This article was downloaded by: [University of Aberdeen]

On: 04 April 2013, At: 15:29

Publisher: Taylor & Francis
Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House,
37-41 Mortimer Street, London W1T 3JH, UK

Critical Reviews in Plant Sciences

Publication details, including instructions for authors and subscription information:
http://www.tandfonline.com/loi/bpts20

Bioassays and Field Studies for Allelopathy in

Terrestrial Plants: Progress and Problems
a b
Inderjit & Erik T. Nilsen
a
Department of Botany, University of Delhi, Delhi 110007, India, e-mail:
allelopathy@satyam.net.in
b
Department of Biology, Virginia Polytechnic and State University, Blacksburg, VA 24061
Version of record first published: 18 Jun 2010.

To cite this article: Inderjit & Erik T. Nilsen (2003): Bioassays and Field Studies for Allelopathy in Terrestrial Plants: Progress
and Problems, Critical Reviews in Plant Sciences, 22:3-4, 221-238

To link to this article: http://dx.doi.org/10.1080/713610857

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions

This article may be used for research, teaching, and private study purposes. Any substantial or systematic
reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to
anyone is expressly forbidden.

The publisher does not give any warranty express or implied or make any representation that the contents
will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should
be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims,
proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in
connection with or arising out of the use of this material.
 Critical Reviews in Plant Sciences, 22(3):221–238 (2003)

Bioassays and Field Studies for Allelopathy in

Terrestrial Plants: Progress and Problems
Inderjit1* and Erik T. Nilsen 2
1Department of Botany, University of Delhi, Delhi 110007, India, e-mail: allelopathy@satyam.net.in; 2Department

of Biology, Virginia Polytechnic and State University, Blacksburg, VA 24061

Referee: Dr. Stella Elakovich, Dept. of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, MS 390406–5043

* Corresponding author
Downloaded by [University of Aberdeen] at 15:29 04 April 2013

ABSTRACT: Bioassays are an integral part of allelopathy research. The unsuitability of laboratory bioassays to
explain field situations is discussed previously. In this article, we discuss progress in bioassay experimental design
and several unresolved problems associated with research on allelopathy. The objectives of this article are to
discuss problems related to (1) collection of allelopathic material for bioassay, (2) allelochemical quantification
in bioassays, (3) selection of concentration of allelochemicals in bioassay, (4) selection of appropriate control,
(5) interaction between allelochemicals and other substances, and (6) in situ allelochemical bioassays. We
concluded that new experimental designs for in situ bioassay are needed that can account for the large number of
confounding factors in a complex field environment, and can be linked to physiological monitoring of target
species and biochemical monitoring of the growth medium.

KEY WORDS: plant phenolics, allelopathic interference, microbial activity, bioassay, experimental design.

I. INTRODUCTION allelopathy. Moreover, Inderjit and Weiner (2001)

Allelopathy can be defined as the effect of
one plant on growth and distribution of other
plants (including microorganisms) through the
release of chemical compounds into the environ-
ment (Rice, 1984). This widely accepted defini-
tion of allelopathy includes both positive and
negative effects. It also includes indirect effects,
that is, via degraded chemicals and through the
effect of chemicals on availability of nutrients. In
a way, the term ‘allelopathy’ is so broadly defined
that it includes everything and causes dissatisfac-
tion among ecologists (Inderjit and Weiner, 2001).
For example, Inderjit and Del Moral (1997) sug-
gested that several mechanisms of plant interfer-
ence (e.g., allelopathy, resource competition, mi-
crobial nutrient immobilization) are likely to
operate in parallel, and Wardle and his colleagues
(1998) advocated that using an ecosystem-level
approach would be a good context for the study of
suggested that progress in the field of allelopathy
could be furthered if allelopathy was studied in
context of soil chemical ecology.
It is generally accepted that allelochemicals
could influence plant growth through their
(1) direct effect on plant growth and establish-
ment (i.e., allelopathy), (2) indirect effect of de-
graded and transformed byproducts, (3) interac-
tion with abiotic and biotic substratum factors,
and (4) induction of release of chemicals by a
third plant species (Inderjit and Keating, 1999).
Allelopathy should be considered a direct plant-
plant chemical interaction within a broader con-
text of chemical interactions among organisms.
However, in this article we discuss plant growth
inhibition due to direct effects of allelochemicals
and their effects through soil chemical ecology.
Bioassays are generally designed to test a
hypothesis (Wolfson, 1988). Laboratory bioas-
says are important tools to understand a particular

0735-2689/03/$.50
© 2003 by CRC Press LLC
221
 component of allelopathy; however, field studies
are needed to test whether different components
are operating together as a unit in field situations
(Inderjit and Weston, 2000). Several workers have
discussed problems with bioassays for allelopa-
thy (Leather and Einhellig, 1986, 1988; Qasem
and Hill, 1989; Inderjit and Dakshini, 1995; Romeo
and Weidenhamer, 1998; Foy, 1999). Although
limited in their application to field situations, labo-
ratory bioassays can be designed to study the
following components of allelopathy.
1. Presence and subsequent release of
allelochemicals.
2. Growth inhibition of the target plant and
interference of allelochemicals with physi-
Downloaded by [University of Aberdeen] at 15:29 04 April 2013
ological processes of the target plant.
3. Fate of allelochemicals in soil.
4. Movement of allelochemicals in the soil
environment and their uptake by the target
plant.
5. Detoxification of allelochemicals taken up
by the target plant.
6. Effects of allelochemicals on soil microbial
ecology and nutrient dynamics.
7. Interaction of promoters (e.g., nitrate), in-
hibitors (e.g., phenolic acids) and neutral
substances (e.g., glucose) in the soil envi-
ronment.
Harper (1994, p. 369) states, “ almost all spe-
cies can, by appropriate digestion, extraction, and
concentration be persuaded to yield a product that
is toxic to one species or another.” Although the
drawback in methodology used to demonstrate
the occurrence of allelopathy has been realized
(Romeo, 2000), the ‘grind and find’ techniques
are still in use. While discussing the shortcoming
of bioassays for allelopathy, Inderjit and Dakshini
(1995) cautioned against several methods used in
allelopathic studies. For example, the grinding of
plant material should be avoided in growth bioas-
says. Instead of artificially sensitive species
(e.g., lettuce, Lactuca sativa), the species co-oc-
curring with the donor plant should be used.
Rimando et al. (2001) developed a bioactivity-
guided method for isolating allelochemicals from
rice (cultivar Taichung Native 1). These authors
argued that data generated through bioassay-
222
guided isolation method can correlate results ob-
tained in laboratory experiments and field activity
of rice allelochemicals. Their method, however,
had some shortcomings. For example, (1) acetone
was used as an extractant, (2) dried and powdered
roots of rice cultivars were used, (3) no soil was
involved in growth experiments, and (4) joint
action of allelochemicals in mixture was not stud-
ied. This bioassay may be suitable for studying
the phytotoxicity of allelochemicals, but it is not
appropriate for investigating their role in the al-
lelopathic potential of rice cutivars. Centaurea
diffusa (Eurasion forb) is an invasive weed in
North America and is reported to suppress native
bunchgrass species such as Festuca ovina,
Koeleria laerssenii, and Agropyron cristatum
(Callaway and Aschehoug, 2000). C. diffusa,
however, did not inhibit the growth of closely
related grass species from the native communi-
ties. Callaway and Aschehoug (2000) argued that
North American grasses may not be adapted to
allelochemical release by C. diffusa, however,
Eurasian species from its native land are adapted.
These results suggest that putative allelopathic
plants may not have a strong allelopathic toxicity
to their neighbors in their native habitat (i.e., neigh-
boring species have evolved resistance mecha-
nisms), whereas that same putative allelopathic
plant may have a strong phytotoxicity to neigh-
boring species in the invaded community. Further
comparisons of alellopathy between co-occurring
species of both native and alien communities will
determine if allelopathy is a common method by
which invasive species gain dominance in the
alien community.
The selection of growth parameters is another
important consideration for bioassay design. Most
studies of allelopathy measure root and/or shoot
length and seedling biomass. Other parameters,
for example, physiological processes and ultra-
structural changes deserve more attention. Inderjit
(2001) discussed the significance of soils in the
expression of allelopathy. In terrestrial systems,
any bioassay without involving soil has no eco-
logical relevance (Foy, 1999). Factors affecting
the expression of allelopathy in laboratory bioas-
say are listed in Table 1. Here we consider some
of the more important issues concerning allelopa-
thy bioassay.
 Downloaded by [University of Aberdeen] at 15:29 04 April 2013

