Annual/Termination Reports:

[01/15/2013] [01/23/2014] [12/28/2014] [03/29/2016] [02/05/2017] [08/10/2017]

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Brief Summary of Minutes

Brief Summary of Minutes of Annual Meeting

Harald Scherm  Administrative Advisor oversaw the annual meeting and provided guidance and information on the renewal process. To date 20 members are signed up for the new S1053. As S1028 has come to a close a termination report will need to be prepared. When discussing future directions for the research group, emphasis on collaborative proposal development was suggested as an area the group could work together to improve on from the last project period.

Those in attendance included:
Sheng Yu - Mississippi State University
Kurt Lamour  University of Tennessee
Kirk Broders  University of New Hampshire
Craig Rothrock  University of Arkansas
Carla Garzon  Oklahoma State University
Craig Canaday  University of Tennessee WTREC
Louisa Santamaria  Oregon State University

After introduction, each member in attendance provided a presentation and update of current ongoing research and results from the past year associated with the project. At the conclusion of the presentation, the group came to a consensus to hold next years meeting of the S1053 multi-state group in Oklahoma City, OK around the first week of November. Kurt Lamour was nominated for Secretary and a unanimous vote confirmed his new post. As secretary, Kurt is also the Chair-elect. Kirk Broders will be the incoming Chair, and Craig Rothrock is the outgoing chair.

The final topic of discussion was how we can better integrate projects by individual investigators to tackle larger problems that require a multi-state effort to accomplish. Some of the idea but forth included:
- Chloride and relationship to Pythium
- P. capsici distribution and management
- Pythium taxonomy and describing non-spore forming species
- Disease complexes symposium/workshop
- Rhizoctonia population resources
- International Potato Center for Rhizoctonia resources

Meeting adjourned.

Accomplishments

Objective 1. Evaluate the population genetic diversity of soilborne pathogens and antagonistic microorganisms in different growing systems and regions using traditional and metagenomic approaches.<br /> <br /> In Mississippi, 900 bacterial isolates were obtained from soils and plant samples and tested for antimicrobial activities and discovery of novel antagonistic bacteria. Approximately 60 isolates showed significant antifungal activities in bioassays against Geotrichum candidum and more than 20 isolates exhibited significant antibiotic activities against Erwinia amylovora. Isolates have been identified to be Bacillus spp., Pseudomonas spp., Burkholderia spp. and Streptomyces spp. using 16S rDNA sequence analysis. Investigations examined the stability and efficacy of antifungal occidiofungin produced by Burkholderia contaminans strain M14. In addition, characterization of genes associated with antimicrobial activities of bacteria Pseudomonas kilonensis, P. chlororaphis and P. vancouverensis were conducted using Tn5 random mutagenesis and cloning genes associated with antimicrobial activities. Genome drafts of three bacterial strains with antimicrobial activities have been generated <br /> <br /> A study of bacterial communities present in Andean soils from Ecuador, Chimborazo Province, suppressive to Phytophthora infestans was conducted in cooperation with Carla Garzon using T-RFLP to assess the microbial diversity present in these soils. Bacteria corresponding to at least 13 phyla were identified. Most bacteria belonged to Actinobacteria, Proteobacteria and Firmicutes. Comparisons of suppression of Rhizoctonia solani and Phytophthora infestans between heat treated and non-treated soils suggest that bacteria of the genera Bacillus and Geobacillus are responsible of most of the suppressive nature of these soils. <br /> <br /> Rhizoctonia populations and diversity are being evaluated by a number of scientists as a part of this project. Enumeration of Rhizoctonia species has been evaluated by the toothpick-baiting technique and selective media across diverse crops and geographic locations. In Arkansas, the diversity of Rhizoctonia spp. in rice soybean cropping systems included R. solani AGs 11, 1-1A, 4 and 2-1, Rhizoctonia oryzae and bi-nucleate Rhizoctonia species. Kirk Borders is examining the diversity of Rhizoctonia solani isolates associated with wheat, canola, soybean, and dry bean by phylogenetic analysis. <br /> <br /> In Arkansas, the spatial distribution of Rhizoctonia spp. in fields undergoing rice and soybean rotations is being characterized. Rhizoctonia aerial blight of soybean is a disease caused by Rhizoctonia solani AG1-IA. This pathogen also causes sheath blight of rice. Populations and disease assessments were characterized in producers field on a spatial scale to represent the topography of the field intermittent of the rice levee positions. Directional distribution ellipses for distribution of R. solani AG1-IA using soil and plant samples indicated agreement with drainage. Across years, distribution of R. solani AG1-IA appears to be controlled by levee position. Where levees do not form logical areas of collection, the greatest concentration of inoculum appears to be in the lower elevations of the field. The spatial distribution of the early-season inoculum of R. solani AG1-IA reiterates the idea that inoculum in the form of sclerotia and hyphae associated with rice residue may be floating and collecting at lower points within the levees. Interestingly, Rhizoctonia solani AG11 has been recovered from both plant and soil samples with greater frequency and does not follow a similar distribution pattern to R. solani AG1-IA. Spatial data for R. solani AG11 indicated isolation from soybean plants correlated spatially to the driest areas of the fields and improved soybean stands. <br /> <br /> Objective 2. Examine the effect of traditional or newly developed management strategies (chemical, cultural, and biological), soil physicochemical properties, or introduced biological control agents on the microbial community and its ability to suppress soilborne pathogens.<br /> <br /> Microbial communities are being characterized from diverse cropping systems as part of this project. In Mississippi, the microbial community present on sweetpotato roots is being characterized to identify the source of the tip/end rot complex using fields planted with Beauregard B-14 cultivar and sampling plant tissues at eight points throughout the growing season and during storage. Symptoms of tip/end rot generally appear in storage roots of sweetpotato after harvest, but have been reported at earlier stages of the growing season. Bacterial cultures were initially subjected to fatty acid methyl ester (FAME) analysis for preliminary identification, followed by molecular analysis using 16S rDNA. Fungal cultures were grouped by general morphology and sequencing of the ITS region for representative isolates or the translation elongation factor 1-alpha (ef1±) DNA region for Fusarium species. Communities appear to shift in relative abundance between the growing cycle and harvest. Bacillus spp., L. enzymogenes, and P. lentimorbus accounted for more than 50% of bacteria identified. No pathogenic bacteria were found, but a number of the species have representatives with known biocontrol activity. The majority of fungi were Macrophomina phaseolina, Aspergillus spp., Trichoderma spp., and a large number of Fusarium spp. In early season samples from seed stock and bedding plants, the community is primarily composed of Fusarium spp., nearly 70%. Post-harvest samples show differences in relative abundance of the dominant species, with M. phaseolina increasing to an average of 6.5% in samples taken from storage, and Fusarium spp. decreasing to an average of 27% between 60 and 90 days post-harvest. To date, isolates found to be pathogenic on sweetpotato are Fusarium species and Macrophomina phaseolina.<br /> <br /> The effect of perennial ryegrass cultivar mixtures on soil microbial communities is being examined in the Northeast US. A multi-location field trial was established in Maine, New Hampshire, New York, and Vermont to examine the effect of perennial ryegrass cultivar mixtures on soil and foliar microbial communities.<br /> <br /> Chloride salts have been demonstrated to increase the incidence of Phytophthora root and stem rot of soybean by changes in the micro-partitioning of calcium in the soybean roots. In Tennessee, the value of supplementary calcium added to a common soybean seed treatment, mefenoxam + fludioxonil + thiamethoxam, on seed germination and seedling growth was examined. Treated seed were planted in solarized field soil and later evaluated for their effect on seedling root rot in greenhouse and field in soils infested with Rhizoctonia solani, Macrophomina phaseolina, Pythium spp. and Fusarium spp. The effects of treatments on the concentration of calcium in the outer cell layers of soybean roots was determined with an Environmental Scanning Electron Microscope (ESEM) using energy-dispersive X-ray analysis (EDX). Best seedling growth was observed when the mefenoxam + fludioxonil + thiamethoxam seed treatment was supplemented with 4% calcium formate, 1% calcium nitrate, 4% calcium lactate, and 2% calcium salicylate in the absence of pathogens. Stands of healthy plants in the field were initially highest when the mefenoxam + fludioxonil + thiamethoxam seed treatment was supplemented with calcium lactate in unfertilized plots but were later highest with the calcium salicylate supplement in plots receiving muriate of potash. While the seed treatments had no effect on the level of many nutrients in seedling roots, the levels of calcium, magnesium, potassium, and phosphorus were significantly increased with the mefenoxam + fludioxonil + thiamethoxam seed treatment compared to the untreated control. Supplementing the mefenoxam + fludioxonil + thiamethoxam seed treatment with calcium nitrate or calcium salicylate significantly increased the level of potassium in seedling roots compared to the control.<br /> <br /> Research on the use of soil amendments and cover crops on disease suppression is continuing as part of the project. In New Hampshire, cover crops for suppression of Verticillium dahliae on strawberry and mint are being evaluated initially by screening strawberry accessions and a variety of cover crops for their ability to resist infection and suppress inoculum levels of the soilborne pathogen V. dahliae. <br /> <br /> The impact of fungicides and heavy metals were also being investigated by project scientists. Fungicide hormesis was assessed in vitro on Pythium irregulare, Sclerotinia homoeocarpa, and Botrytis cinerea, validating the stimulation of pathogen activity by low fungicide levels.<br /> <br /> Heavy metals impact soil biological activity primarily by binding with enzymes and changing the configuration of the enzyme preventing enzymes catalyzing the conversion of substrate to product. Research in Iowa is examining the effects of six heavy metals (Cd, Co, Cr, Cu, Ni, and Pb) of the legumes Vicia faba, Trifolium alexandrium, and Glycine max. Results showed that plant weight, nodulation, and N uptake decreased significantly with increasing heavy metal concentrations, from 0 to as high as 4.3 mmol per kg soil. Plots of the natural log of each of the three parameters against metal concentration were linear. From the slopes of these lines, the concentrations of each metal required to produce 50% reduction in the parameter were calculated. Results showed that values varied among the soils and legumes studied but, in general, the lowest metal concentrations for 50% reduction (i.e., the most toxic) heavy metals were Cd and Pb.

Publications

Peer-reviewed<br /> <br /> Abd-Elmagid, A., Garrido, P.A., Hunger, R., Lyles, J.L., Mansfield, M.A., Gugino, B.K., Smith, D.L., Melouk, H., and Garzon, C.D. Discriminatory simplex and multiplex PCR for four species of the genus Sclerotinia. Journal of Microbiological Methods. Accepted<br /> <br /> Brewer, C. E., Hu, Y., Schmidt-Rohr, K., Loynachan, T. E., Laird, D. A., and Brown, R. C. 2012. Extent of pyrolysis impacts on fast pyrolysis biochar properties. J. Environ. Qual. 41:1115-1122.<br /> <br /> Ellis, D., Gosai, J., Emrick, C., Heintz, R., Romans, L., Gordon, D., Austin, F., Lu, S.-E., and Smith, L. 2012. Occidiofungin's chemical stability and in vitro potency against Candida species. Antimicrobial Agents and Chemotherapy 56:765-769. <br /> <br /> Flores, F.J., and Garzon, C.D. 2012. Detection and assessment of chemical hormesis on the radial growth in vitro of oomycetes and fungal plant pathogens. Dose-Response. Accepted, early view on-line<br /> <br /> Garzon, C.D., Molineros, J.E., Yanez, J.M., Flores, F.J., Jimenez-Gasco, M.M., and Moorman G.W. 2011.Sublethal doses of mefenoxam enhance Pythium damping-off of geraniums. Plant Disease 95:1233-1238.<br /> <br /> Garzon, C.D., and Flores, F.J. 2012. Hormesis: biphasic dose-responses to fungicides in plant pathogens and their potential threat to agriculture. In: Nita M. 2012. <br /> <br /> Tan, W., J. Cooley, F. Austin, S.-E. Lu, L. Smith and S. Pruett. 2012. Nonclinical toxicological evaluation of occidiofungin, a unique glyco-lipopeptide antifungal. International Journal of Toxicology 31:326-36.<br /> <br /> Abstracts<br /> <br /> Haddad, S. A., M. A. Tabatabai, A. A. Abdel-Moneim, and T. E. Loynachan. 2011. Effects of concentration of selected heavy metals on nodulation and nitrogen nutrition of leguminous crops. 363-8 Agron. Abstracts, Madison.<br /> <br /> Spurlock, T., Rothrock, C., and Monfort, W. 2012. Spatial assessment of Rhizoctonia solani in fields undergoing rice and soybean rotations. (Abstr.) Phytopathology 102:S4.113.<br /> <br /> Spurlock, T. N., Rothrock, C. S., and Monfort, S. 2012. Comparison of methods for isolation and quantification of Rhizoctonia spp. from field soil. (Abstr.) Phytopathology 102:S2.9.<br /> <br /> Other publications<br /> <br /> Loynachan, T. E. 2012. Life in the soil: Who cares? pp. 28-30. In Deborah McDonough (ed.) Getting Into Soil and Water. Iowa Water Center, Ames, IA.

Impact Statements

1. New bacterial strains that inhibit Phytophthora infestans and Rhizoctonia solani and other pathogens were isolated and being characterized as biological control agents.
2. Use of perennial ryegrass cultivar mixtures should provide growers with the knowledge they need to extend the grazing season by providing cultivar mixtures for each of the cold hardiness zones as well as reduce losses to plant pathogens.
3. Research on Rhizoctonia solani diversity has characterized a greater diversity in many crops that previously known. Three distinct anastomosis groups are capable of infecting wheat. In addition, AG2-1 of R. solani was found to infect both canola and wheat and therefore lead to the recommendation that rotation with wheat and canola will likely lead to reduced yield due to infection by this fungus, which may limit the benefit for rotation with wheat to reduce the incidence and severity of Sclerotinia stem rot and Blackleg of canola. In rice soybean rotations the majority of the Rhizoctonia populations in soil and colonizing soybean do not cause a recognized problem suggesting members of the group may also play beneficial roles. Better characterization of Rhizoctonia populations will have an impact on growers, and industry and academic researchers as they move forward in developing chemical, biological, and cultural control strategies for these distinct pathogens.
4. Characterizing the spatial distribution of Rhizoctonia populations in soybean and rice rotations demonstrated R. solani AG1-IA was significantly directionally dispersed in agreement with drainage, suggesting that inoculum in the form of sclerotia and hyphae associated with rice residue may be floating and collecting at lower points within the levees. This research suggests the potential for precision management of Rhizoctonia aerial blight of soybean through targeted within field scouting and fungicide sprays.
5. Human activities may contribute to the contamination of soils by heavy metals. Studies suggests that heavy metals in soil can have negative consequences on the nitrogen cycle: limiting plant growth, nodulation by beneficial bacteria, and uptake of the essential plant nutrient N. This research should allow more informed decisions on the impact of heavy metals in the soil environment.

