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ARS Home Northeast Area Beltsville, Maryland (BARC) Beltsville Agricultural Research Center Sustainable Agricultural Systems Laboratory Research





- Lakshman, Dilip
- Roberts, Daniel
- 2023
- Publications
- Plant Diseases (303)

Research Project: Development of Genomic Tools for Control and Characterization of Rhizoctonia solani and Other Soil-borne Plant Pathogens **Location:
                            Sustainable Agricultural Systems Laboratory** 2023 Annual Report **Objectives**
                                

Objective 1. Enhance and utilize Rhizoctonia solani pangenomic database to improve pathogen identification accuracy and characterize pathogenic mechanisms.
Objective 2. Determine the impact of cropping system and soil edaphic factors on populations of pathogenic and non-pathogenic soil-borne fungi.
Objective 3: Develop biological control agents for soil-borne pathogens, including Rhizoctonia solani, of field and nursery crops. **Approach**
                                

Planned research is directed at managing important diseases caused by soil-borne plant pathogens that negatively impact farmers' livelihoods in the Mid-Atlantic region of the United States and elsewhere. A major emphasis of this research is developing sustainable disease management tools for pathogens from the Rhizoctonia solani species complex. 

For Objective 1 the Rhizoctonia solani pangenomic database (RsolaniDB), developed during the prior project, will be enhanced using newly generated near-chromosome-level genome assemblies.  This enhanced RsolaniDB will be used to develop molecular probes for the detection and differentiation of R. solani isolates in farm fields. Specific identification technologies are required to ensure the use of appropriate controls, as isolates from this species complex can be morphologically similar but vary widely in sensitivity to chemical or biocontrol measures. The enhanced RsolaniDB will also be used to develop knowledge concerning mechanisms of pathogenicity of poorly studied isolates from the R. solani species complex.  For this patho-genomic analysis of select genomes in RsolaniDB will be conducted.  Also, RNAseq will be used to carry out temporal, differential gene expression analysis of interactions between a R. solani isolate and known host and non-host crops. Differential expression of R. solani genes during interactions with the host and non-host crops will provide insights into genes involved in pathogenicity and host range of this pathogen.

For Objective 2 we will analyze the impact of corn, wheat, and soybean cropping systems used in the mid-Atlantic region on the ecology of R. solani.  Modern microbiome molecular approaches will be used on DNA extracted from soil samples from fields used for these cropping systems.  Amplicon sequencing of ribosomal markers (ITS for fungi and 16S V4 for bacteria) will provide a description of fungi, including species from the R. solani species complex, and bacteria in the different cropping system fields.  Discoveries will result in best management cropping practices to minimize the inoculum of R. solani and disease in farm fields.

For Objective 3, environmentally friendly biocontrol options for R. solani isolates and the critical soil-borne plant pathogens Pythium ultimum and Sclerotinia sclerotiorum will be developed.  Omics approaches will be used to determine mechanisms of biocontrol.  

Using this multipronged approach of specific identification, development of environmentally sound biocontrol options, and best management practices to minimize inoculum and disease, this project will improve control of important soil-borne diseases and improve sustainability of farming systems in the Mid-Atlantic region and elsewhere. **Progress Report**
                                

This project is in its first year, being approved in September of 2022, three months after the start of the project cycle. All but one of the twelve-month milestones were met or substantially met 9 months after initiation of the project. There were thirteen manuscripts submitted for publication during this period, five of which have been published or accepted for publication. An additional two manuscripts submitted during the FY2022 Annual Report cycle were published. These manuscripts dealt with use of molecular techniques in identification of fungal plant pathogens, the first report of diseases, effectiveness of fungicides for control of sugar beet pathogens, analysis of the resistance response of sugar beet to R. solani AG 2-2IIIB, the impact of cropping system on the corn metabolome, and scalable knowledge management for speeding development and transfer of agricultural technology to the agricultural community. 

