Journal Pre-proof

Soil amendment by composted plant wastes reduces the Verticillium

dahliae abundance and changes soil chemical properties in a bell pepper
cropping system

Ashraf M. Tubeileh, Gregg T.Stephenson

PII: S2214-6628(20)30029-3
DOI: https://doi.org/10.1016/j.cpb.2020.100148
Reference: CPB 100148

To appear in: Current Plant Biology

Received Date: 25 September 2019

Revised Date: 23 March 2020
Accepted Date: 1 April 2020

Please cite this article as: Tubeileh AM, Gregg TStephenson, Soil amendment by composted
plant wastes reduces the Verticillium dahliae abundance and changes soil chemical
properties in a bell pepper cropping system, Current Plant Biology (2020),
doi: https://doi.org/10.1016/j.cpb.2020.100148

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© 2020 Published by Elsevier.

Soil amendment by composted plant wastes reduces the Verticillium dahliae abundance
and changes soil chemical properties in a bell pepper cropping system
Ashraf M. Tubeileh* atubeile@calpoly.edu, Gregg T. Stephenson
California Polytechnic State University, San Luis Obispo, CA 93407, USA
*Corresponding author.

ABSTRACT

Organic amendments can be effective at controlling some soil-borne pathogens. This work
entailed two parts; the first involved two field experiments that investigated the effects of four

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different composts derived from agricultural wastes on soil load of Verticillium dahliae in organic
and conventional bell pepper (Capsicum annuum L.) systems. The composts were prepared using
dairy and horse manure (DC), grape pomace (GC), olive pomace-dairy manure mix (ODC) or mixed

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crop residue (CRC). For each system, one 25 tonne ha-1 compost application was made in May
2018 before bell pepper planting. Soil samples were taken before application and four times after

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application, approximately every six weeks. Plant growth and yield were also monitored. The
second part involved lab experiments that tested the effects of extracts of the above composts
on the growth of V. dahliae on selective media in Petri dishes. All four compost treatments
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significantly suppressed pathogen populations in June, two weeks after the application of
treatments, with plant-based amendments presenting greater effects than DC. These
suppressive effects disappeared within eight weeks (for DC) or 14 weeks (for GC, ODC and CRC)
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after application. The effects of composts on soil chemical properties, V. dahliae abundance and
bell pepper growth and yield parameters were generally similar in both organic and conventional
fields. In the Petri dish assays, plant composts reduced V. dahliae growth by 40-60%, especially
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during the first week after application.

KEYWORDS: DAIRY MANURE COMPOST; GRAPE POMACE COMPOST; OLIVE POMACE COMPOST;
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ORGANIC CROPPING SYSTEM; SOIL-BORNE PLANT PATHOGEN; VEGETABLE PRODUCTION.

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INTRODUCTION

Verticillium dahliae Kleb. is a devastating soil-borne pathogen that has with a wide host range of
over 150 economically important crops (McCain et al., 1981). The host list includes, to various
degrees of susceptibility, several of the top ten crops in California, such as grapes (Vitis vinifera
L.), almonds (Prunus dulcis (Mill.) Webb.) (and other stone fruits), strawberries (Fragaria x
ananassa Duch.), lettuce (Lactuca sativa L.), tomatoes (Solanum lycopersicum L.) (and other
solanaceous plants), and alfalfa (Medicago sativa L.). For Verticillium spp., climate change is

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expected to stimulate fungal growth by increasing soil temperatures towards the biological
optimum in colder soils [1] or by extending the infection period [2]. Models predict an increase
in the frequency and severity of soil-borne fungal pathogens due to climate change [3]. At the
same time, current solutions to soil fungi contribute to climate change. Using soil fumigants such
as chloropicrin and methyl isothiocyanate resulted in a 10-fold increase in topsoil N2O flux rates
that persisted for 48 days with chloropicrin fumigation [4]. Finding less harmful solutions and
adapting to these new climate paradigms are especially important in organic crop production
systems given their limited toolbox. In the meantime, some agricultural wastes have shown some
promise as pest-management tools, instead of presenting environmental burdens [5]. Many
reports show effectiveness of composts against Phytophthora, Pythium and Rhizoctonia root rots

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[6,7]. Grape (Vitis vinifera L.) and olive (Olea europaea L.) pomaces are high in nutrients and
contain antimicrobial and phytotoxic compounds, including polyphenols [8–10]. In a pot study
using peat, a mix of olive leaves and olive pomace composted for three months completely

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eliminated Phytophthora nicotianae and Fusarium oxysporum on tomatoes compared to almost
100% disease incidence in disease-inoculated control plants [11]. The same result was obtained
with grape compost in the same study. However, to our knowledge, there are no studies

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comparing the effects of variable composts on soil load of V. dahliae and phytotoxicity in bell
peppers (Capsicum annuum L.). Bell peppers were selected as they are to be susceptible to V.
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dahliae and California is the top bell pepper producer in the USA with 2017 acreage in excess of
6400 ha [12]. The objectives of this study were
 to determine the effects of soil applications of manure-based compost (DC), grape
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pomace compost (GC), olive pomace-dairy manure mixed compost (ODC), or mixed crop
residue compost (CRC) on soil V. dahliae load and health and yield of a bell pepper crop
under organic and conventional production conditions, and
 to assess the effects of extracts from above-mentioned composts on in vitro V. dahliae
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growth.