TABLE 1
List of Parameters That Influence the Outcome of Bioassays for Allelopathy

223
 Downloaded by [University of Aberdeen] at 15:29 04 April 2013

TABLE 1 (continued)

224
 II. COLLECTION OF ALLELOCHEMICALS
FOR BIOASSAY
Allelochemicals are contributed into the en-
vironment through foliar leaching, root exuda-
tion, volatilization, leaf litter, and other residue
decomposition. An aqueous solution should be the
preferred medium for collecting allelochemicals,
and any use of an organic solvent for extracting
allelochemicals to be used in growth bioassays
should be avoided (Schmidt, 1990). In addition,
allelochemicals should be collected with the least
disruption of the normal mode of release. Such
careful adherence to natural release modes is of-
ten overlooked. For example, Torti et al. (1995)
advocated the use of homogenizer technique for
Downloaded by [University of Aberdeen] at 15:29 04 April 2013
phenolic extracts of leaves compared with con-
ventional sonicator or shaker technique. Any ex-
traction technique that prematurely disrupts (faster
than decomposition) the integrity of tissues al-
lows the extraction of elevated organic constitu-
ents yielding inappropriate results. Tang and
Young (1982) described a method to collect
allelochemicals exuded from an undisturbed root
system of bigalta limpograss (Hemarthria
altissima) (Figure 1). Stolon cuttings of limpograss
were washed and treated with 5% clorox for 15
min. Pots (1-gallon brown glass bottles with bot-
tom removed) were filled with crushed basaltic
rocks and 2:1 (v/v) sand:rock mixture. Containers
wrapped with aluminium foil were heat sterilized
for 2 days at 100°C. After planting cuttings, each
pot was irrigated with 0.1-strength Hoagland’s
solution (100 ml/day). Pots without limpograss
cuttings were identified as control. After 40 days,
limpograss had a well-developed root system. A
column packed with washed (by organic solvents)
XAD-4 resin was attached to the bottom of the
pot with a circulating attachment. Hoagland’s
solution (0.1-strength) was then circulated (1 l/h)
through the pot and column. The nutrient solution
was continuously circulated through the rhizo-
sphere of the living limpograss. To take care of
any water loss due to evaporation, water was
replenished daily. The column was detached after
3 days washed with water and then followed by
methanol. These authors pooled elute from 15
columns. After diluting the elute with water, it
was extracted with CH2Cl2, and identified as the
neutral fraction. The remaining aqueous fraction
was acidified to pH 2 by 1 N HCl and extracted
with CH2Cl2. To obtain the basic fraction, acidi-
fied residue was adjusted by 1 N NaOH to pH
11 and extracted with CH2Cl2. All fractions
were reduced to 3 ml and used for growth bio-
assays.
Different fractions were applied to Whatman
no. 3 paper discs. Organic solvent was dried and
water was applied. Lettuce seeds were used as an
assay species. Radicle length was measured after
48 h of incubation. Although this root exudate
trapping technique of Tang and Young (1982)
minimized the rhizosphere disturbance, several
problems of the technique remained. No natural
soil was used. Further, the medium used to grow
limpograss (basaltic rock and sand: rock mixture)
was heat sterilized. In doing so, the importance of
soil chemistry and microbial ecology was not
appreciated. By selecting sand:rock mixture, au-
thors completely ignored the role of soil texture in
availability of allelochemicals in the substratum.
Several authors have shown the role of soil tex-
ture in allelopathic expression (Del Moral and
Muller, 1969; Del Moral and Cates, 1971; Kuiters
and Denneman, 1987; Oleszek and Jurzysta, 1987;
Inderjit and Dakshini, 1994a). Another major prob-
lem with this trapping technique of Tang and
Young (1982) is their bioassay procedures. Dif-
ferent fractions were extracted in organic solvents
and placed on filter paper. After evaporating or-
ganic solvent, water was added. This is not an
ecologically relevant bioassay method because
allelochemical concentration may be very high
compared with the natural situation. Moreover,
the technique is not appropriate for isolating the
effects of a particular allelochemical because each
fraction may have several compounds and they
may have different polarity.
Czarnota (2001) developed a unique capillary
system for production of large quantities of living
seedling roots and their respective exudates (Fig-
ure 2). The capillary system consisted of growing
100 g sorghum between two double layers of
cheesecloth (60 cm long) that were placed on
aluminium window screen. The screen assembly
was placed over a sandwich of ridged insulation,
capillary mat, and weed mat. The capillary mat,
weed mat, and cheesecloth were draped into a
225
Downloaded by [University of Aberdeen] at 15:29 04 April 2013

FIGURE 1. The hydrophobic root exudates trapping system. (Source: Tang and Young (1982). Reproduced with
permission from American Society of Plant Physiologists.)