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Brief Summary of Minutes

Accomplishments

1. Evaluate the population genetic diversity of soilborne pathogens and antagonistic microorganisms in different growing systems and regions using traditional and metagenomic approaches.<br /> <br /> Oomycetes including Pythium and Phytophthora continue to cause significant damage on a number of economically important vegetable, fruit, ornamental, and row crops in North America and around the world. Members of this multistate research project have continued to provide new insight into the genetic diversity of a number of oomycete pathogens in a variety of production systems. New genetic resources and many new molecular markers were developed for the soilborne vegetable pathogen Phytophthora capsici. The genetic resources include genomic DNA re-sequencing data for 18 P. capsici isolates recovered from locations worldwide and RNA sequencing data for 35 isolates of P. capsici at two time-points in the infection process of tomato (24 and 72 hours). These resources provided the raw material to identify widely distributed and conserved polymorphic single nucleotide polymorphism (SNP) positions in the genome and to develop genotyping assays to assess genetic diversity in populations worldwide. Ongoing analyses of oomycetes baited from Tennessee streams and rivers (predominantly Phytophthora and Pythium) have identified species known to be pathogens of economically important nursery and forest plants.<br /> <br /> Further research on oomycete pathogens have focused on assessing of genetic diversity of Phytopthora infestans inoculum in Andean soils. This work was presented in two B.S. Biotechnology Engineering dissertations written by two undergraduate students from the Universidad de las Fuerzas Armadas ESPE who graduated in Summer 2013 in collaboration with researchers at Oklahoma State University. In related work by researchers at OSU, the multi-locus phylogenetic analysis of the P. irregulare complex was conducted confirming the species status of P. cryptoirregulare and P. vipa. Additionally, the characterization of Pythium isolates collected from ornamental greenhouses in Long Island, NY in 2012 and 2013 by molecular barcoding (ITS) was completed. Current research is focused on the temporal dynamics analysis of these isolates, to understand seasonal changes in Pythium populations in ornamental operations.<br /> <br /> In related work taking place at Oregon State University a new project funded by the Floriculture and Nursery Research Initiative (FNRI) to be developed in a collaborative agreement between NWREC-OSU and USDA-ARS Horticultural Crops Research Laboratory in Corvallis, will identify and characterize the most prevalent soilborne plant pathogens, including Phytophthora, Pythium, and Verticillium affecting commercial nursery production systems; develop novel and rapid assays to detect the most prevalent soilborne pathogens; and characterization of the roles of soil fungal communities in nutrient cycling and soil health.<br /> <br /> Work in Minnesota has also focused on understanding the diversity of Pythium species present in crop soils. Pythium species were surveyed using two different methods for collection of isolates. The two methods differed considerably in their efficiency. In one survey Pythium species were obtained by baiting soil samples collected from 32 locations in Minnesota where stand establishment had been a problem. Only five species were identified in this survey. Pythium irregulare, Pythium recalcitrans, and Pythium ultimum were the predominant species. A related survey for Pythium species was conducted by isolating from symptomatic plants collected in the field. In this survey 30 Pythium species were isolated. The most common species found were Pythium ultimum, Pythium heterothallicum, Pythium sylvaticum, and Pythium oopapillum. In both surveys Pythium species were identified and identity confirmed using morphological and molecular techniques. The isolates collected are being screened for pathogenicity on both corn and soybean.<br /> <br /> Other research activities involved Verticillium dahliae, a fungal species that causes vascular wilt diseases on over 400 host plants. Monocotyledonous crops have been traditionally considered to be non-hosts of V. dahliae, and cereal species have been used in crop rotations as a cultural measure for the management of Verticillium wilts by reducing the amount and/or efficiency of pathogen inoculum, but have had limited or inconsistent success. However, we found that the fungus can establish endophytic relationships with oat plants grown as rotational crops. A population genetics approach using microsatellite markers revealed that the populations from symptomatic and asymptomatic hosts were significantly different. While at least five distinct genotypes were differentiated in the V. dahliae populations obtained from potato, only one of the genotypes was consistently found associated with oats, in different sampling years and grown in different fields. Populations of V. dahliae isolated from two potato cultivars that vary in their response to Verticillium wilt (cvs. Reba and Snowden) were also different, and diversity seemed to be positively correlated with susceptibility to the disease; that is, more diversity in the highly susceptible cultivar, less in the moderately resistant. <br /> <br /> In a related project being conducted in New Hampshire a number of cover crops species were screened to determine whether they harbor asymptomatic infections by the soilborne pathogen V. dahliae. To date we have found that many cover crop species including buckwheat, crimson clover, and winter rye are capable of harboring the fungus, but display no visual symptoms of infection. Only through the use of a strain of V. dahliae that expresses the green fluorescent protein (GFP) was the extensive colonization able to be visualized. Screening of both the commercial strawberry and one of its wild ancestors, Fragaria vesca, for resistance to V. dahliae with the GFP strain of V. dahliae is currently in progress. One of the major obstacles in screening for resistance to soilborne root and wilt pathogens is that visual assessment does not provide an accurate assessment of fungal colonization or host resistance. Therefore two new methods are being evaluated for their ability to dissect resistance, tolerance, and susceptibility. Confocal microscopy using the GFP strain of V. dahliae is being used to measure colonization of susceptible, tolerant (visually healthy but ingress of fungal hyphae present), and resistant (visually healthy no ingress of fungal hyphae) strawberry cultivar. In addition, multispectral and thermal imaging systems are being evaluated for their ability to detect pre-visual stress as a potential phenotyping method. This process was initiated in the past year and protocols for inoculum production, inoculation, and visualization by confocal microscopy have all been standardized and screening of strawberry cultivars with known levels of resistance and susceptibility is in progress. Finally, the sequencing of two strains of V. dahliae (1 that infects strawberry and 1 that infects mint) was initiated. Sequencing was completed at the University of New Hampshire on an Illumina HiSeq 2500. The first assemblies have been completed and we have just started the first comparative analyses against the reference V. dahliae strain, which was recovered from lettuce.<br /> <br /> Research on sweetpotato tip/end rot is focused on defining and evaluating the pathogen(s) associated with this disease problem that was severe and widespread in 2009, but can still be found at high levels following harvesting. An initial study (Phase 1) was established in 2009 to determine the causal agents associated with tip/end rot and completed in 2013. Due to the thousands of isolates collected from traditional culturing, the identifications are now completed. During 2012 it was determined that reniform nematode, Rotylenchulus reniformis, levels were increasing in many fields from a general survey and was suspected that they might form a disease complex resulting in greater tip/end rot in Mississippi. Research was established in 2013 to evaluate those interactions with the most common pathogen isolated, Macrophomina phaseolina and reniform nematode using different populations in the field and in the greenhouse (Phase 2). <br /> <br /> Lastly, several projects have continued to evaluate the genetic diversity of R. solani from a number of important crop species across North America. Researchers at the University of New Hampshire in collaboration with the University of Guelph conducted a phylogenetic comparison of isolates of R. solani collected from canola and wheat growing region of western Canada. A total of 128 multinucleate isolates were identified by ITS sequence and compared to anastomosis group (AG) results. The multinucleate isolates of R. solani were grouped into 8 distinct clades. Two distinct clades were observed for isolates classified as AG2-1 by anastomosis testing, and isolates in a closely related clade to AG2-1 did not successfully fuse with any of the tester strains. While most isolates of AG-5 clustered together according to ITS sequences, three isolates classified by anastomosis grouping as AG-5 grouped with AG2-1, AG-4, and binucleate Rhizoctonia in the phylogenetic analysis. There was no genetic diversity among isolates classified as AG-4. While in most instances the results from AG tests were consistent with ITS sequence, there were several cases where isolates were inconsistently classified or failed to undergo anastomosis with any of the tester strains. In addition, a previously undocumented AG group was identified, and the AG-5 tester strain was found to form hyphal fusions with unrelated groups of multinucleate and binucleate species of Rhizoctonia. Results from this project were submitted for publication in Plant Disease. Current work has begun to focus on the genetic diversity of R. solani infecting soybean and drybean in northern regions with a specific focus on AG2-2. Early results indicate that isolates of R. solani AG2-2 may have as many as six distinct ITS sequences in a single strain. These finding raise a number of questions regarding the frequency at which members of AG2-2 anastomose in the wild and how genetically distinct they need to be before they can no longer anastomose. <br /> <br /> Related research on Rhizoctonia spp. conducted in the southern U.S. has characterized the spatial distribution of Rhizoctonia spp. in fields undergoing rice and soybean rotation in Arkansas. Rhizoctonia aerial blight of soybean is a disease caused by Rhizoctonia solani AG1-IA. This pathogen also causes sheath blight of rice. Populations and disease assessments were characterized in producers' fields on a spatial scale to represent the topography of the field intermittent of the rice levee positions. Directional distribution ellipses for distribution of R. solani AG1-IA using soil and plant samples indicated agreement with drainage. Across years, distribution of R. solani AG1-IA appears to be controlled by levee position. Where levees do not form logical areas of collection, the greatest concentration of inoculum appears to be in the lower elevations of the field. The spatial distribution of the early-season inoculum of R. solani AG1-IA reiterates the idea that inoculum in the form of sclerotia and hyphae associated with rice residue floating and collecting at lower points within the levees. Aerial blight was observed in NE Arkansas in 2013, spatial assessments of incidence of aerial blight and the height up the soybean plant at GPS positions intermittent the levee system validated models indicating the highest amount of disease was in the logical areas of collection (a combination of levee position and elevation). Because the disease occurred in a similar distribution as the inoculum, a predictive model using geographically weighted regression analysis is being constructed for fields with no prior disease assessment. In these fields, elevation data has been provided by USGS (LiDAR) and levee data provided by the grower. Using a quantitative determination of levee bends (m/m2), prediction based on random assignment of geospatially tagged points should allow for the use of handheld GPS devices to guide scouting to areas with the highest likelihood of inoculum collection and aerial blight development. <br /> <br /> In 2013, spatial sampling of soybean plants further confirmed Rhizoctonia solani AG11 as the most populous Rhizoctonia spp. in fields undergoing rice and soybean rotations in Arkansas. While evidence suggests it is weakly pathogenic on rice and soybean no noticeable damage or yield loss was observed. Because prior years' work had determined that R. solani AG11 was associated with areas of greater plant stand, experiments are currently in progress to determine its role in the rice/ soybean system and the potential benefit colonization of soybean may provide.<br /> <br /> R. solani AG1-IA also was observed causing aerial blight of peanut in Arkansas. Currently, there is a renewed interest in peanut production in the state and production practices include irrigation and various rotation schemes with soybean and less frequently rice. Thus, this may be an important disease of peanut in Arkansas.<br /> <br /> 2. Examine the effect of traditional or newly developed management strategies (chemical, cultural, and biological), soil physicochemical properties, or introduced biological control agents on the microbial community and its ability to suppress soilborne pathogens.<br /> <br /> A project established in Iowa is examining the effect of traditional or newly developed management strategies (chemical, cultural, and biological), soil physicochemical properties, or introduced biological control agents on the microbial community and its ability to suppress soilborne pathogens. Researchers are currently doing soil moisture retention curves, and have collected three plant residues (corn, soybean, and alfalfa), dried, ground, and are characterizing them for mineral elements and organics components (lignin, cellulose, and hemicellulose). Dr. Loynachan's group is working with an Iowa company (Hecheng Global, Inc.) and its President (David F. Clark) to laboratory test a Chinese product claimed to enhanced the speed of breakdown of crop residues. His group has obtained the APHIS permit to import these organisms for laboratory testing and are awaiting their arrival.<br /> <br /> Research at The University of Tennessee' West Tennessee Research and Education Center evaluated the effects of potash application, four seed treatment supplements, and in-furrow applications of liquid calcium nitrate fertilizer on snap bean diseases, seedling emergence, plant stand, plant growth, and yield in two experiments in a field naturally infested with several soil-borne plant pathogens. In a test planted April 23, seedling emergence 15 days after planting was greatest when a standard seed treatment (thiamethoxam + mefenoxam + fludioxonil) received a calcium-containing supplement (Treatment A). The number of healthy plants per row was highest when the standard seed treatment was supplemented with Treatment A, particularly in plots receiving muriate of potash. Pathogens isolated from diseased seedlings during this experiment were tentatively identified as Fusarium spp. (60%), Macrophomina phaseolina (17%), Sclerotinia sclerotiorum (6%), and Rhizoctonia solani (5%). Snap bean yield was highest when the standard seed treatment was supplemented (Treatment A). The seed treatment supplements had no effects on seedling vigor, plant height, or plant flowering. In a snap bean test planted August 2, seedling emergence 5 and 7 days after planting was significantly delayed with application of muriate of potash at 100 lb/K2O per acre. Four calcium-containing seed treatment supplements had no effect on seedling emergence. Supplementing the standard snap bean seed treatment with a high rate of another experimental treatment (Treatment E) increased the number of healthy plants per row 2 weeks after planting while a lower rate increased the number of healthy plants 5 weeks after planting. Potash application increased the number of plants lost to seedling diseases by over 56%, decreased plant height by 5%, and decreased snap bean yield by 45%. The seed treatment supplements failed to significantly affect plant height or snap bean yield. Macrophomina phaseolina was isolated from 96% of the diseased plants. In a soybean field experiment, four rates of an in-furrow calcium nitrate spray (0, 1, 2, and 3 lb N/acre) were evaluated with and without muriate of potash for their effects on soybean seedling diseases, plant growth, and yield of untreated soybean seed. Soybean yield was lowest in plots that received the 1 lb N/acre in-furrow spray plus potash fertilization. In a greenhouse planting, a root-knot resistant soybean cultivar had higher levels of calcium and silicon in the outer cell layers of lateral roots than a susceptible cultivar. F3 seed was collected from crosses of potential snap bean breeding lines with horizontal resistance to the seedling disease complex common in Tennessee snap bean growing areas.<br /> <br /> Research conducted at Texas A&M University focused on determining the impact of oilseed meals on soil microbial communities. In the first study, soil microbial community responses to amendments of a glucosinolate-containing brassicaceous oilseed meal (Brassica juncea, mustard), a non-glucosinolate-containing, nonbrassicaceous oilseed meal (Linum usitatissimum, flax), and a nonoilseed biomass (Sorghum bicolor, sorghum-sudangrass) were characterized using a 28-day time series of replicated 16S rRNA gene and fungal ITS gene sequence libraries. Distinct separation occurred by amendment type, with mustard inducing large increases in the abundance of bacterial taxa associated with fungal disease suppression (e.g. Bacillus, Pseudomonas, and Streptomyces spp.). Dramatic shifts were seen among the fungi, too, with phylotype richness decreasing by >60% following mustard addition. Changes in bacterial and fungal community composition were rapid, and distinct community types persisted throughout the study. The results from this study were published in 2013. <br /> <br /> In the second study, the effects of four types of isothiocyanates (ITCs), including allyl, butyl, phenyl, and benzyl ITC, on soil fungal and bacterial communities were determined. Each ITC was added to soil that also received 1% flax seedmeal (to represent organic material added by seedmeals). Microbial communities were analyzed based on the ITS gene for fungi and16S rRNA gene for bacteria using qPCR and tag-pyrosequencing with 454 GS FLX titanium technology. A dramatic decrease in fungal populations (~85% reduction) was observed after allyl ITC addition. Fungal community compositions also shifted following ITC amendments (e.g., Humicola increased in allyl and Mortierella in butyl ITC amendments). Bacterial populations were less impacted by ITCs with community compositions being primarily impacted by time of incubation. A manuscript based on these results was submitted for publication and is currently being revised based upon reviewer comments.<br /> <br /> Current research activities at Penn State University are directed towards beneficial microorganisms for improvement of important crops of the high Andes. Until May, this included both Ecuador and Bolivia, and since May, only Ecuador. With Ecuadorian researchers, we conducted a genetic survey of Peronospora variability on quinoa and its close relatives. This work resulted in a recently accepted article, and also a poster presentation on anthracnose in tree tomato. Similarly, 3 publications on seed borne pathogens of quinoa have appeared in the past 2 years, and point to the fact that live seed of quinoa sold in the U.S. for the food trade has imported exotic pathogens. These are relevant to the U.S. as quinoa is currently being developed as a new crop, particularly suited to the northwestern U.S.<br /> <br /> Experiments were established in Pennsylvania to measure plant and pathogen responses, as well as effects to plant tissue nutrient levels for faba bean and common bean. Significant responses have been recorded following application of plant growth promoting rhizobacteria, and/or the addition of phosphorus solubilizing bacteria. These data are currently being tabulated to adjust next year's treatment lists, and to confirm these results with validation trials in growth chambers. <br /> <br /> With Bolivian counterparts we have been developing sustainable farming practices for low-resource farmers. Plots evaluating numerous biological controls and organisms that enhance nutrient uptake have been established, and are being mirrored in the U.S. for severity of disease, nutrient sufficiency and for plant growth responses. Pennsylvania trials on faba bean, common bean and quinoa were conducted this summer. Plant samples sent to Pennsylvania from Bolivia were processed for beneficial plant-associated microbes, producing more than 300 distinct isolates that have been characterized for production of disease-suppressive antibiotics, phytohormone production, spore production, chitinase production, and phosphorus solubilization. They were also identified to species. We are continuing to evaluate this same pool of beneficial bacteria for effects on faba bean and common bean. An additional study was to isolate DNA from soil samples taken from raised bed farming systems near Lake Titicaca, that have produced sustainable crops for more than 500 years.<br /> <br /> Multiple members of the multistate research project continue to investigate better management strategies for the control of Pythium and the effect different chemical, biological and cultural management strategies have on Pythium community dyanmics. In Oregon an in vitro sensitivity test to determine fungicide resistance on three different species of Pythium (P. irregulare, P. sylvaticum, and P. ultimum) was completed. This research project was carried out in a cooperative agreement between USDA, Corvallis, and the North Willamette Research and Extension Center-Oregon State University, Aurora. A totoal of 119 Pythium isolates from forest nurseries in Oregon were tested for resistance to mefenoxam. Six different concentrations of the fungicide were evaluated. A paper with this data and additional data from evaluations with other fungicides was submitted for review in Plant Disease.<br /> <br /> Pythium is an ecologically diverse group of microorganisms found in virtually all soils, but little is known about the effect of crop rotation on Pythium communities. To understand the effect of crop rotation on species diversity, researcher in Arkansas collected soil from plots following a long-term rotation study including rice, corn, soybean and wheat. Soil from each plot was placed in cups, wetted to saturation, planted with the soybean cultivar Hutcheson, and incubated at 25°C. After 3 days, seeds were collected and washed in running water and placed on 2% water agar to bait out Pythium from soils. Molecular identification was performed by sequencing the ITS region and Blast analysis to a curated reference database. A total of 320 isolates were identified representing 12 species. Overall, the most frequently recovered species were P. spinosum, P. irregulare, P. pereocandrum and P. sylvaticum. In continuous rice production, P. spinosum was the most prevalent species isolated. In the soybean-wheat-rice and rice-wheat-soybean-wheat rotations, P. spinosum and P. irregulare were the most frequently recovered, while P. irregulare and P. sylvaticum were the most frequently recovered species in the soybean-rice and rice-soybean rotations. Pythium species composition reflected the frequency at which a susceptible host was planted in the rotation.<br /> <br /> In related research conducted at Oklahoma State University fungicide hormesis was assessed in vitro on Pythium irregulare, Sclerotinia homoeocarpa, and Botrytis cinerea, validating the stimulation of pathogen activity by low fungicide levels. A new M.S. student, Sumit Pradhan, evaluated a P. ultimum strain for hormetic responses to mefenoxam and 26 S. homoeocarpa isolates for biphasic dose-responses to thiophanate-methyl. Preliminary results on one P. irregulare and one S. homoeocarpa strains were reported at the 2013 APS Annual Meeting.<br /> <br /> A collaborative research project lead by researchers at Oklahoma State University focused on understanding the bacterial communities present in Andean soils from Ecuador, Chimborazo Province, suppressive to Phytophthora infestans, and was conducted using T-RFLP to assess the microbial diversity present in these soils. Bacteria corresponding to at least 13 phyla were identified. Most bacteria belonged to Actinobacteria, Proteobacteria and Firmicutes. Comparisons of suppression of Rhizoctonia solani and Phytophthora infestans between heat treated and non-treated soils suggest that bacteria of the genera Bacillus and Geobacillus are responsible of most of the suppressive nature of these soils. Screening for antifungal activity against Rhizoctonia solani and Phytophthora infestans of diverse bacterial strains from Andean soils was also completed and selected strains are being tested for biological control potential on potato foliage.<br /> <br /> Research conducted at Mississippi State University has focused on understanding genetics and biochemistry of soilborne and endophytic bacteria against plant pathogens. Objective 1 focused on identification of antagonistic bacteria associated with soilborne plant diseases. More than 800 bacterial isolates were obtained from soils and plant samples, and tested for antimicrobial activities using the standard bioassay procedures. Approximately 80 isolates showed significant antifungal activities against Geotrichum candidum and more than 40 isolates exhibited significant antibiotic activities against Erwinia amylovora. These isolates were further purified and preserved in a -80oC freezer. The isolates have been identified to be Bacillus spp., Pseudomonas spp., Burkholderia spp. and Streptomyces spp. using the 16S rDNA sequence analysis. Objective 2 was to investigate the genes associated with production of the antifungal compound occidiofungin produced by the soilborne bacterium Burkholderia contaminans strain M14. In collaboration with Dr. Jim Smith, we have characterized the functions of the ocfC and ocfN genes in antimicrobial activities. The third objective was to isolate endophytic bacteria associated with charcoal rot of soybean. Four pairs of diseased and asymptomatic plants were collected from patches of plants affected by charcoal rot in a soybean field in Leflore County near Sidon, MS. Four additional asymptomatic plants were collected from areas of the field where no symptoms of charcoal rot disease were evident. Plants were surface disinfected and split longitudinally with half of each plant used for isolation of fungal endophytes and half used for isolation of bacterial endophytes. Bacteria were isolated using an enrichment protocol with the majority retained for DNA sequencing and a fraction of each sample cultured on two media in different environments. Analysis of bacterial communities by isolation and Illumina sequencing is under way. <br /> <br /> In New Hampshire the first year of analyses of the fungal and bacteria communities associated with the forage crop perennial ryegrass were completed. The focus of this project has been to evaluate the effect of cultivar mixtures to extend the grazing season. A PhD student is evaluating the ability of these cultivar mixtures to recruit fungal symbionts while excluding pathogens. Soil has been collected from 4 sites in Maine, New Hampshire, Pennsylvannia, and Vermont. Extraction of DNA was successfully completed and he is now in the process of quantifying the fungal and bacterial biomass in each sample and preparing DNA libraries for Illumina sequencing of the 16S (bacteria) and 18S (fungi) for metagenomic analysis.<br /> <br /> Finally, the etiology of root rot caused by Fusarium solani is being investigated in Minnesota. The effects of F. solani on soybean plant germination and growth is influenced by fungal isolate, soybean cultivar, stage of plant development, soil temperature, and soil moisture. In general, soil inoculation with F. solani caused poor germination and reduced plant populations. However, plants that did germinate and flowered appeared to benefit from infection with greater root and above ground growth and greater seed yield. Research was conducted into the effects of inoculum methods and inoculum substrates infested with F. solani or F. virguliforme on disease symptom expression and plant growth. Management of this root rot fungus should emphasize control of its harmful effects on plant populations during seed germination. <br /> <br /> When investigating the etiology of F. solani, spurious symptoms, particularly lesion-like root discoloration and poor root development, were observed when many infested inoculum substrates such as seed of sorghum, rye, barley, or oats are used to inoculate soil during screening experiments. Inoculation with uninfested seed resulted in poor root development and decreased plant growth when compared to either plants root-inoculated with infested substrates or to an uninoculated control. The effect is dependent on species of plant seed used (most severe effects were seen when red sorghum was used as a substrate) but is also determined by the soybean cultivar planted. These effects are particularly relevant for evaluation of soybean cultivars and evaluation of pathogenicity of fungal isolates and can cause misleading symptoms unrelated to the effect of the pathogen.

