6 9 12 15 18 21 24 48
120
D
100
Dissolved oxygen (%)
80
Non-diluted
60 1:10
1:100
1:1000
40
20
0
0 3 6 9 12 15 18 21 24 48
Time (h)
Figure 2. 3. Time course of solution pH, electrical conductivity (dS m-1), temperatures (˚C) and
dissolved oxygen (% of saturation); readings taken at every 3 h intervals until 24 h and final
readings taken at 48 h in rumen compost extracts with different dilutions. Non-diluted - compost
extract with no compost inoculum but with ‗fish and kelp hydrolysate‘ and molasses, 1:10 -
compost extract at the ratio of 1 part compost to 10 parts water (w/v), 1:100 - compost extract at
the ratio of 1 part compost to 100 parts water (w/v), 1:1000 - compost extract at the ratio of 1 part
compost to 1000 parts water (w/v). Values are mean ± SE (n = 3).
70
The higher level of oxygen demand in the 1:10 treatment (Fig. 2.3D) was
also in agreement with the highest relative total microbial activity as indicated by
the highest amount of fluorescein release (Fig. 2.4) and by the greater number of
culturable microorganisms (Table 2.2).
Figure 2. 4. Comparison of total microbial activity at different times (readings taken at 12, 24 and
48 h) following the FDA hydrolysis method in compost extracts with different dilutions. Non-
diluted - compost extract with no compost inoculum but with ‗fish and kelp hydrolysate‘ and
molasses, 1:10 - compost extract at the ratio of 1 part compost to 10 parts water (w/v), 1:100 -
compost extract at the ratio of 1 part compost to 100 parts water (w/v), 1:1000 - compost extract
at the ratio of 1 part compost to 1000 parts water (w/v). Within a time, treatment means with the
same letter do not differ significantly and values are mean ± SE (n = 3).
There was no difference in the microbial activity of ‗non-diluted‘ (i.e. no
compost) and 1:1000 dilution ratios (Fig. 2.4). In the ‗non-diluted‘ treatment,
oxygen levels were maintained above 80% saturation until 15 h of incubation
period (Fig. 2.3D), after which the oxygen level also declined dramatically. As
the cultures were open, and inputs non sterile, the likely sources of this
contamination are either atmospheric, water or the food source. This ready
contamination of the cultures is indicative of a need to maintain a high compost
to water ratio, to maximise inoculum load.
71
Compost extracts based on high dilution ratio, e.g. 1:1000, are being
practiced by many farmers in southern Queensland and northern NSW for broad-
acre production (S. Stevens, 2008 pers. comm.). Based on the results of this
study, this practice is not recommended, given the potential for contamination.
Rather, a compost:water ratio of as high as 1:10 is recommended.
3.4. Experiment 3: Comparison of aerated and non-aerated compost
extracts with and without bags
During incubation, significantly higher values of pH and EC were
recorded in aerated compost extracts compared to the non-aerated compost
extracts, irrespective of the use of bags (Fig. 2.5). Although Pant et al. (2009) did
not find any difference in the pH and EC values between aerated and non-aerated
extracts, Scheuerell and Mahaffee (2004) found significantly higher pH and EC
of aerated compost extracts over non-aerated extracts independent of the use of
bacterial or fungal additives.
72
A 8
7
6
5
pH
4
3
ACE
2 NCE
ACEb
1 NCEb
0
0 3 6 9 12 15 18 21 24 48
B 5.0
4.5
4.0
3.5
EC (dS m-1)
3.0
2.5
2.0
ACE
1.5 NCE
1.0 ACEb
NCEb
0.5
0.0
0 3 6 9 12 15 18 21 24 48
C 32
31
30
29
Temperature (°C)
28
27
26
ACE
25 NCE
24 ACEb
NCEb
23
22
0 3 6 9 12 15 18 21 24 48
D 120
100
80
Dissolved oxygen (%)
60
ACE
40
NCE
ACEb
20 NCEb
0
0 3 6 9 12 15 18 21 24 48
Time (h)
and final readings taken at 48 h) in rumen compost extracts in presence and absence of aeration
with and without the use of bags. ACE - aerated compost extract with compost kept loose in a bag
while incubating, NCE - non-aerated compost extract with compost kept loose while fermenting,
ACEb - aerated compost extract with compost kept in a bag while incubating, NCEb - non-aerated
compost extract with compost kept in a bag while fermenting. Values are mean ± SE (n = 3).
73
The level of oxygen in solution was maintained at > 80% saturation until
9-12 h of incubation under an aerated condition, whereas it was at approximately
60% saturation until 3-6 h of incubation in non-aerated treatments where it
declined to close to zero by 12-15 h (Fig. 2.5D). The dissolved oxygen levels of
the non-aerated extracts, lacking extra supply of oxygen, declined 6-9 h earlier
than the aerated extracts. To improve the aeration rate further, an air delivery
system is required that decreases the size of air bubbles, thereby increasing
bubble surface area to volume ratio, and increases the residency time of the air
bubble in the solution (Garcia-Ochoa et al., 2010).
The non aerated cultures were significantly lower in cultivable
microorganisms and their activities when compared to aerated cultures (Table 2.3
and Fig. 2.6), in agreement with the observations of Scheuerell (2004). This
author postulated that anaerobic conditions could result in the production of
reduced organic compounds that are inhibitory to the microbes. Of course,
population growth of microorganisms is greatly influenced by the concentration
of available dissolved oxygen (Metcalf & Eddy, 2003). Tajuddin et al. (2004)
showed an inverse relationship between dissolved oxygen deficit and bacterial
counts when aerating an effluent of palm oil mill for thirteen days. The authors
found that a continuous supply of oxygen in the effluent enhanced the bacterial
population. Kelley (2004) also noted that microbial growth becomes strictly
limited if supplementary oxygen is not provided to the compost extract
incubation system.
74

incubated for 24 h in presence and absence of aeration with and without the use of bags.
Parameter ACE NCE ACEb NCEb

+ -1
NH4 -N (µg g d wt) 0.72 ± 0.03b 0.65 ± 0.03b 0.72 ± 0.03b 0.47 ± 0.00a
- -1
PO43 -P (µg g d wt) 34.6 ± 1.00b 36.5 ± 0.38b 26.5 ± 0.58a 25.0 ± 0.22a
+ -1
K -K (µg g d wt) 0.83 ± 0.10a 0.66 ± 0.04a 0.73 ± 0.05a 0.75 ± 0.02a
-1
Humic acid (µg g d wt) 0.59 ± 0.02b 0.59 ± 0.07b 0.61 ± 0.07b 0.23 ± 0.02a
-1
Fulvic acid (µg g d wt) 1.26 ± 0.07a 1.12 ± 0.17a 1.11 ± 0.24a 0.74 ± 0.04a
Bacterial population (cfu Log10) @ 24h 8.62 ± 0.03d 7.62 ± 0.03b 8.28 ± 0.03c 7.40 ± 0.02a
Fungal population (cfu Log10) @ 24h 4.83 ± 0.07b 4.10 ± 0.10a 4.59 ± 0.06b 4.00 ± 0.00a
d wt Dry weight. ACE - aerated compost extract with compost kept loose in a bag while incubating,
NCE - non-aerated compost extract with compost kept loose while fermenting, ACEb - aerated compost
extract with compost kept in a bag, NCEb - non-aerated compost extract with compost kept in a bag. Within
rows, means (n = 3) with the same letter are not significantly different according to Tukey's test (p< 0.05).
Figure 2. 6. Comparison of total microbial activity over time (readings taken at 12, 24 and 48 h)
following the FDA hydrolysis method in compost extracts in presence and absence of aeration
with and without the use of bags. ACE - aerated compost extract with compost kept loose in a bag
while incubating, NCE - non-aerated compost extract with compost kept loose while fermenting,
ACEb - aerated compost extract with compost kept in a bag while incubating, NCEb - non-aerated
compost extract with compost kept in a bag while fermenting. Within a time, treatment means
with the same letter do not differ significantly and values are mean ± SE (n = 3).
Aeration also increased mixing, and thus leaching of solutes and
microbiota from the compost (Table 2.3). In accordance with this, Pant et al.
(2009) found higher macronutrient concentrations of total nitrogen, nitrate, nitrite
and potassium with comparable concentrations of secondary nutrients (Ca, Mg,
75
Na) and micronutrients (Fe, Mn, Zn, Cu, B) in aerated compost extracts
compared to the non-aerated compost extracts.
The speed at which dissolved oxygen was consumed (Fig. 2.5D) in the
extraction process was greater for the loose compared to the bagged compost
extract. Limited oxygen at the centre of the compost contained in a bag may have
reduced the microbial activity significantly. Although by 12 h there was a
difference in microbial activity between aerated and non-aerated treatments (Fig.
2.6), no difference between loose and bagged compost extract was noted in the
total microbial activity of aerated compost extracts until after 24 h of incubation,
and then there was only an increase in the bagged aerated treatment. In
accordance with the higher oxygen demand (Fig. 2.5), there was a higher
bacterial count (Table 2.3) in non-aerated compost extract without compared to
with the use of bags. These observations reinforces the conclusions of Garcia-
Ochoa et al. (2010) that oxygen or agitation are needed for the survival and
proliferation of microbes in a given liquid culture.
4. Conclusion
Composted cattle paunch material produced extracts dominated by
bacteria, with approximately 2:1 ratio of bacterial to fungal cfu. To achieve
maximum nutrient extraction and microbial population increase, with bias to
fungal growth over bacterial growth, the following conditions are recommended:
(i) addition of both kelp hydrolysate and molasses; (ii) a compost:water ratio of
1:10, to produce microbially enhanced compost extracts rich in soluble
metabolites; (iii) use of bags to avoid clogging of spray nozzles; and (iv) aeration
76
to maintain bulk solution oxygen levels above 6 ppm. These conditions are used
to produce compost extracts for agronomic trails in companion studies.
77
Chapter 3: Vermiculture based extract - characterisation
Chapter 3
Changes in microbial and nutrient composition
associated with rumen content compost incubation
A version of this chapter has been published in Bioresource Technology by Karuna Shrestha,
Pramod Shrestha, Eric M. Adetutu, Kerry B. Walsh, Keith M. Harrower, Andrew S. Ball and
David J. Midmore
Abstract
Physico-chemical and microbiological investigations were carried out on
rumen content material composted for nine months, fresh vermicasts (obtained
after passing the same compost through the guts of a mixture of three species of
earthworms: Eisenia fetida, Lumbricus rubellus and Perionyx excavates) and
microbially enhanced extracts derived from rumen compost, vermicast and
vermicast leachate incubated for up to 48 h. Compared to composted rumen
contents, vermicast was only improved in terms of microbial biomass C, while
vermicast leached extract was significantly higher in NH4+-N, PO4--P, humic
acid, bacterial counts and total microbial activity compared to rumen compost
extract. Although no difference between treatments was observed in genetic
diversity as indicated by DGGE analysis, community level functional diversity of
vermicast leached extract (BiologTM) was higher than that of composted rumen
78
contents, vermicast and rumen compost extract indicating an enhancement of
microbial activity rather than diversity due to liquid incubation.
1. Introduction
Composting is a process of aerobic decomposition of organic matter under
controlled conditions by microbiota, producing a stable organic end product, with
carbon-dioxide, water and heat produced as by-products (Rynk et al., 1992). For
large scale composting activity, point sources of organic material of uniform
quality is required. One such material is the partially digested rumen contents of
cattle, viewed as a waste in abattoir operations. For example, the two abattoirs in
Rockhampton slaughter approximately 1700 head a day, producing 140 cubic
meters of rumen content per day (R. Lang, pers. comm.).
Vermicomposting is a process of biotransforming and stabilising organic
materials (often waste) into humus (Neuhauser et al., 1988) by the combined
activity of earthworms and microorganisms (Aira & Dominguez, 2008).
Earthworms excrete partially digested materials (Parmelee et al., 1990), known as
vermicasts or castings, which are more homogeneous in composition than the
source material (Albanell et al., 1988), have reduced levels of contamination
(Ndegwa & Thompson, 2000), and contain elevated levels of plant growth
regulators and/or symbiotic microbes (Kale et al., 1992) and organic acids such as
humic and fulvic acids (Edwards et al., 2006).
There is an emerging commercial trend of aerobically incubating an
extract of compost with a carbohydrate and a protein source, producing a
microbially enhanced liquid (Ingham, 1999a; Pant et al., 2009). Known by the
agricultural sector as ‗compost teas‘ in the current study this microbially
79
enhanced product is termed ―Compost Extract‖ or ―CE‖ in short. Compost extract
contains nutrients extracted from compost and thus contributes directly to plant
nutrition, and also contains organic matter, improving soil structure and water
holding capacity by building soil aggregates. Compost extracts derived from
vermicomposts have proven benefits in terms of growth and yield promotion of
various agricultural crops (Pant et al., 2009; Tejada et al., 2008). Moreover,
compost extract adds microorganisms to the soil and ‗brings the soil back to life‘
(Martens, 2001). However, at the typically employed rates of application of 150-
200 L/ha, the fundamental aim of compost extract application is to alter soil
microbial populations towards a more plant beneficial community (Hoitink &
Boehm, 1999b).
Many researchers have characterised the composting process (Singh &
Sharma, 2002; Vivas et al., 2009), however, there is little documentation on the
shift in microbial diversity during the incubation and extraction process.
Therefore, a comparative study was undertaken on the effect of vermicomposting
of rumen content compost and of an extract enhancement process. Physico-
chemical, biochemical and microbial characteristics were monitored on the
following: rumen compost extract (RCE), vermicast extract (VCE) and vermicast
leached extract (VLE), i.e. microbially enhanced vermicast leachate collected in
tanks from vermibeds by continuous recirculation. Microbial enhancement was
effected by amendment of a compost, vermicasts or vermicast leachate with
microbial food sources (fish & kelp hydrolysate and molasses) under aerobic
conditions. The null hypothesis in this experiment is that the microbial population
present in the compost is not altered by earthworm feeding, or by the liquid
extract enhancement process.
80
2.1. Rumen compost
Rumen compost was obtained from a commercial composting operation
(Broadmeadows) which utilises the rumen contents from the Australia Meat
Holding Pty Ltd., Abattoir (maximum daily kill of approximately 1700 cattle) in
Rockhampton, QLD Australia. A windrow composting process was undertaken
over approximately nine months, with windrows turned mechanically at
approximately monthly intervals in order to provide aeration and improve
homogeneity.
Nine month old bulk rumen compost was freshly collected from the
commercial operation to feed the earthworms and for laboratory analysis, several
core samples of which were composited and representative samples were taken.
These compost samples were used immediately or stored in sealed plastic bags at
4 ˚C until use.
2.2. Vermicomposting process, vermicasts and vermicast leachate
A mixture of earthworms (three species; namely, Eisenia fetida,
Lumbricus rubellus and Perionyx excavates) was used in three covered culture
chambers that were supplied from an Australian commercial supplier
(Vermicrobe International Pty LtdTM, Australia, http://www.vermicrobe.com).
These species vary in their adaptation to different climatic conditions, e.g.
Eisenia fetida are hardy and can survive in extreme temperature conditions,
Lumbricus rubellus are well adapted to low temperature (as low as 3-4˚C)
whereas Perionyx excavates are adapted to moderate (e.g. 25˚C) temperature,
which was the reason for using a mixture of three species of earthworms. Mature
81
earthworms were used to seed each chamber at the rate of 6 kg f wt per chamber
containing approximately 60 kg f wt (0.3 m3) of nine month old aged rumen
compost. Feeding occurred every 7-10 days at approximately 20 kg of composted
rumen paunch per chamber. All of the chambers used in this process were
maintained at ambient temperatures during the whole experimental period
(summer of 2009) in Rockhampton (latitude: 23 22‘ 0.345‖ S and longitude: 150 31‘
0.53‖ E). The vermicompost chambers were irrigated twice each day for 15 min at
10:00 a.m. and 2:00 p.m. with vermicast leachate at 120 L for 15 min to maintain
substrate moisture at around 45-70%. Fresh vermicast was removed after twelve
weeks and representative samples (n = 3) were taken for laboratory analysis.
The pH and electrical conductivity (EC) of the rumen compost and
vermicast samples were measured on 1: 20 (w/v) diluted samples. This dilution
ratio was taken considering the high water absorbance of the tested samples.
Moisture content was calculated after drying at 105˚C for 24 h. Inorganic soluble
NPK (NH4+-N, NO3−-N, PO4−-P and K+-K) were determined on triplicate samples
(d wt) using colorimetric test strips (‗RQflex plus‘, Merck, Germany).
2.4. Preparation of compost extracts and sampling regimes
Compost extracts were prepared from rumen compost (RC) and
vermicasts (VC), with 3 kg f wt of material sealed into a cotton (28 g m-2) bag
and submerged into 30 L tap water in a 60 L plastic bucket, and amended with
1% (v/v) ‗fish & kelp hydrolysate‘ and 0.5% (v/v) molasses at the ratio of 1:10
(w/v). Fish & kelp hydrolysate is an organic product of Searles® which consists
82
of 78% fish hydrolysate, 20% kelp, 2% stabiliser and 4% fish oil on w/w basis. It
contains 10.3% N, 2.5% P, 2.5% K, 7% Ca, 0.2% Mg, 0.5% S, 690 mg/kg Fe, 5
mg/kg Cu, 14 mg/kg Mn, 32 mg/kg Zn, 14 mg/kg B, and 1 mg/kg Mo in organic
form. Molasses is a treacle-like by-product of cane processing. The water used
was aerated for 30 minutes to remove chlorine before addition of compost.
Compost extracts were incubated indoors at between 22˚C and 30˚C (diurnal
range). The suspensions were continuously aerated (36 L/ min air delivery per
bucket through air stones) for 48 h to produce rumen compost extract and
vermicast extract. Concurrently, same volumes of vermicast leachate (30 L) with
‗fish and kelp hydrolysate‘ and molasses were also incubated in the same manner
producing vermicast leached extract.
The parameters of pH, EC, temperature and dissolved oxygen were
monitored at 3 h intervals for 24 h and finally at 48 h during preparation of the
extracts. Inorganic nutrient analyses of triplicate samples of the compost extracts
was performed using ‗RQflex plus‘ colorimetric test strips. Humic and fulvic
acids were also determined from samples collected at 24 h using a UV-Vis
spectrophotometer following a slightly modified method by Yamada et al. (1998).
FDA hydrolysis was determined using 1 mL samples collected after 12, 24 and
48 h incubation. Microbial populations were determined from 24 h incubated
samples, using the methods discussed below.
2.5. Microbial determinations
Bacterial and fungal populations of serially diluted samples were
determined by the pour plate method (Harrower, 2006). Bacterial plates were
incubated at 37˚C for 1-2 days, while fungal plates were incubated at 25˚C for 3-
83
5 days, both with three replications. The evolved CO2 of unaltered samples (basal
respiration, BR) and of substrate induced respiration (SIR) was assessed of
triplicate samples as an index of microbial activities of rumen compost and
vermicast samples following the protocol of Anderson (1982). As an alternative
measure of microbial activity, fluorescein diacetate (FDA) hydrolysis was
determined following the method given by Adam and Duncan (2001).
2.6. Microbial biomass carbon analysis
Microbial biomass carbon was determined by the chloroform fumigation
extraction method (Vance et al., 1987). Total and dissolved organic C (TOC and
DOC, respectively) were measured spectrophotometrically following the
dichromate digestion method (Walkley & Black, 1934). The microbial biomass C
was obtained by subtracting DOC from TOC and dividing by a factor used in
converting extracted organic carbon to microbial biomass C, kec, where kec = 0.33
(Sparling & West, 1988).
2.7. Community level physiological profiles of bacteria and fungi
The Ecoplate and FF microplate systems (BiologTM Inc, USA) were used
to identify bacterial and fungal functional diversity, respectively. The rates of
utilisation of 31 different C substrates are assessed in the Ecoplate method, while
the FF microplate involves use of 95 substrates (Insam, 1997). Triplicate samples
of rumen compost and vermicast (1 g f wt) as well as rumen compost extract and
vermicast leached extract samples (1 mL) were serially diluted to 10-3 in 9 mL of
sterile water in triplicate. Solutions were then inoculated to each well of the
Biolog Ecoplates and FF plates, using 100 and 150 μL samples, respectively. The
84
microplates were monitored at 0, 24, 48, 72, 96, 120, 144 and 168 h by ELISA
Reader at 570 nm for the Ecoplate, and at 490 and 750 nm for the FF microplates.
The average well colour development (AWCD) of all absorbance data was
individually calculated for all different C substrates utilised by the microbiota in
both Ecoplate and FF microplate (Konopka et al., 1998), however, the 72 h
absorbance data were chosen for statistical analysis (Garland, 1996).
2.8. PCR and DGGE
Total DNA was extracted by the bead beating method from three samples
of each preparation type (rumen compost, vermicast, rumen compost extract, and
vermicast leached extract), using a DNA extraction kit (MO BIO Laboratories,
Inc., Carlsbad, CA). Polymerase chain reactions (PCR) were performed using a
thermocycler (Bio-Rad, USA) using 50 µL reaction volumes containing
approximately 5 ng of purified genomic DNA, 0.5 µM of each primer, 2.5 mM
MaCl2, 0.2 mM of dNTP, 10 µL of 5X PCR-buffer and 0.3 U of Taq
Polymerase. The thermocycling conditions for PCR were as described by Girvan
et al. (2003). The PCR products from amplifications of 16S rDNA and ITS
regions were analysed with Universal Mutation Detection System (Bio Rad Inc.,
CA, USA) using 9% polyacrylamide gels (the ratio of acrylamide was 37: 1).
Bacterial and fungal communities were profiled by DGGE analysis of the
appropriate amplicons using a denaturing gradient of 40 to 60% for bacteria and
42 to 52% for fungi, electrophoresed at 60 V for up to 20 h at 60˚C. DGGE gels
were then silver stained (Girvan et al., 2003), scanned and saved as tiff files with
Epson Expression V700 Pro and used for subsequent analysis using Phoretix 1D
advanced analysis package (Phoretix Ltd, UK).
85
2.9 Statistical analysis
Statistical significance for all replicated samples was determined by
analysis of variance (ANOVA) following Tukey‘s (HSD) pairwise comparison
using the statistical package (XLSTAT, 2010). Differences in means were
compared at p< 0.05. Data on relative band intensities of microbial communities
from the DGGE profile were calculated with Phoretix 1D advanced analysis
package (Phoretix Ltd, UK). The UPGMA dendrogram was generated and the
digitised image was analysed by the same package to express the relatedness of
microbial communities as similarity clusters. The Shannon Weaver diversity
index H‘ (Shannon & Weaver, 1963) and Equitability index J (Begon et al., 1990)
were calculated from the number and intensities of bands present in each lane.
3.1. Physico-chemical analysis
The rumen compost material contained approximately 0.2% w/w N in
soluble form, and 0.05% soluble P (Table 3.1). Vermicast material was lower in
EC, nitrate, phosphate, humic acid and fulvic acid content, compared to the
original compost material. This result is expected, given extraction of nutrients by
the earthworms (which doubled in mass during the vermicomposting period) and
leaching of the vermicast material (1033 L m-3).
86
Table 3. 1. Initial physico-chemical, microbial and biochemical characteristics of rumen compost

and vermicast (n = 3).
Parameters Rumen compost (RC) Vermicast (VC)

pH (1:20) 6.53 ± 0.02 a 6.62 ± 0.02 a
-1
EC (dS m ) (1:20) 0.35 ± 0.01 b 0.26 ± 0.01 a
Moisture content (%) 58.84 ± 1.51 a 56.56 ± 2.24 a
- -1
NO3 -N (mg kg d wt) 1997.38 ± 29.37 b 1015.25 ± 30.52 a
+ -1
NH4 -N (mg kg d wt) 35.33 ± 2.52 b 21.66 ± 4.17 a
3- -1
PO4 -P (mg kg d wt) 551.46 ± 10.61 b 283.13 ± 10.11 a
+ -1
K -K (mg g d wt) 60.49 ± 11.25 a 50.23 ± 7.23 a
-1
Humic acid (g kg d wt) 45.37 ± 3.14 b 5.67 ± 0.45 a
-1
Fulvic acid (g kg d wt) 21.53 ± 1.56 b 4.54 ± 0.14 a
Bacterial population (cfu Log 10 g-1) 7.88 ± 0.02 b 6.40 ± 0.10 a
Fungal population (cfu Log 10 g-1) 5.19 ± 0.02 b 4.59 ± 0.06 a
-1
Microbial biomass C (mg C g d wt) 3.33 ± 0.45 a 12.56 ± 0.37 b
-1 -1
Basal respiration (µg CO2 g d wt h ) 81.58 ± 3.71 b 38.72 ± 3.52 a
-1 -1
Substrate induced respiration (µg CO2 g d wt h ) 808.39 ± 9.81 b 528.03 ± 3.52 a
-1 -1
Total Microbial activity (µg FDA g d wt h ) 1218.98 ± 176.95 b 611.34 ± 41.30 a
d wt Dry weight
Within rows, means with the same letter are not significantly different according to Paired
T- test (p< 0.05).
3.2. Microbial and biochemical assays
Plate counts of bacteria and fungi, and basal and substrate induced
respiration were significantly higher for the initial rumen compost than for
vermicast, however, microbial biomass was lower (p< 0.0001) and extractable
DNA (data not shown) was not different between the two substrates (Table 3.1).
These results are consistent with a more 'active' microflora in the compost than
the vermicast, the latter in which microbiota were apparently dormant.
3.3. Characterisation of compost extracts
Solid compost materials are bulky to apply in broad scale
horticulture/agriculture, and thus there is a growing interest in liquid extracts or
microbially enhanced liquids based on incubation of source inoculum. In this
study, the initial compost, the vermicast material and the vermicast leachate were
87
used as source inocula; all of which were amended with the ‗fish and kelp
hydrolysate‘ and molasses, producing rumen compost extract, vermicast extract
and vermicast leached extract, respectively.
During incubation significantly higher pH values and temperature were
recorded in vermicast leached extract, while rumen compost extract had
significantly higher EC compared to other liquid extracts (Fig. 3.1). The oxygen
level in vermicast extract was maintained at approximately 80% saturation until 9
h of incubation period. However, dissolved oxygen decreased rapidly after
incubation for 3-6 h in vermicast leached extract, 6-12 h in rumen compost
extract, and 9-12 h in vermicast extract, indicative of the level of microbial
activity during incubation (Fig. 3.1D). The depletion in oxygen recorded during
incubation indicates that the aeration system was not capable of meeting the
oxygen demand of the microbial culture. This could be addressed by either
improving the aeration system (e.g. small bubbles, increased air flow rate) or by
decreasing microbial demand (e.g. provision of less substrate). Such comparative
studies on microbially enhanced compost extracts produced with different levels
of microbial demand have been studied in another experiment (data not
presented).
88
Figure 3. 1. Comparison of pH, EC (dS m-1), fluctuations in temperatures (°C) and dissolved
oxygen (%) per unit time (readings taken at every 3 h intervals until 24 h after commencement of
compost extract incubation and final readings taken at 48 h) in rumen compost extract (RCE),
vermicast leached extract (VLE) and vermicast extract (VCE) (n = 3).
89
The bulk solution oxygen level was < 5% for 6-21 h in vermicast leached
extract, for12-24 h in vermicast extract and for 12-48 h in rumen compost extract,
despite the fact that these solutions were being aerated continuously at the rate of
36 L air/min per 30 L vessel. Given non-ideal mixing and aeration, the culture is
likely to have consisted of a mixture of environments ranging from aerobic to
anaerobic. Such conditions may favour increased microbial diversity.
Vermicast leached extract was high in ammonium, phosphate and humic
acid, relative to the rumen compost extract (Table 3.2), as expected considering
the metabolising activity of earthworms. Potassium and fulvic acid levels were in
the order of rumen compost extract > vermicast leached extract > vermicast
extract, and rumen compost extract = vermicast leached extract > vermicast
extract, respectively. Bacterial and fungal populations as indicated by the plate
counts were in the order vermicast extract > vermicast leached extract > rumen
compost extract and vermicast extract > rumen compost extract > vermicast
leached extract, respectively. In contrary to the pour plate results, highest
microbial activity in vermicast leached extract and least in vermicast extract was
demonstrated by the FDA hydrolysis method (Fig. 3.2). The difference between
the two methods presumably reflects the enumeration of culturable organisms
under the conditions of the pour plate, while the FDA method assesses all active
organisms in the culture at time of assay.
90
Table 3. 2.Comparison of chemical, biochemical and microbial characteristics of rumen compost

extract (RCE), vermicast leached extract (VLE), vermicast extract (VCE) incubated for 24 hours
with 1% kelp & fish hydrolysate and 0.5% molasses as amendments in all treatments (n = 3).
Parameters RCE VLE VCE

+ -1
NH4 -N (mg L ) 1.06 ± 0.05 b 1.42 ± 0.03 c 0.23 ± 0.04 a
3- -1 21.09 ± 0.87 a 26.31 ± 1.15 b 30.22 ± 1.21 b
PO4 -P (mg L )
+ -1
K -K (g L ) 0.68 ± 0.01 b 0.60 ± 0.02 b 0.46 ± 0.02 a
-1
Humic acid (g L ) 0.70 ± 0.11 b 2.23 ± 0.13 c 0.04 ± 0.01 a
-1
Fulvic acid (g L ) 1.12 ± 0.12 b 0.84 ± 0.03 b 0.33 ± 0.02 a
Bacterial population (cfu Log 10 mL-1) 11.50 ± 0.03 a 11.88 ± 0.01 b 11.99 ± 0.01 c
Fungal population (cfu Log 10 mL-1) 5.16 ± 0.04 b 4.89 ± 0.08 a 5.64 ± 0.03 c
d wt Dry weight
Within rows, means with the same letter are not significantly different according to
Tukey's test (p< 0.01).
Figure 3. 2. Comparison of total microbial activity per unit time (readings taken at 12, 24 and 48
h) following the FDA hydrolysis method in rumen compost extract (RCE), vermicast leached
extract (VLE) and vermicast extract (VCE) (n = 3).
The functional diversity of culturable microorganisms of the tested
samples were differentiated on the basis of the bacterial and fungal kinetics and
substrate utilisation patterns (Konopka et al., 1998). Although total microbial
activity (as measured with FDA) in rumen compost was twice that of vermicast
(Table 3.1), no difference was observed between rumen compost and vermicast in
91
terms of the rate of substrate utilisation by bacteria (BiologTM Ecoplate), substrate
utilisation by fungi (FF microplate) was significantly higher in rumen compost
compared to the vermicast (Fig. 3.3) after 72 h of incubation. However, a sudden
decline in optical density (AWCD 490 nm) of rumen compost was noted in the
FF microplate readings at 120 h compared to that measured at 96 h of incubation.
The reason for this could be due to the antagonistic interactions between the
microbiota or due to the toxicity caused to the microorganisms by the redox dyes
used in the Biolog wells as suggested by Ullrich et al. (1996) who studied the
toxic effects of redox dye on bacterial metabolism. However, the exact reason for
this temporary decline is unknown. Biolog plate substrate utilisation by vermicast
leached extract was significantly higher than that by rumen compost extract (Fig.
3.3), consistent with the results obtained from the FDA hydrolysis (Fig. 3.2).
Thus, the incubation of the compost extracts with additional food sources allowed
an increase in the active bacterial and fungal population compared to that of the
original communities of the composts as suggested by other researchers (Naidu et
al., 2010; Scheuerell, 2004).
92
Figure 3. 3. Kinetics of average well colour development (AWCD) and optical densities of (A)
bacterial (570 nm) and (B) fungal (490 nm) communities, originating from rumen compost (RC)
and vermicast (VC) as well as rumen compost extract (RCE) and vermicast leached extract (VLE)
(n = 3).
Principal component analysis of the Biolog data indicated that rumen
compost and vermicast were different in bacterial and fungal substrate utilisation
patterns compared to rumen compost extract and vermicast leached extract (with
separation by PC1, Fig. 3.4). The PCA of Ecoplate data explained 91% (PC1) and
5% (PC2) of the total variance whereas the PCA of FF microplate data explained
84% (PC1) and 7% (PC2) of the total variance (Fig. 3.4). In the present study, we
did not include a statistical control, i.e. rumen compost without earthworms,
because the aim of this experiment was to generate vermicast, which is compost
93
that has passed through the gut of the earthworm and compare that with the initial
compost. It could be that the changes observed in the nutrient and microbial
composition of the vermicast could have been related to the duration of the
incubation and passage through the earthworm. However, substrate utilisation
results cannot confirm the differences in species composition of microbiota as
only the functional physiological profile of culturable microorganisms can be
detected by this method (Widmer et al., 2001).
Figure 3. 4. Principal component analysis of optical densities produced by microbial activities of

rumen compost (RC), vermicast (VC), rumen compost extract (RCE) and vermicast leached
extract (VLE) consuming 32 (Biolog Ecoplate) and 96 (Biolog FF microplate) different food
sources including the control representing (A) bacterial and (B) fungal diversity, respectively (n =
3).
94
3.5. PCR-DGGE analysis
Since it is based on extractable DNA, the PCR-DGGE method should
represent an assessment of microbial diversity, however, DGGE will only detect
the dominant 1% of species in the bacterial population (Murray et al., 1996).
Derived measures of H‘ (diversity), J (evenness) and S (richness) from DGGE gel
pictures were similar for all tested materials (rumen compost, vermicast, rumen
compost extract and vermicast extract), for both bacteria and fungi (Table 3.3).
The only difference was a low result for bacterial S values in rumen compost.
These results suggest a similar species range of microorganisms in all products.
The results of PCR-DGGE analysis were consistent with that of the Biolog, in
that the bacterial community structure of the rumen compost and vermicast were
more similar to each other than to the liquid cultures (Fig. 3.5A). The UPGMA
dendrogram showed 45% homology between all the bacterial communities, while
55% and 57% homology was seen between the solid products (rumen compost
and vermicast) and the liquid extracts (rumen compost extract and vermicast
extract), respectively (Fig. 3.5A). The difference between replicate batches was
low in some cases (e.g. vermicast leached extract and vermicast), but high in
others (e.g. rumen compost) (Fig. 3.5A). Similar results were recorded for the
fungal community analysis (Fig. 3.5B), although amplifiable DNA was
unfortunately not obtained from vermicast leached extract samples.
95
Table 3. 3. Shannon weaver Diversity Index (H‘), Equitability Index (J) and number of bands (S)
for bacteria and fungi present in rumen compost (RC), vermicast (VC), rumen compost extract
(RCE) and vermicast leached extract (VLE) (n = 2).
Bacteria Fungi
Treatments H' J S H' J S
RC 2.42 ± 0.09 a 0.81 ± 0.02 a 20 ± 1.00 a 2.90 ± 0.02 a 0.90 ± 0.01 a 25 ± 0.00 a
VC 2.49 ± 0.02 a 0.76 ± 0.01 a 26 ± 0.00 ab 2.98 ± 0.02 a 0.90 ± 0.00 a 27 ± 0.00 a
RCE 2.51 ± 0.22 a 0.78 ± 0.05 a 25 ± 2.00 ab 2.60 ± 0.28 a 0.87 ± 0.05 a 20 ± 3.00 a
VLE 2.86 ± 0.00 a 0.83 ± 0.00 a 31 ± 0.00 b ND ND ND
ND not determined
Within rows, means with the same letter are not significantly different according to Tukey's test (p< 0.05).
Figure 3. 5. UPGMA (Unweighted Pair Group Method with Arithmetic mean algorithm)
dendrogram generated from responses to 16S rDNA and Internal Transcribed Spacer region based
Denaturing Gradient Gel Electrophoresis (DGGE) analyses for (A) bacteria and (B) fungi present
in rumen compost (RC), vermicast (VC), rumen compost extract (RCE) and vermicast leached
extract (VLE) (n = 2).
Higher functional diversity in vermicast was found compared to the
original compost as revealed by the Shannon-Weaver diversity index H‘ (Sen &
Chandra, 2009). Similarly, Vivas et al. (2009) showed that finished vermicast
96
was richer in bacterial population and functional diversity than the original and
composted olive-mill waste, with increased values of H‘ calculated using DGGE
banding patterns. In contrast, rumen compost extract and vermicast leached
extract were not higher in diversity index (H‘) or equitability index (J) for
bacteria or fungi than the original rumen compost or vermicast although greater
number of bands in the bacterial DGGE profile (band richness, S) was observed
in vermicast leached extract, relative to the original rumen compost (Table 3.3).
This indicates that there was no significant difference in the bacterial or fungal
communities of either solid or liquid phases of rumen compost and vermicast. It
may be that earthworm action results in little change in mirobial species diversity
for well composted material, in comparison to incompletely composted material.
The olive mill waste of Vivas et al. (2009) may have fallen into the latter
category.
4. Conclusions
Coupled with chemical, biochemical and microbial approaches, rumen
compost before and after passing through the guts of earthworms was evaluated
and compared with their subsequent microbially enhanced extracts. While
earthworm activity increased the microbial biomass of the rumen compost,
liquid incubation of composts altered their microbial and nutrient composition.
Although no differences were found in the genetic diversity of microorganisms,
highest functional diversity was observed in vermicast leached extract compared
to rumen composts, vermicast and rumen compost extract. The ability of these
liquid extracts to shift the microbial activity and diversity of the soil is another
fertile area for consideration.
97
Chapter 4: Commercial extract - characterisation
Chapter 4
Comparison of microbially enhanced compost
extracts produced from composted rumen content
material and from commercially available inocula
A version of this chapter has been published in Bioresource Technology by Karuna Shrestha, Eric
M. Adetutu, Pramod Shrestha, Kerry B. Walsh, Keith M. Harrower, Andrew S. Ball and David J.
Midmore
Abstract
A comparative study was performed on compost extracts prepared from
cattle rumen content composted for three and nine months, nine month old
compost inoculated with a Nutri-Life 4/20TM inoculum, and two commercial
preparations (Living SoilTM and Nutri-Life 4/20TM), all incubated for 48 h. Nutri-
Life 4/20TM had the highest concentrations of NO3−-N and K+-K, while rumen
compost extract had higher humic and fulvic acids concentration. The bacterial
and fungal community level functional diversity of three month old compost
extract and of Living SoilTM, assessed with BiologTM, were higher than that of
nine month old rumen compost extract, with or without Nutri-Life 4/20TM
inoculum, or Nutri-Life 4/20TM. No difference in fungal diversity was observed
between treatments, as indicated by Denaturing Gradient Gel Electrophoresis
(DGGE) analysis, however, bacterial diversity was higher in all compost extracts
98
and LivingSoilTM, compared to the Nutri-Life 4/20TM. Criteria for judging the
quality of a microbially enhanced extract are discussed.
1. Introduction
There is an increasing global interest in the use of liquid organic fertilisers
and inoculums of beneficial microbes in support of ‗biological farming‘ and
sustainable agriculture (Naidu et al., 2010). A range of benefits are claimed for
these preparations, including direct nutrient addition, increased mineralisation
and solubilisation, protection against plant pathogens, and mitigation of soil
aluminium toxicity. As an example of an inoculum, compost extracts can be
incubated, either aerobically or anaerobically, with microbial food sources to
create a rich, if uncontrolled, blend of microorganisms. Quite clearly, the quality
of the compost (in terms of minerals and microbiota) will most likely influence
the quality of such extracts. Alternatively, more defined microbial populations
may be used. A number of commercial suppliers of inocula are attempting to
serve this market, typically on a relatively small and local scale. The commercial
products claim to be more consistent than that based on compost, and capable of
being stored (and thus transported). With the increasing use of such amendments
comes a need to establish their benefits, and to set quality criteria.
LivingSoilTM (abbreviated as ―LS‖) (O‘Grady Rural, Lismore, NSW) and
Nutri-Life 4/20TM (abbreviated as ―NL-4/20‖) (Nutri-Tech Solutions, Yandina,
Qld) are examples of microbial formulations that are produced commercially in
Australia. LivingSoilTM is a dry formulation, claimed to contain a blend of
microbiota, plant hormones and humic acids derived from composts or
vermicomposts, with diatomaceous earth acting as a microbial carrier and
99
microbial food (baker‘s yeast) supplied separately. It is reported to be based on
pure cultures of a number of microorganisms, along with compost and
vermicompost, with the final product claimed to contain Azotobacter spp.,
Azospirillum brasilense, Bacillus spp., Cellulomonas cellosea, Chaetomium spp.,
Metarhizium anisopliae, Pseudomonas spp., Polyanguim cellulosum,
Streptomyces spp., Trichoderma spp., together with unidentified microbes from
the compost and vermicompost (www.smartbugs.com.au). These microorganisms
are involved in nitrogen fixation (e.g. Azotobacter spp. and Azospirillum spp.,
Khammas & Kaiser, 1992), phosphate solubilisation and nutrient release (e.g.
Azotobacter spp., Garg et al., 2001), pectin decomposition (e.g. Bacillus spp.,
Halsall & Gibson, 1985), disease suppression/protection against plant pathogens
(e.g. Trichoderma spp., Harman, 2006), soil bioremediation (e.g. Trametes spp.
Atagana, 2009), and cellulose degradation (e.g. Cellulomonas spp., An et al.,
2005). The inoculum is intended to be used to seed a non-sterile culture, with two
food sources (molasses and a solid powder based on yeast). Thus, this product
emulates a ‗compost tea‘, with the LivingSoilTM product replacing fresh compost
as the inoculum source.
Nutri-Life 4/20TM, sourced from Nutri-Tech Solutions Pty Ltd (Yandina,
Qld. Australia, www.nutri-tech.com.au), is a blend of microbes, and is claimed to
contain Trichoderma spp., Rhizobium spp., Bacillus spp. and Pseudomonas spp.
Nutri-Life 4/20TM is recommended for direct application to the soil at the rate of
30 L ha-1, for addition to compost, and to recover previously disease-affected
soils. Nutri-Life 4/20TM is produced by incubating 1 L of Liquid Microbe
FoodTM, 1 L of Dominate (fungi)™ and 50 g of Nutri-Life 4/20TM inoculum per
100 L of non-chlorinated water. Dominate (fungi)™ is claimed to create
100
favourable conditions for a fungal dominated brew. Liquid Microbe FoodTM is
described as a fungal and bacterial food source, and Nutri-Life 4/20TM inoculum
as a freeze-dried formulation of bacterial and fungal source, enriched in
Trichoderma.
Given the claimed benefits of compost extracts and commercial
biofertilisers, the characterisation of such products has become an interesting area
of investigation. Scheuerell (2004) commented on the need to explore the impact
of production process on the abundance, diversity and evenness of
microorganisms in the resultant compost extracts using molecular methods.
Indeed, Soil Foodweb Institute (SFI) Pty Ltd has operated since 2001 in Australia
providing a microscopically based assessment service. Other techniques used in
this study also have the potential to characterise compost extracts. The present
study aimed to compare microbially enhanced compost extracts produced from
cattle rumen content materials (three and nine month old compost, and nine
month compost along with Nutri-Life 4/20TM inoculum) and two commercial
products (LivingSoilTM and Nutri-Life 4/20TM). The chemical, biochemical and
microbial parameters of the given formulations were compared during incubation
using the same techniques as described in Shrestha et al. (2011a).
2.1. Source compost and commercial preparations
Cattle rumen content material, obtained from Broadmeadows Pty Ltd,
Emu Park Road, Australia, was composted following a windrow composting
process over nine months. Those windrows were turned mechanically at monthly
intervals to provide aeration and to improve homogeneity. Rumen compost aged
101
three and nine months, achieved from the same source material, was chosen for
this study and characterised (data not shown here). LivingSoilTM was obtained
from O‘Grady Rural (courtesy of Ray O‘Grady, Lismore, NSW) whereas Nutri-
Life 4/20TM was supplied by Nutri-Tech Solutions Pty Ltd, Yandina, Qld
(courtesy of Graeme Sait, NTS).
2.2. Preparation of compost extracts and commercial extracts
Three and nine month old rumen compost extracts were prepared by
completely submerging the respective composts in tied cotton bags, in the ratio of
1:10 (w/v) in tap water amended with 1% (v/v) ‗fish and kelp hydrolysate‘ and
0.5% (v/v) molasses as described in Shrestha et al. (2011a). LivingSoilTM, a
microbially enhanced solution without the use of compost, was produced by
incubating 450 g of LivingSoil inoculant, 150 g MicroBooster CN30 (microbial
food, revealed to contain yeast cells by microscopic examination) (as supplied by
O‘Grady Rural) and 300 mL of molasses, with an addition of 3 mL of a surfactant
(Oresome Antifoam), per 30 L of water, following the manufacturer‘s directions.
Similarly, Nutri-Life 4/20TM was also used to produce a microbially enhanced
solution, following the recommended incubation instructions (300 mL Liquid
Microbe FoodTM) with 300 mL DominateTM and 15 g Nutri-Life 4/20TM inoculum
per 30 L of culture.
A nine month old compost extract was also amended at the beginning of
the incubation with Nutri-Life 4/20TM inoculum (abbreviated as ‗9MTCE‘) at the
rate of 15 g per 30 L of extract. Nutri-Life 4/20TM inoculum is not marketed as a
compost extract amendment. However, we postulated that it could serve as an
extract inoculum, introducing groups such as Trichoderma.
102
In all the preparations, water was aerated for 30 min for dechlorination
prior to use, and all compost extracts were aerated continuously throughout
incubation with two air stones suspended at the bottom of each bucket, supplying
36 L min-1 of air per 30 L of liquid culture as described in Shrestha et al. (2011a).
All of the preparations were incubated in triplicate for the same duration, up to a
maximum of 48 h, for comparison purpose.
The pH, electrical conductivity (EC), dissolved oxygen, and temperature
of the liquid cultures were monitored at every 3 h interval from 0 to 24 h of
incubation, with an additional measure taken at 48 h. Samples of each culture
were collected in triplicates at 24 h of incubation and concentrations of NH4+-N,
PO4−-P were measured using colorimetric test strips (RQflex plus). For humic
and fulvic acids, samples were centrifuged and filtered through a 0.45 µm
membrane filter and were measured following a slightly modified method by
Yamada et al. (1998).
2.4. Biochemical and microbial analyses
Biological activity in compost extract samples collected at 0, 12, 24, and
48 h was estimated following the FDA hydrolysis method, while microbial
populations were enumerated from samples collected at 24 and 48 h of incubation
using the pour plate count method (Harrower, 2006). For comparative molecular
analyses between treatments, the DNA of the 24 h samples was extracted as
described in Shrestha et al. (2011a) and stored at -20˚C for further use.
103
2.5. Community level physiological profiles
The Ecoplate and FF microplate systems (Biolog Inc, USA) were used to
identify bacterial and fungal functional diversity of the triplicate samples from all
treatments, respectively. The average well colour development (AWCD) was
calculated at 24 h intervals from 0 to 168 h for all different C substrates in both
Ecoplate and FF microplate (Konopka et al., 1998) before subjecting the 72 h
data to statistical analysis (Garland, 1996).
2.6. Microscopic analyses
Samples of LivingSoilTM and nine month old rumen compost extract were
shipped in cooled liquid form (i.e. with ice blocks) to the Soil Foodweb Institute
(Lismore, NSW) for microbial analysis, where samples were analysed using a
microscopic method. Active and total counts of bacteria and fungi, their
respective ratios, total nematodes, protozoa, hyphal diameter of fungi, and plant
available N supply were the major attributes taken into consideration.
2.7. Genetic diversity of microbial communities of compost extracts
For DNA fingerprinting of rumen and commercial cultures, the 16S
rDNA and Internal Transcribed Spacer region (ITS) based DGGE profiles were
analysed. In this process, DNA of respective samples was extracted in duplicate
and PCR-amplified using universal primers for bacteria and fungi separately. The
amplicons (20 µL of PCR products) for both bacterial and fungal PCR were
subjected to polyacrylamide gel electrophoresis using urea formamide (40 to 60%
for bacteria and 42 to 53% for fungi) as the lower and higher denaturant,
respectively (Girvan et al., 2003), and run for 20 h at 60˚C and 60 V for bacteria
104
and for 15 h at 58˚C and 78 V in case of fungi using the Bio-Rad DCode system
(Bio Rad Inc., CA, USA). The UPGMA dendrograms were generated and the
Shannon Weaver diversity index (H‘) (Shannon & Weaver, 1963), equitability
index (J) (Begon et al., 1990) and species richness (S) (Krebs, 1999) were then
calculated.
2.8. Statistical analysis
Statistical analyses for all data including values for microbial diversity,
equitability and richness were performed by one or two way ANOVA using a
statistical package (XLSTAT, 2010). Differences between means were assessed
by Tukey‘s (HSD) pairwise comparison at p< 0.05 (Shrestha et al., 2011a).
3.1. Culture characterisation
The Nutri-Life 4/20TM based culture had two orders of magnitude higher
concentrations of NO3−-N, suggesting the presence of a nitrate salt in this product,
while the compost based products had approximately twice the level of PO4−-P,
humic and fulvic acids than the commercial preparations (Table 4.1). The latter
result is expected as rumen based compost used in this study was rich in
phosphate and organic acids, as presented in Shrestha et al. (2011a). There was
little difference in ammonium levels between treatments.
Cultures based on Living soilTM possessed a high pH, around 8, while
those based on three month old rumen compost extract began at pH 6 and
increased to 8 within 48 h (Fig. 4.1). These observations are consistent with the
presence of alkaline material in the Living soilTM formulation, while the increase
105
in pH with incubation presumably reflects uptake of more anions than cations by
the culture. The pH of nine month old rumen compost extract plus Nutri-Life
4/20TM inoculum decreased from 5.5 to 5 at 48 h (Fig. 4.1), the reason for which
is unclear. It is, however, reported that neutral pH is optimum for the survival of
most microbes isolated from compost (Adegunloye et al., 2007). Future work
should consider pH adjustment of cultures, maintaining a pH of around 6.5.
Table 4.1. Comparison of physico-chemical and microbial characteristics of different liquid

