ECOLOGICAL AND GENETIC DIVERSITY OF SOILBORNE PATHOGENS AND INDIGENOUS MICROFLORA

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1017795
• Multistate No.: S-OLD 1083
• Project Start Date: Oct 1, 2018
• Project End Date: Sep 30, 2023
• Program Code: [(N/A)]- (N/A)

Project Director
Jimenez Gasco, MA, DE.

Recipient Organization
PENNSYLVANIA STATE UNIVERSITY
208 MUELLER LABORATORY
UNIVERSITY PARK,PA 16802

Performing Department
Plant Pathology & Environmental Microbiology

Non Technical Summary
Soilborne diseases caused by plant pathogenic organisms result in major economic losses to agricultural production. These diseases pose a challenge to agriculture since they are difficult to control, as chemical control is usually ineffective and many soilborne pathogens produce resting structures that survive in the soil for a long time, resulting in soils unsuitable for production. Additionally, the emergence of plant pathogenicity in fungal populations is an intriguing matter for which we still do not have a clear understanding, and yet this may hold important clues to designing novel and more efficient practices to manage plant diseases. Plant pathogenic fungi often have complex ecological interactions with the niche being occupied, such as soil and plants. However, these interaction have been largely ignored by plant pathologists when not accompanied by disease. This is specially the case for two fungal species, Fusarium oxysporum and Verticillium dahliae. These fungi cause vascular wilts to hundreds of crop plants, but also have a complex endophytic biology. Sustainable production systems must integrate disease management practices that incorporate disease resistance, nutrient enhancement, and biocontrol, among others, and must target a population of the pathogen and not a single individual. Pathogen populations may be very diverse, in particular regarding their virulence. Therefore, it is of key importance to understand the interactions between host genotypes, biocontrol agents and pathogen populations, within an ecological framework that includes resident and augmented microbial communities, and to incorporate this information into disease management systems.

Animal Health Component

(N/A)

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	4020	1170	100%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
4020 - Fungi;

Field Of Science
1170 - Epidemiology;

Keywords

verticillium dahliae

fusarium oxysporum

disease management

population biology

endophytes

Goals / Objectives
Evaluate the biology and diversity of soil-borne pathogens, associated antagonistic microorganisms, and environmental conditions in the context of the whole-system phytobiome. This objective includes traditional, metagenomics, and spatial/temporal methodologies to understand microbial community dynamics that determine soil-borne disease incidence and severity on economically important crops in the U.S. Evaluate the efficacy of soil-borne disease management strategies (chemical, biorational/biological, cultural) and characterize the associations among microbial community profile, soil physicochemical properties, environmental factors and disease suppression.

Project Methods
Objective 1 will focus on plant- and soil- associated microorganisms, both plant pathogenic and/or endophytic, such as Fusarium oxysporum, Verticillium dahliae, etc. Various techniques will be used to study and characterize diversity in microbial populations, such as sequencing of various conserved genes and other regions useful for species identification and phylogenetic relationships (ITS, IGS, TEF, etc.), microsatellite genotyping, single nucleotide polymorphism (SNP) genotyping or genotyping-by-sequencing (GBS), and comparative genomics of whole genome sequences (WGS) using next-generation sequencing platforms. We will also develop molecular methods to study the presence and quantity of plant pathogenic soilborne fungi in substrates such as soil and planting material (seeds, tubers, etc. These molecular techniques will be coupled with biological assays that include pathogenicity testing, assessment of plant colonization, phenotypic characterization of plant-microbe interactions, benefits to the plant conferred by the microbes, etc.Objective 2 seeks to improve our understanding of microbial interactions (ecologic, metabolic, genomic) with different hosts used in rotations and colonization of the plant by pathogenic and nonpathogenic isolates. These include growth chamber, greenhouse and field experiments. We will study host-specificity or host-adaptation of non-pathogenic, plant-associated fungi (endophytes) and the interaction between pathogen populations and asymptomatic hosts grown in rotation with susceptible and asymptomatic crops. We will evaluate the effect of management practices on the soil population dynamics of plant pathogenic fungi such as Fusarium oxysporum, Verticillium dahliae, etc., as well as the contribution of asymptomatic fungal-plant interactions to the soil inoculum bank. Molecular methods for the detection and quantification of variants of plant pathogenic fungi (such as those in different clonal lineages) will allow us to study the effect of management practices on soil inoculum.

