Final report for GNC18-260

Summary:

Clubroot disease, caused by Plasmodiophora brassicae, is becoming increasingly challenging for cruciferous crop production worldwide. The primary outcome of this project will be a clubroot management strategy that integrates non-host cover crops, biological control agents (BCAs) and an ecofriendly soil disinfestation method. Outreach activities will enhance growers' aptitude to integrate these techniques in their farming systems, which will boost productivity and profitability by minimizing yield losses and dependence on fungicides or soil fumigants and soil liming for disease management. Clubroot is particularly problematic on muck soils in the US Northeast and Midwest due to intensive cultivation and declining soil pH. Most of the currently available management practices disrupt physical, chemical and biological properties of soil or are financially and environmentally expensive. These approaches are also not always effective because of the presence of highly resilient resting spores of the pathogen. However, certain non-host crops can break dormancy of these resting spores in soil, and these germinating resting spores can be killed by abruptly creating an unsuitable environment.
ASD is brought about by incorporation of easily decomposable carbon sources (plant biomass, crop byproducts etc.) into soil, with sufficient moisture to foster microbial activities leading to anaerobicity. ASD alters both chemical and biological properties of soil, which are deleterious to a wide range of crop pests.
Combining ASD with BCAs compatible with the process may provide synergy in P. brassicae population reduction. In this project we will grow non-host cover crops to induce the germination of resting spores and the cover crop biomass will be used as ASD carbon source. Moreover, effective BCAs will suppress the remaining dormant resting spores during the ASD treatment period. The study is expected to provide an effective alternative for organic and conventional growers for clubroot management. On-farm field experiments, field days and a workshop will ensure farmer involvement in the learning process and enable changes in understanding and behavior that will result in sustainable clubroot management. Pre- and post- workshop surveys will be used to evaluate learning outcomes, and publication of a peer-reviewed research article and extension factsheets will be indicators of the quality of project outputs.

Project Objectives:

Outcomes of this project will be helpful to cruciferous crop growers, extension workers and researchers to understand the mechanism and use of non-host cover crops, ASD and biocontrol in clubroot disease management. However, the primary beneficiary will be muck crop growers who have persistent problems with clubroot on their farms.

The learning outcomes of this project are: growers will 1) understand the synergistic roles of non-host cover crops as ASD carbon sources and BCAs in clubroot disease management; and 2) develop the skills to implement these strategies for economically feasible and environmentally safe clubroot management.

The action outcomes of this project are: growers will 1) adopt effective combinations of non-host cover crops, BCAs and ASD to manage clubroot disease; and 2) reduce their dependence on potentially soil damaging fungicides/fumigants and liming practices to manage clubroot.

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Robert P Filbrun (Researcher)
Manager
Muck Crops Agricultural Research Station (1862 Land Grant)

Materials and methods:

Materials and Methods

Effects of inundative biocontrol on clubroot disease of cruciferous vegetables in muck soils. All experiments were conducted in growth chambers in The Ohio State University Department of Plant Pathology, College of Food, Agricultural, and Environmental Sciences (CFAES) Wooster Campus, in muck soil utilizing a randomized complete block design with five replications each of seven treatments. The treatments were five commercial biocontrol products: Trichoderma harzianaun (Rootshield), Bacillus subtilis (Serenade), Gliocladium catenulatum (Prestop), Bacillus amyloliquefaciens (Double Nickel;) and Streptomyces lydicus (Actinovate), the fungicide fluazinam (Omega; standard) and a non-treated inoculated control (Table 1).

Table 1 Details of the biofungicides used in the experiments.

	Product	Manufacturer	Application rate (ha^-1)	Active ingredient
1	Prestop WG	Danstar Ferment Ag. Switzerland (Lallemand Plant Care USA)	500 g	Gliocladium catenulatum J1446, 1 x 10⁹ CFU g^-1
2	Rootshield^® WP	Bioworks USA	6.8 kg	Trichoderma harzianum T-22 1 x 10⁶ c CFU g^-1
3	Omega^®500F	Syngenta USA	2.40 liter	Fluazinam 40%
4	Actinovate	Novozymes BioAg Inc. USA	5.6 kg	Streptomyces lydicus WYEC 108 1 x 10⁷ CFU g^-1
5	Serenade Opti	Bayer CropScience USA	9.6 kg	Bacillus subtilis QST 713 1.31 x 10⁹ CFU g^-1
6	Double Nickel LC	Certis USA L.L.C.	5.6 liter	Bacillus amyloliquefaciens D747* 1×10¹⁰ CFU ml^-1

Experiments were conducted in 350 ml styrofoam cups with muck soil collected from a Plasmodiophora brassicae-infested field on the CFAES Muck Crops Agricultural Research Station, Willard, Ohio. Fifteen seeds of mustard greens 'Green Wave' were planted in each pot, followed by drenching with a resting spore suspension of P. brassicae (1×10⁵spores g^-1soil).

Resting spores of Plasmodiophora brassicae were extracted from clubroot-infected mustard greens plants as suggested by Castlebury et al. (1994) and Mehrabi et al. (2018). Mustard greens plants infected with clubroot were collected from the CFAES - Muck Crops Agricultural Research Station in Willard, OH and stored at -20ºC until spore extraction. Briefly, 250-300 g of frozen mustard greens root galls were surface sterilized by dipping into 70% ethanol for one minute followed by rinsing with sterilized water five times. Then root galls were macerated in a blender (Waring Commercial Blender, Waring Commercial, Torrington, CT) with 500 ml of sterile water at maximum speed for a total of 2 min with a 30 s break in every 30 s. The extract was passed through four layers of cheesecloth. The filtrate was centrifuged for 15 min at 3,650×g at room temperature in 50-ml Falcon tubes. The supernatant was removed and the upper brown layer of pellets containing spores was carefully transferred to new tubes by using a pipet with a 1 ml tip. Spores were resuspended in 40 ml water and centrifuged again. The process was repeated until a white layer of starch in the pellet disappeared. Density gradients were created by adding 15 ml of 32% Ficoll, 10 ml of 16% Ficol and 5 ml of spore suspension, respectively, in a Falcon tube and centrifuging for 15 min at 400×g. The upper layer of liquid was discarded, and the second brown layers were transferred to new Falcon tubes; 40 ml of water was added and the suspension was centrifuged at 3,650×g for 15 min. The supernatant was removed, and the pellet was resuspended in 300 ml water, then spores were counted using a hemocytometer. Suspensions were adjusted to 10⁸spores ml^-1 by adding sterile water and stored at 4ºC until use.

At the same time, biocontrol products were pipetted in each pot as per the manufacturer’s recommendation (Table 1). Light and temperature conditions were 300 photosynthetic photon flux μmol m^-2s^-1 for 12-h, and 25ºC for 12-h and 20ºC for 12-h, respectively. Watering was done daily using a watering can, and no fertilizer was applied. At 30 days after planting, mustard greens plants were uprooted, roots were washed with the tap water and clubroot disease severity was assessed according to a 0 to 3 scale developed by Strelkov et al. (2006), in which 0 corresponds to no galling, 1 corresponds to a small amount of galling, 2 corresponds to a moderate galling, and 3 corresponds to severe galling. Clubroot disease severity indices were calculated ∑(n×0 +n×1+n×2+n×3)×100/(N×3) (where n equals the number of plants with a given rating and N is the total number of plants). After scoring disease, the tops of the plants were separated from roots and their fresh biomass was measured. The experiment was conducted three times.

Effect of cover crops as ASD carbon sources on clubroot suppression. The experiment was conducted twice in growth chambers as described above according to a randomized complete block design with three replications of 12 treatments, including nine cover crops, the wheat bran standard and anaerobic (covered and flooded) and aerobic (uncovered and flooded) controls. The nine cover crops (cowpea, leek, orchardgrass, buckwheat, red clover, ryegrass, Sesbania spp., sudangrass and winter rye) (Table 2) were grown for 45 days in growth chambers in 14.5-cm diameter pots (1.6 liter) containing muck soil collected from a clubroot-infested field on the OSU CFAES Muck Crops Agricultural Research Station, Willard, Ohio. A suspension of P. brassicae resting spores was added to the pots according to the procedure described above. The temperatures ranged between 25 and 30ºC and the photoperiod was set to 12 hours light/dark. The plants were hand-watered daily and fertilized (N-P-K 20-20-20; 3.96 g liter^-1 water) once per week from planting to termination. After 45 days whole plants were uprooted, chopped into small pieces using pruning shears and mixed with soil in the same pot. Fresh plant biomass ranged from 18 to 86 g per pot (Table 2). Wheat bran was mixed with soil at 10 g kg^-1 soil before filling the pots. Each pot was flooded with 300 ml of tap water and allowed to drain for about 2 h. Indicator of Reduction in Soils (IRIS) tubes (1.3 cm in diameter) were inserted in the center of each pot to evaluate reducing conditions during the anaerobic process (Rabenhorst 2008, 2012). Pots were double-sealed utilizing two 3.7-liter Ziploc^® bags (SC Johnson Family Company, Racine, Wisconsin). Pots were incubated in a growth chamber for 30 days with a photoperiod of 12 h light/dark; temperatures were set at 30°C and 25°C, respectively (light/dark).

	Cover crop	Scientific name	Seeding rate (ha^-1)	Above ground biomass per pot (g)
1	Cowpea	Vigna unguiculata	90 kg	35.0 - 51.0
2	Leek	Allium porrum	800 g	18.5 - 30.0
3	Orchardgrass	Dactylis glomerata	7 kg	30.0 - 37.5
4	Buckwheat	Fagopyrum esculentum	65 kg	67.0 -76.0
5	Red clover	Trifolium pratense	13 kg	30.0 - 47.0
6	Ryegrass	Lolium perenne	33 kg	40.0 -57.0
7	Sesbania	Sesbania spp	45 kg	41.0 - 46.5
8	Sudangrass	Sorghum × drummondii	56 kg	65.0 - 86.0
9	Winter rye	Secale cereale	112 kg	38.0 - 48.0

At the end of the incubation period, the Ziploc bags were removed and soil samples (about 100 g) were collected in paper bags for determination of pH and soil moisture content. The IRIS tubes were rinsed with water and blotted with paper towels; the percentage of paint depletion was estimated visually. Soil in the pots was aerated for one week. Before planting, soil was homogenized, and 30 seeds of mustard greens 'Green Wave' were sown in each pot. Pots were kept in a growth chamber for 30 days. Light, temperature and relative humidity were maintained as previously described for mustard greens. Plants were watered daily using a watering can and no fertilizer was applied in the first experiment. Fertilizer (N-P-K 20-20-20; 3.96 g liter^-1 water) was applied once per week from planting to termination of the experiment in the second experiment. At 30 days after planting, plants were uprooted and clubroot disease severity was assessed as previously described, and incidence and disease severity indices were calculated.