Publications

Journal Artiicles:<br /> <br /> Abd-Elmagid A, Garrido PA, Hunger R, Lyles JL, Mansfield MA, Gugino BK, Smith DL, Melouk H, and Garzón CD. 2013. Discriminatory simplex and multiplex PCR for four species of the genus Sclerotinia. Journal of Microbiological Methods 91: 293-300.<br /> <br /> Akshaya Ravichandran, Ganyu Gu, Jerome Escano, Shi-En Lu, and Leif Smit,. 2013. The presence of two cyclase thioesterases expands the conformational freedom of the cyclic peptide occidiofungin. Journal of Natural Products 76:150-156. <br /> <br /> Arif M, Dobhal S, Garrido PA, Orquera GK, Espíndola AS, Young CA, Ochoa-Corona FM, Marek SM, Garzón CD. 2013. Sensitive and rapid detection of Phymatotrichopsis omnivora, causal fungus of cotton root rot. Plant Disease. Accepted.<br /> <br /> Canaday, C. H., 2013, Field evaluation of calcium supplements on soybean seedling diseases, emergence, plant stand, and yield, 2012. Plant Dis. Manag. Rep. 7:FC101. doi: 10.1094/PDMR07<br /> <br /> Dayna Ellis, Akshaya Ravichandran, Gosai Jiten, Shi-En Lu, Donna Gordon and Leif Smith. 2013. The antifungal occidiofungin triggers an apoptotic mechanism of cell death in yeast. Journal of Natural Products 76:829-838.<br /> <br /> Demers JE, Garzón CD, and Jiménez-Gasco MM. 2013. Striking genetic similarity between races of Fusarium oxysporum f. sp. ciceris confirms a monophyletic origin and clonal evolution of the chickpea vascular wilt pathogen. European Journal of Plant Pathology. Accepted.<br /> <br /> Elmer, W.H., Useman, S., Schneider, R.W., Marra, R.E., LaMondia, J.A., Mendelssohn, I.A., Jiménez-Gasco, M. M., and Caruso, F.L. 2013. Sudden vegetation dieback in Atlantic and Gulf Coast salt marshes. Plant Disease 97: 436-445.<br /> <br /> Flores FJ and Garzón CD. 2013. Detection and assessment of chemical hormesis on the radial growth in vitro of oomycetes and fungal plant pathogens. Dose-Response 11: 361-373.<br /> <br /> Geiser, D.M., Akoi, T., Bacon, C.W., Baker, S.E., Bhattacharyya, M.K., Brandt, M.E., Brown, D. W., Burgess, L.W., Chulze, S., Coleman, J.J., Correll, J.C., Covert, S.F., Crous, P.W., Cuomo, C.A., De Hoog, G.S., Di Pietro, A., Elmer, W.H., Epstein, L., Frandsen, R.J.N., Freeman, S., Gagkaeva, T., Glenn, A.E., Gordon, T.R., Gregory, N.F., Hammond-Kosack, K.E., Hanson, L.E., Jiménez-Gasco, M.M., Kang, S., Kistler, H.C., Kuldau, G.A., Leslie, J.F., Logrieco, A., Lu, G., Lysøe, E., Ma, L.-J., McCormick, S.P., Migheli, Q., Moretti, A., Munaut, F., ODonnell, K., Pfenning, L., Ploetz, R.C., Proctor, R.H., Rehner, S.A., Robert, V.A.R.G., Rooney, A.P., Bin Salleh, B., Scandiani, M.M., Scauflaire, J., Short, D.P.G., Steenkamp, E., Suga, H., Summerell, B.A., Sutton, D.A., Thrane, U., Trail, F., Van Diepeningen, A., VanEtten, H.D., Viljoen, A., Waalwijk, C., Ward, T.J., Wingfield, M.J., Xu, J.-R., Yang, J.-B., Yli-Mattila, T., and Zhang, N. 2013. Letter to the Editor: One Fungus, One Name: Defining the genus Fusarium in a scientifically robust way that preserves longstanding use. Phytopathology 104: 400-408.<br /> <br /> Haddad, S., M. A. Tabatabai, and T. E. Loynachan. 2013. Biochemical processes controlling soil nitrogen mineralization under waterlogged conditions. Soil Sci. Soc. of Amer. 77:809-816.<br /> <br /> Hollister, E.B., P. Hu, A.S. Wang, F.M. Hons, and T.J. Gentry. 2013. Differential impacts of brassicaceous and non-brassicaceous oilseed meals on soil bacterial and fungal communities. FEMS Microbiol. Ecol. 83:632-641. <br /> <br /> Hu, J., Diao, Y., Lin, D., Bi, Y., Pang, Z., Fryxell, R. T., Liu, X. and Lamour, K. In Press. Loss of Heterozygosity drives clonal diversity of Phytophthora capsici in China. PLOS ONE.<br /> <br /> Hu, J., Pang, Z., Bi, Y., Shao., Diao, Y., Guo, J., Liu, Y., Lu, H., Lamour, K., and Liu, X. 2013. Genetically diverse long-lived clonal lineages of Phytophthora capsici from pepper in Gansu, China. Phytopathology, doi:10.1094/PHYTO-01-13-0016-R<br /> <br /> Kuan-Chih Chen, Akshaya Ravichandran, Adam Geurrero, Peng Deng, Sonya M. Baird, Leif Smith, and Shi-En Lu. 2013. The ocfC Burkholderia contaminans MS14 gene encodes a xylosyltransferase for the antifungal occidiofungin production. Applied and Environmental Microbiology 79:2899-2905.<br /> <br /> Lamour, K. and Hu, J. Diversity and Phytophthora: a threat to forests, crops and traditional laboratory research - Mini Review. CAB Reviews 8, No. 038.<br /> <br /> Malcolm, G. M., Kuldau, G. A., Gugino, B. K., and Jiménez-Gasco, M. M. 2013. Letter to the Editor: Hidden host plant associations of soilborne fungal pathogens: An ecological perspective. Phytopathology 103: 538-544.<br /> <br /> Remesal, E., Landa, B.B., Jiménez-Gasco, M.M. and Navas-Cortés, J.A. 2013. Sequence variation of protein-coding genes as a diagnostic tool for the identification of mycelial compatibility groups in Sclerotium rolfsii. Phytopathology 104: 479-487.<br /> <br /> Shezeng Li, Xixuan Jin, Jian Chen and Shi-En Lu. 2013. Inhibitory activities of venom alkaloids from the red imported fire ant, Solenopsis invicta against the growth of Clavibacter michiganensis subsp. michiganensis in vitro and the application of piperidine alkaloids to control bacterial canker of tomato in the greenhouse. Pest Management Science. doi:10.1080/09670874.2013.784931.<br /> <br /> Shrestha, S., Zhou, Y., and Lamour, K. 2013. Oomycetes baited from streams in Tennessee 2010-2012. Mycologia 105: 1516-1523. <br /> <br /> Testen, Anna L., Maria del Mar Jimenez-Gasco, Jose B. Ochoa and P. A. Backman. 2014. Molecular detection of Peronospora variabilis in quinoa seeds and phylogeny of the quinoa downy mildew pathogen in South America and the United States. Phytopathology doi:10.1094/PHYTO-07-13-0198-R <br /> <br /> Testen, A.L., J. M. McKemy, P.A. Backman 2013. First Report of Ascochyta Leaf Spot of Quinoa Caused by Ascochyta sp. in the United States. Plant Disease 97(6):844.<br /> <br /> Testen, A.L., McKemy, J.M., and Backman, P.A. 2013. First report of Passalora leafspot of quinoa caused by Passalora dubia in the United States. Plant Disease 97(1):139.<br /> <br /> Urrea, K., Rupe, J. C., and Rothrock, C. S. 2013. Effect of fungicide seed treatments, cultivars, and soils on soybean stand establishment. Plant Dis. 97:807-812.<br /> <br /> <br /> <br /> Book Chapters:<br /> <br /> R.L. Melnick, B.A. Bailey, P.A.Backman. 2013. Bacterial endophytes of perennial crops for management of plant disease. pp 49-76 In: Bacteria in Agrobiology: Disease Management. D.K. Maheshwari Ed. Springer Books, Berlin. <br /> <br /> Loynachan, T. E. 2012 Soil biology: The living component of soil. pp. 49-67. In: D. Lindo et al. (ed.), Know Soil Know Life, Soil Science Society of America, Madison.<br /> <br /> Loynachan, T. E. 2013. Human disease from introduced and resident soilborne pathogens. pp. 107-136. In: E. Brevik and L Burgess (ed.), Soils and Human Health, CRC Press, Boca Raton.<br /> <br /> Garzón CD, Santamaría L, and Moorman GW. 2013. Fungicide resistance development in ornamental crops. In: Stevenson K, McGrath M, and Wyenandt A. Fungicide Resistance in North America. The American Phytopathological Society. Accepted.<br /> <br /> Garzón CD and Flores FJ 2013. Hormesis: biphasic dose-responses to fungicides in plant pathogens and their potential threat to agriculture. Pages 311- 328 in: Nita M ed., Fungicides - Showcases of Integrated Plant Disease Management from Around the World. InTech - Europe. <br /> <br /> Abstracts:<br /> <br /> Díaz CI, Ponce K, Oliva R, Garzón CD, Koch AR, Gia J, Benitez MS. 2013. Microbial communities associated with the suppression of tuber blight infection in soils from Chimborazo province, Ecuador. 2013 Phytopathology 103:S2.35<br /> <br /> Espindola A, Schneider W, Garzon CD. Pythium aphanidermatum strain-discrimination from 454 pyrosequencing metagenomic samples. Phytopathology 103:394-P <br /> <br /> Espíndola A, Schneider W, and Garzón C. 2013. Strain identification of Pythium aphanidermatum in metagenomic samples from 454 pyrosequencing. Oomycete Molecular Genetics Network Meeting. Mar. 10-12, Pacific Grove, CA. <br /> <br /> Faske, T. R., Hurd, K. M., Spurlock, T. N., and Rothrock, C. S. 2013. First detection and pathogenicity of Rhizoctinoa solani AG-1 1A on peanut in Arkansas. Phytopathology 103(Suppl. 1):S2.42.<br /> <br /> Graf-Grachet N, Flores FJ, Pradhan S, Walker NR, Garzón CD. 2013. Stimulation of radial growth in vitro of Sclerotinia homoeocarpa by subinhibitory doses of thiophanate-methyl. Phytopathology 103:S2.52<br /> <br /> Moncrief I, Garzón CD, Marek S, Stack J, Gamliel A, Issac Y, Dehne H, Fletcher J. 2013. Specific discrimination of Fusarium proliferatum using inter-simple sequence repeats (ISSRs) and simple sequence repeats (SSRs). Phytopathology 103:S2.99<br /> <br /> Parker, M., Melzer, M., Boland, G., and Broders, K. 2013. Diversity of Rhizoctonia solani associated with canola, wheat, and pea in Alberta Manitoba, and Saskatchewan. Phytopathology 103: S111<br /> <br /> Spurlock, T. N., Rothrock, C. S. and Monfort, W. S. 2013. Logical areas of collection: A precision concept for management of Rhizoctonia solani AG1-IA. Phytopathology 103(Suppl.1):S2.137.<br /> <br /> Spurlock, T. N., Rothrock, C. S. and Monfort, W. S. 2013. Rhizoctonia solani AG11: Spatial analyses reveal strategies of a successful parasite. Phytopathology 103(Suppl. 1):S1.10.<br /> <br /> Stetina, T. J., and Rothrock, C. S. 2013. The effects of salinity on Pythium seed and root rots of soybean. Phytopathology 103(Suppl. 1):S1.10.<br /> <br /> Urrea, K. E., Rupe, J. C., Rothrock, C. S., Steger, A. J., Anders, M. M., Rojas, A. J., and Chilvers, M. I. 2013. Effect of crop rotation on Pythium spp. population composition in Arkansas soybean fields. Phytopathology 103(Suppl. 1):S2.150-151.