226
Downloaded by [University of Aberdeen] at 15:29 04 April 2013
FIGURE 2. A unique capillary system for the production of large quantities of living seedling roots and their exudates.
(Source: Czarnota [2001]. Reproduced with permission from Cornell University, Ithaca.)
water trough. The entire system was covered with
plastic and topped with two sheets of egg crate
light baffles pressed under 10 kg weight in order
to keep the screen in contact with the capillary
mat and to keep the system components intact as
seeds germinated. The solution does not recircu-
late, but is maintained at a consistent level so that
the capillary mat is uniformly but not overly moist.
Roots are then cut off and extracted with a sol-
vent, methylene chloride. Solenoid valves main-
tain a constant water level in the trough. Matting
is present to absorb water and keep the overall
layers moist. The capillary mat system was able
to produce exudates within 10 to 14 days without
contamination by fungal or bacterial pathogens.
Currently, this capillary mat system is used to
extract large amounts of sorgoleone from differ-
ent sorghum accessions (see also Czarnota et al.,
2001).
III. ALLELOCHEMICAL QUANTIFICATION
IN BIOASSAY MEDIUM
Quantitative and qualitative estimation of
allelochemicals in bioassays is the most difficult part of
allelopathic research. Examining the quantification of
phenolics in bioassay can represent some of these
difficulties. Inderjit et al. (1997) investigated the allelo-
pathic potential of certain terpenoid and phenolic sub-
stances. These were p-hydroxybenzoic acid (benzoic
acid derivative), ferulic acid (cinnamic acid derivative),
umbelliferone (coumarin), catechin (flavonoid), emo-
din (anthraquinone), 1,8-cineole (monoterpenoid),
carvone (monoterpenoid), and betulin (triterpenoid). A
mixture of these chemicals was prepared to simulate a
natural extract or “leachate”, which contained chemi-
cals from different classes of organic compounds. Some
of these chemicals are UV-absorbing (p-hydroxybenzoic
acid, ferulic acid, umbelliferone, catechin, emodin, and
carvone), while others are not (1,8-cineloe and betulin).
Furthermore, two different wavelengths were required
to quantify UV-absorbing compounds, that is, 254 for
umbelliferone, catechin, emodin, and carvone, and 275
nm for p-hydroxybenzoic acid and ferulic acid. Also,
HPLC was not the most appropriate quantification
technique for all compounds (e.g., these authors used
GC spectroscopy to quantify monoterpenes). This study
demonstrated that several analytical tools (i.e., HPLC,
RI-HPLC, or GC) are required to identify and quantify
allelochemicals in many extracts. Haig (2001) reviewed
the significance of hyphenated chromatography–mass
spectroscopy techniques in allelopathy research. The
LC-MS technique is considered better compared with
227
 GC-MS because it can separate labile heat-sensitive,
nonvolatile, and high-molecular-weight compounds that
account for 80% of all known compounds. The re-
maining 20% are volatiles and more stable. These
volatile compounds can be best separated by capillary
GC-MS because it has higher chromatography resolu-
tion compared with LC-MS. However, GC-MS has
another advantage over LC-MS, that is, the existence
of large commercial libraries of computer-searchable
standard electron-impact mass spectra. Moreover, Haig
(2001) gave an important list of allelochemical identi-
fications using chromatography. These include (1) stud-
ies using nonhyphenated techniques, (2) allelochemical
identifications using hyphenated chromatography–mass
spectrometry, and (3) allelochemical quantifications
using hyphenated chromatography-mass spectrometry.
Downloaded by [University of Aberdeen] at 15:29 04 April 2013
He stressed the need for identifying individual com-
pounds within a complex plant-related sample that
possesses biological activity.
Due to the difficulty of identifying allelochemical
species in a complex natural mixture, the estimation
of total phenolics is a preferred parameter in allel-
opathy research (Inderjit, 1996; Dalton 1999).
Waterman and Mole (1994) summarized different
methods used to estimate total phenolics (Table 2).
The Folin-Ciocalteu method (Swain and Hillis, 1959)
is often used to estimate total phenolics. Although
measuring total phenolics is easier than identifying
and quantifying the specific allelochemicals in a
complex coctail, the measurement of total phenolics
is not trouble free. For example, the techniques for
measuring total phenolics may have the problem of
reacting with several oxidizable nonphenolic com-
pounds (Van Alstyne, 1995). Moreover, Blum and
his co-workers (1994) opined that there is no phenol
reagent or HPLC method currently developed that
can calculate the absolute concentration of total phe-
nolics in soils. For example, water and EDTA can
extract only certain types of phenolic compounds,
not all types. Because there are chances of reactivity
with nonphenolic compounds, and extractions are
incomplete, methods for measuring total phenolics
should be used only to estimate changes in relative
concentrations among treatments not absolute con-
centrations (Box, 1983; Waterman and Mole, 1994).
Van Alstyne (1995) compared three methods for
quantifying phloroglucinol in the presence of protein,
bovine serum albumin (BSA). First, Folin-Ciocalteu
assay was used for compounds extracted in 80%
228
methanol. Second, Folin-Ciocalteu assay was used
for compounds detected in 75% methanol-25% trichlo-
roacetic acid. Third, a polyvinylpolypyrrolidone
(PVPP) method was used. It was found that the Folin-
Ciocalteu method for compounds extracted with 80%
methanol provided the most consistent results be-
cause of the least interference by proteins present in
the extract. Although these authors recommended
this method to be more consistent and efficient, whether
it generates ecological relevant data from an allelopa-
thy perspective needs further research.
IV. CONCENTRATION OF ALLELOPATHIC
MATERIAL IN BIOASSAY
Many times two terms — concentration and
content — are used as synonym (Farhoomand
and Peterson, 1968). However, ‘content’ is cor-
rectly defined as the total amount of compound
present in a specific amount of plant tissue, while
‘concentration’ is the amount of compound present
in a unit amount of plant tissue. Care should be
taken to use these terms appropriately. In bioas-
say for allelopathy, careful attention to matching
natural allelochemical concentrations is critical.
Moreover, in soil amendment experiments it is
important to select an appropriate ratio of plant
debris to soil mass. Inderjit and Dakshini (1992),
for example, added leaves of Pluchea lanceolata
to the soil (1.6%, w/w) based on the amount of
debris collected from clipped quadrats. However,
a range of concentration (four or more) should be
selected in plant debris-soil bioassays for allel-
opathy. Moreover, any addition of plant debris
into soil will result in changes of organic and
inorganic soil chemical components (Inderjit and
Dakshini, 1999). Another important question is
what soil:leachate ratio should be considered for
interpreting growth experiments? Inderjit and
Dakshini (1994a) amended 200 g soil with 100 ml
of different strength (full-strength, 1:4, 1:8, and
1:12) of P. lanceolata leaf leachates. Chemical
characteristics of all amended soils were altered.
These authors compared chemical characteristics
of amended soil with those of natural P. lanceolata
soils. It was found that soils amended with mini-
mum and maximum amounts of P. lanceolata
leachate were significantly different from natural
 TABLE 2
Different Methods Commonly Used to Estimate Total Phenolics. Source: Waterman and
Mole (1994). Reproduced after permission from Blackwell Scientific publications
Downloaded by [University of Aberdeen] at 15:29 04 April 2013
P. lanceolata soil, and therefore should not be
used for growth bioasasys. The soil:leachate ratio
that made insignificantly altered characteristics
from those of natural soils should be used.
Just as in soil amendment bioassay, concen-
tration is critical for filter paper bioassay. If pre-
cipitation (throughfall or stemflow) is tested for
allelochemical toxicity, then no concentration
process should be employed. A dilution series can
be useful for both determining the effective con-
centration for toxicity and for separating allelo-
pathic inhibition from inhibition by resource limi-
tation. If growth inhibition decreases with
increasing dilution, then the inhibition is likely to
be allelopathic. However, if inhibition increases
with dilution then the inhibition is likely caused
by resource (nutrient) limitation. If there is no
change of inhibition with dilution, then allelopa-
thy and resourse limitation may be counteracting
each other. However, if heavy metal toxicity (or
anthropogenic toxic component) is the cause of
inhibition this cannot be differentiated from allel-
opathy by a dilution experiment.
Selecting an appropriate leachate concentra-
tion made from litter or humus may not be possible
unless the concentration of putative allelochemical
in the medium is known. Determining the concen-
tration of a putative allelochemical in litter or hu-
mus material is difficult because that concentration
is likely to be very heterogeneous in time (as the
material repetitively wets and dries) and space (lo-
cal pockets of higher concentration). Some impor-
tant considerations when making a leachate from
litter or humus should be: (1) the ratio of water to
organic matter mass, (2) duration of soaking before
filtering, (3) temperature of the extraction, and (4)
necessity for shaking.
V. SELECTION OF APPROPRIATE
CONTROLS
The selection of an appropriate control may
be one of the most critical aspects of an allelopa-
thy bioassay. Most commonly the control bioas-