cultures sampled at 24 h and at 48 h of incubation. 3MCE - three month old rumen compost
extract, 9MCE - nine month old rumen compost extract, 9MTCE - nine month old rumen compost
extract plus Nutri-Life 4/20TM inoculum, LS - Living soilTM, NL-4/20 - Nutri-Life 4/20TM. Within
rows, means (n = 3) with the same letter are not significantly different according to Tukey‘s test
(p< 0.05).
Parameter 3MCE 9MCE 9MTCE LS NL-4/20
- -1
NO3 -N (mg L ) 1.20 ± 0.88a 3.61 ± 0.60a 2.56 ± 0.75a 1.13 ± 0.00a 223 ± 14.17b
+ -1
NH4 -N (mg L ) 0.83 ± 0.03b 0.36 ± 0.03a 0.41 ± 0.05a 0.54 ± 0.04ab 0.54 ± 0.13ab
- -1
PO43 -P (mg L ) 25.0 ± 2.69c 14.8 ± 1.25b 25.0 ± 0.78c 6.74 ± 1.39a 5.33 ± 0.58a
+ -1
K -K (g L ) 0.74 ± 0.03a 0.79 ± 0.03a 0.80 ± 0.03a 0.84 ± 0.07a 1.09 ± 0.06b
-1
Humic acid (g kg d wt) 1.31 ± 0.01c 0.61 ± 0.08b 0.49 ± 0.13b 0.06 ± 0.01a 0.03 ± 0.01a
-1
Fulvic acid (g kg d wt) 1.54 ± 0.01c 1.43 ± 0.02c 1.46 ± 0.05c 1.03 ± 0.10b 0.13 ± 0.03a
Bacterial population (cfu Log 10) @ 24h 7.90 ± 0.03c 6.00 ± 0.00a 6.00 ± 0.00a 7.21 ± 0.05b 6.10 ± 0.10a
Bacterial population (cfu Log 10) @ 48h 8.98 ± 0.00c 8.41 ± 0.04ab 8.53 ± 0.04b 8.50 ± 0.06b 8.22 ± 0.05a
Fungal population (cfu Log 10) @ 24h 5.18 ± 0.05a 5.95 ± 0.03bc 5.93 ± 0.04b 6.11 ± 0.04c 6.40 ± 0.01d
Fungal population (cfu Log 10) @ 48h 6.81 ± 0.03a 7.14 ± 0.02b 6.81 ± 0.01a 6.74 ± 0.03a 7.27 ± 0.02c
d wt Dry weight
106
Figure 4.1. Comparison of (A) pH, (B) electrical conductivity (dS m-1), (C) temperatures (˚C) and
(D) dissolved oxygen (ppm) over time (readings taken at every 3 h intervals until 24 h and final
readings taken at 48 h) in different cultures. 3MCE - three month old rumen compost extract,
9MCE - nine month old rumen compost extract, 9MTCE - nine month old rumen compost extract
plus Nutri-Life 4/20TM inoculum, LS - Living soilTM, NL-4/20 - Nutri-Life 4/20TM (n = 3).
Garcia et al. (1991) reported an increase in EC during composting,
presumably representing a release of mineral ions as organic matter decays.
Although Living soilTM started with the lowest EC, it increased with time in the
Living soilTM incubation (Fig. 4.1), which most likely represents solubilisation or
mineralisation of substrate. The EC of other extracts were relatively stable, after
107
an increase during the first 3 h of incubation. This observation is consistent with
an extraction of soluble salts from the compost within 3 h, despite the
containment of the compost in a mesh bag. The final extract EC values of around
4 dS m-1 are well within the range tolerated by most plants, indicating that plants
could be grown on neat extracts (‗organic hydroponics‘).
Solution temperature was not significantly different between treatments
(data not shown), except in the last measurement period, at which point three
month old rumen compost extract and Living soilTM treatments were of higher
temperature, suggesting higher microbial growth and activity than present in
Nutri-Life 4/20TM, nine month old rumen compost extract, and nine month old
rumen compost extract plus Nutri-Life 4/20TM inoculum.
A sharp decline in solution dissolved oxygen, reflecting a microbial
oxygen demand greater than the oxygen transfer rate into the solution, occurred
in the three month old rumen compost extract treatment after 6 h of incubation,
after 9 h in the Living soilTM treatment, and later in other treatments (Fig. 4.1D).
This oxygen demand will parallel microbial growth as suggested by Shrestha et
al. (2011a). Here, although equal amount of oxygen was supplied in each
treatment, the level of dissolved oxygen varied among them indicating variation
in microbial population and their activities. The delayed start of microbial activity
in nine month old rumen compost extract, nine month old rumen compost extract
plus Nutri-Life 4/20TM inoculum and Nutri-Life 4/20TM could be due to lower
density of microorganisms in those treatments. The food supply must have
sustained their growth and activity until 24 h of incubation after which the
dissolved oxygen dropped significantly.
108
3.2. Microbial and biochemical analyses
Plate counts of bacteria were significantly higher in three month old
rumen compost extract, whereas fungal counts were significantly higher in Nutri-
Life 4/20TM samples, whether collected at 24 and 48 h of incubation (Table 4.1).
Higher bacterial counts in three month old rumen compost extract compared to
nine month old rumen compost extract could be due to the aging of source
compost during which the readily available fractions of carbon gets depleted
(Garcia-Gomez et al., 2003; Wu and Ma, 2002). The addition of beneficial fungi
and microbial foods favouring fungal growth, such as Liquid Microbe FoodTM,
Dominate (fungi)TM, and Nutri-Life 4/20TM inoculum, must be the reason for
higher fungal counts in Nutri-Life 4/20TM treatment. Adam and Duncan (2001)
reported a potential bias of the FDA hydrolysis method towards enzymatic
activities of bacteria rather than that of fungi, and total microbial activity as
measured by the FDA hydrolysis was highest by far in three month old rumen
compost extract, followed by that of Living soilTM at 12, 24 and 48 h of
incubation, compared to other treatments (Fig. 4.2). However, the oxygen
demand of the three month old rumen compost extract culture was also higher
than that of any other treatment (Fig. 4.1). Higher microbial activity in ‗young‘
(three month) compost (i.e. higher plate counts and FDA hydrolysis) is expected,
with a decrease in extractable organic carbon during the latter curing phase of
composting, as demonstrated by Wu and Ma (2002). The reason behind low
values in nine month old rumen compost extract and nine month old rumen
compost extract plus Nutri-Life 4/20TM inoculum compared to three month old
rumen compost extract could be due to aging of the compost. The aged compost
must have low organic component compared to the young compost. However,
109
after 48 h of incubation, the density of microorganisms (Table 4.1) as well as
microbial activity (Fig. 4.2) increased dramatically. This must have resulted in
the large oxygen consumption at 48 h of incubation in the respective cultures as
shown in Fig. 4.1.
Figure 4.2. Comparison of total microbial activity at different times (readings taken at 12, 24 and
48 h) following the FDA hydrolysis method in different cultures. 3MCE - three month old rumen
compost extract, 9MCE - nine month old rumen compost extract, 9MTCE - nine month old rumen
compost extract plus Nutri-Life 4/20TM inoculum, LS - Living soilTM, NL-4/20 - Nutri-Life 4/20TM
(n = 3). Within a time, treatment means with the same letter do not differ significantly and values
are mean ± SE (n = 3).
The addition of Nutri-Life 4/20TM inoculum to the nine month compost
did not result in higher bacterial or fungal plate counts nor enzymatic activities of
microbiota compared to the extract based on nine month compost alone (Fig.
4.2). The plateau population can be expected to be limited by food supply, not
inoculums. Certainly the food sources provided to the culture will influence
microbial species composition. For example, Naidu et al. (2010) reported that
humic acid and yeast extract in the ratio of 4:7 w/w, added as 1 g per 100 g of
compost, enhanced microbial growth (total bacteria, fungi and actinomycetes
measured as cfu mL-1 using spread plate method) during incubation of compost
110
extracts (prepared at the dilution of 1:5 w/v compost to water). The authors found
that humic acid favoured fungal (e.g. Trichoderma spp., yeast, actinomycetes and
other filamentous fungi) growth up to 61%, while the yeast extract contributed to
bacterial proliferation (e.g. Pseudomonas spp., lactic acid bacteria). There was no
evidence of a quicker population growth in the Nutri-Life 4/20TM inoculum
amended culture. Presumably microbes present in the additive were outcompeted
by those in the compost.
Although total microbial activity of three month old rumen compost
extract was the highest amongst all extracts from 12 h onwards (Fig. 4.2),
bacterial communities of Living soilTM showed equal functional diversity to that
of three month old rumen compost extract (Fig. 4.3) as indicated by their similar
patterns and non-significant differences in AWCD values at 72 h of incubation in
wells (Fig. 4.3). The pattern of microbial activity represented by the FDA was
consistent with the pattern of activity indicated by the FF microplate analysis.
The Nutri-Life 4/20TM treatment showed the lowest metabolic rates of substrate
utilisation of both bacteria and fungi (Fig. 4.3). This is consistent with the
bacterial plate counts but inconsistent with the results of fungal plate counts with
high fungal cfu recorded (Table 4.1). The single medium used in the Petri plates
may have favoured a few types of fast growing fungi, compared to the FF
microplate (which contains 95 different carbon substrates that allows the growth
of different fungal species leading into a distinct growth fingerprint).
111
Figure 4.3. Kinetics of average well colour development (AWCD) and optical densities of (A)
bacterial (570 nm) and (B) fungal (490 nm) communities, respectively, from different compost
extracts and microbial preparations. 3MCE - three month old rumen compost extract, 9MCE -
nine month old rumen compost extract, 9MTCE - nine month old rumen compost extract plus
Nutri-Life 4/20TM inoculum, LS - Living soilTM, NL-4/20 - Nutri-Life 4/20TM (n = 3) sampled
after 24 h of incubation.
The average well colour for bacterial (Ecoplate) growth of the compost
extract amended with Nutri-Life 4/20TM inoculum was not different to that of
unamended compost extract, however, higher fungal (FF microplate) growth was
noted in nine month old rumen compost extract plus Nutri-Life 4/20TM inoculum
compared to nine month old rumen compost extract (Fig. 4.3). The added
bacterial species in nine month old rumen compost extract plus Nutri-Life 4/20TM
inoculum might have been outcompeted and/or eliminated by the microbiota
already present in the compost extract. However, higher fungal (FF microplate)
112
growth was noted in nine month old rumen compost extract plus Nutri-Life
4/20TM inoculum compared to nine month old rumen compost extract (Fig. 4.3B).
Significant differences among treatments in average utilisation of specific
substrate guilds were evident when BiologTM (Ecoplate and FF microplate) data
were analysed by guild categories (Fig. 4.4). The Living soilTM culture displayed
the most even growth across all carbon substrates on the Ecoplate cultures. Of all
guild categories, no difference in bacterial substrate utilisation among nine month
old rumen compost extract, nine month old rumen compost extract plus Nutri-
Life 4/20TM inoculum and Nutri-Life 4/20TM was apparent except nine month old
rumen compost extract plus Nutri-Life 4/20TM inoculum averaged higher
utilisation of polymers than the other treatments (Fig. 4.4A). Similarly, the FF
microplate data of three month old rumen compost extract and Living soilTM
averaged higher utilisation of all guild categories of carbon substrates than the
other treatments (Fig. 4.4B). The fungal substrate utilisation of nine month old
rumen compost extract and nine month old rumen compost extract plus Nutri-
Life 4/20TM inoculum as shown by FF microplate data did not vary, however,
they averaged higher utilisation of amino acids, amines/amides and miscellaneous
substrates.
113
Figure 4.4. Average carbon substrate utilisation from different substrates by samples from three
month old rumen compost extract (3MCE), nine month old rumen compost extract (9MCE), nine
month old rumen compost extract plus Nutri-Life 4/20TM inoculum (9MTCE), Living soilTM (LS)
and Nutri-Life 4/20TM (NL-4/20) consuming (A) 32 (Biolog Ecoplate) and (B) 96 (Biolog FF
microplate) different food sources. Samples were collected from 72 h incubations of either Biolog
plate and utilisation was measured as the average optical density across all substrates witihin each
guild which included 7 different carbohydrates, 9 carboxylic acids, 4 polymers, 6 amino acids, 2
amines/amides and 3 miscellaneous for Ecoplate while 44 different carbohydrates, 17 carboxylic
acids, 5 polymers, 13 amino acids, 6 amines/amides and 10 miscellaneous for FF microplate.
Biolog data represent the physiological profile of culturable
microorganisms (Garland & Mills, 1991; Widmer et al., 2001; Zak et al., 1994).
The PCA of Ecoplate data explained 66% (PC1) and 10% (PC2) of the total
variance, whereas the PCA of FF microplate data explained 89% (PC1) and 2%
(PC2) of the total variance (Fig. 4.5). Principal component analysis revealed two
114
distinct clusters clearly for both bacteria and fungi (Fig. 4.5), with Living soilTM
and three month old rumen compost extract separated from nine month old rumen
compost extract, nine month old rumen compost extract plus Nutri-Life 4/20TM
inoculum and Nutri-Life 4/20TM. This suggests a large difference in catabolic
capacity between the microbial communities of these cultures (Fig. 4.5).
Relatedness of liquid cultures within either group indicates that the functional
groups of microbiota present in each culture are similar, possessing similar
potential for substrate utilisation. However, the overall shift in community
structure could not be attributed to the changes in the abundance of any particular
functional group in accord with Garland (1999), as indicated by the loadings of
the first principle component (data not shown).
115
Figure 4.5. Principal component analysis (PCA) of optical densities produced by microbial
activities of five different cultures consuming 32 (Biolog Ecoplate) and 96 (Biolog FF microplate)
different food sources including the control; A and B representing bacterial and fungal diversity.
3MCE - three month old rumen compost extract, 9MCE - nine month old rumen compost extract,
9MTCE - nine month old rumen compost extract plus Nutri-Life 4/20TM inoculum, LS - Living
soilTM, NL-4/20 - Nutri-Life 4/20TM (n = 3), all of which sampled at 24 h after incubation.
Since the Living soilTM formulation was derived from a compost based on
cow manure (which was further vermicomposted), it was not surprising to find
similarity of its bacterial communities with those of three month old compost
extract, although the fungal communities between those two varied (Fig. 4.5).
While the fungal substrate utilisation pattern of Living soilTM clustered with that
of nine month old rumen compost extract, nine month old rumen compost extract
plus Nutri-Life 4/20TM inoculum and Nutri-Life 4/20TM, the fungal community of
116
three month old compost extract was distinctly separated from those of other
treatments, suggesting a dissimilarity in species composition of microflora
between them (Fig. 4.5). Relatedness of nine month old rumen compost extract
and nine month old rumen compost extract plus Nutri-Life 4/20TM inoculum was
understandable, with little influence from the addition of the microbial
amendment to the compost extract. The functional diversity of Nutri-Life 4/20TM
was similar to that of nine month old compost extracts. This result was
unexpected, and no explanation is offerred.
3.4. Microscopic analyses
The methods used in the Soil Foodweb Institute (SFI) analysis are not
defined, but presumably are based around microscopic assessment of cell
numbers (from indications in SFI tutorial information). The SFI results suggest
that rumen based compost extract is bacterial dominated as compared to the
fungal dominated Living soilTM, with both extracts supplying only minimal
amounts of available N to the plants (Table 4.2).
Table 4.2. Sample analysis data of nine month old rumen compost extract (9MCE) and LS
(Living SoilTM) provided by Soil Foodweb Institute, NSW, Australia.
Expected range
Attributes 9MCE Comment LS Comment Low High
Sample size (mL) 1 - 1 - - -
Active bacteria (µg/mL) 179 above range 80.4 in range 10 150
Total bacteria (µg/mL) 11136 above range 17408 above range 150 3000
Active fungi (µg/mL) 123 above range 417 above range 2 10
Total fungi (µg/mL) 162 above range 427 above range 2 20
Hyphal diameter (µm) 4.5 - 4.5 - - -
Protozoa
Flagellates (numbers/g) 7 Low 0 Low 1000 -

Amoebae (numbers/g) 0 Low 0 Low 1000 -
Ciliates (numbers/g) 0 Low 0 Low 20 50
Total nematodes 0 Low 0 Low 2 10
Total fungi to total bacteria 0.01 Good 0.02 Good 0.01 0.1
Active to total fungi 0.76 High 0.98 High 0.1 0.25
Active to total bacteria 0.02 Low 0.005 Low 0.1 0.25
Active fungi to active bacteria 0.69 Low 5.18 High 0.9 1.1
Plant available N supply (lbs/ac) <5 - <5 - - -
117
The SFI results, however, are not consistent with the results of the
bacterial and of the fungal plate counts (Table 4.1). This apparently pinpoints the
limitations of direct count method using a microscope (Scheuerell, 2004),
although it is possible that the samples had altered during shipment. Additionally,
Bloem et al. (1995) indicated that living and dead cells could not be differentiated
by direct count method, thereby leading to overestimation.
3.5. PCR and DGGE
A UPGMA dendrogram was generated to express the relatedness of
microbial communities as similarity clusters (Fig. 4.6). Replicate variability of
both bacteria and fungi was higher in nine month old compost extracts than that
of other cultures, the latter showing more than 90% similarity in their replicated
DNA (Fig. 4.6).
118
Figure 4.6. UPGMA (Unweighted Pair Group Method with Arithmetic mean algorithm)
dendrogram for (A) bacteria and (B) fungi present in 3MCE, 9MCE, 9MTCE, LS and NL-4/20.
3MCE - three month old rumen compost extract, 9MCE - nine month old rumen compost extract,
9MTCE - nine month old rumen compost extract plus Nutri-Life 4/20TM inoculum, LS - Living
soilTM, NL-4/20 - Nutri-Life 4/20TM (n = 2), all of which sampled at 24 h after incubation.
A dissimilarity in bacterial community composition between nine month
old compost extract and that amended with Nutri-Life 4/20TM inoculum was
revealed, indicating that although overall activity and functional diversity were
not altered by the amendment, species composition was.
Curiously, the amended nine month old compost extract was more similar
to three month old rumen compost extract with approximately 85% homology,
than to the non-amended extract (Fig. 4.6A). The addition of Nutri-Life 4/20TM
inoculum presumably added bacterial species similar to those present in three
month old rumen compost extract and to a lesser extent to those present in Living
119
soilTM. These species must have been absent or below limit of detection by the
DGGE method in the nine month old rumen compost extract.
The 16S rDNA and Internal Transcribed Spacer region based DGGE
analyses showed significantly greater bacterial community diversity of rumen
based compost extracts and Living soilTM compared to that of Nutri-Life 4/20TM;
while fungal communities were more similar in their diversity between treatments
(Table 4.3). The calculated values for Shannon diversity index (H‘) and
equitability index (J) obtained from the bacterial DGGE profile (Table 4.3) were
not different between the treatments of three month old rumen compost extract,
nine month old rumen compost extract, nine month old rumen compost extract
plus Nutri-Life 4/20TM inoculum and Living soilTM, but this group had
significantly higher values for diversity and evenness compared to those of Nutri-
Life 4/20TM. This suggests the lowest diversity of bacterial community in Nutri-
Life 4/20TM.
Table 4.3. Diversity and equitability of microbial communities in different liquid cultures
compared. Shannon-Weaver diversity index, H‘ (Shannon and Weaver, 1949) and equitability, J
(Pielou, 1966) and number of bands, S were generated from DGGE profiles of PCR amplified
16S rDNA and ITS genes from all samples collected at 24 h of incubation. 3MCE - three month
old rumen compost extract, 9MCE - nine month old rumen compost extract, 9MTCE - nine month
old rumen compost extract plus Nutri-Life 4/20TM inoculum, LS - Living soilTM, NL-4/20 - Nutri-
Life 4/20TM (n = 2), all of which sampled at 24 h after incubation. Within columns, means with
the same letter are not significantly different according to Paired T-test (p< 0.05).
Bacteria Fungi
3MCE 2.95 ± 0.03b 0.82 ± 0.00b 36 ± 1.00c 2.93 ± 0.00a 0.87 ± 0.00a 29 ± 0.50b
9MCE 2.38 ± 0.29b 0.74 ± 0.06b 25 ± 3.00b 2.56 ± 0.28a 0.86 ± 0.05a 20 ± 3.00ab
9MTCE 2.81 ± 0.03b 0.80 ± 0.01b 33 ± 0.00bc 2.17 ± 0.36a 0.80 ± 0.05a 15 ± 4.00a
LS 2.83 ± 0.05b 0.80 ± 0.01b 34 ± 0.50c 2.07 ± 0.12a 0.74 ± 0.03a 17 ± 0.50ab
NL-4/20 1.09 ± 0.13a 0.39 ± 0.05a 16 ± 0.00a 2.33 ± 0.00a 0.81 ± 0.00a 18 ± 0.00ab
However, the significantly higher number of bands, as indicated by
species richness (S), in the DGGE gels of three month old rumen compost extract
and Living soilTM implies greater diversity of the bacterial communities of these
120
two treatments in comparison to the remaining treatments (Table 4.3). Higher
values of S representing greater bacterial diversity in three compared to nine
month compost extract agreed with the results of Biolog data as discussed earlier.
Unlike bacterial community analysis, no differences in Shannon-Weaver
diversity and evenness were found between the treatments for fungi, however,
higher fungal species richness was evident in three month old rumen compost
extract compared to nine month old rumen compost extract plus Nutri-Life
4/20TM inoculum (Table 4.3). The addition of Nutri-Life 4/20TM inoculum, which
is claimed to be rich in beneficial fungi including Trichoderma, to nine month
compost extract failed to increase the species richness (S) of fungal species
compared to three month old rumen compost extract (Table 4.3). This result can
be explained as indicating either that the species added were already present in
the compost extract, or that the added species were outcompeted and eliminated
by the microbiota of the compost extract, as indicated by other measures of
microbial activity, and Biolog measure of diversity or they were not detected by
DGGE.
Unlike the production of a pure culture (e.g. of Rhizobium, for legume
inoculation; or of a fungal plant pathogen, for disease studies), the production of
microbially enhanced ‗soil health‘ products is an ‗inexact science‘ due to the
enormous possible microbial variation. It is difficult to offer criteria as to the
‗quality‘ of the product. Rather, there is an inherent assumption that the microbial
assemblage present in aerobic compost is beneficial for plant growth (e.g.
Ingham, 2005; Pant et al., 2009). However, this assemblage will differ with
compost condition (e.g. age) and incubation conditions (e.g. aeration, food
sources) (Carpenter, 2005). Commercial products, such as Nutri-Life 4/20TM and
121
Living soilTM, seek to provide storable forms of a set of beneficial microbiota,
although changes in the assemblage during storage are likely. Further, incubation
conditions (e.g. anaerobic vs. anaerobic) are also likely to result in a change in
the microbial assemblage.
4. Conclusions
The Biolog and DGGE tools were useful in characterising the microbial
assemblages of the liquid cultures. The three month old rumen compost extract
and Living soilTM were closely related and functionally more active compared to
nine month old rumen compost extract, nine month old rumen compost extract
plus Nutri-Life 4/20TM inoculum and Nutri-Life 4/20TM which showed
relatedness to each other. The addition of Nutri-Life 4/20TM inoculum in nine
month old rumen compost extract plus Nutri-Life 4/20TM inoculum did not alter
the functional microbial activity compared to the original extract. Fungal
diversity did not vary, but bacterial diversity was higher in all cultures compared
to Nutri-Life 4/20TM. The potential role of such biofertilisers in soil functioning
and crop health needs to be explored.
122
Chapter 5: Compost extract and plant nutrition
Chapter 5
Microbially enhanced compost extract: does it
increase solubilisation of minerals and
mineralisation of organic matter and thus improve
plant nutrition?
A version of this chapter has been submitted to Crop and Pasture Science (currently under
review)
Abstract
Microbially enhanced compost extracts are increasingly being used to
improve soil and crop ‗health‘. The aim of this research was to assess potential
mechanisms by which compost extracts may influence plant growth. Tomato
(Lycopersicum esculentum cv. Tiny Tim) and sorghum (Sorghum bicolor cv.
Sweet Jumbo LPA) were used as test crops. The effect of rumen content compost
extracts (aerated and non-aerated, sterilised or not) on plant growth was
compared to inorganic fertiliser and a water-control in greenhouse and growth
chamber pot trials using various growing media. Soil type was the primary factor
in determining growth rate of crops. Compost extracts produced by either
extraction method had similar effects on plant growth, and there was no
difference between sterilised and non-sterilised treatments. Thus the noted growth
benefit of compost extracts was not directly biological in nature. The positive
123
impact of compost extract application on plant growth was ascribed to a
nutritional effect, attributed to the high doses of compost extract application used
(2 L pot-1, equivalent to 34,000 L ha-1), delivering 3.4 g of N pot-1.
1. Introduction
Compost extract, popularly known as ‗compost tea‘, is an extract of
compost incubated with or without a microbial food source with the aim of
extracting soluble nutrients and plant beneficial microbes into the solution (Diver,
2002). Although there is an extensive anecdotal evidence to support the claim of
improved plant growth in response to compost extract application, studies
conducted under controlled conditions are limited. For example, Egamberdiyeva
(2007) reported evidence of higher nutrient uptake and a growth stimulatory
effect of microbial inoculation (with growth promoting rhizobacteria, namely,
Pseudomonas alcaligenes, Bacillus polymyxa and Mycobacterium phlei) on
maize in nutrient deficient soils. However, the direct cause of this response was
not elucidated – the effect could be due amongst others to a direct fertilisation
effect, increased mineralisation/solubilisation, or a disease suppression effect.
For example, Ekin (2010) reported increased shoot biomass and seed
yield of sunflowers associated with a soil application of phosphate solubilising
bacteria (Bacillus M-13) and a phosphorus fertiliser, compared to fertiliser alone.
This result was ascribed to the effect of soil microbiota on soil mineral
solubilisation at a level significant to plant growth.
The extracted mineral nutrients can improve soil fertility directly
(Campbell, 2006; Merrill et al., 1997; Scheuerell & Mahaffee, 2002). Other
claims include a role for the extracted microbiota in improved mineralisation of
124
soil organic matter and solubilisation of soil minerals, chelation of ions (Janzen et
al., 1995), suppression/biocontrol of certain plant root and foliar diseases (Bernal-
Vicente et al., 2008; Haggag & Saber, 2007), and microbial production of plant
growth promoting hormones such as auxins (Garcia et al., 2002), or cytokinin-
like substances (Arthur et al., 2001).
However, many reports of the use of compost extract involve high rates of
application, such that a direct nutritional effect will dominate. For example,
Hargreaves et al. (2008) reported that compost extract (produced at the ratio of 1:
10 w/v) applied at weekly intervals to a raspberry crop at the rate of 150-300 mL
per plot (2.4 m2) per application (NOSB, 2004) promoted the growth of
raspberries as effectively as applying compost at the rate of 150 kg total N per ha
in 2004 and 75 kg N per ha in the following years to the soil. However, 27
applications made over 3 years will have delivered approximately 4000 - 8000
-2
mL 2.4m (i.e. equivalent to 17,000 - 34,000 L ha-1, derived from 1.7 – 3.4 t
compost), however, the actual supply of N from these compost extracts has not
been indicated. In another study, Hargreaves et al. (2009) produced compost
extracts by steeping compost for 72 h at the ratio of 1: 5 (w/v), with application at
the rate of 1 L and 2 L per plot (of 2 m2 containing six strawberry plants) per
week continuously for twelve weeks in 2006 and for five weeks in 2007 (i.e.
120,000 L ha-1, or 24 ton ha-1 of compost). Compost extracts may thus be used to
maintain soil nutrient levels by growers who are restricted from using inorganic
fertilisers by the norms of organic farming, with the advantage of easier
distribution (e.g. through irrigation systems) than is possible for solid compost.
Compost extracts, however, can vary in composition. There are limited
studies comparing methods of preparation of compost extracts (Pant et al., 2009).
125
For example, compost extracts can be produced following aerobic (aerated
compost extract) or anaerobic methods (non-aerated compost extract),
presumably resulting in distinctly different microbial populations, however there
is a lack of evidence as to the superiority of one or other method for agricultural
purposes (Brinton et al., 2004). Ingham (1999a) has advocated the use of aerobic
compost extracts, whereas other studies have found a significantly positive effect
of non-aerated compost extracts on plant growth due to disease suppression
(Scheuerell & Mahaffee, 2004; 2006; Tranker, 1992; Weltzien, 1991).
The present experiment was designed to test two hypotheses: (a) that
application of compost extract to the soil results in an enhancement of the
microbial population of the soil, and (b) that microorganisms present in compost
extract may enhance solubilisation of relatively inaccessible inorganic pools and
increase mineralisation of organic matter to release mineral nutrients, thereby
improving soil fertility. Two pot trials were conducted to determine the effects of
extraction methods (aerobic and non-aerobic) on the growth of tomato and
sorghum supplied with extracts (either sterilised or non-sterilised) derived from a
cattle rumen content compost. In one trial, the soil medium contained sand and
leached compost (to test mineralisation capacity), while in the second trial two
soils were used, both of low nutrient availability and organic matter content (to
test solubilisation capacity), with a rotation crop planted into the root residues of
the first crop (to test mineralisation capacity).
Tomato was chosen as a likely horticultural crop for ‗compost tea‘
application, being Australia‘s second largest commercial vegetable crop, with
Queensland being the main producer of fresh market tomatoes (DPIF, 2000).
126
2.1. Compost and compost extract
Cattle rumen content (paunch material) was used for compost given the
relative uniformity of this material (e.g. relative to urban green waste) and its
local abundance (with up to 1700 head of cattle slaughtered per day in
Rockhampton abattoirs). A commercial composting operation in Rockhampton
utilises a thermophilic windrow composting process, with the windrows (2 m
height, 2.5 m width and 50 m length of material) turned mechanically at monthly
intervals, for nine months to ensure homogeneity and aeration. Four
consignments of nine month old compost were used in the course of this study.
Compost extracts were produced at the ratio of 1:10 (w/v) of compost
submerged in dechlorinated tap water following aerated and non-aerated methods
of extraction (at between 18˚C and 25˚C). Dissolved oxygen, measured with a
TPS WP 82 Dissolved Oxygen Meter (EnviroEquip, Australia), of aerated and
non-aerated compost extracts at 24 h of the incubation period was 71 % and 1.5
% of air-saturation, respectively. Addition of 0.5 % v/v of molasses and 1 % v/v
of soluble ‗fish and kelp hydrolysate‘ (Fish and Kelp Product, Australia) was
made prior to application of the compost extract to soil/growing media. Compost
extracts were prepared on the day of soil application.
Nutrient analysis of composite samples of the aerated and the non-aerated
compost extracts, i.e. representatives of all extracts used, was undertaken by the
Soil and Plant Laboratory, Wesfarmers CSBP Limited, Australia. The samples
were shipped in cooled liquid form (i.e. with ice blocks).
127
2.2. Experiment 1
The hypothesis was tested that non-sterile compost extract would enhance
plant growth over an equivalent amount of sterile compost extract through the
enhancement of soil mineralisation in an ‗artificial‘ soil of organic matter and
sand mix.
The growing media was prepared by mixing the thoroughly washed (i.e.
leached) sterilised rumen compost with sterilised sand at the ratio of 1: 5 v/v.
Materials (sand, compost, compost extract) were sterilised by autoclaving at
121˚C for 15 minutes. Three-week old tomato seedlings (Lycopersicum
esculentum cv. Tiny Tim) were transplanted one each into 13 cm diameter pots
and maintained in a growth chamber. Each pot contained 1 kg of the growing
media.
A completely randomised experimental design was implemented, with
five treatments: (i) control (i.e., equivalent amount of water only), (ii) aerated
compost extract (ACE), (iii) non-aerated compost extract (NCE), (iv) sterilised
aerated compost extract (S ACE) and (v) sterilised non-aerated compost extract
(S NCE). Pots were placed on a 45 cm by 25 cm grid, with eight replicate pots
per treatment. Treatments, including the water control, were applied twice at the
rate of 100 mL kg-1 compost-sand mix, the first being applied one week after
transplanting and the second a week later.
Two replicate trials (I and II) were conducted. In trial I, the growth
chamber was set at 28˚C, 720 µmol m-2 s-1 PAR and 75 % relative humidity
(RH). In trial II, the growth chamber was set with lower temperature and light
intensity, but with the same relative humidity as in trial I (24˚C, 480 µmol m-2 s-1
PAR and 75 % RH).
128
The growth parameters of plant height, stem diameter, number of leaves
and leaf chlorophyll concentration were measured at 10 day intervals starting
from 18 and 31 days after transplanting in trials I and II, respectively. Dry weight
of all plants (roots and shoot biomass) was measured following harvest at 45 and
51 days after transplanting in trials I and II, respectively.
2.3. Experiment 2
The hypothesis was tested that microbial enhancement of the soil could
serve to improve solubilisation of nutrients (and mineralisation of any soil
organic matter) and so improve plant growth.
A vertisol was sourced (courtesy of Eric Coleman, QDPIF) from a
Gracemere site (latitude: 23.44˚and longitude: 150.46˚) that had been maintained
as uncultivated grassland for over thirty years. A ferrosol was sourced (courtesy
of farmer Tony Wolfenden) from an organic sweet potato farm at Rossmoya
(latitude: 23.05˚ and longitude: 150.48˚), from a site that had been cultivated
regularly over the past decade. At both sites, soil was collected to a depth of 30
cm, and was transported as a single, homogenised sample (soil mixed using a
small front end loader). Prior to starting the experiment, replicated samples from
both soils were collected, homogenised individually and combined to obtain
representative samples and sent for nutrient analysis (Wesfarmers CSBP Limited,
Soil and Plant Laboratory, Australia).
Soil was air dried and each pot (30 cm diameter, 40 cm height and 20 L
volume) was filled with 20 kg of vertisol or 22 kg of ferrosol. All pots were
weighed and watered to achieve field capacity (40 % v/v and 38 % v/v for the
vertisol and ferrosol, respectively). Pots were maintained in a greenhouse
129
(ambient temperature ranging from 18 to 40˚C with 60 to 100 % relative
humidity). Pots were spaced at 75 x 50 cm. Tomato (cv. Tiny Tim) seeds were
germinated and seedlings transplanted to achieve one plant per pot.
The experiment used a factorial design within a randomised complete
block design, with four application treatments applied to the two soil types, and
four replicate pots per treatment. Treatments were (i) aerated compost extract, (ii)
non-aerated compost extract, (iii) chemical fertiliser and, (iv) a water control. All
treatments were applied as a soil drench, with 0.5 L solution applied weekly to
each pot for four weeks (equivalent to approximately 8,500 L ha-1 per
application), beginning from one week after transplanting. The chemical fertiliser
treatment utilised the same volume of solution containing 0.9 g of ―Thrive‖
(Yates, NSW Australia), which consisted of 15: 4: 26 NPK and all other essential
nutrients. Culturable heterotrophic bacterial and fungal counts (cfu Log 10 mL-1)
of representative samples of aerated and non-aerated compost extracts (n = 3),
sampled immediately prior to the application to the pots, were determined
following the pour plate method (Harrower, 2006).
Tomato plants (the same cultivar as used in Experiment 1) were grown for
76 days, being watered to field capacity at weekly intervals. Soil respiration and
soil temperature were measured per pot (n = 3) 76 days after transplanting, to
estimate plant root and microbial activity in the soil using an EGM3 respirometer
(PP Systems International Ltd., Hertfordshire, UK). Plant height, stem diameter,
leaf number, leaf chlorophyll concentration (SPAD-502 meter, Minolta
Corporation, Japan), plant nutrient status, fruit yield and above ground biomass
were assessed at harvest on all plants (76 days after transplanting).
130
The microbial population of the soil was quantified in triplicate at 36 days
after tomato transplanting, that is, one day after the last compost extract
application, using a plate count method (Table 5.4) (Harrower, 2006). Soil was
sampled with a 2 cm diameter core sampler from the top 10 cm depth,
composited, and 1 g of representative soil sample was taken from each treatment.
A nitrogen budget was constructed. Shoot material, compost extract and
fertiliser, dried at 65˚C, were ground using a CQUniversity manufactured ball
mill grinder. Subsamples (10 mg) were packed in tin capsules. Sample nitrogen
elemental and isotopic content (natural abundance) was determined using an
automated continuous flow isotope cube utilising Dumas flash combustion
(Elementar, Germany) coupled with a continuous flow mass spectrometer
(Isoprime, England) at the University of Melbourne. Machine precision and
standardisation were established with acetanilide (Merck, Germany) as a tertiary
standard (10.36 %N and 0.2 ‰ δ15N) with analytical precision (1σ) of < 0.1 ‰.
2.4. Experiment 3
The hypothesis was tested that microbial enhancement of the soil could
serve to improve mineralisation of residual tomato roots, and hence improve plant
growth.
Following harvest of the tomato crop (removal of shoot system only, with
the soil left undisturbed) and a fallow period of one month, 10 sorghum seeds
(Sorghum bicolor cv. Sweet Jumbo LPA) were directly sown in each pot, and
thinned out after one week to maintain six plants per pot. From the previous
tomato trial, there was no significant effect of blocks on yield (above-ground
biomass). In that trial, blocks were arranged down the length of the greenhouse.
131
Blocks were rearranged across the width of the greenhouse to test for an
environmental gradient in this direction (i.e. a possible wind speed gradient away
from the windsock located at one edge of the greenhouse).
Two treatments, namely, aerated compost extract and sterilised aerated
compost extract, were applied at three week intervals at the rate of 5 mL per kg of
soil. The rate of compost extract application was reduced to minimise nutrient
input (equivalent to approximately 1700 L ha-1 per application). These treatments
were applied to two pots each of the original four replicates of the first (tomato)
rotation (i.e. across the two soils (vertisol and ferrosol) and four previous
treatments (control, aerated compost extract, non-aerated compost extract, and
chemical). The pots were randomly assigned treatments within three blocks.
Percent light interception at the base of the sorghum plant per pot was
measured using a Ceptometer, LAI-2000 (Decagon, USA), and leaf chlorophyll
concentration of the top third leaf per plant per pot was indexed using a SPAD-
502 meter (Minolta Corporation, Japan). Soil respiration (root + microbial
respiration) per pot (n = 3) was measured with an EGM3 respirometer just before
harvest. The vegetative growth (shoot biomass), as measured by above-ground
plant dry weight, of the forage sorghum crop was measured on all pots at 50 days
after sowing.
2.5. Statistical analyses
Data were analysed using the GLM procedure of GENSTAT (2008) and
statistical significance was evaluated at p< 0.05 and p< 0.001. The effect of
treatments on different parameters including plant biomass was analysed using
either one-way or two-way analysis of variance (ANOVA). For the glasshouse
132
trial, tomato shoot biomass (plant dry weight in g) was used as a covariance-
factor on the sorghum shoot biomass for the analysis of variance.
3. Results
3.1. Characterisation of compost extract
The soluble nutrient contents of compost extracts produced under aerated
and non-aerated conditions were similar (Table 5.1).
Table 5. 1. Nutrient (macro and micro) concentrations for aerated and non-aerated compost
extract samples analysed by Wesfarmers CSBP Limited, Australia. ‗Thrive‘ nutrients based on the
chemical composition supplied by manufacturer Yates and Co. Limited, Australia.
Nutrients (mg L-1) ACE NCE Thrive