Impact Statements

1. Our results indicate that the type of ITC released from seedmeals may result in differential impacts on soil microorganisms. This information will likely impact the selection and breeding of plants for biofumigation-based control of soil-borne pathogens while minimizing the impacts on non-target microorganisms.
2. Loynachan serves as the regional coordinator for Ask a Soil Scientist maintained by the Soil Science Society of America: https://www.soils.org/ask. Loynachan maintains a URL on Soil Biology (http://agron-www.agron.iastate.edu/~loynachan/mov/) and responds weekly to questions asked by users of the information. These inquires vary from individuals in grade school to fellow scientists worldwide.
3. Thus far, 48 SNP genotyping assays have been developed for loci that span the P. capsici genome and these have been applied to laboratory populations of P. capsici (sexual crosses and single zoospore progeny) and field populations from China. Application to laboratory populations revealed extensive asexual plasticity within the P. capsici genome during growth on typical agar media in a laboratory setting. In addition, the SNP markers illuminated extensive asexually derived diversity of P. capsici at locations across China.
4. Some isolates of bacteria have showed a great potential as biological control agents in plant disease management. Discovery of the ocfN gene, which promotes biosynthesis of more variants of antimicrobial compounds, provides a possibility of increasing functional spectra of biological control agents.
5. In 2013, aerial blight of soybean and peanut, banded sheath blight of corn, and sheath blight of rice were observed in NE Arkansas. In rice and soybean, the sheath blight disease was prolific enough to warrant fungicide applications to prevent further yield loss. Given the likelihood that many fields will be rotated to soybean from rice and that the previous season allowed the buildup of a substantial amount of inoculum, the findings of this work to date should arm the farmer and consultants with the ability to detect and manage aerial blight sooner and more precisely than before if environmental conditions favor development of disease.

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Report Information

Participants

Lamour, Kurt - University of Tennessee;

Canaday, Craig - University of Tennessee;

Scherm, Harald - University of Georgia (Administrative Advisor);

Broders, Kirk - University of New Hampshire;

Garzon, Carla - Oklahoma State University;

Spurlock, Terry - University of Arkansas;

Kurle, Jim - University of Minnesota;

Lu, Shien - Mississippi State University;

Ma, Din-Pow - Mississippi State University;

Brief Summary of Minutes

Accomplishments

1. Evaluate the population genetic diversity of soilborne pathogens and antagonistic microorganisms in different growing systems and regions using traditional and metagenomic approaches.<br /> <br /> In Minnesota, researchers aim to identify important seedling pathogens found in soybean and corn production fields, characterize etiology of disease development, determine species using traditional and molecular approaches, determine pathogenicity on corn and soybean, characterize sensitivity to commonly used seed treatment fungicides. Infection and lesion development caused by Fusarium solani was characterized microscopically. Infection occurred when lateral roots emerged through the root epidermis and simultaneously with lateral root development. Root lesions caused by F. solani originated at the point of lateral root primordium penetration through the epidermis and appeared to be enabled by disruption of the epidermis. Lesions then progressed both into the root cortex and laterally under the epidermis away from the emerging lateral root. The lesions were distinctive. Fields symptomatic for seed, seedling, or root rots in 22 Minnesota counties were sampled for Pythium species using one of two different methods. In the first method Pythium was isolated directly from symptomatic soybean seedlings collected in the field. In the second, soil from the field was collected, dried, and in the laboratory baited with soybean seeds. Isolates were identified using both morphological and molecular techniques. Thirty-four Pythium or Phytopythium species were found in samples from 13 counties. After collection, the pathogenicity of the most 18 common Pythium and Phytopythium species was tested at three different temperatures, 15, 20, and 25°C, on both soybean and corn seed using an in-vitro assay and on seedlings planted in growth media. A wide range of lesion development and root growth reduction was observed depending on the Pythium species and host plant. The effect of temperature on seed germination was dependent on Pythium species tested. For example P. intermedium was most damaging at 15°C while P. ultimum var sporangiferum and ultimum were more damaging on both corn and soybean at 25°C. P. conidiophorum, P. intermedium, P. irregulare, P. sylvaticum, and P. ultimum var sporangiforum and ultimum were most pathogenic on soybean. P. irregulare, P. sylvaticum, and P. ultimum var sporangiforum and ultimum also reduced corn seed germination. Pythium irregulare and P. ultimum var ultimum were pathogenic on developing soybean seedlings. The data show that some of the most common Pythium species found in Minnesota, including P. ultimum and P. sylvaticum, are detrimental to germination and growth of both corn and soybean. In addition Pythium seedling damage could be caused by Pythium spp. at either 15 or 25°C. <br /> <br /> In Tennessee, researchers developed novel genomic resources, including hundreds of new SNP markers for the downy mildew of spinach (Peronospora farinosa), the late blight of potato and tomato (Phytophthora infestans) and the fungal pathogen Cercospora sojina which causes frogeye leaf spot. The work with the markers and organisms is ongoing.<br /> <br /> In Mississippi, research focused on understanding genetics and biochemistry of soilborne and endophytic bacteria against plants pathogens. More than 200 bacterial isolates were obtained from soils and plant samples, and tested for antimicrobial activities using standard bioassay procedures. Approximately 60 isolates showed significant antifungal activities against Geotrichum candidum and more than 20 isolates exhibited significant antibiotic activities against Erwinia amylovora. These isolates were further purified and preserved at -80°C. The isolates were preliminarily identified to be Bacillus spp., Burkholderia spp. and Pseudomonas spp. using 16S rDNA sequence analysis. The genes associated with production of the antibacterial compounds synthesized by the bacterium Burkholderia contaminans strain M14 and Pseudomonas sp. strain YL23 were investigated. The two genes associated with antibacterial activities of strain MS14 have been identified, which encode a LuxR regulatory protein and a nonribosomal peptide synthase. secG is predicted to code a transporter protein and is required for antibiotic activities of Pseudomonas sp. YL23 against Erwinia amylovora and Dickeya chrysanthemi. The pqqC gene is essential for antifungal activity of Pseudomonas kilonensis JX22 against Fusarium oxysporum. Draft genome sequences of the bacteria Pseudomonas sp. YL23 and Burkholderia pyrrocinia strain Lyc2 were completed and published. Some useful gene clusters have been identified from the genome sequence drafts and the sequence data provide important insights into understanding antimicrobial activities of the bacteria. To understand the interaction between rice and the bacterial pathogen, the causative agent of bacterial panicle blight, transcriptomic analyses using next-generation sequencing and bioinformatics were conducted to identify differentially expressed transcripts between resistant and susceptible interactions and formulate a model for rice resistance to the disease. A few gene candidates were identified, which may be associated with plant resistance to the bacteria. <br /> <br /> The emergence and spread of Verticillium wilt was recently observed in cotton plants at the R.R. Foil Plant Science Research Center at Mississippi State during the summer of 2013 and 2014. This disease could pose a considerable threat to cotton growers because some breeding lines appeared to be extremely susceptible to the disease. Two soilborne fungi with different morphology and growth characteristics were isolated from diseased cotton plants by culturing infected stem tissues on potato dextrose agar (PDA) plates. The two fungi species were identified as Verticillium sp. and Diaporthe phaseolorum via ribosomal ITS DNA sequencing. Two ITS regions, ITS1 (between the 18S rRNA and 5.8S rRNA) and ITS2 (between 5.8S rRNA and 28S rRNA), are present in the fungal rRNA operon. The two ITS regions are removed during pre-rRNA cleavage. The isolated Verticillium sp. could be Verticillium dahliae, Verticillium longisporum or Verticillium albo-atrum and will be further identified using a PCR-based genotyping method with species-specific primers.<br /> <br /> In Arkansas, The spatial distribution of Rhizoctonia spp. in fields undergoing rice and soybean rotations was characterized in two producer fields in 2014 with the goal of management of R. solani AG1-IA, the cause of aerial blight on soybean and sheath blight on rice. In 2014, spatial sampling of soybean plants further confirmed Rhizoctonia solani AG11 as the most populous Rhizoctonia spp. in fields undergoing rice and soybean rotations in Arkansas. Colonization of soybean plants at the three-leaf stage were greater than 15% of the plants near Dumas and 37% of plants near Wiener, Arkansas. Isolates of R. solani were almost exclusively AG-11 at both sites. Application of the fungicide azoxystrobin limited the increase in colonization of soybean plants by R. solani compared to points not sprayed. The research reinforces the idea that populations of R. solani in soil colonize crops within the field and are dominated by populations that are not considered pathogenic.<br /> <br /> In New Hampshire, a number of cover crops species were screened to determine whether they harbor asymptomatic infections by the soilborne pathogen V. dahliae. To date we have found that many cover crop species including buckwheat, crimson clover, and winter rye are capable of harboring the fungus, but display no visual symptoms of infection. Only through the use of a strain of V. dahliae that expresses the green fluorescent protein (GFP), was the extensive colonization able to be visualized. Screening of both the commercial strawberry and one of its wild ancestors, Fragaria vesca, for resistance to V. dahliae with the GFP strain is currently in progress. One of the major obstacles in screening for resistance to soilborne root and wilt pathogens is that visual assessment does not provide an accurate assessment of fungal colonization or host resistance. Therefore two new methods are being evaluated for their ability to dissect resistance, tolerance, and susceptibility. Confocal microscopy using the GFP strain of V. dahliae is being used to measure colonization of susceptible, tolerant (visually healthy but ingress of fungal hyphae present), and resistant (visually healthy no ingress of fungal hyphae) strawberry cultivar. In addition, multispectral and thermal imaging systems are being evaluated for their ability to detect pre-visual stress as a potential phenotyping method. This process was initiated in the past year and protocols for inoculum production, inoculation, and visualization by confocal microscopy have all been standardized and screening of strawberry cultivars with known levels of resistance and susceptibility is in progress. Finally, the sequencing of two strains of V. dahliae (2 that infect strawberry and 1 that infects mint) was completed. The first assemblies have been completed and the comparative analyses against the reference V. dahliae strain, which was recovered from lettuce, have been completed. An additional 12 strains of V. dahliae from other host including tomato, potato, pistachio and the soil were included in the analysis. Preliminary analyses indicate that the isolate from strawberry recovered in California is more closely related to lettuce than the strawberry isolated recovered in New Hampshire. In addition the isolate from mint recovered in Oregon grouped together with the strawberry and lettuce strains from California but still differed at over 16,000 SNPs. Preliminary synteny analysis also indicate significant genomic rearrangement both within chromosome and between chromosomes. Future work will focus on identifying transposable element that may be responsible for the genome rearrangements. <br /> <br /> Research on sweetpotato tip/end rot is focused on defining and evaluating the pathogen(s) associated with this disease problem that was severe and widespread in 2009, but can still be found at high levels following harvesting. An initial study (Phase 1) was established in 2009 to determine the causal agents associated with tip/end rot and completed in 2013. Due to the thousands of isolates collected from traditional culturing, the identifications are now completed. During 2012 it was determined that reniform nematode, Rotylenchulus reniformis, levels were increasing in many fields from a general survey and was suspected that they might form a disease complex resulting in greater tip/end rot in Mississippi. Research was established in 2013 to evaluate those interactions with the most common pathogen isolated, Macrophomina phaseolina and reniform nematode using different populations in the field and in the greenhouse (Phase 2). <br /> <br /> Lastly, several projects have continued to evaluate the genetic diversity of R. solani from a number of important crop species across North America. Researchers at the University of New Hampshire in collaboration with the University of Guelph conducted a phylogenetic comparison of isolates of R. solani collected from canola and wheat growing region of western Canada. A total of 128 multinucleate isolates were identified by ITS sequence and compared with anastomosis group (AG) results. The multinucleate isolates of R. solani were grouped into 8 distinct clades. Two distinct clades were observed for isolates classified as AG2-1 by anastomosis testing, and isolates in a closely related clade to AG2-1 did not successfully fuse with any of the tester strains. While most isolates of AG-5 clustered together according to ITS sequences, three isolates classified by anastomosis grouping as AG-5 grouped with AG2-1, AG-4, and binucleate Rhizoctonia in the phylogenetic analysis. There was no genetic diversity among isolates classified as AG-4. While in most instances the results from AG tests were consistent with ITS sequence, there were several cases where isolates were inconsistently classified or failed to undergo anastomosis with any of the tester strains. In addition, a previously undocumented AG group was identified, and the AG-5 tester strain was found to form hyphal fusions with unrelated groups of multinucleate and binucleate species of Rhizoctonia. Results from this project were published in Plant Disease. Current work has begun to focus on the genetic diversity of R. solani infecting soybean and dry bean in northern regions with a specific focus on AG2-2. Early results indicate that isolates of R. solani AG2-2 may have as many as six distinct ITS sequences in a single strain. These finding raise a number of questions regarding the frequency at which members of AG2-2 anastomose in the wild and how genetically distinct they need to be before they can no longer anastomose.<br /> <br /> Objective 2. Examine the effect of traditional or newly developed management strategies (chemical, cultural, and biological), soil physicochemical properties, or introduced biological control agents on the microbial community and its ability to suppress soilborne pathogens<br /> <br /> An Iowa project is evaluating a Chinese product claimed to enhance the speed of breakdown of crop residues. The work was coordinated with an Iowa company hoping to market the product in the US. The objectives of the study were to quantitatively measure the efficacy of the product in decomposing plant residue. Corn residue and a Typic Hapludoll soil were characterized for essential elements (C, N, P, K, and pH). Residues were chopped and mixed with the soil in individual mesocosms (air-tight jars with NaOH vials to the collect carbon dioxide evolved); soil moisture was kept at field capacity and temperature at 25°C.Treatments were as follows: (1) no soil, no residue, no product (control), (2) soil, no residue, no product (3) soil, residue, no product, (4) soil, no residue, product, (5) soil, residue, product, and (6) soil, residue, sterilized product. After data analysis, it was determined there were no significant differences among treatments. We concluded that the product does not affect the rate of residue decomposition breakdown when the residue is chopped into small pieces and mixed with a warm, moist soil. Further work is planned for surface-applied residues and residues not finely chopped into smaller pieces.<br /> <br /> In Arkansas, Meloidogyne incognita, the root-knot nematode, and Thielaviopsis basicola, the cause of black root rot, and their interaction on cotton were examined for soil textures that ranged from 48 to 87% sand in microplots. Each soil texture was infested with 0, 4, or 8 M. incognita eggs and 0 or 20 T. basicola chlamydospore chains per gram of soil. Plant growth was suppressed early in the season and midseason by T. basicola and M. incognita suppressed plant growth and delayed plant development late in the season across all soil textures. Cotton yield was lower in the presence of either T. basicola or M. incognita. An interaction between M. incognita and T. basicola, which decreased plant growth and yield, occurred in one year when neither pathogen alone caused substantial plant damage. Root colonization by T. basicola and fungal reproduction and survival decreased in soil having 87% sand. M. incognita generally caused more galling and reproduction in soils as sand content increased. Root galling severity and M. incognita reproduction were suppressed by the presence of T. basicola in soil at sand contents lower than 87%. Soil texture had a greater impact on T. basicola than on M. incognita in this study.<br /> <br /> The findings support work on T. basicola over a 10-year period in a texturally diverse cotton field near Portland, Arkansas, where populations were quantified at planting and harvest in 512 contiguous plots between 2001 and 2013. The objective was to determine the spatial distribution of T. basicola and if these distributions had changed over time. Data were subjected to exploratory spatial analysis and then rigorous spatial regression to determine the distribution and correlations for each sampling time. For each year sampled, the distributions of T. basicola were aggregated. In all years, a significant negative correlation existed between T. basicola and percent sand fraction and a significant positive correlation existed between the fungus and percent silt. Areas corresponding to the highest clay content also had the highest T. basicola populations. Over the time of the study, the spatial distribution of T. basicola was relatively stable and dependent on soil texture.<br /> <br /> Meloidogyne incognita and Rotylenchulus reniformis are economically important nematode species in cotton production. Evidence from 2001-2003, in a texturally diverse cotton field near Portland, Arkansas, suggested that populations of M. incognita had the greatest effect on yield in soils with greater sand content. Additionally, exhaustive nematode assays indicated R. reniformis was increasing in population and distribution in the field. Since that work, the field has continued to be planted in cotton until being planted in soybean in 2013. From the original 512 plots, nematode population densities were measured in the fall immediately after the crop was harvested in 2011 and 2013. Over the 10 year period, populations of M. incognita declined sharply while R. reniformis increased. Univariate Moran’s I indicated significantly aggregated distributions for both pathogens in all years except 2011, and trend surface models indicated Range values decreased for M. incognita but increased for R. reniformis prior to 2013. Bivariate exploratory spatial analysis and spatial regression indicated a significantly dispersed distribution and inverse relationship in 3 of 4 years and 2 of 4 years respectively confirming competition and displacement of M. incognita by R. reniformis. Neither R. reniformis nor M. incognita populations were directly related to yield loss across soil textures. The highest yield impact of nematode populations occurred in the sandier areas of the field indicating site specific management in these textural zones should have the greatest economic return.<br /> <br /> Pythium spp. are important seed and seedling disease pathogens of soybean in Arkansas. Resistance to seedling diseases caused by Pythium spp. has been identified in the soybean cultivar ‘Archer’. To characterize and genetically map this resistance to Pythium aphanidermatum, 84 F2:7 recombinant inbreed lines (RIL) from an ‘Archer’ X ‘Hutcheson‘ cross were evaluated for resistance to P. aphanidermatum by using seed plate and greenhouse assays. In the seed plate assay, germination ranged from 4 to 85% and, in the greenhouse assay, stands ranged from 21 to 94%. Archer and Hutcheson germination averaged 37 and 4%, and stands averaged 73 and 21% for the seed plate and greenhouse assays, respectively. Single nucleotide polymorphisms (SNP) analysis using a 6K SNP chip is being done to examine inheritance.<br /> <br /> In the Mississippi region, Soybean Charcoal Rot research with the following specific objectives was conducted; 1) Characterization of endophytic bacterial and fungal communities associated with the soybean-charcoal rot disease system using both culture-dependent and independent analyses; and 2) Investigation of the effects of inoculation of bacteria and/or fungi on disease development and soybean growth.<br /> <br /> Four pairs of diseased and asymptomatic plants were collected from patches of plants affected by charcoal rot in a soybean field in Leflore County near Sidon, MS. Four additional asymptomatic plants were collected from areas of the same field where no symptoms of charcoal rot disease were evident. Soil from the rhizosphere of only one plant had a plant parasitic nematode present at greater than threshold levels. Each plant was prepared for bacterial and fungal endophyte examination through a series of washing steps and longitudinal division of the plant. One half of the plant was processed for isolation of bacterial endophytes and the other half was used for isolation of fungal endophytes. Fungal endophytes were isolated from each plant part (root, stem, leaf) separately. Three sets of plates were prepared and incubated in the three different oxygen environments. Plates were incubated in the dark at 28oC for 48 to 72 hours, at which time emerged mycelium was subcultured onto sterile plates containing the appropriate medium and incubated in the same environment as the original isolation plate for approximately 10 days. Plates were initially grouped by morphological similarities (color, texture, pattern of growth, etc.). The ITS region was amplified using primers ITS1f and ITS4 and the amplified product sequenced. Identification of culturable fungal isolates has been completed from the 2013 field samples. The most commonly occurring species are Phoma sp., Penicillium chryseogenum, Diaporthe phaseolorum, Fusarium sp., Aspergillus flavus, Diaporthe sojae, Fusarium oxysporum, Trichoderma reesei, Hypocrea lixii, and Alternaria alternata. Continuing work includes the extraction of DNA from whole soybean plant tissues (plant DNA and DNA of associated microorganisms), followed by amplification of the ITS1 gene region of any fungi associated with these tissues. The reverse ITS primers (ITS4) used for this procedure contain unique barcodes, which will allow separation and distinction of any non-culturable species that are present within the plant tissue via Illumina whole-community sequencing. DNA extraction is currently underway and PCR required for identification of fungi using Illumina sequencing of ITS DNA.<br /> <br /> Sweetpotato Tip/End rot Research: Studies are ongoing to define and evaluate the pathogen(s) associated with this disease problem that was severe and widespread in 2009, but can still be found at high levels following harvesting. An initial study (Phase 1) was established in 2009 to determine the causal agents associated with tip/end rot and completed in 2013. Due to the thousands of isolates collected from traditional culturing, the identifications are now completed. During 2012 it was determined that reniform nematode, Rotylenchulus reniformis, levels were increasing in many fields from a general survey and was suspected that they might form a disease complex resulting in greater tip/end rot in Mississippi. Research was established in 2013 to evaluate those interactions with the most common pathogen isolated, Macrophomina phaseolina and reniform nematode using different populations in the field and in the greenhouse (Phase 2). <br /> <br /> Phase 1: Multivariate analysis was conducted on the fungal isolate population to determine species richness, evenness, and diversity. The relative species richness for each confirmed fungi was compared using PC-ORD multivariate analysis software. Results show a wide distribution of species across many management periods, but the harvest and post-harvest sampling periods show the greatest species richness over both years. A number of fungi, Macrophomina phaseolina, Aspergillus flavus, A. niger, A. tubingens, A. japonicus, and six species of Fusarium, were pathogenic in trials on disease-free sweetpotato root tissue. In addition, F. oxysporum and F. solani consistently produced necrotic lesions in root tissue. These two species accounted for nearly 70% of the overall isolates from early-season seed stock and bedding plant samples. Microbial populations in post-harvest tissues also differed in relative abundance from prior sampling dates. Macrophomina phaseolina increased to 27% occurrence and F. oxysporum and F. solani decreased to 15.5% isolation frequencies between 60 and 90 days post-harvest. The relative occurrence of each of the identified pathogens was also analyzed using PC-ORD. Fusarium species were the dominant pathogenic isolate recovered from plant tissues during early season sampling, but in harvest through post-harvest sampling, Macrophomina phaseolina became the dominant pathogenic fungi recovered from plant tissues. <br /> <br /> Phase 2: We have used data from year 1 of our ongoing study for 2013-14, funded by the USDA Specialty Crops Research Initiative grant, to clearly define the relationship between the organisms, categorize risks to sweet potato growers, and propose management strategies to minimize assessed risks. In both 2013 and 2014 growing seasons, two fields with known histories of high nematode densities were sampled using plots of 1/1000 acre. Treatments within each field site were based on reniform counts including low range and high range. Twelve replicate plots per treatment were established per location. Data collection included destructive sampling of plants within each of the small plots at intervals. Plant tissues collected during destructive sampling were examined for nematode damage and fungi from samples are being cultured on PDA. Identified fungi will be recorded and placed in long-term storage. Harvestable yield data will be collected for each plot at the end of the growth cycle when the field is harvested by the grower. <br /> <br /> In Tennessee, a provisional patent application for one or more seed treatment supplements for improved control of seedling diseases of snap bean, soybean, and other agricultural or horticultural crops was submitted. Research at the University of Tennessee’s West Tennessee Research and Education Center evaluated the effects 12 seed treatment supplements with four rates of potash fertilization for their effects on seedling diseases of soybean, plant growth, and soybean yield in a field naturally infested with several soil-borne plant pathogens. Pathogens isolated from diseased seedlings during this experiment were tentatively identified as Rhizoctonia solani (57%). Fusarium spp. (39%), and Macrophomina phaseolina (4%). Increasing the rate of potash fertilization increased seedling disease incidence and decreased seedling emergence, seedling vigor, plant stand, plant growth and height, and soybean yield. Adding a supplement to a common soybean seed treatment decreased the overall loss of plant stand and increased early flowering. Adding a seed treatment supplement to the common soybean seed treatment increased soybean yield by over 320 kg/ha. Energy-dispersive X-ray analyses of the outer cell layers of lateral roots of 6-day-old soybean seedlings grown from seed with a seed treatment supplement had higher levels of calcium + magnesium than seedlings grown without the supplement.<br /> <br /> Research conducted at Texas A&M University focused on determining the impact of oilseed meals on fungal community composition and Fusarium wilt of chili pepper. Soil was amended with various seed meals [Camelina sativa ‘Crantz’ (CAME), Brassica juncea ‘Pacific Gold’ (P), Sinapis alba cv. ‘IdaGold’(I) and PI (mixture of P and I with 1:1 ratio)], incubated for 25 days, and grown with chili pepper in pots for 35 days. Soil microbial populations were characterized using qPCR and pyrosequencing. Fungal abundance was significantly increased by CAME, P and PI amendments, but did not correspond to disease incidence. The seed meal amendments also affected the fungal community composition. Specifically, CAME enriched Mortierellales including the genus Mortierella, whose abundance was not correlated disease incidence. P and PI enriched Chaetomium, whose abundance was negatively correlated with disease incidence, and also inhibited Hypocreales, including the genus of Fusarium, whose abundance was positively correlated with disease incidence. A manuscript based on these results was submitted for publication.