MATERIALS AND METHODS

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General field characteristics

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Two field experiments were conducted during the summer growing season of 2018 from
May to November, on the California Polytechnic State University campus in San Luis Obispo, CA
(35°18'15"N, 120°40'21"W). The two fields were 500 m apart; Field 1 was located within the Cal
Poly Organic Farm and under organic management since 2004, while Field 2 is conventionally
managed. Previous crops in the organic field include vegetable row crops such as kale (Brassica
oleracea var. sabellica L.), broccoli (B. oleracea var. italica L.), and cauliflower (B. oleracea var.
botrytis L.). Previous crops in the conventional field include pumpkins (Cucurbita pepo pepo L.)
and broccoli. Throughout the duration of the experiment both fields were managed organically

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in order to eliminate any management effects. The soil texture is classified as clay loam with 45%
sand, 23% silt and 32% clay. Prior to the treatment application, three composite samples were
taken from each field to determine soil chemical properties. The methods used are standard
methods recommended by the Soil Science Society of America. A summary of the analysis results
from both fields is presented in Table 1.

Table 1. Soil chemical properties in our experimental fields.

Parameter Organic field Conventional field
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Soil organic matter (g kg ) 37.0 ± 3.4 29.7 ± 2
Olsen P (mg kg-1) 75.3 ± 7.4 65.3 ± 3.8

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K (mg kg-1) 509 ± 88.2 565 ± 56.6
Mg (mg kg-1) 960 ± 82.1 881 ± 33.8
Ca (mg kg-1)

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2057 ± 199 2979 ± 103
Na (mg kg-1) 51.0 ± 8.5 49.6 ± 2.5
pH 7.23 ± 0.1 7.67 ± 0.1

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C.E.C. (meq/100 g) 19.7 ± 1.9 23.7 ± 0.5
N (NO3-N) (mg kg-1) 18.7 ± 2.5 4.7 ± 1.5
S (SO4-S) (mg kg-1) 9.3 ± 4.4 12.0 ± 1.7
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Zn (mg kg-1) 3.23 ± 0.21 1.60 ± 0.10
Mn (mg kg-1) 3.00 ± 1.00 6.33 ± 2.08
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Fe (mg kg-1) 18.0 ± 1.0 8.67 ± 0.6

Cu (mg kg-1) 3.60 ± 0.10 1.50 ± 0.10
B (mg kg-1) 0.63 ± 0.06 0.70 ± 0.10
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Soluble Salts (dS m-1) 0.53 ± 0.40 0.60 ± 0.10

Values represent means ± standard deviation (n = 3).
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Organic amendment sources

The organic amendment treatments applied were dairy manure-based compost (DC),
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grape pomace compost (GC), olive pomace-dairy manure mixed compost (ODC) or mixed crop
residue compost (CRC), in addition to a control treatment with no compost application. The DC
consisted of mainly dairy manure mixed with horse manure and wood chips to achieve target C:N
ratio, around 25, and pathogen reduction goals. Fresh olive pomace was provided by La Panza
Ranch in San Luis Obispo County, CA. The olive pomace was mixed (1:1 vol.) with dairy manure
to achieve target C:N ratio between 25 and 30, and was windrow composted from December
2017 to application in June 2018 on the Cal Poly campus. These two composts were processed at
the California Polytechnic State University Composing Unit according to California Department of

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Food and Agriculture and organic certification guidelines. The GC consisted of ‘Pinot Noir’ seeds
and skins. It was windrow composted and provided by the Cagliero Ranch in San Miguel, CA. The
CRC was produced from a mixture of plant waste materials including strawberry crowns, grape
pomace, general green and vegetable waste, and was provided by Engel and Gray, Inc. in Santa
Maria, CA. Compost analysis is given in Table 2.

Table 2. Characterization of the composts used in the experiments.