Combined effect of selected inundative BCAs and ASD with different cover crops as carbon sources on clubroot suppression in mustard greens. The third set of experiments was conducted to evaluate the combined effects of promising biocontrol products and ASD with different cover crops for clubroot suppression in mustard greens. Experiments were conducted utilizing a factorial randomized complete block design with three replications, where the first factor was cover crop (winter rye, ryegrass, sudangrass, and leek), the standard wheat bran and anaerobic and aerobic controls. The second factor was the biocontrol treatment (Prestop, Actinovate, and Serenade) and control (without biocontrol). Cover crops were grown in 10-cm pots inoculated with the pathogen (P. brassicae) and incorporated into the soil before ASD treatment as described above. Biocontrol products were inoculated into soil after inserting IRIS tubes and flooding as described. Mustard greens plants were grown and disease assessments were done as mentioned. The experiment was conducted two times.

Evaluation of ASD with different carbon sources and BCA application in field trials. A field experiment was conducted at OSU CFAES Muck Crops Agricultural Research Station, Willard, Huron County, Ohio from April to September 2019 and 2020. The experimental design was an incomplete factorial randomized complete block design with four replications. In 2019, the first factor was ASD amendment (winter rye, ryegrass, or leek cover crop, the standard wheat bran, and aerobic and anaerobic controls. The second factor was biocontrol treatment (Prestop, Actinovate, Serenade or no biocontrol product) applied to the amended ASD treatments only. The individual plot size was 2.1 m × 1.8 m, with 0.91-m buffers between the plots, and 1.8-m buffers between the blocks. Cover crops were seeded over prepared beds by using a seed driller (John Deer 8250 adjusted with notches at 26 for ryegrass and 11 for winter rye) in 12 rows 17.8 cm apart. The field was broadcast with fertilizer12-0-0 (N-P-K) at 7.8 kg ha^-1 at sowing and 65.0 kg ha^-1 14 days later. The seeding rate was 112 kg ha^-1 for winter rye and 33.6 kg ha^-1for ryegrass. For leek, 49-day old seedlings grown in 50-cells plug trays containing commercial soil mix (Baccto Professional Growers Mix, Houston, TX) were planted manually in 7 rows per bed at 30×15 cm spacing. Both aerobic and anaerobic controls remained fallow for the entire cover crops growing period while aerobic control plots also remained fallow during ASD treatment period. The cover crops were incorporated into the soil at 52 days after sowing (DAS) using a tractor-mounted chopper and to a depth of 15 to 20 cm using a rototiller. Soil was rototilled until the chopped cover crop biomass was completely buried in the soil. Wheat bran (22.2 Mg ha^-1) was broadcast by hand on the surface of the plots and incorporated in the soil to a depth of 15 to 20 cm with a rototiller. Overhead irrigation was applied to saturate the field to a depth of 20 cm.

HOBO Pendant temperature data loggers (Onset Computer Corporation, Bourne, MA, USA) were buried 15 cm deep to measure the soil temperature in one plot per treatment. The data logger was set to measure the soil temperature at 30-min intervals. Two IRIS tubes were inserted into guide holes created by using 2-cm diameter PVC pipes in the middle of the plot 1 m apart from each other before initiation of ASD.

Biocontrol products were diluted in water and drenched into the soil using watering cans as per the manufacturer’s recommendation (Table 1) 2 h after overhead irrigation was complete. Each plot was covered with 1.5-mm embossed non-permeable black plastic sheeting (PolyExpert, Inc., Laval, QC, Canada) except for the aerobic control. The edges of plastic sheeting were buried in the soil to prevent the exchange of gases. The plastic mulch was removed 35 days after covering, IRIS tubes were carefully collected to avoid mechanical removal of paint, and soil samples were collected from three to five different spots in the plot to make a composite sample for bioassay and pH measurements. The soil pH was measured in 1:1 soil-water slurry using a Hanna Halo wireless pH probe (HI12922, Smithfield, RI, USA). IRIS tubes were rinsed with tap water, blotted with paper towels and the percentage of paint removal was quantified by visual observation.

Mustard greens ‘Green Wave’ were direct seeded one week after removal of plastic mulch. One-time overhead sprinkler irrigation was applied; otherwise, the crop relied on rainfall. At 40 DAS, 25 mustard greens plants were uprooted from the center of the middle rows, placed in plastic bags and transported to the laboratory for disease estimation. Disease scoring and measurement of fresh and dry above-ground biomass were done as mentioned above.

In 2020 the experimental design, field management and treatments were similar to the 2019 field experiment with a few modifications. There were four replications, leek was replaced by sudangrass and only Prestop and Serenade were used. The sudangrass seeding rate was at 56 kg ha^-1. Cover crops were terminated 58 DAS. Plastic mulch (2.43 m wide x 1.25 mm thickness, Berry Global Inc, Washington, GA, USA) was removed 37 days after initiation of ASD. Light pre-irrigation was done before planting mustard greens. Due to poor germination of mustard greens in the first planting, the variety ‘Southern Curl’ was sown 3 weeks after plastic removal. Mustard greens were uprooted 41 days after planting for disease assessment.

Post-ASD bioassay. Ohio muck soil is very fine and there is a chance of cross-contamination among plots due to flooding and air drift (Testen and Miller 2019). Therefore, a post-ASD bioassay was conducted in growth chambers using composite soil samples collected from each field plot immediately after plastic removal. Soil from each plot was placed in 14.5 cm diameter plastic pots and seeded with mustard greens ‘Green Wave’. Pots were kept in a growth chamber for 30 days. Light, temperature and relative humidity were maintained as previously described for mustard greens. Plants were watered daily using a watering can. Fertilizer (N-P-K 20-20-20; 3.96 g liter^-1 water) was applied once per week from planting to termination. At 30 days after planting, plants were uprooted and clubroot disease severity, fresh and dry above-ground biomass were determined as described above.

Validation trial. In 2020 one additional trial was conducted to validate promising treatments on the Muck Crops Research Station. The experiment was conducted in a randomized complete block design with three replications of six treatments. The treatments were ASD-winter rye, ASD-winter rye + Prestop, ASD-winter rye + Serenade, ASD-wheat bran standard and aerobic and anaerobic controls. The plot size and field management were similar to 2020 field experiment. Winter rye was planted and incorporated on the same days as the 2020 field evaluation trial. Plots were established and maintained as described above. Soil samples were collected after ASD termination for bioassays as described above. Mustard greens ‘Green Wave’ seeds were sown on the same day as in the 2020 field evaluation trial and was replanted 21 days after ASD termination due to poor germination. Mustard greens were uprooted 41 days after planting and assessed for clubroot disease and biomass as described above for the field evaluation trials.

Data analysis. Data were subjected to the Shapiro-Wilk test followed by the Bartlett test for testing normality of residuals and homogeneity for variance, respectively, before proceeding to the analysis of variance (ANOVA). Datasets that deviated from normal distribution were either arcsine(square-root)-, square root- or log-transformed before performing ANOVA. Data were analyzed using the linear model function "lm" in RStudio (R-3.2.5; RStudio Support, 2018). When there was a significant difference between the treatment means the Fisher tests of least significant difference (LSD) were applied in the ‘predictmeans’ package (Luo et al. 2014).

The Bliss independence model was applied assuming BCAs and ASD - carbon sources act independently to suppress clubroot severity in mustard greens (Yan et al. 2010; Xu et al. 2011; Willyerd et al. 2011). The combined effect of BCA and ASD carbon source on clubroot severity indicates the union of two probabilistically independent events. The combined effects (F_UA) were calculated as the product of individual effects of BCA (F_UA1) and ASD carbon source (F_UA2)

F_UA = F_UA1× F_UA2

where F_UA is the remaining fraction of severity control relative to non ASD (covered control) and non-BCA treatments (unaffected fraction of disease severity reduction, for example if disease severity reduction is 0.17, F_UAwill be 1-0.17 = 0.83). According to the Bliss independence assumption, F_UA is the expected effect of combined treatments and synergistic, additive and antagonistic relationships between the treatments were determined as follows:

If the observed combined effect of BCA and ASD-carbon source is equal to F_UA,the relationship is additive and there is no interaction between ASD-carbon source and BCAs
If the observed combined effect is greater than F_UA,the relationship is synergistic
If the observed combined effect is less than F_UA,the relationship is antagonistic

Research results and discussion:

Results

Biocontrol of clubroot of mustard greens. Clubroot severity indices ranged from 11.8% in plants treated with fluazinam to 64.3% in the non-treated control. Prestop, Actinovate, and Serenade significantly (P < 0.001) reduced clubroot severity compared to the non-treated control but were less effective than the fluazinam treatment (Table 3). Rootshield and Double Nickel did not reduce the clubroot severity index compared to the non-treated control. The biocontrol treatments did not affect fresh plant biomass in two of three experimental runs, or when data were combined for all three runs (P = 0.1) (Table 4).

Table 3 Suppression of clubroot in mustard greens by inundative application of biological control products.

Treatment^w	Combined analysis^x				First experiment		Second experiment		Third experiment
	Disease index (%)^y			Control (%)^z	Disease index (%)		Disease index (%)		Disease index (%)
Non-treated control	64.3	a	-		86.9	a	59.4	a	36.6	a
Prestop	31.0	b	51.8		33.8	cd	28.2	bcd	29.1	a
Rootshield	44.5	ab	-		59.4	abc	36.6	bc	36.6	a
Omega (fluazinam)	11.8	c	81.6		23.0	d	17.4	d	0.5	b
Actinovate	35.7	b	44.5		55.4	bc	36.6	bc	13.8	ab
Serenade	35.7	b	44.5		72.6	ab	22.1	cd	13.8	ab
Double Nickel	47.5	ab	-		69.0	ab	42.5	ab	29.1	a
P-value	<0.001				0.004		0.004		0.03

^wMeans followed by the same lower-case letter within a column are not significantly different at P < 0.05. Analysis of variance was performed on the arcsine (square-root) transformation of disease index (%) for combined, first and second experiments and log (1+disease index) transformation for third experiment. Means were separated using Fisher’s least significant difference test on transformed data. Presented means are original means.

^xCombined ANOVA of three independent experiments.