Publications

Broders, K.D., Parker, M., and Boland G. J. 2014. Diversity of Rhizoctonia solani associated with canola and wheat in Alberta, Manitoba, and Saskatchewan. Plant Disease. 98:1695-1701.<br /> <br /> Canaday, C. H., 2014, Effects of experimental seed treatment supplements on seedling diseases of spring-planted snap bean, 2013, Plant Disease Management Reports 8:ST024.<br /> <br /> Canaday, C. H., 2014, Effects of potash and experimental seed treatment supplements on seedling diseases of summer-planted snap bean, 2013, Plant Disease Management Reports 8:ST025.<br /> <br /> Cochran, K.A., and Rothrock, C.S. 2015. Brassica green manure amendments for management of Rhizoctonia solani in two annual ornamental crops in the field. HortScience (accepted).<br /> <br /> Donald, P., K. Lawrence, T. Kirkpatrick, B.Kemerait, J. Bond, D. Herschman, C. Overstreet, A. Wrather, G. Lawrence, S. Koenning, P. Adugelo and C. Canaday. 2014. Occurrence, distribution, and impact of nematodes in soybean fields in the southern United States. Journal of Nematology 46(2):154.<br /> <br /> Jaraba, J., Rothrock, C. S., Kirkpatrick, T. L., and Brye, K. R. 2014. Soil texture influence on Meloidogyne incognita and Thielaviopsis basicola and their interaction on cotton. Plant Dis. 98:336-343.<br /> <br /> Jianhong Xu, Peng Deng, Kurt C. Showmaker, Hui Wang, Sonya M. Baird, and Shi-En Lu. 2014. The pqqC gene is essential for antifungal activity of Pseudomonas kilonensis JX22 against Fusarium oxysporum f. sp. lycopersici. FEMS Microbiology Letters 353(2):98-105.<br /> <br /> Kamoun S, Furzer O, Jones JD, Judelson HS, Ali GS, Dalio RJ, Roy SG, Schena L, Zambounis A, Panabières F, Cahill D, Ruocco M, Figueiredo A, Chen XR, Hulvey J, Stam R, Lamour K, Gijzen M, Tyler BM, Grünwald NJ, Mukhtar MS, Tomé DF, Tör M, Van den Ackerveken G, McDowell J, Daayf F, Fry WE, Lindqvist-Kreuze H, Meijer HJ, Petre B, Ristaino J, Yoshida K, Birch PR, Govers F. 2014. The Top 10 oomycete pathogens in molecular plant pathology. Molecular Plant Pathology doi: 10.1111/mpp.12190. <br /> <br /> Loynachan, T. E. 2013. Experience in teaching a distance agronomy soil-plant graduate course. 275-9 Agron. Abstracts, Madison.<br /> <br /> Magbanua V. Zenaida, Arick Mark, Buza Teresia, Hsu Chuan-Yu, Showmaker C Kurt, Chouvarine Philippe, Deng Peng, Peterson G. Daniel, Lu Shi-En. Transcriptomic dissection of the rice-Burkholderia glumae interaction. BMC Genomics 15:755; doi:10.1186/1471-2164-15-755.<br /> <br /> Sandesh Shrestha, Jian Hu, Rebecca Trout Fryxell, Joann Mudge and Kurt Lamour. 2014. SNP markers identify widely distributed clonal lineages of Phytophthora colocasiae in Vietnam, Hawaii, and Hainan Island, China. Mycologia (in press).<br /> <br /> Spurlock, T. N., Kirkpatrick, T. L., Rothrock, C. S., and Monfort, W. S. 2014. Displacement of Meloidogyne incognita by Rotylenchulus reniformis during a 10 year cotton monoculture and implications for site-specific management. Journal of Nematology 46:238 (Abstr.) <br /> <br /> Spurlock, T. N., Kirkpatrick, T. L., Rothrock, C. S., and Monfort S. 2014. Evaluation of methods to quantify populations of Rhizoctonia spp. in soil. Plant Dis. (accepted).<br /> <br /> Spurlock, T. N., Greer, A. M. Tolbert, A. C., Kirkpatrick, T. L., Rothrock, C. S., and Monfort S. 2014. Spatiotemporal distribution of Thielaviopsis basicola after a ten year cotton monoculture. Phytopathology 104(Suppl.):S3.113.<br /> <br /> Urrea, K. E., Rupe, J. C., Rothrock, C.S., and Chen, P. 2014. Characterization of soybean resistance to Pythium aphanidermatum. Phytopathology 104 (Suppl.):S3.121.<br /> <br /> Wang X-Q, Showmaker KC, Yu X-Q, Bi T, Hsu C-Y, Baird SM, Peterson DG, Li X-D, Lu S.-E. 2014. Draft genome sequence of Burkholderia pyrrocinia Lyc2, a biological control strain that can suppress multiple plant microbial pathogens. Genome Announcements 2(5):e00991-14. doi:10.1128/genomeA.00991-14.<br /> <br /> Yan Li, Gary W. Lawrence, Shi-En Lu, Clarissa Balbalian, and Vincent Klink. 2014. Quantitative field testing Heterodera glycines from metagenomic DNA samples isolated directly from soil under agronomic production. PLoS One. 9(2): e89887. doi:10.1371/ journal.pone.0089887.<br /> <br /> Youzhou Liu, Shi-En Lu, Sonya Baird, Junqing Qiao, and Yan Du. 2014. Draft genome sequence of Pseudomonas chlororaphis YL-1, a biocontrol strain suppressing plant microbial pathogens. Genome Announcements 2(1): e01225-13.<br />

Impact Statements

1. The impact of R. reniformis on soybean is not well understood; however, the findings of a 10-year study indicated that R. reniformis could displace M. incognita where cotton was predominantly grown. Currently, nematode sampling in soybean is being subsidized by the Arkansas Soybean Promotion Board. Determinations of nematode populations based on soil textural maps and yield maps from previous years will allow estimations of impact to yield on a larger (spatial) scale and will aid farmers in management decisions moving forward.
2. Loynachan serves as the regional coordinator for ?Ask a Soil Scientist? maintained by the Soil Science Society of America: https://www.soils.org/ask. Loynachan maintains a URL on Soil Biology (http://agron-www.agron.iastate.edu/~loynachan/mov/) and responds weekly to questions asked by users of the information. These inquires vary from individuals in grade school to fellow scientists worldwide.
3. Some isolates of bacteria from Mississippi have showed a great potential as biological control agents in plant disease management. Discovery of the genes, gene clusters and genome sequencing will help us understand molecular mechanisms of antimicrobial activities and develop biological control agents for plant disease management. The model for rice resistance to the bacterial panicle blight would be beneficial to development of disease resistant varieties of rice.
4. Novel markers for fungal and oomycete plant pathogens were developed in Tennessee.

