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

Final report for GNC18-260

Project Type: Graduate Student
Funds awarded in 2018: $11,995.00
Projected End Date: 09/30/2021
Grant Recipient: The Ohio State University-Wooster Campus
Region: North Central
State: Ohio
Graduate Student:
Faculty Advisor:
Dr. Sally Miller
The Ohio State University, Dept of Plant Pathology
Expand All

Project Information

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.

Cooperators

Click linked name(s) to expand/collapse or show everyone's info
  • Robert P Filbrun (Researcher)

Research

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 (1×105 spores g-1 soil).

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 108 spores 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-2 s-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-1 for 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 (FUA) were calculated as the product of individual effects of BCA (FUA1) and ASD carbon source (FUA2)

FUA = FUA1× FUA2

where FUA 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, FUA will be 1-0.17 = 0.83). According to the Bliss independence assumption, FUA is the expected effect of combined treatments and synergistic, additive and antagonistic relationships between the treatments were determined as follows:

  1. If the observed combined effect of BCA and ASD-carbon source is equal to FUA, the relationship is additive and there is no interaction between ASD-carbon source and BCAs
  2. If the observed combined effect is greater than FUA, the relationship is synergistic
  3. If the observed combined effect is less than FUA, 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.

 

Treatmentw

Combined analysisx

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.

 

Treatmenty

Fresh weight (g)

Combinedz

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 productu

Paint loss from IRIS tubes (%)v

pHw

 

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

Biocontrolv

Paint loss from IRIS tubes (%)w

pHx

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 sourcev

Biocontrol product

Paint loss from

IRIS tubes (%)w

pHx

 

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/Az

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 sourcex

Biocontrol product

Paint loss

from IRIS tubes (%)y

pHz

 

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 sourcew

 

Biocontrol product

Fresh

shoot biomass (g)x

Dry shoot biomass (g)

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 sourcey

Biocontrol product

Fresh shoot biomass (g)z

Dry shoot weight (g)

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 sourcex

 

Biocontrol product

Disease index (%)y

Disease incidence (%)z

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 productw

Disease index (%)xy

Disease incidence (%)z

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.

Parameterz

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.

Biocontrolu

ASD carbon source

Disease severity (%)

Reductionx (observed)u

FUA1v

FUA2w

Expected disease control (E)  

(FUA)x

O-Ey

Remarksz

 

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.

vFUA1 = 1- Percent reduction in clubroot severity for biocontrol products under aerobic control conditions compared to no-biocontrol inoculated aerobic control.

wFUA1 = 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 (FUA) =1- (FUA1 for biocontrol product under aerobic conditions×FUA2 for ASD carbon source under non-biocontrol inoculated conditions).

yO - observed percent reduction in clubroot severity, E- expected percent disease control (FUA).  

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.

Biocontrolu

ASD carbon source

Disease severity (%)

Reductionx (observed)u

FUA1v

FUA2w

Expected disease control (E)  

(FUA)x

O-Ey

Remarksz

 

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.

vFUA1 = 1- Percent reduction in clubroot severity for biocontrol products under aerobic control conditions compared to no-biocontrol inoculated aerobic control.

wFUA1 = 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 (FUA) =1- (FUA1 for biocontrol product under aerobic conditions×FUA2 for ASD carbon source under non-biocontrol inoculated conditions).

yO - observed percent reduction in clubroot severity, E- expected percent disease control (FUA).  

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.

Biocontrolu

ASD carbon source

Disease severity (%)

Reductionx (observed)u

FUA1v

FUA2w

Expected disease control (E)  

(FUA)x

O-Ey

Remarksz

 

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.

vFUA1 = 1- Percent reduction in clubroot severity for biocontrol products under aerobic control conditions compared to no-biocontrol inoculated aerobic control.

wFUA1 = 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 (FUA) =1- (FUA1 for biocontrol product under aerobic conditions×FUA2 for ASD carbon source under non-biocontrol inoculated conditions).

yO - observed percent reduction in clubroot severity, E- expected percent disease control (FUA).  

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.

Treatmentw

Paint loss from IRIS tubes (%)x

Soil pHy

Fresh plant biomass (g)z

 

Shoot-root biomass ratio

Fieldz

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.