^yClubroot disease index was assessed on mustard greens roots using a 0-3 scale and clubroot disease indices were calculated by , where n equals the number of plants with a given rating.

^zPercent control values were calculated according to the formula: [(SC – ST)/SC]*100 where SC is the average severity of the non-treated control and ST is the average severity of the treatment.

Table 4 Fresh weight of mustard greens tops produced in soil infested with Plasmodiophora brassicae and treated inundatively with biocontrol products.

Treatment^y	Fresh weight (g)
Treatment^y	Combined^z		First experiment	Second experiment		Third experiment
Non-treated control	7.0	b	8.5	4.1	bc	9.0
Prestop	7.3	ab	6.5	5.8	a	10.1
Rootshield	7.9	ab	10.5	3.5	c	10.3
Fluazinam	8.8	a	9.7	7.0	a	9.9
Actinovate	6.3	b	6.5	4.2	abc	8.9
Serenade	7.4	ab	7.8	5.0	ab	9.9
Double Nickel	7.8	ab	7.8	6.4	a	9.5
P-value	0.1		0.2	0.01		0.8

^yMeans followed by the same lower-case letter within a column are not significantly different at P < 0.05.

^zCombined ANOVA of three independent experiments.

Effect of ASD with different cover crops as carbon sources on clubroot suppression. Soil reducing conditions as indicated by iron oxide paint loss from IRIS tubes were highest in ASD-treated soil amended with wheat bran or sudangrass biomass (Fig. 1). Soils treated with ASD with buckwheat, ryegrass, cowpea and winter rye, but not with leek, Sesbania, red clover or orchardgrass as carbon sources also indicated significantly higher reducing conditions than the anaerobic control. Reducing conditions were higher in all ASD-treated soils except those amended with leeks or orchardgrass than in the aerobic control. Mean soil pH in the anaerobic control was 5.9, significantly higher than in the aerobic control (pH = 5.7) (Fig. 2). Soil pH was significantly reduced in wheat bran-amended ASD-treated soil compared to both controls. However, ASD treatment with any of the cover crops except Sesbania spp., leek and buckwheat as carbon sources increased soil pH compared to the aerobic control.

Figure 1 Percentage paint removal from Indicator of Reduction in Soils (IRIS) tubes during anaerobic soil disinfestation (ASD) with different cover crops or wheat bran (standard) as carbon sources.

Figure 2 pH of soils treated with anaerobic soil disinfestation (ASD) with cover crops or wheat bran (standard) as carbon sources.

The clubroot disease severity index was significantly (P < 0.001) lower in mustard greens plants grown in winter rye- or wheat bran-amended ASD-treated soil compared to those grown in the anaerobic or aerobic control soils (Fig. 3A). The clubroot index was significantly lower in mustard greens plants grown in Sesbania spp., ryegrass, leek, or sudangrass biomass-amended ASD-treated soil than in plants grown in the aerobic control soils. The trends were similar for clubroot incidence (Fig. 3B). None of the ASD treatments except ASD-wheat bran significantly (P = 0.002) reduced clubroot incidence in mustard greens compared to anaerobic control.

Figure 3 Clubrot index (A) and incidence (B) in mustard greens plants grown in anaerobic soil disinfestation (ASD)-treated soil amended with cover crops or wheat bran (standard) as carbon sources.

Combined effect of BCAs and ASD with cover crop carbon sources on clubroot suppression in mustard greens. Soil inoculation prior of various biocontrol products, averaged across ASD treatments, did not result in significant changes in soil pH, reducing conditions or soil gravimetric moisture percentage (Table 5). Soil reducing conditions as indicated by depletion of iron oxide paint from IRIS tubes were significantly (P < 0.001) higher in all the cover crop- and wheat bran-amended ASD treatments than in either non-amended control, averaged across the biocontrol treatments. Iron oxide paint loss was significantly higher in ASD-treated plots amended with sudangrass or winter rye compared to ASD-treated plots with leek as the carbon source. Soil amendment with carbon sources did not reduce soil pH compared to the aerobic and anaerobic controls; rather the pH increased slightly but significantly (P < 0.001) towards neutrality compared to the aerobic control (Table 5). Soil gravimetric moisture was lowest in the aerobic control, however there were no significant differences among the ASD treatments including the non-amended anaerobic control.

Table 5 Effects of anaerobic soil disinfestation (ASD) with cover crop carbon sources averaged across biocontrol treatments and biocontrol products across ASD treatments on soil parameters, plant growth and clubroot (Plasmodiophora brassicae) development in mustard greens in growth chamber experiments.

ASD carbon source	Biocontrol product^u	Paint loss from IRIS tubes (%)^v		pH^w		Gravimetric moisture (% )^x		Shoot weight	Shoot/root ratio		Disease index (%)^y		Disease incidence (%)^z
	None	48.2	b	6.9		38.9		31.0	15.1		55.4	a	69.5	a
	Actinovate	60.3	ab	6.9		38.2		29.0	18.5		35.7	b	55.5	b
	Prestop	56.8	ab	7.0		37.3		27.9	18.4		31.9	b	52.5	b
	Serenade	61.6	a	6.9		39.6		30.1	20.8		31.0	b	50.7	b
	P-value	0.02		0.2		0.2		0.2	0.3		<0.001		<0.001
Aerobic control		2.5	c	6.6	c	1.5	b	26.0	8.6	b	68.0	a	81.5	a
Anaerobic control		6.9	c	6.9	b	43.5	a	27.0	18.6	ab	52.5	b	78.7	a
Leek		61.8	b	7.0	ab	45.4	a	28.4	16.2	ab	43.5	bc	68.0	ab
Ryegrass		77.3	ab	7.1	a	45.5	a	30.2	20.0	a	30.0	cd	50.7	bc
Sudangrass		84.2	a	7.0	ab	45.1	a	32.2	21.5	a	29.1	cd	28.2	d
Winter rye		84.8	a	7.0	ab	45.9	a	34.1	20.6	a	28.2	d	41.3	cd
Wheat bran		79.6	ab	6.9	ab	42.4	a	28.7	22.2	a	19.7	d	51.0	bc
	P-value	<0.001		<0.001		<0.001		0.6	<0.001		<0.001		<0.001

^uMeans followed by the same lower-case letter within a column are not significantly different at P < 0.05. P: Probability value of Analysis of variance. Analysis of variance was performed on the arcsine (square-root) transformation of disease index (%) and disease incidence (%) and square-root transformation of paint loss from IRIS tubes. Means were separated using Fisher’s least significant difference test on transformed data. Presented values are original means. Values are the means of two experiments each with three replications.

^vIron oxide paint loss from IRIS (Indicator of Reduction in Soil) tubes, estimated visually.

^wpH was measured in 1:1 ratio of soil: water slurry using a Hanna HALO® wireless soil pH meter.

^xGravitational moisture percentage was determined by drying the soil collected just after removal of pots from Ziploc bags at 80°C for 48 h.

^yClubroot disease severity was assessed on mustard greens roots using a 0-3 scale and clubroot disease indices were calculated using the formula , where n = the number of plants with a given rating.

^zClubroot disease incidence on mustard greens roots was calculated as the total number of plants with clubroot symptoms×100/total number of plants assessed.

Soil inoculation with biocontrol products did not affect the above-ground fresh biomass (P = 0.2) or shoot/root ratio (P = 0.3) in mustard greens across the ASD treatments (Table 5). The ASD treatments also did not affect above-ground fresh biomass (P = 0.6) across the biocontrol treatments, however the shoot/root biomass ratio was significantly (P < 0.001) higher in mustard greens plants grown in ASD-treated soil amended with ryegrass, sudangrass, winter rye, or wheat bran than in plants grown in the non-amended aerobic control across the biocontrol treatments.

All biocontrol treatments significantly (P < 0.001) reduced the clubroot disease severity index and incidence in mustard greens compared to those of plants grown in non-biocontrol inoculated soils across all ASD treatments (Table 5). The clubroot disease index was significantly (P < 0.001) lower in mustard greens grown in all ASD-treated soils compared to the aerobic control, and significantly lower than the anaerobic control in all ASD treatments except ASD-leek. All ASD treatments except ASD-leek significantly (P < 0.001) reduced clubroot incidence compared to both controls.

Interactions between biocontrol products and ASD with different carbon sources were not significant for soil reducing conditions (P = 1) or soil pH (P = 0.3). However, interactions were marginally significant for above ground fresh biomass (P = 0.09), clubroot severity (P = 0.08) and incidence (P = 0.1) in mustard greens. The combination of ASD treatment and biocontrol product application did not increase above-ground fresh mustard greens biomass when combined compared to ASD treatment alone, although fresh biomass was significantly (P < 0.001) reduced in ASD-sudangrass or AS-wheat bran plus Actinovate than in the respective ASD treatments alone (Table 6). Mustard greens plants grown in ASD-sudangrass-treated soil inoculated with any of the biocontrol products had significantly (P < 0.001) lower clubroot severity indices and incidence than plants grown in ASD-treated soil amended with sudangrass biomass alone. Mustard greens plants grown in ASD-wheat bran-treated soil and inoculated with Actinovate or Serenade had significantly lower clubroot severity indices and incidence than plants grown in ASD-wheat bran-treated soil alone. Plants grown in ASD-winter rye-treated soil and inoculated with Prestop or Serenade had significantly lower clubroot severity indices and incidence than plants grown in ASD-winter rye-treated soil alone. The addition of Actinovate to the anaerobic control resulted in significantly lower clubroot severity index in mustard greens plants compared to that of plants grown in the Actinovate-treated aerobic control and the aerobic controls with and without Actinovate. Under aerobic conditions Prestop and Serenade, but not Actinovate reduced clubroot severity and incidence significantly.

Table 6 Effects of anaerobic soil disinfestation (ASD) with cover crop carbon sources and biocontrol product application on soil parameters, plant growth and clubroot (Plasmodiophora brassicae) development in mustard greens.