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Report Information

Participants

Fulya Baysal-Gurel, Tennessee State University
Carla Garzon, Oklahoma State University
Franz Lichtner, Colorado State University (representing Kirk Broders)
Craig Rothrock, University of Arkansas
Luisa Santamaria, Oregon State University
Harald Scherm, University of Georgia (Administrative Advisor)

Invited and student attendees:
George Abawi, Cornell University
Maria Soledad Benitez, USDA-ARS Brookings, South Dakota
Nathan Walker, Oklahoma State University
Stephen Marek, Oklahoma State University
Fernanda Proaño-Cuenca, Oklahoma State University
Andres Espindola, Oklahoma State University

Brief Summary of Minutes

Multi-State Project Meeting S-1053

Minutes and notes from the Annual Meeting

December 4th, 2015

 

Time: 9 am- 4 pm CST

Location: Noble Research Center – Oklahoma State University, Stillwater, OK

 

Member attendees:

Fulya Baysal-Gurel                Tennessee State University

Carla Garzon                          Oklahoma State University

Franz Lichtner                       Colorado State University (representing Kirk Broders)

Craig Rothrock                      University of Arkansas

Luisa Santamaria                  Oregon State University

Harald Scherm,                      University of Georgia (Administrative Advisor)

 

Invited and student attendees:

George Abawi                                    Cornell University

Maria Soledad Benitez          USDA-ARS  Brookings, South Dakota

Nathan Walker                      Oklahoma State University

Stephen Marek                      Oklahoma State University

Fernanda Proaño-Cuenca    Oklahoma State University

Andres Espindola                  Oklahoma State University

 

Luisa Santamaria (Chair) provided a brief overview of her applied research projects on root rot diseases in ornamentals caused by Phytophthora plurivora and Pythium. Also presented her current extension and outreach programs in and effort to educate the workforce at the nursery industry in Oregon using technology and hybrid classes.

 

Harald Scherm, the Administrative Adviser for S-1053, reported on a few national issues of interest. He mentioned the chartering of the public-private Foundation for Food and Agriculture Research which emerged out of the 2014 Farm Bill. Once matching private funds are secured, the Foundation will have $200 million to invest in research in two priority areas, 1) More productive, sustainable agriculture, and 2) Better health through food. The former priority area may be of particular interest to the group since it includes a sub-theme titled "Transforming soil health."

Scherm also mentioned the discussions that are currently ongoing at the national level regarding USDA's new Open Access implementation plan, which will have important implications for making both publications and the digital data that led to the conclusions in these papers available in publicly accessible and curated data bases. The details of how, where, and in what format these data should be deposited, and who pays for long-term duration, is still being worked out at this time.

 

With regard to NIFA funding programs, Scherm reported that the Center of Excellence provision will be carried forward into the FY16 RFAs, although the language may change based on review panel feedback during the previous funding cycle. If multiple members of S-1053 participated in a grant proposal, it should be relatively straightforward to make the case for a Center of Excellence.

 

Carla Garzon and her students provided information regarding their current research on characterization and identification of Pythium and Globisporangium species present in ornamental greenhouses from Long Island, NY in 2014 and the draft genome of Sclerotinia minor.

 

Fulya Baysal-Gurel provided information regarding current research on soil- borne diseases of woody ornamentals in nursery production systems in Tennessee. 

 

Craig Rothrock provided information on Rhizoctonia and spatial soilborne disease pattern research in Arkansas.

 

Franz Lichtner on behalf of Kirk Broders provided an overview of work related to soil microbial community change over time and space relative to plant variety mixtures.

 

Further discussions:

Possibility of meeting at the 2016 APS Annual Meeting in Tampa, Florida as a group to enhance collaboration and interaction in the group. Harald Scherm, Carla Garzon and Fulya Baysal-Gurel will be sending out e-mail in March to determine the meeting details.

 

Possibility of pre-application for USDA-NIFA SCRI Research and Extension Planning Project in December 2016.  Fulya Baysal-Gurel will be sending out request to set up conference call in June 2016.

Business meeting:

Location for the next meeting was discussed and the meeting will be held in Tennessee (Nashville or McMinnville). The next chair (Craig Rothrock) and the new secretary (Fulya Baysal-Gurel) will be informing the group members regarding meeting date and location.

 

Fulya Baysal-Gurel was elected as the new secretary of the group for 2016.