Treatmentx

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.

 

 

References

Bornman, J. J., Bornman, L., and Barnard, R. O. 1998. The effects of calcium carbonate and calcium hydroxide on plant growth during overliming. Soil Sci. 163:498–507.

Castlebury, L. A., Maddox, J. V., and Glawe, D. A. 1994. A technique for the extraction and purification of viable Plasmodiophora brassicae resting spores from host root tissue. Mycologia. 86:458–460.

Chai, A. L., Xie, X. W., Shi, Y. X., and Li, B. J. 2014. Research status of clubroot (Plasmodiophora brassicae) on cruciferous crops in China. Can. J. Plant Pathol. 36:142–153

Dixon, G. R. 2014. Clubroot (Plasmodiophora brassicae Woronin) – an agricultural and biological challenge worldwide. Can. J. Plant Pathol. 36:5–18.

Djian-Caporalino, C., Mateille, T., Bailly-Bechet, M., Marteu, N., Fazari, A., Bautheac, P., et al. 2019. Evaluating sorghums as green manure against root-knot nematodes. Crop Prot. 122:142–150.

Friberg, H., Lagerlöf, J., and Rämert, B. 2005. Germination of Plasmodiophora brassicae resting spores stimulated by a non-host plant. Eur. J. Plant Pathol. 113:275.

Friberg, H., Lagerlöf, J., and Rämert, B. 2006. Usefulness of nonhost plants in managing Plasmodiophora brassicae. Plant Patho. 55:690–695.

Heinrich, A., Kawai, S., and Myers, J. 2017. Screening Brassica cultivars for resistance to Western Oregon clubroot pathotypes. HortTechnology. 27:510–516.

Hewavitharana, S. S., Ruddell, D., and Mazzola, M. 2014. Carbon source-dependent antifungal and nematicidal volatiles derived during anaerobic soil disinfestation. Eur. J. Plant Pathol. 140:39–52.

Jiang, Y., Kang, Y., Han, C., Zhu, T., Deng, H., Xie, Z., et al. 2019. Biochar amendment in reductive soil disinfestation process improved remediation effect and reduced N2O emission in a nitrate-riched degraded soil. Arch. Agron. Soil Sci. 66 (7):983-991.

Kasinathan, H. 2012. Influence of pH, temperature, and biofungicides on clubroot of canola. Thesis Master of Science in Plant Agriculture, The University of Guelph.

Khadka, R. B., and S. A. Miller.  2021.  Synergy of anaerobic soil disinfestation (ASD) and Trichoderma spp. in Rhizoctonia root rot suppression. Front. Sustain. Food Syst. DOI: 10.3389/fsufs.2021.645736.

Korthals, G. W., Thoden, T. C., van den Berg, W., and Visser, J. H. M. 2014. Long-term effects of eight soil health treatments to control plant-parasitic nematodes and Verticillium dahliae in agro-ecosystems. Appl. Soil Ecol. 76:112–123.

Lahlali, R., Peng, G., Gossen, B. D., McGregor, L., Yu, F. Q., Hynes, R. K., et al. 2013. Evidence that the biofungicide Serenade (Bacillus subtilis) suppresses clubroot on canola via antibiosis and induced host resistance. Phytopathology. 103:245–254.

Liu, L., Huang, X., Zhao, J., Zhang, J., and Cai, Z. 2019. Characterizing the key agents in a disease-suppressed soil managed by reductive soil disinfestation. Appl. Environ. Microbiol. 85:e02992-18.

Luo, D., Ganesh, S., Koolaard, J., and Luo, M. D. 2014. Package ‘predictmeans. https://cran. r-project. org/web/packages/predictmeans/index. html.

Mehrabi, S., Stjelja, S., and Dixelius, C. 2018. Root gall formation, resting spore isolation and high molecular weight DNA extraction of Plasmodiophora brassicae. Bio-101: e2864.

Moxham, S. E., and Buczacki, S. T. 1983. Chemical composition of the resting spore wall of Plasmodiophora brassicae. Trans. Br. Mycol. Soc. 80:297–304.

Narisawa, K., Shimura, M., Usuki, F., Fukuhara, S., and Hashiba, T. 2005. Effects of pathogen density, soil moisture, and soil pH on biological control of clubroot in Chinese cabbage by Heteroconium chaetospira. Plant Dis. 89:285–290.