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:This research is intended to fulfill the needs of U.S. agricultural producers for new methods targeting the management of soilborne diseases. Results of this project target the academic community in general, including general plant pathologists, diagnosticians, vegetable growers, population biologists and mycologists. Results are delivered through scientific publications, professional meetings and other gatherings, and consultation, both nationally and internationally. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided opportunities to train one Ph.D. student, one postdoctoral researcher, and provided research opportunities for one undergraduate student. How have the results been disseminated to communities of interest?Results were disseminated to academic communities through scientific publications and presentations in professional societies. We also presented this research at a meeting of multistate project S1083. What do you plan to do during the next reporting period to accomplish the goals?Goal 1:We will study populations of F. oxysporum associated to asymptomatic plants (banana and tomato) and V. dahliae in Pennsylvania potato crops using them to understand the diversity associated with these crops. Goal 2:We will establish if the different genotypes of V. dahliae in Pennsylvania potato crops have the same virulence towards potatoes and can colonize oats asyptomatically. This will be done in growth chamber experiments.

Impacts
What was accomplished under these goals? Goal 1: We evaluated the diversity and biology or Fusarium oxysporum (a very significant soilborne pathogen) associated to banana in Ecuador. Soilborne diseases result in major economic losses to agricultural production because they are difficult to control. For many of such diseases, sustainable management strategies largely rely on crop resistance which target a population of the pathogen and not a single individual. Pathogen populations can be very diverse, and this may be correlated to their virulence. It is of key importance to understand the diversity of pathogen populations and to integrate this information into disease management. The continued dispersal of Fusarium oxysporum f. sp. cubense Tropical race 4 (FocTR4), a quarantine soil-borne pathogen that kills banana plants, has placed this worldwide industry on alert and triggered enormous pressure on National Plant Protection (NPPOs) agencies to limit new incursions. Accordingly, biosecurity plays an important role while long-term control strategies are developed. We studied the population biology of F. oxysporum f. sp. cubense (Foc) in Ecuador, one of the top producers for export banana in the world. This study included phylogenetics, mating type, vegetative compatibility group analyses (VCG), and pathogenicity testing. We sampled banana plants displaying symptoms of Fusarium wilt from several provinces in the coastal area of Ecuador, including Esmeraldas, Manabí, Guayas, Los Rios, El Oro, Santo Domingo, and Bolivar; the most important banana-producing areas in Ecuador. The search for symptomatic plants followed the main routes connecting the provinces mentioned above. Sampling sites included backyards, home-gardens, roadsides, abandoned farms, cacao plots, and small banana farms. Cavendish plantations were scouted for the disease, but no Fusarium wilt was identified. A total of 298 isolates of F. oxysporum were obtained and subjected to phytogenetic analyses based on partial sequences of the translation elongation factor 1-alpha (TEF) gene. A subset of isolates were also subjected to phytogenetic analysis based on the intergenic spacer region of the Ribosomal DNA intergenic spacer (IGS), and to VCG and mating type analyses. Pathogenicity testing was done on Cavendish 'Williams,''Bluggoe' and 'Gros Michel' banana plants in growth chambers. We employed a soil inoculation method using an infested cornmeal-sand mixture. Our results revealed that Foc populations in Ecuador comprise a single clonal lineage, associated with VCG0120. The lack of diversity observed in Foc populations is consistent with a single introduction event from which secondary outbreaks originated. The predominance of VCG0120, together with previous reports of its presence in Latin American countries, suggests this group as the main cause of the devastating Fusarium wilt epidemics that occurred in the 1950s associated to the demise of 'Gros Michel' bananas in the region. The isolates sampled from Ecuador caused disease in cultivars that are susceptible to races oneand twounder greenhouse experiments, although Fusarium wilt symptoms in the field were only found in 'Gros Michel.'Isolates belonging to the same VCG0120 have historically caused disease on Cavendish cultivars in the subtropics. Overall, this study shows how Foc can be easily dispersed to other areas if restriction of contaminated materials is not well enforced. Goal 2: We studied Metarhizium robertsii, a soilborne fungus that is commonly found in agricultural soils. Fungi in the genus Metarhizium (Hypocreales: Clavicipitaceae) are entomopathogens that can establish as endophytes and benefit