Assessment of beneficial microorganisms: Trichoderma, Actinomycetes, and Bacillus in anaerobic soil disinfestation (ASD)

Final Report for GS14-128

Project Type: Graduate Student
Funds awarded in 2014: $10,993.00
Projected End Date: 12/31/2015
Grant Recipient: The University of Tennessee
Region: Southern
State: Tennessee
Graduate Student:
Major Professor:
Dr. David Butler
University of Tennessee, Knoxville
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Project Information


Studies on anaerobic soil disinfestation (ASD), a non-chemical alternative to soil fumigants for controlling many soilborne diseases, have shown that it enhances populations of beneficial microorganisms against plant pathogens, including increased presence of the biocontrol agent Trichodermaas sclerotial parasites of Sclerotium rolfsii. However, studies on ASD effectiveness paired with beneficial mycoparasites and commercial biofungicide applications are lacking. This study compared the effect of ASD and incorporation of antagonists separately or in combination, at the initiation of ASD treatment, against the sclerotial germination and parasitism. The effect of ASD amendment on soil populations of endophytic isolates of Trichoderma,actinomycetes, and Bacillus spp. were also assessed. The anaerobic condition was also determined during ASD treatment in growth chamber studies. The root nodules of cowpea and plant biomass (cowpea and tomato) after ASD treatment were also recorded in greenhouse study. In contrast to the negative effect of ASD on sclerotial population, we observed positive or no effect on the population of beneficial microorganisms. Further, ASD enhanced the mycoparasitic and bacterial colonization of sclerotia;however, ASD followed by addition of antagonists did not increase sclerotial mortality or parasitism of sclerotia.


Anaerobic soil disinfestation (ASD) or biological soil disinfestation is a practical and relevant soil treatment to replace chemical fumigant pesticides to control soil borne pathogens. It is also applicable to places where other environmentally friendly approaches, such as flooding, solarization, and biofumigation techniques, are not effective or economically feasible. ASD uses locally available organic materials as a carbon source and can improve soil quality, increase populations of beneficial soil microorganisms, in addition to numerous other agronomic and environmental benefits. In ASD, the carbon source from organic amendments increases microbial activity leading to consumption of available soil oxygen; microbial populations shift to those adapted to oxygen-limited environments and release various compounds along with organic acids, e.g. butyric acid and acetic acid that significantly contribute to the soil disinfestation process (Momma et al., 2006).

Anaerobic condition that result from ASD mainly increases the number of anaerobic bacteria of clostridia groups (Mowlick et al., 2012), while there is some research on effect of ASD on bacterial communities, studies on ASD impact on beneficial fungal populations are lacking. It is important to find effective ASD treatment methods to control plant pathogens without effecting the population of beneficial soil organisms. Hence, the SARE-funded graduate research project based at The University of Tennessee examined the ASD treatment effect on the soil beneficial microbial organisms such as Trichoderma, Actinomycetes, Bacillus and root colonizers (Rhizobia) and investigated combined effect of ASD and microbial biocontrols at the initiation of ASD treatment on germination and parasitism of sclerotia of Sclerotium rolfsii.

Project Objectives:
  1. Examine the efficacy of incorporation of endophytic Trichoderma and two commercially available biofungicides Trichoderma harzianum (RootShield®) and Streptomyces riseoviridis (Mycostop®) in ASD treatment against sclerotial germination and parasitism.
  2. Evaluate the effect of ASD amendment on populations of Trichoderma, actinomycetes Bacillus and nodulation by Rhizobia
  3. Compare yield of plant biomass among ASD, biocontrols, and ASD in combination with biocontrols.