NO3-N 0.80 1.10 157
+
NH4 -N 80.6 70.2 64.0
N as Urea - - 46.0
Total soluble N 81.4 71.3 267
P 186 182 71.0
K 334 334 463
Ca 146 166 -
Mg 29.1 26.5 9.00
S 95.5 101 -
Cu 0.34 0.15 0.09
Mn 0.42 0.54 0.71
Zn 1.28 1.86 0.36
Fe 8.24 9.36 3.20
B 0.24 0.24 0.09
Na 411 418 -
Cl 374 375 -
Mo - - 0.04
ACE - Aerated compost extract, NCE - Non-aerated compost
extract, Thrive - water soluble nutrient (chemical fertiliser).
In each compost extract, 3 kg f wt of compost at 60 % moisture (i.e. 1.2
kg d wt) was added to 30 L of water. Given a N content of the compost of 1.9 %
d wt, 22.8 g N was added to the 30 L extract in the form of compost. Each
compost extract also contained 1 % of Searles Fish and Kelp. Given a N content
of 10.3 % w/v in this product (as reported on label), 30.9 g N was added to the 30
L brew in the form of this product. Thus, a 30 L compost extract is expected to
133
contain 53.7 g N. In supplying the extract at the rate of 2 L per pot, 3.58 g N
should have been delivered.
3.2. Experiment 1: Tomato growth in sand-compost mix
3.2.1. Plant biomass
In both trials, total plant biomass (dry weight, shoot + root) was
significantly different (p< 0.001) between imposed treatments and the control
(Fig. 5.1A), however there was a non-significant difference among the compost
extract treatments (both sterilised and non-sterilised).
In all treatments receiving compost extracts, tomato plant shoot to root
ratio was increased compared to the control, but no difference was found between
compost extract treatments (Fig. 5.1B).
134
Figure 5. 1. Effect of compost extracts on (A) total plant biomass (g d wt) and (B) shoot: root
biomass of tomato plants in Experiment 1 grown in a thoroughly washed compost-sand mix at 1:
5 ratio in two replicate trials, harvested at 48 and 51 days after transplanting, respectively. Cont -
control, ACE - aerated compost extract, NCE - non-aerated compost extract, S ACE - sterilised
aerated compost extract and S NCE - sterilised non-aerated compost extract. Within a trial, means
with different letters indicate significant difference (p< 0.05). Vertical bars represent the standard
error of the mean (n = 8).
3.2.2. Tomato leaf chlorophyll concentration
In trial I (Fig. 5.2A), chlorophyll concentrations (SPAD Units) were
significantly higher in compost extract treated plants than in the control at 18, 28
(except for aerated compost extract) and 38 days after transplanting (p< 0.001, p<
0.01 and p< 0.05, respectively). In trial II (Fig. 5.2B), higher chlorophyll
concentrations were found in leaves of plants treated with both sterilised and non-
sterilised compost extracts (aerated and non-aerated) compared to the control at
31 (p< 0.001) and 41 (p< 0.05) days after transplanting.
135
However, in trial II, no difference in leaf chlorophyll concentrations
between treatments was recorded at 51 days after transplanting.
Figure 5. 2. Effect of compost extracts in Experiment 1 on chlorophyll concentration in leaves in

(A) trial I, and (B) trial II. Cont - non-fertilised, ACE - aerated compost extract, NCE - non-
aerated compost extract, S ACE - sterilised aerated compost extract and S NCE - sterilised non-
aerated compost extract. Means with different letters within a sampling date indicate significant
difference (p< 0.05) and vertical bars represent the standard error of the mean (n = 8).
3.2.3. δ15N of fertilisers (compost and compost extracts) and plants
grown in compost-sand mix
Mean values for δ15N content of the compost (at 15 ‰), used in the
compost-sand mix, were lower than that for the two compost extracts (at 17 ‰), a
result consistent with denitrification and volatilisation in the compost extracts,
however this difference was not significantly different due to replicate variability
136
(typical standard error of approx 0.5 ‰) (Fig. 5.3). However, plant δ15N was
similar in all three treatments, and significantly lower than that of the compost
extract.
Figure 5. 3. δ15N values of fertilisers applied including the rumen compost and of the tomato
plants grown in compost-sand mix (1: 5) under growth chamber conditions (Experiment 1). Cont -
δ15N of non-fertilised tomato plants and rumen compost, NCE - non-aerated compost extract, and
S ACE - sterilised aerated compost extract. Means with different letters indicate significant
3.3. Experiment 2: Tomato growth on soil
3.3.1. Soil
The two soils were characterised with respect to their basic properties
(Table 5.2). The ferrosol was comparatively nutrient poor, in terms of P and K,
and slightly more acidic than the vertisol.
137
Table 5. 2. Characterisation of vertisol and ferrosol used in the greenhouse trial.
Soil characterisitcs Vertisol Ferrosol

Total N (%) 0.15 0.22
NO3-N (mg kg-1) 9.00 7.00
+
NH4 -N (mg kg-1) 3.00 3.00
Total P (mg kg-1) 138 29.0
P Olsen (mg kg-1) 48.9 7.20
K (mg kg-1) 506 256
Organic C (%) 1.96 2.52
Fe (mg kg-1) 2613 2285
S (mg kg-1) 3.60 10.7
pH (H2O) 7.40 6.40
pH (CaCl2) 6.10 5.40
EC (dS m-1) 0.06 0.06
BD (kg L-1) 1.10 1.20
Texture 3.00 3.50
Gravel Nil Nil
Colour Dark brown Orange
Classification Heavy clay Clay
Soil texture was determined by the "Feel method" (CSBP lab)
3.3.2. Tomato growth, yield and chlorophyll concentration
At harvest (i.e. 76 days after transplanting), shoot biomass (g d wt) was
significantly lower (p< 0.001) for plants grown in the ferrosol than those grown
in the vertisol, irrespective of the amendment treatments (Fig. 5.4A). In both soil
types, shoot biomass was significantly lower in the control treatment (p< 0.001)
compared to the other three treatments, however there was no significant
difference among the three (two compost extract and one inorganic) treatments in
both vertisol and ferrosol soils (Fig. 5.4A). Although soil type showed a
significant effect (p< 0.001) on fruit yield per plant, there was no significant
difference in fruit yield among any of the treatments (Fig. 5.4B). There was a
proportionally greater benefit of non-aerated compost extract on leaf chlorophyll
concentration on the ferrosol than the vertisol (Fig. 5.4C). Chlorophyll
concentration was increased in plants treated with either compost extract, relative
to the control and the chemically treated plants (Fig. 5.4C).
138
Figure 5. 4. Data from Experiment 2 on (A) total above-ground biomass (g d wt per plant) of
tomato subject to four treatments after four months of planting on two soil types, (B) fruit yield of
tomato (g d wt per plant), (C) leaf chlorophyll concentration (SPAD unit) measured at 76 days
after transplanting of tomato. (i) Cont - non-fertilised, (ii) ACE - aerated compost extract, (iii)
NCE - non-aerated compost extract and (iv) Chem - chemical fertiliser. Within a given soil type,
means with different letters indicate significant difference (p< 0.05) and vertical bars represent the
standard error of the mean (n = 6).
3.3.3. δ15N of fertilisers and plants grown on soil
The δ15N isotopic composition of the inorganic fertiliser used was low, at
5 ‰, while that of the compost extract was high, at 18 ‰ (Fig. 5.5). The latter
139
result is consistent with denitrification occurring in the composting and extraction
processes, with 14N preferentially lost.
Figure 5. 5. δ15N values of tomato plants grown in ferrosol conducted in a greenhouse

(Experiment 2). Cont - non-fertilised, Chem - chemical fertiliser, ACE - aerated compost extract.
Means with different letters indicate significant difference (p< 0.0001) and vertical bars represent
the standard error of the mean (n = 3).
Tomato plants grown on a ferrosol without amendment possessed a δ15N
isotopic composition of approximately 12 ‰, while that of plants grown on the
same substrate but with addition of inorganic fertiliser was 6 ‰, and that of the
fertiliser itself was 5 ‰. This result indicates that (12-6)/ (12-5)*100 = 86 % of N
sourced by the plant was from the chemical fertiliser, with the remainder sourced
from the soil. The observed low contribution of N from the soil is consistent with
a low rate of N mineralisation and thus availability.
In contrast, the δ15N isotopic composition of plants treated with compost
extract (but no inorganic fertiliser) was 15 ‰, with that of the compost extract
itself, 18 ‰, indicating that (15-12)/ (18-12)*100 = 50 % of N sourced by the
plants in this treatment was sourced from the extract, although the δ15N values of
aerated compost extract treated plants was not significantly different to that of the
140
control plants (Fig. 5.5). Note that these calculations assume that the compost
extract was homogenous in its δ15N composition (e.g. soluble and insoluble
pools).
3.3.4. Microbial enumeration of aerated and non-aerated compost
extracts
The bacterial colony forming units (cfu) load of non-aerated compost
extract was approximately two orders of magnitude lower than that of aerated
compost extract, at all assessment dates, while the fungal load was similar in the
two extract preparations (Table 5.3). The addition of 2 L of aerated compost
extract per pot will have involved the addition of approximately 2 x 1012 cfu of
bacteria and approximately 7 x 107 cfu of fungi to each pot.
Table 5. 3. Enumeration of bacteria and fungi populations in representative samples of ACE and
NCE. The compost extracts were produced at the ratio of 1:10 w/v and amended with 0.5 % v/v of
molasses and 1 % v/v of soluble ‗fish and kelp hydrolysate‘, and were sampled immediately prior
to the application to the pots. Results are expressed as cfu Log 10 mL-1.
Application of compost extracts

CEs Population Week 1 Week 2 Week 3 Week 4
ACE Bacterial (cfu Log 10 mL-1) 7.24 ± 0.03a 9.47 ± 0.01b 9.46 ± 0.01b 10.40 ± 0.01c
Fungal (cfu Log 10 mL-1) 4.40 ± 0.10a 4.52 ± 0.04a 4.20 ± 0.10a 6.10 ± 0.10b
NCE Bacterial (cfu Log 10 mL-1) 5.81 ± 0.13a 9.13 ± 0.05c 8.83 ± 0.05c 8.46 ± 0.04b
Fungal (cfu Log 10 mL-1) 4.16 ± 0.16a 4.20 ± 0.10a 6.36 ± 0.23b 6.00 ± 0.00b
± values are SE of the means, n = 3 and means with different letters within rows are significantly
different at p< 0.05 in compost extracts (ACE and NCE)
3.3.5. Soil microbial populations
The population (log values) of culturable bacteria was significantly higher
in compost extract (both aerated compost extract and non-aerated compost
extract) treated soils, 36 days after the transplanting of tomato, compared to
control and chemical fertiliser treatments (p< 0.001) (Table 5.4). Consistent with
the fungal count in the compost extract itself, there were no significant
141
differences in either soil in terms of fungal populations in the two compost
extract treatments. Additionally, there was no significant difference in fungal load
between any of the four treatments including control and inorganic chemical
fertilisation (Table 5.4).
Table 5. 4. Effect of compost extracts on microbial (bacterial and fungal) populations (cfu Log 10
mL-1) in two soil types sampled at 36 days after transplanting (a day after the last compost extract
application) from the tomato experiment conducted in the greenhouse (Experiment 2). Means
with different letters within rows are significantly different at p< 0.05 (n = 3) in different
treatments, namely, Cont - control, ACE - aerated compost extract, NCE - non-aerated compost
extract, and Chem - chemical fertiliser. Results are expressed as cfu Log 10 mL-1.
Treatments
Soils Microorganisms Cont ACE NCE Chem
Vertisol Bacterial (cfu Log 10 mL-1) 4.67 ± 0.03a 6.27 ± 0.01c 6.19 ± 0.01c 4.88 ± 0.04b
Fungal (cfu Log 10 mL-1) 4.10 ± 0.10a 4.26 ± 0.14a 4.20 ± 0.10a 4.16 ± 0.16a
Ferrosol Bacterial (cfu Log 10 mL-1) 5.28 ± 0.03b 6.25 ± 0.01c 6.20 ± 0.01c 5.16 ± 0.01a
Fungal (cfu Log 10 mL-1) 4.10 ± 0.10a 4.20 ± 0.10a 4.36 ± 0.18a 4.10 ± 0.10a
± values are SE of the means, n = 3 and means with different letters within rows are significantly different
at p< 0.05 in various treatments, Cont - control, ACE - aerated compost extract, NCE - non-aerated
compost extract, Chem - chemical fertiliser.
3.3.6. Soil respiration and temperature
The measurement of soil respiration at harvest (76 days after
transplanting) represents a sum of both soil microbial and root respiration. There
was no significant difference evident in respiration rate, either between soil types
or treatments (data not shown).
Soil temperature (monitored 30 cm below the soil surface) did not differ
significantly between any of the treatments and soil types (data not shown).
3.3.7. Mass balance of nutrients
The four applications of 0.5 L of aerated compost extract (total of 2 L per
pot) resulted in an application of approximately 162 mg soluble N as NH4+ and
142
NO3−, 371 mg soluble P and 668 mg soluble K, respectively to each pot. An
additional 3580 mg insoluble N was present in the compost extract.
A mass balance of N, as an example, for each treatment in the vertisol was
calculated and is presented in Table 5.5. The aerated compost extract treatment,
for which there was an above-ground biomass of 130 g d wt, and given an
estimated root to shoot ratio of 1 and N content of 1.1 %, the calculated plant N
content was 2860 mg (Table 5.5). Thus, the N provided in the compost extract
was equivalent to approximately 125 % (N) of that in the plant.
Table 5. 5. Mass balance of N in compost extract and tomato crop grown in vertisol.
N added per pot Cont ACE NCE Chem

Soluble N added (mg) 0.00 162 143 534
Insoluble N added (mg) 0.00 3418 3437 0.00
Total N added (mg) 0.00 3580 3580 534
N in Plants
Aboveground biomass(d wt in g) 104 130 135 133
Assumed root: shoot ratio 1.00 1.00 1.00 1.00
% N in plants 1.00 1.10 1.70 1.10
Calculated plant N (mg)* 2080 2860 4590 2926
Added N/plant N X 100 (%) 0 125 78 18
*calculated as aboveground biomass X 2 X % N, Cont - control, ACE - aerated compost extract,
NCE - non-aerated compost extract, Chem - chemical fertiliser.
3.4. Experiment 3: Sorghum crop rotation
3.4.1. Plant growth, shoot biomass, chlorophyll concentration and light
interception
There were no visual differences between the growth of plants treated
with aerated and sterilised aerated compost extracts in either soil type while a
dramatic difference (p< 0.001) was observed in above-ground sorghum biomass
(harvested 50 days after sowing) between soil types (Fig. 5.6A).
143
Figure 5. 6. Data on sorghum from Experiment 3 on (A) above-ground dry weight biomass per
plant, (B) chlorophyll concentrations of leaves and (C) percent light interception (45 days after
sowing) grown in vertisol or ferrosol, according to treatments applied to the sorghum crop (x axis)
and to treatments applied to the previous rotation (tomato crop). Cont - non-fertilised, ACE -
aerated compost extract, NCE - non-aerated compost extract and Chem - chemical fertiliser.
Means with different letters across soil type and previous treatments indicate significant
144
However, above-ground biomass did not differ between treatments
imposed on the sorghum plants (Fig. 5.6A), or between the treatments imposed
on the preceding tomato crop as shown by the covariance test (p = 0.98). For
percent light interception (Fig. 5.6C), significant difference was again found only
between soil types (p< 0.001).
There was no significant difference in sorghum leaf chlorophyll
concentration between any treatments (Fig. 5.6B).
4. Discussion
4.1. Effects of treatments on growth and nutrition of tomato plants
In the tomato growth trial (Experiment 1), conducted in sand-compost
mix, the lack of a plant growth difference between the sterilised and non-
sterilised treatments is consistent with the interpretation of a direct nutritional
benefit from the applied compost extract, rather than from a secondary benefit of
the microbial activity, such as mineralisation of organic matter in the sand-
compost mix.
The mass balance of N (Table 5.5) is again consistent with the
interpretation that the differences in plant growth between the applied treatments
is due to the nutrients supplied in the compost extract itself, rather than an
enhancement of soil mineralisation by the enhanced soil microbiota (Experiment
2).
The soluble and insoluble nutrient concentration of compost extracts
produced under aerated and non-aerated conditions was similar (Table 5.1, 5.5).
However, the N present in insoluble components of the compost extract (e.g.
microbial biomass) was approximately 20 times higher than in the soluble
145
fraction (Table 5.5). Potentially the nutrients in the microbial biomass would not
be immediately available for plant growth, and a greater plant response might be
expected from autoclaved extracts. As there was no such difference, the majority
of the applied microbial biomass presumably dies on application to the compost-
sand mix, freeing nutrients for plant growth.
No differences between aerated and non-aerated compost extract
treatments were observed in the current study in terms of growth (e.g. d wt
biomass) of tomato plants (Fig. 5.1 and Fig. 5.4). Pant et al. (2009) tested the
growth of pak choi under greenhouse conditions treated with aerated, non-aerated
and microbially enhanced vermicompost extracts prepared at the ratio of 1: 10
(w/v) and applied at the rate of 150 mL pot-1 for four continuous weeks,
compared to an aerated water-control. They also observed non-significant
differences in growth for plants treated with aerated and non-aerated compost
extracts, with all vermicompost regimes increasing above-ground fresh weight of
pak choi compared to the water-control.
In Experiment 1, tomato plants receiving compost extracts had a higher
shoot to root ratio compared to the control plants (Fig. 5.1B). An increased shoot:
root ratio is consistent with a relief of a nutrient limitation in the extract treated
plants (e.g. Ericsson, 1995).
The higher shoot to root ratio of tomato plants in the first trial compared
to the second trial could be attributed to the lower light level employed in the
second experiment.
146
4.2. Effects of treatments on tomato leaf chlorophyll concentration
In both trials I and II of Experiment 1, chlorophyll concentrations were
significantly higher in both sterilised and non-sterilised compost extract treated
plants compared to control. Fayed (2010a) also observed higher leaf chlorophyll
concentration in pomegranate trees treated with two foliar applications of
compost extract mixed with antioxidants at the rate of five litres per tree. Such
observations are consistent with a N or Fe fertilisation effect by the compost
extract.
However, leaf chlorophyll concentrations between treatments did not
differ 51 days after transplanting in trial II. The gradual increase in chlorophyll
concentration in plants of the control treatment must reflect the mineralisation of
organic matter present in the soil mix.
Higher leaf chlorophyll concentration was found in the first trial
compared to that in the second trial (Fig. 5.2B), a result which could be due to
higher light intensity and higher temperature employed in the first trial. Low
temperature and low light intensity can lead to an inhibition or impairment of
formation of chlorophyll in tomato (Allen & Ort, 2001; Hu et al., 2006).
Plants supplied with compost extract had a significantly lower δ15N level
than that of the compost extracts itself, suggesting that plants sourced N from the
mineralisation of the solid rumen compost in addition to the liquid compost
extracts (Fig. 5.3). Presumably the N of the compost extracts remained
unavailable to the plant as it was within microbial biomass.
In Experiment 2, the effect of compost extract on leaf chlorophyll
concentration (measured 76 days after transplanting) varied with soil type, with a
significant interaction (p< 0.001) between soil type and treatments noted (Fig.
147
5.4C). The significant interaction effect was attributed to the lack of response of
tomato (in terms of chlorophyll concentration) to the chemical fertiliser treatment
on the ferrosol. Chlorophyll concentration in tomato plants grown in the vertisol
was higher compared to those grown in the ferrosol (Fig. 5.4C), which could be
related to the higher nutrient availability in the former, especially, phosphorus
and potassium (Table 5.2).
4.3. Influence of soil types on crop growth
Soil type had a stronger influence on plant growth than the influence of
soil amendments used in the present study. In Experiment 2, vertisol produced
higher shoot biomass of tomato plants compared to those grown in the ferrosol,
which was consistent with the higher fertility (especially P) of the former soil.
Chemical fertiliser supported increased growth on ferrosol, however only 36 mg
P was given while the difference in soil P in the pots was 2200 g.
The grazing soil from which vertisol was collected has no known history
of fertiliser addition and is likely to be deficient in both Zn and B. Since compost
extracts have concentrations of Zn and B that are substantially higher than those
of the commercial fertiliser, plants grown in this soil are highly likely to respond
to the addition of the trace elements present in the extracts.
The vertisol also supported greater sorghum plant growth (above-ground
dry weight biomass) and high light interception compared to the ferrosol (Fig.
5.6A, 5.6C). This result is again attributed to the significantly greater nutritional
status of the vertisol in comparison to the ferrosol (Table 5.2).
148
4.4. Nutritional and microbial supply from compost extracts
The measured dissolved N (as ammonium and nitrate) content of the
extract represented only a small fraction (5 % and 4 % for aerated and non-
aerated compost extract, respectively) of the total N content (Table 5.1). The
difference (e.g. 3418 N mg per 2 L of aerated compost extract) must either be
present as microbial biomass, or insoluble or soluble but organic forms of N in
the compost extract, and/or this N was lost from the compost extract through
15
denitrification and volatilisation. The observed microbial load and the high N
values of the compost extract are consistent with both processes occurring.
Given the rate of extract application (2 L pot-1), the level of plant nutrients
in the compost extracts was certainly significant in terms of plant nutrition. For
example, the harvested (above-ground) biomass in Experiment 2 was
approximately 100 g d wt per pot. The average N concentration of above-ground
biomass across all treatments was 1.2 % (from elemental analyzer – mass
spectrometry analysis, data not shown). Given this, harvested plant biomass
contained 1.2 g N (per pot). Assuming a conservative shoot root ratio of 1: 1, the
total plant N content was 2.4 g per pot. Thus, a direct fertilisation effect is
expected for the extract treatments (3.4 g N delivered per pot).
The chemical fertiliser treatment was intended as a control exercise on the
effect of nutrient addition, as opposed to other possible effects of extract addition
(e.g. mineralisation). The chemical fertiliser application involved a similar order
of magnitude of soil inorganic elements per pot as the compost extract treatments,
except for N (which at 534 mg N pot-1 was much higher than the soluble
inorganic N pool in the compost extracts (Table 5.1), but also much lower than
149
the total N pool of the extracts). Little difference existed in the level of assessed
soluble nutrients between aerated and non- aerated compost extracts (Table 5.1).
However, the four compost extracts, prepared at weekly intervals, were
not consistent in their bacterial or fungal loads, with differences of up to two
orders of magnitude between batches. Of course, some variation is expected,
given the non-sterile, non-controlled incubation conditions and variation in the
compost inoculum, however this level of variation is large. Further, such
variation in bulk bacterial and fungal numbers also hints at variation in species
composition as well. More attention to the incubation process is recommended to
reduce the observed variation in final microbial load.
4.5 Effects of treatments on soil microbial properties
In Experiment 2, although there was a difference in bacterial counts in
compost extract treated soils compared to the control and chemical treatments, no
differences existed in fungal loads between the treatments (Table 5.4). This
indicates that while compost extract application may have changed fungal species
composition; there was no immediate effect on total fungal (cfu) load.
Soil temperature and soil respiration did not differ between any of the
treatments and soil types. The level of soil microbiota activity was unlikely to be
sufficiently high to raise soil temperature. Soil type (as related to solar
absorptivity) is likely to impact soil temperature, and therefore perhaps soil
respiration, but sample variability was sufficiently high to disguise any such
relationship.
150
5. Conclusions
It was hypothesised that compost extracts could increase soil
solubilisation and mineralisation rates, and thus increase plant growth. In the
experiments undertaken in this study, data suggest that the impact of compost
extract on plant nutrition and growth was apparently though the direct addition of
nutrients, rather than through an indirect effects of soil microbiota. This result is
ascribed to the high rate of extract addition, at 2 L per pot (equivalent to 34,000 L
ha-1). Compost extracts produced using the two extraction methods (with and
without aeration), did not vary in their effect on plant growth as demonstrated by
the responses of tomato and sorghum crops. Further experimentation is needed to
assess compost extract contribution to plant nutrition through increasing soil
microbiota, adding microbiota key to certain nutrient cycles.
151
Chapter 6: Compost extract and biodegradation
Chapter 6
Biodegradation of sugarcane trash through the use
of enhanced compost extracts

A revised version of this chapter has been submitted to Compost Science and Utilisation
(currently under review)
Abstract
Compost extracts were produced from rumen waste composted for one,
three, six and ten months, and from nine month old rumen compost and
sugarcane stubble (1:1 v/v mix) further composted for one month. The extracts
produced from sugarcane stubble amended compost enhanced the rate of
degradation of intact trash mat lying over a red ferrosol over a six month period
as indicated by the integral of CO2 release, with a significantly higher total
microbial activity recorded on sugarcane amended compost extract compared to
other extracts. Increased concentrations of total soil C and dissolved organic C
were found in soil samples covered with harvest residue of sugarcane compared
to the bare soil. The results of the current study indicate that compost extracts
have potential benefits to the ‗green trash‘ sugarcane production system.
152
1. Introduction
Sugarcane is a crop economically important to Australia. The Australian
sugar industry is mainly concentrated along the coastal Queensland and northern
New South Wales regions. Traditional management involved burning of the
sugarcane crop before harvest, with consequent loss of dry matter and N (ranging
from 77 to 97%) and other mineral nutrients (Mitchell et al., 2000). The
alternative, ‗greentrashing‘, involves topping out the leaves from the stalk during
harvest and shredding into pieces and leaving to dry in the field (Hall et al.,
2006). The green cane trash blanketing system has been increasingly adopted by
Australian cane growers over the past two decades (Meier, 2007; Wood, 1991);
with around 70% of the sugarcane currently grown under this system (Kingston
& Norris, 2001). This approach provides mulch, resulting in reduced soil
compaction, improved soil structure, greater water and nutrient retention,
improvement of the physical and microbial status of the soil, reduction of
fertiliser demand and ultimately increased cane yield (Ball-Coelho et al., 1993;
Ball-Coelho et al., 1992; Barzengar et al., 2002; Blair & Crocker, 2000;
Casagrande et al., 1995; Graham et al., 2002; Robertson & Thorburn, 2007).
Agricultural management practices, such as stubble retention systems directly
impact microbial community structure of soil and affect the biogeochemical
processes, for example, cycling of major nutrients, rate of decomposition and
other important ecological functions (Swift & Anderson, 1994; Wakelin et al.,
2007). Trash decomposition allows for N mineralisation and incorporation of
organic C into the soil (Sutton et al., 1996; Wood, 1991). Additional benefits of
the green trash blanketing system, that attracted growers to adopt this system,
153
include better weed control, reduced irrigation in dry periods, reduced soil
erosion rates and reduced labour requirement (Norrish, 1996; Small, 2000).
In the Australian sugarcane production system, green waste from the
harvest is left as mulch. It can not be incorporated into the soil if there is a rattoon
crop. The covered soils are cooler and the second (rattoon) crop is delayed. Some
disadvantages include decreased soil temperatures under the trash blanket, with
subsequent delay in ratooning, and an increase in various pests (e.g. rats) and
diseases between crops (Stewart & Wood, 1987). Rapid decomposition of the
trash is, therefore, desirable.
Soil microbiota play a central role in decomposition of organic residue in
soils, and the rate of this turnover can be increased by microbial enhancement
using microbially enhanced compost extracts (Ingham, 2005; Ryan, 2003). Some
of the fungal species known to aid in degradation of organic matter, and prevalent
in most soils, are Trichoderma spp. and cellulose digesting brown rot fungi, such
as Coniophora prasinoides, C. puteana (Highley, 1980) and Cellulomonas spp.
(Lines-Kelly, 2004). A compost extract from an inoculum source of compost
which is likely to favour proliferation of relevant organisms (cellulolytic bacteria
or fungi) may act to speed-up sugarcane trash decomposition. Some examples of
dominant bacteria, such as lactic acid bacteria, Bacillus spp., Micrococcus spp.,
Pseudomonas spp., and dominant fungi, such as Penicillium spp., Trichoderma
spp., Aspergillus spp., yeast, actinomycetes and Streptomyces spp. are reported in
microbially enhanced compost extracts (Naidu et al., 2010).
Microbial populations are likely to change during the composting process.
Presumably early successional stage composts, that are microbially active, are
needed to produce compost extracts for spraying onto the trash mat. Hall et al.
154
(2006) applied extracts prepared from a compost mix of aquaculture pond
sediment, ground sugarcane, wood chips, bagasse and corn silage / cotton seed
blend over the trash mat of sugarcane. They reported increased microbial
respiration (over 168 h) of the trash mat following treatment with extracts from
eight week old compost, as compared to extracts from four, twelve and sixteen
week-old composts. This result was ascribed to an increase in bacterial
population load.
The compost mix used by Hall et al. (2006) was, however, complex. For
adoption of the use of compost extract for enhanced trash breakdown on a broad
scale basis by the sugar industry, a readily available, consistent compost
inoculum is required. Consider that if 100 L of compost extract were applied per
hectare, across the 300,000 ha of the Australia sugarcane crop, 30 ML of extract
would be required. If produced using a 1:10 water: compost mix, approximately
3,000 m3 of compost would be required annually. The beef industry overlaps
geographically with that of the sugar industry, with abattoirs located within
reasonable distance to the cane growing areas.
An experiment was, therefore, established to test the efficacy of rumen
content compost extract in degradation of harvest residue. The experiment
utilised the microbially enhanced extracts of rumen waste composted for one,
three, six, and ten months and compared these with an extract produced from a
feedstock mix of nine month old compost and the same sugarcane residue, which
was further composted for one month, and with a water control. The hypothesis
set for the possible benefits of compost extract to plants is that application of
compost extract to the sugarcane trash covered soil could enhance the
degradation of harvest residue due to the microbial action of compost extracts.
155
This may subsequently increase soil C content through the addition of organic
matter via partial decomposition of plant residues.
2.1. Soil, plant and compost material
Ferrosol soil was obtained from an organic sweet potato farm of
Rossmoya (from a depth of 30 cm) for this experiment. The principal
characteristics of this soil were previously studied and discussed in Chapter 5.
The rumen waste, obtained from an abattoir (Broadmeadows, Qld), is a
lignocellulosic by-product rich in C/N ratio.
Sugarcane trash was collected from a freshly harvested sugarcane field in
Bundaberg, Qld region and stored air dry at 4˚C. Typically, this trash contains
lignocellulosic materials and comprises of cellulose (approximately 40%),
hemicellulose (25%), lignin (18-20%) and protein (34 mg g-1 ) (Singh et al.,
2008). I chose intact over chopped trash mat for the experiment because Hall et
al. (2006) did not find any difference between the biodegradation of either
chopped or intact trash mat. Further, use of intact trash mat is more field relevant
compared to chopped or ground material.
2.2. Biodegradation of trash mat
Different aged rumen content composts from one (1MC), three (3MC), six
(6MC) and nine month old were acquired from Broadmeadows Pty Ltd,
Rockhampton. Some of the nine month old compost was further processed and
manipulated by adding sugarcane trash at the ratio of 1:1 (v/v) and allowed to
compost further for one month (10SC) in a partially enclosed condition with
156
water sprinkled occasionally. The remaining portion of nine month old compost
was incubated under the same condition for a month but without the addition of
sugarcane trash (10MC) for comparison purpose.
Microbially enhanced extracts were prepared as in Chapter 2 from the five
composts. Extracts were misted over equal amounts of sugarcane trash
(approximately 100 g f wt) laid over 5 kg f wt of soil (ferrosol) in partially
enclosed plastic containers (0.28 m2 surface area and 20 L volume) in an
evaporatively-cooled greenhouse from April to October, 2009. Equal volumes of
a water-control were similarly applied. All of the treatments were applied twice at
the rate of 200 L ha-1 (i.e. 1.4 mL per container). Negative controls for all the
treatments were also established using containers with soil only. During the
experimental period, other than during respiration measurement, the containers
were covered with perforated plastic sheet to reduce water loss while maintaining
an aerobic condition. Water was sprayed over the trash or soil surface once a
month at 50 mL per pot to maintain humid conditions inside the pots. There was
no significant difference in the moisture content of soil collected from each
treatment at the end of the study (data not presented). The perforated plastic
covers were replaced with airtight plastic lids for 24 h in order to trap the CO2
produced through respiration inside the containers on days 1, 7, 14, 21, 28, 56,
84, 112, 140 and 168 of the incubation period. In terminating the experiment (168
day), the trash blanket was manually removed and weighed, allowing calculation
of weight loss. The experiment was conducted using a completely randomised
design with three replications. Total organic C, dissolved organic C and microbial
biomass C of the soil core samples collected from the top 5 cm at the end of the
157
experiment were determined by the chloroform-fumigation method (Vance et al.,
1987).
2.3. Analytical methods
2.3.1 Physico-chemical analyses
The pH and EC were measured on the differently aged composts (1:20
w/v suspension), and on soil samples collected at the end of the experiment
(prepared as a 1:5 w/v suspension). Compost extracts (1:10 w/v mix) were
prepared indoors and the parameters, such as pH, EC, temperature and dissolved
oxygen were measured at 3 h intervals over a 24 h period and a final reading was
taken at 48 h, following the methods as described in detail in Chapter 2. The
moisture content of the soil samples was determined following drying of the
samples at 105˚C for 24 h. Inorganic nutrient analyses of the same composts and
compost extracts thus produced, were performed using ‗RQflex plus‘ colorimetric
test strips (Merck, Germany). All samples were analysed in triplicate.
2.3.2. Microbial analyses
Serially diluted populations of bacteria and fungi of the different aged
composts as well as compost extracts were enumerated by the pour plate count
method (Harrower, 2006). Basal and substrate induced respiration of the compost
samples were analysed following the methods as described in Chapter 3.
Microbial activities in composts, compost extracts and soil samples were assessed
by measuring fluorescein diacetate (FDA) hydrolysis following the protocol of
Adam and Duncan (2001) as described in Chapter 2.
158
Microbial respiration was determined by the amount of CO2 trapped in 15
mL of 0.5 M NaOH solution inside the sealed containers over 24 h, as measured
by the acid titration method described in Chapter 3 (Anderson, 1982). Cumulative
respiration rates of sugarcane trash materials were calculated by subtracting the
respiration values generated by bare soil from those by soil with trash cover.
Data were analysed using factorial ANOVA of XLSTAT (2010), and
statistical significance of differences was evaluated at p< 0.05 using Tukey‘s test.
3.1. Physico-chemical, biochemical and microbial analyses of different
aged composts
Samples were taken from rumen content piles that had been composting
for different periods (one, three, six and nine months), representing a pseudo-time
series of the composting process. There was a trend for pH to increase, EC to
decrease (except for an inconsistent result at nine months), NH4-N to increase to
three months, then decrease, basal respiration to decrease, and microbial activity
(FDA), bacterial plate count and fungal plate count to decrease with time of
composting (Table 6.1). The pH rise could be attributed to proteolysis as
suggested by Garcia et al. (1991). The EC decrease is attributed to leaching of the
open to atmosphere piles, and incorporation of mineral salts by microbes. NH4-N
increase to three month was consistent with proteolytic activity. The subsequent
decrease in one month old compost and nine month old compost amended with
equal volume of sugarcane trash, which was further composted for a month
159
compared to other composts (Table 6.1) could be due to the presence of yet to be
mineralised organic substrates, which were highly lingo-cellulosic in nature. The
decrease in microbial activity is consistent with ‗maturation‘ of the compost
process.
Addition of sugarcane trash to nine month old rumen content compost
effectively ‗set the clock back‘ on these indices of the composting process, with
pH akin to six month, basal respiration to one month, microbial activity (FDA) to
six month, fungal plate count to one month rumen content compost, while
bacterial activity exceeded any sample of rumen content compost. The order of
magnitude of bacterial populations follows different phases of composting and
indicates that early aged compost extracts have higher metabolic activity than that
of the later phases of composting, the phases associated with the depletion of
readily available fractions of carbon from aged compost (Garcıa-Gomez et al.,
2003; Herrmann & Shann, 1997; Wu & Ma, 2002). However, addition of
sugarcane substrates in nine month old compost and further composting it for a
month must have added water soluble C and nitrogen and thus enhanced
propagation of bacteria and increased their plate counts of bacteria compared to
three, six and ten month old composts.
The basal respiration was higher in one month old compost and nine
month old compost amended with equal volume of sugarcane trash, which was
further composted for a month, while substrate induced respiration was higher in
one month old compost compared to other composts (Table 6.1). Increased
evolution of CO2 is indicative of the presence of higher populations of active
microorganisms in the immature compost (e.g. one month old compost) as
compared to the mature composts. Respiration decreases during the maturation or
160
stabilisation processes (Wu et al., 2000). Increased values of bacterial and fungal
plate counts, basal respiration, and microbial activity in nine month old compost
amended with equal volume of sugarcane trash, which was further composted for
a month compared to ten month old compost (Table 6.1) might be due to the
similar reason discussed earlier.
Table 6. 1. Physico-chemical, biochemical and microbial comparisons of different aged, and

sugarcane stubble amended, composts. 1MC - one month old compost, 3MC - three month old
compost, 6MC - six month old compost, 10MC - ten month old compost, 10SC - nine month old
compost amended with equal volume of sugarcane trash and further composted for a month.
Within rows, means (n = 3) with the same letter are not significantly different according to
Tukey‘s test (p< 0.05).
Parameters 1MC 3MC 6MC 10MC 10SC
pH (H2O) 6.49 ± 0.04b 7.57 ± 0.03d 7.64 ± 0.04d 6.20 ± 0.02a 7.01 ± 0.02c
EC (dS m-1) 0.81 ± 0.01c 0.62 ± 0.01b 0.53 ± 0.01a 1.55 ± 0.01d 0.64 ± 0.00b
+
NH4 -N (mg kg-1 d wt) 63.4 ± 1.48a 676 ± 35.3d 271 ± 5.79c 273 ± 9.41c 168 ± 18.2b
Basal respiration (µg CO2 g-1 d wt h-1) 157 ± 3.80b 62.7 ± 5.17a 66.4 ± 6.27a 47.6 ± 9.10a 138 ± 10.2b
SIR respiration (µg CO2 g-1 d wt h-1) 575 ± 28.1d 290 ± 17.9b 137 ± 10.9a 456 ± 3.97c 257 ± 21.3b
Total microbial activity (µg FDA g-1 d wt h-1) 564 ± 85.5bc 853± 118.3c 518 ± 68.3b 76.3 ± 15.0a 540 ± 14.0b
Bacterial population (cfu Log10) 7.06 ± 0.04b 7.00 ± 0.03b 7.86 ± 0.01c 6.10 ± 0.10a 11.1 ± 0.01d
Fungal population (cfu Log10) 5.33 ± 0.05c 4.95 ± 0.03b 4.77 ± 0.04ab 4.55 ± 0.07a 5.52 ± 0.01c
3.2. Characterising the incubation process using different aged
compost extracts as inoculum
Dissolved oxygen declined rapidly in extracts seeded with one month old
compost and nine month old compost amended with equal volume of sugarcane
trash, which was further composted for a month, indicative of high respiratory
demand (Fig. 6.1). This finding broadly agrees with the data from the microbial
plate counts, basal respiration and total microbial activity for those composts
(Table 6.1). The pH was significantly higher in ten month old sugarcane amended
compost extract than other treatments at 24 and 48 h of incubation, however EC
of six month old compost extract and ten month old compost extract remained
higher throughout the incubation period, with few exceptions, compared to the
remaining treatments (Fig. 6.1). This agrees with the findings of Garcia et al.
161
(1991) who reported higher EC in mature compost extracts compared to the
extracts of immature composts. Although temperature of ten month old sugarcane
amended compost extract increased significantly above that of other treatments at
certain times during incubation, no difference was noted between treatments at 24
and 48 h of incubation (Fig. 6.1).
162
9
A
8
7
6
5
pH
4
1MCE
3 6MCE
3MCE
2 10MCE
10SCE
1
0
0 3 6 9 12 15 18 21 24 48
5.0
B
4.5
4.0
3.5
EC (dS m -1)
3.0
2.5
2.0 1MCE
6MCE
1.5 3MCE
10MCE
1.0 10SCE
0.5
0.0
0 3 6 9 12 15 18 21 24 48
35
C
30
25
Temperature (°C)
20
15
1MCE
6MCE
10 3MCE
10MCE
5 10SCE
0
0 3 6 9 12 15 18 21 24 48
D 120
100
Dissolved Oxygen (%)
80
1MCE
6MCE
60 3MCE
10MCE
10SCE
40
20
0
0 3 6 9 12 15 18 21 24 48
Time (h)
and final readings taken at 48 h) between differently aged and sugarcane stubble amended
compost extracts. 1MCE - one month old compost extract, 3MCE - three month old compost
extract, 6MCE - six month old compost extract, 10MCE - ten month old compost extract, 10SCE
- compost extract produced from nine month old compost amended with equal volume of
sugarcane trash and further composted for a month. Values are mean ± SE (n = 3).
163
3.3. Comparison of chemical, microbial and biochemical
characteristics of different aged and sugarcane stubble amended
compost (24 h incubated) extracts
Plate counts of compost extracts revealed that bacterial populations were
found in the order; one month old compost extract > ten month old sugarcane
amended compost extract > three month old compost extract > six month old
compost extract > ten month old compost extract (Table 6.2), consistent with the
bacterial load of the inoculum composts. This indicates that the incubation
conditions supported bacterial growth. In contrast, there was little difference
between the treatments in terms of culturable fungal load, despite differences in
the composts per se, suggesting that the incubation conditions did not favour
fungal growth.
No differences were found in phosphate, potassium and fulvic acids, but
the ammonium and humic acid concentrations were found to be higher in ten
month old sugarcane amended compost extract compared to all other compost
extracts (Table 6.2). These increments must have been sourced from sugarcane
stubble added to the nine month old compost. Garcia et al. (1991) found
significant reduction in NH4+-N during composting and maturation, however
ammonium concentration increased initially then declined after three months of
composting (Table 6.2), the reason for which is unclear.
164
Table 6. 2. Comparison of chemical, biochemical and microbial characteristics of different aged