ASD carbon source	Biocontrol^v	Paint loss from IRIS tubes (%)^w		pH^x		Shoot fresh weight (g)		Disease index (%)^y		Disease incidence (%)^z
Aerobic control	None	2.2	d	6.6	ij	18.1	fg	84.1	a	97.9	a
Aerobic control	Actinovate	1.0	d	6.6	j	23.7	c-g	76.7	ab	93.3	ab
Aerobic control	Prestop	1.8	d	6.8	e-j	23.8	c-g	52.4	c-f	70.3	b-f
Aerobic control	Serenade	1.7	d	6.7	g-j	25.3	c-g	35.5	e-j	64.6	c-h
Anaerobic control	None	3.7	d	6.8	c-j	29.3	b-e	68.5	a-c	87.4	abc
Anaerobic control	Actinovate	5.3	d	6.8	d-j	20.2	efg	50.6	c-f	67.5	b-f
Anaerobic control	Prestop	4.3	d	6.7	g-j	21.1	d-g	56.7	b-e	74.0	b-e
Anaerobic control	Serenade	3.3	d	6.8	f-j	22.8	c-g	65.5	a-d	86.0	a-d
Leek	None	31.2	c	7.1	a-d	28.4	b-e	48.2	c-g	68.4	b-f
Leek	Actinovate	48.4	c	7.0	a-e	26.9	b-g	47.8	c-g	68.5	b-g
Leek	Prestop	42.5	c	7.1	a-d	20.0	efg	48.8	c-g	69.9	b-f
Leek	Serenade	35.8	c	7.0	a-g	24.1	c-g	42.0	d-i	65.0	b-g
Ryegrass	None	67.5	b	7.0	a-g	27.6	b-f	37.1	e-j	50.0	e-i
Ryegrass	Actinovate	81.7	ab	7.0	a-d	29.2	b-e	37.6	e-j	56.3	d-i
Ryegrass	Prestop	68.3	ab	7.1	ab	24.3	c-g	31.1	f-k	45.9	e-j
Ryegrass	Serenade	87.0	ab	7.1	a-d	29.0	b-e	27.8	g-m	50.5	e-i
Sudangrass	None	79.2	ab	6.9	b-i	39.2	a	38.5	e-j	50.7	e-i
Sudangrass	Actinovate	92.7	ab	6.7	hij	29.1	b-e	12.2	lm	22.1	j
Sudangrass	Prestop	85.0	ab	6.9	a-h	32.2	abc	17.9	k-m	21.6	j
Sudangrass	Serenade	93.3	ab	7.1	ab	27.2	b-g	9.4	m	18.5	j
Winter rye	None	80.3	ab	7.1	ab	29.4	b-e	43.8	c-h	57.5	d-i
Winter rye	Actinovate	87.2	ab	7.1	ab	30.0	a-d	18.9	i-m	30.2	hij
Winter rye	Prestop	90.8	ab	7.2	a	27.7	b-e	26.9	f-l	48.7	e-i
Winter rye	Serenade	88.8	ab	7.1	ab	35.8	ab	19.7	j-m	28.8	ij
Wheat bran	None	82.5	ab	7.0	a-f	30.2	a-d	55.7	b-e	74.9	b-e
Wheat bran	Actinovate	95.0	a	7.1	abc	17.3	g	37.8	d-j	49.8	e-i
Wheat bran	Prestop	86.3	ab	7.1	ab	28.4	b-e	27.6	g-m	36.9	g-j
Wheat bran	Serenade	87.5	ab	7.1	ab	28.1	b-e	24.7	h-m	41.9	f-j
P-value		<0.001		<0.001		<0.001		<0.001		<0.001

^vMeans followed by the same lower-case letter within a column are not significantly different at P < 0.05. P: Probability value of Analysis of variance. Analysis of variance was performed on the arcsine (square-root) transformation of disease index (%) and disease incidence (%) and square-root transformation of paint loss from IRIS tubes. Means were separated using Fisher’s least significant difference test on transformed data. Presented values are original means. Values are the means of two experiments each with three replications.

^wIron oxide paint loss from IRIS (Indicator of Reduction in Soil) tubes, estimated visually.

^xpH was measured in 1:1 ratio of soil: water slurry using a Hanna HALO® wireless soil pH meter.

^yClubroot disease severity was assessed on mustard greens roots using a 0-3 scale and clubroot disease indices were calculated using the formula , where n = the number of plants with a given rating.

^zClubroot disease incidence was calculated as the total number of plants with clubroot symptoms×100/total number of plants assessed.

Evaluation of ASD with different cover crops and BCAs in the field.

Effects on soil parameters. In 2019, ASD with wheat bran or leek as the carbon source increased average soil temperature by 1.7ºC and 1.0ºC, respectively, compared to the aerobic and anaerobic controls (Table 7). No change in temperature was observed in the ASD-treated soil with winter rye and ryegrass biomass as carbon sources compared to the controls. In 2020 field trial, ASD with sudangrass and winter rye raised soil temperatures by 2.3ºC compared to aerobic control soil (data not shown). Data loggers used for ASD-wheat bran and anaerobic control plots failed during treatment.

Table 7 Percentage of iron oxide paint loss from Indicator of Reduction in Soils (IRIS) tubes, soil pH and soil temperature during anaerobic soil disinfestation (ASD) field trial, Huron County, OH, 2019.

ASD carbon source^v	Biocontrol product	Paint loss from IRIS tubes (%)^w		pH^x		Soil temperature (ºC)^y
	None	14.2		6.0
	Actinovate	13.4		6.1
	Prestop	17.5		6.0
	Serenade	17.6		6.0
P-value		0.28		0.4
Anaerobic control		1.7	c	6.1	ab	26.1
Aerobic control		2.1	c	6.0	b	26.0
Leek		15.7	b	6.0	b	27.1
Ryegrass		9.1	b	6.0	b	25.8
Winter rye		12.3	b	6.0	b	26.0
Wheat bran		41.4	a	6.2	a	27.9
P-value		<0.001		<0.001		N/A^z
Anaerobic control	None	1.7	ef	6.1
Aerobic control	None	0.9	f	5.9
Aerobic control	Actinovate	2.6	f	6.1
Aerobic control	Prestop	3.5	d-f	6.1
Aerobic control	Serenade	1.3	f	6.0
Leek	None	17.2	cd	5.9
Leek	Actinovate	14.9	c	6.0
Leek	Prestop	14.6	bc	6.1
Leek	Serenade	16.1	a-c	6.0
Ryegrass	None	11	cd	5.9
Ryegrass	Actinovate	6.1	c-e	6.1
Ryegrass	Prestop	6.1	c-e	5.9
Ryegrass	Serenade	13.3	cd	6.0
Winter rye	Actinovate	24.4	a-c	6.1
Winter rye	None	3.8	d-f	6.0
Winter rye	Prestop	7.1	c-e	5.9
Winter rye	Serenade	13.8	a-c	6.0
Wheat bran	None	43	ab	6.2
Wheat bran	Actinovate	39.6	a	6.2
Wheat bran	Prestop	39.4	ab	6.1
Wheat bran	Serenade	43.8	a	6.2
P-value		<0.001		0.9

^vMeans followed by the same lower-case letter within a column are not significantly different at P < 0.05. P: Probability value of Analysis of variance. Analysis of variance was performed on the square-root transformation of paint loss from IRIS tubes. Means were separated using Fisher’s least significant difference test on transformed data. Presented values are original means.

^wIron oxide paint loss from IRIS (indicator of reduction in soil) tubes, estimated visually.

^xpH was measured in 1:1 ratio of soil: water slurry using a Hanna HALO® wireless soil pH meter.

^yTemperature was measured at 30-min interval during ASD treatment period using HOBO Pendant temperature data loggers.

^zNot applicable.

Soil reducing conditions as indicated by percent iron oxide paint loss from IRIS tubes were not significantly affected by the inoculation of biocontrol products across the ASD treatments in both years of testing (Tables 7 and 8). However, ASD with all cover crops and wheat bran significantly (P < 0.001) increased soil reducing conditions compared to the anaerobic and aerobic controls in both years. Reducing conditions were consistently higher in plots treated with ASD-wheat bran than in those amended with any cover crop and subjected to ASD across the biocontrol treatments in both years of testing.

Inundative inoculation of soil with biocontrol products did not change the soil pH compared to non-inoculated soils (Tables 7 and 8). However, soil amendment with wheat bran followed by ASD treatment slightly but significantly (P < 0.001) increased soil pH compared to the aerobic control only in the 2019 field trial. Soil pH was not changed by ASD treatments across biocontrol inoculations in the 2020 field trial.

Table 8 Percent of iron oxide paint loss from Indicator of Reduction in Soils (IRIS) tubes placed in anaerobic soil disinfestation (ASD) field trial, soil pH and soil temperature during ASD treatment period in Huron County, OH in 2020.

ASD carbon source^x	Biocontrol product	Paint loss from IRIS tubes (%)^y		pH^z
	None	9.1		6.2
	Prestop	14.8		6.2
	Serenade	13.2		6.4
	P-value	0.4		0.4
Anaerobic control		0.0	c	6.0
Aerobic control		0.0	c	6.5
Ryegrass		10.2	b	6.3
Sudangrass		13.5	b	6.3
Winter rye		9.2	b	6.1
Wheat bran		31.9	a	6.3
	P-value	<0.001		0.3
Anaerobic	Non-biocontrol	0.0	e	6.0
Aerobic	None	0.0	e	6.6
Aerobic	Prestop	0.0	e	6.3
Aerobic	Serenade	0.0	e	6.5
Ryegrass	Non-biocontrol	4.5	d	6.5
Ryegrass	Prestop	8.5	d	6.1
Ryegrass	Serenade	17.5	bcd	6.2
Sudangrass	Non-biocontrol	9.4	cd	6.2
Sudangrass	Prestop	15.6	bcd	6.3
Sudangrass	Serenade	15.6	bcd	6.3
Winter rye	Non-biocontrol	14.4	bcd	6.0
Winter rye	Prestop	7.3	d	6.1
Winter rye	Serenade	6.0	d	6.2
Wheat bran	Non-biocontrol	26.3	abc	6.1
Wheat bran	Prestop	42.5	a	6.3
Wheat bran	Serenade	26.9	ab	6.5
	P-value	<0.001		0.7

^xMeans followed by the same lower-case letter within a column are not significantly different at P < 0.05. P: Probability value of Analysis of variance. Analysis of variance was performed on the square-root transformation of paint loss from IRIS tubes. Means were separated using Fisher’s least significant difference test on transformed data. Presented values are original means.

^yIron oxide paint loss from IRIS (indicator of reduction in soil) tubes, estimated visually.

^zpH was measured in 1:1 ratio of soil: water slurry using a Hanna HALO® wireless soil pH meter

Effects on plant biomass. Above-ground fresh and dry mustard greens biomass was not significantly (P = 0.3) affected by the inoculation of biocontrol products across ASD treatments in either year (Tables 9, and 10). In 2019, ASD with wheat bran but not with any of the cover crops as carbon sources significantly (P < 0.001) increased the above-ground fresh and dry plant biomass of mustard greens across biocontrol treatments compared to the aerobic control in the field trial and post-ASD bioassay. However, in 2020 the above-ground fresh and dry plant biomass were significantly higher in mustard greens plants grown in ASD-wheat bran-treated soils only in the post-ASD bioassay.