say is performed with the extraction medium (usu-
ally distilled water). However, it is important to
control for some other variables. Leachate osmo-
lality (concentration of dissolved osmotically ac-
tive species) can vary dramatically from distilled
water. Moreover, pH and nutrient availability can
be very different from the control imbibing mate-
rial. Therefore, it is advisable to match the control
solution to the pH and osmolality of the leachate.
Germination and radical elongation in controls of
filter paper bioassay are often less than leachate
229
 treatments because distilled water is used without
any added nutrients. Utilizing a weak Hoagland’s
solution may cause the germination and growth
of controls to be equal to or above that of the
leachate treatment. Unfortunately, the added nu-
trients also may increase the speed by which molds
can develop in the bioassay shortening the length
of time for growth responses.
Plant debris incorporated into soil is often
used in bioassays for allelopathy (Blum, 1995,
1999; Inderjit and Daksini, 1994a, b). However,
the design of the control in these experiments is
not uniform. The selection of control is an impor-
tant step to evaluate allelopathic responses
(Williamson and Richardson, 1988) in debris ad-
dition studies. While some workers have used
Downloaded by [University of Aberdeen] at 15:29 04 April 2013
peat moss to balance organic matter (Pardales et
al., 1992; Ahmed and Wardle, 1994), others have
not added any inert material in their control
(Inderjit and Dakshini, 1992, 1994a, b; Inderjit et
al., 1999). In some regions, peat moss may be
inappropriate as an additive to control soils be-
cause of the absence of peat in the source region
for the test soil (Inderjit and Dakshini, 1998).
Moreover, Inderjit and Mallik (1997) found that
chemical characteristics of soil from regions where
peat is found (boreal forests) significantly changed
when amended with peat moss when compared
with unamended boreal forest soil. Peat moss had
higher values for total phenolics, nitrogen, Al3+,
Ca2+, Mg2+, and lower values for pH, phosphate,
Mn2+, K+, and Na+. Therefore, peat moss is not an
appropriate control in their study. Inderjit and
Dakshini (1998) suggested that test debris mate-
rial repeatedly washed with water might serve as
an appropriate control. In fact, only one or two
washing of test allelopathic debris would be suf-
ficient to create organic matter that was insignifi-
cantly different from that of the control soil. An-
other possible design for checking the allelopathic
potency of the organic matter produced by the test
species is to use material from a number of differ-
ent putatively nonallelopathic species in other
treatments. In a study of the allelopathic potential
of Rhododendron maximum leaves to the growth
of mycorrhizae in agar medium, initial studies
demonstrated toxicity compared with mycorrhizae
growing on medium without R. maximum leaves
(Nilsen et al., 1999). However, when the study
230
was repeated with leaves from other species in the
growth medium, phytotoxicity of R. maximum
leaves was minimal compared with that for other
species of the region (Nilsen et al., 1999). There-
fore, the design of the control and a comparison
with material from putatively nonallelopathic spe-
cies are critical steps to generating ecologically
relevant data.
VI. INTERACTIONS BETWEEN
ALLELOCHEMICALS AND OTHER
SUBSTANCES
In field situations, allelochemicals are always
released in combination with other substances,
for example, inorganic ions, carbohydrates, and
amino acids (Tang et al., 1995). Blum et al. (1993)
suggested that allelopathic effects could be due to
the interaction of promoters (e.g., nitrate), inhibi-
tors (e.g., phenolic acids), and neutral substances
(e.g., glucose) in the soil environment. It may not
be ecologically relevant to compare growth inhi-
bition due to root exudate/foliar leachate to that
obtained with synthetic chemicals. Here is a ma-
jor impasse in bioassay-based research on allel-
opathy. Natural product chemists try to isolate
biologically active compounds from the suspected
allelopathic plant, and after doing bioassays in
artificial media these results are equated with al-
lelopathic potential of the plant in a field situa-
tion. By doing so the intricate interaction of pro-
moters, inhibitors, and neutral substances in the
complex soil system is completely ignored. While
studying the modification of allelopathic activity
of p-coumaric acid on seedling biomass of
morningglory (Ipomoea hederacea), Blum and
his co-workers (1993) found that at increasing
levels of glucose, the influence of phenylalanine
and p-hydroxybenzoic acid on morning glory bio-
mass decreased; however, as the amounts of leu-
cine, methionine, and p-coumaric acid increased,
morningglory biomass decreased. These authors
suggested that methionine, glucose, and nitrate
influence the allelopathic potential of p-coumaric
acid. Higher levels of nitrate decreased the con-
centration of methionine and increased the con-
centration of p-coumaric acid required to inhibit
morningglory biomass. These authors have con-
 vincingly shown that the amount of allelopathic
compound needed for a given inhibition of
morningglory biomass reduced when another
source of carbon was added. Therefore, the pres-
ence of another carbon source is likely to influ-
ence the inhibitory potential of an allelochemical
through its influence on microbial utilization and
sorption of allelochemicals (Blum et al., 1993;
Pue et al., 1995).
When studying the interference potential of
Kalmia angustifolia, Inderjit and Mallik (1996)
amended different amounts of Kalmia leaf litter
into mineral soil. A concentration dependent in-
crease in the amounts of water-soluble phenolics,
available phosphate, potassium, and iron was ob-
served (Figure 3). Interestingly, root growth inhi-
Downloaded by [University of Aberdeen] at 15:29 04 April 2013
bition of black spruce (Picea mariana) grown in
the amended soils did not show any trend (Figure 4).
However, shoot growth of black spruce showed
concentration-dependent inhibition. Any effect on
black spruce seedling growth could be either due
to higher levels of phenolics, inorganic ions, or
both. It is possible that the increase of available
phosphate intensifies the inhibitory potential of
phenolics leached out of K. angustifolia, or the
increase in nutrients influences microbial ecology
that secondarily intensifies the inhibition.
Microbial populations can also act as inhibi-
tors or promoters of allelopathic effects. In fact,
most allelopthic situations are influenced by a
microbial population in one way or another. For
example, it is clear that phenolic acids are often
implicated in allelopathy (Inderjit, 1996; Dalton,
1999), and phytotoxic effects of phenolic acids
are modified by neutral substances (e.g., glucose)
and promoters (e.g., nitrate) (Blum et al., 1993).
However, phytotoxicity of phenolic acids is also
influenced by microbial populations (Kaminsky,
1981). Schmidt et al. (2000) concluded that simple
phenolic compounds such as salicylate are un-
likely to persist for long time in soil due to micro-
bial consumption. Consequently, they have lim-
ited role in allelochemical interference. The
substrate-induced growth response (SIGR) method
(originally proposed by Schimdt, 1992) was em-
ployed to quantify salicylate-mineralizing mi-
crobes and total microbial biomass in soils below
Salix brachycarpa or its surrounding meadow
dominated by Kobresia myosuroides. Soil was
incubated with substrate at a concentration that
was determined earlier to induce the growth of a
specific microbial functional group (Schmidt et
al., 2000). Salicylate (200 µg C/g), glucose/
glutamate (2000 µg C/g) and 14C-labeled sub-
strate (< 0.1 µCi/flask) were added. Carbon diox-
ide emitted from the soil (an index of microbial
activity) was captured in 0.5 N NaOH. A shift in
the rate of carbon dioxide emission was used as
an index of mineralizing microbial biomass and
the maximum specific growth rate of that biom-
ass. Populations of salicylate-mineralizing mi-
crobes were higher in soils beneath S. brachycarpa
than soils beneath K. myosuroides based on the
SIGR assay. These authors concluded that biom-
ass and activity of salicylate mineralizing mi-
crobes can be greatly enhanced by salicylate-pro-
ducing plants in the field. In addition, Schmidt
(1988, 1990) argued that juglone may not accu-
mulate at phytotoxic levels in soils beneath black
walnut (Juglans nigra). This was due primarily to
the presence of a bacterium, Pseudomonas putida,
that degrades juglone (Rettenmaier et al., 1983).
The degradation reaction is below: Juglone →
3-hydroxyjuglone → 2,3,-dihydroxybenzoate →
2-hydroxymuconic acid semialdehyde (Rettenmaier
et al., 1983). Therefore, it is desirable to carry out
experiments on soil microbiology to convincingly
argue the accumulation of allelochemicals at phy-
totoxic levels and to understand the role of mi-
crobes in the alelopathic process.
VII. IN SITU ALLELOPATHY BIOASSAY
One of the best ways to increase assurance
that any test of allelopathy reflects the natural
(field) conditions is to perform a bioassay in situ.
However, natural systems are fraught with com-
plicating factors that can reduce the credibility of
the bioassay as a test of allelopathic interference.
When performing and reporting an in situ bioas-
say, limitations imposed by the complex environ-
ment on the interpretation of the bioassay must
always be kept in mind. Here we discuss a few
possible field bioassays, their utility, and their
inadequacies.
Just as in the lab bioassay, field soil can be
amended with debris or leachate from the putative
231
Downloaded by [University of Aberdeen] at 15:29 04 April 2013