Parameter Compost
DC GC ODC CRC
pH Value 8.40 8.20 8.71 8.10

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Electrical Conductivity (dS m ) 3.60 1.40 2.60 4.40
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Density (kg m ) 705 545 561 625

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Organic Matter (mg g ) 307 446 476 359
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Organic Carbon (mg g ) 150 250 280 170
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Ash (mg g ) 693 554 524 641

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C/N Ratio (g g ) 10.7 14.7 16.5 12.1
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Carbonates (mg g ) 16 6.0 7.5 12.5
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Total N (mg g ) 14.0 17.0 17.0 14.0
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+ -1
NH4 (mg kg ) 13.0 11.0 21.0 17.0
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NO3 (mg kg ) 430 70 15 240
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P (mg g ) 5.7 2.5 4.0 3.9

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K (mg g ) 14 14 12 11
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Ca (mg g ) 25 18 18 24
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Mg (mg g ) 16 11 11 6
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SO4 (mg kg ) 1200 60 530 2500
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Cu (mg kg ) 53 29 42 71
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Zn (mg kg ) 210 85 130 190
-1
Fe (mg kg ) 18000 13000 11000 11000
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Mn (mg kg ) 500 290 300 300
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B (mg kg ) 21 22 27 22
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Na (mg g ) 3.3 0.71 1.9 1.6
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Cl (mg g ) 2.8 0.61 2.2 3.7
Values represent one composite sample per treatment. DC: dairy manure-based compost,
GC: grape pomace compost, ODC: olive pomace compost, CRC: crop residue compost.

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Experimental design and field set-up

A completely randomized design with five replicates was laid out in each field. The plot
size was 7.5 m x 3 m, comprising three 1 m wide beds. Each compost was applied at the rate of
25 tonnes dry matter per hectare, based upon recommendations of Bonanomi et al. [13], and
spread on 12 June 2018. Then, each plot was fertilized before planting with a feather meal
fertilizer (13-0-0) at the rate of 225 kg N ha-1. After compost and fertilizer application the fields
were disked to a depth of 10 cm to incorporate the compost and raised beds were shaped. On
26 June 2018 the field was planted with bell pepper cv. ‘Huntington’ transplants with one row
per bed at a spacing of 20 cm. All plots were watered at the same rate as per UC Cooperative
Extension guidelines for peppers so that plants were not subject to water stress [14]. The

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transplanted plants were irrigated with sprinklers for the first two weeks after planting for
establishment. Then one line of 1.5 cm high-flow drip tape was laid per bed, and irrigation ran 4

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hours at a time twice per week through July, three times per week through August and
September, and twice per week through final harvest in November.
Treatment effects on soil chemical properties were evaluated from composite samples

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collected on 30 June 2018; approximately two weeks after application of compost treatments.
Composite soil samples were collected from three randomly chosen plots of each treatment from
each field. Each composite sample was made up of three subsamples collected from random
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locations within the plot at a depth of 15 cm with a soil auger then mixed thoroughly.
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Verticillium dahliae soil load

The abundance of the pathogenic fungus V. dahliae was assessed following the methods
reported in Kabir et al. [15]. For each plot, three subsamples were collected using a soil auger
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from random locations within the plot from the top 15 cm then mixed thoroughly to make one
composite sample. Random locations were chosen by breaking plots into a grid with spacing of
10 cm and using a random number generator to choose which grid blocks to sample. The first
sample was collected on 18 May 2018 before treatments were applied and prior to tillage and
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plant establishment. Preliminary testing indicated that Verticillium dahliae load in both fields was
high and sufficiently uniform that inoculation was not required. The second sample collection
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occurred two weeks after the application of the treatments on 30 June 2018. Subsequent sample
collections occurred every six weeks after the treatment application; 11 August, 21 September,
and 1 November 2018. Samples were air dried for 14 days then were mixed again and ground
with a mortar and pestle. For each sample, five separate 0.1 gram subsamples were taken, mixed
with 0.9 ml deionized water, and plated individually onto Sorenson’s NP-10 selective media in
100 mm x 15 mm polystyrene Petri dishes. Sorenson’s NP-10 is a selective media including agar
and antibiotics developed specifically to grow cultures of V. dahliae.

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 Once plated, the samples were cultured for two weeks in the dark at room temperature;
between 19°C and 22°C. Each plate was examined under a dissecting microscope and
microsclerotia or colony forming units (CFU’s) of the fungi were counted as per Goud et al. [16].
As each microsclerotia in the field could begin a new colony, the count from each dish is a sample
number of V. dahliae CFU’s per 0.1 gram of soil from the sample site.

Verticillium dahliae in vitro growth

A lab assay was conducted to assess the effects of the compost extracts, also referred to
as compost extracts, on V. dahliae growth. Extracts of each compost were prepared from a
compost to deionized water ratio of 0.2 L L-1 left at 20°C for five days. Extractions were stirred

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once per day for 60 seconds throughout the 5-day extraction period. Extracts were then poured
through Whatman 1 filter paper (Sigma-Aldrich, Darmstadt, Germany) and run through a 0.45

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m syringe filter (Corning Inc., New York, USA) to obtain the final compost extracts used in this
experiment as per Curlango-Rivera et al. [17].
Compost extracts were added during preparation of the media. First, 20 ml of each