their host plant through growth promotion and suppression of insect pests. In previous research under field conditions, we observed a positive correlation between the prevalence of M. robertsii and legume cover crops, and a negative relationship with brassicaceous cover cropswith increasing proportion of cereal rye in mixtures. We reported the effects of endophytic M. robertsii on three cover crop species under greenhouse conditions. To understand these observations, we conducted experiments in the greenhouse where we inoculated seeds of Austrian winter pea, cereal rye, and winter canola with conidia of M. robertsii to assess the effects of endophytic colonization on cover crop growth. We recovered M. robertsii from 59%, 46%, and 39% of seed-inoculated pea, cereal rye, and canola plants, respectively. Endophytic M. robertsii significantly increased height and above-ground biomass of pea and cereal rye, but not canola; and colonization did not affect chlorophyll content of any of the cover crop species. Among inoculated plants from which we recovered M. robertsii, the above-ground biomass of pea plants was positively correlated with the proportion of colonized root but not leaf tissue sections. Our results suggest that winter cover crops may help to conserve Metarhizium spp. in annual cropping systems. We also assessed the effects of endophytic M. robertsii on growth of maize and larval black cutworm, Agrotis ipsilon, an economically-important early-season pest of maize. The specific objectives of this study were to assess the endophytic colonization of M. robertsii in V4 maize plants grown from inoculated seed, to measure the effect of established endophytic colonization of M. robertsii on V4 maize growth, including chlorophyll content, and expression of key defense genes, and to examine the effect of endophytic M. robertsii on the growth rate of secondinstar black cutworm. We inoculated maize seeds with spores of Metarhizium robertsii and evaluated V4 maize plant for endophytic colonization of leaves and roots, measured plant height, chlorophyll content and above-ground biomass, and relative growth rate of black cutworm, Agrotis ipsilon. In addition, we studied the expression of selected plant defense genes. We recovered M. robertsii from 91% of plants grown from inoculated seeds and detected M. robertsii more frequently in roots compared with leaves. Colonized plants were greater in plant height and aboveground biomass compared to control plants. Leaf tissue from colonized plants showed changes in the expression of genes involved in plant defense. Genes in the JA biosynthesis pathway lox1 and opr7 were up-regulated while mpi was down-regulated in leaf tissue from maize plants grown from M. robertsii-inoculated seed compared with control plants. We suggest that the up-regulation of the JA biosynthesis pathway in the absence of feeding by insects in M. robertsii-endophytic maize plants may be due to the accumulation of relatively higher levels of precursors of JA as a priming effect. This response to endophytic M. robertsii could improve plant protection due to modulation of plant defense mechanisms. The gene pr5 (SA response pathway) was up-regulated in leaf tissue from plants grown from M. robertsii-inoculated seeds. As the SA-dependent defense response pathway is considered effective against biotrophic pathogens and sapfeeding insects, the plant may have perceived M. robertsii as a biotrophic pathogen and responded by eliciting the SA response pathway. In feeding bioassays, the relative growth rate of black cutworm was lower on leaves from endophytic plants compared to control plants. Endophytic colonization of maize plants by M. robertsii promoted plant growth and altered defense gene expression in maize, and suppressed growth rate of black cutworm larvae.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Ahmad, I., Jimenez-Gasco, M. M., Luthe, D. S., and Barbercheck, M. E. 2020. Systemic colonization by Metarhizium robertsii enhances cover crop growth. Journal of Fungi, 6, 64: 1-16.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Ahmad, I., Jim�nez-Gasco, M. M., Luthe, D. S., Shakeel, S.N., and Barbercheck, M. E. 2020. Endophytic Metarhizium robertsii promotes maize growth, suppresses insect growth, and alters plant defense gene expression. Biological Control, 144: 104167.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Magdama, F., Monserrate-Maggi, L., Serrano, L., Garcia-Onofre, J., and Jim�nez-Gasco, M. M. 2020. Genetic diversity of Fusarium oxysporum f. sp. cubense, the Fusarium wilt pathogen of banana, in Ecuador. Plants, 9: 1133.
• Type: Journal Articles Status: Published Year Published: 2020 Citation: Crandall, S. G., Gold, K. M., Jim�nez-Gasco, M. M., Filgueiras, C. C., and Willett, D. S. A multi-omics approach to solving problems in plant disease ecology. 2020. PLoS ONE, 15(9): e0237975.
• Type: Theses/Dissertations Status: Published Year Published: 2020 Citation: Bautista-Jalon, L. S. Population biology of Verticillium dahliae associated with potato agroecosystems. Ph.D. Plant Pathology and International Agriculture and Development. The Pennsylvania State University, 2020. 242 pp.