FIGURE 3. Total phenolics and inorganic ion concentration of kalmia-free (CS) and soil amended with different
amounts of Kalmia litter leachate. Mineral soil was amended with 200 ml Kalmia litter leachate, which was prepared
by soaking 5 g (MS1), 10 g (MS2), 15 g (MS3), and 25 g (MS4) kalmia litter. (Source: Inderjit and Mallik (1996).
Reproduced with permission from National Research Council Canada.)

allelopathic species. Amendment can be made on
a large scale such as in an agricultural field. The
incorporation of post-harvest rice debris into the
field caused a reduction in crop growth compared
with a nonamended field (Chou and Lin, 1976;
Chou et al., 1981). Amendment can also be done
on a small scale such as in 2 × 2 m quadrats in
natural ecosystems. On the smaller scale dose
response studies can be performed by adding dif-
ferent quantities of debris into the topsoil of dif-
ferent quadrats. Moreover, debris from the puta-
tive allelopathic species can be added to some
quadrats and debris from nonallelopathic species
can be added to other quadrats. Some of the ad-
vantages of these in situ soil amendment experi-
ments are that appropriate target species can be
used, many characteristics of the environment are
unaffected by the bioassay design, and natural
rainfall is used to moisten the bioassay system.
However, there are also complicating factors. Soil
texture, aeration, water-holding capacity, and
nutrient availability will be altered by the amend-
ment. Searching for a control that maximizes simi-
larity to these changes without the allelochemical
is a critical effort.
Instead of amending native soil with debris a
soil replacement experiment can be performed in
the field. Small areas of soil (double the size of
the bioassay species root system) can be removed
from the location impacted by an allelopathic
species. A plastic ring or pot can be inserted in the
hole and benign soil (hopefully similar in texture,
nutrient availability, bulk density, and organic
matter to the removed soil) is added to fill the
plastic sleeve. After an appropriate time for the
soil to settle (and be refilled), a bioassay species
can be planted. The control to this experiment is
prepared by replacing the soil that was removed
back into the plastic-lined soil pit. Modifications
of this experiment can be done by reciprocal trans-

232
Downloaded by [University of Aberdeen] at 15:29 04 April 2013

FIGURE 4. Relative (%) increase or decrease in root and shoot length of black spruce when grown in soil amended
with 200 ml Kalmia litter leachate, which was prepared by soaking 5 g (MS1), 10 g (MS2), 15 g (MS3), and 25 g
(MS4) kalmia litter. (Source: Inderjit and Mallik (1996). Reproduced with permission from National Research Council
Canada.)

planting of soil between affected and nonaffected
areas, or a combination experiment, including the
presence and absence of surface debris. Of course,
this experiment is only appropriate for species
releasing water-soluble allelochemicals. Other
problems with this experiment are that the bioas-
say species are released from root competition
with other species, soil nutrient availability will
increase relative to the undisturbed soil, and there
may be changes in water availability because
overland flow is disrupted.
If the source of allelochemicals is hypoth-
esized to reside in litter and decomposing humus
on top of the soil in a natural ecosystem, a recip-
rocal transplant experiment can be a useful in situ
bioassay (Nilsen et al., 1999, 2001; Walker et al.,
1999). Litter and humus layers are removed from
plots affected by the putative allelopathic species.
These materials from all quadrats are pooled into
litter and humus piles. In a similar manner, litter
and organic matter is removed from quadrats in
regions unaffected by the allelopathic species.
These materials are also pooled into separate piles.
The litter and organic matter is then mixed within
piles and replaced into specified plots (layered as
in the natural condition) in a reciprocal replace-
ment design. All the quadrats are planted with the
same bioassay species, and growth of the bioas-
say species is followed over time. The reciprocal
replacement design is an excellent way of estab-
lishing statistically significant results (compari-
son by ANOVA). If significant toxicity is associ-
ated with the litter or humus of the putative
allelopathic species in all quadrat locations, then
one could assign allelopathic potential to the litter
and humus. This would also be verified if inhibi-
tion were negated by utilizing litter and humus
from a nonallelopathic species in regions affected
by the putative allelopathic species. However,
this study only can verify if the toxicity resides in
litter or humus. If inhibition in areas affected by
the allelopathic species is unresponsive to litter or