Table 9 Aboveground fresh and dry mustard greens biomass after anaerobic soil disinfestation (ASD) with different carbon sources and application of biocontrol products in field trials in Huron County 2019 and post ASD bioassay in a growth chamber.

ASD carbon source^w	Biocontrol product	Fresh shoot biomass (g)^x				Dry shoot biomass (g)
ASD carbon source^w	Biocontrol product	Field		Bioassay		Field
	None	1146		26.9		98.3
	Actinovate	1361		29.4		107.7
	Prestop	1187		26.7		98.9
	Serenade	1061		26.3		84.0
	P-value	0.36		0.5		0.14
Anaerobic control		1080	b	30.1	ab	91.8	b
Aerobic control		1063	b	24.6	b	89.9	b
Leek		910	b	24.1	b	74.0	b
Ryegrass		1178	b	27.5	ab	89.4	b
Winter rye		1106	b	28.5	ab	93.4	b
Wheat bran		1692	a	31.5	a	140.9	a
P-value		<0.001		0.08		<0.001
Anaerobic control	None	1080	b-e	30.1		91.8	cd
Aerobic control	None	1017	c-e	22.7		93.4	cd
Aerobic control	Actinovate	1125	b-e	28.8		89.2	cd
Aerobic control	Prestop	1034	c-e	26.6		84.2	cd
Aerobic control	Serenade	1066	b-e	20.3		92.9	cd
Leek	None	912	c-e	18.8		82.6	cd
Leek	Actinovate	1115	b-e	28.9		84.4	cd
Leek	Prestop	868	de	25.4		70.3	cd
Leek	Serenade	745	de	23.5		58.8	d
Ryegrass	None	1018	c-e	32		83.0	cd
Ryegrass	Actinovate	1305	b-e	30.3		100.9	cd
Ryegrass	Prestop	1128	b-e	24.2		87.9	cd
Ryegrass	Serenade	1261	b-e	24.5		85.7	cd
Winter rye	Actinovate	1446	a-d	25.4		101.4	cd
Winter rye	None	666	e	27.4		85.5	cd
Winter rye	Prestop	1261	b-e	27.1		104.8	bc
Winter rye	Serenade	1053	c-e	36.1		81.8	cd
Wheat bran	None	2133	a	31.6		152.0	a
Wheat bran	Actinovate	1812	ab	33.5		162.9	a
Wheat bran	Prestop	1643	a-c	30		147.4	ab
Wheat bran	Serenade	1181	b-e	30.7		101.2	cd
P-value		<0.04		0.3		0.01

^wMeans followed by the same lower-case letter within a column are not significantly different at P < 0.05. P: Probability value of Analysis of variance. Means were separated using Fisher’s least significant difference test on transformed data. Presented values are original means.

^xFresh and dry aboveground biomass (g) average of 25 plants.

Table 10 Aboveground fresh and dry mustard greens biomass affected by anaerobic soil disinfestation (ASD) with different carbon sources and inoculation of biocontrol products in field trials in Huron County 2020 and post ASD bioassay in 2020 in a growth chamber.

Carbon source^y	Biocontrol product	Fresh shoot biomass (g)^z				Dry shoot weight (g)
Carbon source^y	Biocontrol product	Field		Bioassay		Field	Bioassay
	None	622.0		24.0		35.6	1.6
	Prestop	594.0		21.4		34.1	1.6
	Serenade	558.0		22.4		32.3	1.5
	P-value	0.4		0.7		0.4	0.9
Anaerobic		647.0		21.9	bc	39.4	1.5	bc
Aerobic		598.0		16.8	c	33.9	1.2	c
Ryegrass		518.0		19.4	bc	31.7	1.4	bc
Sudangrass		544.0		16.8	c	31.3	1.2	c
Winter rye		627.0		24.2	b	36.5	1.7	b
Wheat bran		661.0		36.7	a	35.3	2.2	a
	P-value	4		<0.001		0.3	<0.001
Anaerobic	None	647.0	a-c	21.9	bcd	39.4	1.5	c-g
Aerobic	None	544.0	bc	18.3	cd	31.6	1.3	c-g
Aerobic	Prestop	603.0	bc	12.2	d	36.0	0.8	g
Aerobic	Serenade	647.0	a-c	20.0	bcd	34.2	1.3	d-g
Ryegrass	None	442.0	c	17.4	cd	27.6	1.2	efg
Ryegrass	Prestop	589.0	bc	25.2	bc	33.6	2.0	a-d
Ryegrass	Serenade	524.0	bc	15.7	cd	33.9	1.1	fg
Sudangrass	None	609.0	bc	19.4	cd	34.8	1.3	d-g
Sudangrass	Prestop	576.0	bc	15.6	cd	32.8	1.2	efg
Sudangrass	Serenade	446.0	c	15.4	cd	26.3	1.2	efg
Winter rye	None	823.0	a	26.3	bc	44.3	1.9	a-e
Winter rye	Prestop	510.0	bc	23.3	b-d	29.9	1.7	a-f
Winter rye	Serenade	550.0	bc	22.9	b-d	31.7	1.5	b-g
Wheat bran	None	668.0	ab	40.9	a	35.8	2.2	ab
Wheat bran	Prestop	691.0	ab	30.8	ab	38.4	2.0	abc
Wheat bran	Serenade	624.0	a-c	38.2	a	35.5	2.4	a
	P-value	0.089		<0.001		0.2	0.002

^yMeans followed by the same lower-case letter within a column are not significantly different at P < 0.05. P: Probability value of Analysis of variance.

^zFresh and dry aboveground biomass (g) per 15 plants.

Interactions between biocontrol products and ASD with different carbon sources were not significant for fresh (P = 0.62) or dry (P = 0.82) mustard greens biomass in the field or fresh biomass (P = 0.5) in the post-ASD bioassay in 2019. Significantly (P = 0.04) higher fresh biomass was observed in mustard greens grown in ASD-treated soil amended with wheat bran alone or wheat bran plus Actinovate compared to plants grown in ASD-treated plots amended with winter rye alone or plus Serenade, ryegrass alone, leek alone or plus Serenade, or the aerobic control or inoculated with Prestop in the 2019 field trial (Table 9). Dry shoot biomass was significantly (P = 0.01) higher in mustard greens grown in ASD-treated soil amended with wheat bran with or without biocontrol products compared to plants grown in anaerobic control plots, aerobic control plots with or without biocontrol products or all other plots with ASD/biocontrol combinations.

Interactions between biocontrol products and ASD with different carbon sources were marginally significant for fresh (P = 0.06), and dry biomass (P =0.13) in 2020 field experiment but interaction were not significant for fresh (P = 0.60) or dry (P = 0.78) mustard greens biomass in the post-ASD bioassay experiments in 2020.

In the 2020 field trial, the above-ground fresh biomass was marginally significantly (P = 0.089) higher in mustard greens grown in soil treated with ASD-winter rye alone than for plants grown in soils treated with ASD-winter rye plus Prestop or Serenade, ASD-ryegrass and ASD-sudangrass alone or with biocontrol products, and in aerobic control soils alone or with Prestop (Table 10). In the 2020 post-ASD bioassay averaged across biocontrol product treatments, above-ground fresh biomass was significantly (P<0.001) higher for mustard greens plants grown in ASD-wheat bran-treated soil than in soils amended with any of the cover crops or control soils (Table 10). Soil treatment with ASD-wheat bran alone or with Serenade resulted in higher shoot biomass than in all other treatments and controls, except ASD-wheat bran with Prestop. Dry plant biomass was not affected by any ASD treatment/biocontrol combination in the 2020 field trial. However, significantly (P = 0.002) higher above-ground dry biomass was observed in mustard greens grown in soil treated with ASD-wheat bran with or without biocontrol products compared to mustard greens grown in aerobic control soil with Prestop or Serenade, soil treated with ASD-ryegrass alone or plus Serenade, or ASD-sudangrass with or without biocontrol products in the 2020 post-ASD bioassay.

Effects on clubroot severity and incidence. Averaged across all ASD treatments, clubroot disease severity and incidence in the field trial and post-ASD bioassay did not differ significantly from the controls due to the addition of any of the biocontrol products in either year (Tables 11 and 12). However, ASD-wheat bran treatment significantly reduced clubroot severity and incidence compared to the controls and ASD with cover crops as carbon sources across the biocontrol treatments in both years.

Table 11 Clubroot disease severity index and incidence in mustard greens affected by anaerobic soil disinfestation (ASD) with different carbon sources and application of biocontrol products in field trials in Huron County 2019 and post ASD bioassay in a growth chamber.

ASD carbon source^x	Biocontrol product	Disease index (%)^y				Disease incidence (%)^z
ASD carbon source^x	Biocontrol product	Field		Bioassay		Field		Bioassay
	None	65.8		57.7		80.3		77.6
	Actinovate	56.1		46.0		75.2		63.3
	Prestop	54.4		44.3		73.6		61.6
	Serenade	57.9		48.3		79.2		66.0
	P-value	0.47		0.2		0.8		0.3
Anaerobic control		80.6	a	57.0	a	98.4	a	80.1	a
Aerobic control		69.5	a	62.8	a	86.5	a	78.3	a
Leek		65.4	a	55.4	a	80.7	a	74.4	a
Ryegrass		61.9	a	50.7	a	82.8	a	69.7	a
Winter rye		65.4	a	49.7	a	85.8	a	67.6	a
Wheat bran		27.8	b	24.9	b	46.2	b	43.2	b
P-value		<0.001		<0.001		<0.001		0.01
Anaerobic control	None	80.6	a	57.0	a-c	98.4	a	80.1	a
Aerobic control	None	77.7	a	73.8	a	88.1	ab	81.8	a
Aerobic control	Actinovate	78.9	a	67.6	ab	91.5	a	85.8	a
Aerobic control	Prestop	60.3	a-d	55.6	a-c	82.4	ab	74.5	ab
Aerobic control	Serenade	61.0	a-d	54.4	a-c	84.0	ab	71.2	ab
Leek	None	70.6	a	60.1	a-c	83.6	ab	75.3	ab
Leek	Actinovate	56.4	a-e	50.6	a-c	75.4	a-d	70.6	ab
Leek	Prestop	67.0	ab	43.9	b-d	82.5	ab	64.3	ab
Leek	Serenade	67.6	ab	67.1	ab	81.3	ab	87.6	a
Ryegrass	None	70.6	ab	61.9	a-c	87.2	ab	76.0	ab
Ryegrass	Actinovate	61.7	a-c	49.6	bc	83.1	ab	79.5	a
Ryegrass	Prestop	52.9	a-e	52.7	a-c	70.7	a-d	67.2	ab
Ryegrass	Serenade	62.4	a-d	41.6	b-d	90.3	a	57.5	ab
Winter rye	None	78.4	a	60.4	a-c	93.9	a	85.2	a
Winter rye	Actinovate	55.0	a-e	51.9	a-c	77.6	a-c	64.7	ab
Winter rye	Prestop	66.7	ab	44.3	b-d	87.5	ab	62.8	ab
Winter rye	Serenade	61.5	a-d	39.6	b-d	84.4	ab	54.4	a-c
Wheat bran	None	20.3	e	34.0	b-d	35.2	e	66.8	ab
Wheat bran	Actinovate	28.7	c-e	10.3	d	48.2	c-e	15.9	c
Wheat bran	Prestop	25.2	de	24.9	cd	45.1	de	39.2	bc
Wheat bran	Serenade	37.1	b-e	32.5	b-d	56.1	b-e	53.5	a-c
P-value		<0.001		0.004		<0.001		0.06