Accomplishments

<ol><br /> <li><strong><em> Evaluate the population genetic diversity of soilborne pathogens and antagonistic microorganisms in different growing systems and regions using traditional and metagenomic approaches.</em></strong></li><br /> </ol><br /> <p>Members of this multistate research project continue to provide new insight into the genetic diversity of a number of oomycete pathogens in a range of production systems. Oomycetes including <em>Pythium</em> and <em>Phytophthora</em> continue to cause significant damage on economically important vegetable, fruit, ornamental, and row crops in North America and around the world. New genetic resources and many new molecular markers were developed for the soilborne vegetable pathogen <em>Phytophthora capsici</em>. These include genomic DNA re-sequencing data for 18 <em>P. capsici</em> isolates recovered from locations worldwide and RNA sequencing data for 35 isolates of <em>P. capsici</em> at two time-points in the infection process of tomato (24 and 72 h). These resources provided the raw material to identify widely distributed and conserved polymorphic single nucleotide polymorphism (SNP) positions in the genome and to develop genotyping assays to assess genetic diversity in populations worldwide. Ongoing analyses of oomycetes baited from Tennessee streams and rivers (predominantly <em>Phytophthora</em> and <em>Pythium</em>) have identified species known to be pathogens of economically important nursery and forest plants.</p><br /> <p>Population genetic analyses have been completed for <em>P. capsici</em> in central and northern Mexico, indicating the populations are likely sexual and survive fallow or non-host periods as sexual oospores in the soil. In addition, novel SNP markers have been developed for the spinach downy mildew pathogen <em>Peronospora effusa</em> and were recently applied to populations from spinach production areas in Yuma, AZ and the Salinas Valley in CA.</p><br /> <p>Further research on oomycete pathogens has focused on assessing the genetic diversity of <em>Phytopthora infestans</em> inoculum in Andean soils. This work was presented in two B.S. Biotechnology Engineering dissertations written by two undergraduate students from the Universidad de las Fuerzas Armadas ESPE, who graduated in Summer 2013 in collaboration with researchers at Oklahoma State University (OSU). In related work at OSU, the multi-locus phylogenetic analysis of the <em>Pythium irregulare</em> complex was conducted confirming the species status of <em>P. cryptoirregulare</em> and <em>P. vipa</em>. Additionally, characterization of <em>Pythium</em> isolates from ornamental greenhouses in Long Island, NY in 2012 and 2013 by molecular barcoding (ITS) was completed. Current research is focused on the temporal analysis of these isolates, to understand seasonal changes in <em>Pythium</em> populations in ornamental operations.</p><br /> <p>Also at OSU, primers for PCR amplification of SSR markers for <em>Phymatotrichopsis omnivora</em> (cotton root rot) were developed. The genetic diversity and phylogenetic status of <em>Fusarium proliferatum</em> isolates from diverse continents and hosts was evaluated and confirmed to be monophyletic. SSR markers for characterization of <em>F. proliferatum</em> were validated and reported. A study on the genetic diversity of <em>F. proliferatum</em> in onions in Ecuador is in progress. A bioinformatics tool for detection and discrimination among <em>Pythium</em> spp. strains from metagenomic data from infected plant samples was developed and validated. Primers for multiplex PCR detection and discrimination of <em>Ophiosphaerella</em> spp. were designed and validated. A draft genome of <em>Sclerotinia minor</em> was assembled and is currently being annotated. E-probes for detection of expressed genes during the <em>S. minor</em> infection were generated and are being validated.</p><br /> <p>In related work taking place at Oregon State University, a new project funded by the Floriculture and Nursery Research Initiative (FNRI) to be developed in a collaborative agreement with the USDA-ARS Horticultural Crops Research Laboratory &ndash; Corvallis, will identify and characterize the most prevalent soilborne plant pathogens, including <em>Phytophthora, Pythium,</em> and<em> Verticillium</em> affecting commercial nursery production systems; develop novel and rapid assays to detect the most prevalent soilborne pathogens; and characterization of the roles of soil fungal communities in nutrient cycling and soil health.</p><br /> <p>Work in Minnesota has also focused on understanding the diversity of <em>Pythium</em> species present in crop soils using two different methods for collection of isolates. The two methods differed considerably in their efficiency. In one survey, <em>Pythium</em> species were obtained by baiting soil samples collected from 32 locations in Minnesota where stand establishment had been a problem. Only five species were identified in this survey. <em>Pythium irregulare, Pythium recalcitrans,</em> and <em>Pythium ultimum</em> were the predominant species. A related survey for <em>Pythium</em> species was conducted by isolating from symptomatic plants collected in the field. In this survey 30 <em>Pythium</em> species were isolated. The most common species found were <em>Pythium ultimum,</em> <em>Pythium heterothallicum, Pythium sylvaticum, </em>and<em> Pythium oopapillum.. </em>In both surveys <em>Pythium</em> species were identified and identity confirmed using both morphological and molecular techniques. These isolates are being screened for pathogenicity on corn and soybean.</p><br /> <p>During 2014-2015 the Minnesota researchers sampled seedling and soil from 49 soybean production fields that yielded 30 <em>Pythium </em>species and four <em>Phytopythium</em> species. The species were identified using ITS and COX1 sequences. Pathogenicity was evaluated in an in-vitro assay at three temperatures. Seven of the 21 <em>Pythium </em>spp. were pathogenic on both soybean and corn, reducing root growth by 20% or more, while two <em>Pythium</em> and one <em>Phytopythium</em> spp. were pathogenic only on soybeans. This is the first report of <em>P. recalcitrans</em> as a pathogen of soybean and of <em>Phytopythium</em> spp. as a pathogen on either corn or soybean in Minnesota. <em>P. ultimum</em> and <em>P. irregulare</em> were the most pathogenic <em>Pythium</em> species on either soybeans or corn. <em>P. conidiophorum</em>, <em>P. irregulare</em>, <em>P. intermedium</em>, <em>P. ultimum</em>, and <em>P. sylvaticum</em> were pathogenic on soybean at the three temperatures. <em>P. acrogynum</em>, <em>P. inflatum</em>, <em>P. sylvaticum, P. ultimum</em> increased in pathogenicity with increasing temperature. On corn <em>P.antheridium, P. conidiophorum, P. intermedium, P. irregulare, P. heterothallicum, P. minus, P. recalcitrans, P. sylvaticum, and P. ultimum</em> were pathogenic at 15 and 20^oC. At 25^oC pathogenicity of <em>P. heterothallicum, P. minus, P recalcitrans, P. schmitthenneri,P. torulosum</em> increased further.</p><br /> <p>Research on sweetpotato tip/end rot is focused on defining and evaluating the pathogen(s) associated with this disease problem. In 2012 it was determined that reniform nematode, <em>Rotylenchulus reniformis</em>, levels were increasing in many fields from a general survey and was suspected that they might form a disease complex resulting in greater tip/end rot in Mississippi. Research was established in 2013 to evaluate those interactions with the most common pathogen isolated, <em>Macrophomina phaseolina</em> and reniform nematode using different populations in the field and in the greenhouse. In 2014 and 2015, field and greenhouse trials were established and new trials for 2015-2016 are currently ongoing to study <em>M. phaseolina</em> isolates separately from the nematodes and together. Data from 2014-2015 (year 1 of interaction study) have not been analyzed and will be discussed during the next annual report.</p><br /> <p><strong>Also at Mississippi State University, research on soybean charcoal rot is ongoing with the </strong>specific objectives 1) Characterization of endophytic bacterial and fungal communities associated with the soybean-charcoal rot disease system using both culture-dependent and independent analyses; and 2) Investigation of the effects of inoculation of bacteria and/or fungi on disease development and soybean growth.</p><br /> <p>Four pairs of diseased and asymptomatic plants were collected from patches of plants affected by charcoal rot in a soybean field in Leflore County near Sidon, MS in 2013 and 2014 (completed really in 2015). Four additional asymptomatic plants (controls) were collected from areas of the same field both years where no symptoms of the disease were evident. Each plant was prepared for bacterial and fungal endophyte examination through a series of washing steps and longitudinal division of the plant. One half of the plant was processed for isolation of bacterial endophytes and the other half was used for isolation of fungal endophytes. Fungal endophytes were isolated from each plant part (root, stem, leaf) separately. Three sets of plates were prepared and incubated in the three different oxygen environments. Plates were incubated in the dark at 28^oC for 48 to 72 h, at which time emerged mycelium was subcultured onto sterile plates containing the appropriate medium and incubated in the same environment as the original isolation plate for 10 days. Plates were initially grouped by morphological similarities. Cultural identifications for the two years of isolations continued into 2015 to further distinguish the groupings prior to molecular determinations. The ITS region of grouped isolates were amplified using primers ITS1f and ITS4 and products sequenced. Approximately 70% of the isolate sequences had acceptable reads for NCBI Library Blast determinations thus far and the other 30% are currently being regrown and re-sequenced. Approximately 14 taxa were identified with the most common ones being <em>Fusarium solani </em>(complex) generally found on the healthy plants, two <em>Alternaria </em>species and <em>Fusarium chlamydosporum </em>common on diseased soybeans, whereas two species of <em>Phoma&nbsp; </em>and two <em>Trichoderma </em>species had greatest frequencies on healthy plants. &nbsp;Many of the other fungal species found are antagonistic or may be biological controls. Other species of <em>Fusarium </em>can vary in their ability to derive nutrition but, for example, <em>Fusarium solani </em>strains have been shown to be effective in biological control of root pathogens. Furthermore, <em>Verticillium lecanii</em> identified here can be antagonistic to insects and is being used commercially for their control, but the fungus has been reported to be a pathogen of other fungi. <em>Cladosporium sphaerospermum</em> present in plant tissues is known to produce compounds that increase plant growth. However, the fungus was found equally on diseased and healthy plants. Additionally, over 20 other fungi identified across tissue types were primarily considered plant pathogens. Among the most abundant fungus species identified using the NCBI database, six belonged to the <em>Diaporthe</em> spp. complex and five to <em>Fusarium </em>spp. with the most common taxon <em>Fusarium solani</em>.</p><br /> <p>In 2015, library preparations were conducted for Illumina MiSeq whole-community study. From all tissues evaluated using cultural isolates, a portion of the cultured pieces was saved at -80&deg;C. Data analysis training was done in November and actual analysis using the Mothur program is currently ongoing. Since the same tissue pieces were used for culture study, an attempt will be made statistically to compare those data with whole-community results. These efforts will go into 2016 and will be reported during the annual reporting cycle of next year.</p><br /> <p>Research on <em>Rhizoctonia</em> spp. conducted in the southern U.S. has characterized the spatial distribution of <em>Rhizoctonia</em> spp. in fields undergoing rice and soybean rotation in Arkansas. Rhizoctonia aerial blight of soybean is a disease caused by <em>Rhizoctonia solani</em> AG1-IA. This pathogen also causes sheath blight of rice. Populations and disease assessments were characterized in producers&rsquo; fields on a spatial scale to represent the topography of the field intermittent of the rice levee positions. Directional distribution ellipses for distribution of <em>R. solani</em> AG1-IA using soil and plant samples indicated agreement with drainage. Across years, distribution of <em>R. solani</em> AG1-IA appears to be controlled by levee position. Where levees do not form logical areas of collection, the greatest concentration of inoculum appears to be in the lower elevations of the field. The spatial distribution of the early-season inoculum of <em>R. solani</em> AG1-IA reiterates the idea that inoculum in the form of sclerotia and hyphae associated with rice residue floating and collecting at lower points within the levees. Spatial assessments of incidence of aerial blight and the height up the soybean plant at GPS positions intermittent the levee system validated models, indicating the highest amount of disease was in the logical areas of collection (a combination of levee position and elevation). Because the disease occurred in a similar distribution as the inoculum, a predictive model using geographically weighted regression analysis is being constructed for fields with no prior disease assessment. In these fields, elevation data has been provided by USGS (LiDAR) and levee data provided by the grower. Using a quantitative determination of levee bends (m/m²), prediction based on random assignment of geospatially tagged points should allow for the use of handheld GPS devices to guide scouting to areas with the highest likelihood of inoculum collection and aerial blight development.</p><br /> <p>Spatial sampling of soybean plants further confirmed <em>R. solani</em> AG11 as the most populous <em>Rhizoctonia</em> spp. in fields undergoing rice and soybean rotations in northeast Arkansas, on 25 to 34% of soybean plants. However, a new site in southeast Arkansas found higher level of <em>R. solani</em> AG7 (12 to 16 % colonization of soybean) than AG11 (6 to 10%). AG1 was rarely or never recovered. Application of the fungicide azoxystrobin limited the increase in colonization of soybean plants by <em>R. solani</em> compared with points not sprayed at both sites. The research reinforces the idea that populations of <em>R. solani</em> in soil colonize crops within the field and are dominated by populations that are not considered pathogenic.</p><br /> <p>Because prior years&rsquo; work had determined that <em>R. solani</em> AG11 was associated with areas of greater plant stand, experiments are currently in progress to determine its role in the rice/ soybean system and the potential benefit colonization of soybean may provide. <em>R. solani </em>AG1-IA also was observed causing aerial blight of peanut in Arkansas. Currently, there is a renewed interest in peanut production in the state, and production practices include irrigation and various rotation schemes with soybean and less frequently rice. Thus, this may be an important disease of peanut in Arkansas.</p><br /> <p>In 2015, the Arkansas research group also evaluated other soilborne pathogens. <em>Meloidogyne incognita</em> and <em>Rotylenchulus reniformis</em> are economically important plant pathogenic nematode species in cotton production. <em>Thielaviopsis basicola</em> causes black root rot of cotton. For cool and wet soils, black root rot results in stunting and reduced vigor that can negatively impact yield. When either of these nematodes feed on cotton roots, black root rot can worsen. Evidence from 2001-2003, in a texturally diverse cotton field near Portland, AR, suggested that populations of <em>M. incognita</em> had the greatest effect on yield in soils with greater sand content. Additionally, exhaustive nematode assays indicated that <em>R. reniformis</em> was increasing in population and distribution in the field. Since that work, the field has continued to be planted in cotton until being planted in soybean in 2013. From the original 512 plots, nematode and <em>T. basicola</em> population densities were measured through 2013. Over the 10+ year period, the population and distribution of <em>M. incognita</em> decreased while that of <em>R. reniformis</em> increased. <em>Thielaviopsis basicola</em> populations and distributions were consistent. There was significantly aggregated distributions of all pathogens in all years except 2011, and trend surface models indicated <em>Range</em> values decreased for <em>M. incognita</em> but increased for <em>R. reniformis</em> prior to 2013. The analyses further indicated a significantly dispersed distribution and inverse relationship in 3 of 4 years and 2 of 4 years respectively confirming competition and displacement of <em>M. incognita</em> by <em>R. reniformis</em>. In all years, a significant negative correlation existed between <em>T. basicola</em> and percent sand fraction and a significant positive correlation between the fungus and percent silt and clay. Pathogen populations were not related to yield loss across soil textures. The greatest yield consequence occurred in the sandier areas of the field, indicating these three pathogens should be managed site-specifically by textural zone.</p><br /> <p><strong><em>&nbsp;</em></strong></p><br /> <ol start="2"><br /> <li><strong><em> Examine the effect of traditional or newly developed management strategies (chemical, cultural, and biological), soil physicochemical properties, or introduced biological control agents on the microbial community and its ability to suppress soilborne pathogens.</em></strong></li><br /> </ol><br /> <p>In Iowa, work is being conducted on the importance of the soil microbial community in carbon cycling as plant residues are decomposed. The researchers studied the partitioning of carbon contained in corn residue during decomposition, affecting the sequestration of carbon in soil, into microbial biomass and carbon dioxide respiration. They found weak evidence that pH-stressed bacterial cells use more carbon for cell maintenance and produce less biomass in an incubation study.</p><br /> <p>Corn residue was collected from the field and characterized for carbon, nitrogen, and other elements. They isolated three bacilli (<em>Bacillus pasteurii, B. circulans, and B. sphaericus</em>). A Webster soil (Typic Endoaquolls) was sampled and analyzed for nutrients and moisture tension. The pH of the soil was adjusted from 6.5 to 7.5 and 5.5 to test the hypothesis that stressed cells (in this case pH-stressed) would use more carbon during residue decomposition for cell maintenance and less for new cell production. Corn residue was added and mixed into the test soil in Mason jar mesocosms and autoclaved. A known quantity of each <em>Bacillus</em> sp. was individually added and incubated at room temperature (20-25&deg;C). <em>B. pasteurii</em> degraded the corn residue most rapidly, with the rate of decomposition being greater at pH 7.5 than 6.5 and 5.5. All other treatments followed a similar pattern having greater rates of decomposition at pH 7.5. Microbial biomass C for all treatments also followed a similar pattern and increased to a peak on day 21, where microbial biomass carbon (MBC -- the amount of the carbon in the residue that ends up in the living tissue of the soil) was significantly different. The lowest MBC values were observed at pH 5.5 with <em>B. circulans</em>, followed by <em>B. sphaericus</em>, and then <em>B. pasteurii</em>. The least amount of MBC was observed when the rate of CO₂ evolution was at its greatest. Our data provided weak evidence that stressed cells required more metabolized C for cell maintenance, but differences existed among the three isolates and an overall generalization could not be made.</p><br /> <p>The project has provided for the training of an M.S. student in soil microbiology and biochemistry. Furthermore, students in an undergraduate/graduate dual-listed course (Agron 485/585) helped evaluate the technique used to measure carbon dioxide evolutions. Additionally, one undergraduate student gained laboratory experiences during the summer of 2015.</p><br /> <p>Multiple members of the multistate research project continue to investigate better management strategies for the control of <em>Pythium</em> and the effect different chemical, biological and cultural management strategies have on <em>Pythium</em> community dynamics. In Oregon, an <em>in vitro</em> sensitivity test to determine fungicide resistance of three species of <em>Pythium </em>(<em>P. irregulare, P. sylvaticum, and P. ultimum</em>) was completed. A total of 119 <em>Pythium</em> isolates from forest nurseries in Oregon were tested for resistance to mefenoxam. Six different concentrations of the fungicide were evaluated. A paper with this data and additional results from evaluations with other fungicides was submitted for review in Plant Disease.</p><br /> <p>During 2015, at Oregon State University &ndash; NWREC, efforts to establish guidelines for developing and implementing disease control strategies in managing Phytophthora root rot continues. We have an ongoing trial to determine effectiveness of varying concentrations of calcium amendments, in the form of gypsum, against inoculum of two soilborne pathogens, <em>Phytophthora cinnamomi</em> and <em>P. plurivora</em>. These two species are of most concern in terms of crop loss at nurseries (Parke et al., 2014). Evidence suggests that calcium amendments in plant growth substrates can be effective in controlling <em>P. cinnamomi</em> root rot of avocado (Messenger, et al. 2000). <em>P. plurivora</em> is a recently discovered species causing severe root rot on many plant hosts in European nurseries. Both pathogens have wide host ranges. However, growers do not know the economic risk associated with these pathogens in their nurseries, and there is little information about disease control treatments that are effective for managing these pathogens.</p><br /> <p><em>Pythium </em>is an ecologically diverse group of microorganisms found in virtually all soils, but little is known about the effect of crop rotation on <em>Pythium </em>communities. To understand the effect of crop rotation on species diversity, researcher in Arkansas collected soil from plots following a long-term rotation study including rice, corn, soybean and wheat. Soil from each plot was placed in cups, wetted to saturation, planted with the soybean cultivar Hutcheson, and incubated at 25&deg;C. After 3 days, seeds were collected and washed in running water and placed on 2% water agar to bait <em>Pythium </em>from soils. Molecular identification was performed by sequencing the ITS region and conducting a Blast analysis to a curated reference database. A total of 320 isolates were identified representing 12 species. Overall, the most frequently recovered species were <em>P. spinosum</em>, <em>P. irregulare</em>, <em>P. pereocandrum </em>and <em>P. sylvaticum</em>. In continuous rice production, <em>P. spinosum </em>was the most prevalent species isolated. In the soybean-wheat-rice and rice-wheat-soybean-wheat rotations, <em>P. spinosum </em>and <em>P. irregulare </em>were the most frequently recovered, while <em>P. irregulare </em>and <em>P. sylvaticum </em>were the most frequently recovered species in the soybean-rice and rice&ndash;soybean rotations. <em>Pythium </em>species composition reflected the frequency at which a susceptible host was planted in the rotation.</p><br /> <p>Sudden death syndrome of soybean (SDS) is caused by the fungus <em>Fusarium virguliforme</em>. The fungus infects and initially causes deterioration of the crown and upper taproot. As the plants enter the reproductive stages of development, foliar symptoms are often observed where reddish brown lesions form on the leaves and defoliation occurs after significant leaf damage. In 2014, a field near Dumas, AR and a field near Yancopin, AR were confirmed to have SDS. At R5, aerial imagery was obtained by flying a Cessna 172 with a Geovantage Geoscanner sensor package having a 4-band multispectral unit utilizing blue, green and red light wavelengths in the visible part of the spectrum and near-infrared (NIR) beyond the red visible light bands. The near infrared imagery was georeferenced and added as a layer in ArcGIS 10.2. Yield data was collected on a John Deere 9870 combine with a factory installed yield monitor and stored as a georeferenced file for the field near Yancopin. Soil EC was collected for both fields with a Veris 3150 soil EC mapping system on 12-ft centers and stored as a georeferenced file. The yield and soil EC data were added to the same .mxd as the NIR aerial imagery. A field boundary was digitized in ARCMap and 5000 random points assigned within the boundary using the random points tool in ArcToolbox. The NIR, yield, and soil EC were sampled at each random point using the spatial join tool in ArcToolbox. Data were then analyzed using Moran&rsquo;s <em>I</em> to determine spatial autocorrelation and distribution and spatial regression to determine spatial dependence and relationships in GeoDa 1.6.6. In both fields, NIR revealed severe localized defoliation. Spatial regression analysis indicated a significant (<em>P</em>=<em>0.05</em>) relationship between soil texture and defoliation where the majority of the disease was correlated with soil EC readings in the 20-35 ds/m range. Yield data was only obtained and analyzed for one field, with yield correlating negatively to both soil texture and defoliation. The evidence suggests that SDS could be managed site-specifically. This method could be applied to determine the distributions of other soilborne diseases or potential interactions with SDS.</p><br /> <p>Brassica green manure soil amendments were investigated as a possible alternative to chemical management of soilborne diseases in ornamental landscape and bedding plants. The objective was to determine the importance of crop selection and application rate of brassica green manures for disease caused by <em>Rhizoctonia solani</em> on impatiens and petunia. Microplot experiments were conducted over 2 years using brassica green manure from <em>Brassica juncea</em> 'Fumus' and 'Bionute', and <em>Brassica napus</em> 'Jetton', at the application rates of 700, 1400, and 4200 g/m² fresh weight aboveground biomass. Microplots were artificially infested to evaluate disease on these ornamentals, with a second set of experiments using noninfested plots to examine effects of the green manure alone on plant growth. All brassica green manure crops reduced disease symptoms in both impatiens and petunias. Rate of brassica application was more important than brassica crop variety for use as a green manure. The highest rate of the brassica green manure decreased crown lesions by 21 and 24%, root discoloration by 9 and 7%, and <em>R. solani</em> isolation by 15 and 8% for impatiens and petunias, respectively, for 4200 g/m² compared with 700 g/m² rates of application. No phytotoxic effects were observed from the brassica green manures following a waiting period of 4 weeks between amending the soil and planting the ornamental crops.</p><br /> <p>At OSU in 2015, Fungicide hormesis was examined on <em>Fusarium proliferatum</em> using multiple modes of action. <em>F. proliferatum</em> radial growth in vitro was significantly and consistently stimulated by one contact fungicide. The effect of hormesis on fumonisin production was assessed and statistical analysis of the data is in progress.</p><br /> <p>Research at Mississippi State University has focused on understanding the genetics and biochemistry of soilborne and endophytic bacteria against plants pathogens. More than 800 bacterial isolates were obtained from soil and plant samples, and tested for antimicrobial activities using standard bioassay procedures. Approximately 80 isolates showed significant antifungal activities against <em>Geotrichum candidum</em> and more than 40 isolates exhibited significant antibiotic activities against <em>Erwinia amylovora</em>. These isolates were further purified and preserved at -80^oC. The isolates have been identified to be <em>Bacillus</em> spp., <em>Pseudomonas</em> spp<em>.,</em> <em>Burkholderia</em> spp. and <em>Streptomyces</em> spp. using 16S rDNA sequence analysis. In a separate study, we investigated the genes associated with production of the antifungal compound occidiofungin produced by the soilborne bacterium <em>Burkholderia contaminans</em> strain M14. In collaboration with Dr. Jim Smith, we have characterized the functions of the <em>ocfC</em> and <em>ocfN</em> genes in antimicrobial activities.</p><br /> <p>The etiology of root rot caused by <em>Fusarium solani</em> is being investigated in Minnesota. The effects of <em>F. solani </em>on soybean plant germination and growth is influenced by fungal isolate, soybean cultivar, stage of plant development, soil temperature, and soil moisture. In general, soil inoculation with <em>F. solani </em>caused poor germination and reduced plant populations. However, plants that did germinate and flowered appeared to benefit from infection with greater root and aboveground growth and greater seed yield. Research was conducted into the effects of inoculation methods and inoculum substrates infested with <em>F. solani</em> or <em>F. virguliforme </em>on disease symptom expression and plant growth. Management of this root rot fungus should emphasize control of its harmful effects on plant populations during seed germination. When investigating the etiology of <em>F. solan</em>i, spurious symptoms, particularly lesion-like root discoloration and poor root development, were observed when many infested inoculum substrates such as seed of sorghum, rye, barley, or oats are used to inoculate soil during screening experiments. Inoculation with uninfested seed resulted in poor root development and decreased plant growth when compared with either plants root-inoculated with infested substrates or with an uninoculated control. The effect is dependent on species of plant seed used (most severe effects were seen with red sorghum as a substrate) but is also determined by the soybean cultivar planted. These effects are particularly relevant for evaluation of soybean cultivars and evaluation of pathogenicity of fungal isolates and can cause misleading symptoms unrelated to the effect of the pathogen.</p><br /> <p>Sensitivity to azoxystrobin, ethaboxam, mefenoxam, pyraclostrobin, and trifloxystrobin of isolates of ten pathogenic <em>Pythium</em> species was tested in an in-vitro assay. Isolates of <em>Pythium</em> and <em>Phytopythium</em> species varied in sensitivity to the fungicides, but most were highly sensitive to mefenoxam and ethaboxam, with EC50 values &lt;10^-2 of that observed for the strobilurin fungicides. Only <em>P. acrogynum</em> did not display any sensitivity to ethaboxam at rates of 0.0, 0.5 or 5 &micro;g/ml. Treatment with 100 &micro;g/ml decreased growth by only 24%. However, only one isolate of <em>P. acrogynum</em> was available for testing and it is unknown if these results are representative of the species. The results indicate that <em>Pythium</em> spp. in Minnesota soybean fields are diverse, vary in sensitivity to fungicides, and may be significant causes of seedling disease on soybean and corn.</p>