233
 humus transplantation, then the mechanism of
inhibition cannot be determined. This is because
there remain many possible mechanisms of inhi-
bition such as nutrient limitation, soil toxicity
(e.g., heavy metal), toxicity from root exudates,
throughfall toxicity, volatile compound toxicity,
light competition, mycorrhizal interference, etc.
Another possible mechanism of relieving al-
lelopathy in situ is by site amendment with acti-
vated charcoal (Nilsson, 1994; Callaway and
Aschehoug, 2000; Ridenour and Callaway, 2001).
The philosophy behind this in situ bioassay is that
water-soluble organic constituents (some of which
are allelochemicals) are removed from the site by
a layer of activated charcoal. If bioassay species
perform better in the presence of the activated
Downloaded by [University of Aberdeen] at 15:29 04 April 2013
charcoal (layered on top of the mineral soil below
the litter later) then inhibition by soluble organic
compounds has been relieved. Bioassay species
are planted with and without activated charcoal in
areas affected or not affected by the alleopathic
species. If inhibition is released by the activated
charcoal only in regions affected by the
allelophathic species, then allelopathy is a prob-
able mechanism of inhibition. However, if bioas-
say species grow better in the presence of char-
coal in both affected and unaffected regions, then
allelopathy is not a probable mechanism of inhi-
bition. This study is complicated by many factors
associated with the activated charcoal. In order to
determine if there is enough charcoal to make a
significant decrease in soluble organic constitu-
ents, some mechanism of collecting soil solution
and measuring soluble organic materials is re-
quired. Activated charcoal can alter other aspects
of soil chemistry in addition to any removal of
soluble organics. Therefore, the experiment must
be designed with enough statistical robustness to
assay quantitative differences in release between
affected and unaffected regions.
Several types of in situ germination trials are
possible. Seed germination can be reduced by
inappropriate physiological cues, pathogens, her-
bivores, resource availability, or allelochemicals.
The trick with in situ bioassay of germination is
to negate all other mechanisms of seedling inhibi-
tion except allelochemicals. One possibility is to
plant seeds in random locations (some of which
are affected by a putative allelopathic species) in
234
cages that prevent access by seed herbivores. Seeds
are examined often to determine mortality by
pathogens, and watered as necessary. Germina-
tion in the lab under sterile conditions is required
to establish the maximum potential germination
rate. Allelochemical inhibition of seed germina-
tion is hypothesized if in situ % germination (- the
effect of pathogens) in areas affected by the puta-
tive allelopathic species is less than that in areas
unaffected. This type of study is best performed
with bioassay seed of species that are experienc-
ing negative interference in the field. These in situ
germination trials are problematic because the
germination of native seed is often low even with-
out any external inhibition, and seed may take
several years to have internal inhibitors removed.
Moreover, other important environmental pertur-
bations, such as ingestion by animals, floods, or
fires, may be required for maximum germination
in the field. Therefore, these field in situ germina-
tion trials are complicated by the species-specific
physiological and ecological requirements for seed
germination.
In situations where inhibition occurs, but it is
unknown if inhibition is due to resource limita-
tion (by competition or otherwise), in situ release
from resource limitation is a possible experimen-
tal design. In resource release experiments, lim-
ited resources are added to the system, or the
mechanism of resource limitation is removed. This
can be done by a number of possible site manipu-
lations depending of the experimental system: for
example, competitive species removed, mycor-
rhizae added, above-ground shade removed, fer-
tilizer added, water added, site trenched to re-
move nutrient absorption by competing roots,
fugicide or bacteriacide added to inhibit micro-
bial sequestering of nutrients. If there is a positive
response to the resource supplementation, then
one could suggest that inhibition of growth was
due to resource limitation not allelopathy. In any
of these studies an insignificant response of the
bioassay species to resource supplementation does
not necessarily imply that allelopathy is the mecha-
nism of inhibition. For example, the specific re-
sources that have been augment might not be the
resources that are limiting growth, the resource
supplementation may not have been adequate to
stimulate a response, the resource supplementa-
 tion might not have reached the absorptive sur-
faces, other nontarget organisms (perhaps mi-
crobes) accumulated all the supplementary re-
sources, or the resource supplement may have
caused some toxicity to target species that are
adapted to low resource availability. Therefore, it
is necessary to understand the target plant re-
sponses to resources and have a good mechanism
of determining resource availability in the treat-
ment system. Designing a control can be very
difficult in these systems because changing re-
source availability also alters many other ecosys-
tem and community processes.
These are just a few of the possible field
manipulation experiments that address allelopa-
thy in situ. All of these designs have serious
Downloaded by [University of Aberdeen] at 15:29 04 April 2013
flaws, yet much can be learned from field studies
of plant-plant interference. New designs of field
experiments are needed to advance our apprecia-
tion for the significance of allelopathy to agricul-
tural and natural systems.
VIII. SUMMARY OF IMPORTANT
CONSIDERATIONS
Allelochemicals may not be toxic per se.
Therefore, laboratory-based bioassays that isolate
allelochemicals from natural community processes
are inadequate for evaluating if allelopathy oc-
curs in nature. Negative results in a lab bioassay
should not be taken as definitive proof that allel-
opathy does not occur in a particular situation.
Factors playing an important role in the toxicity
of allelochemicals include their concentration,
residence time, fate in the environment, and their
effect on substratum ecology. Most studies on
allelochemicals or ecotoxins are carried out at the
population level or in laboratory bioassay, but
differences between temporary and long-term
changes, due to ecotoxins, at the species and com-
munity level is not well understood. It is impor-
tant to design studies to: (1) explore quantitative
and qualitative differences between temporary vs.
long-term ecological changes due to ecotoxins,
(2) define the changes at the population and com-
munity level, (3) study the fate of ecotoxins in the
ecosystem, and (4) find the ecotoxin-organismic
equilibrium, that is, at what concentration level in
the environment, the selected ecotoxins have ad-
verse effects on organisms and ecosystem com-
ponents.
Performing bioassays in field situations are
critical to validating the significance of allelopa-
thy observed in lab bioassay to natural and agri-
cultural systems. New experimental designs for
in situ bioassay are required that can account for
the large number of complicating factors in a
complex environment. New field bioassay ex-
periments linked to physiological monitoring of
target species and biochemical monitoring of the
growth medium may be one important approach
for the future of allelopathy research.
REFERENCES
Ahmed, M. and Wardle, D. A. 1994. Allelopathic potential
of vegetative and flowering ragwort (Senecio jacobaea L.)