^xMeans followed by the same lower-case letter within a column are not significantly different at P < 0.05. P: Probability value of Analysis of variance. Analysis of variance was performed on the arcsine (square-root) transformation of disease index (%), and disease incidence (%). Means were separated using Fisher’s least significant difference test on transformed data. Presented values are original means

^yClubroot disease severity index was assessed on mustard greens roots using a 0-3 scale and clubroot disease indices were calculates by ( where n equals the number of plants with a given rating)

^zClubroot disease incidence was assessed on mustard greens roots were calculated by (total number of plants with clubroot symptoms×100/total number of plants assessed)

Table 12 Clubroot disease index and incidence in mustard greens plants affected by anaerobic soil disinfestation (ASD) with different carbon sources and inoculation of biocontrol products in field trials in Huron County, OH, 2020 and post ASD in a growth chamber.

ASD carbon source	Biocontrol product^w	Disease index (%)^xy				Disease incidence (%)^z
ASD carbon source	Biocontrol product^w	Field		Bioassay		Field		Bioassay
	None	36.0		59.3		58.4		68.4
	Prestop	38.7		51.4		64.0		65.6
	Serenade	32.2		43.8		54.1		52.9
	P-value	0.7		0.2		0.5		0.2
Anaerobic control		66.8	a	91.5	a	93.8	a	97.5	a
Aerobic control		40.1	b	64.2	b	63.6	b	76.2	ab
Ryegrass		40.8	b	53.1	b	65.6	ab	62.9	b
Sudangrass		45.4	ab	63.5	b	72.5	ab	76.8	ab
Winter rye		33.3	b	54.0	b	60.6	b	65.8	b
Wheat bran		8.3	c	12.1	c	20.1	c	20.1	c
	P-value	<0.001		<0.001		<0.001		<0.001
Anaerobic control	None	66.8	a	91.5	a	93.8	a	97.5	a
Aerobic control	None	49.0	ab	79.09	ab	71.9	ab	89.7	ab
Aerobic control	Prestop	43.6	ab	66.62	abc	70.9	ab	76.3	ab
Aerobic control	Serenade	27.8	bc	46.8	cd	48.1	bc	62.6	bc
Ryegrass	None	32.5	bc	54.97	bcd	49.7	bc	59.4	bc
Ryegrass	Prestop	42.0	ab	47.28	cd	67.6	ab	67.0	bc
Ryegrass	Serenade	47.9	ab	56.91	bc	79.6	ab	62.3	bc
Sudangrass	None	32.4	bc	59.16	bc	58.8	abc	78.3	abc
Sudangrass	Prestop	55.3	ab	67.37	abc	80.1	ab	77.4	ab
Sudangrass	Serenade	48.5	ab	63.86	abc	78.4	ab	74.8	ab
Winter rye	None	26.4	bc	62.68	bc	58.7	bc	70.6	abc
Winter rye	Prestop	42.9	ab	52.14	bcd	72.8	ab	66.8	bc
Winter rye	Serenade	30.6	bc	47.08	bcd	50.5	bc	60.0	bc
Wheat bran	None	9.2	c	8.22	e	17.9	c	14.8	d
Wheat bran	Prestop	9.5	c	23.73	de	28.5	c	40.8	cd
Wheat bran	Serenade	6.3	c	4.37	e	13.8	c	4.8	d
	P-value	0.006		<0.001		0.009		<0.001

^wMean values in a column sharing the same letter do not differ significantly at P < 0.05. P: Probability value of Analysis of variance.

^xAnalysis of variance was performed on the arcsine (square-root) transformation of disease index (%) and disease incidence (%), means were separated using Fisher’s least significant difference test on transformed data. Presented values are original means),

^yClubroot disease severity index was assessed using a 0-3 scale and clubroot disease indices were calculated according to the formula where n equals the number of plants with a given rating.

^zClubroot disease incidence was calculated as (total number of plants with clubroot symptoms×100/total number of plants assessed).

Interactions between biocontrol products and ASD with different carbon sources were not significant for clubroot index (P = 0.86, 0.9) and incidence (P = 0.96, 0.7) in mustard greens in the field and the post-ASD bioassay, respectively in 2019 (Table 9). Interactions between biocontrol products and ASD with different carbon sources were not significant for clubroot severity index (P = 0.94, 0.46), incidence (P =0.91, 0.52) mustard greens in the field and the post-ASD bioassay respectively in 2020.

Clubroot severity indices were significantly lower in mustard greens plants grown in wheat bran-amended ASD-treated soil regardless of biocontrol products used compared to the mustard greens plants grown in anaerobic and aerobic control plots in both years field trials (Tables 11 and 12). Under aerobic conditions, inoculation with Serenade resulted in significantly lower clubroot severity in mustard greens plants compared to those grown in the anaerobic control in the field (P = 0.006) and both aerobic and anaerobic controls in the bioassay (P < 0.001) in 2020. No significant reductions in disease severity or incidence were observed for any ASD-carbon source treatment/ biocontrol combination compared to their individual applications.

Correlation analysis. Field conditions were conducive for clubroot disease development. A strong positive correlation with P-value <0.001 and r = 0.93 was observed between disease severity and disease incidence (Table 13). Soil pH and paint loss from IRIS tubes (indicator of soil reducing conditions) were also significantly correlated (P = 0.01, r = 0.28). A significant (P <0.001) negative correlation between disease severity and soil reducing conditions was observed with an r value -0.51. A similar relationship was obtained between disease incidence and soil reducing conditions (P = 0.001, r =-0.48).

Table 13 Pearson correlation between above ground fresh mustard greens biomass, above-ground dry plant biomass, clubroot index, clubroot incidence, soil pH and paint loss in Indicator of Reduction in Soils (IRIS) tubes (%) in a field trial in Huron County, OH, 2019.

Parameter^z	Fresh biomass	Dry biomass	Clubroot index (%)	Clubroot incidence (%)	Paint loss in IRIS tubes (%)	Soil pH
Fresh biomass		0.89	-0.31	-0.17	0.15	-0.32
Dry biomass	<0.001**		-0.31	-0.18	0.19	-0.27
Clubroot index (%)	<0.01*	<0.001**		0.93	-0.51	-0.51
Clubroot incidence (%)	0.13	0.10	<0.001		-0.48	-0.60
Paint loss in IRIS tubes (%)	0.17	0.08	<0.001	<0.001**		0.28
Soil pH	0.001**	0.01*	<0.001**	<0.001**	0.01*

^zThe value above the diagonal indicates Pearson correlation coefficient (r) and values below diagonal indicate correlation probability values, * significant, **highly significant

Synergy analysis. Synergy analysis using the Bliss independence model indicated synergy in the combinations of Actinovate with ASD-leek, -ryegrass and -winter rye treatments in reducing clubroot severity indices and additivity between ASD-rye grass and Prestop, ASD-winter rye and Serenade, and ASD-winter rye and Prestop in the 2019 field trial (Table 14). ASD-wheat bran in combination with any of the biocontrol products or ASD-leek with Prestop were antagonistic in reducing clubroot disease under field conditions.

Table 14 Bliss independence test for clubroot index in mustard greens between treatment combinations of anaerobic soil disinfestation (ASD) with different carbon sources and biocontrol products in field trials in Huron County, OH, 2019.

Biocontrol^u	ASD carbon source	Disease severity (%)	Reduction^x (observed)^u	F_UA1^v	F_UA2^w	Expected disease control (E) (F_UA)^x	O-E^y	Remarks^z
None	Aerobic control	78.0
Actinovate	Aerobic control	79.0	-0.01	1.01
Prestop	Aerobic control	60.0	0.23	0.77
Serenade	Aerobic control	61.0	0.22	0.78
None	Leek	71.0	0.09		0.91
None	Ryegrass	71.0	0.09		0.91
None	Wheat bran	20.0	0.74		0.26
None	Winter rye	78.0	0.00		1.00
Actinovate	Leek	56.0	0.28			0.08	0.20		Synergistic
Actinovate	Ryegrass	62.0	0.21			0.08	0.13		Synergistic
Actinovate	Winter rye	55.0	0.30			-0.01	0.31		Synergistic
Actinovate	Wheat bran	29.0	0.63			0.74	-0.11		Antagonistic
Prestop	Leek	67.0	0.14			0.08	-0.16		Antagonistic
Prestop	Ryegrass	52.0	0.33			0.08	0.03		Additive
Prestop	Winter rye	67.0	0.14			-0.01	-0.09		Additive
Prestop	Wheat bran	25.0	0.68			0.74	-0.12		Antagonistic
Serenade	Leek	68.0	0.13			0.08	-0.16		Antagonistic
Serenade	Ryegrass	63.0	0.19			0.08	-0.10		Antagonistic
Serenade	Winter rye	61.0	0.22			-0.01	0.00		Additive
Serenade	Wheat bran	37.0	0.53			0.74	-0.27		Antagonistic
^uPercent reduction in clubroot severity in mustard greens compared to non-biocontrol inoculated aerobic control. ^vF_UA1 = 1- Percent reduction in clubroot severity for biocontrol products under aerobic control conditions compared to no-biocontrol inoculated aerobic control. ^wF_{UA1 =}1- Percent reduction in clubroot severity for ASD carbon source under non-biocontrol inoculated conditions compared to non-biocontrol inoculated aerobic control. ^xExpected percent disease control (F_UA) =1- (F_{UA1 for biocontrol product under aerobic conditions}×F_{UA2 for ASD carbon source under non-biocontrol inoculated conditions}). ^yO - observed percent reduction in clubroot severity, E- expected percent disease control (F_UA). ^zRemarks: O>E: synergistic, O<E: antagonistic, otherwise: additive.