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compost extract was added to 200 ml of Sorenson’s NP-10 selective media. Each mix was then
stirred for 2 minutes and poured into five Petri dishes. An additional five Petri dishes were filled
with 200 ml Sorenson’s NP-10 selective media and 20 ml deionized water to function as the
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control; making a total of 25 dishes. Media solidified after 48 hours and inoculations were made
with a 6-week-old colony of V. dahliae isolated from our soils. Each dish was inoculated from the
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same colony and left to culture in the dark at room temperature, between 19°C and 22°C. The
maximum linear growth within each plate was measured every 48 hours for 13 days. The
maximum radius of the colony (maximum linear growth) was considered as the measurement
from the center site of inoculation to the furthest growth within each plate as suggested by De
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Corato et al. [18].

Plant growth and yield parameters
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Bell pepper ‘huntington’ health was measured through the height and the measure of
relative chlorophyll content of the plants, in addition to visual plant observations for disease or
abiotic symptoms. Height and relative chlorophyll content were measured from five randomly
chosen plants within each plot on 2 September 2018. Height was recorded from ground level to
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the apical meristem. The relative chlorophyll content was determined on a randomly chosen
young and fully expanded leaf on selected plants using a SPAD 502 chlorophyll meter (Konica
Minolta Inc., Tokyo, Japan).
Peppers were harvested on three separate dates; 3 September, 4 October, and 5
November 2018. On each harvest date a crew worked through both fields to harvest all ripe bell
peppers. Peppers were considered ripe based on maturity, which was determined in the field
based on size and development of the shoulder ridge above the calyx [19]. At the final harvest in
November 2018, all fruits were collected including immature fruits to be included in the yield

6
biomass assessment. For analysis, all harvest fresh weights from each plot were pooled into a
season total harvest weight per plot.

Statistical analyses

Statistical analyses were carried out with JMP 14 software (SAS Institute, Cary, North
Carolina, USA). Treatment and sampling date were analyzed as main effects while field
management (organic vs. conventional) was considered random effect. When no significant
interaction between treatment and field was observed, data between fields were pooled for
analysis. Soil V. dahliae CFU numbers were square-root transformed to maximize consistency in
residuals of the model. Differences in least square means were tested by ANOVA then p-values

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from pairwise comparisons were generated by way of Student’s t-test. Benjamini-Hochberg
procedure [20] was utilized for assessment of significance from p-values to ensure maximum

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statistical power and maintain type one error rate at 0.05.

RESULTS AND DISCUSSION

Effect of composts on soil chemical properties

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In the organic farm, soil organic matter (SOM) and some microelements such as boron (B)
and zinc (Zn) tended to increase with all compost treatments, but no significant treatment effect
was observed due to high variability (Table 3). Soil pH and cation exchange capacity (CEC) were
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not affected by the treatments. DC and CRC treatments increased the levels of soil phosphorus
(P), potassium (K), sodium (Na), and sulfur (S), compared to the control, while treatments GC and
ODC had no significant effect on these elements. DC had the largest impact on soil Na with an
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average of 221 mg kg-1, four times the level observed in the control, while CRC almost tripled the
Na levels compared to the control. None of the four compost treatments had a significant effect
on soil concentrations of nitrate N (NO3-N), magnesium (Mg), calcium (Ca), copper (Cu), iron (Fe),
manganese (Mn), or soluble salts compared to the control.
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Table 3: Effect of compost treatments on soil chemical properties in the organic field.
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Soil Treatment P value

parameter
Control DC GC ODC CRC
SOM (g kg-1) 39.0 ± 1.73 49.0 ± 9.54 49.3 ± 10.1 51.7 ± 7.02 53.3 ± 11.9 0.368
pH 6.93 ± 0.21 7.17 ± 0.15 7.07 ± 0.12 7.07 ± 0.06 7.10 ± 0.10 0.384
C.E.C. (meq 22.2 ± 1.23 23.4 ± 0.53 22.3 ± 0.84 22.6 ± 0.90 23.9 ± 1.91 0.384
100 g-1)

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 NO3 (mg kg- 89.3 ± 22.1 123 ± 39.9 89.0 ± 17.1 81.7 ± 29.5 112 ± 41.8 0.472
1
)
Olsen P (mg 82.3 ± 6.66 161 ± 36.4 a 94.0 ± 8.72 99.7 ± 9.29 129 ± 25.9 0.006*
kg-1) c bc bc ab
K (mg kg-1) 613 ± 65.2 1094 ± 224 a 816 ± 13.8 ab 916 ± 149 ab 1124 ± 313 0.040*
b b
Mg (mg kg-1) 1059 ± 30.2 1004 ± 50.1 1037 ± 32.6 1047 ± 29.0 1038 ± 0.444
26.4
Ca (mg kg-1) 2272 ± 106 2275 ± 43.4 2280 ± 113 2223 ± 62.1 2373 ± 161 0.552
S (SO4-S) (mg 26.7 ± 4.62 70.0 ± 22.9 30.3 ± 6.03 c 35.7 ± 7.64 83.0 ± 40.8 0.029*