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:This research is intended to fulfill the needs of U.S. agricultural producers for new methods targeting the management of soilborne diseases. Results of this project target the academic community in general, including general plant pathologists, diagnosticians, vegetable growers, population biologists and mycologists. Results are delivered through scientific publications, professional meetings and other gatherings, and consultation, both nationally and internationally. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided opportunities to train two Ph.D. students, one M.S. student and provided research opportunities for one undergraduate student. How have the results been disseminated to communities of interest?Results were disseminated to academic communities through scientific publications and presentations in professional societies, specifically meetings of the American Phytopathological Society and Fungal Genetics. We also presented this research at the Mid-Atlantic Fruit and Vegetable Convention, held in Hershey, PA in January 2019. This has become the premier grower education meeting in the Northeast US with over 2,000 professionals in agriculture attending each year. The research was also presented at the USDA Symposium on Phytobiomes and Population Biology held at the USDA-ARS Beltsville Agricultural Research Campus, Beltsville, MD in June 6, 2019. Over 50 USDA researchers participated in this event. What do you plan to do during the next reporting period to accomplish the goals?Objective 1. We will study populations of V. dahliae in Pennsylvania potato crops using SNP markers to understand the diversity associated with this crop. Objective 2. We will establish if the different genotypes of V. dahliae in Pennsylvania potato crops have the same virulence towards potato and can colonize oats asymptomatically. This will be done in growth chamber experiments. Publications expected in next reporting period/In Progress: Bautista-Jalon, L.S., Malcolm, G.M., Gugino, B.K., Frenkel,O., Tsror (Lahkim), L., Lebiush, S., Hazanovsky, M.L., Milgroom, M.G., and Jiménez-Gasco, M.M. Verticillium dahliae lineages infecting symptomatic and asymptomatic plant hosts in potato fields. Phytopathology Bautista-Jalon, L.S., Malcolm, G.M., Gugino, B.K., and Jiménez-Gasco, M.M. Verticillium dahliae infecting potatoes and asymptomatic rotational oats in a field affected by Verticillium wilt: pathogen in one host, endophyte in another. Plant Disease Demers, J., and Jimenez-Gasco, M.M. Phylogenetic analysis reveals lineages within Fusarium oxysporum adapted to endophytism in chickpea. Fungal Ecology