In the 2020 field trials, the outcome of combining ASD-wheat bran with Prestop or Serenade on reduction of clubroot disease severity was additive, but the other combinations were antagonistic (Table 15). However, the post-ASD bioassay on soil collected from the 2020 field trials indicated additive outcomes for clubroot suppression with the combinations of ASD-wheat bran or ASD-winter rye with Serenade, or ASD-winter rye or ASD-ryegrass with Prestop (Table 16). There were no synergistic interactions in any of the ASD and biocontrol treatment combinations in 2020.

Table 15 Bliss independence test for clubroot index in mustard greens between treatment combinations of anaerobic soil disinfestation (ASD) with different carbon sources and biocontrol products in field trials in Huron County, OH, 2020.

Biocontrol^u	ASD carbon source	Disease severity (%)	Reduction^x (observed)^u	F_UA1^v	F_UA2^w	Expected disease control (E) (F_UA)^x	O-E^y	Remarks^z
None	Aerobic control	49.0
Prestop	Aerobic control	43.6	0.11	0.89
Serenade	Aerobic control	27.8	0.43	0.57
None	Ryegrass	32.5	0.34		0.66
None	Sudangrass	32.4	0.34		0.66
None	Winter rye	26.4	0.46		0.54
None	Wheat bran	9.2	0.81		0.19
Prestop	Ryegrass	42.0	0.14			0.41	-0.27	Antagonistic
Prestop	Sudangrass	55.3	-0.13			0.41	-0.54	Antagonistic
Prestop	Winter rye	42.9	0.12			0.52	-0.40	Antagonistic
Prestop	Wheat bran	9.5	0.81			0.83	-0.02	Additive
Serenade	Ryegrass	47.9	0.02			0.41	-0.39	Antagonistic
Serenade	Sudangrass	48.5	0.01			0.41	-0.40	Antagonistic
Serenade	Winter rye	30.6	0.38			0.52	-0.14	Antagonistic
Serenade	Wheat bran	6.3	0.87			0.83	0.04	Additive
^uPercent reduction in clubroot severity in mustard greens compared to non-biocontrol inoculated aerobic control. ^vF_UA1 = 1- Percent reduction in clubroot severity for biocontrol products under aerobic control conditions compared to no-biocontrol inoculated aerobic control. ^wF_{UA1 =}1- Percent reduction in clubroot severity for ASD carbon source under non-biocontrol inoculated conditions compared to non-biocontrol inoculated aerobic control. ^xExpected percent disease control (F_UA) =1- (F_{UA1 for biocontrol product under aerobic conditions}×F_{UA2 for ASD carbon source under non-biocontrol inoculated conditions}). ^yO - observed percent reduction in clubroot severity, E- expected percent disease control (F_UA). ^zRemarks: O>E: synergistic, O<E: antagonistic, otherwise: additive.

Table 16 Bliss independence test for clubroot disease severity index in mustard greens between treatment combinations of anaerobic soil disinfestation (ASD) with different carbon sources and biocontrol products in a bioassay with the soil collected from a field trial in Huron County, OH, 2020.

Biocontrol^u	ASD carbon source	Disease severity (%)	Reduction^x (observed)^u	F_UA1^v	F_UA2^w	Expected disease control (E) (F_UA)^x	O-E^y	Remarks^z
None	Aerobic control	79.1	0
Prestop	Aerobic control	66.6	0.16	0.83
Serenade	Aerobic control	46.8	0.41	0.59
None	Ryegrass	55.0	0.30		0.70
None	Sudangrass	59.2	0.25		0.75
None	Winter rye	62.7	0.21		0.79
None	Wheat bran	8.2	0.90		0.10
Prestop	Ryegrass	47.3	0.4			0.42	-0.02	Additive
Prestop	Sudangrass	67.4	0.15			0.37	-0.22	Antagonistic
Prestop	Winter rye	52.1	0.34			0.33	0.01	Additive
Prestop	Wheat bran	23.7	0.7			0.91	-0.21	Antagonistic
Serenade	Ryegrass	56.9	0.28			0.59	-0.31	Antagonistic
Serenade	Sudangrass	63.9	0.19			0.56	-0.37	Antagonistic
Serenade	Winter rye	47.1	0.4			0.53	-0.13	Additive
Serenade	Wheat bran	4.4	0.94			0.94	0.00	Additive
^uPercent reduction in clubroot severity in mustard greens compared to non-biocontrol inoculated aerobic control. ^vF_UA1 = 1- Percent reduction in clubroot severity for biocontrol products under aerobic control conditions compared to no-biocontrol inoculated aerobic control. ^wF_{UA1 =}1- Percent reduction in clubroot severity for ASD carbon source under non-biocontrol inoculated conditions compared to non-biocontrol inoculated aerobic control. ^xExpected percent disease control (F_UA) =1- (F_{UA1 for biocontrol product under aerobic conditions}×F_{UA2 for ASD carbon source under non-biocontrol inoculated conditions}). ^yO - observed percent reduction in clubroot severity, E- expected percent disease control (F_UA). ^zRemarks: O>E: synergistic, O<E: antagonistic, otherwise: additive.

Validation field trial. Soil reducing conditions indicated by iron oxide paint loss from IRIS tubes were significantly (P < 0.001) higher in ASD-wheat bran and ASD-winter rye-treated soils compared to anaerobic control soils (Table 17). Soil pH was not affected by any of the treatments.

Table 17 Percent of iron oxide paint loss on Indicator of Reduction in Soils (IRIS), soil pH, aboveground mustard greens biomass, shoot/root biomass ratio affected by anaerobic soil disinfestation (ASD) with different carbon sources and inoculation of biocontrol products in a field validation trial in Huron County 2020 and post ASD bioassay in 2020 in the growth chamber.

Treatment^w	Paint loss from IRIS tubes (%)^x		Soil pH^y	Fresh plant biomass (g)^z			Shoot-root biomass ratio
Treatment^w	Paint loss from IRIS tubes (%)^x		Soil pH^y	Field^z	Bioassay		Field	Bioassay
Anaerobic control	1.2	c	6.8	609.4	8.5	b	17.5	6.6
ASD-winter rye	22.5	a	6.6	576.9	18.4	b	14.5	10.5
ASD-winter rye + Prestop	4.2	bc	6.1	639.5	20.4	b	19.9	7.5
ASD-winter rye + Serenade	16.7	ab	6.2	638.6	17.5	b	13.7	9.1
ASD-wheat bran	35.0	a	6.2	618.6	45.1	a	18.3	15.2
P-value	0.001		0.6	0.9	0.04		0.8	0.4

^wMeans followed by the same lower-case letter within a column are not significantly different at P < 0.05. P: Probability value of Analysis of variance. Analysis of variance was performed on the square-root transformation of paint loss from IRIS tubes. Means were separated using Fisher’s least significant difference test on transformed data. Presented values are original means.

^xIron oxide paint loss from IRIS (indicator of reduction in soil) tubes, estimated visually.

^ypH was measured in 1:1 ratio of soil: water slurry using a Hanna HALO® wireless soil pH meter.

^zFresh and dry aboveground biomass (g) average of 15 plants in field and total plants in bioassay.

Plant biomass was not affected by any of treatments in the field experiment (Table 17). However, biomass was significantly (P = 0.04) higher in mustard greens grown in ASD-wheat bran-treated soil than in the anaerobic control and ASD-cover crop-treated soils in the post-ASD bioassay. The shoot/root biomass ratio in mustard greens plants was not significantly affected by treatment in both field and bioassay experiments.

Clubroot severity (P = 0.002) and incidence (P = 0.001) were significantly lower in mustard greens grown in ASD-wheat bran-treated soils than in aerobic and anaerobic control or the winter rye-amended ASD-treated soils in the field trial (Table 18). No differences in clubroot severity were observed between mustard greens plants grown in anaerobic and aerobic control soils and any of the winter rye cover crop-amended ASD-treated plots. However, in the post-ASD bioassay, clubroot severity (P = 0.018) and incidence (P = 0.003) were significantly lower in mustard greens grown in any ASD-treated soil than in the aerobic or anaerobic control soils.

Table 18 Clubroot disease severity index and incidence in mustard greens affected by anaerobic soil disinfestation (ASD) with different carbon sources and inoculation of biocontrol products in field trials in Huron County, OH in 2020 and a post ASD bioassay in a growth chamber.

Treatment^x	Disease index (%)^y				Disease incidence (%)^z
	Field		Bioassay		Field		Bioassay
Aerobic control	31.1	a	62.5	ab	61.6	a	100.0	a
Anaerobic control	30.8	a	72.1	a	68.3	a	88.6	a
ASD-winter rye	30.6	a	18.8	bc	68.9	a	22.9	bc
ASD-winter rye + Prestop	27.1	a	30.0	bc	68.9	a	45.9	b
ASD-winter rye + Serenade	29.1	a	14.9	c	45.9	a	23.1	bc
ASD-wheat bran	0.0	b	0.6	c	0.0	b	1.8	c
P-value	0.002		0.018		0.001		0.003

^xMeans followed by the same lower-case letter within a column are not significantly different at P < 0.05. P: Probability value of Analysis of variance. Analysis of variance was performed on the arcsine (square-root) transformation of disease index (%) and disease incidence (%) data. Means were separated using Fisher’s least significant difference test on transformed data. Presented values are original means.

^yClubroot disease severity index was assessed using a 0-3 scale and clubroot disease indices were calculated according to the formula where n equals the number of plants with a given rating).

^zClubroot disease incidence was calculated as the total number of plants with clubroot symptoms×100/total number of plants assessed).

Discussion

Clubroot, caused by Plasmodiophora brassicae, is a major production threat to cruciferous crops worldwide and management is challenging due to the lack of available options. Liming to increase the soil pH is the most widely adopted method worldwide, however long-term use of lime disrupts soil quality and leads to detrimental effects on soil productivity and nutrient availability (Bornman et al. 1998). Due to their high organic matter content, muck soil systems are highly resilient to soil pH change by external agents. Therefore, a large amount of lime is required to increase the soil pH and liming might not be always economically feasible. The efficacy of soil fungicides might also be disrupted by the buffering capacity of muck soil. Thus, alternative techniques are required for clubroot disease management in muck soil systems. For successful management of clubroot, the integration of multiple tools is recommended rather than depending on a single technique.