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kg-1) c ab bc a
Na (mg kg-1) 54.0 ± 6.08 221 ± 78.2 a 75.7 ± 1.53 124 ± 29.0 150 ± 55.4 0.008*

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c bc bc ab
B (mg kg-1) 0.73 ± 0.06 1.37 ± 0.38 0.87 ± 0.06 1.03 ± 0.23 1.33 ± 0.42 0.066
Cu (mg kg-1) 4.40 ± 0.46 4.43 ± 0.46 4.77 ± 0.32 4.37 ± 0.21 4.63 ± 0.51 0.713
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Fe (mg kg ) 18.3 ± 1.53
Mn (mg kg-1) 7.33 ± 4.51
18.7 ± 0.58
10.0 ± 0.00
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18.3 ± 0.58
7.00 ± 3.46
18.3 ± 2.08
8.33 ± 3.51
20.7 ± 1.53
9.67 ± 1.15
0.248
0.677
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Zn (mg kg ) 3.50 ± 0.26 5.70 ± 0.82 4.10 ± 0.26 4.30 ± 0.61 6.43 ± 1.72 0.015*
c ab bc bc a
Soluble salts 1.70 ± 0.36 2.63 ± 0.45 1.77 ± 0.29 2.07 ± 0.81 3.23 ± 1.54 0.200
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(dS m )
Values represent means ± standard deviation (n = 3). *Means sharing the same letter within a row
are not significantly different (P ≤ 0.05). DC: dairy manure-based compost, GC: grape pomace
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compost, ODC: olive pomace/dairy manure compost, and CRC: mixed crop residue compost.
In the conventional field, SOM, P, and some microelements (B, Mn and Zn) tended to
increase with compost applications, although no significant differences were detected (Table 4).
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Furthermore, the four compost treatments had no significant effect on soil pH, cation exchange
capacity levels, Mg, Ca, NO3, S, Zn, Mn, Fe, Cu, B, or soluble salts compared to control. On the
other hand, soil K significantly increased by the ODC and CRC treatments compared to the
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control. Additionally, soil Na increased by DC, ODC and CRC compared to control. GC had no
significant effect on soil K or Na. In the conventional field, DC only had a significant effect on Na,
which was 150% higher than control. CRC and ODC had similar effects on soil K and Na; K being
125% of control and Na over 150% of control.

Table 4: Effect of each compost treatment on soil chemical properties in the conventional field.

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 Soil Treatment P value
parameter
Control DC GC ODC CRC
Organic
matter (g 31.0 ± 5.29 33.3 ± 2.08 37.0 ± 5.20 46.7 ± 11.6 38.0 ± 7.81 0.153
kg-1)
pH 7.50 ± 0.15 7.53 ± 0.12 7.43 ± 0.12 7.47 ± 0.15 7.63 ± 0.15 0.493
C.E.C. (meq
23.8 ± 0.56 22.3 ± 1.86 24.5 ± 0.36 23.3 ± 0.61 24.5 ± 0.47 0.082
100 g-1)
NO3 (mg kg-

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42.3 ± 24.5 28.7 ± 27.7 52.0 ± 21.0 44.3 ± 11.5 45.0 ± 23.6 0.779
)
Olsen P
63.0 ± 5.20 82.7 ± 12.4 71.3 ± 4.16 80.3 ± 11.9 80.3 ± 7.77 0.106

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(mg kg-1)
556 ± 22.8 619 ± 76.4 668 ± 88.8 672 ± 57.6
K (mg kg-1) 520 ± 35.0 b 0.044*
b ab a a
Mg (mg kg-1) 852 ± 143

Ca (mg kg-1) 3049 ± 136

874 ± 49.7

2652 ± 333
2949
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962 ± 63.5 882 ± 128
± 2790 ±
910 ± 62.9 0.692

2982 ± 116 0.107

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96.8 41.2
S (SO4-S)
31.0 ± 7.81 21.7 ± 11.6 27.3 ± 6.43 30.7 ± 2.52 32.0 ± 8.54 0.528
(mg kg-1)
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96.3 ± 27.0 73.3 ± 7.57 99.7 ± 13.3 87.3 ± 16.0