and sugarcane stubble amended compost extracts incubated for 24 h with 1% kelp and 0.5%
molasses as amendments. 1MCE - one month old compost extract, 3MCE - three month old
compost extract, 6MCE - six month old compost extract, 10MCE - ten month old compost extract,
10SCE - compost extract produced from nine month old compost amended with equal volume of
sugarcane trash and further composted for a month. Within rows, means (n = 3) with the same
letter are not significantly different according to Tukey‘s test (p< 0.05).
Parameters 1MCE 3MCE 6MCE 10MCE 10SCE
+
NH4 -N (mg L-1) 0.54 ± 0.04a 0.91 ± 0.05b 0.67 ± 0.05a 0.65 ± 0.03a 1.11 ± 0.03c
PO43--P (mg L-1) 25.7 ± 1.09a 22.0 ± 1.43a 22.0 ± 1.70a 22.0 ± 0.58a 25.7 ± 1.15a
K+-K (g L-1) 0.57 ± 0.06a 0.58 ± 0.01a 0.63 ± 0.02a 0.62 ± 0.03a 0.56 ± 0.02a
Humic acid (g L-1) 0.17 ± 0.01a 0.22 ± 0.05a 0.13 ± 0.03a 0.18 ± 0.03a 0.41 ± 0.01b
Fulvic acid (g L-1) 0.23 ± 0.01a 0.26 ± 0.03a 0.28 ± 0.00a 0.28 ± 0.00a 0.24 ± 0.00a
Bacterial population (cfu log 10) @ 24 h 12.2 ± 0.02e 11.1 ± 0.02c 10.5 ± 0.03b 10.3 ± 0.02a 11.4 ± 0.01d
Fungal population (cfu log 10) @ 24 h 5.63 ± 0.03b 5.70 ± 0.09b 5.20 ± 0.04a 5.54 ± 0.06b 5.45 ± 0.04ab
The least microbial activity in ten month old compost extract as compared
to one, three and six month old compost extracts suggests that active microbiota
decline as the compost matures (Fig. 6.2). Nevertheless, the addition of sugarcane
trash in nine month old compost greatly stimulated and revived the microbial
activity of the resultant compost extract (Fig. 6.2). Higher microbial activity in
compost extract produced from ten month old sugarcane amended compost
extract as compared to ten month old compost extract implies that the addition of
sugarcane substrate might have supplied energy (in the forms of the labile and
total organic C) to the less active microbiota of the ten month old compost. This
could be related to a priming effect or substrate stimulation causing
mineralisation of organic matter, as found with the addition of glucose to soil
(Shen & Bartha, 1996).
165
Figure 6. 2. Comparison of total microbial activity at different times (readings taken at 12, 24 and
48 h) following the FDA hydrolysis method in different aged (one, three, six and ten month old)
and sugarcane stubble amended compost extracts. 1MCE - one month old compost extract, 3MCE
- three month old compost extract, 6MCE - six month old compost extract, 10MCE - ten month
old compost extract, 10SCE - compost extract produced from nine month old compost amended
with equal volume of sugarcane trash and further composted for a month. Within a given time,
treatment means with the same letter do not differ significantly and values are mean ± SE (n = 3).
When solid composts were compared with liquid forms of compost
extracts incubated with microbial food sources and continuous supply of aeration,
the bacterial populations increased tremendously in the extracts, irrespective of
the treatment effects (compare data in Tables 6.1 and 6.2). Given the bacterial
counts of ten month old compost (1.3 x 106 cfu g-1) and ten month old compost
extract (2.1 x 1010 cfu mL-1), there was a significant increase in the number of
bacteria when transformed from solid to liquid form. Similarly, fungal
populations were slightly increased by liquid incubation in all treatments with an
exception of ten month old sugarcane amended compost extract, but not by as
much as bacteria. Total microbial activity (FDA hydrolysis) likewise increased
by varying amounts in liquid extracts compared to their original forms, except in
six month old compost extract which was similar in both solid and liquid forms
(Table 6.1 and Fig. 6.2). These results suggest that conversion of solid composts
166
into liquid forms under the stated conditions is beneficial for microbial
enrichment in the resulting extracts.
It seems that the benefit of compost extract over compost is in the
multiplication of microbes. The next question is survival of these microbes in the
soil. For example, rhizobia are liquid-cultured and can be applied directly to plant
roots/soil. But in the field, survivability of these microbes is low (storage plus in-
field survival). Therefore, a delivery system, such as peat is normally used.
Nevertheless, previous studies have shown that rhizobia was successfully
delivered into the soil via drip irrigation system (biofertigation) compared to a
conventional seed pelleting system (Mussaddak & Fawaz, 2008). Inoculation of a
liquid culture of rhizobia through this system showed enhanced N2 fixation by
soybean plants. This system could also apply to compost extracts as they deliver
beneficial microorganisms into a field soil.
3.4. Effects of compost extract treatments on sugarcane trash
degradation
After 168 d, soil pH was lower in treatments involving cane trash (Table
6.3) which is in accordance with the findings of Robertson (2003). There was no
difference in soil pH between treatments receiving different compost extract but
without trash, in contrast to when trash was present. Although no difference was
noted between the non-covered bare soil treated with different compost extracts,
total and dissolved organic C were higher in soils covered with sugarcane trash
(Table 6.3). Mulching generally increases microbial load and respiration in soil as
indicated by the release of CO2-C (measured with fumigation extraction) and by
the stimulation of substrate induced respiration (SIR), respectively (Wardle et al.,
167
1999). For microbial biomass C, there was only one major difference between
treatments. There was a significant increase in microbial biomass C in ten month
old sugarcane amended compost extract applied to bare soil as compared to the
water-control treatment with sugarcane stubble (Table 6.3). The literature shows
that numerous factors, such as soil-trash contact (Magid et al., 2006), and climate
and soil interaction (Young & Ritz, 2000) influence increases of microbial
biomass (Robertson & Thorburn, 2007).
There was no significant difference in total microbial activities of soil
samples collected across treatments either with or without a trash cover, however
soil microbial activity was comparatively higher (two-fold or more), though not
at significant level, when sugarcane stubble was retained compared to the bare
soil (Table 6.3). Higher microbial activity (as shown by the respiration data) in
the compost extract treated trash indicated that once the microbes are able to
degrade the lingo-cellulosic secondary cell walls, the decomposition should
proceed quickly. A reason for lower soil biological activity without trash is
generally linked to the decline in dissolved organic C (labile C) (Bell et al.,
2007), which is in line with the findings of the current study.
168
Table 6. 3. Chemical, biochemical and microbial characteristics of soil samples (collected after 168 days of incubation) covered with or without sugarcane trash and treated
with compost extracts produced from differently aged and sugarcane stubble amended compost and incubated for 24 h with 1% kelp and 0.5% molasses as amendments.
1MCE - one month old compost extract, 3MCE - three month old compost extract, 6MCE - six month old compost extract, 10MCE - ten month old compost extract, 10SCE
- compost extract produced from nine month old compost amended with equal volume of sugarcane trash and further composted for a month. Within rows, mean (n = 3) ±
SE with the same letter are not significantly different according to Tukey‘s test (p< 0.05).
with trash without trash

Parameters Cont 1MCE 3MCE 6MCE 10MCE 10SCE C Cont C 1MCE C 3MCE C 6MCE C 10MCE C 10SCE
pH (H2O) 5.10 ± 0.10a 5.51 ± 0.01cd 5.53 ± 0.02cd 5.44 ± 0.02bc 5.46 ± 0.04bc 5.21 ± 0.12ab 5.56 ± 0.04cd 5.68 ± 0.02cd 5.70 ± 0.02cd 5.75 ± 0.01d 5.64 ± 0.05cd 5.70 ± 0.01cd
EC (dS m-1) 0.11 ± 0.00ab 0.14 ± 0.00b 0.12 ± 0.00ab 0.11 ± 0.01a 0.12 ± 0.01ab 0.11 ± 0.01ab 0.12 ± 0.00ab 0.11 ± 0.01ab 0.11 ± 0.00ab 0.11 ± 0.00a 0.11 ± 0.01ab 0.11 ± 0.01ab
Total microbial activity (µg FDA g-1 d wt h-1) 674 ± 70.8a 732 ± 35.5a 589.2 ± 34.9a 489 ± 331.4a 553 ± 228a 723 ± 73.6a 276 ± 28.7a 207 ± 12.8a 267 ± 40.7a 224 ± 12.0a 266 ± 58.1a 401 ± 82.7a
-1
Total organic C (mg C g d wt) 0.07 ± 0.00c 0.07 ± 0.00c 0.08 ± 0.00c 0.08 ± 0.00c 0.08 ± 0.00c 0.08 ± 0.00c 0.02 ± 0.00a 0.04 ± 0.01b 0.03 ± 0.00ab 0.02 ± 0.00a 0.02 ± 0.00a 0.03 ± 0.00a
-1
Dissolved organic C (mg C g d wt) 0.07 ± 0.00b 0.06 ± 0.00b 0.06 ± 0.01b 0.07 ± 0.00b 0.07 ± 0.00b 0.07 ± 0.01b 0.01 ± 0.00a 0.03 ± 0.00a 0.01 ± 0.00a 0.01 ± 0.00a 0.01 ± 0.00a 0.01 ± 0.00a
-1
Microbial biomass C (mg C g d wt) 0.02 ± 0.01a 0.03 ± 0.00ab 0.05 ± 0.01ab 0.04 ± 0.00ab 0.02 ± 0.00a 0.05 ± 0.01ab 0.03 ± 0.00ab 0.05 ± 0.03ab 0.04 ± 0.00ab 0.03 ± 0.01ab 0.04 ± 0.00ab 0.09 ± 0.03b
Trash weight (g f wt) 98.2 ± 0.88a 95.2 ± 5.64a 98.0 ± 2.02a 97.5 ± 2.29a 95.2 ± 2.46a 90.8 ± 1.17a N/A N/A N/A N/A N/A N/A
N/A - Not applicable
169
3.5 Weight loss of trash and cumulative respiration rate
Although ten month old sugarcane amended compost extract showed the
maximum loss in weight (f wt) of sugarcane trash, there was no significant
difference among the treatments (Table 6.3). In contrast, Witkamp (1966) found a
significant loss in weight (i.e. 40-90%), using a mesh-bag technique, of four
tested leaf litters (mulberry, redbud, oak and pine) within a year under a natural
forest environment. This is logical considering the one-year long trial period
compared to the current six month biodegradation trial. This weight loss of
various leaf species in their study was attributed to the leaching of water soluble
nutrients from their surfaces and was highly correlated with the microbial load
present in the litter. In contrary, perhaps there was no loss of soluble nutrients
from the surfaces of sugarcane litter in the present study.
In the current experiment, however, sugarcane trash treated with compost
extracts did show visual signs of degradation, such as discolouration, compared
to those treated with water only (Fig. 6.3). This could be due to leaching of
nutrients caused by the mineralisation of organic matter of trash or due to
degradation caused by microbial attack or cellulose utilisation by the microbes
(Parsons & Congdon, 2008).
170
Figure 6. 3. On the left (A) showing sugarcane trash treated with water control and on the right
(B) showing the same trash treated with compost extract produced from nine month old compost
amended with equal volume of sugarcane trash and further composted for a month (10SCE).
Although there was no significant weight loss by ten month old sugarcane
amended compost extract, cumulative respiration as measured in the ten month
old sugarcane amended compost extract treated sugarcane trash was significantly
higher (p< 0.05) from day 1 to day 56 compared to that of the trash control
treatment, after which no significant difference was noted in the cumulative
respiration between treatments (Fig. 6.4). This could be due to the loss of
moisture inside the containers in the due course of time that there was no
difference in weight loss of sugarcane trash between treatments. It could also be
because the time period was insufficient to allow a difference in trash weight to
be expressed. Indeed the cumulative respiration of 230000 ug CO2 pot-1 is
equivalent to 230 x 12 / 44 = approximately 63 mg C pot-1 lost in respiration, a
trivial amount compared to the trash weight of approximately 95 g pot-1.
Higher cumulative respiration from ten month old sugarcane amended
compost extract treated sugarcane trash could possibly be due to addition and
stimulation of microbiota present in the sugarcane layer or the soil through the
application of compost extracts. Another possible reason for this could simply be
due to the combined synergistic effect of soil indigenous and exogenous
171
microbiota, the latter applied in the form of organic amendments or biofertilisers
(Prasanna et al., 2008). Synergistic or antagonistic effects of microbial diversity
in litter decomposition of the terrestrial ecosystems have been reviewed by
Hattenschwiler et al. (2005). Synergistic interaction, in general, enhances the rate
of decomposition unlike the antagonistic interaction, in which microorganisms
compete for the similar resources, thereby slowing the rate of litter decay.
Nevertheless, no such antagonistic effect has been noticed in the current study
because there was a higher cumulative respiration in each treatment compared to
that of the water control.
172
Figure 6. 4. (A) Time course of respiration rates which was assessed as accumulation of CO2
over a 24 h period and (B) comparison of cumulative respiration rates of sugarcane trash materials
lying on top of the ferrosol soil over six month (28 weeks) from 22nd April to 28th October, 2009
(readings taken at 1, 7, 14, 28, 56, 84, 112, 140 and 168 days of incubation period) applied with
different aged and sugarcane stubble amended compost extracts as measured by trapping CO2
evolved. Respiration rates and cumulative respiration rates of sugarcane trash materials were
calculated by subtracting the respiration values generated by bare soil from those by soil with
trash cover. 1MCE - one month old compost extract, 3MCE - three month old compost extract,
6MCE - six month old compost extract, 10MCE - ten month old compost extract, 10SCE -
compost extract produced from nine month old compost amended with equal volume of sugarcane
trash and further composted for a month. Values are mean ± SE (n = 3).
There was distinct seasonal variation in microbial respiration; during the
winter (weeks 8 to 16), the rate of respiration was much reduced compared to the
warmer summer (Fig. 6.4) although the containers were kept in the greenhouse
(i.e. in a semi-controlled environment). Other studies have also shown a direct
influence of environmental variables, for example, ambient temperature and
moisture, on soil respiration (Bingrui & Guangsheng, 2009; Han et al., 2007;
173
Lloyd & Taylor, 1994) and ultimately on the rate of decomposition and nutrient
cycling (Davidson et al., 2000; Heal et al., 1997; Zak et al., 1999).
4. Conclusions
Microbially enhanced compost extract produced from a mixture of
composted rumen waste and sugarcane stubble has the potential to accelerate
degradation of a sugarcane trash mat, as indicated by the enhanced cumulative
respiration in this study. It is evident that none of the differently aged compost
extracts enhanced degradation of an intact trash mat, unless sugarcane stubble
was incorporated into the compost. This suggests that incorporation of the harvest
residue into the rumen content compost selected for microorganisms specific for
degradation of cane trash. Increased levels of organic C in soil covered with
harvest residue, irrespective of the compost extract treatments, highlighted the
benefits of mulching. Future research should focus on optimising conditions for
degradation, such as comparing trash particle size and incorporation of trash into
soil, besides the use or not of compost extract in situ.
174
Chapter 7: Compost extract and disease suppression
Chapter 7
Suppressing fungal wilt diseases of tomato caused
by Fusarium oxysporum, F. solani and Rhizoctonia
solani using compost extracts in vitro and in
hydroponics culture
A revised version of this chapter has been accepted in Biological Control, prepared by Karuna
Shrestha, Pramod Shrestha, Kerry B. Walsh, Keith M. Harrower and David J. Midmore
Abstract
The efficiency of compost extracts prepared aerobically or anaerobically,
with three or nine month old compost, and subsequently sterilised or not, on
disease suppression of pathogenic fungi was studied. Fresh, but not sterilised,
compost extracts suppressed mycelial growth of three fungal pathogens, namely,
Fusarium oxysporum, Fusarium solani, and Rhizoctonia solani by 82, 79 and
88%, respectively, compared to the non-treated control. The highest inhibition
was demonstrated by aerated extract based on three month composted material.
Sporulation of F. oxysporum and F. solani was also reduced by all non-sterilised
compost extracts aged three and nine months, with overall spore reduction by up
to 98%. Sterilised compost extracts enhanced mycelial growth and sporulation in
most cases. Hydroponically cultured tomato plants (cv. Tiny Tim) inoculated
with conidial suspensions (1 x 106 spores per plant) of the pathogenic fungi F.
175
oxysporum had less severe fungal wilt symptoms when compost extract was
added to the medium, however disease suppression on inoculation with F. solani
and R. solani was not significantly reduced. In the hydroponics studies, there was
little difference in terms of benefit between aeration of compost extracts, or age
of composts.
1. Introduction
Fungal pathogens, such as Fusarium oxysporum, Fusarium solani, and
Rhizoctonia solani are important plant pathogens, which cause severe damage to
several fruit and vegetable crops, including tomato. Control of these
phytopathogenic fungi typically relies on use of the synthetic fungicides Thairam,
Dichlofluanid, and Fluazinam (Elad et al., 2007). These fungicides are persistent,
and have a level of toxicity to wild life and humans (Crnko et al., 1992). Their
widespread use is implicated in the development of pathogen resistance to these
pesticides (Elad et al., 1992), thereby threatening the stability of crop production.
Elevated concentrations can cause reduced soil biological activity in agricultural
lands (Dumestre et al., 1999), and in addition, these biocides are not confined to
target species; their application can also negatively impact beneficial organisms
(Zwieten et al., 2007). Therefore, alternatives to these are extensively being
explored, so avoiding use of synthetic fungicides in order to sustainably produce
safe food.
Organic soil amendments, such as compost have been reported to be
effective in controlling several fungal diseases, such as Sclerotium rolfsii (Danon
et al., 2007; Hadar & Gorodecki, 1991), Fusarium spp. (Chef et al., 1983;
Cotxarrera et al., 2002; Henis et al., 1984) and Rhizoctonia solani (Nelson &
176
Hoitink, 1983). Suppressive activities against various pathogens are considered to
be due to antagonism by beneficial microflora (Hoitink et al., 1997), including
strains of Trichoderma spp. (Cotxarrera et al., 2002), Bacillus subtilis (Phae et al.,
1990), or due to the production and release of allelochemicals (Bailey &
Lazarovits, 2003).
Compost extract is likewise reported to suppress the root diseases in
economically important crops, including tomato, caused by fungal pathogens, for
example, F. oxysporum f. sp. lycopersici (Hibar et al., 2006; Ma et al., 2001b;
Utkhede & Koch, 2004). Studies evaluating the effects of various compost
extracts on Pythium debaryanum, Fusarium oxysporum f. sp. lycopersici and
Sclerotium bataticola carried out in situ and in vitro showed that microflora
present in compost extract suppressed the growth of tested fungi by parasitism
(El-Masry et al., 2002). Research studies conducted in vitro demonstrated up to
98% inhibition of Venturia inequalis conidia germination by the antibiotic effect
of compost extract as compared to the water control (Cronin et al., 1996). The
antagonistic action of the microorganisms present in compost extracts against
pathogenic fungi may be due to direct competition for nutrients and space (Al-
Mughrabi et al., 2008), production of antimicrobial compounds or induced
systemic resistance in plants (Zhang et al., 1998). Some researchers have
suggested that the antagonistic effect may be due to compounds in the compost
extract (Hoitink et al., 1997; Siddiqui et al., 2008b).
Other studies have also reported an effectiveness of compost extracts in
suppressing Fusarium wilt of sweet pepper (Fusarium oxysporum f. sp.
vasinfectum) by up to 89% (Ma et al., 2001b). Similarly, an approximately 85%
reduction in Choanephora wet rot severity in a field situation was demonstrated
177
on okra when treated with Trichoderma-fortified extract produced from a mixture
of rice straw and composted oil palm inflorescence stalks. The extract showed a
comparable effect to the conventional fungicide Dithane M-45R (Siddiqui et al.,
2008b). Scheuerell and Mahaffee (2004) reported that aerated compost extract
amended with kelp and humic acid was more effective than non-aerated compost
extract in controlling damping-off in cucumber seedlings caused by Pythium
ultimum. In contrast, some studies has shown that compost extracts produced
with continuous aeration fail to suppress fungal diseases, such as grey mold
(Botrytis cinerea) on geranium (Elad & Steinberg, 1994) and late blight
(Phytopthora infestans) on potato (Olanya & Larkin, 2006). Cronin et al. (1996)
also showed that aeration decreased the efficacy of compost extracts compared to
the non-aerated controls and ascribed this to the possible production of major
inhibitory metabolites in non-aerated extracts, which are heat stable, non-protein
in nature, and low in molecular weight. Nevertheless, Scheuerell (2003) did not
find any difference between aerated and non-aerated compost extracts in
controlling fungal diseases, both were effective against grey mold on greenhouse
grown geraniums, damping-off of basil seedlings.
In vitro studies have also been employed to test the effect of compost
extracts on fungal growth. El-Masry et al. (2002) reported that compost extracts
based on fruit waste, garden waste and crop waste suppressed growth of various
fungal species, including Fusarium oxysporum f. sp. lycopersici. Compost
extracts at concentrations of 5%, 10% and 15% (v/v) to PDA-Sigma growth
medium suppressed the hyphal growth of F. oxysporum f. sp. lycopersici by
94.4%. However, there was no antagonistic effect against the tested fungi using
autoclaved compost water extracts.
178
McQuilken et al. (1994) also noted that filter sterilisation or autoclaving
of compost extracts negated the disease suppression effect, in their case against
the plant pathogen Botrytis cinerea. Furthermore, stimulatory effects of sterilised
compost extracts have been reported by Hardy and Sivasithamparam (1991) on
sporangia formation of Phytopthora spp. while non-sterilised extracts showed
inhibitory effects and induced lysis of sporangia.
Based on previous work I, therefore, expect compost extract to suppress
fungal pathogen growth, but not all extracts may have the same effect due to
different forms of compost extracts, their being microbially enhanced, aerated,
non-aerated, different aged and so on.
The aim of this study was to compare the disease suppressive effects of
compost extracts from different aged composts in vitro and in situ, and to assess
their ability to reduce disease severity on a tomato crop caused by Fusarium
oxysporum f. sp. lycopersici, F. solani DAR 66102 and Rhizoctonia solani DAR
61830. The hypothesis to be tested is that compost extract suppresses fungal root
diseases of tomato, reducing mycelial growth, spore production and total biomass
of these pathogens through its biological and/or non-biological action, thus
offering crop protection against these fungal diseases.
The efficacy of compost extracts in suppressing fungal wilt diseases of
tomato was tested in vitro, in terms of fungal hyphal growth (trial 1and 2), and in
hydroponics culture, in terms of tomato root disease expression (trial 3 and 4).
For trials 1 and 3, Fusarium oxysporum f. sp. lycopersici (sourced from
CQUniversity, Qld) was employed.
179
For trials 2 and 4, the same Fusarium oxysporum f. sp. lycopersici, and
two additional fungal pathogens: Fusarium solani DAR 66102 and Rhizoctonia
solani DAR 61830 (both sourced from Department of Primary Industries, NSW),
were used, as causal agents of fungal wilt of tomato. These strains were cultured
on Petri plates using a basal media (Harrower, 2006), consisting of Na2HPO4
0.725, KH2PO4 0.725, MgSO4.7H2O 0.12, NaCl 0.1, NH4NO3 0.4, glucose 1.8,
yeast extract 1.1, malt extract 1.0 and agar 15.0 in g per litre of water. In both
experiments, the plates were incubated at 25˚C until heavy sporulation was
observed and then stored at 4˚C until use.
Compost extract was produced by incubating nine month old rumen
compost in water at the ratio of 1: 10 (w/v), both with continuous aeration for 24
h and without aeration. The process followed was as in Chapter 2. All compost
extracts in both experiments were amended with 1% ‗fish and kelp hydrolysate‘
from Searle Pty Ltd, Australia, and 0.5% molasses at the onset of incubation
(Shrestha et al., 2011b).
2.1. In vitro hyphal growth trials
Two in vitro trials were undertaken. In the first trial, two concentrations
(50 ml L-1 and 100 ml L-1) of aerated and non-aerated extracts prepared from nine
month old rumen content compost were compared with the same concentrations
of sterilised aerated and sterilised non-aerated compost extracts and the control
(equal volumes of distilled water). In trial 2, three and nine month old compost
extracts were produced with continuous aeration and nine month old compost
extract was also produced without aeration, as described above. Sterilisation of
one half of each compost extract was achieved by autoclaving the extracts at
180
121˚C for 15 minutes at 15 psi (0.1034 MPa) pressure followed by filtration
through 0.45 µm-pore membrane filters (Millipore, Australia) to assure the
absolute elimination of live microorganisms. Three parameters, namely, mycelial
growth, spore count and total biomass were assessed to evaluate the efficacy of
compost extracts in suppressing the fungi in both in vitro trials.
In both trials, aliquots of 100 mL of non-sterilised and sterilised compost
extracts and a distilled water control were added and blended thoroughly with the
melted basal medium (1 L) before pouring into plates. Following solidification, a
one cubic mm agar block with cultured fungal pathogen was placed at the centre
of each plate. Plates were then incubated at ± 25˚C for a week. Five (trial 1) and
four (trial 2) replicates were established for each treatment. Plates were randomly
placed within an incubator. Mycelial growth rate was quantified by measuring the
radial extension of the mycelia in millimeters at six days (in trial 1), and three and
six days (in trial 2) after plate inoculation, before growth reached the edge of the
Petri plates (Harrower, 2006).
Following a week after the measurement of radial extension at six days
after plate inoculation in both experiments, spores produced on each Petri plate
were collected by swirling 6 mL of distilled water with 0.1% Tween 80 and
0.01% chloramphenicol. The collected spore suspensions were stained separately
with two drops of lactophenol cotton blue dye per container. Due to the presence
of phenol in the stain, the microorganisms were killed. Spore density was
assessed by microscopic observation using a haemocytometer.
To determine the fungal biomass, in trial 1, 200 μL of spore suspension of
F. oxysporum was inoculated into 70 mL liquid basal medium contained in Schott
bottles. In trial 2, the same volume of spore suspensions of three fungal
181
pathogens was inoculated into 35 mL liquid basal medium contained in Falcon
tubes under the same conditions as described for trial 1. At the same time of
pathogen inoculation, compost extracts at the rate of 50 mL L-1 and 100 mL L-1
(in trial 1) and 100 mL L-1 (in trial 2) were added to the liquid basal media, and
the mixtures were shaken constantly at 150 rpm for two weeks in the laboratory
at room temperature. The cultures were then harvested, oven dried at 55˚C for 72
hours and weighed to obtain mean mycelial biomass (d wt) in either trial. Each
treatment was replicated five and four times for trials 1 and 2, respectively.
2.2. Hydroponics trials
A non-recirculating hydroponics system (Midmore & Wu, 1999) with
eight plants per styrofoam hydroponics container was established. Tomato seeds
(cultivar Tiny Tim) were sown in vermiculite and two weeks later tomato
seedlings were transplanted individually into perforated poly-pots (3.5 cm
diameter) containing pieces of nylon net at the bottom and filled with vermiculite
to support the plant.
The pots containing the tomato seedlings were inserted into holes in the
styrofoam lids of the hydroponics containers, which were filled with reverse
osmosis (RO) water with (in trial 3) and without (in trial 4) nutrients.
Hydroponics nutrients (Manutec Pty Ltd, Australia) was used in trial 3 at the rate
of 120 g (part I) plus 80 g (part II) per 100 litre of water. Nutrient addition was
avoided in trial 4 because it could mask the effect of compost extracts.
A month after transplanting, tomato roots were inoculated with spore
suspensions using a plastic sprayer with a nozzle at the rate of 1 x 106 spores per
plant (Ramsay et al., 1992) of F. oxysporum in trial 3 and of the three pathogenic
182
strains in trial 4. A second inoculation was followed a week later. As these fungal
wilt diseases are prevalent in warm weather, air temperature inside the
greenhouse was increased to 28˚C in shaded conditions in both experiments. The
pH of the solution culture was also adjusted to 4.5 by adding either HCl or
NaOH.
The compost extract treatment involved approximately 10 mL per
container, equivalent to an application rate of 725 L ha-1. The treatments were
first applied four weeks after transplanting, just prior to the first inoculation of the
tested pathogens and then secondly a week later.
A completely randomised design was used in both experiments. In trial 3,
there were three treatments, namely, control (water only), aerated compost
extract, and sterilised aerated compost extract, all with and without pathogen
inoculation to hydroponics tomato plants. Each treatment was applied to three
replicate containers, each container holding eight plants. In trial 4, six treatments
were employed: nine month old aerated compost extract (9M ACE) and nine
month old sterilised aerated compost extract (9M SACE), nine month old non-
aerated compost extract (9M NCE) and nine month old sterilised non-aerated
compost extract (9M SNCE), and three month old aerated compost extract (3M
ACE) and the water control. Each treatment was applied to three replicate
containers, each container holding eight plants. As no difference was noted in
growth of tomato plants between pathogen inoculated and non-inoculated plants
in trial 3, only pathogen inoculated plants were used in trial 4.
The presence of F. oxysporum in root tissue was assessed in trial 3. Root
samples were collected (n = 3 per treatment), and surface sterilised in a 10%
bleach solution for 30 seconds. This was followed by incubation of root samples
183
(1 cm long segments) for 48 hours at 25˚C on Petri plates containing a selective
culture media. The selective culture media ‗malachite green agar‘, suitable for
isolating F. oxysporum (Castella et al., 1997), was prepared by mixing peptone (1
g), KH2PO4 (1 g), MgSO4.7H2O (0.5 g), malachite green oxalate (2.5 mg), agar
bacteriological (20 g) and malachite green agar (2.5 ppm) in one litre of RO
water. This stimulated production of fungal spores from conidia and fungal
invasion from inside of the roots, and the presence or absence of the pathogen in
the tomato root samples was noted.
In both trials, disease severity was assessed a week after the second
inoculation of pathogen in the plants, with a disease severity rating of 0-5
(Ramsay et al., 1992) (0 - no wilt, healthy plant; 1 - slightly stunted, <5% leaves
affected; 2 - slight yellowing and wilting, <25% leaves affected; 3 - moderate
wilting, 25-50% leaves affected; 4 - severe wilting, 50-75% leaves affected; and
5- >75% leaves wilted). Leaf chlorosis (yellowing) is a major symptom of wilt
disease; therefore, chlorophyll concentration of the leaves was also measured in
trial 4 as an index of disease severity using a SPAD-502 meter (Minolta
Corporation, Japan), a meter measuring absorbance at 663 nm (chlorophyll a) and
645 nm (chlorophyll b).
In both experiments, data were analysed using the GLM procedure of
XLSTAT (2010) and statistical significance was evaluated at p< 0.05.
184
3.1. In vitro trials
3.1.1. Mycelial growth of fungi
In trial 1, the effect of two concentrations (50 mL L-1 and 100 mL L-1) of
sterilised and non-sterilised aerated and non-aerated compost extracts of nine
month old compost were compared to a water control, in terms of impact on
mycelial growth of F. oxysporum f. sp. lycopersici. The mycelial growth rate of
F. oxysporum (p< 0.001) was markedly suppressed (32.8%) by the non-sterilised
aerated compost extract treatment at 100, but not 50 mL L-1 nutrient agar after six
days of inoculation as compared to the control (Fig. 7.1). Therefore, the
application rate of 100 mL L-1 was chosen for trial 2.
El-Masry et al. (2002) reported that concentration of 5% (v/v), which is
equivalent to 50 mL L-1, compost extract based on crop compost to PDA-Sigma
medium suppressed the mycelial growth of Sclerotium bataticola by 83%.
Similarly, concentration of 10% (v/v) and 15% (v/v) compost extracts based on
leafy fruit compost or garden compost suppressed the mycelial growth of the
same pathogen by 94%. In the same experiment, they found that the mycelial
growth of F. oxysporum was suppressed by 94% using either concentration or
compost source.
185
Figure 7. 1. Effect of compost extracts in trial 1 on mycelial growth rates (mm d -1) of Fusarium
oxysporum, f. sp. lycopersici six days after inoculation. Cont - control (water), S ACE1 - sterilised
aerated compost extract 50 mL L-1, S ACE2 - sterilised aerated compost extract 100 mL L-1, S
NCE1 - sterilised non-aerated compost extract 50 mL L-1, S NCE2 - sterilised non-aerated
compost extract 100 mL L-1, ACE1 - aerated compost extract 50 mL L-1, ACE2 - aerated compost
extract 100 mL L-1, NCE1 - non-aerated compost extract 50 mL L-1, NCE2 - non-aerated compost
extract 100 mL L-1. Means with different letters indicate significant difference (p< 0.05) across
treatments and vertical bars represent standard errors of the means (n = 5).
In trial 2, all ‗live‘ compost extracts significantly inhibited the radial
growth of the three tested pathogens, compared to the water control. The mycelial
growth (six days after inoculation) was inhibited by approximately 82%, 79% and
88%, respectively for F. oxysporum, F. solani and R. solani on addition of three
month old extract (Fig. 7.2). In accord with these results, Hibar et al. (2006)
reported an inhibition (up to 70%) of mycelial growth of F. oxysporum f. sp.
radicis-lycopersici incubated at 25˚C for six days by addition of compost extract
produced at 1:1 (v/v) compost to water ratio and applied at the rate of 10 mL per
100 mL (which is equivalent to 100 mL L-1) of potato dextrose broth.
No significant inhibition was evident on the mycelial radial extension of
F. oxysporum or R. solani with autoclaved and microfiltered compost extracts,
indicating that the disease suppression effect is linked to the living microbiota.
This is consistent with the findings of McQuilken et al. (1994) and El-Masry et
186
al. (2002) who showed a loss of antagonistic effect in autoclaved compost
extracts against the tested phytopathogenic fungi (Botrytis cinerea, Fusarium
oxysporum, Sclerotium bataticola, Pythium debaryanum). Indeed, mycelial
growth of F. solani was enhanced dramatically with both sterilised extracts (Fig.
7.2).
Figure 7. 2. Effect of compost extracts in trial 2 on mycelial growth rates (mm d -1) of Fusarium
oxysporum, f. sp. lycopersici, F. solani DAR 66102, and Rhizoctonia solani DAR 61830 over (A)
three and (B) six days after inoculation. Cont - control (water), 3M ACE - three month old
aerated compost extract, 9M ACE - nine month old aerated compost extract, 9M NCE - nine
month old non-aerated compost extract, 9M SACE - nine month old sterilised aerated compost
extract, 9M SNCE - nine month old sterilised non-aerated compost extract. Means with different
letters indicate significant difference (p< 0.05) across a given species and vertical bars represent
standard errors of the means (n = 4).
187
3.1.2. Spore count
Consistent with the mycelial growth results, the sporulation of F.
oxysporum was decreased (p< 0.05) when the agar medium was amended with
either aerated or non-aerated compost extracts applied at the rate of 100 mL L-1,
in comparison to the 50 mL L-1 treatment which had much less suppressive effect
(Fig. 7.3). There was no significant difference between sterilised compost
extracts and the control on sporulation by F. oxysporum (Fig. 7.3). Again, this
result is consistent with a role for living microbiota in the suppressive effect, as
opposed to allelochemicals present in the compost extract.
Fusarium oxysporum, f. sp. lycopersici six days after inoculation. Cont - control (water), S ACE1
- sterilised aerated compost extract 50 mL L-1, S ACE2 - sterilised aerated compost extract 100
mL L-1, S NCE1 - sterilised non-aerated compost extract 50 mL L-1, S NCE2 - sterilised non-
aerated compost extract 100 mL L-1, ACE1 - aerated compost extract 50 mL L-1, ACE2 - aerated
compost extract 100 mL L-1, NCE1 - non-aerated compost extract 50 mL L-1, NCE2 - non-aerated
compost extract 100 mL L-1. Means with different letters indicate significant difference (p< 0.05)
across treatments and vertical bars represent standard errors of the means (n = 5).
In trial 2, a significant reduction in sporulation by F. oxysporum and F.
solani was achieved by all non-sterilised compost extracts, with no effect of
compost age (three and nine months) or aeration during the extraction process
188
(Fig. 7.4). This result is consistent with that of Scheuerell (2002), and Scheuerell
and Mahaffee (2000) who noted that both aerated and non-aerated compost
extracts significantly reduced the extent of powdery mildew on rose
(Sphaerotheca pannosa var. rosae), with no difference noted between their
efficacy. No reductions were noted in spore numbers when sterilised compost
extracts (both aerated and non-aerated) were compared to the water control for F.
oxysporum (Fig. 7.4). However, increased numbers of spores of F. solani were
found when treated with nine month old sterilised aerated compost extract (Fig.
7.4). In like manner, sporangia production of the tested fungi (Phytopthora spp.)
was reduced by non-sterilised extracts based on composted Eucalyptus-bark,
while the sterilised extracts stimulated its production (Hardy & Sivasithamparam,
1991).
Fusarium oxysporum, f. sp. lycopersici and F. solani DAR 66102, Cont - control (water), 3M
ACE - three month old aerated compost extract, 9M ACE - nine month old aerated compost
extract, 9M NCE - nine month old non-aerated compost extract, 9M SACE - nine month old
sterilised aerated compost extract, 9M SNCE - nine month old sterilised non-aerated compost
extract. Means with different letters indicate significant difference (p< 0.05) across a given
species and vertical bars represent standard errors of the means (n = 4).
189
3.1.3. Total fungal biomass
In trial 1, no treatments reduced the total dry weight of F. oxysporum
compared to the control (Fig. 7.5). Rather, sterilised aerated compost extract
increased the total fungal biomass as compared to that of the water control (Fig.
7.5), possibly because of the loss of antagonistic effect due to sterilisation of
compost extracts on the tested pathogen, as reported by McQuilken et al. (1994).
The sterilised extract might have supplied nutrients or growth promoting
hormones, which presumably enhanced the growth of the tested fungus.
f. sp. lycopersici six days after inoculation. Cont - control (water), S ACE1 - sterilised aerated
compost extract 50 mL L-1, S ACE2 - sterilised aerated compost extract 100 mL L-1, S NCE1 -
sterilised non-aerated compost extract 50 mL L-1, S NCE2 - sterilised non-aerated compost extract
100 mL L-1, ACE1 - aerated compost extract 50 mL L-1, ACE2 - aerated compost extract 100 mL
L-1, NCE1 - non-aerated compost extract 50 mL L-1, NCE2 - non-aerated compost extract 100 mL
L-1. Means with different letters indicate significant difference (p< 0.05) across treatments and
vertical bars represent standard errors of the means (n = 5).
In trial 2, although three and nine month old compost extracts showed
similar effects in reducing the total biomass of F. oxysporum, F. solani and R.
solani, three month old aerated compost extract was the most effective in
reducing the total dry weight biomass of the former two pathogens (Fig. 7.6). In
190
contrast, Winterscheidt et al. (1990), cited in Weltzien (1991), reported that non-
aerated extract based on six month aged horse manure compost was more
effective than that aged one year in suppressing downy mildew of cucumber.
Another study reported reduced efficacy of non-aerated compost extract based on
18 to 24 month aged cattle manure and straw compost, compared to that based on
12 month old compost, in inhibiting the germination of Venturia inequalis
(Andrew, 1993).
The non-aerated sterilised compost extracts (nine month old compost)
stimulated the growth of F. solani and R. solani, with total biomass higher by
44% and 84%, respectively, compared to the control treatment (Fig. 7.6). This
result is consistent with the observed increase in linear mycelial growth and spore
counts associated with the sterilised extract treatment. Other studies have also
reported the stimulation of fungal growth with the application of autoclaved or
filter sterilised compost extracts (e.g. Hardy & Sivasithamparam, 1991;
McQuilken et al., 1994). However, no difference was noted between the effects
of non-aerated compost extract and sterilised aerated compost extract in terms of
the total biomass of the tested pathogens (Fig. 7.6).
Of interest, the non-aerated compost extract (nine month old) did not
reduce the biomass of F. oxysporum and F. solani compared to the control; rather
the total biomass of R. solani treated with nine month old non-aerated compost
extract showed an increased biomass compared to the control (Fig. 7.6). This
clearly showed that non-aerated compost extracts did not combat the fungal
disease organism R. solani, whereas the aerated extracts did. In contrast, Ma et al.
(2001b) showed a mycolytic effect of non-aerated compost extracts on
chlamydospores and microspores of F. oxysporum f. sp. vasinfectum.
191
Scheuerell and Mahaffee (2002) summarized a number of studies,
although not peer reviewed, conducted in controlled conditions on aerated
compost extracts and found considerable variation in the efficacy of aerated
extracts against various plant diseases.
f. sp. lycopersici (black bar), F. solani DAR 66102 (light grey bar), and Rhizoctonia solani DAR
61830 (dark grey bar), six days after inoculation. Cont - control (water), 3M ACE - three month
old aerated compost extract, 9M ACE - nine month old aerated compost extract, 9M NCE - nine
extract, 9M SNCE - nine month old sterilised non-aerated compost extract. Means with different
letters indicate significant difference (p< 0.05) across a given species and vertical bars represent
standard errors of the means (n = 4).
3.2. Hydroponics trials
In trial 3, compost extracts (both sterilised and non-sterilised) were
effective in reducing the disease severity caused by F. oxysporum of tomato
plants grown in the hydroponics compared to the control treatment (Fig. 7.7).
There was no significant difference (p=0.63) in disease severity between tomato
plants treated with either sterilised or non-sterilised compost extract although
there was a clear difference in the performance of either extract on mycelial
growth, spore count and fungal biomass in vitro. Several studies have
192
demonstrated that sterilisation or micro-filtration does not necessarily affect the
suppressive activity of compost extracts against various phytopathogens (Cronin
et al., 1996; Elad & Steinberg, 1994; Yohalem et al., 1994). Presumably sterilised
compost extract contains an antibiotic that acts to suppress the disease organism,
as discussed by Brinton (1995) and Scheuerell and Mahaffee (2002), or acts to
induce a systemic acquired resistance within the treated plants (Cook et al., 1995;
Litterick et al., 2004; Zhang et al., 1998). The live compost extracts could also
inhibit pathogen growth through parasitism or competition.
Figure 7. 7. (A) A graph showing an effect of application of compost extracts in hydroponics

trial 3 on two month old tomato plants against the severity of fungal wilt caused by F. oxysporum
f. sp. lycopersici grown in the greenhouse (B) pictures of pathogen inoculated control plants (on
the left) and plants treated with compost extracts (on the right). Disease severity based on rating
scale 0-5, 0, no wilt, healthy plant; 1, slightly stunted, <5 % leaves affected; 2, slight yellowing
and wilting, <25 % leaves affected; 3, moderate wilting, 25-50% leaves affected; 4, severe
wilting, 50-75% leaves affected; and 5, >75 % leaves wilted. Compost extracts were applied to
tomato roots until runoff. Cont - control, ACE - Non-sterilised aerated compost extract, S ACE -
Sterilised aerated compost extract (n = 3). Means with different letters indicate significant
difference (p< 0.05) across treatments and vertical bars represent standard errors of the mean (n =
3).
193
The presence of F. oxysporum was confirmed through axenic culture of
root segments. Roots of tomato plants not treated with compost extract were
found to be significantly infected with F. oxysporum, compared to roots treated
with either non-sterilised or sterilised aerated compost extracts (p< 0.001). Roots
of aerated compost extract treated tomato, however, were assessed to have a low
level of infection, possibly indicating cross contamination (Table 7.1).
Table 7. 1. Fusarium infection in roots based on rating scale of 0 (pathogen absent) to 1