plants against associated pasture species. Plant Soil 164:
61–68.
Bell, D. T. and Koeppe, D. E. 1972. Noncompetitive effects
of giant foxtail on the growth of corn. Agron. J. 64:
321–325.
Blum, U., Gerig, T. M., Worsham, A. D., and King, L. D.
1993 Modification of allelopathic effects of p-
Coumaric acid on morning-glory seedling biomass
by glucose, methionine, and nitrate. J. Chem. Ecol.
19: 2791–2811.
Blum, U., Shafer, S. R., and Lehman, M. E. 1999. Evidence
for inhibitory allelopathic interactions involving phe-
nolic acids in field soils: concepts vs. experimental
model. Crit. Rev. Plant Sci. 18: 673–693.
Blum, U. 1995. The value of model plant-microbe-soil sys-
tems for understanding processes associated with al-
lelopathic interactions: one example. In: Allelopathy:
Organisms, Processes and Applications. pp. 127–131.
Inderjit, Dakshini, K. M. M. and Einhellig, F. A.,
Eds., American Chemical Society, Washington, DC.
Blum, U. 1996. Allelopathic interactions involving phenolic
acids. J. Nematol. 28: 259–267.
Blum, U. 1999. designing laboratory plant debris-soil bioas-
says: somD reflections. In: Principles and Practices
in Plant Ecology: Allelochemical Interactions. pp.
17–23. Inderjit, Dakshini, K. M. M. and Foy, C. L.,
Eds., CRC Press, Boca Raton, FL.
Blum, U. and Shafer, S. R. 1988. Microbial populations and
phenolic acids in soils. Soil Biol. Biochem. 20: 793–
800.
Blum, U., Worsham, A. D., King, D. L., and Gerig, T. M.
1994. Use of water and EDTA extraction to estimate
available (free and reversibly bound) phenolic acids
in Cecil soils. J. Chem. Ecol. 20: 341–359.
235
 Box, J. D. 1983. Investigations of the Folin-Ciocalteu phenol
reagent for the determination of polyphenolic sub-
stances in natural water. Water Res. 17: 511–525.
Callaway, R. M. and Aschehoug, E. T. 2000. Invasive plant
versus their new and old neighbors: a mechanism for
exotic invasion. Science 290: 521–523.
Cheng, H. H. 1995. Characterization of the mechanisms of
allelopathy: Modeling and experimental approaches.
In: Allelopathy: Organisms, Processes and Applica-
tions. pp. 132–141. Inderjit, Dakshini, K. M. M. and
Einhellig, F. A., Eds., American Chemical Society,
Washington, DC.
Chou, C. H. and Lin, H. J. 1976. Autointoxication of mecha-
nism of Oryza sativa. I. Phytotoxic effects of decom-
posing rice residues in soil. J. Chem. Ecol. 2: 353–
367.
Chou, C. H., Chiang, Y. C., and Cheng, H. H. 1981. Auto-
intoxication mechanism of Oryza sativa. III. Effect of
temperature on phytotoxin production during rice straw
Downloaded by [University of Aberdeen] at 15:29 04 April 2013
decomposition in soil. J. Chem. Ecol. 7: 741–752.
Czarnota, M. A. 2001. Sorghum (Sorghum spp.) Root Exu-
dates: Production, Localization, Chemical Composi-
tion and Mode of Action. Ph.D. thesis. Cornell Uni-
versity, Ithaca, NY.
Czarnota, M. A., Buxton, J. W., and Weston, L. A. 2001.
Mode of action, localization of production, chemical
nature and activity of sorgoleone: a potent PS II in-
hibitor in Sorghum spp. root exudates. Weed Technol.
15: 813–825.
Dalton, B. R. 1999. The occurrence and behavior of plant
phenolic acids in soil environment and their potential
involvement in allelochemical interference interac-
tions: methodological limitations in establishing con-
clusive proof of allelopathy. In: Principles and Prac-
tices in Plant Ecology: Allelochemical Interactions.
pp. 57–74. Inderjit, Dakshini, K. M. M., and Foy, C.
L., Eds., CRC Press, Boca Raton, FL.
Del Moral, R. and Muller, C. H. 1970. The allelopathic
effects of Eucalyptus camaldulensis. Am. Midl. Nat.
83: 254–82.
Del Moral, R. and Muller, C. H. 1969. Fog drip: a mecha-
nism of toxin transport from Eucalyptus globulus.
Bull. Torrey Bot. Club 96: 467–475.
Del Moral, R. and Cates, R. G. 1971. Allelopathic potential
of the dominant vegetation of western Washington.
Ecology 52: 1030–1037.
Farhoomand, M. B. and Peterson, L. A. 1968. Concentration
and content. Agron. J. 60: 708–709.
Fischer, N. H. 1986. The function of mono and sesquiterpenes
as plant germination and growth regulators. In: The
Science of Allelopathy. pp. 203–218. Putnam, A. R. and
Tang, C. S., Eds., John Wiley & Sons, New York.
Foy, C. L. 1999. How to make bioassays for allelopathy
more relevant to field conditions with particular refer-
ence to cropland weeds. In: Principles and Practices
in Plant Ecology: Allelochemical Interactions. pp.
25–33. Inderjit, Dakshini, K. M. M. and Foy, C. L.,
Eds., CRC Press, Boca Raton, FL.
236
Haig, T. 2001. Application of hyphenated chromatorgraphy-
mass spectrometry techniques to plant allelopathy
research. J. Chem. Ecol. 27: 2363–2396.
Harper, J. L. 1994. Population Biology of Plants. Academic
Press, London.
Horsley, S. B. 1990. Allelopathy In: Biophysical Research
for Asian Agroforestry. pp. 167–183. Avery, M. E.,
Cannell, M. G. R., and Ong, C. K., Eds., Winrock
International, USA.
Huang, P. M., Wang, M. C., and Wang M. K. 1999. Catalytic
transformation of phenolic compounds in the soil. In:
Principles and Practices in Plant Ecology: Allelochemical
Interactions. pp. 287–306. Inderjit, Dakshini, K. M. M.
and Foy, C. L., Eds., CRC Press, Boca Raton, FL.
Inderjit. 1996. Plant phenolics in allelopathy. Bot. Rev. 62:
182–202.
Inderjit. 2001. Soils: environmental effect on allelochemical
activity. Agron. J. 93: 79–84.
Inderjit and Dakshini, K. M. M. 1992. Interference potential
of Pluchea lanceolata (Asteraceae): growth and physi-
ological responses of asparagus bean, Vigna
unguiculata var. sesquipedalis. Am. J. Bot. 79: 977–
981.
Inderjit and Dakshini, K. M. M. 1995. On laboratory bioas-
says in allelopathy. Bot. Rev. 61: 28–44.
Inderjit and Dakshini, K. M. M. 1994a. Allelopathic effect of
Pluchea lanceolata (Asteraceae) on characteristics of
four soils and tomato and mustard growth. Am. J. Bot.
81: 799–804.
Inderjit and Dakshini, K. M. M. 1994b. Allelopathic poten-
tial of phenolics from the roots of Pluchea lanceolata.
Physiol. Plant. 92: 571–576.
Inderjit and Dakshini, K. M. M. 1998. The use of washed test
material as control in laboratory bioassay for allelopa-
thy. Trop. Agric. 75: 396–400.
Inderjit and Dakshini, K. M. M. 1999. Bioassay for allelopa-
thy: interactions of soil organic and inorganic con-
stituents. In: Principles and Practices in Plant Ecol-
ogy: Allelochemical Interactions. pp. 35–44. Inderjit,
Dakshini, K. M. M. and Foy, C. L., Eds., CRC Press,
Boca Raton, FL.
Inderjit and Del Moral, R. 1997. Is separating resource com-
petition from allelopathy realistic? Bot. Rev. 63: 221–
230.
Inderjit, and Keating, K. I. 1999 Allelopathy: principles,
procedures, processes, and promises for biological
control. Adv. Agron. 67: 141–231.
Inderjit and Mallik, A. U. 1996. The nature of interference
potential of Kalmia angustifolia. Can. J. For. Res. 26:
1899–1904.
Inderjit and Mallik, A. U. 1997. Effect of phenolic com-
pounds on selected soil properties. For. Ecol. Man-
age. 92: 11–18.
Inderjit and Weiner, J. 2001. Plant allelochemical interfer-
ence or soil chemical ecology? Perspect. Plant Ecol.
Evol. System. 4: 3–12.
Inderjit, Cheng, H. H., and Nishimura, H. 1999. Plant phe-
nolics and terpenoids: transformation, degradation,
 and potential for allelopathic interactions. In: Prin-
ciples and Practices in Plant Ecology: Allelochemical
Interactions. pp. 255–266. Inderjit, Dakshini, K. M.
M. and Foy, C. L., Eds., CRC Press, Boca Raton, FL.
Inderjit, Muramatsu, M., and Nishimura, H. 1997. On allelo-
pathic potential of terpenoids, phenolics and their
mixture, and their recovery in soil. Can. J. Bot. 75:
888–891.
Inderjit, Striebig, J., and Olofsdotter, M. 2002. Joint action
of phenolic acid mixtures and its significance in allel-
opathy research. Physiol. Plant. 114: 422–428.
Inderjit and Weston, L. A. 2000. Are laboratory bioassays
