Location: Biological Control of Pests Research
2022 Annual Report
Objectives
Objective 1: Optimize aerial and seed treatment application strategies for biological and chemical pest control agents for row crops such as corn, peanut, and soybean.
Sub-objective 1.A: Managing aflatoxin in corn by biochar and non-toxigenic Aspergillus flavus.
Sub-objective 1.B: Managing aflatoxins in peanut by biochar and non-toxigenic Aspergillus flavus.
Sub-objective 1.C: Highly economical deployment of non-aflatoxigenic A. flavus for corn production in Guatemala.
Sub-objective 1.D: Managing mycotoxin in soybean infected with charcoal rot disease caused by Macrophomina phaseolina.
Objective 2: Utilize Aspergillus genomics for population studies, and improve risk models.
Sub-objective 2.A: Genetic characterization of the A. flavus population within niches of the corn production ecosystem.
Sub-objective 2.B: Improve emerging weather-based aflatoxin risk models with field level data of pathogen genotypes.
Approach
Mycotoxin contamination in row crops like corn, peanut and soybean contributes to significant economic losses for farmers and industry. Mycotoxin contamination (e.g., aflatoxins in corn and peanuts) poses substantial food and feed safety risks. The project goal is to continue work on reducing and managing major toxins in row crops by improving biological control and other methods using more effective formulations incorporating bioplastic and biochar. Bioplastic formulations for delivering biological agents will be refined to improve plant health by increasing fungal control and broadening to other plant diseases. Biochar seed coating formulations will also be developed with bioplastic that provide nutrients for plant seedlings, improve nutrient-holding capacity of soil, and prevent fungal pathogens from infecting seeds by binding substances needed for infection. Research will be expanded to Macrophomina phaseolina, a fungus which produces multiple mycotoxins (e.g., botryodiplodin, moniliformin, others) and causes significant yield losses in soybean and other row crops. Assays for routine laboratory use have been developed for M. phaseolina mycotoxins, which will be used to test soybean seeds harvested from infected plants to determine their impact on food and feed quality and safety. Knowledge of Aspergillus flavus population biology and aflatoxin risk models will be improved by exploring agroecosystems with high aflatoxin occurrence, including isolating non-aflatoxigenic A. flavus and field testing as regionally adapted biocontrol products for high stress environments. Atoxigenic fungi will also be evaluated for compatibility with seed-applied pesticides needed for improved yield with more economical deployment strategies. New formulations will be evaluated by mycotoxin analyses (ELISA, HPLC, LC-MS/MS) on plant samples with and without treatment. Mycotoxin control allows US farmers to grow non-toxic grains with improved quality, safety, and value, benefiting farmers and the public. Data obtained will enable optimization of industrial quality biological control delivery tools during the next five years, improving the efficiency and practicality of bioagent products in agriculture.
Progress Report
The project falls under National Program 303 and will be centered on reduction of harmful mycotoxins and fungal diseases in row crops such as maize, peanut and soybean. The proposed research has two research objectives and six research support objectives and is relevant to Component 2 - Biology, Ecology, and Epidemiology of Plant Pathogens; Problem Statement 2A: Advance an Understanding of Fundamental Pathogen Biology; Problem Statement 2C: Characterize Microbial Ecology and Epidemiology of Plant Diseases; Component 3– Plant Health Management; Problem Statement 3B: Advance Biologically-based and Integrated Disease Management Strategies; and Problem Statement 3C: Develop Pre-plant Approaches to Reduce Pathogen Pressure for Commercial Crop Production Systems. This is the first year of this five-year plan. In fiscal year 2022 (to date) has contributed to 3 peer-reviewed scientific publications. Research was conducted by ARS researcher in Stoneville, Mississippi, with collaborators from other institutions.
Objective 1 Progress: In Spring 2022, experiments for conventional corn seed treatment formulation of non-toxigenic Aspergillus flavus (Strains K49 and Afla-Guard®) formulated in starch-based bioplastic have continued to be conducted in the field. In Fall 2021, field samples including corn seeds and soil were collected, processed by drying, weighing, and grinding (seed only), and evaluated by counting the number of A. flavus colony forming units (CFU) and determining the percentage of toxigenic isolates using cultural methods (UV, pigmentation, and ammonia vapor). More than 1000 isolates of A. flavus were collected from soil, corn (soil and seed), and peanuts (soil and seed), and the isolates were sequenced for genetic characterization.