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  • Dr. Bonnie Ownley
  • Utsala Shrestha


Materials and methods:

The repeated completely randomized design with the ASD and non-ASD treatments (List 1) were carried out in a growth chamber maintained at 15-25°C at the University of Tennessee. ASD treatments included a organic amendment, dry molasses maintained at optimum C:N ratio of 30:1 (mixed with corn starch) at C rates of 4 mg C/g soil. Soil to fill pots (~ 2.5 L) was collected from the ‘Ap’ horizon of soil at the Plateau Research and Education Center, Crossville, TN, where previously ASD treatment has been carried out and sieved (< 10-mm) to remove organic debris. Oxidation-reduction potential (ORP) electrodes installed at 15-cm depth to monitor redox potential continuously using an automatic data logging system over the 3-week ASD treatment for calculation of accumulation of anaerobic soil conditions. Soil temperature and soil moisture was continuously monitored at 10 cm using temperature /moisture probes and a data logger. Three S. rolfsii inoculum bags with ten sclerotia were buried at 5-, 10- and 15-cm depths in each pot and bag with 100 hundred sclerotia buried at 2 cm depth. Studies started on March 2015 and repeated on June 2015. At completion of ASD, soil samples were collected for estimation of soil moisture, soil pH and biocontrol agents. Cumulative soil anaerobic condition was calculated as described in (Butler et al., 2012b; McCarty et al., 2014).

List 1. Treatments      

  1. ASD alone
  2. ASD + endophytic Trichoderma
  3. ASD + RootShield®
  4. ASD + Mycostop®
  5. ASD + RootShield® + Mycostop®
  6. endophytic Trichoderma (T. asperellum)
  7. RootShield® (T. harzianum Rifai strain KRL-AG2)
  8. Mycostop®(Streptomyces griseoviridis Strain K61)
  9. RootShield® + Mycostop®   
  10. Non amended covered control  
  11. Non amended uncovered control      


After 3 weeks of ASD treatment, packets containing sclerotia were retrieved and sclerotia were plated onto 24-well plates containing either antibiotic-amended potato dextrose agar (PDA), actinomycete isolation agar (AIA), or Trichoderma-selective medium (TSM) to assess germination and parasitism of sclerotia. Quantification of Trichoderma, Actinomycetes and Bacillus were assessed by serial dilution methods on media TSM, AIA and media for facultative anaerobic spore formers (BIA). Three-week-old tomato seedlings of ‘Florida Lanai’ and germinated Cowpea seeds if ‘Calfornia blackey pea) were planted in pots, and transferred to the greenhouse. Sclerotial survival and foliar disease pressure evaluated weekly. After 8 weeks, root nodules dry biomass of plants was determined. Data were analyzed using a mixed model in SAS (Glimmix procedure, SAS Institute, Cary, NC) to determine the impact on sclerotial germination, quantification of beneficial organism and disease development and crop performance.

To identify Trichoderma, the fungus with green appearance on PDA was observed under microscope. Further identification of Trichoderma spp. was done by extracting Genomic DNA from colonies of isolate grown on potato dextrose broth using the QiagenDNeasy DNA extraction kit. The ITS region 1 and 2 of Trichoderma isolates (White et al. 1990) amplified by PCR using the appropriate primers pairs (5'-TCCGTAGGTGAACCTGCGG-3' / ITS2: 5'-GCTGCGTTCTTCATCGATGC-3'). PCR was carried out in a 50-μl reaction mixture containing 50ng genomic DNA, 5μl of 0.5 μM primers, 1 μL dimethylsulfoxide and 25 μL of 5 PRIME HotMasterMix  (VWR International). The PCR conditions consisted of an initial denaturation of 9 min at 94°C followed by 42 cycles of 1 min denaturation at 94°C, 1 min of annealing at 42 °C and 2 min extension at 72°C, and final extension of 3 min at 72°C. The PCR products was purified using ExoSAP-IT® PCR Product Cleanup ExoSAP-IT (Affymetrix, Santa Clara, CA), and  sequencing was provided by Molecular Biology Resource Facility, UTK, Knoxville, TN. The obtained sequences were used to blast in the NCBI GenBank database and were aligned with Sequncer (v 5.1) configured for highest accuracy.