Impacts
What was accomplished under these goals? Soilborne diseases result in major economic losses to agricultural production, mainly because they are difficult to control. Crop resistance is often not available, chemical control is usually ineffective and many soilborne pathogens produce resting structures that survive in the soil for a long time. Sustainable production systems must integrate disease management practices that incorporate disease resistance, nutrient enhancement, and biocontrol, among others, and must target a population of the pathogen and not a single individual. Pathogen populations can be very diverse, and this may be correlated to their virulence. It is of key importance to understand the diversity of pathogen populations and to integrate this information into disease management. One of these soilborne diseases is Verticillium wilt caused by the soilborne fungus Verticillium dahliae. V.dahliae has a highly clonal population structure. There are numerous molecular markers that have been applied to study populations of V. dahliae, such as microsatellites. In our research we compared clusters defined by microsatellite markers with clonal lineages defined by single-nucleotide polymorphisms (SNPs) and vegetative compatibility groups (VCGs). All but one cluster corresponded to a known clonal lineage, which now allows researchers to analyze and compare correlations of phenotypes with different markers. As shown in previous research, most race 1 isolates (that carry the effector gene Ave1 and is avirulent on hosts that carry resistance gene Ve1 or its homologues) are in lineage 2A, and most isolates with the defoliating pathotype are in lineage 1A. Microsatellite genotyping of lineage 1A isolates collected from cotton and olive in Spain over a 29-year period revealed remarkably little variation (Objective 1). We also studied V. dahliae isolates recovered from cotton, pistachio and Prunus spp. in Iran using microsatellites and SNPs generated through Genotype-by-Sequencing (GBS). We found that genotypes of Prunus were different from those isolates from cotton and pistachio although they all were placed in the same lineage (2B824), concluding that these hosts are affected by different lineages of the fungus (Objective 2). We also found that V. dahliae populations from cotton and pistachio cannot be differentiated, consistent with the hypothesis that Verticillium wilt of cotton epidemics are likely the source of inoculum for the disease in pistachio. Overall, this research showed that microsatellite genotyping and SNPs can be used to identify clonal lineages in V. dahliae, which has predictive power for inferring phenotypes of phytopathological relevance such as race, pathotype and virulence level. In other words, the type of V. dahliae matters significantly, and this information is crucial for designing effective management.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Rafiei, V., Banihashemi, Z., Jim�nez-D�az, R.M., Navas-Cort�s, J.A., Landa, B.B., Jim�nez-Gasco, M.M., Turgeon, B.G., and Milgroom, M.G. 2018. Comparison of genotyping by sequencing and microsatellite markers for unravelling population structure in the clonal fungus Verticillium dahliae. Plant Pathology 67: 76-86.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Rafiei, V., Banihashemi, Z., Bautista-Jal�n*, L.S., Jim�nez-Gasco, M.M., Turgeon, B.G., and Milgroom, M.G. 2018. Genetic relationships among populations of Verticillium dahliae from cotton and pistachio in Iran using microsatellite markers and genotyping by sequencing. Phytopathology 108: 780-788.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Bell, T.H., Hockett, K.L., Alcal�-Brise�o, R.I., Barbercheck, M., Beattie, G.A., Bruns, M.A., Carlson, J.E., Chung, T., Collins, A., Emmet, B., Esker, P., Garrett, K.A., Glenna, L., Gugino, B., Jim�nez-Gasco, M.M., Kinkel, L., Kovac, J., Kowalski, K.P., Kuldau, G., Leveau, J.H., Michalska-Smith, M.J., Myrick, J,. Peter, K., Shade, A., Stopnisek, N., Tan, X., Welty, A.T., Wickings, K., and Yergeau, E. 2019. Manipulating Wild and Tamed Phytobiomes: Challenges and Opportunities. Phytobiomes Journal 3: 3-12. https://doi.org/10.1094/PBIOMES-01-19-0006-W.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2019 Citation: Aynardi, B. A., Jim�nez-Gasco, M.M. and Uddin, W. 2019. Effects of Type I and Type II isolates of Clarireedia spp. on severity of dollar spot in turfgrasses by host type. European Journal of Plant Pathology in press. https://doi.org/10.1007/s10658-019-01813-z.
• Type: Theses/Dissertations Status: Published Year Published: 2018 Citation: Tesdall, G. R. An ecological lens on the emergence of plant pathogenicity: comparative studies of pathogenic and non-pathogenic Fusarium oxysporum." M.S. Plant Pathology and International Agriculture and Development Dissertation. University Park (PA), The Pennsylvania State University, 2018. 107 p.
• Type: Theses/Dissertations Status: Published Year Published: 2019 Citation: May, S. R. Celery leaf disease: Unravelling the causal agent, population genetics, symptomology and fungicide performance for improved diagnostics and management. Ph.D. Plant Pathology. University Park (PA), The Pennsylvania State University, 2019. 125 p.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Bautista-Jalon, L. S., Gugino, B.K., and Jim�nez-Gasco, M.M. 2019. Verticillium wilt of potato caused by Verticillium dahliae: The story of a soilborne pathogen that hides as an endophyte (Abstr.). Phytopathology 109:S1.23. https://doi.org/10.1094/PHYTO-109-9-S1.23. North East Division American Phytopathological Society meeting. State College, PA. April 3-5, 2019.
• Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Miller, C., Bautista-Jalon, L. S., and Jim�nez-Gasco, M.M. 2019. Genetic diversity and movement of Verticillium dahliae lineage 4A: A lineage highly aggressive to potatoes. (Abstr.). Phytopathology 109:S1.29. https://doi.org/10.1094/PHYTO-109-9-S1.29. North East Division American Phytopathological Society meeting. State College, PA. April 3-5, 2019.