Na (mg kg-1) 57.0 ± 6.56 b 0.043*
a ab a a
B (mg kg-1) 0.70 ± 0.12 0.77 ± 0.15 0.80 ± 0.00 0.97 ± 0.31 0.93 ± 0.15 0.428
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Cu (mg kg-1) 1.60 ± 0.06 1.63 ± 0.25 1.73 ± 0.12 1.73 ± 0.12 1.70 ± 0.17 0.864
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Fe (mg kg ) 8.00 ± 1.00 9.67 ± 0.58 9.33 ± 1.15 8.00 ± 0.00 10.0 ± 1.73 0.124
Mn (mg kg-
3.00 ± 1.53 6.33 ± 1.15 6.33 ± 3.21 5.67 ± 1.53 4.00 ± 1.00 0.247
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)
Zn (mg kg-1) 1.60 ± 0.10 1.97 ± 0.45 2.10 ± 0.20 2.33 ± 0.67 2.60 ± 0.61 0.170
Soluble salts
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1.10 ± 0.30 0.90 ± 0.44 1.20 ± 0.26 1.20 ± 0.20 1.17 ± 0.35 0.761
(dS m-1)
Values represent means ± standard deviation (n = 3). *Means sharing the same letter within a
row are not significantly different (P ≤ 0.05). DC: dairy manure-based compost, GC: grape
pomace compost, ODC: olive pomace/dairy manure compost, and CRC: mixed crop residue
compost.
The effects of composts observed on both organic and conventional fields were generally
similar, leading to slightly higher SOM, especially with ODC treatment, which increased SOM by
12 and 15 g kg-1 in the organic and conventional fields, respectively. Literature studies often show
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that composts increase soil carbon for extended periods due to the highly stable nature of
compost amendments [21]. For example, in Italy, a 30 t ha-1 ODC application significantly
increased SOM from 16.2 to 27.2 g kg-1, and the increase in SOM was correlated to the
amendment amount [22]. A different study has shown that with continuous application over 8
years, 50.9% of all carbon applied was retained in the soil when the waste was composted versus
a mere 6.84% when fresh residue was applied [23]. Our composts have not changed soil pH,
which confirms the results of Hachicha et al. who did not find an effect on soil pH as a result of
application of olive pomace/poultry manure compost [24]. The higher P and K values obtained
with the compost treatments are due to the relatively high content of these elements in this
material. This results is in line with previous findings for dairy manure compost [25] and olive

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compost [23]. An increase in soil Na is also observed with dairy manure compost [25].

Effect of the composts on soil V. dahliae abundance

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There was no significant difference in the V. dahliae numbers, expressed in number of
CFU (or microsclerotia) g-1 soil, between the two fields tested. Additionally, there was no

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significant interaction between the amendment treatments and the field management (i.e.,
conventional vs. organic) (treatment x field: P = 0.477). Therefore, the field factor was analyzed
as a random factor to increase the power of the test, and further analyses of V. dahliae were
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performed by pooling the data from both fields [26].
Soil V. dahliae abundances demonstrated clear trends both across treatments and
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through time. The initial samplings for V. dahliae CFU g-1 soil were taken in May 2018 before any
field preparation or treatments. At that time, pathogen loads were not significantly different
between treatments (treatment: P = 0.063) with a mean of 52.4 ± 29.6 CFU g-1 soil (n = 250). This
pathogen level is considered sufficient to cause disease in certain crops such as strawberries,
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which are particularly susceptible and will begin to show symptoms with as little as 3 CFU g-1 soil
[27]. Bell peppers are less susceptible, thus requiring a higher field pathogen load than
strawberries to become infected [28].
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By treating date as a fixed effect there was a consistent pattern in V. dahliae pathogen
load through the season with no two sequential sampling dates being equivalent (time: P <
0.0001). The first sampling, on 18 May 2018, had the highest mean pathogen load at 52.4 ± 29.6
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CFU g-1 soil (Table 5). By the second sampling on 30 June 2018, pathogen loads had dropped in
all treatments, including control. This drop was expected due to several soil disturbance events
in early June: the four organic amendment treatments were applied, and the fields were both
disked, the beds were shaped and planted. All these practices cause major disruption to soil
microbes, particularly fungi, as hyphae are susceptible to such mechanical disturbances [29].
Thus, it is expected to see a significant drop in V. dahliae soil load between these times. There
was a universal pathogen load decrease between May and June; all organic amendment-treated

10
plots had a greater reduction in pathogen load when compared to untreated control plots (Table
5). All four compost treatments significantly reduced the pathogen populations in June, two
weeks after the application of treatments, with GC, ODC, and CRC amendments presenting the
largest reduction in soil V. dahliae. In the third sampling on 11 August 2018, the average number
of microsclerotia for all treatments increased by 40% compared to June, with the smallest
increase observed in the ODC treatment. Nevertheless, the significant effect of the GC, ODC, and
CRC amendments on reducing soil pathogen load, compared to control plots remained through
this sampling date. However, the effect of the DC amendment was no longer significantly
different from the control. By the fourth sampling date, 21 September 2018, overall field
pathogen loads had nearly returned to pretreatment levels from May with an overall mean of

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45.4 CFU g-1 soil. There were no significant differences in the V. dahliae soil pathogen load
between any treatments. This was the first sampling date with no reduction of soil pathogen load
observed by any treatment. Thus, reduction in V. dahliae CFU’s from applied compost treatments

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was measurable only up to eight weeks post-application. At the final sampling date, 1 November
2018, overall V. dahliae pathogen loads dropped down to 29.6 CFU g-1 soil with no significant
differences between any treatments on this date.