Research results and discussion:
  1. ASD treatments with or without biocontrol significantly lowered germination of artificially inoculated sclerotia retrieved from 5-, 10- and 15-cm depths.
  2. Mortality of sclerotia was lower at 5-cm than greater depths (10- and 15-cm).
  3. Biocontrols alone did not reduce sclerotial germination compared to the plastic covered or non-covered control.
  4. At 5-cm, sclerotial germination was highest for Mycostop®+ RootShield® (88%) followed by two controls and endophytic Trichoderma.
  5. Sclerotia retrieved from 5-cm depth, plated on PDA, showed relatively higher percentage Trichoderma parasitism of sclerotia in ASD treatments compared to the control and incorporation of biocontrols with ASD did not increase sclerotial parasitism by Trichoderma.
  6. Sclerotia retrieved from 10-cm, examined on AIA, showed significantly highest bacterial parasitism in ASD alone and ASD with biocontrols, except Mycostop®, when compared with non-ASD with biocontrols (72-83%) and actinomycete parasitism of sclerotia was significantly higher in Mycostop® treatments (65-88%).
  7. Sclerotia retrieved from the 15-cm depth examined on TSM showed significantly higher parasitism of sclerotia by Trichoderma in non-ASD and control (32-72%) than ASD treatment with or without biocontrols.
  8. Trichoderma populations were similar across the ASD treatments and was highest in ASD when compared to ASD with biocontrol, except for T. asperellum with and without ASD.
  9. ASD treatments enhanced populations of Bacillus with highest CFU recorded for ASD with asperellum and the combination of Mycostop® and RootShield® (5.6 log CFU+1 g-1 of soil).
  10. We observed that incorporation of biocontrols (endophytic Trichoderma, Mycostop® and RootShield) reduced the populations of actinomycetes during ASD treatment as compared to ASD alone and biocontrol alone.
  11. Different species of Trichoderma ( harzianum, T. citrinovirde, T.spirale, and T.asperellum) were found parasitizing sclerotia.
  12. ASD with asperellum showed highest total (cowpea + tomato) dry biomass and nodule mass when compared to other treatments.
Participation Summary

Educational & Outreach Activities

Participation Summary:

Education/outreach description:

Shrestha U, B Ownley, M Dee, and DM Butler (Submitted Sep 2016) Interaction of anaerobic soil disinfestation and introduced biocontrol agents on Sclerotium rolfsii germination and parasitism. 2016 Annual International Research Conference on Methyl Bromide Alternatives and Emissions Reductions, Orlando, FL.

Shrestha, U, "Anaerobic Soil Disinfestation: Meta-analysis and Optimization of Amendment Carbon Rate and C:N Ratio to Control Key Plant Pathogens and Weeds. " PhD diss., University of Tennessee, 2016. Available at

Shrestha U, M Dee, B Ownley, and DM Butler (2016) Effect of anaerobic soil disinfestation on survival of S. rolfsii sclerotia and soil populations of Trichoderma. 2016 APS Southern Division Meeting, Balm, FL. Available at

Project Outcomes

Project outcomes:

S. rolfsii is one of the major soil borne pathogens of vegetable crops that results in the significant economic loss to the growers. Control of southern blight disease caused by S. rolfsii is much more challenging for organic growers as well as to commercial growers after phase out of methyl bromide fumigation as feasible alternatives are underprovided. ASD treatment is well-suited for farmers using raised-bed-plasticulture systems. As we observed low germination and high parasitism of sclerotia of S. rolfsii in ASD study (with or without any biocontrols), application of ASD in vegetable farms could be an alternative option to control southern blight disease. Further, after application of ASD population of beneficial organisms i.e. Trichoderma spp. and Bacillus spp. increased and no negative effect of ASD on actinomycetes and root nodule forming bacteria were observed indicating ASD has no harmful impact on beneficial organisms. However, application of commercial bio-fungicides such as Mycostop® or RootShield®, or endophytic isolate - T. asperellum under covered condition did not promote suppression of sclerotial germination, compared to ASD only treatment.

Economic Analysis


Farmer Adoption



Areas needing additional study

Because the incorporation of commercial and endophytic biocontrols before ASD treatment did not significantly suppress sclerotial germination and parasitism, we suggest studying the effect of the biocontrols in ASD by adding them after the completion of ASD. Further investigation of the impact of ASD on beneficial organisms in field condition is needed to enhance the effects of ASD to control soil borne pathogens.

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.