(pathogen present) on eight tomato plant roots in each hydroponics container (trial 3). Cont -
control, ACE - Non-sterilised aerated compost extract, S ACE - Sterilised aerated compost
extract. Means with different letters within rows indicate significant difference (p< 0.05) across
pathogen inoculated and non-inoculated treatments (n = 3).
Treatments Cont ACE S ACE
Pathogen inoculated 0.54 ± 0.10a 0.25 ± 0.12b 0.08 ± 0.08b
Pathogen non-inoculated 0.00 ± 0.00a 0.04 ± 0.04a 0.00 ± 0.00a
In trial 4, across all three pathogens, there was an effect of all compost
extracts in maintaining the level of severity below that of the water control. This
was evident by the third week of the trial but only reached significance in the
fifth week, and then only for F. oxysporum and for nine month old aerated
compost extracts (Fig. 7.8). Earlier in vitro studies (trials 1 and 2) had shown
disease suppression with the use of compost extracts when compared to the
control as revealed by mycelial growth, spore counts as well as total biomass of
all fungal pathogens. Significant inhibition of mycelial growth (up to 70%) was
also shown by Hibar et al. (2006) when the efficacy of compost extract was tested
in vitro on F. oxysporum f. sp. radicis-lycopersici.
No difference was demonstrated between aerated and non-aerated extracts
or between sterilised and non-sterilised extracts in terms of reducing disease
194
severity (Fig. 7.8). Evidently, the beneficial effect of compost extract was not
clear because severity response of tomato plants varied with different fungal
pathogens despite the strong effect of extracts against all the tested pathogens in
vitro. Hibar et al. (2006) demonstrated an inhibitory role of compost extract
against fusarium crown and root rot disease of tomato crop.
195
A 4.5 Cont
3M ACE
4.0 9M ACE
9M NCE
9M SACE
3.5 9M SNCE
3.0
Severity (%)
2.5
2.0
1.5
1.0
0.5
0.0
1 2 3 4 5
B 4.5 Cont
3M ACE
4.0 9M ACE
9M NCE
9M SACE
3.5 9M SNCE
3.0
Severity (%)
2.5
2.0
1.5
1.0
0.5
0.0
1 2 3 4 5
C 4.5 Cont
3M ACE
4.0 9M ACE
9M NCE
9M SACE
3.5 9M SNCE
3.0
Severity (%)
2.5
2.0
1.5
1.0
0.5
0.0
1 2 3 4 5
Week after treatment
Figure 7. 8. Effect of application of compost extracts in hydroponics trial 4 on two month old
tomato plants against the severity of fungal wilt diseases caused by (A) F. oxysporum, (B) F.
solani and (C) R. solani in greenhouse assays. Disease severity was based on a rating scale of 0 –
5: 0, no wilt, healthy plant; 1, slightly stunted, <5% leaves affected; 2, slight yellowing and
wilting, <25% leaves affected; 3, moderate wilting, 25 – 50% leaves affected; 4, severe wilting,
50 – 75% leaves affected; and 5, >75% leaves wilted. Compost extracts were applied to tomato
roots until runoff. Cont - control (water), 3M ACE - three month old aerated compost extract, 9M
ACE - nine month old aerated compost extract, 9M NCE - nine month old non-aerated compost
extract, 9M SACE - nine month old sterilised aerated compost extract, 9M SNCE - nine month
old sterilised non-aerated compost extract. Values are mean ± SE (n = 3).
196
In trial 4, as expected, tomato plants responded well to all of the compost
extracts in terms of chlorophyll concentration, starting from three weeks after
treatment, with significantly higher values in comparison to those of the control
treatment which decreased with time (Fig. 7.9). It was not until the third week
after treatment application that tomato plants inoculated with F. oxysporum and
R. solani showed any greater chlorophyll than in the control (Fig. 7.9A and C).
Four weeks after treatment application, chlorophyll concentration was
significantly higher in the leaves of nine month old aerated compost extract
treated plants which was inoculated with R. solani compared to the inoculated
control (Fig. 7.9). Thereafter, all of the F. oxysporum inoculated plants responded
positively to compost extract application when compared to the water-control
plants (Fig. 7.9). This could simply be attributed to the increased uptake of
nutrients needed for chlorophyll development or to the bio-efficiency of compost
extracts as reported by Siddiqui et al. (2008b), who studied the incidence of wet
rot in okra using Trichoderma-fortified compost extracts. Such a bio-efficiency
could also be attributed to the production of antibiotic compounds, such as
siderophores (Brinton et al., 1996; Potera, 1994) or to induced systemic resistance
developed by tomato plants (Cook et al., 1995; Zhang et al., 1998).
197
A 40
Cont
9M ACE
Chlorophyll concentration per plant (SPAD units)

9M NCE
9M SACE
35 9M SNCE
3M ACE
30
25
20
15
10
0
1 2 3 4 5
B 45
Cont
9M ACE
9M NCE
40 9M SACE
9M SNCE
3M ACE
35
30
25
20
15
10
0
1 2 3 4 5
C 45 Cont
9M ACE
9M NCE
40 9M SACE
9M SNCE
3M ACE
35
30
25
20
15
10
0
1 2 3 4 5
Week after treatment
Figure 7. 9. Effect of application of compost extracts in hydroponics trial 4 on the leaf

chlorophyll concentration of two month old tomato plants grown in the greenhouse and inoculated
with three different fungal pathogens, namely, (A) F. oxysporum, (B) F. solani and (C) R. solani,
measured one week after the second inoculation. Cont - control (water), 3M ACE - three month
old aerated compost extract, 9M ACE - nine month old aerated compost extract, 9M NCE - nine
extract, 9M SNCE - nine month old sterilised non-aerated compost extract. Values are mean ± SE
(n = 3).
198
4. Conclusions
In vitro studies show that non-sterilised compost extracts have the ability
to suppress fungal wilt of tomatoes, mostly due to the biotic components of
compost extracts while sterilised extracts in the main enhanced the in vitro
growth of the three wilt pathogens. These results suggest that microbes present in
compost extracts are inhibitory and possibly produce a biochemical that reduces
linear growth of mycelia and inhibits conidia production by the fungal pathogens.
Results of hydroponics trials in vivo showed that inhibition by compost extract
occurs directly or indirectly through nutrient supplement and subsequent
strengthening of the plants on the host-pathogen system. However, the variable
results from the hydroponics trials show that all fungal diseases cannot be
suppressed absolutely by reliance upon compost extract alone. Further
experiments are necessary to identify the mechanisms responsible for the
suppression of diseases observed in the current study.
199
Chapter 8: Compost extract and aluminium toxicity
Chapter 8
The effect of compost extract on root elongation of
maize (Zea mays L.) in the presence of aluminium
Abstract
The aim of this study was to assess the ameliorative effect of compost
extract, used either in solution culture or in seed imbibitions, and either ‗fresh‘ or
‗sterilised‘, on the root growth of maize (Zea mays L. cv. Hycorn 675 IT)
seedlings. Root elongation of maize was inhibited to 43% of the control in the
presence of 200 µM of trivalent aluminium at pH 4.5. Organic acids can chelate
aluminium ions, but the inhibition was not alleviated by either the addition of a
1:500 dilution of microbially enhanced extract of a compost based on rumen
waste or of leachate from vermicasts produced from the same material
(containing 1.6 and 4.4 mg L-1 humic acid, and 2.0 and 1.2 mg L-1 fulvic acid,
respectively). However, when seeds were imbibed in the neat extracts, and then
germinated in the absence of further extract, root elongation was significantly
enhanced in seeds that were imbibed with a fresh compost extract, but not by the
sterilised extract or the vermicast leachate compared to those imbibed in water
(control) and germinated in the same condition. It could be that compost extract
contains an auxin or gibberellic acid-like growth regulator (that is, destroyed by
200
heat in a sterilised extract), and this could have improved root elongation or
increased germination rate of maize.
1. Introduction
Acidification of agricultural lands threatens sustainable production of
crops and pastures, primarily through increased bio-availability of phytotoxic
aluminium in the soil solution (Clune & Copeland, 1999). The problem becomes
acute at soil pH below 5.0 (Foy, 1974; Foy et al., 1978). Aluminium is available
in multiple forms (e.g. dissolved, bound, free, monomeric, polymeric) in the soil,
and pH can determine its speciation (Imray et al., 1995). The freely available
form of trivalent aluminium (Al+++), largely restricted to acidic conditions, is
phytotoxic (Delhaize & Ryan, 1995).
Nosko et al. (1988) reported that aluminium does not inhibit seed
germination but it inhibits growth of roots after the seeds have germinated,
primarily through an effect on the root apex (Bennet & Breen, 1991; Ryan et al.,
1993). Toxicity symptoms are usually visible as ‗stunted‘ root tips and
‗thickened‘ lateral roots, later turning brown in colour (Roy et al., 1988).
Aluminium ions bind to the DNA of root meristematic cells, hindering mitotic
cell division and root elongation (Clarkson, 1969; Matsumoto et al., 1976;
Matsumoto et al., 1977). Thus, root growth is more sensitive to soil aluminium
than is shoot growth (Taylor, 1988). Affected roots are inefficient in absorbing
nutrients and water from the soil (Foy, 1992), and hence they will become
sensitive to drought and nutrient deficiency. The failure in efficient uptake of
nutrients, particularly of Ca2+ and P, is the secondary physiological effect
associated with aluminium toxicity (Henning, 1975).
201
In general, the acid-aluminium stress also negatively impacts the growth
and multiplication of microorganisms, for example, Rhizobium (Keyser &
Munns, 1978). Nearly dividing cells show sensitivity to toxic aluminium, while
non-dividing cells can survive an acid-aluminium stress (Munns & Keyser,
1981). Aluminium tolerance has been reported to vary widely between microbial
strains, but remains stable within a given strain (Munns, 1977).
Plant roots are reported to release organic acids in the soil in response to a
range of environmental stresses (Ma et al., 2001). Amelioration of aluminium
toxicity in both soil and solution culture has been achieved through chelation of
the aluminium ions by organic acids, for example, by fulvic acid (65 mg L-1 of
organic C per 3.5 L of nutrient solution), as evidenced by tap root elongation of
seedlings of soybean, cowpea, and green gram grown in solution culture treated
with 50 µM aluminium at pH 4.5 (Suthipradit et al., 1990). Tan and Binger
(1986) conducted a greenhouse experiment to test the effect of humic acid on
corn seedlings grown in sand culture in the presence of 0, 390 and 820 µM
aluminium. In the absence of humic acid, total dry weight of corn seedlings was
reduced by 41% at 820 µM aluminium compared to the control treatment.
However, addition of humic acid at 100 mg kg-1 and 350 mg kg-1 sand increased
the total dry weight of treated corn plants (820 µM aluminium) by 32.5% and
42.5%, respectively, compared to the treatment that had no humic acid.
Harper et al. (1995) tested the effects of 40, 120, 360 mg C L-1 humic and
fulvic acids, extracted from the leaves of Eucalyptus camaldulensis, on root
elongation of Zea mays in solution culture at pH 4.5 with 30 µM aluminium. The
negative effect of aluminium on root elongation was nullified by the addition of
any concentration of humic and fulvic acids. The studies of Harper et al. (1995)
202
involved solution culture and employed ‗pure‘ humates. Hue and Amien (1989)
reported a soil (pot culture; soil-water pH of 4.0) experiment which demonstrated
the efficacy of different green manures (ground leaves of cowpea, leucaena and
guinea grass) in alleviating aluminium toxicity. They found that aluminium was
detoxified more efficiently by cowpea and leucaena than by guinea grass as
indexed by the biomass of a test crop (Sesbania cochinchinensis) and soil
chemical composition. They concluded that an increment in soil pH with the
addition of manure and chelating action of organic molecules could be the
possible mechanism for aluminium detoxification.
Root elongation is also reported to be stimulated by humic and fulvic
acids at concentrations ranging from 25 to 250 mg C L-1 in the absence of
aluminium (Linehan, 1976; Rauthan & Schnitzer, 1981; Schnitzer & Poapst,
1967; Vaughan & Linehan, 1976). However, Harper et al. (1995) found that
while low concentration (40 mg C L-1) of humic acid stimulated root elongation,
a high concentration of humic acid (360 mg C L-1) reduced root length, both in
the presence or absence of aluminium.
Vaughan and Linehan (1976) suggested that humic and fulvic acids can
have a ‗biochemical or hormonal function‘, while Maggioni et al. (1987)
suggested that these acids can directly affect ATPase activity and influence
nutrient (Mg2+, K+) uptake by plants. Low concentrations of humic and fulvic
acids (0 to 10 mg C L-1) were associated with enhanced in vivo levels of
indoleacetic acid oxidase activity (Mato et al., 1972a; b). However, activity of
this enzyme decreased with higher concentrations of soil humic acid fractions (10
to 40 mg C L-1) (Mato et al., 1972a). They established a direct relationship
between the extent of enzyme activity inhibition and the number of phenolic
203
groups, ratio of C to H, and the C and N concentrations of humic acids. Muscolo
et al. (1999) reported a stimulation of nitrogen metabolism and cell growth in
Daucus carota by humates. Others (Aibuzio et al., 1986; Piccolo et al., 1992)
found that nutrient uptake and plant growth regulation are triggered by humic
substances.
Compost extracts (‗compost teas‘) are in relatively common use in a range
of production systems (from biodynamic through organic to broadacre grain
cropping, see Chapter 1), primarily for their perceived role in enhancing soil
microbial activity (i.e. ‗soil health‘). However, such extracts appear to be a rich
source of organic acids, such as humates and fulvates in rumen compost extract
(Shrestha et al., 2011a), as measured and judged from their visual deep dark
coloured appearance, and thus may play a role in alleviation of aluminium
toxicity in acid soils. Leachates from earthworm processed composts appear even
darker in colour than extracts of the raw compost. Therefore, experiments were
undertaken to evaluate the effectiveness of compost and vermicast extracts in
alleviating aluminium toxicity, in the context of the presence of humic and fulvic
acids in the extracts. The measured levels of humates (0.7 g L-1) and fulvates (1.1
g L-1) (Chapter 3, also reported as Shrestha et al., 2011a) are one to three orders
of magnitude lower than those reported in studies employing addition of ‗pure‘
humates.
Compost extracts (e.g. vermicast leachates) contain a high microbial load,
as well as organic acids (Shrestha et al., 2011a). It has been suggested that
earthworms promote microbial activity in compost producing significant amounts
of plant growth hormones such as indole-acetic acids (auxins), gibberellins and
cytokinins (Edwards, 1998). Casenave de Sanfilippo et al. (1990) also found
204
contents of such plant growth hormones in humic acids produced from
earthworm activities. It is possible that the active microbiota of compost extracts
may directly take up aluminium ions, thus reducing the toxic aluminium
concentrations in the root zone, or compost extract may consist of plant growth
regulators that enhance root growth.
The current study assesses the ameliorative effects of compost extract,
used either in solution culture or in seed imbibitions, and either ‗fresh‘ or
‗sterilised‘, on the growth of maize roots.
2.1. Rumen compost extract and vermicast leachate
A microbially enhanced extract of rumen content (cattle paunch) compost
was produced as described in Chapter 3 (briefly, a 1:10 ratio of compost to water
was amended with 1% ‗fish and kelp hydrolysate‘ and 0.5% molasses and
incubated with aeration for 24 h to produce rumen compost extract, ‗RCE‘). The
vermicast leachate, ‗VL‘ used in this experiment was obtained from feeding the
above mentioned compost to a mixture of three species of earthworms (as
described in Chapter 3). The umen compost extract and vermicast leachate were
used ‗fresh‘ or ‗sterilised‘ (121˚C for 15 min at 0.1034 MPa). The extracts were
filtered through 0.45 µm-pore membrane filters (Millipore, Australia) to ensure
elimination of live microbes in the extracts.
2.2. Chemical and microbial analyses of compost extracts
Inorganic nutrients, humic and fulvic acid concentrations of both sterilised
and non-sterilised rumen compost extract and vermicast leachate collected after
205
24 h of incubation were analysed following the methods as described in Chapter
2.
Culturable populations of bacteria and fungi of serially diluted samples
were determined by the pour plate count method (Harrower, 2006) as described
in Chapter 2.
2.3. Plant material and experimental set up
Root weight is reported to be less sensitive to aluminium damage than
root length (Jackson, 1967). Thus, only tap root elongation was considered in the
present study. The bioassay procedures used are based on those of Clune and
Copeland (1999) and Harper et al. (1995), with modifications noted below.
Seeds of maize (Zea mays L. cv. Hycorn 675 IT; courtesy of John Eggins,
Pacific Seeds, Australia) were washed in running tap water for 15 minutes and
rinsed with distilled water to remove fungicide. They were then soaked in a
solution of 200 μM CaSO4 and 50 μM H3BO3 for 30 minutes.
The experimental set up consisted of a 1 L plastic tray containing 500 mL
of reverse osmosis (RO) water, upon which a styrofoam block (13 cm long x 9
cm wide x 1.5 cm thick) was floated. The styrofoam was covered with autoclaved
paper towels (20.2 x 28.5 cm2) (Kimsoft Kleenex brand, Australia), and five
seeds of maize were placed onto the raft. Solution pH was adjusted to and
maintained at 4.5 by addition of 0.1 M HCl or NaOH. Seeds were allowed to
germinate and grow on the raft for 96 h, with trays held under ambient laboratory
conditions (approximately 22˚C).
In a preliminary trial, treatments consisted of amendment of the tray water
to achieve 0, 25, 50, 100, 200, 400, 600, 800 and 1000 μM Al (pH 4.5), through
206
addition of a stock solution of 0.5 M AlCl3. After imposition of the treatments,
seedlings were allowed to grow for a further 96 h and tap root length was then re-
measured, with data expressed as the average root length extension per seed, per
tray. Four replicate trays were maintained for each treatment. This preliminary
experiment was undertaken to establish the concentration of aluminium that
suppressed root extension under the conditions of the bioassay. A single
concentration was then imposed as a treatment relative to a zero aluminium
control in two further experiments.
In the main experiment, seedling root length was measured, and then the
aluminium treatments imposed. Then the seedlings were allowed to grow for a
further 96 h, during which the seedling root extension was recorded.
In the first experiment, the compost extracts (rumen compost extract and
vermicast leachate, both non-sterilised and sterilised) were added to the solution
culture at 1 mL per 500 mL of aluminium contaminated solution (200 µM Al) or
pure water. In the second experiment, maize seeds were imbibed overnight (12 h)
in the undiluted compost extracts (non-sterilised or sterilised) or water (control),
before placing them on styrofoam blocks contained in the plastic trays. Seedlings
were grown on the rafts floating on water (pH 4.5) for 96 h but without further
addition of compost extract.
Experimental set up was the same for all experiments, including the
preliminary trial, however the treatments differed.
2.4. Statistical analysis
Significant differences among treatments were determined using simple
analysis of variance using XLSTAT (2010) at a confidence level of 95%.
207
3.1. Effect of Al on root growth of maize seedlings - Preliminary trial
Maize seedlings exposed to > 25 μM aluminium showed a reduction in
root extension, with the effect leveling off at concentrations above 200 μM (at
55% of control; Fig. 8.1). Clune and Copeland (1999) reported a reduction in
relative root extension of canola seedlings when exposed to aluminium (from 40
to 100 μM) at a pH of 4.5 in nutrient solutions as compared to non-treated
control, with a decrease to 55% of control noted at 80 μM Al.
Figure 8. 1. Relative root extension (% of control, where control plants exhibited a root extension
of 14 cm) of maize (Zea mays L. cv. Hycorn 675 IT) grown in a solution culture (pH 4.5) at a
range of aluminium concentrations. Vertical bars represent standard errors of the means (n = 4).
The 200 μM aluminium was chosen as the base treatment in assessing
the effects of compost extract treatments on maize seedling root growth.
208
3.2. Composition of compost extract
The humic acid concentration of vermicast leachate was twice as that of
rumen compost extract (at approximately 2 mg L-1). In contrast, fulvic acid
concentration of vermicast leachate was lower than that of rumen compost extract
(Table 8.1). Presumably earthworm activity selectively enhances humic acid
content. Bacterial and fungal loads were higher in the rumen compost extract, as
expected for a microbially enhanced product (i.e. food sources and aeration
provided), relative to the vermicast leachate (Table 8.1). Thus, microbial load
also varied between these treatments. Inorganic ion concentrations (NH4+-N,
PO4−-P, and K+-K) were higher in rumen compost extract than in vermicast
leachate (Table 8.1), and hence it is possible that the osmolality of the solutions
could impact on germination rates. Following seed germination, however, root
elongation should be relatively independent of rhizosphere nutrient availability, at
least for the first few days of growth, as the seeds rely upon available seed
reserves.
Table 8. 1. Comparison of physico-chemical and microbial characteristics of sterilised and non-

sterilised rumen compost extract and vermicast leachate. RCE - rumen compost extract, S RCE -
sterilised rumen compost extract, VL - vermicast leachate, S VL - sterilised vermicast leachate.
Within columns, mean ± SE (n = 3) with the same letter are not significantly different according
to Tukey‘s test (p< 0.001).
Parameters RCE S RCE VL S VL
+ -1
NH4 -N (mg L ) 1.03 ± 0.04a 0.98 ± 0.05a 1.45 ± 0.04b 1.42 ± 0.03b
- -1
PO43 -P (mg L ) 21.3 ± 0.78a 20.0 ± 1.52a 27.0 ± 1.21b 27.6 ± 0.78b
+ -1
K -K (g L ) 0.67 ± 0.02b 0.66 ± 0.02b 0.44 ± 0.03a 0.45 ± 0.02a
-1
Humic acid (g L ) 0.77 ± 0.06a 0.79 ± 0.08a 2.15 ± 0.29b 1.95 ± 0.02b
-1
Fulvic acid (g L ) 0.99 ± 0.02b 0.92 ± 0.08b 0.63 ± 0.03a 0.64 ± 0.04a
Bacterial population (cfu Log 10 mL-1) 11.5 ± 0.03c 0.00 ± 0.00a 6.72 ± 0.07b 0.00 ± 0.00a
Fungal population (cfu Log 10 mL-1) 5.29 ± 0.06c 0.00 ± 0.00a 4.20 ± 0.10b 0.00 ± 0.00a
209
3.3. Experiment 1
The presence of vermicast leachate in the hydroponic solution (1:500
dilution; both ‗fresh‘ and ‗sterilised‘) significantly (p< 0.05) decreased root
extension, compared to the water control (Fig. 8.2). This is particularly
pronounced in the presence of Al. Compost extract did not significantly improve
or retard root extension (Fig. 8.2). Vermicast leachate, which contains substances
that decrease root growth, affected root extension even at a dilution of 1:500.
This response is consistent with that noted by Churilova (2010) in pak choi plants
treated with the same vermicast leachate (1:1, 1:2 dilutions) compared to that of
the control plants.
seedlings (n = 4) following growth for 96 h on a solution culture with or without 200 µM of Al at
pH 4.5, with treatments of non-sterilised and sterilised rumen compost extracts and vermicast
leachate (1:500 dilution). The control consisted of water only. Cont - control, RCE - rumen
compost extract, S RCE - sterilised rumen compost extract, VL - vermicast leachate, S VL -
sterilised vermicast leachate. Bars with the different letters indicate significant difference (p<
0.05). Error bar represents one standard error of the mean (n =4).
Neither vermicast leachate nor compost extract treatments alleviated the
inhibition of root extension in the presence of 200 μM aluminium, relative to the
210
control (Fig. 8.2). At a dilution of 1:500, the compost extract treatment would
have contained 1.6 and 2.0 mg L-1, that is, 0.6 and 0.8 mg C L-1 (assuming these
compounds have 40% C), or 0.8 and 1.0 mg tray-1 humic and fulvic acids,
respectively. These levels are low compared to those used in published literature
(40-360 mg C L-1) on root elongation of maize (Harper et al., 1995). Indeed, the
500 mL of 200 μM aluminium solution in each tray contained 100 µmoles of
aluminium. Assuming a chelation capacity or cation exchange capacity of 300
cmol+ kg-1, for example, as for soil organic matter or humus (DPIPWE, 2010),
the humates and fulvates present in each tray could potentially complex 1.8 mg
tray-1 x 0.3 x 0.001 mg g-1 = 5.4 µmoles. Thus, on a stoichiometric basis, it
appears that the applied level of humates was inadequate to complex the available
Al. To achieve stoichiometry, the compost extract should ideally for this
proposed effect have been used at a dilution of at least 1:25 (instead of 1:500).
The level of compost extract addition employed in the current study was
chosen as a level that had practical relevance (in soil, roots will not be exposed to
‗neat‘ compost extracts). These results show that the above extracts do not
ameliorate soil aluminium toxicity. However, humates may accumulate in the soil
from successive additions of compost extracts and this accumulation may lead to
reduced aluminium toxicity. Tejada et al. (2010) compared the effects of various
organic amendments (e.g. municipal solid waste compost, poultry manure and
cow manure) on aluminium contaminated soil using an earthworm incubation
experiment. The authors found that the amendments with higher level of humic
acids were more effective in reducing aluminium toxicity in soil (as indicated by
the earthworm response) compared to those with lower levels. Any future work in
this area should, therefore, attempt to achieve at least an order of magnitude high
211
level of humic and fulvic acids (e.g. through use of a different compost or
through concentration of the extract), and should consider the issue of soil
accumulation of humates.
3.4. Experiment 2
Pre-imbibed seed in compost extract showed an increased tap root
elongation (Fig. 8.3) compared to that in Experiment 1 (Fig. 8.2). Seed priming is
well known to improve seedling growth (Clark et al., 2001; Rashid et al., 2004).
Presumably the 12 h imbibition treatment of Experiment 2 allowed for quicker
imbibition than that the wet towel-raft conditions of Experiment 1.
Root elongation of maize seeds imbibed in non-sterilised rumen compost
extract (non-diluted) and grown in 200 µM aluminium solution was increased by
51%, compared to the control, whereas that in sterilised compost extract or
vermicast leachate was not significantly different to that of the control treatment
(Fig. 8.3). The increase in root extension with the compost extract treatment
cannot be explained by a ‗fertilisation‘ effect, given the lack of response to the
vermicast leachate treatments.
212
seedlings (n = 4) measured 96 h after imbibition in solution culture in the presence or absence of
200 µL Al (solution pH 4.5). Seeds were imbibed for 12 h in non-sterilised and sterilised rumen
compost extracts, vermicast leachate and the water control. Cont - control, RCE - rumen compost
extract, S RCE - sterilised rumen compost extract, VL - vermicast leachate, S VL - sterilised
vermicast leachate. Bars with the different letters indicate significant difference (p< 0.05), and
error bar represents one standard error of the mean (n = 4).
The lack of response of the sterilised compost extract may be ascribed to
either the active role that microorganisms might have in the uptake of aluminium
from the solution, to the loss of chelating capacity of the extract due to
sterilisation, or to the loss of a growth regulator (e.g. auxin) activity.
During imbibition, solution is osmotically drawn into the seed. The
physical flow would result in the movement of solutes contained in the solution
(including humic and fulvic acids) into the seed. However, these chemicals are
unlikely to move within the plant. After 96 h of germination, roots were
approximately 6-8 cm long (i.e. in the presence of Al). The Al sensitive root tips
presumably at this stage, and somewhat earlier, did not contain humic or fulvic
acid from the initial imbibition treatment. Rather any difference between
treatments must be ascribed to a physiological effect of these chemicals on the
germinating seedling, for example, through an increase in gibberellins or auxin
213
levels, leading to an earlier germination or an increase in the root extension rate
(Mato et al., 1972a; b).
4. Conclusion
Although organic acids like humic and fulvic acids within compost
extract were expected to chelate aluminium ions, the inhibition of root elongation
by trivalent aluminium ions was not improved by either microbially enhanced
extracts of compost based on rumen waste or of leachate from vermicasts
produced from the same material. In contrast, maize seeds imbibed with fresh
compost extract demonstrated significantly enhanced root elongation in the
presence of aluminium ions, whereas seeds imbibed with sterilised extracts or the
vermicast leachate did not. The influence of compost extract, possibly via growth
regulators present in the compost extracts, on rate of germination should
be considered.
214
Chapter 9: Compost extract and soil microbial activity
Chapter 9
Enhancing microbial activity of a field soil with
long-term use of liquid biofertilisers
Abstract
The influence of several agricultural management practices, including
liquid biological inocula, green manures, liquid fertiliser and conventional
chemical fertiliser, on soil physico-chemical and microbial properties of a
cultivated soil at Baralaba in central Queensland, Australia, was investigated with
comparison to soil properties of an adjacent site with the original native
vegetation (brigalow, Acacia harpophylla). The effect of these management
practices was minor compared to that of seasonal changes, and compared to the
difference between the cultivated soil and the brigalow soil. For example, nitrate
levels were 87% higher in brigalow soil than in conventionally cultivated soils
during the dry season. Average soil nitrate level in cultivated soils (average of all
treatments) was 16% higher in the wet compared to the dry season. Microbial
activity was higher in the brigalow soil than soil of any cultivation treatment,
though activity was enhanced by all treatments, including the application of
compost extract, compared to the control. Substrate utilisation patterns
(BiologTM) of bacterial and fungal community functional diversities did not differ
215
between treated field soils and the brigalow soil, however bacterial community
diversity was higher in biologically treated soils compared to that of the
conventionally cultivated soils in the dry-winter season.
1. Introduction
Traditionally, ecosystem health has been assessed in terms of the ‗macro‘
components of the ecosystem, such as land form, vegetation and macrofauna; but
attention is now being given to the diversity of soil microbiota. Generally, soil
quality is characterised by its abiotic factors (e.g. cation exchange capacity/water
holding capacity), however increasing recognition is being given to biotic factors
– the ‗health‘ of the soil (Doran & Safley, 1997; Pankhurst et al., 1997).
Microorganisms are considered to be useful indicators of soil quality due to their
fundamental roles in organic matter decomposition, nutrient recycling and
maintenance of soil structure (Anderson, 2003; Lalande et al., 2000; Robertson et
al., 1994; Sparling, 1997).
Accelerated and intensified use of agricultural soils has negatively
impacted ‗soil health‘ (McCaig et al., 2001; Nusslein & Tiedje, 1999; Ovreas et
al., 1998). Indeed up to 40% of world‘s agricultural land has been estimated to be
seriously degraded (Sample, 2007), with degradation in Australia also reported to
be substantial (Mabbutt, 1992; Woods, 1983). Intensive farming practices, for
example, tillage and biomass removal from fields, result in increased
mineralisation of soil organic matter and thus its depletion (Dalal & Mayer, 1986;
Golchin et al., 1995; Lemenih et al., 2005; Mann, 1986; Oldeman et al., 1990;
Spaccini et al., 2001). Buckley and Schmidt (2001) found lower proportions of
microbial communities (e.g. Proteobacteria, Actinobacteria, as shown by the T-
216
RLFP patterns), in soils subjected to long-term cultivation practices compared to
undisturbed soils. Similarly, Steenwerth et al. (2003) observed variation in soil
microbial community composition associated with cultivation practices, such as
fertiliser application, tillage frequency, grazing, irrigation, and herbicide
application.
Conversely, addition of biological materials (e.g. farm yard manure) has
shown to improve bacterial community composition in a long-term cropping-field
experiment (Widmer et al., 2006). Change in microbial community structure has
also been noted by Peacock et al. (2001), who found that application of dairy
manure increased soil organic C, increased microbial biomass, and enhanced the
community of typical Gram-negative bacteria compared to the control and
ammonium nitrate fertilised treatments. It is generally accepted that increasing
soil organic matter will result in improved soil structure (Pulleman et al., 2003),
and enriched soil biodiversity (Tu et al., 2006).
To restore degraded soils, techniques, such as mulching, crop rotation,
green manuring, use of compost, and more recently, amendment of soils with
liquid biological fertilisers are commonly used (Garcia-Gil et al., 2000;
Hernandez et al., 2007; Omay et al., 1998). The resulting change in soil
microbiota is also considered to convey resistance to soilborne plant pathogens
(Cotxarrera et al., 2002; Zwieten et al., 2007). Microbially enhanced compost
extracts (‗compost tea‘) are one type of liquid biological fertiliser.
The impact of a ‗compost extract‘ is likely to fall into one of two
categories: (i) where the extract is applied in high volume, or in a localised
manner (e.g. with seed), there will be a capacity to increase the overall soil
microbial population in that soil volume; (ii) where the ‗extract‘ is applied at a
217
very low rate, but it contains microbiota that play key roles in various nutrient
cycles, or in protection of plants from pathogens, but are lacking from the soil.
Addition of rhizobia to soil is an example in which the overall soil microbial load
may be unaltered, and overall diversity number not measurably increased, and yet
the performance of a matched legume crop measurably enhanced.
The effect of organic and microbial amendment of soil is expected to be
greater in moist soil. Central Queensland broadacre cropping on black cracking
clays is largely reliant on rainfall, and the soil profile can be effectively emptied
of water by the end of a crop, and through the dry season. In such a system, soil
microbial populations will vary dramatically between seasons, and it is not clear
if a microbial soil amendment alone can have a sustained impact on soil
microbial population.
In 2007, a project (Central Queensland Regenerative Farming Initiative)
was established to conduct a farm based trial (coordinated by Scott Stevens,
funded by the Fitzroy Basin Association ‗FBA‘) of seven management options at
Baralaba on the central Queensland highlands, on a site typical of this cropping
region (cracking clay soils, originally vegetated by brigalow, Acacia
harpophylla). This trial was commenced in 2007, with grain sorghum planted on
3rd September, 2007, followed by wheat, sown on 17th June, 2008, then
mungbean, sown on 19th January, 2009. The trial site management followed the
normal local practice of opportunity cropping under rainfed conditions and zero
tillage, where practical, with cereal (wheat, sorghum) alternated with pulse crops
(mungbean, chickpeas) or green manures (mixture of forages, legumes and
cereals). The current study was designed to ‗value add‘ to the ongoing field study
218
in central Queensland, crop yield assessment work being undertaken, in terms of
a focus on soil microbiological properties.
2.1. Study site and treatments
Field samples were obtained from a farm trial site located at Baralaba,
central Queensland (latitude: 24.24˚ S and longitude: 149.86˚ W). This site has
been continuously cropped for thirty years, following clearing of the original
brigalow (Acacia harpophylla) community. The aim of the trial was to compare
biological farming systems with the current conventional cropping system in
terms of their effect on soil ‗health‘. Seven soil amendment systems were
implemented : (i) ‗best-bet biology‘ (BBB), (ii) ‗biology boom spray‘ (BBS), (iii)
‗biology direct injection‘ (BDI), (iv) feather-top Rhodes (FTR), (v) green manure
(GM), (vi) ‗best-bet conventional‘ (BBC) and the (vii) control (Cont), with
details of each treatment shown in Table 9.1.
These treatment applications were made in 2009 to the mungbean crop
(Table 9.1). The control treatment did not receive any fertiliser. In the best-bet
conventional treatment, recommended rates of fertilisers (Starter Z, Incitec Pivot
Ltd, Vic., Australia) were applied at planting. ‗Starter Z‘ is a zinc-fortified
granular fertiliser consisting of a balance of nitrogen, phosphorus and sulphate
sulphur. In the best-bet biology treatment, compost extract along with additional
nutrients and microbial food was applied via a liquid injection method during
planting, at the rate of 30 L ha-1, while foliar applications of a microbial inoculant
(Twin N diazotrophs, AgriBiotec Pty Ltd, Qld, Mapleton, Australia, which are
marketed as free-living rhizosphere N2 fixers) were made just before onset of
219
flowering at the rates mentioned in Table 9.1. The same soil injection
amendments (compost extract along with additional nutrients and microbial food)
were made for the biology direct injection treatment, but the foliar application
was omitted. In the biology boom spray treatment, the same liquid amendments
as in the best-bet biology and biology direct injection treatments were applied
during planting but the compost extract was omitted, and a foliar (boom spray)
application of microbial inoculant (Twin N) was made prior to the onset of
flowering. The green manure and feather-top Rhodes treated plots were planted
with panorama millet, cowpea, lab lab and forage sorghum as a shotgun mix at
the rate of 4 kg ha-1 each on the same day of other treatment applications. This
was then turned back in, using a disc plough, 7 weeks after planting. The green
manure treatment was specifically set up to look at the effects of introducing
green manures into a biological program. In the feather-top Rhodes treatment,
strategies were adopted to control feather-top Rhodes grass. Both treatments used
similar products (biology and liquid nutrients) to best-bet biology, biology direct
injection and biology boom spray. There was no difference in management
between both these treatments between the sampling dates used to generate the
results within this study. However, prior to soil sampling, these two treatments
differed in that the green manure treatment received Starter Z at planting and
Twin N as a foliar spray while the feather-top Rhodes treatment received calcium
nitrate as a foliar spray for the preceding wheat crop. Rhizobial inoculant
‗Nodulaid‘, from Becker and Underwood (www.beckerunderwood.com), was
applied at the same rate to all treatments. All treatments received equal rates of
herbicide (January, 2009) and insecticide (March, 2009) during the growth of
mungbean crop because the weeds and insect pressure was considered the same
220
across the treatments. The herbicides applied were Haloxyfop 520 (HerbiGuide
Pty Ltd, WA) at 520 g L-1 ha-1, 2, 4 - D MCPA sprays (KensoAgcare, Qld) and 2,
4 - D Amine 625 (Dow AgroSciences Australia Ltd, NSW) at 625 g L-1 ha-1each,
while insecticide, Dimethoate 400 (Conquest Agrochemicals Pty Ltd, WA) was
applied at 0.4 g L-1 ha-1.
Table 9. 1. Details of treatments and fertility programs of a long-term field trial (commenced in
2007) conducted in Baralaba, Qld. Table showing treatment applications made in 2009 to the
mungbean crop.
Treatments Fertility program Application Rate