Goal 1.1 of Objective 1 of the project concerns enhancing the Rhizoctonia solani pangenomic database (RsolaniDB) that was developed by ARS scientists in Beltsville, Maryland, in collaboration with scientists at King Abdullah University of Science and Technology (KAUST). Improvement of RsolaniDB through long-read sequencing of the R. solani species complex genomes is necessary to increase the utility of RsolaniDB regarding developing technologies for detection and identification of morphologically similar R. solani pathogens and for characterization of R. solani pathogenic genes and mechanisms. For Goal 1.1, thirteen long-read-sequence-quality genomes of R. solani isolates covering seven Anastomosis Groups (AGs) and selected subgroups were purified in preparation for shipment to CD Genomics for long-read sequencing. An agreement is in place with CD genomics to perform the long-read sequencing. Goal 1.2 of Objective 1 concerns using RsolaniDB to identify and differentiate R. solani AG4 and AG2-2IIIB isolates. This is necessary since AGs of R. solani differ regarding sensitivity to fungicides, biocontrol agents, and plant immune responses. To distinguish the genomes of AG4 and AG2-2IIB, differential primer regions were selected using the ShuString protocol (http://adenine.biz.fh-weihenstephan.de/shustring/). Unfortunately, progress towards Goal 1.2 stalled due to Covid restrictions and the student collaborator from North Dakota State University (NDSU), entrusted with this work, leaving the graduate program. Going forward primers will be designed from the differential genomic regions and PCR tested on genomes of AG4 and AG2-2IIIB isolates infecting sugar beet, for selectivity. There are no milestones for Goal 1.3 of Objective 1 until thirty-six months after project initiation.

Objective 2 is designed to determine the impact of cropping system and resulting differences in soil edaphic factors on populations of pathogenic and non-pathogenic soil-borne fungi, including isolates from the R. solani species complex. For this, the Farming Systems Project (FSP) located at the ARS facility in Beltsville, Maryland, is to be used. FSP has been in place since 1997 and compares five corn-wheat-soybean cropping systems (two conventional, three organic) regarding various agronomic and economic factors. FSP is an excellent platform to compare impacts of cropping system on populations of pathogenic, beneficial, and other soil-borne microbes. Sampling of the FSP field site was delayed one year due to the late start of the project. All milestones for the project should still be able to be met as two years at the end of the project were budgeted for data analysis and manuscript preparation. Data analysis and manuscript preparation will now be compressed into the final year of the project. 

Objective 3 is designed to develop biological control agents for the soil-borne pathogens R. solani, Pythium ultimum, and Sclerotinia sclerotiorum. For Objective 3, Goal 3.1, in vitro antagonistic potential of 13 myxobacterial strains was evaluated against four soil-borne plant pathogens, two isolates each from R. solani AG2-2IIIB and R. solani AG4, and one isolate each from S. sclerotiorum and P. ultimum. Of the 13 myxobacterial strains tested in vitro, eight strains (ATCC  25194, ATCC 29617, ATCC 51243, ATCC 29616, ATCC 53080, DK 801, BS 247, BS 249) showed in vitro antagonism against all four pathogens and relative levels of antagonism were noted. Survival of one myxobacterial isolate (BS 249) in greenhouse potting mix and garden soil is being investigated to find a suitable planting medium for biocontrol assays against R. solani, P. ultimum, and S. sclerotiorum isolates. Genome-sequence-quality DNA was extracted from 11 selected myxobacterial isolates and genome sequencing and contig generation for three isolates (DK 836, DK 897, BS 247) completed by CD Genomics. In collaboration with scientists from NDSU two inoculation methods (i.e., crown vs. root inoculations) were compared to determine a suitable inoculation technique to screen sugar beet germplasm for resistance against damping-off caused by R. solani AG2-2IIIB.  For Objective 3, Goal 3.2, a library of mutants of Serratia marcescens N4-5 was completed. This library will be screened for mutants that no longer produce compounds necessary for suppression of damping-off caused by P. ultimum. In this way compounds from this strain responsible for suppression of disease will be identified. In the future, manipulation of genes responsible for synthesis of these compounds will be used to improve disease control. For Objective 3, Goal 3.3 data resulting from two years of field trials testing biocontrol isolate combinations for control was partially organized for analysis. Analysis of this data will be able to be completed within 24 months to meet the 24-month milestone.

First reports of diseases were contributed for Fusarium solani seedling damping-off of hemp (Cannabis sativa) in North Dakota and Stemphylium vesicarium leaf spot on sugar beet (Beta vulgaris) in Minnesota. In collaboration with scientists from NDSU, the identification and characterization of several more pathogens on sugar beet in the Red River Valley region of North Dakota, Minnesota, and Wyoming are in progress. In collaboration with scientists from NDSU, we determined that virulence of Cercospora baticola isolates on sugar beet is not altered due to resistance development against QoI and DMI fungicides in the north central region of the United States. In collaboration with scientists from Rani Lakshmi Bai Central Agricultural University, Jhansi, India, we assessed the virulence, morphologically characterized, and molecularly identified soil-borne Fusarium species causing post-flowering stalk rot of maize. In another investigation with collaborators from the Indian Agricultural Research Institute, New Delhi, India, we determined the best time for whitefly (Bemisia tabaci) vector interception with crop cover to minimize losses due to chili leaf curl virus (ChiLCV) on chilli (Capsicum annuum). **Accomplishments**
                                