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Table 5. Number of Verticillium dahliae colony-forming units g-1 soil for as affected by four
compost treatments over time.
Number of colony-forming units g-1 soil
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Date P value

Control DC GC ODC CRC

18 May 51.4 43.8 49.2 56.4 59.8 0.063
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30 June 34.4 a 25.6 b 14.6 c 16.8 c 14.0 c 0.001*

11 August 53.6 a 42.6 a 29.8 b 23.8 b 24.6 b 0.01*
21 September 40.8 44.2 53.6 48.0 40.6 0.443
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1 November 36.2 23.8 29.0 30.6 28.6 0.194

*Within a date, values followed by the same letter are not significantly different (P ≤ 0.05, n = 250).
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DC: dairy manure-based compost, GC: grape pomace compost, ODC: olive pomace/dairy manure
compost, and CRC: mixed crop residue compost.

The reduction in V. dahliae load observed after the compost applications confirms
previous literature findings other pathogens. Dairy manure composts have been shown to have
suppressive qualities on soil-borne pathogens Pythium spp., Phytophthora spp., Fusarium spp.,
and Rhizoctonia solani [6]. Similarly, in a pot study, a single olive pomace compost application (at
150 g kg-1 soil) significantly reduced the density of V. dahliae microsclerotia in an inoculated soil

11
grown to olives and eggplants (Solanum melongena L.) compared to the untreated control,
especially within the first 20 days after application [30].
Olive pomace and leaves compost, and grape waste-based composts to a lesser extent,
have been shown to be effective against P. nicotianae, with the effectiveness decreasing with the
age of the compost [11]. A similar effect was detected with olive mill wastewater [10].
Suppression mechanisms in previous studies point to either chemical suppression via
introduction of toxic compounds, induced systemic resistance, or biological suppression from
stimulation of the microbial community including fungal antagonistic genera [9,31].
The suppressive effects of composts disappeared within eight weeks (DC) or 14 weeks
(GC, ODC and CRC) after application. In Oregon (USA), Darby et al. reported that suppressive
effects by dairy manure compost on three root diseases lasted for two months but disappeared

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upon the second sampling after 12 months [32]. The concentrations of cellulose and lignins in
the composted manure define the longevity of the suppressive effect [6].

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Effect of the composts on bell pepper health and yield

There was no phytotoxicity symptoms from any of the treatments on bell pepper plants.

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Furthermore, none of the compost treatments tested had a significant effect on the plant height
(Fig. 1). Similarly, SPAD absorbance readings were not significantly affected by any treatment
(Fig. 2).
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30
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25
Plant height (cm)

20
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15

10
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0
Control DC GC ODC CRC
Compost treatments

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 Fig. 1. Bell pepper plant height as affected by four compost treatments over time. Error bars
represent ± one standard error of the mean (P = 0.833, n = 50). DC: dairy manure-based
compost, GC: grape pomace compost, ODC: olive pomace/dairy manure compost, and CRC:
mixed crop residue compost.

70

60
Leaf SPAD absorbance reading

50

40

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30

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20

10

0
Control DC GC
Compost treatments
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ODC CRC
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Fig. 2. SPAD absorbance in bell pepper leaves as affected by four compost treatments over time.
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Error bars represent ± one standard error of the mean (P = 0.554, n= 50). DC: dairy manure-based
compost, GC: grape pomace compost, ODC: olive pomace/dairy manure compost, and CRC:
mixed crop residue compost.
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There was no treatment effect on pepper yield between the fields (data not shown) or by
treatment on yield (Fig. 3). Despite a significant effect of treatment on pathogen load there was
no significant effect on the yield of peppers. Although the pathogen load of V. dahliae was above
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the threshold for disease development, the pepper plants had no observable disease symptoms
throughout the trial. The chosen bell pepper variety is known to be susceptible to verticillium
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wilt. A possible explanation for the absence of disease is the fact that this piece of land has not
had any pepper crops at least within the last five years. Bhat et al. have shown that in order for
V. dahliae to develop on bell peppers, it requires pathogen isolates from another bell pepper
crop (or eggplant), confirming earlier reports on the high specificity of V. dahliae in peppers [33].
This is not the case with other nightshade family plants such as tomato, potato and eggplant,
which can accept isolates from other nightshade crops.
Despite increasing soil concentrations of P, K and some trace elements, composts did not
increase crop yield. All our treatments (control and compost) received recommended fertilizer

13
applications, so the compost treatments were not necessarily expected to increase yields from a
nutrition standpoint. In addition, nutrients found in composts are often slow to become available
to plants making composts function as soil amendments rather than effective fertilizers. In a 10-
year study with three different rates of yearly applications of composted municipal waste, Erhart
et al. found that compost significantly increased yields only in one year and at the highest rate
only [34].