Control (Cont) Sodium molybdenate (kg ha-1) 0.01
Peat inoculant (Nodulaid) (kg ha-1) 0.10
Best-bet biology (BBB) Compost extract (L ha-1) 30.0
CalSap (L ha-1) 3.00
Liquid N (L ha-1) 2.00
Sodium molybdenate (kg ha-1) 0.05
Fish hydrolysate (L ha-1) 2.00
Humic acid (L ha-1) 0.50
Sea minerals (L ha-1) 1.00
Inoculant (Twin N) (kg ha-1) 0.10
Biology direct injection (BDI) Compost extract (L ha-1) 30.0
Fish hydrolysate (L ha-1) 2.00
Humic acid (L ha-1) 0.50
Best-bet conventional (BBC) Starter Z (kg ha-1) 10.0
Biology boom spray (BBS) CalSap (L ha-1) 3.00
Inoculant (Twin N) (kg ha-1) 0.10
Green manure (GM) Panorama millet seed (kg ha-1) 4
Cowpea seed (kg ha-1) 4
Lab lab seed (kg ha-1) 4
Forage sorghum seed (kg ha-1) 4
Feather-top Rhodes (FTR) Panorama millet seed (kg ha-1) 4
Cowpea seed (kg ha-1) 4
Lab lab seed (kg ha-1) 4
Forage sorghum seed (kg ha-1) 4
221
The compost extract used in the best-bet biology and biology direct
injection treatments was based on a mixture (1:1:1 ratio) of three different
composts, two from CQ compost Pty Ltd in Emerald (aerobic compost) and the
third (vermicompost) from Nutrigold, Mareeba. The thoroughly mixed compost
(4 kg) was amended with 15 mL each (diluted in 200 mL water) of Aloe-TechTM,
Sea-Change KFFTM liquid fertiliser (Nutri-Tech Solutions products, Yandida,
Qld) and Fish Hydrolysate (South Australian Marine Product Industries, SAMPI,
Fremantle, WA) five days before the extract incubation. The following recipe
was then used for preparing compost extract: 4 kg of compost, 1 L of Sampi Fish,
0.5 L of Aloe-TechTM, and 250 g of oat bran were added to 700 L of rainwater at
the onset of incubation. Compost extract was aerated for 24 h and used within 2
to 4 h following incubation.
Treatments were applied to three replicate plots each, except for the
treatments of green manure and feather-top Rhodes, for which only two replicate
plots were established. Each plot was 600 m by 11.4 m in size. The treatments
were imposed in a randomised complete block design (seven treatments, three
replicate plots).
2.2. Soil sampling
Soil sampling occurred on 19th January, 2009 (wet-summer season) and
on 27th July, 2009 (dry-winter season) (for rainfall data, see Australian Bureau of
Meteorology www.bom.gov.au/climate/glossary/seasons.shtml). Soil was
sampled from each replicate plot. Soil samples (two replications) were also
procured from a nearby intact virgin brigalow community. A 35 cm long
cylindrical stainless-steel corer was used to collect to a depth of 10 cm from the
222
soil surface. Three soil cores were collected from random locations within each
plot. After transport to the laboratory, soil cores from individual plots were well
composited and representative soil samples were taken following the cone
sampling method. The soil samples collected in dry-winter were incubated for
five days after addition of 10% (w/v) water, sieved (2 mm) and stored at 4˚C for
further analysis. No artificial incubation was carried out for samples collected in
the wet-summer.
2.3. Soil characterisation
Soil pH and EC of the samples were measured of 1:5 (w/v) suspensions of
soil in distilled water. Soil moisture and inorganic ion levels were determined
(n=3) as described in Chapters 2 and 3.
Microbial load of triplicate soil samples was assessed using a culture-
based method from serially diluted soil samples according to the method of
Harrower (2006). Microbial activity was determined for each soil sample (i.e.
three analyses per plot) using the fluorescein diacetate hydrolysis method, as
described by Adam and Duncan (2001), and by the basal and substrate induced
respiration (SIR) methods described by Anderson (1982). All of these methods
are described in greater detail in Chapters 2 and 3.
Microbial biomass C was estimated on all soil samples using the
chloroform fumigation extraction method (Vance et al., 1987) (10 g dry weight
each of triplicate soil samples as described in Chapter 3). Total and dissolved
organic C (TOC and DOC) were determined spectrophotometrically at 590 nm
following the dichromate digestion method (Walkley & Black, 1934) and
223
microbial biomass C was calculated following the protocol of Sparling and West
(1988) as described in detail in Chapter 3.
Bacterial and fungal substrate utilisation was assessed of samples
collected in the dry season, using the Ecoplate and FF microplate systems
(BiologTM Inc, USA), respectively. A 100 μL aliquot of a 1 to 10 soil: water
extract was used for each well of the Ecoplate system, while a 150 μL aliquot was
used for the FF microplates. The average well colour development (AWCD) was
calculated at 24 h intervals from 0 h to 168 h for all different C substrates in both
Ecoplate and FF microplate (Konopka et al., 1998), with the 72 h data used for
statistical analysis (Garland, 1996), as described in Chapter 1.
The change in density and diversity of the soil microbial community was
investigated using DGGE, based on bacterial 16S rDNA and fungal ITS regions
amplified from total soil DNA. ‗Total‘ DNA was extracted from 5 g fresh weight
of composite soil samples (i.e. one sample per plot) of control and best-bet
biology treatments in wet-summer and dry-winter seasons (all treatments) using
a soil commercial DNA isolation kit (PowerMaxTM Soil) following the
manufacturer‘s instruction (Mo Bio Laboratories, Inc., Carlsbad, CA), and using
a bead beating method. The extracted DNA from each sample was PCR-
amplified and DGGE analysis was performed for bacterial and fungal
communities (Girvan et al., 2003).
Differences between means were compared for different physical,
chemical and microbial parameters as well as indices of species richness,
evenness and diversity at p< 0.05 using analysis of variance (ANOVA) of the
224
statistical software package (XLSTAT, 2010). The Shannon-Weaver diversity
index H‘ (Shannon & Weaver, 1963) and the equitability index J (Begon et al.,
1990) were also calculated (Dilly et al., 2004; Krebs, 1999) and subjected to
ANOVA.
3. Results
3.1. Physico-chemical, microbial and biochemical analyses
Variations in physico-chemical, biochemical and biological characteristics
were noted among the treatments, with dramatic differences observed between
the two seasons (Table 9.2). Soil pH was not affected by season, but increased by
treatments. EC was only weakly increased by treatment, particularly by green
manure. Moisture content was not different, even between seasons, because
moisture content of soil collected in dry-winter season was measured after adding
water. Nitrate levels were higher in brigalow than the control during the dry
season, but average nitrate level in cultivated soils was 16% higher in the wet
compared to the dry season. Biology direct injection and green manure treatments
were higher in ammonium than in the control, while best-bet conventional
treatment was higher in phosphate compared to other treatments in the summer
season.
In soils of the control treatment, plate counts of bacterial and fungal
populations were higher in the wet-summer than in the (5 day incubated) dry-
winter samples (Table 9.2). Bacterial counts (cfu Log 10) were significantly
higher in best-bet biology, best-bet conventional, biology boom spray and green
manure treatments than in the control in the wet-summer, while counts were
higher in best-bet conventional and biology boom spray treatments, and the
225
brigalow soil, than the control in the (5 day incubated) dry-winter samples (Table
9.1). Fungal counts (cfu Log 10) did not differ significantly between the
treatments in either season; but were significantly higher in the brigalow
community soil. The average microbial biomass C of the seven treatments
(excluding brigalow community) was 47% higher in the wet-summer than in the
dry-winter, as expected given increased temperatures and plant biomass.
However, there was no significant difference in microbial biomass C between
treatments in either season, although the level of the brigalow soil was almost
double than that of the cropping treatments. The attributes of soil microbial
activity, as indicated by FDA, basal and substrate induced respiration, were all
higher in the incubated dry-winter season samples than in the field samples of the
previous wet-summer season (Table 9.2).
In the wet-summer season, basal respiration was significantly higher in
best-bet conventional and green manure treated soils, compared to feather-top
Rhodes, whereas substrate induced respiration was significantly higher in biology
direct injection and feather-top Rhodes soils compared to the control (Table 9.2).
The total microbial activity, however, was the highest in best-bet conventional
treatment, with least microbial activity in green manure during the wet-summer
season (Table 9.2). There were no differences among treatments in terms of total
microbial activity, or basal and substrate induced respiration of soil in the dry-
winter season.
The soil from the brigalow community differed from the other treatments
by virtue of its significantly elevated pH, nitrate, bacterial and fungal populations,
microbial biomass C, substrate induced respiration and total microbial activity in
226
the dry-winter season (Table 9.2). The brigalow forest soil was not sampled in the
wet-summer season.
227
Table 9. 2. Variation in the physico-chemical, microbial and biochemical characteristics of soil samples collected from plots treated with different management practices at
the Baralaba trial site in two different seasons. Cont - control, BBB - best-bet biology, BBC - best-bet conventional, BDI - biology direct injection, BBS - biology boom
spray, FTR - feather-top Rhodes , GM - green manure, BRI - brigalow soil. Values are mean ± SE (n = 3) for all treatments except for green manuring, biological weed
control and brigalow soil samples (n = 2) collected in the dry season. Within rows, within a season, means with the same letter are not significantly different according to
Tukey‘s test (p< 0.05). Moisture content was assessed after addition of 10% (w/v) water in the dry season. ‗-‘ indicates missing values lower than the detectable limit of the
RQflex meter.
Wet season (Summer) Dry season (Winter)

Biochemical and microbial characteristics Cont BBB BBC BDI BBS FTR GM Cont BBB BBC BDI BBS FTR GM BRI
pH (1:5) 5.59 ± 0.0a 7.02 ± 0.0d 6.64 ± 0.0c 7.08 ± 0.1d 7.02 ± 0.0d 6.58 ± 0.0c 6.28 ± 0.1b 6.02 ± 0.1a 6.04 ± 0.1a 6.39 ± 0.0b 6.47 ± 0.0bc 6.72 ± 0.0cd
6.93 ± 0.0d 6.97 ± 0.0d 6.91 ± 0.1d
-1
EC (dS m ) 0.11 ± 0.0a 0.13 ± 0.0ab 0.11 ± 0.0a 0.14 ± 0.0bc 0.14 ± 0.0bc 0.12 ± 0.0ab 0.15 ± 0.0c 0.08 ± 0.0a 0.30 ± 0.0cd 0.08 ± 0.0a 0.35 ± 0.0d 0.25 ± 0.0bcd
0.10 ± 0.0ab 0.11 ± 0.0ab 0.16 ± 0.1abc
Moisture content (%) 13.0 ± 0.1a 14.2 ± 0.0a 13.8 ± 0.4a 13.3 ± 0.2a 13.2 ± 0.5a 12.8 ± 0.1a 14.1 ± 0.4a 13.4 ± 0.2a 13.4 ± 0.2a 13.5 ± 0.1a 13.8 ± 0.3a 14.7 ± 0.5a
14.0 ± 0.2a 13.3 ± 0.5a 19.6 ± 1.3b
- -1 7.79 ± 1.3a 33.3 ± 2.4ab 7.79 ± 0.7a 61.4 ± 27.5b
NO3 -N (mg kg d wt) 26.0 ± 2.0a 26.7 ± 1.6a 21.4 ± 3.6a 27.3 ± 2.0a 23.4 ± 2.0a 36.3 ± 4.7a 29.3 ± 4.6a 29.3 ± 3.2ab 30.6 ± 4.1ab
19.0 ± 8.5ab 32.4 ± 6.5ab
+ -1
NH4 -N (mg kg d wt) 0.45 ± 0.0a 1.35 ± 0.3a 1.80 ± 0.5a 4.91 ± 0.5b 0.59 ± 0.1a 1.78 ± 0.3a 3.61 ± 0.3b 3.87 ± 0.3c 1.19 ± 0.3a 2.68 ± 0.5abc 1.80 ± 0.5abc 1.52 ± 0.3ab
3.61 ± 0.9bc 1.78 ± 0.0abc 2.43 ± 0.5abc
-1
PO43- -P (mg kg d wt) 10.0 ± 1.3a 6.32 ± 0.7a 20.5 ± 2.0b 8.00 ± 0.9a 5.00 ± 0.2a 6.00 ± 0.0a 6.57 ± 0.7a 7.25 ± 1.1a 4.25 ± 0.2a 4.50 ± 0.4a - - 9.48 ± 1.9a 4.50 ± 0.8a 8.15 ± 3.3a
Bacterial population (cfu Log 10) 6.00 ± 0.0a 6.62 ± 0.1c 6.63 ± 0.0c 6.36 ± 0.1b 6.69 ± 0.1c 6.30 ± 0.0b 6.52 ± 0.0bc 5.30 ± 0.2a 5.56 ± 0.0a 6.61 ± 0.0b 5.49 ± 0.1a 6.24 ± 0.1b 5.59 ± 0.1a 5.47 ± 0.2a 6.29 ± 0.0b
Fungal population (cfu Log 10) 4.20 ± 0.1a 4.36 ± 0.1a 4.36 ± 0.1a 4.10 ± 0.1a 4.00 ± 0.0a 4.16 ± 0.2a 4.42 ± 0.1a 3.52 ± 0.0a 3.33 ± 0.2a 3.10 ± 0.1a 3.30 ± 0.2a 3.42 ± 0.1a 3.16 ± 0.2a 3.16 ± 0.2a 4.27 ± 0.1b
-1
Microbial biomass C (mg C g d wt) 0.70 ± 0.4a 1.18 ± 0.3a 1.06 ± 0.5a 0.79 ± 0.1a 0.97 ± 0.5a 0.86 ± 0.2a 1.09 ± 0.2a 0.33 ± 0.0a 0.70 ± 0.3a 0.45 ± 0.2a 0.59 ± 0.1a 0.76 ± 0.2a 0.44 ± 0.0a 0.28 ± 0.1a 1.94 ± 0.3b
-1 -1
Basal respiration (µg CO2 g d wt h ) 16.0 ± 1.8ab 21.6 ± 3.6ab 30.3 ± 3.1b 21.3 ± 4.7ab 17.8 ± 3.5ab 10.7 ± 1.8a 26.0 ± 0.9b 14.1 ± 4.6a 31.0 ± 5.5a 33.6 ± 19a 31.9 ± 7.1a 32.2 ± 1.8a 39.1 ± 1.9a 30.8 ± 2.2a 44.7 ± 7.7a
-1 -1
Substrate induced respiration (µg CO2 g d wt h ) 17.6 ± 4.6a 27.6 ± 2.4ab 33.7 ± 4.7ab 38.8 ± 4.7b 35.2 ± 1.8ab 40.3 ± 4.6b 31.1 ± 0.9ab 24.7 ± 3.0a 33.5 ± 7.7a 35.3 ± 11a 47.9 ± 1.8ab 39.4 ± 1.8ab 35.5 ± 6.2ab 37.9 ± 19ab 78.5 ± 13b
-1 -1
Total Microbial activity (µg FDA g d wt h ) 114 ± 6.3ab 131 ± 2.9ab 150 ± 7.8b 111 ± 4.7ab 119 ± 3.2ab 115 ± 12ab 106 ± 15a 224 ± 13a 254 ± 23a 216 ± 11a 311 ± 13a 275 ± 28a 237 ± 58a 269 ± 116a 508 ± 110b
d wt Dry weight
228
A clear differentiation in substrate utilisation pattern was visible between
treatments (Fig. 9.1A and 9.1B). No difference was observed in bacterial or
fungal substrate utilisation patterns (BiologTM ecoplate) between treated field
soils and the virgin brigalow soil until 72 h of incubation (Fig. 9.1). Although the
metabolic activity of fungi in the control treatment seems slightly higher than the
other treatments, it was not significantly different. The rate of substrate utilisation
was higher in brigalow soil than in cultivated soil, suggesting higher metabolic
activities of both bacterial and fungal communities in the brigalow soil compared
to those of other treatments.
Significant differences among treatments in average utilisation of
substrate guilds were evident within BiologTM (Ecoplate and FF microplate) data
(Fig. 9.2). Samples from the brigalow treatment averaged higher utilisation of
carbohydrates in the Ecoplate than those of other treatments while biology boom
spray samples averaged higher utilisation of miscellaneous substrates than the
feather-top Rhodes treatment. Similarly, brigalow samples averaged higher
utilisation of carbohydrates than biology direct injection, and higher utilisation of
polymers compared to other treatments, except feather-top Rhodes, in the FF
microplate (Fig. 9.2B).
229
0.30 A
Control
Best-bet conventional
Biology boom spray
0.25
Biology direct injection
Best-bet biology
Green manure
Feather-top Rhodes
0.20
Brigalow soil
AWCD 570 nm
0.15
0.10
0.05
0.00
0 24 48 72 96 120 144 168
0.30
Control B
Best-bet conventional
Biology boom spray
0.25 Biology direct injection
Best-bet biology
Green manure
Feather-top Rhodes
0.20 Brigalow soil
AWCD 490 nm
0.15
0.10
0.05
0.00
0 24 48 72 96 120 144 168
Time (h)
Figure 9. 1. Kinetics of average well colour development (AWCD) of (A) bacterial (570 nm) and
(B) fungal (490 nm) communities originating from soil samples collected from the trial site during
the dry-winter season at Baralaba. Values are mean ± SE (n = 3) for all treatments except for
green manure, feather-top Rhodes and brigalow soil (n = 2).
230
Figure 9. 2. Average carbon substrate utilisation for different substrate categories in terms of (A)
32 (Biolog Ecoplate) and (B) 96 (Biolog FF microplate) different food sources. Samples were
collected from 72 h incubations of both Biolog plate types and utilisation was measured as the
average optical density across all substrates within each guild (i.e. across 7 different
carbohydrates, 9 carboxylic acids, 4 polymers, 6 amino acids, 2 amines/amides and 3
miscellaneous for Ecoplate, and 44 different carbohydrates, 17 carboxylic acids, 5 polymers, 13
amino acids, 6 amines/amides and 10 miscellaneous) for FF microplate.
Principal components analysis (PCA) of the Biolog Ecoplate assay data
explained 43% and 20% of the total variance in the first two principal
components (Fig. 9.3A). A plot of PC1 against PC2 revealed that the bacterial
communities present in the brigalow soil were quite different to that of the
cropping soils (Fig. 9.3A). The communities in the cropping treatments were
231
clustered, although a decrease in principal component 2 characterised some of the
best-bet conventional and feather-top Rhodes treatment replicates.
The PCA of FF microplate data explained 38% (PC1) and 21% (PC2) of
the total variance (Fig. 9.3B). In a PC2 against PC1 plot, the brigalow soil
samples were separated from the main cluster of cropped soil samples, except for
one sample from the control group (with an extreme PC2 value) and one sample
from the best-bet biology treatment (extreme PC1 value) (Fig. 9.3B).
Figure 9. 3. Principal components analysis of optical densities produced by microbial activities of