Olanrewaju, O. S., and Babalola, O. O. 2019. Streptomyces: Implications and interactions in plant growth promotion. Appl. Microbiol Biotechnol. 103:1179–1188.

Peng, G., Lahlali, R., Hwang, S. F., Pageau, D., Hynes, R. K., McDonald, M. R., et al. 2014. Crop rotation, cultivar resistance, and fungicides/biofungicides for managing clubroot (Plasmodiophora brassicae) on canola. Can. J. Plant Pathol. 36:99–112.

Peng, G., McGregor, L., Lahlali, R., Gossen, B. D., Hwang, S. F., Adhikari, K. K., et al. 2011. Potential biological control of clubroot on canola and crucifer vegetable crops. Plant Pathol. 60:566–574.

Rabenhorst, M. C. 2008. Protocol for using and interpreting IRIS tubes. Soil Surv. Horiz. 49:74–77.

Rabenhorst, M. C. 2012. Simple and reliable approach for quantifying IRIS tube data. Soil Sci. Soc. Am. J. 76:307–308.

Schipanski, M. E., Barbercheck, M., Douglas, M. R., Finney, D. M., Haider, K., Kaye, J. P., et al. 2014. A framework for evaluating ecosystem services provided by cover crops in agroecosystems. Agric. Syst. 125:12–22.

Sharma, K., Gossen, B. D., and McDonald, M. R. 2011. Effect of temperature on cortical infection by Plasmodiophora brassicae and clubroot severity. Phytopathology. 101:1424–1432.

Shrestha, U., Dee, M. E., Ownley, B. H., and Butler, D. M. 2017. Anaerobic soil disinfestation reduces germination and affects colonization of Sclerotium rolfsii sclerotia. Phytopathology. 08:342-351.

Strelkov, S. E., Tewari, J. P., and Smith-Degenhardt, E. 2006. Characterization of Plasmodiophora brassicae populations from Alberta, Canada. Can. J. Plant Pathol. 28:467–474.

Testen, A. L., and Miller, S. A. 2019. Anaerobic soil disinfestation to manage soilborne diseases in muck soil vegetable production systems. Plant Dis. 103:1757-1762.

Wheeler, J., Mulcahy, C., Walcott, J., and Rapp, G. 1990. Factors affecting the hydrogen cyanide potential of forage sorghum. Aust. J.  Agric. Res. 41:1093.

Widmer, T. L., and Abawi, G. S. 2000. Mechanism of suppression of Meloidogyne hapla and its damage by a green manure of sudangrass. Plant Dis. 84:562–568.

Widmer, T. L., and Abawi, G. S. 2002. Relationship between levels of cyanide in sudangrass hybrids incorporated into soil and suppression of Meloidogyne hapla. J. Nematol. 34:16–22.

Willyerd, K. T., Li, C., Madden, L. V., Bradley, C. A., Bergstrom, G. C., Sweets, L. E., et al. 2011. Efficacy and stability of integrating fungicide and cultivar resistance to manage Fusarium head blight and deoxynivalenol in wheat. Plant Dis. 96:957–967.

Xu, X.-M., Jeffries, P., Pautasso, M., and Jeger, M. J. 2011. Combined use of biocontrol agents to manage plant diseases in theory and practice. Phytopathology. 101:1024–1031.

Yan, H., Zhang, B., Li, S., and Zhao, Q. 2010. A formal model for analyzing drug combination effects and its application in TNF-α-induced NFκB pathway. BMC Syst Biol. 4:50.

Zhu, T., Dang, Q., Zhang, J., Müller, C., and Cai, Z. 2014. Reductive soil disinfestation (RSD) alters gross N transformation rates and reduces NO and N2O emissions in degraded vegetable soils. Plant Soil. 382:269–280.

Participation Summary

Educational & Outreach Activities

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
300 Ag professionals participated
Education/outreach description:
  1. 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.
  2. 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).
  3.  Talked at OSU Congressional Assistants Tour - Muck Crops Agricultural Experiment Station; described project achievements and ongoing field experiments. 
  4. 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.
  5. 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.
  6. 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

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. 

 

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.