In Spring 2022, field studies were done in Stoneville and Leland, Mississippi, for the effects of biochar and bioplastic on mycotoxins and the health of the plant on corn and soybean. This research is conducted with other ARS researchers in Stoneville, Mississippi, and their teams. Samples were given to an ARS researcher in the Crop Genetics Researcher Unit in Stoneville, Mississippi, to analyze for seed quality and composition. We also conducted studies in Stoneville with using 80 germplasms of corn to screen them for resistance to mycotoxins (aflatoxin and fumonisin) by artificial injection with toxigenic isolates of Aspergillus and Fusarium under field condition. This study is conducted in cooperation with ARS researcher in Starkville, Mississippi.
Field studies in Dawson, Georgia, in collaboration with the ARS researchers at the National Peanut Laboratory, were conducted for managing levels of aflatoxins in peanut using seed treated in our laboratory with biocontrol strains of A. flavus (Afla-Guard®) formulated with bioplastic and biochar. Later, (156) peanut and (180) soil samples were received back from Dawson, Georgia, to analyze for mycotoxins and isolate Aspergillus isolates from soil. Samples were also given to ARS researcher in the Crop Genetics Researcher Unit in Stoneville, Mississippi, to analyze nutritional components. Lab evaluations, including host viability, germination, concentration, and quality control, of K49 and Afla-Guard spores were conducted to determine the efficacy of the spores. Another study is conducted annually with ARS researchers with corn and peanuts to look for resistant lines for aflatoxin and heat stress and sends samples (848 corn and 40 peanut) to our laboratory for chemical analysis for the presence of aflatoxins.
We also collected over 1500 isolates of Aspergillus flavus from peanut, corn and soil and evaluated them for toxigenicity. These isolates were sent to other ARS researchers for DNA sequencing evaluation. All samples received in fall 2021 to present, for other people inside and outside the ARS were analyzed for mycotoxins and some were analyzed for the presence of fungi.
A locally indigenous, genetically distinct, non-aflatoxigenic isolate was used in testing commercially acceptable, low-cost seed treatments. Planting this seed resulted in a population shift of the soil and rhizosphere communities towards a less aflatoxigenic state. Follow-up work in 2022 has included alternative seed-applied pesticides to evaluate the compatibility with other products in case neonicotinoidinsecticides are not available.
Objective 2 Progress: To monitor the Aspergillus flavus population in the corn-producing agroecosystem we have collected A. flavus isolates from a field near Stoneville, Mississippi, and Corpus Christi, Texas. We are collecting isolates from grain and soil as well as airborne spores and spores that are insect associated. The genetic composition of this population is being contextualized by comparing the SSR haplotypes with over 20,000 isolates in the AflaSat database as well as our own growing collection of A. flavus isolates. In 2021 the survey of A. flavus isolates from Mississippi Delta corn and soil continued. This collection has been supplemented with over 100 isolates from Guatemala and 300 from Texas and a smaller number from highly contaminated corn collected by the Federal Grain Inspection Service. This collection now stands at over 3,000 isolates in storage that have been georeferenced and scored for morphotype and aflatoxin production. Over 2,500 DNA extractions are complete and genetic characterization is underway.
Accomplishments
1. Development and release of improved MG IV Soybean Germplasm Line with tolerance to mature seed damage and free of mycotoxins. A new soybean germplasm (DS31-243) released in 2022 by ARS researchers in Stoneville, Mississippi, to have less mature seed damage (mold, insect damage, wrinkled green seed) was tested by the incumbent for its ability to resist toxin contamination produced by Fusarium species (spp.). Fusarium spp. are well known to produce toxins such as the group of toxins called trichothecenes, including T-2 and nivalenol, in soybean and other crops. ARS researchers showed that seed from DS31-243 produced less of these toxins than current commercial cultivars, such as Progeny 4211. DS31-243 had less seed damage, higher seed germination, and competitive yield and could save growers financial losses from the toxins and damage that develop prior to harvest. Seed was provided to Southern soybean breeders through MTAs to develop improved cultivars and will be deposited in the USDA Soybean Germplasm Collection for use by scientists around the world.