soil samples collected from the trial site at Baralaba consuming (A) 32 (Biolog Ecoplate) and (B)
96 (Biolog FF microplate) different food sources; representing bacterial and fungal diversity,
respectively. Values are mean (n = 3) for all treatments except for green manure, feather-top
Rhodes and brigalow soil (n = 2).
232
3.3. PCR-DGGE analysis for bacteria and fungi
Extracted soil DNA from each treatment (n=2) were run on the DGGE
gels to obtain microbial community patterns. Replicate variability was higher in
fungal compared to bacterial communities.
A UPGMA (Unweighted Pair Group Method with Arithmetic mean
algorithm) dendrogram was based on the DGGE generated DNA profiles (Fig.
9.4). A shift in microbial communities occurred between seasons (Fig. 9.4),
although the change was more marked in the bacterial than the fungal community
(Fig. 9.4).
The UPGMA dendrogram for bacterial community formed four distinct
clusters, where best-bet biology treatment grouped with control in either season
whereas brigalow grouped with biology direct injection, biology boom spray and
best-bet conventional treatments (Fig. 9.4A). Similarly, feather-top Rhodes and
green manure treatments were clustered together showing relatedness of bacterial
communities between the two treatments. Unlike the PCA analysis of the Biolog
(Ecoplate) substrate utilisation data, the UPGMA dendrogram did not separate
the brigalow communities with those of the field treated soils.
Likewise, the dendrogram for fungal communities formed five clusters
where best-bet biology grouped with control samples collected in the wet-
summer season whereas in the dry-winter season, fungal communities of biology
boom spray, biology direct injection, best-bet biology and control treatments
clustered together (Fig. 9.4B). Brigalow soil formed a separate cluster whereas
feather-top Rhodes and green manure clustered together. The best-bet
conventional treatment, however, showed replicate variability and formed a
separate cluster.
233
Figure 9. 4. Unweighted Pair Group Method with Arithmetic mean algorithm dendrogram
constructed from the DGGE profiles of (A) bacterial 16S rDNA and (B) fungal ITS genes
amplified from duplicate DNA samples of soils collected in the dry-winter season, except control
(I) and best-bet biology (I) which were additionally collected in the previous wet-summer season
(highlighted in the figure), from the trial site and the nearby virgin brigalow soil at Baralaba. The
scale bar represents percent similarity.
The UPGMA dendrogram generated for bacterial communities also
showed that best-bet biology and biology direct injection treatments were
clustered with approximately 60% homology forming a sub group (Fig. 9.4A).
234
The fungal dendrogram showed approximately 65% homology between best-bet
biology and biology direct injection treatments (Fig. 9.4B). Both of these
treatments involved compost extract applied via liquid injection during planting,
but with and without commercial microbial inoculant (Twin N ‗diazotrophs‘),
respectively. The results of the PCR-DGGE analysis (Fig. 9.4A) were consistent
with the principal components analysis (BiologTM) data, in that the bacterial
community structure of the biology direct injection and best-bet conventional
treated soils demonstrated relatedness (Fig. 9.3A). The fungal community
structure of soils treated with biology boom spray, biology direct injection and
best-bet biology treatments showed relatedness to one another (Fig. 9.3B and
9.4B), but not to the degree shown by the bacterial communities.
Bacterial community diversity of best-bet biology as denoted by Shannon-
Weaver diversity index (H‘) was signifcantly higher compared to the best-bet
conventional treatment (Table 9.3). Although the UPGMA dendrogram for either
bacteria and fungi showed considerable differences, no changes in their diversity,
evenness and richness were evident in the control treatments when compared
between the two seasons (Table 9.3). Nevertheless, significantly higher bacterial
diversity was found in the best-bet biology system in the dry-winter compared to
the wet-summer season (Table 9.3).
235
Table 9. 3. Shannon-Weaver Diversity Index (H‘), Equitability Index (J) and number of bands (S)
for bacteria and fungi present in soil samples collected in the dry-winter season from plots treated
with different management practices including compost extract treatments with two methods of
application at Baralaba trial site. All soil samples were collected in the dry-winter season, except
control (I) and best-bet biology (I) which were also collected in the previous wet-summer season.
Within columns, mean ± SE (n = 2) with the same letter are not significantly different according
to Tukey‘s test (p< 0.05).
Bacteria Fungi
Control 2.26 ± 0.10b 0.88 ± 0.04a 13 ± 0.00b 2.88 ± 0.04cd 0.81 ± 0.01bc 35 ± 0.50cd
Best-bet biology 2.74 ± 0.10c 0.91 ± 0.01a 21 ± 1.50c 2.46 ± 0.02abc 0.74 ± 0.01abc 28 ± 0.50ab
Best-bet conventional 2.33 ± 0.01b 0.92 ± 0.01a 13 ± 0.50b 2.65± 0.21abcd 0.77 ± 0.04abc 31 ± 3.00abc
Biology boom spray 2.39 ± 0.03bc 0.92 ± 0.00a 14 ± 0.50b 2.32 ± 0.11a 0.71 ± 0.03a 26 ± 0.00ab
Biology direct injection 2.39 ± 0.01bc 0.96 ± 0.01a 12 ± 0.00b 2.54 ± 0.05abcd 0.75 ± 0.01abc 29 ± 1.00abc
Green manure 2.43 ± 0.08bc 0.92 ± 0.01a 14 ± 1.00b 2.79 ± 0.03bcd 0.80 ± 0.01abc 32 ± 0.00bcd
Feather-top Rhodes 2.32 ± 0.13b 0.90 ± 0.02a 13 ± 1.00b 2.37 ± 0.07ab 0.73 ± 0.01ab 26 ± 1.50a
Brigalow soil 2.35 ± 0.01bc 0.94 ± 0.01a 12 ± 0.00b 2.96 ± 0.02d 0.82 ± 0.00bc 38 ± 0.50d
Control (I) 2.35 ± 0.08bc 0.93 ± 0.01a 13 ± 0.50b 2.94 ± 0.01d 0.83 ± 0.00c 35 ± 0.00cd
Best-bet biology (I) 1.86 ± 0.02a 0.95 ± 0.01a 7 ± 0.00a 2.82 ± 0.02cd 0.80 ± 0.00abc 34 ± 0.00cd
No differences were recorded among treatments in terms of evenness (J)
of bacterial communities, however richness (S) in bacterial communities was
higher in best-bet biology than that of other treatments including biology direct
injection. Both biology direct injection and best-bet biology received similar
treatments but best-bet biology was treated with Twin N inoculant (free-living
rhizosphere N2 fixers) prior to the onset of flowering (Table 9.3). Bacterial
community diversity (H‘) and richness (S) in best-bet biology treatment were
higher in the wet-summer compared to the dry-winter. The lack of difference in
evenness scores among all treatments indicates the uniformity of band intensities
in the DGGE profiles for bacteria (Table 9.2). A shift in soil bacterial
communities was produced by best-bet biology treatment, in contrast to the best-
bet conventional treatment, although fungal communities of those treatments
remain unchanged.
The ITS-DGGE data showed no alterations in the fungal diversity and
evenness of control and biological treatments between the two seasons, however
fungal richness was higher in best-bet biology treatment in the wet-summer than
236
in the dry-winter season (Table 9.3). ITS-DGGE profiles revealed the presence of
few common bands in all soil treatments along with the virgin brigalow land with
considerable variation in relative intensity of common bands between treatments.
In the dry-winter season, best-bet biology, biology boom spray and feather-top
Rhodes treatments demonstrated lower fungal diversity as assessed by Shannon-
Weaver diversity values compared to the virgin brigalow soil, and even compared
to the control (Table 9.3).
The fungal communities in the soil samples, however, showed greater
diversity (H‘) and relative abundance or richness (S) of fungi, as indicated by the
higher number of bands, than equivalent bacterial communities irrespective of the
treatment effects (Table 9.3).
4. Discussion
4.1. Seasonal variation
The summer season enjoys higher temperatures and rainfall, and thus soil
moisture, and higher plant biomass, conditions expected to favour soil microbial
activity and mineralisation. For example, Kaiser et al. (1995) also observed
temporal variation in microbial biomass C of -15% to +15% of the annual mean
in winter and summer seasons, respectively, for an arable soil (luvisol). In the
current experiment (PWP), the soil field capacity of the vertisol soil was 33%
w/v, while permanent wilting point is associated with a moisture content of 19%
w/v (Chinn & Pillai, 2008). In the current study, the moisture content of the
surface (0-10 cm) soil in the wet season was around 13% w/v, or well below
PWP. The moisture content of the soil collected in the dry season was adjusted to
a similar moisture level for a 5 d incubation period. However, plate counts of
237
bacteria, fungi, and microbial biomass C (in average) were higher in the wet-
summer than the following dry-winter (Table 9.2).
Although higher plate counts and microbial biomass were found in soils
collected in summer season, it was not reflected in the microbial activity. Soil
microbial activity and soil respiration (both basal and substrate induced) were
higher in soil collected from winter compared to that from the previous summer
season (Table 9.2). It could be that the fungal spores were present in resting
stages in soil during summer given that the moisture content of the soil collected
was well below PWP. Although winter soil was wet up to this level, wetting was
never uniform. Therefore, it is possible that there were pockets of soil at water
potentials closer to zero, supporting microbial activity.
4.2. Comparison of brigalow and cultivated soils
The brigalow forest soil was significantly higher in nitrate, moisture
content, bacterial and fungal populations, microbial biomass C, substrate induced
respiration and microbial activity in the dry-winter season compared to the
cultivated soil (Table 9.2). This could be ascribed to the fact that the forest soil
has benefit of continual vegetation and mulch cover, with fire acting in removal
of organic matter and deposition of carbon. There are many studies documenting
changes in soil over time since brigalow clearing (Radford et al., 2007; Solomon
et al., 2000; Thornton et al., 2007). Collard and Zammit (2006) reported reduced
levels of total organic as well as labile carbon in intensively used agricultural
land compared to the remnant brigalow soil. Labile carbon and microbial biomass
in soil are noted to be strongly correlated, with microbial biomass linked to the
rate of turnover of organic matter (Bell et al., 1998). Spaccini et al. (2001) also
238
demonstrated that land cultivation reduced soil aggregate stability, with forest
soil richer in organic carbon.
The PCA of the Biolog data of the present study (Fig. 9.3) suggests that
original bacterial communities of the virgin brigalow soil have been shifted by
cultivation practices. However, culture based methods, such as Biolog neglect the
presence of non-culturable microorganisms, and as such are not totally true
guides to the microbial species composition of the sample (Widmer et al., 2001).
The culture limitation of the Biolog technique is likely to affect slow growing
organisms, and organisms inhabiting different ecological niches to that tested in
the Biolog procedure (e.g. obligate anaerobes). Smalla et al. (1998) characterised
the microbial communities of activated sludge samples using Biolog GN plates
followed by DGGE and TGGE techniques in order to explore which microbial
populations of the communities generate the Biolog patterns. Using the molecular
techniques, they revealed that the highly adaptive and fast-growing communities
of bacteria, dominant in the Biolog wells, were responsible for the pattern
observed. Therefore, the PCR-DGGE technique was applied in the current study
in order to verify the results obtained from Biolog.
DGGE profiles for the treated plots revealed distinct shifts between
biological and conventional treatments, however few bands were present in all
treatments, and some bands were unique either to the cultivated land or to the
virgin brigalow soil. No difference between the bacterial communities in
brigalow and the field-treated soils as suggested by the UPGMA dendrogram and
the DGGE profile indicate that both cultivated and brigalow soils share the
common bacterial community structure. Although not performed in this study, the
DGGE generated bands can be excised from the gels, sequenced, and analysed
239
phylogenetically to species level through comparison with sequences available in
GeneBank database (e.g. www.ncbi.nlm.nih.gov) (Jing et al., 2010).
In the dry-winter season, fungal diversity as assessed by Shannon-Weaver
diversity was higher in the virgin brigalow soil compared to best-bet biology,
biology boom spray and feather-top Rhodes treatments (Table 9.3). Higher fungal
diversity in the brigalow soil may be related to the presence of the recalcitrant
forest litter, compared to the plant debris of cultivated soils (Lauber et al., 2008).
This is consistent with the results of Biolog where brigalow sample performed
better on polymers in FF microplates (Fig. 9.2).
The greater diversity of soil fungal communities than equivalent bacterial
communities in brigalow soil, noted in this study (Table 9.3), is in accordance
with the findings of Bailey et al. (2002), who also observed greater fungal to
bacterial biomass, as measured by phospholipid fatty acid analysis (PLFA), in
forest soil compared to the agriculturally manipulated soils. Unfavorable climatic
conditions, such as insufficient rainfall might have stimulated production of
fungal spores, which can remain viable in dry soil for longer periods (Elliot et al.,
2002). In the biology boom spray treatment, the compost extract is sprayed on the
surface of the soil which could be one of the reasons why the soil exhibited the
least fungal diversity, while the bacterial communities remained uniform to the
other treatments (Table 9.3).
4.3. Effects of management practices on soil properties
Both Twin-N inoculant (rhizosphere N2 fixers) and green manuring crops
could have contributed to a significant increase in ammonium concentration in
biology direct injection and green manure treatments (Table 9.2) during summer.
240
Similarly, the granular fertiliser ‗Starter Z‘ (19.5% P) could be the source of
higher phosphate concentration in best-bet conventional treated soils compared to
other treatments in the same season.
Increased soil respiration is a direct indicator of increased microbial
activity (Gomez et al., 2001; Parkin et al., 1996). In the present study,
significantly higher basal respiration was noted in best-bet conventional and
green manure, compared to feather-top Rhodes, whereas substrate induced
respiration was significantly higher in biology direct injection and feather-top
Rhodes compared to the control in the summer season (Table 9.2). This could be
due to stimulation of soil microbiota and increase in microbial biomass with the
addition of green manures (Stark et al., 2007) and mineral fertilisers (Chu et al.,
2007), or it could simply be due to the synergistic effect of soil indigenous and
exogenous microbiota supplied through the organic amendments and
biofertilisers (Prasanna et al., 2008).
5. Conclusions
The observation that soil microbial diversity is different in undisturbed
brigalow soil to cultivated soils is interesting, but not unexpected. After all, the
above-ground community structure is also quite different to that of cultivated
land.
The agricultural management practices applied in this study demonstrated
little impact on soil microbial diversity relative to a seasonal effect. However,
compost extract application in soil along with foliar applications of microbial
inoculant and nutrients improved bacterial communities in soil compared to the
conventional treatment.
241
Future exploration of the practical benefits of compost extracts should
focus on the status of key microbes – key to various soil processes, for example,
Rhizobium for legume nodulation and N2 fixation, rhizobacteria (such as Bacillus,
and Nitrobacter) for organic phosphate mineralisation and inorganic phosphate
solubilisation, making nitrogen and phosphorus available to plants. Other groups
such as ammonia oxidizers or nitrifiers, sulphur oxidizing bacteria, mycorrhizal
fungi, which are considered important in nutrient cycling, nutrient transport and
chelating of toxic elements should also be considered for future work. Some
agronomic information to suggest some target species which help supply these
nutrients limiting for crop production in central Queensland, where ferrosol is
generally limited in P, should also be explored.
242
Chapter 10: Summary and future directions
Chapter 10
Summary and future directions
Microbially enhanced compost extract (compost tea), obtained after
incubating compost in water with microbial food sources, is claimed to have
benefits in improving soil and crop ‗health‘ (Ingham, 2005), but there is relatively
little in the scientific literature on the effectiveness and mode of action of this
product (as reviewed in Chapter 1). The following hypotheses were established as
to the benefit of compost extract to plant growth: (a) direct improvement of plant
nutrition; (b) improvement of plant nutrition through an indirect action, via
microbial activities in mineralising or solubilising of organic and inorganic
components of the soil; (c) acceleration of litter decomposition; (d) bio-protection
against fungal pathogens (e.g. Fusarium oxysporum f. sp. lycopersici); and (e)
protection of root growth against high levels of toxic soil substances (e.g. Al+++),
via chelation of Al ions by humic acids or uptake by microbes.
The results of this thesis indicated that compost extracts (a) improved
plant growth via direct nutrient addition, (b) but not via mineralisation or
solubilisation of organic or inorganic matter in soil, and (c) accelerated litter
decomposition to some extent, (d) reduced root fungal diseases, and (e) reduced
243
aluminium toxicity possibly via the action of auxin or gibberellic acid-like
growth regulators.
The effect of compost extract on plant growth in the tomato and sorghum
pot experiments was largely attributed to the nutrients inherent in the high dose of
compost extract applied (equivalent to 34,000 L ha-1). Of course, field application
of such large quantities is not practical in most production systems. However,
such compost extracts may find as an organic liquid fertiliser, for example,
‗organic hydroponics‘, combining two higher values, but normally diametrically
opposed categories, in the supply chain.
There was no evidence of an increase in solubilisation of soil minerals or
mineralisation of soil organic matter, sufficient to impact on plant nutrition.
Compost extracts sprayed onto leaf litter did result in a long term respiration rate
increase, however the impact on litter breakdown was limited over the 168 day
trial. Acceleration of stubble breakdown is a logical application of compost
extract. Use of biological sprays to assist stubble breakdown is well documented
(e.g. Freeman et al., 1948; Greathouse, 1949; Nilsson, 1974; Nilsson & Ginns,
1979), and several commercial providers exist (e.g. supplying cellulose digesting
fungi, specifically Chaetomium brasiliense and Trichoderma harzianum;
www.bionutrient.com.au/products/full-product-list-46/stubble-digest-57.htm).
The relatively poor rate of sugarcane litter breakdown observed in the present
study might be addressed by using compost based on sugarcane trash, or by
species specific enrichment of the compost extract. Attention could also be given
to the degradation environment (e.g. moisture levels; trash particle size; trash
turning). This application is worthy of further attention, being of significance to
244
the Australian sugarcane industry as it adopts the practice of green cane
harvesting.
Further study using lower dilutions of compost extract with respect to the
amelioration of aluminium toxicity is warranted, and on the accumulation of
humates in the soil from successive additions of compost extract. The observed
effect of amelioration of toxicity (200 μM) on maize roots following imbibitions
in compost extract hints at the involvement of auxin or gibberellic acid-like
growth regulators. Chemical identification of these agents would be useful, in
order to allow compost extracts to be tailored to favour this function.
Compost extracts have been reported to be of use in suppressing plant
diseases. Although the results of the present studies conducted in vitro on the
efficacy of compost extracts in suppressing fungal wilt diseases were promising,
the effect of compost extracts on disease suppression in tomato crops in
hydroponics culture was not consistent for the three tested pathogens. Future
effort should focus on identifying techniques to reduce such inherent variability,
which may require the identification of suppressive microbiota, ensuring their
survival during the production process, and ensuring the delivery of desired
microbes onto the roots or foliage of plants (e.g. through various application
techniques along with wetting agents, spray adjutants, and emulsifiers).
Integrated approaches such as multiple cropping, crop rotations and fallow
cropping should be used along with the use of compost extracts at the field scale,
the latter needing further optimisation to control such diseases effectively. It
should, however, be understood that compost extract is not a panacea for all
diseases, and therefore growers need to employ integrated plant health
management tools in parallel to the use of compost extracts.
245
Rumen based compost extracts were compared with two commercial
products with respect to nutritional and microbial characteristics. The question of
whether a relatively uncontrolled microbial species mix (as present in enhanced
compost extract) or controlled single or multiple species tailored to specific
applications should be recommended for agronomic use remains to be answered.
Certainly, the Rhizobium industry has developed around the use of single strains
targeted to specific crops (in Australia) (Brandon et al., 1998) or a mix of a
number of strains of the one species, for use across a number of crop types (in the
USA) (Stacey & Upchurch, 1984). Similarly, relatively pure cultures of
microorganisms, such as Azotobacter, Trichoderma, and Azospirillum are
commercially available as ‗biofertilisers‘ (e.g.
www.biofertilizer.com/organic_garden_biofertilizers/biofertilizer). Arguably, an
extract could be tailored for an application if it were inoculated with specific
microbial species selected for a specific task (litter breakdown, disease
protection, phosphate solubilisation, or similar). Indeed, this is the approach taken
by SmartBugs Pty Ltd, who markets a freeze-dried inoculum of beneficial
microbes (e.g. Azotobacter spp., Azospirillum spp., Bacillus spp., Trichoderma
spp., Cellulomonas spp.) instead of compost, for use in production of ‗compost
tea‘. Conversely, the ‗low tech‘ option of producing a compost ‗tea‘ from locally
produced compost holds appeal to those who favour a ‗natural‘ approach.
The compost extract incubation process attempts to idealise microbial
growth conditions - but there is the risk of growth of pathogenic microorganisms.
Although not investigated in this study, human-health-related issues of compost
extracts is also of concern and should also be addressed in future studies.
246
In this thesis, an open air incubation system was characterised in terms of
the incubation history of pH, EC, temperature and bacterial-fungal population
level. This type of basic description could be undertaken to compare the range of
commercially available incubators that have become available (e.g. fitted with
advanced aerators). Further study on assessing and increasing the storability or
shelf-life of compost extracts for storage to allow for long distance transport is
also recommended.
The quality of the compost is one of the determinants of the quality of the
compost extract. Vermicomposting, followed by separation and collection of
leachate from the solid waste, was seen in the present study to increase the
microbial biomass of the rumen compost. Further, liquid incubation of composts
to produce an extract altered their nutrient and microbial composition with
highest functional diversity observed in vermicast leached extract compared to
the original composts. Thus, there is good scope for the utilisation of earthworms
as a means of improving the quality of the inoculum.
In the field study, although little effect was found on the fungal
communities, total and active bacterial communities were enhanced by compost
extract application compared to the other treatments. It is likely that compost
extracts will find an expanded role in broadscale agriculture.
The ability of these liquid extracts to favourably shift the activity and
diversity of desirable microorganisms in field soil, when applied at low
application rates (e.g. 200 L ha-1) is another interesting area for consideration.
Application of key ‗missing‘ microbiota can have a large scale impact from a
small scale application (e.g. as with rhizobia or mycorrhizae), but addition of
microbiota already present in the soil is unlikely to have such an impact. Further
247
research on field survival and establishment of such microbiota added to the soil
in compost extracts is warranted, combined with consideration of application
frequency, rate, and method (liquid injection vs. fertigation or foliar spray, seed
priming vs. soil drench). Attention should be given to studies on combinations of
potential plant growth promoting microorganisms for improved results. Perhaps
ultimately there could be a small set of ‗indicator‘ species one would quantify as
an index of a given soil‘s capacity for mineralisation, phosphate solubilisation,
and plant disease protection.
Other potential areas for further study include:
1. the ability of compost extract to alter microbial populations on the
phylloplane of plants following low rates (e.g. 20-50 L ha-1) of foliar
application.
2. the ability of compost extract to improve plant nutrition through
mineralisation of the soil organic fraction, especially to P nutrition, through
P-solubilisation by the action of bacteria and mycorrhizae.
3. the ability of compost extract to result in increased N2 fixation in the soil by
free living soil bacteria.
4. the role of compost extract in detoxifying organic compounds, for example,
pesticides, and inorganic ions other than Al+++ (e.g. Mn) in acid soils by
organic acids or sequestration by microbiota.
5. the effect of compost extract in improving soil structure through direct
addition of humates to the soil and through addition of organic matter via
partial decomposition of plant residues.
248
6. the role of compost extract in improving water holding capacity of soil
through improvement in soil structure and through addition of organic matter
via partial decomposition of plant residues.
7. the role of compost extract with respect to increasing plant resistance to
insects and insect deterrence or control.
The findings of this research have provided an insight to the
users/producers of microbially enhanced compost extract or ‗compost tea‘. At the
same time, it has generated more questions to be answered and will hopefully
encourage future researchers to investigate the other possible roles of microbially
enhanced compost extracts.
249
References
References
Abbott, L.K., Murphy, D.V. 2003. Soil Biological Fertility: A Key to Sustainable
Land Use in Agriculture, Dordrecht; Boston: Kluwer Academic
Publishers, pp. 264.
Adam, G., Duncan, H. 2001. Development of a sensitive and rapid method for the
measurement of total microbial activity using fluorescein diacetate (FDA)
in a range of soils. Soil Biol. Biochem., 33, 943-951.
Adams, J.D.W., Frostick, L.E. 2009. Analysis of bacterial activity, biomass and
diversity during windrow composting. Waste Management, 29, 598-605.
Adams, P.B. 1990. The potential of mycoparasites for biological control of plant
diseases. Annu. Rev. Phytopathol., 28, 59-77.
Adegunloye, D.V., Adetuyi, F.C., Akinyosoye, F.A., Doyeni, M.O. 2007.
Microbial analysis of compost using cowdung as booster. Pakistan J.
Nutr., 6, 506–510.
Aibuzio, A., Ferrari, G., Nardi, S. 1986. Effects of humic substances on nitrate
uptake and assimilation in barley seedlings. Can. J. Soil Sci., 66, 731-736.
Aira, M., Dominguez, J. 2008. Optimising vermicomposting of animal wastes:
Effects of rate of manure application on carbon loss and microbial
stabilisation. J. Environ. Management, 88, 1525-1529.
Al-Mughrabi, K.I., Berthelerne, C., Livingston, T., Burgoyne, A., Poirier, R.,
Vikram, A. 2008. Aerobic compost tea, compost and a combination of
both reduce the severity of common scab (Streptomyces scabies) on
potato tubers. J. Plant Sci., 3(2), 168-175.
250
References
Albanell, E., Plaixats, J., Cabrero, T. 1988. Chemical changes during
vermicomposting (Eisenia fetida) of sheep manure mixed with cotton
industrial wastes. Biol. Fertil. Soils, 6, 266-269.
Alexander, M. 1994. Biodegradation and Bioremediation. Academic Press, New
York.
Allard, A.S., Neilson, A.H. 1997. Bioremediation of organic waste sites: a critical
review of microbiological aspects. Int. Biodeterioration &
Biodegradation, 39(4).
Allen, D.J., Ort, D.R. 2001. Impact of chilling temperatures on photosynthesis in
warm-climate plants. Trends Plant Sci., 6, 36–42.
Alms, M. 2002. Castings tea: Panacea or fantasy? in: Vermico’s Bimonthly
Newsletter, Vol. 7.
Altomare, C., Norvell, W.A., Björkman, T., Harman, G. 1999. Solubilization of
phosphates and micronutrients by the plant growth-promoting and
biocontrol fungus Trichoderma harzianum Rifai 1295-22. Appl. Environ.
Microbiol., 65, 2926–2933.
An, D.S., Im, W.T., Yang, H.C., Kang, M.S., Kim, K.K., Jin, L., Kim, M.K., Lee,
S.T. 2005. Cellulomonas terrae sp. nov., a cellulolytic and xylanolytic
bacterium isolated from soil. Int. J. Syst. Evol. Microbiol., 55(4), 1705-
1709.
Anderson, J.P.E. 1982. Soil respiration. Second ed. in: Methods of Soil Analysis,
Am. Soc. Agron. Madison, WI, pp. 831–871.
Anderson, T.-H. 2003. Microbial eco-physiological indicators to assess soil
quality. Agric. Ecosyst. Environ., 98, 285-293.
251
References
Andrew, J.H. 1993. Compost extracts and the biological control of foliar plant
disease.
Antonio, G.F., Carlos, C.R., Reiner, R.R., Miguel, A.A., Angela, O.L.M., Cruz,
M.J.G., Dendooven, L. 2008. Formulation of a liquid fertiliser for
sorghum (Sorghum bicolour (L.) Moench) using vermicompost leachate.
Bioresour. Technol., 99, 6174-6180.
Arthur, G.D., A.K., J., J., V.S. 2001. The release of cytokinin-like compounds
from Gingko biloba leaf material during composting. Environ. Exp. Bot.,
45, 55-61.
AS4454. 1999. Australian standard - composts, soil conditioners and mulches
standards, Association of Australia Homebush, NSW.
Atagana, H.I. 2009. Biodegradation of PAHs by fungi in contaminated-soil
containing cadmium and nickel ions. Afr. J. Biotechnol., 8(21), 5780-
5789.
Avnimelech, Y., Eilat, R., Porat, Y., Kottas, P.A. 2004. Factors affecting the rate
of windrow composting in field studies. Compost sci. util., 12(2), 114-
118.
Bailey, K.L., Lazarovits, G. 2003. Suppressing soil-borne diseases with residue
management and organic amendments. Soil Tillage Res., 72, 169-180.
Bailey, V.L., Smith, J.L., Bolton, H. 2002. Fungal-to-bacterial ratios in soils
investigated for enhanced C sequestration. Soil Biol. Biochem., 997–1007,
34.
Ball-Coelho, B., Tiessen, H., Stewart, J.W.B., Salcedo, I.H., Sampaio, E.V.S.B.
1993. Residue management effects on sugarcane yield and soil properties
in northeastern Brazil. Agron. J., 85, 1004-1008.
252
References
Ball-Coelho, B.R., Sampaio, E.V.S.B., Tiessen, H., Stewart, J.W.B. 1992. Root
dynamics in plant and ratoon crops of sugarcane. Plant Soil, 142, 297-
305.
Barzengar, A.R., Yousefi, A., Daryashenas, A. 2002. The effect of addition of
different amounts and types of organic materials on soil physical
properties and yield of wheat. Plant Soil, 247, 295-301.
Begon, M., Harper, J.L., Townsend., C.R. 1990. Ecology: individuals,
populations, and communities. Blackwell Sci. Pub., 945.
Bell, M.J., Moody, P.W., Connolly, R.D., Bridge, B.J. 1998. The role of active
fractions of soil organic matter in physical and chemical fertility of
Ferrosols. Aust. J. Soil Res., 36, 809-819.
Bell, M.J., Stirling, G.R., Pankhurst, C.E. 2007. Management impacts on health
of soils supporting Australian grain and sugarcane industries. Soil Tillage
Res., 97, 256-271.
Bennet, R.J., Breen, C.M. 1991. The aluminium signal: new dimensions of
aluminium tolerance. Plant Soil, 134, 153-166.
Bernal-Vicente, A., Ros, M., Tittarelli, F., Intrigliolo, F., Pascual, J.A. 2008.
Citrus compost and its water extract for cultivation of melon plants in
greenhouse nurseries. Evaluation of nutriactive and biocontrol effects.
Bioresour. Technol., 99, 8722–8728.
Berner, A., Hildermann, I., Fließbach, A., Pfiffner, L., Niggli, U., Mader, P.
2008. Crop yield and soil fertility response to reduced tillage under
organic management. Soil Tillage Res., 101, 89–96.
Bess, V.H. 2000. Understanding compost tea. BioCycle, 41 (10), 71-72.
253
References
Bingrui, J., Guangsheng, Z. 2009. Integrated diurnal soil respiration model during
growing season of a typical temperate steppe: Effects of temperature, soil
water content and biomass production. Soil Biol. Biochem., 41, 681–686.
Blair, N., Crocker, G.J. 2000. Crop rotation effects on soil carbon and physical
fertility of two Australian soils. Aust. J. Soil Res., 38, 71–84.
Bloem, J., Bolhuis, P.R., Veninga, M.R., Wieringa, J. 1995. Microscopic methods
for counting bacteria and fungi in soil. in: Methods in Applied Soil
Microbiology and Biochemistry, (Eds.) K. Alef, P. Nannipieri, Academic
Press. London, pp. 162–191.
Bloem, J., Lebbink, G., Zwart, K.B., Bouwman, L.A., Burgers, S.L.G.E., de Vos,
J.A., de Ruiter, P.C. 1994. Dynamics of microorganisms, microbivores
and nitrogen mineralisation in winter wheat fields under conventional and
integrated management. Agric. Ecosys. Environ., 51(1-2), 129-143.
Bourn, D., Prescott, J. 2002. A comparison of the nutritional value, sensory
qualities, and food safety of organically and conventionally produced
foods. Crit. Rev. Food Sci. Nutr., 42(1), 1–34.
Bourne, M., Nicotra, A.B., Colloff, M.J., Cunningham, S.A. 2008. Effect of soil
biota on growth and allocation by Eucalyptus microcarpa. Plant Soil,
305(1-2), 145-156.
Brandon, N.J., Date, R.A., Clem, R.L., Robertson, B.A., Graham, T.W.G. 1998.
Growth responses of Desmanthus virgatus to inoculation with Rhizobium
strain CB3126. II. A field trial at 4 sites in south-east Queensland.
Tropical Grasslands, 32, 20-27.
Brinton, W.F. 1995. The control of plant pathogenic fungi by use of compost
teas. Biodynamics, 12-15.
254
References
Brinton, W.F., Storms, P., Evans, E., Hills, J. 2004. Compost teas: Microbial
hygiene and quality in relation to method of preparation. J. Biodynamics,
1-9.
Brinton, W.F., Tranker, A., Droffner, M. 1996. Investigation into liquid compost
extracts. BioCycle, 37(11), 68-70.
Bryant, T. 2008. When under attack, plants can signal microbial friends for help.
in: Science News, University of Delaware.
Buckley, D.H., Schmidt, T.M. 2001. The structure of microbial communities in
soil and the lasting impact of cultivation. Microbial Ecol., 42, 11–21.
Bulluck, L.R., Brosius, M., Evanylo, G.K., Ristaino, J.B. 2002. Organic and
synthetic fertility amendments influence soil microbial, physical and
chemical properties on organic and conventional farms. Appl. Soil Ecol.,
19, 147–160.
Buswell, J.A., Eriksson, K.-E.L. 1994. Effect of olignin-related phenols and their
methylated derivatives on the growth of eight white-rot fungi. World J.
Microbiol. Biotech., 10, 169-174.
Campbell, A. 2006. Overview of compost tea use in New South Wales. Recycled
Organics Unit, Department of Environment and Conservation, The
University of New South Wales.
Canullo, G.H., Rodrıguez-Kabana, R., Kloepper, J.W. 1992. Changes in the
populations of microorganisms associated with the application of soil
amendments to control Sclerotium rolfsii. Plant Soil, 144, 59–66.
Carpenter, L. 2005. Diving into compost tea. BioCycle, 46(7), 61.
Casagrande, A.A., Melo, W.J.D., Pereira, R., Mutton, M.A., Barbosa, J.C.,
Campos, M.S. 1995. Influence of residues from mechanical harvesting
255
References
and vinasse on soil nitrogen in sugarcane ratoon crops. Proc. Int. Soc.
Sugarcane Technol., 22, 67-69.
Casenave de Sanfilippo, E., Arguello, J.A., Abdala, G., Orioli, G.A. 1990.
Content of auxin-, inhibitor- and gibberellin-like substances in humic
acids. Biologia Plantarum, 32, 346–351.
Castella, G., Bragulat, M.R., Rubiales, M.V., Cabañes, F.J. 1997. Malachite
green agar, a new selective medium for Fusarium spp. Mycopathol., 137,
173–178.
Chef, D.G., Hoitink, H.A.J., Madden, L.V. 1983. Effects of organic components
in container media on suppression of Fusarium wilt of crysanthemum and
flax. Phytopathol., 73, 279-281.
Chinn, C., Pillai, U.P.P. 2008. Self-repair of compacted vertisols from Central
Queensland, Australia. Geoderma, 144(3-4), 491-501.
Chu, H., Lin, X., Fujii, T., Morimoto, S., Yagi, K., Hu, J., Zhang, J. 2007. Soil
microbial biomass, dehydrogenase activity, bacterial community structure
in response to long-term fertilizer management. Soil Biol. Biochem., 39,
2971–2976.
Churilova, E.V. 2010. Vermicompost leachate (Vermiliquer) as a liquid fertilizer
for hydroponically-grown pak choi (Brassica chinensis L.) in the tropics.
in: Masters Thesis, Centre for Plant and Water Science, CQUniversity.
Rockhampton, Australia, pp. 215.
Clark, L.J., Whalley, W.R., Ellis-jones, J., Dent, K., Rowse, H.R., Finch-Savage,
W.E., Gatsai, T., Jasi, L., Kaseke, N.E., Murungu, F.S., Riches, C.R.,
Chiduza, C. 2001. On-farm seed priming in maize: a physiological
256
References
evaluation. Seventh Eastern and Southern Africa Regional Maize
Conference. pp. 268-273.
Clarkson, D.T. 1969. Metabolic aspects of aluminium toxicity and some possible
mechanisms for resistance. in: Ecological Aspects of Mineral Nutrition of
Plants, (Ed.) I.H. Rorison, Blackwell Sci. Publ., Oxford and Edinburgh.
Clune, T.S., Copeland, L. 1999. Effects of aluminium on canola roots. Plant Soil,
216, 27-33.
Collard, S.J., Zammit, C. 2006. Effects of land-use intensification on soil carbon
and ecosystem services in Brigalow (Acacia harpophylla) landscapes of
southeast Queensland, Australia. Agri. Ecosys. Environ., 117, 185–194.
Cook, R.J., Baker, K.F. 1983. The nature and practice of biological control of
plant pathogens. Am. Phytopathol. Soc.
Cook, R.J., Thomashow, L.S., Weller, D.M., Fujimoto, D., Mazzola, M.,
Bangera, G., Kim, D.S. 1995. Molecular mechanisms of defense by
rhizobacteria against root disease, Vol. 92, Proc. Natl. Acad. Sci. USA,
pp. 4197-4201.
Cotxarrera, L., Trillas-Gay, M.I., Steinberg, C., Alabouvette, C. 2002. Use of
sewege sludge compost and Trichoderma asperellum isolates to suppress
Fusarium wilt of tomato. Soil Biol. Biochem., 34, 467-476.
Crnko, G.S., Stall, W.M., White, J.M. 1992. Sweet corn weed control evolutions
on mineral and organic soils. pp. 326-330.
Cronin, M.J., Yohalem, D.S., Harris, R.F., Andrews, J.H. 1996. Putative
mechanism and dynamics of inhibition of the apple scab pathogen
Venturia inequalis by compost extracts. Soil Biol. Biochem., 28(9), 1241-
1249.
257
References
Dalal, R.C., Mayer, D.G. 1986. Long-term trends in fertility of soils under
continuous cultivation and cereal cropping in southern Queensland. I.
Overall changes in soil properties and trends in winter cereal yields. Aust.
J. Soil Res., 24, 265–279.
Danielle, O.P., Rai, K.S. 2006. On-farm management practices to minimise off-
site movement of pesticides from furrow irrigation. Pest Management
Sci., 62, 899–911.
Danon, M., Zmora-Nahum, S., Chen, Y., Hadar, Y. 2007. Prolonged compost
curing reduces suppression of Sclerotium rolfsii. Soil Biol. Biochem., 39,
1936-1946.
Davidson, E.A., Verchot, L.V., Henrique, C., Ackerman, I.L., Carvalho, J.E.M.
2000. Effects of soil water content on soil respiration in forests and cattle
pastures of eastern Amazonia. Biogeochem., 48, 53-69.
Dees, P.M., Ghiorse, W.C. 2001. Microbial diversity in hot synthetic compost as
revealed by PCR-amplified rRNA sequences from cultivated isolates and
extracted DNA. FEMS Microbiol. Ecol., 35(207–216).
Delhaize, E., Ryan, P.R. 1995. Aluminum toxicity and tolerance in plants. Plant
Physiol., 107, 315-321.
Deportes, I., Benoit-Guyod, J.L., Zmirou, D.E., Bouvier, M.C. 1995. Hazard to
man and environment posed by the use of urban waste compost: A
review. Sci. Total Environ., 172, 197–222.
Deportes, I., Benoit-Guyod, J.L., Zmirou, D.E., Bouvier, M.C. 1998. Microbial
disinfection capacity of municipal solid waste (MSW) composting. J.
Appl. Microbiol., 85, 238–46.
258
References
Dianez, F., Santos, M., Boix, A., Cara, M., Trillas, I., Avelis, M., Tello, J.C.
2006. Grape marc compost tea suppressiveness to plant pathogenic fungi:
Role of siderophores. Compost Sci. Util., 14(1), 48-53.
Diatloff, E., Harper, S.M., Asher, C.J., Smith, F.W. 1998. Effects of humic and
fulvic acids on the rhizotoxicity of lanthanum and aluminium to corn.
Aust. J. Soil Research, 36, 913-919.
Dilly, O., Bloem, J., Vos, A., Munch, J.C. 2004. Bacterial diversity in agricultural
soils during litter decomposition. Appl. Environ. Microbiol., 70, 468-474.
Diver, S. 2002. Notes on compost teas: A supplement to the ATTRA publication:
compost teas for plant disease control. in: Pest Management Technical
Note, Appropriate Technology Transfer for Rural Areas (ATTRA).
University of Arkansas, Fayetteville, pp. 1-19.
Döbereiner, J., Pedrosa, F.O. 1987. Nitrogen Fixing Bacteria in Non-leguminous
Crop Plants. Science Tech. Madison and Springer Verlag, Berlin.
Dominy, C.S., Haynes, R.J. 2002. Influence of agricultural land management on
organic matter content, microbial activity and aggregate stability in the
profiles of two Oxisols. Biol. Fertil. Soils, 36, 298–305.
Dominy, C.S., Haynes, R.J., van Antwerpen, R. 2002. Loss of soil organic matter
and related soil properties under long-term sugarcane production on two
contrasting soils. Biol. Fertil. Soils, 36, 350–356.
Doran, J.W., Safley, M. 1997. Defining and assessing soil health and sustainable
productivity. in: Biological Indicators of Soil Health, (Eds.) C.E.
Pankhurst, B.M. Doube, V.V.S.R. Gupta, CAB International. New York,
pp. 1–28.
259
References
Doran, J.W., Zeiss, M.R. 2000. Soil health and sustainability: managing the biotic
component of soil quality. Appl. Soil Ecol., 15, 3-11.
DPIF. 2000. Compost. in: Agriculture Notes, State of Victoria, Department of
Primary Industries. Farm Diversification Information Service, Bendigo,
pp. 1-3.
DPIPWE. 2010. Soil Organic Matter, Department of Primary Industries, Parks,
Water and Environment. Hobart, Tasmania, Australia.
Dubeikovsky, A.N., Mordukhova, E.A., Kochetkov, V.V., Polikarpova, F.Y.,
Boronin, A.M. 1993. Growth promotion of blackcurrant softwood cuttings
by recombinant strain Pseudomonas fluorescens BSP53a synthesizing an
increased amount of indole-3-acetic acid. Soil Biol. Biochem., 25, 1277–
1281.
Dumestre, A., Sauve, S., McBride, M., Baveye, P., Berthelin, J. 1999. Copper
speciation and microbial activity in long-term contaminated soils.
Archives Environ. Contamination Toxicol., 36(2), 124-131.
Dursun, A.Y., Aksu, Z. 1999. Biodegradation kinetics of Ferrous (II) cyanide
complex ions by immobilised P. fluorescens in a packed bed column
reactor. Process Biochem., 35, 615-622.
Edwards, C.A. 1998. The use of earthworms in the breakdown and management
of organic wastes. in: Earthworm Ecology, CRC Press. Boca Raton, FL,
pp. 327–354.
Edwards, C.A., Arancon, N.Q., Graytak, S. 2006. Effects of vermicompost teas
on plant growth and disease. BioCycle, 47(5), 28-31.
260
References
Egamberdiyeva, D. 2007. The effect of plant growth promoting bacteria on
growth and nutrient uptake of maize in two different soils. Appl. Soil
Ecol., 36, 184-189.
Eiland, F., Leth, M., Klamer, M., Lind, A.M., Jensen, H.E.K., Iversen, J.J.L.
2001. C and N turnover and lignocellulose degradation during composting
of miscanthus straw and liquid pig manure. Compost Sci. Util., 9(3), 186.
Ekin, Z. 2010. Performance of phosphate solubilizing bacteria for improving
growth and yield of sunflower (Helianthus annuus L.) in the presence of
phosphorus fertilizer. Afr. J. Biotechnol., 9(25), 3794-3800.
El-Masry, M.H., Khalil, A.I., Hassouna, M.S., Ibrahim, H.A.H. 2002. In situ and
in vitro suppressive effect of agricultural composts and their water
extracts on some phytopathogenic fungi. World J. Microbiol. Biotech., 18,
551-558.
Elad, Y., Malathrakis, N.E., A.J.D. 1996. Biological control of Botrytis-incited
diseases and powdery mildews in greenhouse crops. Crop Protection,
15(3), 229-240.
Elad, Y., Steinberg, D. 1994. Effect of compost water extract on grey mould
(Botrytis cinerea). Crop Protect., 13(2), 109-114.
Elad, Y., Williamson, B., Tudzynski, P., Delen, N. 2007. Botrytis: Biology,
Pathology and Control. Kluwer academic publishers, Dordrecht,
Netherlands.
Elad, Y., Yunnis, H., Katan, T. 1992. Multiple fungicide resistance to
benzimidazoles, dicarboximides and diethofencarb in field isolates of
Botrytis cinerea in Israel. Plant Pathol., 41, 41-46.
261
References
Elfstrand, S., Hedlund, K., Martensson, A. 2007. Soil enzyme activities,
microbial community composition and function after 47 years of
continuous green manuring. Appl. Soil Ecol., 35, 610–621.
Elliot, S.L., Mumford, J.D., Moraes, G.J.d. 2002. The role of resting spores in the
survival of the mite-pathogenic fungus Neozygites floridana from
Mononychellus tanajoa during dry periods in Brazil. J. Invertebrate
Pathol., 81, 148–157.
Elliott, L.F., Lynch, J.M. 1994. Biodiversity and soil resilience. in: Soil
Resilience and Sustainable Land Use, (Eds.) D.J. Greenland, I. Szabolc,
CAB International. Wallingford, UK, pp. 353–364.
Erhart, E., Burian, K., Hartl, W., Stich, K. 1999. Suppression of Pythium ultimum
by biowaste composts in relation to compost microbial biomass, activity
and content of phenolic compounds. J. Phytopathol., 147, 299–305.
Ericsson, T. 1995. Growth and shoot: root ratio of seedlings in relation to nutrient
availability. Plant Soil, 168-169, 205-214.
Etter, L. 2008. Lofty prices for fertiliser put farmers in a squeeze. Wall Street J.
Ezzi, M.I., Lynch, J.M. 2002. Cyanide catabolising enzymes in Trichoderma spp.
Enzyme Microb. Tech., 31, 1042-1047.
Fayed, T.A. 2010a. Effect of compost tea and some antioxidant applications on
leaf chemical constituents, yield and fruit quality of pomegranate. World
J. Agric. Sci., 6(4), 402-411.
Fayed, T.A. 2010b. Optimizing, yield fruit quality and nutrition status of
Roghiani olives grown in Libya using some organic extracts. J. Hort. Sci.
Ornamental Plants, 2(2), 63-78.
262
References
Feng, X., Simpson, M.J. 2009. Temperature and substrate controls on microbial
phospholipid fatty acid composition during incubation of grassland soils
contrasting in organic matter quality. Soil Biol. Biochem., 41, 804–812.
Ferreras, L., Gomez, E., Toresani, S., Firpo, I., Rotondo, R. 2006. Effect of
organic amendments on some physical, chemical and biological properties
in a horticultural soil. Bioresour. Technol., 97, 635–640.
Finlay, R.D., Rosling, A. 2006. Integrated nutrient cycles in boreal forest
ecosystems – the role of mycorrhizal fungi. in: Fungi in Biogeochemical
Cycles, (Ed.) G.M. Gadd, Published by Cambridge.
Fontvieille, D.A., Outaguerouine, A., Thevenot, D.R. 1992. Fluorescein diacetate
hydrolysis as a measure of microbial activity in aquatic systems:
application to activated sludges. Environ. Technol., 13, 531-540.
Foy, C.D. 1974. Effects of aluminium on plant growth. in: The plant root and its
environment, (Ed.) E.W. Carson, Charlottesville Univ. Press. Virginia.
Foy, C.D. 1992. Soil chemical factors limiting plant root growth. Adv. Soil Sci.,
19, 97–149.
Foy, C.D., Chaney, R.L., M.C., W. 1978. The physiology of metal toxicity in
plants. Annu. Rev. Plant Physiol., 29, 511-566.
Franche, C., Lindström, K., Elmerich, C. 2009. Nitrogen-fixing bacteria
associated with leguminous and non-leguminous plants. Plant Soil, 321,
35–59.
Freeman, G.G., Baillie, A.J., Macinnes, C.A. 1948. Bacterial degradation of
CMC and methyl ethyl cellulose. Chem. & Industry, 279-282.
263
References
Garcia-Gil, J.C., Plaza, C., Soler-Rovira, P., Polo, A. 2000. Long-term effects of
municipal solid waste compost application on soil enzyme activities and
microbial biomass. Soil Biol. Biochem., 32, 1907-1913.
Garcıa-Gomez, A., Roig, A., Bernal, M.P. 2003. Composting of the solid fraction
of olive mill wastewater with olive leaves: organic matter degradation and
biological activity. Bioresour. Technol., 86, 59–64.
Garcia-Ochoa, F., Gomez, E., Santos, V.E., Merchuk, J.C. 2010. Oxygen uptake
rate in microbial processes: An overview. Biochem. Eng. J., 49, 289–307.
Garcia, C., Hernandez, T., Costa, F. 1991. Study on water extract of sewage
sludge composts. Soil Sci, Plant Nutr., 37(3), 399-408.
Garcia, M.I., Cruz Sosa, F., Saavedra, A.L., Hernandez, M.S. 2002. Extraction of
auxin-like substances from compost. Crop Research, 24, 323-327.
Garg, S.K., Bhatnagar, A., Kalla, A., Narula, N. 2001. In vitro nitrogen fixation,
phosphate solubilization, survival and nutrient release by Azotobacter
strains in an acquatic systems. Bioresour. Technol., 80, 101-109.
Garland, J.L. 1997. Analysis and interpretation of community-level physiological
profiles in microbial ecology. FEMS Microbiol. Ecol., 24, 289-300.
Garland, J.L. 1996. Analytical approaches to the characterization of samples of
microbial communities using patterns of potential C source utilization.
Soil Biol. Biochem., 28, 213–221.
Garland, J.L. 1999. Potential and limitations of BIOLOG for microbial
community analysis. Microbial Biosystems: New Frontiers: Proceedings
of the 8th International Symposium on Microbial Ecology, Halifax,
Canada. Atlantic Canada society for microbial ecology.
264
References
Garland, J.L., Mills, A.L. 1991. Classification and characterization of
heterotrophic microbial communities on the basis of patterns on
community-level, sole-carbon-source utilization. Appl. Environ.
Microbial., 57, 2351–2359.
GENSTAT. 2008. GenStat Procedure Library Release PL16. Eleventh ed.
Gerhardt, R.A. 1997. A comparative analysis of the effects of organic and
conventional farming systems on soil structure. Biol. Agricultur.
Horticol., 14, 139-157.
Giller, K.E., Beare, M.H., Lavelle, P., Izac, A.M.N., Swift, M.J. 1997.
Agricultural intensification, soil biodiversity and agroecosystem function.
Appl. Soil Ecol., 6, 3–16.
Girvan, M.S., Bullimore, J., Ball, A.S., Pretty, J.N., Osborn, A.M. 2004.
Responses of active bacterial and fungal communities in soils under
winter wheat to different fertilizer and pesticide regimens. Appl. Environ.
Microbiol., 70, 2692–2701.
Girvan, M.S., Bullimore, J.N., Osborn, A.M., Ball, A.S. 2003. Soil type is the
primary determinant of the composition of the total and active bacterial
communities in arable soil. Appl. Environ. Microbiol., 69, 1800-1809.
Golchin, A., Clarke, P., Oades, J.M., Skjemstad, J.O. 1995. The effects of
cultivation on the composition of organic matter and structural stability of
soils. Aust. J. Soil Res., 33, 975–993.
Gomez, E., Ferreras, L., Toresani, S., Ausilio, A., Bisaro, V. 2001. Changes in
some soil properties in a vertic argiudoll under shortterm conservation
tillage. Soil Till. Res., 61, 179–186.
265
References
Graham, M.H., Haynes, R.J. 2005. Catabolic diversity of soil microbial
communities under sugarcane and other land uses estimated by Biolog
and substrate-induced respiration methods. Appl. Soil Ecol., 29, 155–164.
Graham, M.H., Haynes, R.J., Meyer, J.H. 2002. Soil organic matter content and
quality: Effects of fertilizer applications, burning and trash retention on a
long-term sugarcane experiment in South Africa. Soil Biol. Biochem., 34,
93-102.
Grayston, S.J., Campbell, C.D., Bardgett, R.D., Mawdsley, J.L., Clegg, C.D.,
Ritz, K., Griffiths, B.S., Rodwell, J.S., Edwards, S.J., Davies, W.J.,
Elston, D.J., Millard, P. 2004. Assessing shifts in microbial community
structure across a range of grasslands of differing management intensity
using CLPP, PLFA and community DNA techniques. Appl. Soil Ecol., 25,
63–84.
Greathouse, G. 1949. Microbiological degradation of cellulose. Am. Chem.Soc.
Meeting.
Green, V.S., Stott, D.E., Diack, M. 2006. Assay for fluorescein diacetate
hydrolytic activity: Optimisation for soil samples. Soil Biol. Biochem., 38,
693-701.
Griffiths, E. 1965. Micro-organisms and soil structure. Biol. Rev., 40(1), 129–
142.
Grobe, K. 2003. California landscape contractor calls it compost tea time.
BioCycle, 44(2), 26-27.
Grundy, A.C., Green, J.M., Lennartsson, M. 1998. The effect of temperature on
the viability of weed seeds in compost. Compost Sci. Util., 6, 26–33.
266
References
Gurbuz, F., Ciftci, H., Akcil, A. 2009. Biodegradation of cyanide containing
effluents by Scenedesmus obliquus. J. Hazard. Mater., 162, 74-79.
Ha, K.V., Marschner, P., Bünemann, E.K. 2008. Dynamics of C, N, P and
microbial community composition in particulate soil organic matter
during residue decomposition. Plant Soil 303, 253–264.
Haaba, D., Hagspiela, K., Szakmarya, K., Kubicek, C.P. 1990. Formation of the
extracellular proteases from Trichoderma reesei QM 9414 involved in
cellulase degradation. J. Biotechnol., 16(3-4), 187-198.
Hadar, Y., Gorodecki, B. 1991. Suppression of germination of sclerotia of
Sclerotium rolfsii in compost. Soil Biol. Biochem., 23, 303-306.
Haggag, W.M., Saber, M.S.M. 2007. Suppression of early blight on tomato and
purple blight on onion by foliar sprays of aerated and non-aerated
compost teas. J. Food Agri. Environ., 5(2), 302-309.
Hall, S.G., Schellinger, D.A., Carney, W.A. 2006. Enhancing sugarcane field
residue biodegradation by grinding and use of compost tea. Compost Sci.
Util., 14, 32-38.
Halsall, D.M., Gibson, A.H. 1985. Cellulose decomposition and associated
nitrogen fixation by mixed cultures of Cellulomonas gelida and
Azospirillum Species or Bacillus macerans. Appl. Environ. Microbiol.,
50(4), 1021-1026.
Han, G.X., Zhou, G.S., Xu, Z.Z., Yang, Y., Liu, J.L., Shi, K.Q. 2007. Soil
temperature and biotic factors drive the seasonal variation of soil
respiration in a maize (Zea mays L.) agricultural ecosystem. Plant Soil,
291, 15–26.
267
References
Hardy, G.E., Sivasithamparam, K. 1991. Effects of sterile and non-sterile
leachates extract from composted Eucalyptus-bark and Pine-bark
container on Phytophthora spp. Soil Biol. Biochem., 23, 25-30.
Hargreaves, J.C., Adl, M.S., Warman, P.R. 2009. The effects of municipal solid
waste compost and compost tea on mineral element uptake and fruit
quality of strawberries. Compost Sci. Util., 17(2), 85-94.
Hargreaves, J.C., Adl, M.S., Warman, P.R., Rupasinghe, H.P.V. 2008. The
effects of organic amendments on mineral element uptake and fruit
quality of raspberries. Plant Soil, 308, 213-226.
Harman, G.E. 2006. Overview of mechanisms and uses of Trichoderma spp.
Phytopathol., 96(2), 190-194.
Harper, S.M., Edwards, D.G., Kerven, G.L., Asher, C.J. 1995. Effects of organic
acid fractions extracted from Eucalyptus camaldulensis leaves on root
elongation of maize (Zea mays) in the presence and absence of
aluminium. Plant Soil, 171, 189-192.
Harrower, K.M. 2006. Assessing fungal growth in the undergraduate teaching
laboratory. Aust. Mycol., 25(1), 1-4.
Hattenschwiler, S., Tiunov, A.V., Scheu, S. 2005. Biodiversity and litter
decomposition in terrestrial ecosystems. Annu. Rev. Ecol. Evol. Syst., 36,
191–218.
Heal, O.W., Anderson, J.W., Swift, M.J. 1997. Plant litter quality and
decomposition: an historical overview. in: Driven by Nature: Plant Litter
Quality and Decomposition, (Eds.) G. Cadisch, K.E. Giller, CAB
International. Wallingford, UK, pp. 3-30.
268
References
Henis, Y., Lewis, J.A., Papavizas, G.C. 1984. Interactions between Sclerotium
rolfsii and Trichoderma spp. - relationship between antagonism and
disease control. Soil Biol. & Biochem., 16, 391-395.
Henning, S.J. 1975. Aluminium toxicity in the primary meristem of wheat roots,
Vol. PhD, PhD thesis, Oregon State University. Oregon, United States.
Hernandez, D., Fernandez, J.M., Plaza, C., Polo, A. 2007. Water-soluble organic
matter and biological activity of a degraded soil amended with pig slurry.
Sci. Tot. Environ., 378, 101-103.
Herrmann, R.F., Shann, J.F. 1997. Microbial community changes during the
composting of municipal solid waste. Microb. Ecol., 33, 78–85.
Hibar, K., Daami-Remadi, M., Jabnoun-Khiareddine, H., Znaïdi, I.E., El-
Mahjoub, M. 2006. Effect of compost tea on mycelial growth and disease
severity of Fusarium oxysporum f. sp. radicis-lycopersici. Biotechnol.
Agron. Soc. Environ., 10(2), 101-108.
Highley, T.L. 1980. Cellulose degradation by cellulose clearing and non-cellulose
clearing brown rot fungi. Appl. Environ. Microbiol., 40(6), 1145-1147.
Hoitink, H.A.J., Boehm, M.J. 1999a. Biocontrol within the context of soil
microbial communities: A substrate-dependent phenomenon. Annu. Rev.
Phytopathol., 37, 427–446.
Hoitink, H.A.J., Boehm, M.J. 1999b. Biocontrol within the context of soil
microbial communities: A substrate-dependent phenomenon. Annu.
Rev.Phytopathol., 37, 427–446.
Hoitink, H.A.J., Stone, A.G., Han, D.Y. 1997. Suppression of plant diseases by
composts. Hort. Sci., 32, 184-187.
269
References
Holtan-Hartwig, L., Dorsch, P., Bakken, L.R. 2002. Low temperature control of
soil denitrifying communities: kinetics of N2O production and reduction.
Hoyos-Carvajal, L., Orduz, S., Bissett, J. 2009. Growth stimulation in bean
(Phaseolus vulgaris L.) by Trichoderma. Biol. Cont., 51, 409–416.
Hu, W.H., Zhou, Y.H., Du, Y.S., Xia, X.J., Yu, J.Q. 2006. Differential response
of photosynthesis in greenhouse- and field-ecotypes of tomato to long-
term chilling under low light. J. Plant Physiol., 163, 1238—1246.
Hue, N.V., Amien, I. 1989. Aluminium detoxification with green manures.
Commun. Soil Sci. Plant Anal., 20(15 & 16), 1499-1511.
Imray, P., Moore, M.R., Callan, P.W., Lock, W. 1995. Aluminium.
Ingham, E.R. 1998. Anaerobic bacteria and compost tea. BioCycle, 39(6), 86-86.
Ingham, E.R. 2000. Brewing Compost Tea. Kitchen Gardener, 29.
Ingham, E.R. 2003. Compost tea – promises & practicalities. in: Reprinted from
ACRESUSA, a voice for eco-agriculture, Vol. 33.
Ingham, E.R. 2005. The Compost Tea Brewing Manual. Fifth ed. US Printings,
Soil Foodweb Incorporated, Oregon.
Ingham, E.R. 2004. Compost Tea Quality: Light Microscope Methods. Soil
Foodweb Inc, Corvallis, Oregon
Ingham, E.R. 1999a. Making a high quality compost tea, Part II. BioCycle, 40(4),
94.
Ingham, E.R. 1999b. What is compost tea? Part I. BioCycle, 40(3), 74-75.
Ingham, E.R., Alms, M. 1999. Compost Tea Manual. Soil Food Web, Growing
Solutions Inc., Corvallis, OR.
270
References
Insam, H. 1997. Substrate utilisation test in microbial ecology: A preface to the
special issue. J. Microbial Methods, 30, 1-2.
Jackson, W.A. 1967. Physiological effects of soil acidity. in: Soil Acidity and
Liming, (Eds.) R.W. Pearson, F. Adams, Vol. 12, Am. Soc. agron.
Madison, Wis, pp. 43-124.
Janvier, C., Villeneuve, F., Alabouvette, C., Edel-Hermann, V., Mateille, T.,
Steinberg, C. 2007. Soil health through soil disease suppression: Which
strategy from descriptors to indicators? Soil Biol. Biochem., 39, 1–23.
Jany, J., Barbier, G. 2008. Culture-independent methods for identifying microbial
communities in cheese. Food Microbiol., 25, 839-848.
Janzen, R.A., Cook, F.D., McGill, W.B. 1995. Compost extract added to
microcosms may stimulate community-level controls on soil
microorganisms involved in element cycling. Soil Biol. Biochem., 27(2),
181-188.
Jesus, E.d.C., Marsh, T.L., Tiedje, J.M., Moreira, F.M.d.S. 2009. Changes in land
use alter the structure of bacterial communities in Western Amazon soils.
Int. Soc. Micro. Ecol. J., 3, 1004–1011.
Jing, H., Liu, H., Wong, T., Chen, M. 2010. Impact of mesozooplankton grazing
on the microbial community revealed by denaturing gradient gel
electrophoresis (DGGE). J. Experimental Marine Biol. Ecol., 383, 39–47.
Jones, D., Wyatt, D. 2005. Super Brewing Guidelines: The Vermicrobe System
Manual.
Jones, K., Bangs, D. 1985. Nitrogen fixation by free-living heterotrophic bacteria
in an oak forest: The effect of liming. Soil Biol. Biochem., 17(5), 705-709.
271
References
Jones, P., Martin, M. 2003. A review of the literature on the occurance and
survival of pathogens of animals and humans in green compost. Institute
for Animal Health, Compton.
Joshi, D., Hooda, K.S., Bhatt, J.C., Mina, B.L., Gupta, H.S. 2009. Suppressive
effects of composts on soil-borne and foliar diseases of French bean in the
field in the western Indian Himalayas. Crop Protec., 28, 608–615.
Kahindi, J.H.P., Woomer, P., George, T., Moreira, F.M.d.S., Karanja, N.K.,
Giller, K.E. 1997. Agricultural intensification, soil biodiversity and
ecosystem function in the tropics: the role of nitrogen-fixing bacteria.
Appl. Soil Ecol., 6, 55-76.
Kai, H., Udea, T., Sakaguchi, M. 1990. Antimicrobial activity of bark compost
extracts. Soil Biol. Biochem., 7, 983-986.
Kaiser, E.-A., Martens, R., Heinemeyer, O. 1995. Temporal changes in soil
microbial biomass carbon in an arable soil:consequences for soil
sampling. Plant Soil, 170, 287-295.
Kale, R.D., Mallesh, B.C., Bano, K., Bagyaraj, D.J. 1992. Influence of
vermicompost applications on the available macro nutrients and selected
microbial population in a paddy field. Soil Biol. Biochem., 24(12), 1317-
1320.
Kelley, S. 2004. Building a knowledge base for compost tea. BioCycle, 45(6), 30-
34.
Ketterer, N., Fisher, B., Weltzien, H.C. 1992. Biological control of Botrytis
cinerea on grapevine by compost extracts and their microorganisms in
pure culture. Proceedings of the 10th International Botrytis Symposium,
Wageningen, The Netherlands. pp. 179-186.
272
References
Keyser, H.H., Munns, D.N. 1978. Tolerance of rhizobia to acidity, aluminum and
phosphate. Soil Sci. Soc.Am. J., 43, 519-523.
Khammas, K.M., Kaiser, P. 1992. Pectin decomposition and associated nitrogen
fixation by mixed cultures of Azospirillum and Bacillus species. Can. J.
Microbiol., 38, 794-797.
Kingston, G., Norris, C. 2001. The green cane harvesting system - an Australian
perspective. in: Innovative approaches to sugarcane productivity in the
new millennium, American Society of Sugar Cane Technologists. Miami,
Florida, pp. 9.
Kloepper, J.W., Zehnder, G.W., Tuzun, S., Murphy, J.F., Wei, G., Yao, C.,
Raupach, G.S. 1996. Toward agricultural implementation of PGPR-
mediated induced systemic resistance against crop pests. in: Advances in
Biological Control of Plant Diseases, (Eds.) T. Wenhua, R.J. Cook, A.
Rovira, China Agricultural University Press. Beijing, pp. 165-174.
Koné, S.B., Dionne, A., Tweddell, R.J., Antoun, H., Avis, T.J. 2010. Suppressive
effect of non-aerated compost teas on foliar fungal pathogens of tomato.
Biol. Cont., 52, 167–173.
Konopka, A., Oliver, L., Turco, J. 1998. The use of carbon substrate utilization
patterns in environmental and ecological microbiology. Microb. Ecol., 35,
103-115.
Koschinsky, S., Peters, S., Schwieger, F., Tebbe, C.C. 1999. Applying molecular
techniques to monitor microbial communities in composting processes. in:
Microbial Biosystems: New Frontiers, Microbial Processes during
Composting, (Eds.) C.R. Bell, M. Brylinsky, P. Johnson-Green. Atlantic
Canada Society for Microbial Ecology, Halifax, Canada.
273
References
Krebs, C.J. 1999. Ecological Methodology, (Ed.) B. Cummings. Menlo Park,
California, pp. 620.
Lalande, R., Gagnon, B., Simard, R.R., Cote, D. 2000. Soil microbial biomass
and enzyme activity following liquid hog manure application in a long-
term field trial. Can. J. Soil Sci., 80, 263-269.
Lampkin, N. 1990. Organic Farming. in: Farming Press Books, Ipswich, UK.
Larkin, R.P. 2008. Relative effects of biological amendments and crop rotations
on soil microbial communities and soilborne diseases of potato. Soil Biol.
Biochem., 40, 1341-1351.
Lauber, C.L., Strickland, M.S., Bradford, M.A., Fierer, N. 2008. The influence of
soil properties on the structure of bacterial and fungal communities across
land-use types. Soil Biol. Biochem., 40, 2407–2415.
Lee, K.E., Pankhurst, C.E. 1992. Soil organisms and sustainable productivity.
Aust. J. Soil Research, 30(6), 855-892.
Lemenih, M., Karltun, E., Olsson, M. 2005. Assessing soil chemical and physical
property responses to deforestation and subsequent cultivation in
smallholders farming system in Ethiopia. Agric. Ecosyst. Environ., 105,
373–386.
Leu, A. 2006. Organics and soil carbon: increasing soil carbon, crop productivity
and farm profitability. Third OFA National Organic Conference,
“Organics – Solutions to Climate Change”, Sydney, Australia. pp. 4-12.
Liang, Y., Nikolic, M., Peng, Y., Chen, W., Jiang, Y. 2005. Organic manure
stimulates biological activity and barley growth in soil subject to
secondary salinization. Soil Biol. Biochem., 37, 1185-1195.
274
References
Linehan, D.J. 1976. Some effects of a fulvic acid component of soil organic
matter on the growth of cultured excised tomato roots. Soil Biol.
Biochem., 8, 511-517.
Lines-Kelly, R. 2004. Current research into soil biology in agriculture. NSW.
Department of Primary Industries.
Litterick, A.M., Harrier, L., Wallace, P., Watson, C.A., Wood, M. 2004. The role
of uncomposted materials, composts, manures, and compost extracts in
reducing pest and disease incidence and severity in sustainable temperate
agricultural and horticultural crop production - A review. Critical Rev.
Plant Sci., 26(3), 453-479.
Liu, B., Tu, C., Hu, S., Gumpertz, M., Ristaino, J.B. 2007. Effect of organic,
sustainable, and conventional management strategies in grower fields on
soil physical, chemical, and biological factors and the incidence of
Southern blight. Appl. Soil Ecol., 37, 204-214.
Liu, L., Kloepper, J.W., Tuzun, S. 1995. Induction of systemic resistance in
cucumber against bacterial angular leaf spot by plant growth-promoting
rhizobacteria. Phytopathol., 85, 843-847.
Lloyd, J., Taylor, J.A. 1994. On the temperature dependence of soil respiration.
Functional Ecol., 8, 315–323.
Ma, J.F., Ryan, P.R., Delhaize, E. 2001. Aluminium tolerance in plants and the
complexing role of organic acids. Trends Plant Sci., 6(6), 273-278.
Ma, L., Xiongwu, Q., Fen, G., Bianqing, H. 2001b. Control of sweet pepper
Fusarium wilt extracts and compost with its mechanism. Chin. J. Appl.
Env. Biol., 7(1), 84–87.
275
References
Mabbutt, J.A. 1992. Degradation of the Australian drylands: a historical
approach. in: Degradation and Restoration of Arid Lands, (Ed.) H.E.
Dregne, pp. 27–98.
Maggioni, A., Varanini, Z., Nardi, S., Pinton, R. 1987. Action of soil humic
matter on plant roots: stimulation of ion uptake and effects on (Mg 2+ + K
+) ATPase activity. Sci. Total Environ., 62, 355-363.
Magid, J., De Neergaard, A., Brandt, M. 2006. Heterogenous distribution may
substantially decrease initial decomposition, long-term microbial growth
and N-immobilization from high C-to-N ratio resources. Eur. J. Soil Sci.,
57(517-529).
Mann, L.K. 1986. Changes in soil carbon storage after cultivation. Soil Sci., 142,
279–288.
Marcote, I., Hernandez, T., Garcia, C., Polo, A. 2001. Influence of one or two
successive annual applications of organic fertilizers on the enzyme
activity of a soil under barley cultivation. Bioresour. Technol., 79(2), 147-
154.
Marmonier, P., Fontvieille, D., Gibert, J., Vanek, V. 1995. Distribution of
dissolved organic carbon and bacteria at the interface between the Rhone
River and its alluvial aquifer. J. North Am. Benthol. Soc., 14, 382-392.
Martens, M.-H.R. 2001. Compost tea - Just what the doctor ordered. A Voice for
Eco-Agriculture, 31(2), 13.
Mato, M.C., Gonzalez-Alonso, L.M., Mendez, J. 1972a. Inhibition of enzymatic
indoleacetic acid oxidation by soil fulvic acids. Soil Biol. Biochem., 475-
478.
276
References
Mato, M.C., Olmedo, M.G., Mendez, J. 1972b. Inhibition of indoleacetic acid-
oxidase by soil humic acids fractionated on sephadex. Soil Biol. Biochem.,
4, 469-473.
Matsumoto, H., Hirasawa, R., Toricari, H., Takahashi, E. 1976. Localisation of
absorbed aluminium in pea roots and its binding to nucleic acids. Plant
Cell Physiol., 18, 325-335.
Matsumoto, H., Morimura, S., Takahashi, E. 1977. Less environment of pectin in
the precipitation of aluminium in pea roots. Plant Cell Physiol., 17(127-
137).
Mazzola, M. 2004. Assessment and management of soil microbial community
structure for disease suppression. Annu. Rev. Phytopathol., 42, 35-59.
McCaig, A.E., Grayston, S.J., Prosser, J.I., Glover, L.A. 2001. Impact of
cultivation on characterisation of species composition of soil bacterial
communities. FEMS Microbiol. Ecol., 35, 37-48.
McQuilken, M.P., Whipps, J.M., Lynch, J.M. 1994. Effects of water extracts of a
composted manure-straw mixture on the plant pathogen Botrytis cinerea.
World J. Microbiol. Biotech., 10, 20-26.
Meier, E. 2007. Availability of nitrogen in green cane trash blanketed soils in the
wet tropics and its impact on productivity / profitability: a systems
analysis. in: School of Natural and Rural Systems Management, Vol. PhD,
The University of Queensland. Brisbane, pp. 231.
Melero, S., Porras, J.C.R., Herencia, J.F., Madejon, E. 2006. Chemical and
biochemical properties in a silty loam soil under conventional and organic
management. Soil Tillage Res., 90, 162–170.
277
References
Merrill, R., Hoberechi, K., McKeon, J. 1997. Organic teas from composts and
manures. Cabrillo Community College.
Metcalf, Eddy, I. 2003. Wastewater Engineering: Treatment and Reuse. Fourth
ed. McGraw-Hill International editions, New York.
Michel, F.C., Forney, L.J., Huang, A.J.F., Drew, S., Czuprenski, M., Lindeberg,
J.D., Reddy, C.A. 1996a. Effects of turning frequency, leaves to grass mix
ratio and windrow vs. pile configuration on the composting of yard
trimming. Compost Sci. Util., 4(1), 26-43.
Michel, F.C., Huang, A.J.F., Forney, L.J., Reddy, C.A. 1996b. Field scale study
of the effect of pile size, turning regime and leaf to grass mix ratio on the
composting of yard trimmings. in: The Science of Composting Part 1,
(Eds.) M.D. Bertoldi, P. Sequi, P. Lemmes, T. Papi. London, U.K., pp.
577-584.
Midmore, D.J. 2006. Intensive organic production systems and issues of
sustainability. Primary Industries Research Centre, Central Queensland
University, Australia.
Midmore, D.J., Wu, D.L. 1999. Work that water! Hydroponics made easy.
Waterlines, 17(4), 28-30.
Misra, R.V., Roy, R.N., Hiraoka, H. 2003. On-farm composting methods.
Mitchell, R.D.J., Thorburn, P.J., Larsen, P. 2000. Quantifying the loss of
nutrients from the immediate area when sugarcane residues are burnt.
Proceedings of the Australian Society of Sugar Cane Technologists. pp.
206-211.
Moeskops, B., Sukristiyonubowo, Buchan, D., Sleutel, S., Herawaty, L., Husen,
E., Saraswati, R., Setyorini, D., De Neve, S. 2010. Soil microbial
278
References
communities and activities under intensive organic and conventional
vegetable farming in West Java, Indonesia. Appl. Soil Ecol., 45, 112-120.
Moon, P. 1997. Final report on basic on-farm composting manual. CM-97-3.
Munns, D.N. 1977. Soil acidity and related factors. in: Exploiting the Legume-
Rhizobium Symbiosis in Tropical Agriculture, (Eds.) J.M. Vincent, A.J.
Whitney, J.B. Niftal. Hawaii, pp. 211-236.
Munns, D.N., Keyser, H.H. 1981. Response of Rhizobium strains to acid and
aluminium stress. Soil Biol. Biochem., 13, 115-118.
Murray, A.E., Hollibaugh, J.T., Orrego, C. 1996. Phylogenetic compositions of
bacterioplankton from two Californian estuaries by denaturing gradient
gel electrophoresis of 16S rDNA fragments. App. Environ. Microbiol., 59,
2676-2680.
Muscolo, A., Bovalo, F., Gionfriddo, F., Nardi, S. 1999. Earthworm humic matter
produces auxin-like effects on Daucus carota cell growth and nitrate
metabolism. Soil Biol. Biochem., 31, 1303-1311.
Mussaddak, J., Fawaz, K. 2008. Assessment of injection of liquid rhizobial
inoculum and traditional inoculation of soybean under furrow and drip
irrigation. Agrochimica, 52(1), 1-11.
Muyzer, G. 1999. DGGE/TGGE a method for identifying genes from natural
ecosystems. Curr. Opin. Microbiol., 2(3), 317-322.
Muyzer, G., Smalla, K. 1998. Application of denaturing gradient gel
electrophoresis (DGGE) and temperature gradient gel electrophoresis
(TGGE) in microbial ecology. Antonie Van Leeuwenhoek, 73(1), 127-41.
Muyzer, G., Wall, E.C.D., Uitterlinden, A.G. 1993. Profiling of complex
microbial populations by denaturing gradient gel electrophoresis analysis
279
References
of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl.
Environ. Microbiol., 59, 695–700.
Naidu, Y., Meon, S., Kadir, J., Siddiqui, Y. 2010. Microbial starter for the
enhancement of biological activity of compost tea. Int. J. Agric. Biol., 12,
51–56.
Ndegwa, P.M., Thompson, S.A. 2000. Effects if C-to-N ratio on
vermicomposting of biosolids. Bioresour. Technol., 75, 7-12.
Nehls, U. 2008. Mastering ectomycorrhizal symbiosis: the impact of
carbohydrates. J. Experimental Bot., 59(5), 1097–1108.
Nelson, E.B., Hoitink, H.A.J. 1983. The role of microorganisms in the
suppression of Rhizoctonia solani in container media amended with
composted hardwood bark. Phytopathol., 73, 274-278.
Nelson, P.N., Oades, J.M. 1998. Organic matter, sodicity, and soil structure. in:
Sodic Soils, (Eds.) M.E. Summer, R. Naidu, Oxford University Press.
New York, pp. 51-75.
Neuhauser, E.F., Loehr, R.C., Malecki, M.R. 1988. The potential of earthworms
for managing sewage sludge. in: Earthworms in Waste and Environmental
Management, (Eds.) C.A. Edwards, E.F. Neuhauser, SPB Academic
Publishing. Netherlands, pp. 9–20.
Nilsson, T. 1974. Comparative study on the cellulolytic activity of white-rot and
brown-rot fungi. Mater. Org., 9, 173-198.
Nilsson, T., Ginns, J. 1979. Cellulolytic activity and the taxonomic position of
selected brown-rot fungi. Mycologia, 71, 170-177.
280
References
Norrish, S. 1996. Constraints to the adoption of green cane trash blanketing in
central and southern districts. Sugar Research and Development
Corporation. BS109S.
NOSB. 2004. Compost tea task force report. National Organic Standards Board.
Nosko, P., Brassard, P., Kramer, J.R., Kershaw, K.A. 1988. The effect of
aluminium on seed germination and early seedling establishment growth
and respiration of white spruce (Picea glauca). Can. J. Bot., 66, 2305-
2310.
Ntougias, S., Ehaliotis, C., Papadopoulou, K.K., Zervakis, G. 2006. Application
of respiration and FDA hydrolysis measurements for estimating microbial
activity during composting processes. Biol. Fertility Soil, 42, 330-337.
Ntougias, S., Zervakis, G.I., Kavroulakis, N., Ehaliotis, C., Papadopoulou, K.K.
2004. Bacterial diversity in spent mushroom compost assessed by
amplified rDNA restriction analysis and sequencing of cultivated isolates.
Systematic Appl. Microbiol., 27, 746-754.
Nusslein, K., Tiedje, J.M. 1999. Soil bacterial community shift correlated with
change from forest to pasture vegetation in a tropical soil. Appl. Environ.
Microbiol., 65, 3622-3626.
O‘Grady, R., Alexander, M. 2008. Healthy Soils Workshop. Australia.
Okur, N. 2002. Response of soil biological and biochemical activity to salination.
The Journal of Agricultural Faculty of Ege University, 39(1), 87-93.
Olanya, O.M., Larkin, R.P. 2006. Efficacy of essential soils and biopesticides on
Phytopthora infestans suppression in laboratory and growth chamber
studies. Biocontrol Sci.Technol., 16(9), 901-917.
281
References
Oldeman, L., van Engelen, V., Pulles, J. 1990. The Extent of Human-induced Soil
Degradation. International Soil Reference and Information Centre,
Wageningen.
Omay, A.B., Rice, C.W., Maddux, L.D., Gordon, W.B. 1998. Corn yield and
nitrogen uptake in monoculture and in rotation with soybean. Soil Sci.
Soc. Am. J., 62, 1596-1603.
OSUE. 1999. Ohio’s Livestock and Poultry Mortality Composting Manual. Ohio
State Univ. Ext. Pub.
Ovreas, L., Jensen, S., Daae, F.L., Torsvik, V. 1998. Microbial community
changes in a perturbed agricultural soil investigated by molecular and
physiological approaches. Appl. Environ. Microbiol., 64, 2739-2742.
Pankhurst, C.E., Doube, B.M., Gupta, V.V.S.R. 1997. Biological indicators of
soil health: synthesis. in: Biological indicators of soil health, (Eds.) C.E.
Pankhurst, B.M. Doube, V.V.S.R. Gupta, CAB International. New York,
pp. 419–435.
Pant, A.P., Radovich, T.J.K., Hue, N.V., Talcott, S.T., Krenek, K.A. 2009.
Vermicompost extracts influence growth, mineral nutrients,
phytonutrients and antioxidant activity in pak choi (Brassica rapa cv.
Bonsai, Chinensis group) grown under vermicompost and chemical
fertiliser. J. Sci. Food Agri.
Parkin, T.B., Doran, J.W., Franco-Vizcaino, E. 1996. Field and laboratory tests of
soil respiration. in: Methods for Assessing Soil Quality, (Eds.) J.W. Doran,
A.J. Jones, SSSA Special Publication, pp. 231–245.
Parmelee, R.W., Beare, M.H., Cheng, W., Hendrix, P.F., Rider, S.J., Crossley,
D.A., Coleman, D.C. 1990. Earthworms and enchytraeids in conventional
282
References
and no tillage agroecosystems: a biocide approach to assess their role in
organic matter breakdown. Biol. Fertility Soils, 10, 1-10.
Parsons, S.A., Congdon, R.A. 2008. Plant litter decomposition and nutrient
cycling in north Queensland tropical rain-forest communities of differing
successional status. J. Tropical Ecol., 24, 317–327.
Pascual, J.A., Hernandez, T., Garcia, C., Ayuso, M. 1998. Enzymatic activities in
an arid soil amended with urban organic wastes: laboratory experiment.
Pe´rez-Piqueres, A., Edel-Hermann, V., Alabouvette, C., Steinberg, C. 2006.
Response of soil microbial communities to compost amendments. Soil
Biol. Biochem., 38, 460–470.
Pe´rez, J., Munõz-Dorado, J., de la Rubia, T., Martıńez, J. 2002. Biodegradation
and biological treatments of cellulose, hemicellulose and lignin: an
overview. Int. Microbiol., 5, 53–63.
Peacock, A.D., Mullen, M.D., Ringelberg, D.B., Tyler, D.D., Hedrick, D.B.,
Gale, P.M., White, D.C. 2001. Soil microbial community responses to
dairy manure or ammonium nitrate applications. Soil Biol. Biochem., 33,
1011-1019.
Phae, C.G., Sasaki, M., Shoda, M., Kubota, H. 1990. Characteristics of Bacillus
subtilis isolated from composts suppressing phytopathogenic
microorganisms. Soil Sci. Plant Nutr., 36, 575-586.
Piccolo, A., Nardi, S., Concheri, G. 1992. Structural characteristics of humic
substances as related to nitrate uptake and growth regulation in plant
systems. Soil. Biol. Biochem., 24, 373-380.
283
References
Postma, J., Schilder, M.T., Bloem, J., van Leeuwen-Haagsma, W.K. 2008. Soil
suppressiveness and functional diversity of the soil microflora in organic
farming systems. Soil Biol. Biochem., 40, 2394-2406.
Potera, C. 1994. From bacteria: A new weapon against fungal infection. Science,
265, 605.
Prasanna, R., Jaiswal, P., Singh, Y.V., Singh, P.K. 2008. Influence of
biofertilizers and organic amendments on nitrogenase activity and
phototrophic biomass of soil under wheat. Acta Agron. Hung, 56(2), 149-
159.
Preston-Mafham, J., Boddy, L., Randerson, P.F. 2002. Analysis of microbial
community functional diversity using sole-carbon-source utilisation
profiles – a critique. FEMS Microbiol. Ecol., 42, 1-14.
Pulleman, M., Jongmans, A., Marinissen, J., Bouma, J. 2003. Effects of organic
versus conventional arable farming on soil structure and organic matter
dynamics in a marine loam in the Netherlands. Soil Use and Management,
19(2), 157-165.
Radford, B.J., Thornton, C.M., Cowie, B.A., Stephens, M.L. 2007. The Brigalow
Catchment Study: III. Productivity changes on brigalow land cleared for
long-term cropping and for grazing. Australian Journal of Soil Research,
45, 512–523.
Raghothama, K.G. 1999. Phosphate acquisition. Ann. Rev. Plant Physiol.
Molecular Biol., 50, 665–693.
Ramsay, M.D., O‘Brien, R.G., Pegg, K.G. 1992. Fusarium oxysporum associated
with wilt and root rot of tomato in Queensland; races and vegetative
compatibility groups. Aust. J. Exp. Agri., 32, 651-655.
284
References
Rao, D.L.N., Pathak, H. 1996. Ameliorative influence of organic matter in the
biological activity of salt affected soils. Arid Soil Res. Rehab., 10, 311-
319.
Rashid, A., Harris, D., Hollington, P., Ali, S. 2004. On-farm seed priming
reduces yield losses of mungbean (Vigna radiata) associated with
mungbean yellow mosaic virus in the North West Frontier Province of
Pakistan. Crop Protection, 23, 1119–1124.
Raupach, G.S., Kloepper, J.W. 1997. Integrated pest management of multiple
cucumber pathogens through PGPR-mediated induced systemic
resistance. Plant growth-promoting rhizobacteria—Present status and
future prospects. Proceedings of the fourth international workshop on
plant growth-promoting rhizobacteria, Sapporo, Japan. Nakanishi
Printing. pp. 281-282.
Raupach, G.S., Liu, L., Murphy, J.F., Tuzun, S., Kloepper, J.W. 1996. Induced
systemic resistance in cucumber and tomato against cucumber mosaic
cucumovirus using plant growth-promoting rhizobacteria (PGPR). Plant
Dis., 80, 891-894.
Rauthan, B.S., Schnitzer, M. 1981. Effects of a soil fulvic acid on the growth and
nutrient content of cucumber (Cucumis sativus) plants. Plant Soil, 63,
491-495.
Reeve, J.R., Carpenter-Boggs, L., Reganold, J.P., York, A.L., Brinton, W.F.
2010. Influence of biodynamic preparations on compost development and
resultant compost extracts on wheat seedling growth. Bioresour. Technol.,
101(Article in press), 5658–5666.
285
References
Rincon, A., Ruız-Dıez, B., Fernandez-Pascual, M., Probanza, A., Pozuelo, J.M.,
de Felipe, M.R. 2006. Afforestation of degraded soils with Pinus
halepensis Mill.: Effects of inoculation with selected microorganisms and
soil amendment on plant growth, rhizospheric microbial activity and
ectomycorrhizal formation. Appl.Soil Ecol., 34, 42–51.
Robertson, F. 2003. Sugarcane trash management: consequences for soil carbon
and nitrogen. CRC for Sustainable Sugar Production.
Robertson, F.A., Myers, R.J.K., Saffigna, P.G. 1994. Dynamics of carbon and
nitrogen in a long term cropping system and permanent pasture system.
Aust. J. Soil Res., 45, 211-221.
Robertson, F.A., Thorburn, P.J. 2007. Decomposition of sugarcane harvest
residue in different climatic zones. Aust. J. Soil Research, 45, 1-11.
Rodríguez, H., Fraga, R. 1999. Phosphate solubilizing bacteria and their role in
plant growth promotion. Biotechnol Adv., 17(4-5), 319-39.
Romantschuk, M., Sarand, I., Peta¨nen, T., Peltola, R., Jonsson-Vihanne, M.,
Koivula, T., Yrja la, K., Haahtela, K. 2000. Means to improve the effect
of in situ bioremediation of contaminated soil: an overview of novel
approaches. Environ. Pollution, 107, 179–185.
Rousk, J., Brookes, P.C., Bååth, E. 2010. The microbial PLFA composition as
affected by pH in an arable soil. Soil Biol. Biochem., 42, 516-520.
Roy, A.K., Sharma, A., Talukder, G. 1988. Some aspects of aluminium toxicity
in plants. Bot. Rev., 54, 145-177.
Ryan, M. 2003. Compost tea production, application, and benefits, The Rodale
Institute.
286
References
Ryan, M., Wilson, D., Hepperly, P., Travis, J., Halbrendy, N., Wise, A. 2005.
Compost tea potential is still brewing. BioCycle, 46, 30–32.
Ryan, P.R., Ditomaso, J.M., Kochian, L.V. 1993. Aluminium toxicity in roots: an
investigation of spatial sensitivity and the role of the root cap. J. Exp.
Bot., 44, 437-446.
Rynk, R., van de Kamp, M., Willson, G.B., Singley, M.E., Richard, T.L., Kolega,
J.J., Gouin, F.R., Jr, L.L., Kay, D., Murphy, D.W., Hoitink, H.A.J.,
Brinton, W.F. 1992. On-Farm Composting Handbook, Northeast Regional
Agricultural Engineering Service. NY.
Sackenheim, R., Weltzien, H.C., Kast, W.K. 1994. Effects of microflora
composition in the phyllosphere on biological regulation of grapevine
fungal diseases. Vitis, 33, 235–240.
Sample, I. 2007. Global food crisis looms as climate change and population
growth strip fertile land. in: The Guardian.
Scheuerell, S. 2004. Compost tea production practices, microbial properties, and
plant disease suppression. . in: I International Conference on Soil and
Compost Eco-Biology. Spain, pp. 41-51.
Scheuerell, S.J. 2002. Compost teas and compost-amended container media for
plant disease control, Vol. PhD, Oregon State University.
Scheuerell, S.J. 2003. Understanding how compost tea can control disease.
BioCycle, 44(2), 20-25.
Scheuerell, S.J., Mahaffee, W.F. 2000. Assessing aerated and non-aerated watery
fermented compost and Trichoderma harzianum T-22 for control of
powdery mildew (Sphaerotheca pannosa var. rosae) of rose in the
Willamette Valley. Phytopathol., 90, 69.
287
References
Scheuerell, S.J., Mahaffee, W.F. 2004. Compost tea as a container medium
drench for suppressing seedling damping-off caused by Pythium ultimum.
Phytopathol., 94, 1156-1163.
Scheuerell, S.J., Mahaffee, W.F. 2002. Compost tea: principles and prospects for
plant disease control. Compost Sci. Util., 10, 313-338.
Scheuerell, S.J., Mahaffee, W.F. 2006. Variability associated with suppression of
gray mold (Botrytis cinerea) on geranium by foliar applications of non-
aerated and aerated compost teas. Plant Dis., 90, 1201-1208.
Schnitzer, M., Poapst, P. 1967. Effects of a soil humic compound on root
initiation. Nature, 213, 598-599.
Schnurer, J., Rosswall, T. 1982. Fluorescein diacetate hydrolysis as a measure of
total microbial activity in soil and litter. Appl. Environ. Microbiol., 43(6),
1256-1261.
Sebti, K.E.H. 2005. Compost tea effects on soil fertility and plant growth of
organic tomato (Solanum lycopersicum Mill) in comparison with different
organic fertilizers. in: International centre for advanced mediterranean
agronomic studies, Vol. Master Thesis, Istituto Agronomico Mediterraneo
di Bari. Morocco, pp. 67.
Segarra, G., Reis, M., Casanova, E., Trillas, M.I. 2009. Control of powdery
mildew (Erysiphe polygoni) in tomato by foliar applications of compost
tea. J. Plant Pathol., 91(3), 683-689.
Sen, B., Chandra, T.S. 2009. Do earthworms affect dynamics of functional
response and genetic structure of microbial community in a lab-scale
composting system? Bioresour. Technol., 100, 804–811.
288
References
Shannon, E.E., Weaver, W. 1963. The Mathematical Theory of Communication.
The University of Illinois Press, Urbana, Illinois.
Shen, J., Bartha, R. 1996. Priming effect of substrate addition in soil based
biodegradation tests. Appl. Environ. Microbiol., 62, 1428-1430.
Shi, W., Norton, J.M., Miller, B.E., Pace, M.G. 1999. Effects of aeration and
moisture during windrow composting on the nitrogen fertilizer values of
dairy waste composts. Appl. Soil Ecol., 11, 17-28.
Shrestha, K., Shrestha, P., Adetutu, E.M., Walsh, K.B., Harrower, K.M., Ball,
A.S., Midmore, D.J. 2011a. Changes in microbial and nutrient
composition associated with rumen content compost incubation.
Bioresour. Technol., 102, 3848–3854.
Shrestha, K., Shrestha, P., Walsh, K.B., Harrower, K.M., Midmore, D.J. 2011b.
Microbial enhancement of compost extracts based on cattle rumen content
compost - Characterisation of a system. Bioresour. Technol., 102, 8027-
8034.
Siddiqui, Y., Meon, S., Ismail, M.R., Ali, A. 2008a. Trichoderma-fortified
compost extracts for the control of Choanephora wet rot in okra
production. Crop Protec., 27, 385–390.
Siddiqui, Y., Meon, S., Ismail, R., Rahmani, M. 2009. Bio-potential of compost
tea from agro-waste to suppress Choanephora cucurbitarum L. the causal
pathogen of wet rot of okra. Biol. Cont., 49, 38–44.
Siddiqui, Y., Meon, S., Ismail, R., Rahmani, M., Ali, A. 2008b. Bio-efficiency of
compost extracts on the wet rot incidence, morphological and
physiological growth of okra (Abelmoschus esculentus L. Moench).
Scientia Hort., 117, 9-14.
289
References
Singh, A., Sharma, S. 2002. Composting of a crop residue through treatment with
microorganisms and subsequent vermicomposting. Bioresour. Technol.,
85, 107–111.
Singh, P., Suman, A., Tiwari, P., Arya, N., Gaur, A., Shrivastava, A.K. 2008.
Biological pretreatment of sugarcane trash for its conversion to
fermentable sugars. World J. Microbiol. Biotechnol., 24, 667–673.
Skladany, G.J., Metting, F.B. 1992. Bioremediation of contaminated soil. in: Soil
Microbial Ecology: Applications in Agricultural and Environmental
Management, (Ed.) F.B. Metting Jr., Marcel Dekker. New York, pp. 483–
513.
Small, F.G. 2000. Quantifying the socio-economic impacts of harvesting residue
retention systems – Growers‘ survey on burnt and green cane trash
blanket farming systems in the Burdekin and Proserpine districts. Sugar
Research and Development Corporation. BSS173.
Smalla, K., Wachtendorf, U., Heuer, H., Liu, W.T., Forney, L. 1998. Analysis of
Biolog GN substrate utilisation patterns by microbial communities. Appl.
Environ. Microbiol., 64, 1220-1225.
Solomon, D., Lehmann, J., Zech, W. 2000. Land use effects on soil organic
matter properties of chromic luvisols in semi-arid northern Tanzania:
carbon, nitrogen, lignin and carbohydrates. Agric. Ecosyst. Environ., 78,
203–213.
Spaccini, R., Zena, A., Igwe, C.A., Mbagwu, J.S.C., Piccolo, A. 2001.
Carbohydrates in water-stable aggregates and particle size fractions of
forested and cultivated soils in two contrasting tropical ecosystems.
Biogeochem., 53(1), 1-22.
290
References
Sparling, G.P. 1997. Soil microbial biomass, activity and nutrient cycling as
indicators of soil health. in: Biological Indicators of Soil Health, (Eds.)
C.E. Pankhurst, B.M. Doube, V.V.S.R. Gupta, CAB International. New
York, pp. 97–119.
Sparling, G.P., West, A.W. 1988. A direct extraction method to estimate soil
microbial C: calibration in situ using microbial respiration and 14C
labelled cells. Soil Biol. Biochem., 20, 337-343.
Stacey, G., Upchurch, R.G. 1984. Rhizobium inoculation of legumes. Trends in
Biotechnol., 2(3), 65-70.
Stark, C., Condron, L.M., Stewart, A., Di, H.J., Callaghan, M.O. 2007. Influence
of organic and mineral amendments on microbial soil properties and
processes. Appl. Soil Ecol., 35, 79–93.
Steenwerth, K.L., Jackson, L.E., Calderoń, F.J., Stromberg, M.R., Scow, K.M.
2003. Soil microbial community composition and land use history in
cultivated and grassland ecosystems in coastal California. Soil Biol.
Biochem., 35, 489–500.
Stewart, R.L., Wood, A.W. 1987. Proceedings of green cane symposium. CSR
Ltd.
Suarez-Estrella, F., Vargas-Garcia, M.C., Elorrieta, M.A., Lopez, M.J., Moreno,
J. 2003. Temperature effect on Fusarium oxysporum f. sp. melonis
survival during horticultural waste composting. J. Appl. Microbiol., 94,
475–482.
Sundberg, C., Smårs, S., Jönsson, H. 2004. Low pH as an inhibiting factor in the
transition from mesophilic to thermophilic phase in composting.
Bioresour. Technol., 95(2), 145-150.
291
References
Supanjani, Shim, H.H., Sung, J.J., Lee, K.D. 2006. Rock phosphate-potassium
and rock-solubilising bacteria as alternative, sustainable fertilisers. Agron.
Sustain. Dev., 26, 233-240.
Suresh, A., Pallavi, P., Srinivas, P., Praveen Kumar, V., Chandra, S.J., Reddy,
S.R. 2010. Plant growth promoting activities of fluorescent
pseudomonads associated with some crop plants. Afric. J. Microbiol. Res.,
4(14), 1491-1494.
Suthipradit, S., Edwards, D.G., Ascher, C.J. 1990. Effects of aluminium on tap-
root elongation of soybean (Glycine max), cowpea (Vigna unguiculata)
and green gram (Vigna radiata) grown in the presence of organic acids.
Plant Soil, 124, 233-237.
Sutton, M.R., Wood, A.W., Saffigna, P.G. 1996. Long Term Effects of Green
Cane Trash Retention on Herbert River Soils, (Eds.) J.R. Wilson, D.M.
Hograth, J.A. Campbell, A.L. Garside, CSIRO Division of Tropical Crops
and Pastures. Brisbane, pp. 178-180.
Swift, M.J., Anderson, J.M. 1994. Biodiversity and ecosystem function in
agricultural ecosystems. in: Biodiversity and Ecosystem Function, (Eds.)
E.-D. Schulze, H.A. Mooney. New York, pp. 15–41.
Swift, M.J., van der Meer, J., Ramakrishnan, P.S., Anderson, J.M., Ong, C.K.,
Hawkins, B.A. 1996. Biodiversity and agroecosystem function. in:
Functional Roles of Biodiversity: A Global Perspective, (Eds.) H.A.
Mooney, H. C.J., E. Medina, O.E. Sala, E.D. Schulze, John Wiley and
Sons. Chichester, pp. 261–298.
Sylvia, E.W. 2004. The effect of compost extract on the yield of strawberries and
severity of Botrytis cinerea. J. Sustainable Agri., 25(1).
292
References
Tajuddin, R.M., Ismail, A.F., Salim, M.R. 2004. Effects of oxygen concentration
on microbial growth in aerated palm oil mill effluent using oxygen
enriched air membrane system. In: Regional symposium on membrane
science and technology, Johor Bahru, Johor, Malaysia.
Tan, K.H., Binger, A. 1986. Effect of humic acid on aluminum toxicity in corn
plants. Soil Sci., 141, 20-25.
Tang, J.C., Shibata, A., Zhou, Q., Katayama, A. 2007. Effect of temperature on
reaction rate and microbial community in composting of cattle manure
with rice straw. J. Biosci. Bioeng., 104(4), 321–328.
Taylor, G.J. 1988. The physiology of aluminium tolerance in higher plants.
Commum. Soil Sci. Plant Anal., 19, 1179-1194.
Tejada, M., Gomez, I., Hernandez, T., Garcıa, C. 2010. Response of Eisenia
fetida to the application of different organic wastes in an aluminium-
contaminated soil. Ecotoxicol. Environ. Safety, 73, 1944–1949.
Tejada, M., Gonzalez, J.L., Hernandez, M.T., Garcia, C. 2008. Agricultural use
of leachates obtained from two different vermicomposting processes.
Thompson, J.P. 1996. Correction of dual phosphorus and zinc deficiencies of
linseed (Linumusita tissimum L.) with cultures of vesicular arbuscular
mycorrhizal fungi. Soil Biol. Biochem., 28(7), 941-951.
Thompson, J.P. 1987. Decline of vesicular-arbuscular mycorrhizae in long fallow
disorder of field crops and its expression in phosphorus deficiency of
sunflower. Aust. J. Agric. Res., 38, 847-67.
Thornton, C.M., Cowie, B.A., Freebairn, D.M., Playford, C.L. 2007. The
Brigalow Catchment Study: II. Clearing brigalow (Acacia harpophylla)
293
References
for cropping or pasture increases runoff. Australian Journal of Soil
Research, 45, 496–511.
Tiquia, S.M. 1996. Further composting of pig-manure disposed from the pig-on-
litter (POL) system in Hong, Vol. PhD thesis, The University of Hong
Kong. Pokfulam Road, Hong Kong.
Tiquia, S.M., Tam, N.F.Y., Hodgkiss, I.J. 1996. Microbial activities during
composting of spent pig-manure sawdust litter at different moisture
contents. Bioresour. Technol., 55, 201-206.
Touart, A.P. 2000. Time for compost tea in the Northwest. BioCycle, 41(10), 74-
77.
Tranker, A. 1992. Use of agricultural and municipal organic wastes to develop
suppressiveness to plant pathogens. in: Biological Control of Plant
Diseases, (Eds.) E.S. Tjamos, G.C. Papavizas, R.J. Cook, Plenum press.
NY pp. 35-42.
Tu, C., Ristaino, J.B., Hu, S. 2006. Soil microbial biomass and activity in organic
tomato farming systems: Effects of organic inputs and straw mulching.
Soil Biol. & Biochem., 38, 247–255.
Tuomela, M., Vikman, M., Hatakka, A., Itavaara, M. 2000. Biodegradation of
lignin in a compost environment: a review. Bioresour. Technol., 72, 169 -
183.
Ullrich, S., Karrasch, B., Hoppe, H.-G., Jeskulke, K., Mehrens, M. 1996. Toxic
effects on bacterial metabolism of the redox dye 5-cyano-2,3-ditolyl
tetrazolium chloride. Appl. Environ. Microbial., 62, 4587-4593.
294
References
Unkovich, M., Herridge, D., Peoples, M., Cadisch, G., Boddey, R., Giller, K.,
Alves, B., Chalk, P. 2008. Measuring plant-associated nitrogen fixation in
agricultural systems.
Urban, J., Trankner, A. 1993. Control of gray mold (Botrytis cinerea) with
fermented compost/water extracts. Biological control of foliar and post-
harvest diseases: proceedings of a workshop. West Palearctic Regional
Section. pp. 8-11.
Utkhede, R., Koch, C. 2004. Biological treatments to control bacterial canker of
greenhouse tomatoes. Biocontrol Sci. Technol., 49, 305-313.
Uvarov, A.V., Tiunov, A.V., Scheu, S. 2006. Long-term effects of seasonal and
diurnal temperature fluctuations on carbon dioxide efflux from a forest
soil. Soil Biol. Biochem., 38, 3387–3397.
Vadakattu, G., Paterson, J. 2006. Free living bacteria lift soil nitrogen supply. in:
Farmind Ahead, Vol. 169, pp. 40.
van Elsas, J.D., Garbeva, P., Salles, J.F. 2002. Effect of agronomical measures on
the microbial diversity of soil as related to the suppression of soil-borne
plant pathogens. Biodegradation, 13, 29–40.
van Peer, R., Numann, G.J., Schippers, B. 1991. Induced resistance and
phytoalexin accumulation in biological control of Fusarium wilt of
carnation by Pseudomonas sp. strain WCS417r. Phytopathol., 81, 728-
734.
Vance, E.D., Brookes, P.C., Jenkinson, D.S. 1987. An extraction method for
measuring soil microbial biomass C. Soil Biol. Biochem., 19, 703–707.
295
References
Vásquez, P., Holguín, G., Puente, M.E., López-Cortéz, A., Bashan., Y. 2000.
Phosphate solubilizing microorganisms associated with the rhizosphere of
mangroves in a semiarid coastal lagoon. Biol. Fert. Soils, 30, 460–468.
Vaughan, D., Linehan, D.J. 1976. The growth of wheat plants in humic acid
solutions under axenic conditions. Plant Soil, 44, 445-449.
Vivas, A., Moreno, B., Garcia-Rodriguez, S., Benitez, E. 2009. Assessing the
impact of composting and vermicomposting on bacterial community size
and structure, and microbial functional diversity of an olive-mill waste.
Wakelin, S.A., Colloff, M.J., Harvey, P.R., Marschner, P., Gregg, A.L., Rogers,
S.L. 2007. The effects of stubble retention and nitrogen application on soil
microbial community structure and functional gene abundance under
irrigated maize. FEMS Microbiol. Ecol., 59, 661–670.
Walkley, A., Black, I.A. 1934. An examination of the Degtjareff method for
determining organic carbon in soils: effect of variations in digestion
conditions and of inorganic soil constitutions. Soil Sci., 63, 251-263.
Wardle, D.A., Yeates, G.W., Nicholson, K.S., Bonner, K.I., Watson, R.N. 1999.
Response of soil microbial biomass dynamics, activity and plant litter
decomposition to agricultural intensification over a seven-year period.
Watanabe, K. 2001. Microorganisms relevant to bioremediation. Current Opinion
in Biotechnol., 12, 237–241.
Wei, G., Kloepper, J.W., Tuzun, S. 1991. Induction of systemic resistance of
cucumber to Colletotrichum orbiculare by select strains of plant growth-
promoting rhizobacteria. Phytopathol., 81, 1508-1512.
296
References
Welke, S. 2000. Effectiveness of compost extracts as disease suppressants in
fresh market crops in British Columbia. Organic Farming Research
Foundation.
Weltzein, H.C. 1992. Biocontrol of foliar fungal diseases with compost extracts.
in: Microbial Ecology of Leaves, (Eds.) J.H. Andrews, S.S. Hirano,
Springer-Verlag. New York, pp. 430-450.
Weltzein, H.C. 1989. Some effects of composted organic materials on plant
health. Agric. Ecosystems Environ., 27, 439–446.
Weltzein, H.C., Ketterer, N. 1986. Control of Phytophthora infestans on tomato
leaves and potato tubers through water extracts of composted organic
wastes. Phytopathol., 76, 1104.
Weltzien, H.C. 1991. Biocontrol of fungal foliar disease with compost extracts.
Microbial Ecol. Leaves, 430-450.
Weltzien, H.C. 1990. The use of composted materials for leaf disease suppression
in field crops. . in: Monograph – British Crop Protection Council, Vol.
45, pp. 115-120.
Widmer, F., Flieβbach, A., Laczko, E., Schulze-Aurich, J., Zeyer, J. 2001.
Assessing soil biological characteristics: a comparison of bulk soil
community DNA-, PLFA-, and Biolog- analyses. Soil Biol. Biochem., 33,
1029-1036.
Widmer, F., Rasche, F., Hartmann, M., Fliessbach, A. 2006. Community
structures and substrate utilization of bacteria in soils from organic and
conventional farming systems of the DOK long-term field experiment.
Appl. Soil Ecol., 33, 294–307.
Winter, C.K., Davis, S.F. 2006. Organic foods. J. Food Sci., 71(9), 117–124.
297
References
Winterscheidt, H., Minassian, V., Weltzein, H.C. 1990. Untersuchungen zur
biologischen Bekampfung des Falschen Mehltaus der Gurke -
Pseudoperonospora cubensis (Berk. et Curt.) Rost.- mit
Kompostextrakten. Gesunde Pflanzen, 42, 235-238.
Witkamp, M. 1966. Decomposition of leaf litter in relation to environment,
microflora, and microbial respiration. Ecology, 47(2), 194-201.
Wood, A.W. 1991. Management of crop residues following green harvesting of
sugarcane in north Queensland. Soil Till. Res, 20(1), 69-85
Woods, L.E. 1983. Land Degradation in Australia. Department of Home Affairs
and Environment, Canberra: Australian Government Printers.
Wright, R.L. 1989. Soil aluminium toxicity and plant growth. Commun. Soil Sci.
Plant Anal., 20, 1479-97.
Wu, L., Ma, L.Q. 2002. Relationship between compost stability and extractable
organic carbon. J. Environ. Qual., 31, 1323–1328.
Wu, L., Ma, L.W., Martinez, G.A. 2000. Comparison of methods for evaluating
stability and maturity of biosolids compost. J. Environ. Qual., 29, 424–
429.
XLSTAT. 2010. XLSTAT-Pro (Win), Addinsoft, Thierry Fahmy, 40 rue
Damrémont, 75018. Paris, France.
Xu, H. 2000. Effects of a microbial inoculant and organic fertilizers on the
growth, photosynthesis and yield of sweet corn. J. Crop Prod., 3, 183–
214.
Yamada, E., Ozaki, T., Kimura, M. 1998. Determination and behaviour of humic
substances as precursors of trihalomethane in environmental water.
Analytical Sci., 14, 327-332.
298
References
Yan, F., Schubert, S., Mengel, K. 1996. Soil pH increase due to biological
decarboxylation of organic anions. Soil Biol. Biochem., 28(4), 611-624.
Yohalem, D.S., Harris, R.F., Andrews, J.H. 1994. Acquous extracts of spent
mushroom substrate for foliar disease control. Compost Sci. Util., 2, 67-
74.
Young, I.M., Ritz, K. 2000. Tillage, habitat space and function of soil microbes.
Soil Tillage Res., 53, 201-213.
Zak, D.R., Holmes, W.E., MacDonald, N.W., Pregitzer, K.S. 1999. Soil
temperature, matric potential, and the kinetics of microbial respiration and
nitrogen mineralization. Soil Sci. Soc. Am. J., 63, 575-584.
Zak, J.C., Willig, M.R., Moorhead, D.L., Wildman, H.G. 1994. Functional
diversity of microbial communities: a quantitative approach. Soil Biol.
Biochem., 26, 1101–1108.
Zhang, W., Han, D.Y., Dick, W.A., Davis, K.R., Hoitink, H.A.J. 1998. Compost
and compost water extract-induced systemic acquired resistance in
cucumber and arabidopsis. Phytopathol., 88(5), 450-455.
Zmora-Nahum, S., Danon, M., Hadar, Y., Chen, Y. 2008. Chemical properties of
compost extracts inhibitory to germination of Sclerotium rolfsii. Soil Biol.
Biochem., 40, 2523–2529.
Zwieten, M.V., Stovold, G., Zwieten, L.V. 2007. Alternatives to copper for
disease control in the Australian organic industry. RIRDC publication.
299