Testen and Miller (2019) reported a consistent reduction of clubroot in mustard greens planted in muck soil treated with ASD after amendment with wheat bran, molasses and a mixture of wheat bran + molasses. ASD is a local resource-based pre-plant soil rehabilitation process with a broad-spectrum effect on soilborne pathogens, nematodes and weeds. A substantial amount of literature indicates that ASD helps to improve soil quality (Jiang et al. 2019), increase soil suppressiveness to pathogens (Liu et al. 2019), enhance nutrient and organic matter availability for plants and enhance crop productivity (Zhu et al. 2014; Korthals et al. 2014) with no known environmental impacts. However, ASD requires a relatively large amount of carbon inputs, which might not always be economically feasible. Cover crops have multiple benefits in farming systems such as soil conservation, soil conditioning by promoting the growth of beneficial microbes, reducing weeds, insects and pathogen populations and addition of plant nutrients (Schipanski et al. 2014). Furthermore, certain cover crop species enhance the germination of P. brassicae resting spores but do not provide sites for infection, which leads to dying out of the resting spores (Friberg et al. 2005). Thus, the use of cover crops as carbon sources for ASD is potentially attractive for clubroot management. We tested nine different cover crops as ASD amendments under growth chamber conditions. We raised the cover crops in pots, incorporated them, and treated the soil with ASD. We found a substantial reduction of clubroot disease with cover crop-amended ASD treatment and selected the four most effective cover crops for further investigation.

The best suppression of clubroot was observed with winter rye, ryegrass, sesbania, leek and sudangrass cover crops as ASD carbon sources. Ryegrass, winter rye and leek stimulate of the germination of P. brassicae resting spores (Friberg et al. 2005). Sudangrass was the fastest-growing cover crop, building a considerable amount of biomass in a short period of time. Higher plant biomass promotes anaerobic conditions in the soil, which as indicated by the percentage of iron oxide paint loss from IRIS tubes, was generally higher in sudangrass biomass-amended ASD treatments than with other cover crop amendments in our experiments. Sudangrass tissues contain a cyanogenic glucoside (dhurrin) and during the decomposition process, they release hydrogen cyanide (HCN), which is deleterious to a wide range of pathogens and nematodes (Widmer and Abawi 2002; Wheeler et al. 1990; Widmer and Abawi 2000; Djian-Caporalino et al. 2019). This might have also contributed to the lower clubroot severity after ASD-sudangrass compared to other cover crops.

Serenade, Prestop, and Actinovate consistently reduced clubroot severity in all three growth chamber experiments. The efficacy of other biocontrol products was not consistent across the experiments. The effectiveness of Serenade, Prestop, and Actinovate in reducing clubroot in canola under growth chamber conditions has also been reported in Canada, but the results were inconsistent under field conditions (Peng et al. (2011, 2014); Lahlali et al. (2013)). Lahlali et al. (2013) suggested antibiosis against zoospores of P. brassicae and induced systemic resistance in plants are responsible for reducing clubroot severity after the treatment with Serenade. They reported 2.2 to 23-fold upregulation of genes responsible for jasmonic acid, ethylene and phenylpropanoid pathways in Serenade-treated plants compared to non-treated plants.

In field experiments, wheat bran-amended ASD suppressed clubroot disease severity indices and disease incidence and enhanced mustard greens biomass considerably compared to ASD with cover crops as carbon sources irrespective of the application of biocontrol products. It was surprising that none of the biocontrol products reduced clubroot severity indices, disease incidence or plant biomass under field conditions. Environmental conditions were optimal for clubroot disease development and the correlation between clubroot severity and incidence was high (r = 0.91, P < 0.001). The effectiveness of biocontrol products depends on the soil, environment and plant genotype. Therefore, their efficacy is often not consistent across fields and over time. Kasinathan (2012) reported 40% reduction of clubroot in canola after inoculation of Prestop in muck and mineral soils; Serenade was only effective in mineral soil. However, in our case the soil used in growth chamber experiments was collected from the same field in which the field experiment was conducted. Therefore, soil factors might not be the major contributors to the inconsistent results. Harsh field conditions including fluctuating temperatures, sunlight and soil moisture and interaction/competition with native soil microbes could be more important factors for low efficacy of inundative biocontrol products (Narisawa et al. 2005). Furthermore, viable spore density of P. brassicae and soil moisture conditions also greatly impact the efficacy of biocontrol products for clubroot suppression under field conditions (Narisawa et al. 2005). Average daily soil temperature ranged from 26 to 30ºC and total rainfall was 198.1 mm during crop period in the 2019 field trial. Average daily soil temperature ranged from 19.4 to 25.5ºC and total rainfall was 170 mm during the crop period in the 2020 field trial. We also irrigated the field weekly to ensure conducive conditions for disease development.

The Bliss independence model was used to analyze the combined response of BCA and ASD treatments by comparing the observed combination response with the predicted combination response for clubroot suppression (Yan et al. 2010; Xu et al. 2011; Willyerd et al. 2011). We tested possible outcomes of synergy, additivity or antagonism between ASD with different carbon sources and biocontrol products in the field experiments. In the 2019 field trial most of the ASD-cover crop and biocontrol product combinations demonstrated synergy or additivity in reducing clubroot disease severity in mustard greens. In particular, Actinovate acted synergistically with ASD with any of the three cover crops tested to suppress clubroot disease severity more than expected for either alone. Therefore, there is a potential benefit from combining ASD-cover crop with Actinovate. However, using ASD amended with any cover crop plus Actinovate did not improve suppression of clubroot disease compared to ASD-wheat bran alone. There was no apparent advantage in combining ASD-wheat bran, the most effective treatment across all trials for clubroot suppression, with any biocontrol products. Since Actinovate was not tested in 2020, its consistency in acting synergistically with ASD-cover crops is unknown. The synergy shown in combinations of Actinovate with ASD with different cover crops could be due to better survivability and higher colonizing capacity of Streptomyces lydicus during ASD since some of the Streptomyces spp. can grow in anaerobic conditions (Olanrewaju and Babalola 2019), however this needs to be verified. This is also supported by our observation of consistently lower clubroot severity in Actinovate-inoculated anaerobic control treatments than Actinovate- inoculated aerobic controls in growth chamber experiments.

Overall, ASD with wheat bran was effective in suppressing clubroot in mustard greens in a series of growth chamber and field experiments. Though efficacy in clubroot suppression of biocontrol products and ASD with different cover crops was limited under field conditions, several treatments were promising in growth chamber experiments. It may be possible to improve the efficacy of ASD with cover crops as carbon sources by applying higher rates or combining cover crops with reduced rates of wheat bran, which could reduce the cost of the ASD carbon source while retaining the benefits of both in clubroot suppression and crop management.

Acknowledgments

We thank Amilcar Vargas, Alex Taylor, Madelyn Horvat, Emma Piaget, Josh Amrhein and the OSU CFAES - Muck Crops Research Station team, B. Filbrun and H. Perez for assistance with the field experiments. We would also like to thank the USDA NIFA North Central Region- Sustainable Agriculture Research and Education Graduate Student Research Program (Award ID 20173864026916/H06607411) and the Storkan-Hanes-McCaslin Research Foundation for financial support.

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Participation Summary

1 On-farm demonstrations

4 Webinars / talks / presentations

3 Workshop field days

2 Other educational activities: Talked at OSU Congressional Assistants Tour

Participation Summary:

100 Farmers participated

300 Ag professionals participated

Education/outreach description:

Increase awareness of growers and other stakeholders of Ohio Muck Crops about ongoing activities and project achievements at three Muck Crop breakfasts (OSU Extension), between June to September 2019.
Increase awareness of growers, researchers and other stakeholders of Ohio Muck Crops about ongoing activities and project achievements at OSU Muck Crops Agricultural Experiment Station Field Day (July 2019).
Talked at OSU Congressional Assistants Tour - Muck Crops Agricultural Experiment Station; described project achievements and ongoing field experiments.
Virtual research talk based on this project was presented at Annual Meeting of American Phytopathological Society, Plant Health 2020, August 10-14. Students, researchers, and agriculture professionals from government, industry, academia and nonprofit organizations across the US were the targeted audience.
Virtual research talk based on this project was presented at the 2nd NAPA (Association of Nepalese Agricultural Professionals in Americas) Biennial International Scientific Conference, 2020 on 22-24 May 2020. Students, researchers, and agriculture professionals from government, industry, academia and nonprofit organizations across the US and Nepal were the targeted audience.
Virtual research talk based on this project was presented at Ph.D. defense seminar. Students, researchers, faculty and staffs of the Ohio State University were the target audience.

Project outcomes:

Soilborne diseases including clubroot in vegetable crops are very difficult to manage. We successfully demonstrated ASD with wheat bran can suppress the clubroot in mustard greens in series of growth chambers and field experiments. ASD is a local resource-based environmentally benign soil disinfestation technique with no known health and environmental effects. Growers are mostly using soil liming to increase the pH for clubroot management. However, overuse of lime can disrupt the soil, physical, chemical and biological conditions. Besides, ASD also helps to condition the soil pH, enhance organic matter and ultimately positive impact on plant growth and yield has been recorded. Thus the adoption of ASD with wheat barn can be utilized to suppress clubroot in mustard greens by reducing the farmer's dependencies in soil liming and other chemical fumigations.

Knowledge Gained:

ASD combined with cover crops and selected biocontrol successfully reduced the clubroot severity in mustard greens in series of experiments in growth chamber conditions but results are inconsistent in field conditions. However, ASD with wheat bran consistently reduced clubroot severity and incidence in field trials compared to controls. Therefore it might be possible to improve the efficacy of ASD with cover crops as carbon sources by applying higher rates or combining cover crops with reduced rates of wheat bran, which could reduce the cost of the ASD carbon source while retaining the benefits of both in clubroot suppression and crop management.

This project provided the opportunities to test and verify the efficacy of integration of several sustainable techniques together to manage soilborne diseases. We learn to analyze the combined effects of several techniques together using the Bliss independence model. It also helped us to build our confidence in testing these techniques in field conditions. Finally, we got several opportunities to interact with growers, educators, and researchers in several workshops, seminars, and conferences to share our research outputs and get insight from other participants.

Combined Effects of Inundative Biocontrol and Anaerobic Soil Disinfestation (ASD) Using Non-Host Cover Crops as Carbon Sources for Clubroot Management in Cruciferous Crops

Project Information

Cooperators

Research