2. In culture and instrumental (liquid chromatography tandem mass spectrometry; LC-MS/MS) methods developed that measure (-)-Botryodiplodin Produced by Macrophomina phaseolina. An ARS researcher in Stoneville, Mississippi, established and lead a multi-institutional team studying the mycotoxins produced by M. phaseolina, the cause of charcoal rot in soybean and many other economically important crops. Charcoal rot is the major cause of economic losses in the Mid-South and an increasing problem world-wide due to climate change. The team developed an in-culture assay that detects production of the mycotoxin (-)-botryodiplodin by reaction with glycine in the medium to form a red pigment. This simple method does not require specialized technical expertise or equipment and can be used by laboratories with limited resources. Results with this method were confirmed by LC-MS/MS. (-)-Botryodiplodin is believed to play an important role in the root infection process of the fungus.The in-culture assay is being used by the team to study the mechanisms by which M. phaseolina in the soil reservoir finds soybean roots, enters them and spreads to adjacent plants. The results of these studies will be useful for growers, industry and scientists working in this area of research.
3. Bioplastic as a binder, a sprayable carrier and a seed coating for the delivery of agricultural chemicals and biocontrol agents. Bioplastic made from cornstarch has properties that give it many advantages over previous delivery systems for agricultural agents, including both biocontrol agents and conventional agrochemicals, such as fungicides, insecticides, and nematicides, either individually or together. Studies by a multi-institutional team established by an ARS researcher in Stoneville, Mississippi, have shown that bioplastic has a variety of valuable properties that enable it to be used as granules, sprayable liquid suspensions and seed coatings. It is sufficiently hydrophilic to absorb water to form a hydrogel, particles of which deform sufficiently to pass through a sprayer head. It is sufficiently lipophilic so that particles in sprayable formulations bind agrochemicals and biocontrol microorganisms as well as stick to the cuticle on leaf surfaces after spraying. It provides nutrition for the survival and proliferation of biocontrol agents after application to leaf surfaces. It is inert, stable and compatible with most agrochemicals and biocontrol microorganisms. Suspensions of bioplastic particles dry to form hard, durable coatings on seeds that can incorporate agrochemicals and biocontrol microorganisms in a form the resists dust-off of incorporated agents. It is completely biodegradable and environmentally friendly. This research has had major impacts in agricultural biotechnology nationally and internationally.
Review Publications
Alam, S., Abbas, H.K., Sulyok, M., Khambhati, V.H., Okunowo, W.O., Shier, T.W. 2022. Pigment produced by glycine-stimulated macrophomina phaseolina is a (-)-botryodiplodin reaction product and the basis for an in-culture assay for (-)-botryodiplodin production. Pathogens. 11(3):280. https://doi.org/10.3390/pathogens11030280.
Bellaloui, N., Mengistu, A., Smith, J.R., Abbas, H.K., Accinelli, C., Shier, T.W. 2021. Effects of charcoal rot on soybean seed composition in soybean genotypes that differ in charcoal rot resistance under irrigated and non-irrigated conditions. Plants. 10(9):1801. https://doi.org/10.3390/plants10091801.
Accinell, C., Abbas, H.K., Bruno, V., Khambhati, V.H., Little, N., Bellaloui, N., Shier, T.W. 2022. Field studies on the deterioration of microplastic films from ultra-thin 1 compostable bags in soil. Journal of Environmental Management. 305:114407. https://doi.org/10.1016/j.jenvman.2021.114407.
Weaver, M.A., Mizra, N., Boyette, C.D., Brown, S.P., Mandel, J.R. 2022. Whole genome sequence and draft assembly of the biocontrol fungal pathogen Albifimbria verrucaria CABI-IMI 368023. Microbiology Resource Announcements. 11:1. https://doi.org/10.1128/MRA.00909-21.
Weaver, M.A., Hoagland, R.E., Boyette, C.D., Brown, S.P. 2021. Taxonomic evaluation of a bioherbicidal isolate albifimbria verrucaria, formerly myrothecium verrucaria. The Journal of Fungi. 7(9):694. https://doi.org/10.3390/jof7090694.