Cqu 7854+SOURCE1+SOURCE1.3

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Cqu 7854+SOURCE1+SOURCE1.3

Uploaded by

Copyright:

Available Formats

CHARACTERISATION AND POTENTIAL USES

OF MICROBIALLY ENHANCED COMPOST

Submitted in Partial Fulfillment of the

CENTRE FOR PLANT AND WATER SCIENCE

Faculty of Sciences, Engineering and Health

First and foremost, I would like to thank my principal supervisor, Professor

either referenced or acknowledged.

I authorise CQUniversity to reproduce this thesis in whole or in part for

Chapter 3: Changes in microbial and nutrient composition associated with rumen

Chapter 4: Comparison of microbially enhanced compost extracts produced from

Chapter 5: Microbially enhanced compost extract: does it increase solubilisation of

Chapter 6: Biodegradation of sugarcane trash through the use of enhanced compost

Chapter 7: Suppressing fungal wilt diseases of tomato caused by Fusarium

Chapter 10 ........................................................................................................................ 243

A literature review on the use and efficacy of

compost extracts in crop production

70s (the ‗green revolution‘) was supported by improved genetics and by

increased chemical inputs (inorganic fertilisers, herbicides and pesticides).

production systems) chemical inputs, on the basis of reducing harm to the

food web‘) as well as physico-chemical attributes (e.g. soil aeration, cation

exchange capacity) (Elfstrand et al., 2007; Supanjani et al., 2006).

A wide array of formulations and products are now marketed in

developed countries in support of this focus on soil ‗health‘. Typically, these

regenerating soil microbial communities, with typical claims including action as a

bio-fertiliser, a bio-decomposer and/or a disease suppressor (Antonio et al., 2008;

specifically the mechanism of action, of these products is typically very poor.

known as ‗compost tea‘. Production of ‗compost extract‘ has progressed from a

simple, anaerobic extraction process to an ‗aerobic bio-fermenter‘ approach,

expanding from small, simple on-farm activity (cow manure soaking in a 44

gallon drum) to large, relatively elaborated activity (e.g. specialised incubating

and aeration apparatus, addition of microbial food sources, enhancement with

specific microbial strains, centralised production with distribution to farms, and

sophisticated distribution/application systems on farm). The term ‗microbially

will be used in this thesis.

Compost is bulky and hence is costly to transport. Use in broad-acre

growth of a crop to be left in the field to decompose). Compost extract can be

seen as a value-add to compost. As a liquid, it is easily applied. Further, compost

foliage and plant remains, either by drenching or by spraying (Litterick et al.,

Compost extracts are gaining popularity, particularly, among those who

are seeking substitutes to agrochemicals (Bess, 2000; Touart, 2000). Organic

petrochemical-derived fertiliser products, as prices soar due to increasing global

demand and limited supply. As an example, the US Department of Agriculture

reported a 65% increase in price of synthetic fertiliser, such as nitrate, potassium

and phosphate over 12 months from date to date (Etter, 2008).

However, there is relatively little verification of these claims

extract is a unique biological community), to the scope of the claims (from

may be required (multiple years).

of action of compost extracts in agriculture.

system, sustaining life (plants, animals including microorganisms) and

the essential mineral elements. In hydroponic systems these mineral requirements

are typically completely met by supplying them in a soluble inorganic form. In

insoluble forms. Soil biota are essential to functions of mineralisation of organic

matter and solubilisation of inorganic minerals.

ascomycete and basidiomycete fungi is well established to improve plant access

enhanced populations of free living N2 fixers, such as Azotobacter and

Azospirillum are examples of microbiota found in the rhizosphere or phylloplane)

(Kahindi et al., 1997; Unkovich et al., 2008).

nevertheless contribute to processes that favour plant growth. Examples of

organisms that contribute to mineral solubilisation in the soil include the

phosphate solubilising bacteria (Ekin, 2010; Rodríguez & Fraga, 1999).

Examples of organisms that contribute to organic matter breakdown, and thus

nutrient availability through mineralisation include bacteria (e.g. Nitrococcus,

Nitrobacter), fungi (e.g. Rhizopus, Fusarium spp.), flagellates, amoebae and