1. Root inoculation with Rhizoctonia solani is optimal for screening sugar beet resistance. Sugar beet crown rot and root rot caused by R. solani are serious threats to sugar beet production and processing. Prior to the adoption of Roundup Ready® sugar beet (RRSB) cultivars, crown rot was a serious problem resulting from mechanical tillage operations required for weed control. Following the introduction and large-scale cultivation of RRSB, however, crown rot was reduced but root rot became severe. This necessitated reassessment of screening methods for the development of Rhizoctonia-resistant cultivars. In a collaborative study between USDA-ARS scientists from Beltsville, Maryland, and scientists from North Dakota State University, crown inoculation and root inoculation methods were evaluated for the development of Rhizoctonia root rot and assessed regarding efficacy of differentiating the disease reaction of sugar beet cultivars. Results demonstrated that the root inoculation method is optimal for consistent disease rating of sugar beet germplasm in the greenhouse. It was concluded that use of the root inoculation method is convenient and of acceptable accuracy for screening RRSB cultivars in a resistance breeding program. 2. Resistance to QoI and DMI fungicides does not alter virulence of Cercospora beticola. Cercospora leaf spot (CLS) is a destructive disease limiting sugar beet production and is managed using resistant cultivars, crop rotation, and timely applications of effective fungicides. Since 2016, its causal agent, C. beticola, (Cb) has been reported to be resistant to Quinone outside inhibitors (QoIs) and to have reduced sensitivity to Demethylation inhibitors (DMIs) in sugar beet growing areas in North Dakota and Minnesota. USDA scientists from Beltsville, Maryland, in collaboration with scientists from North Dakota State University and other USDA scientists (Fargo, North Dakota), showed that resistant Cb isolates had significantly less mycelial growth and spore production than sensitive isolates, while no significant difference in spore germination was detected. Also, resistant isolates had significantly smaller “Area Under Disease Progress Curve” (AUDPC), but still caused high disease severity as the sensitive ones. This information needs to be factored into CLS management practices. **Review Publications**
                            *Short, N.M.,Woodward-Greene, M.J., Buser, M.D., Roberts, D.P. 2023. Scalable knowledge management to meet global 21st century challenges in agriculture. Land. 12:588.*
                                *Mattoo, A.K., Cavigelli, M.A., Misic, D., Gasic, U., Maksimovic, V., Kramer, M., Kaur, B., Matekalo, D., Nestorovic Zivkovic, J., Roberts, D.P. 2023. Maize metabolomics in relation to cropping system and growing year impacts. Frontiers in Sustainable Food Systems. https://doi.org/10.3389/fsufs.2023.113008910.3389/fsufs.2023.1130089.*
                                *Khan, M.F., Bhuiyan, Z.M., Lakshman, D.K., Mosher, P., Knoke, S. 2022. First report of damping off and seedling rot of hemp (Cannabis sativa L.) caused by Fusarium solani (Mart.) Sacc. in North Dakota, USA. Plant Disease. https://doi.org/10.1094/PDIS-12-21-2681-PDN.*
                                *Bhuiyan, Z.M., Lakshman, D.K., Del Rio Mendoza, L.E., Qi, A., Khan, M.F. 2022. Comparison of crown and root inoculation methods for evaluating resistance response of sugar beet cultivars to R. solani AG 2-2IIIB. Crop Protection. https://doi.org/10.1016/j.cropro.2022.106120.*
                                *Harish, J., Jambhulkar, P., Bajpai, R., Meenakshi, A., Babele, P., Chaturvedi, S., Kumar, A., Lakshman, D.K. 2023. Morphological characterization, pathogenicity screening, and molecular identification of Fusarium spp. isolates causing post flowering stalk rot in maize. Frontiers in Microbiology. https://doi.org/10.3389/fmicb.2023.1121781.*
                                *Roy, B., Venu, E., Sathlyaseelan, K., Dubey, S., Lakshman, D.K., Mandal, B., Sinha, P. 2023. Leaf curl epidemic risk in chilli as a consequence of vector migration rate and contact rate dynamics: A critical guide to management. Viruses. https://doi.org/10.3390/v15040854.*
                                *Khan, M., Bhuiyan, K., Lakshman, D.K., Luis, D., Azizi, A., Ameen, G., Sarwar, A. 2023. First report of leaf spot of sugar beet caused by Stemphylium vesicarium (Wallr.) E.G. Simmons in Minnesota, USA. Plant Disease. https://doi.org/10.1094/PDIS-02-23-0256-PDN.*


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