16000
Fresh weight production (kg ha-1)

14000

12000

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10000

8000

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6000

4000

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2000

0
Control DC GC ODC CRC
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Compost treatments
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Fig. 3. Effect of four compost treatments on fresh weight of bell pepper fruit per plot. Error bars
represent ± one standard error of the mean (P = 0.869, n = 10). DC: dairy manure-based compost,
GC: grape pomace compost, ODC: olive pomace/dairy manure compost, and CRC: mixed crop
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residue compost.
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Effect of compost extracts on V. dahliae in vitro growth

In vitro assays were carried out using filter-sterile compost extracts to identify the most
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probable mechanisms of action by which the tested composts inhibited V. dahliae growth. After
15 days of incubation all V. dahliae colonies in control plates had grown to the edge of the Petri
dish with a 40 mm radius (Fig. 4). The DCE-treated colonies had an average maximum linear
growth of 36.2 mm, not significantly different from the control (P = 0.288). GCE, ODCE, and CRCE
all demonstrated a significant reduction of V. dahliae in vitro growth compared to the control
from day 3 through day 15 with average maximum linear growth of 29.7, 30.3, and 29.5 mm,
respectively, at day 15 (P = 0.022).

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 45
40
V. dahliae linear growth (mm)

35
30
25
20
Control
15
DCE

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10 GCE
ODCE
5

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CRCE
0
1 3 5 7 9 11 13 15

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Days after application

Fig. 4. Mean V. dahliae linear growth in vitro on selective NP-10 media by treatment over time.
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Error bars represent ± standard error of the mean (n = 5). DCE: dairy manure-based compost
extract, GCE: grape pomace compost extract, ODCE: olive pomace/dairy manure compost
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extract, and CRCE: mixed crop residue compost extract.

All plant-based compost extracts significantly reduced V. dahliae growth compared to the
control while the DCE was intermediate between the control and the other treatments. These
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results confirm the findings of our field trial showing a reduction of soil pathogen load with plant-
based compost applications. The sterile nature of the extracts supports the hypothesis of
chemical inhibition through antifungal compounds in the compost extracts. This is especially true
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with grape and olive wastes that are known to be high in fungicidal phenolic compounds. Cayuela
et al. have reported that olive waste compost extract suppressed Phytophthora capsici, Pythium
ultimum and Botrytis cinerea, although did not affect Rhizoctonia solani [35]. Similarly, aqueous
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plant-based compost extracts inhibited the growth of pathogens P. ultimum (on cucumber
(Cucumis sativus L.)), Phytophthora nicotianae (on tomato), R. solani (on beans), Sclerotinia
sclerotiorum (on lettuce), Fusarium oxysporum f. sp. melonis (on melon), F. oxysporum f. sp.
lycopersici (on tomato), and V. dahliae (on eggplant) [18]. Evidence for both biological and
chemical suppression mechanisms against V. dahliae was also provided by Arriagada et al. [36]
who isolated 12 saprophytic fungi capable of suppressing this pathogen and demonstrated that
sterile aqueous extract of uncomposted olive residue was able to control the pathogen.

15
CONCLUSIONS

A significant reduction of the soil-borne fungal pathogen V. dahliae was observed up to

14 weeks after the application of olive, grape, and mixed crop residue-based composts with an
average decrease between 29% and 42% of the V. dahliae population for those three composts
compared to the control. Additionally, a significant inhibition of V. dahliae in vitro growth was
observed after treatment with extracts from these same materials; reducing fungal growth by
25% - 50% compared to the control. This study provides evidence that V. dahliae growth
inhibition is, at least in part, due to chemicals introduced with the organic amendments having a
deleterious effect on the pathogen. The materials studied here present promise in controlling V.
dahliae and should be tested on other serious soil-borne pathogens.

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Author statement

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Ashraf Tubeileh: Proposal writing, Conceptualization, Methodology,
Supervision, Writing
Gregg Stephenson: Data collection, Statistical analysis, Writing
original draft preparation.

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Declaration of interests
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
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ACKNOWLEDGEMENTS

This research was made possible thanks to funding by the California State University Agricultural
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Research Institute, grant number 20-03-112. The support and training provided by Dr. Kelly Ivors
and the Cal Poly Strawberry Center were instrumental for the success of this project. Dr. Andrew
Schaffner’s help with the statistical analysis is greatly acknowledged. In-kind donations of
pomace/compost materials by La Panza Ranch, Cagliero Vineyards and Cal Poly Compost Unit are
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appreciated. Thanks are due to Kevin Piper and Mike Bridgman for their help with the composting
process.
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