for allelopathy suitable for prediction of field re-
sponses? J. Chem. Ecol. 26: 2111–2118.
Kaminsky, R. 1981. The microbial origin of the allelopathic
potential of Adenostoma fasiculatum H & A. Ecol.
Monogr. 51: 365–382.
Kuiters, A. T. and Denneman, C. A. J. 1987. Water-soluble
phenolic substances in soils under several coniferous
Downloaded by [University of Aberdeen] at 15:29 04 April 2013
and deciduous tree species. Soil Biol. Biochem. 19,
765–769.
Leather, G. R. and Einhellig, F. A. 1986. Bioassays in the
study of allelopathy. In: The Science of Allelopathy.
pp. 133–145. Putnam, A. R. and Tang, C. S., Eds.,
John Wiley & Sons, New York.
Leather, G. R. and Einhellig, F. A. 1988. Bioassay in natu-
rally occurring allelochemicals for phytotoxicity. J.
Chem. Ecol. 14: 1821–1828.
Leather, G. R., and Einhellig, F. A. 1985. Mechanisms of
allelopathic action in bioassay. In: The Chemistry of
Allelopathy. pp. 197–205. Thompson, A. C., Ed.,
American Chemical Society, Washington, D.C.
Nilsen, E. T., Walker, J. F., Miller, O. K., Semones, S. W.,
Lei, T. T., and Clinton, B. D. 1999. Inhibition of
seedling survival under Rhododendron maximum
(Ericaceae): could allelopathy be a cause? Am. J. Bot.
86: 1597–1605.
Nilsen E.T., Lei, T. T., Semones, S. W., Walker, J. F., Miller,
O. K., and Clinton, B. 2001. Does Rhododendron
maximum L. (Ericaceae) reduce the availability of
resources above and belowground for canopy tree
seedlings. Am. Midl. Natu. 145: 324–343.
Nilsson, M. C. 1994. Separation of allelopathy and resource
competition by the boreal dwarf shrub Empetrum
hermaphroditum hagerup. Oecologia 98: 1–7.
Oleszek, W. and Jurzysta, M. 1987. The allelopathic poten-
tial of alfalfa root medicagenic acid glycosides and
their fate in soil environment. Plant Soil 98: 67–80.
Pardales, J. R., Jr., Kono, Y., Yamauchi, A., and Iijima, M.
1992. Seminal root growth in sorghum (Sorghum bi-
color) under allelopathic influences from residues of
taro (Colocasia esculenta). Ann. Bot. 69: 493–496.
Pedersen, G. A. 1986. White clover seed germination in agar
containing tall fescue leaf extracts. Crop Sci. 26: 1248–
1249.
Pue, K. J., Blum, U., Gerig, T. M., and Shafer, S. R. 1995.
Mechanism by which noninhibitory concentrations of
glucose increase inhibitory activity of p-coumaric acid
on morning-glory seedling biomass accumulation. J.
Chem. Ecol. 21: 833–847.
Putnam, A. R. and Duke, W. B. 1978. Allelopathy in
agroecosystems. Ann. Rev. Phytopathol. 16: 431–451.
Qasem, J. R. and Hill, T. A. 1989. On difficulties with
allelopathy methodology. Weed Res. 29: 345–347.
Rettenmaier, H., Kupas, U., and Lingens, F. 1983. Degrada-
tion of juglone by Pseudomonas putida J1. FEMS
Microbiol. Lett. 19: 193–197.
Rice, E. L. 1984. Allelopathy. Academic Press, Orlando, FL
Ridenour, W. M. and Callaway, R. M. 2001. The relative
importance of allelopathy in interference: the effect of
an invasive weed on a native bunchgrass. Oecologia
126: 444–450.
Rimando, A. M., Olofsdotter, M., Dayan, F. E., and Duke, S. O.
2001. Searching for rice allelochemicals: an example of
bioassay-guided isolation. Agron. J. 93: 16–20.
Romeo, J. T. 2000. Raising the beam: moving beyond phy-
totoxicity. J. Chem. Ecol. 26: 2011–2014.
Romeo, J. T. and Weidenhamer, J. D. 1998 Bioassays for
allelopathy in terrestrial plants. In: Methods in Chemi-
cal Ecology. Vol. 2. Bioassay Methods. pp. 179–211.
Haynes, K. F. and Millar, J. G., Eds., Kluwer Aca-
demic Publishing, Norvell, MA.
Schmidt, S. K. 1988. Degradation of juglone by soil bacteria.
J. Chem. Ecol. 14: 1561–1571.
Schmidt, S. K. 1990. Ecological implications of the destruc-
tion of juglone (5–hydroxy-1,4–naphthoquinone) by
soil bacteria. J. Chem. Ecol. 16: 3547–3549.
Schmidt, S. K. 1992. A substrate-induced growth-response
(SIGR) method for estimating the biomass of micro-
bial functional groups in soil and aquatic systems.
FEMS Microbiol. Ecol. 101: 1970–206.
Schmidt, S. K., Lipson, D. A., and Raab, T. A. 2000. Effects
of willows (Salix brachyearpa) on populations of sali-
cylate-mineralizing microorganisms in alpine soils. J.
Chem. Ecol. 26: 2049–2057.
Schmidt, S. K. and Ley, R. E. 1999. Microbial competition and soil
structure limit the expression of phytochemicals in nature.
In: Principles and Practices in Plant Ecology: Allelochemical
Interactions. pp. 339–351. Inderjit, Dakshini, K. M. M. and
Foy, C. L., Eds., CRC Press, Boca Raton, FL.
Schumacher, W. J., Thill, D. C., and Lee, G. A. 1983. Allelo-
pathic potential of wild oat (Avena fatua) on spring
wheat (Triticum aestivum) growth. J. Chem. Ecol. 9:
1235–1246.
Stowe, L. G. 1979. Allelopathy and its influence on distribu-
tion of plants in an Illinois old-field. J. Ecol. 67:
1065–1085.
Stowe, L. G. and Osborn, A. 1980. The influence of nitrogen
and phosphorus levels on the phytotoxicity of phe-
nolic compounds. Can. J. Bot. 58: 1149–1153.
Streibig, J. C., Dayan, F. E., Rimando, A. M., and Duke, S.
O. 1999. Joint action of natural and synthetic photo-
system II inhibitors. Pestic. Sci. 55: 137–146.
Swain, T. and W. E. Hillis. 1959. The phenolic constituents
of Prunus domestica. I. The quantitative analysis of
phenolic constituents. J. Sci. Food Agric. 10: 63–68.
237
 Tang, C. S. and Young, C. C. 1982. Collection and identifi-
cation of allelopathic compounds from the undisturbed
root system of bigalta limpograss (Hemarthria
altissima). Pl. Physiol. 69: 155–160.
Tang, C. S., W. F. Cai, K. Kohl, and R. K. Nishimoto. 1995.
Plant stress and allelopathy. In: Allelopathy: Organ-
isms, Processes, and Applications. pp. 142–157.
Inderjit, Dakshini, K.M.M. and Einhellig, F. A., Eds.,
ACS Symp. Ser. 582, American Chemical Society,
Washington, DC.
Tanrisever, N., Fronczek, F. R., Fischer, N. H., and
Williamson, G. B. 1987. Ceratiolin and other fla-
vonoids from Ceratiola ericoides. Phytochemistry 26:
175–179.
Thijs, H., Shann, J. D., and Weidenhamer, J. D. 1994. The
effect of phytotoxins on competitive outcome in a
model system. Ecology 75: 1959–1964.
Tinnin, R. O. and Muller, C. H. 1971. The allelopathic
potential of Avena fatua: Influence on herb distribu-
Downloaded by [University of Aberdeen] at 15:29 04 April 2013
tion. Bull. Torrey Bot. Club 98: 243–50.
Torti, S. D., Dearing, M. D., and Kursar, T. A. 1995. Extrac-
tion of phenolics compounds from fresh leaves: a
comparison of methods. J. Chem. Ecol. 21: 117–125.
Van Alstyne, K. L. 1995. Comparison of three methods for
quantifying brown algal polyphenolic compounds. J.
Chem. Ecol. 21: 45–58.
238
Walker J., Miller, O. K., Lei, T. T., Semones, S. W., Nilsen, E.
T., and Clinton, B. D. 1999. Inhibition of ectomycorrhizal
synthesis in subcanopy seedlings of canopy trees below
Rhododendron maximum. Mycorrhiza 9: 49–56.
Wardle, D. A., Nilsson, M. C., Gallet, C., and Zackrisson, O.
1998. An ecosystem level perspective of allelopathy.
Biol. Rev. 73: 305–319.
Waterman, P. G., and Mole, S. 1994. Methods in Ecology:
Analysis of Phenolics Plant Metabolities. Blackwell
Scientific Publication, Oxford.
Weidenhamer, J. D., Morton, T. C., and Romeo, J. T. 1987.
Solution volume and seed number: often overlooked factor
in allelopathic bioassays. J. Chem. Ecol. 13: 1481–1491.
Weidenhamer, J. D. 1996. Distinguishing resource competi-
tion and chemical interference: overcoming the meth-
odological impasse. Agron. J. 88: 866–875.
Williams, R. D. and Hoagland, R. E. 1982. The effects of
naturally occurring phenolic compounds on seed ger-
mination. Weed Sci. 30: 206–212.
Williamson, J. B. and Richardson, D. R. 1988. Bioassays for
allelopathy: measuring treatment responses with inde-
pendent controls. J. Chem. Ecol. 14: 181–187.
Willis, R. J. 1985. The historical bases of the concept of
allelopathy. J. Hist. Biol. 18: 71–102.
Wolfson, J. L. 1988. Bioassay techniques: an ecological
perspective. J. Chem. Ecol. 14: 1951–1963.