Publications

<p>Castro-Rocha, A., Shrestha, S., Lyon, B., Grimaldo-Pantoja, G., Juan Pedro Flores-Marges, Jos&eacute; Valero-Galv&aacute;n, Marisela Aguirre-Ram&iacute;rez, Pedro Osuna-&Aacute;vila, Nuria G&oacute;mez-Dorantes, Graciela &Aacute;vila-Quezada, Jos&eacute; de Jes&uacute;s Luna-Ru&iacute;z, Gerardo Rodr&iacute;guez-Alvarado, Sylvia Patricia Fern&aacute;ndez-Pav&iacute;a and Kurt Lamour. 2016. An initial assessment of genetic diversity for <em>Phytophthora capsici</em> in northern and central Mexico. Mycological Progress 15:1-12.</p><br /> <p>Abd-Elmagid, A., Hunger, R., Garzon, C.D., Jub H, Payton M, and Melouk H. 2015. Effect of osmotic and matric potentials on <em>Sclerotinia minor</em> and <em>Sclerotinia sclerotiorum</em> virulence on peanut. Int. J. Phytopathol. 4:147-158.</p><br /> <p>Cochran, K. A., and Rothrock, C. S. 2015. Brassica green manure amendments for management of Rhizoctonia solani in two annual ornamental crops in the field. Hortscience 50:555&ndash;558.</p><br /> <p>Esp&iacute;ndola AS, Schneider W, Hoyt P, Marek SM, Garzon CD. 2015. A new approach for detecting fungal and oomycete plant pathogens in next generation sequencing metagenome data utilizing electronic probes. International Journal of Data Mining and Bioinformatics 12(2):115-128.</p><br /> <p>Greer, A. M., Spurlock, T. N., Coker, C. M. 2015. First report of Neocosmospora stem rot of soybean caused by <em>Neocosmospora vasinfecta</em> in Arkansas. Plant Dis. 99:554.</p><br /> <p>Liu, Y., Baird, S.M., Qiao, J., Du, Y., and Lu, S.E. 2015. SecG is required for antibiotic activities of <em>Pseudomonas</em> sp. YL23 against <em>Erwinia amylovora</em> and <em>Dickeya chrysanthemi</em>. Journal of Basic Microbiology 55(5):617-624.</p><br /> <p>Parke, J.L., Knaus, B.J., Fieland, V.J., Lewis, C., and N.J. Grunwald. 2014. Phytophthora community structure analyses in Oregon nurseries inform systems approaches to disease management. Phytopathology 104(10):1052-1062.</p><br /> <p>Rothrock, C. S., Avanzato, M. V., and Rupe, J. C. 2015. Pythium seed rot, damping-off and root rot. Pages 76-79 in: Compendium of Soybean Diseases, 5th ed. G. L. Hartman, J. C. Rupe, E. J. Sikora, L.L. Domier, J. A. Davis, and K. L. Steffey, eds. APS Press, St. Paul.</p><br /> <p>Spurlock, T. N., Rothrock, C. S., Monfort, W. S. 2015. Evaluation of methods to quantify populations of <em>Rhizoctonia</em> in soil. Plant Dis. 99:836-841.</p><br /> <p>Spurlock, T. N., Kirkpatrick, T. L., Rothrock, C. S., Monfort, W. S. 2015. Displacement of <em>Meloidogyne incognita</em> by <em>Rotylenchulus reniformis</em> during a 10 year cotton monoculture&nbsp;&nbsp; and implications for site-specific management. (Abstr.) Phytopathology 105:S2.10.</p><br /> <p>Wang, X.Q., Bi, T., Li, X., Zhang, L., and Lu, S.E. 2015. First report of corn whorl rot caused by <em>Serratia marcescens</em> in China. Journal of Phytopathology 163(11-12):1059-1063.</p><br /> <p>Wang, X.Q., Showmaker, K.C., Yu, X.Q., Bi, T., Hsu, C.Y., Baird, S.M., Peterson, D.G., Li, X.D., and Lu, S.E.. 2014. Draft genome sequence of <em>Burkholderia pyrrocinia</em> Lyc2, a biological control strain that can suppress multiple plant microbial pathogens. Genome Announcements 2(5):e00991-14. doi:10.1128/genomeA.00991-14.</p><br /> <p>Wilson, K. D., Rothrock, C. S., Spurlock, T. N. 2015. Spatial examination of soil factors on cotton seedling disease pressure. ACPA Research Conference 2015, Fayetteville, AR.</p><br /> <p>Wilson, K. D., Rothrock, C. S., Spurlock, T. N. 2015. Identifying spatial distribution of seedling disease pressure in cotton fields. (Abstr.) Phytopathology 105:S4.149.</p><br /> <p>Wilson, K. D., Rothrock, C. S., Spurlock, T. N. 2015. Spatial variability of seedling pathogens and diseases on cotton; influence of soil factors and cultural practices. Page 613 in: Proceedings of the 2015 Beltwide Cotton Conferences.</p><br /> <p>Zenaida, M.V., Arick, M., Buza, T., Hsu,&nbsp; C.Y., Showmaker, C.K., Chouvarine, P., Deng, P., Peterson, G.D., and Lu,&nbsp; S.E. 2014. Transcriptomic dissection of the rice&ndash;<em>Burkholderia glumae</em> interaction. <em>BMC Genomics</em> 15:755; doi:10.1186/1471-2164-15-755.</p><br /> <p>&nbsp;</p>

Impact Statements

1. Educational Aids and Curricula. Loynachan serves as the regional coordinator for “Ask a Soil Scientist” maintained by the Soil Science Society of America: https://www.soils.org/ask. He maintains a URL on Soil Biology (http://agron-www.agron.iastate.edu/~loynachan/mov/) and responds weekly to questions asked by users of the information. These inquires vary from individuals in grade school to fellow scientists worldwide.

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Report Information

Participants

Fulya Baysal-Gurel - Tennessee State University
Craig Rothrock - University of Arkansas
Terry Spurlock - University of Arkansas
Luisa Santamaria - Oregon State University
Tony Adesemoye - University of Nebraska-Lincoln
Carla Garzon - Oklahoma State University (by video conference)
Harald Scherm - University of Georgia (Administrative Adviser)

Brief Summary of Minutes

Accomplishments

<ol><br /> <li><strong><em> Evaluate the population genetic diversity of soilborne pathogens and antagonistic microorganisms in different growing systems and regions using traditional and metagenomic approaches.</em></strong></li><br /> </ol><br /> <p>Members of this multistate research project have continued to provide new insight into the genetic diversity of a number of soilborne pathogens in a variety of production systems.</p><br /> <p>A disease dissimilar to other reported diseases of soybean has been found in the Mississippi River Delta. In fields where soybeans are R5, plants appear chlorotic, stunted, and when extracted from the soil, have a malformed and necrotic taproot that is often black. The estimated yield loss from affected plants was approximately 30% in 2014. The occurrence of symptomatic plants was sporadic and it was estimated that the impact to overall yield was less than 1%.&nbsp; However, in 2015, disease was more severe in some fields and widespread regionally. Additionally, in areas of symptomatic plants, gaps in stand were evident with dead plants between the chlorotic plants. When dead plants were extracted from the soil, the taproot was malformed and black if present.&nbsp; Symptoms were similar in Mississippi and Louisiana. Recently, a team of scientists from the University of Arkansas, Mississippi State University, and Louisiana State University have named the new disease of soybean prevalent in those three states, taproot decline (TRD). The regional distribution of disease occurrence and yield loss is unclear at this time.&nbsp; However, as of harvest in 2016, it has been found as far north as Jonesboro, Arkansas and some farmer and consultant reports indicate losses could be as high as 10 bu/acre. In 2016, fields in Mississippi and Louisiana suffered substantial yield losses from this disease.&nbsp; Currently, seed treatment fungicide or varietal recommendations for growers are unavailable. Further, understanding the regional distribution, commercially available seed treatment efficacy, and varietal susceptibilities are necessary for successful management of this disease. Two fields were chosen and 100 positions marked by GPS encompassing 0.70 and 0.67 ha in 2014 and 2015 respectively. Yellow plants were counted within 3 m of the GPS position in both years. In 2015, the number of dead plants and overall stand within 3 m was determined for each GPS position.&nbsp; Data were analyzed spatially using Moran&rsquo;s I in GeoDa.&nbsp; In both years, chlorotic plants were aggregated (<em>P</em>=0.05) indicative of a soilborne disease. The major ranges for yellow plants were 61.5 m and 128.4 m in 2014 and 2015, respectively. In 2015, the spatial distributions of yellow chlorotic plants and dead plants were related (<em>P</em>=0.004) and the relationship of yellow and dead plants was inversely related to overall healthy stand (<em>P</em>&lt;0.0001). These findings indicate the cause of the disease is likely soilborne and symptoms range from plants dead in the vegetative stages to plants chlorotic and stunted in the reproductive stages, likely due to timing of infection and colonization. Repeatedly, similar fungal isolates were being isolated from infested plant material in all three states. These isolates were used in each laboratory to complete Koch&rsquo;s postulates, essentially confirming this fungus as the cause of the disease by recreating the disease symptoms as they are observed in the field. Molecular methods were used in addition to observation of morphological characters to confirm these fungi were in fact similar to other fungi in the genus <em>Xylaria</em>. However, similarity with a particular species could not be determined. Through phylogenetic analysis of isolates collected from Arkansas, Louisiana, and Mississippi, as well as other known species of <em>Xylaria</em>, it has been determined that this fungus is previously undescribed and that the causal agent of TRD is a member of the <em>Xylaria arbuscula</em> species aggregate and strongly related in the phylogeny to <em>Xylaria striata</em>.</p><br /> <p>Isolates of <em>Rhizoctonia solani</em> recovered from major row crops in the southern United States as part of a Rhizoctonia survey, representing anastomosis groups (AG) 2, 4, 7, and 11, were evaluated for virulence using stand and hypocotyl/coleoptile ratings on soybean, corn, rice and cotton. At planting isolates of AG2 caused significant stand loss and brown-reddish discoloration of coleoptiles in corn, and AG4 isolates were highly virulent caused significant stand loss on cotton. In all four crops, AG4 and AG11 isolates caused reddish brown lesions on hypocotyls or coleoptiles. When AG4 isolates were used to infest soil that contained isolates of other AGs the severity of symptoms were reduced compared with soils infested only with AG4, even with isolates that demonstrated some level of virulence.</p><br /> <p>Mycovirus epidemiology in <em>Rhizoctonia solani </em>on important row crops was examined using 190 geotagged isolates from five different fields in Arkansas using cellulose affinity chromatography. The cDNA from double-stranded RNA enriched material was sequenced and sequences analyzed to determine whether they belong to known or undescribed viruses. Detection primers were designed for each virus and then used in all geotagged isolates. Results indicate mycoviruses are commonly found in isolates of <em>R. solani</em>. More than 20 new viruses, belonging to at least three different virus families have been discovered. Future experiments will determine the effects of mycovirus presence on growth and virulence of the fungus. Spatial analysis should help reveal whether interactions exist between <em>Rhizoctonia </em>spp., AGs, and mycovirus distribution in fields, as well as cropping and soil properties.</p><br /> <p>Work in Mississippi has focused on characterization of endophytic microbial communities associated with the soybean charcoal rot disease system. Symptomatic and asymptomatic soybean plants in a field known to be affected by charcoal rot were collected in 2013 and 2014. Final identification of the fungi was conducted in 2015 using traditional morphological methods and supported by molecular ITS sequence data and TEF-1&alpha;² for <em>Fusarium</em> spp. Fourteen common fungi either pathogenic, endophytic or saprophytic were identified in over 77% of isolations. &nbsp;The most commonly occurring species from soybean tissues in order of frequency were<em> Alternaria</em> spp<em>, Fusarium solani</em> (species complex with two taxa), <em>Penicillium chryseogenum</em>, <em>Cladosporium sphaerospermum, Fusarium chlamydosporum, Clonostachys rosea </em>(teleomorph=<em>Bionectria ochroleuca</em>)<em>, Trichoderma harzianum </em>(= <em>Hypocrea lixii</em>)<em>, Fusarium oxysporum, Fusarium</em> sp. (=<em>Fusarium proliferatum</em>) and <em>Trichoderma </em>sp.&nbsp; Using the common isolates obtained in the study, bioassays were conducted using <em>Macrophomina phaseolina</em> 151 (charcoal rot pathogen). Select species isolates were evaluated in preliminary bioassays to determine which isolates may be involved in charcoal rot disease suppression. From those studies <em>Clonostachys rosea</em> (=<em>Bionectria ochroleuca</em>) and <em>Fusarim solani </em>(two taxa within complex) showed some level of antifungal activity. In another research project, the Mississippi researchers focused on development of baseline data on sweetpotato root microorganisms and determination of the causal agent(s) associated with a newly emerging rot disease complex. Tissue samples from select plant parts (e.g. storage roots) across 2 years and eight growth stages were collected to determine variations in fungal populations. These results attempted to provide indication of potential timing and causes of tip/end rot disease of sweetpotato. Results for fungal diversity analysis show a wide distribution of species across many management periods, but the harvest and post-harvest sampling periods show the greatest species richness over both years.&nbsp;<em>Macrophomina phaseolina</em>, <em>Aspergillus flavus</em>, <em>A. niger</em>, <em>A. tubingens, A. japonicus</em>, and six species of <em>Fusarium</em> were pathogenic in trials on disease-free sweetpotato root tissue. In addition, <em>F. oxysporum</em> and <em>F. solani</em> consistently produced necrotic lesions in root tissue. These two species accounted for nearly 78% of the overall isolates from early season seed stock and bedding plant samples. Microbial populations in post-harvest tissues also differed in relative abundance from prior sampling dates. <em>M. phaseolina</em> increased to 30% occurrence and <em>F.</em> <em>oxysporum</em> and <em>F. solani</em> decreased to 15.5% isolation frequencies between 60 and 90 days post-harvest. During a previous study, a possible interaction between <em>M. phaseolina</em> and high populations of the reniform nematode, <em>Rotylenchulus reniformis</em>, was discovered. Three-year studies evaluating both organisms separately and together in greenhouse and field trials did not show any direct correlations to levels of tip/end rot in field or later in stored storage roots.&nbsp; It was determined that other management practices should be considered as cause of the increase in tip/end rot of sweetpotato. Other research and preliminary results showed that biochar, a byproduct of the fast pyrolysis of organic matter to create biofuels, may have disease control potential and support plant growth. Using <em>M. phaseolina</em> as the tester pathogen, chitosan and biochar preliminary culture plate studies confirm that the pathogen had decreased mycelial growth compared with the control cultures <em>in vitro</em>. Chemical signatures or MVOCs (Microbial Volatile Organic Compounds) of <em>M. phaseolina</em> and other tip/end rot studies are being determined to develop equipment for sensory detection of infected soybean seeds and sweetpotato roots held in storage. Initial studies center on pure cultures of the pathogen and various forms to confirm results contain consistent compounds. In spring 2017, sterile storage roots will be available and the MVOCs will be compared from those microbial clean tissues and with the fungal pathogen. To ensure that MVOC data is unique to <em>M. phaseolina</em> from the pure culture studies, we are now looking at closely related fungal species (<em>Botrysphaeriaceae</em>) common to tip/end rot and soybean diseased necrotic tissues, and other Phyla of fungi common to sweetpotato and soybean plants. Further research has focused on detection of pre-storage and packing identifications of fungi infecting tip/end rot sweet potato roots using portable MVOC detection machinery within warehouses. The researchers collected new <em>M. phaseolina</em> isolates from identical hosts of sweet potatoes. The isolates were separated, verified, and prepared for MVOC analysis. The MVOC results from the GC/MS were unexpected due to high background noise. Therefore, further work is planned to reduce the analytical noise and improve the accuracy of MVOC identification.&nbsp;</p><br /> <p>Additional research conducted at Mississippi State under this project include the folowing. (1) Identification of antagonistic bacteria associated with plant diseases. More than 200 bacterial isolates were obtained from soils and plant samples, and tested for antimicrobial activities using the standard bioassay procedures. Approximately 30 isolates showed significant antifungal activities against <em>Geotrichum candidum</em> and more than 20 isolates exhibited significant antibiotic activities against <em>Erwinia amylovora</em>. These isolates were further purified and preserved at -80^oC. The isolates were preliminarily identified to be <em>Bacillus</em> spp., <em>Paenibacillus</em> spp., <em>Burkholderia</em> spp., <em>Enterobacter</em> spp. and <em>Pseudomonas</em> spp<em>. </em>using 16S rDNA sequence analysis. (2) Investigation of the genes associated with production of the antibacterial activities. RNAseq analysis was employed to understand the regulation network of <em>Burkholderia contaminans</em> strain MS14. In addition to the LuxR gene and non-ribosomal biosynthesis gene (NRPS) for siderophore biosynthesis, we have identified an additional two genes predicted to synthesize bactericidal compound(s). Confirmations of their functions is underway. We have confirmed that occidiofungin is an important component responsible for the antifungal activity of <em>Burkholderia pyrrocinia</em> strain Lyc2. In addition, strain MS586 possesses significant antibacterial activity against the plant-wilting bacterium <em>Ralstonia solanacearum</em>, and was proposed as a novel species of the genus <em>Pseudomonas</em>. Two genes were confirmed to be critical for its antibacterial activity. (3) Genome sequencing of <em>Burkholderia contaminans </em>strain MS14 and <em>Pseudomonas chlororaphis</em> strain UFB2 were completed and the findings have been published. The genome sequence data provide important insights into understanding antimicrobial activities of these bacteria. (4) Evaluation and characterization of endophytic bacteria of soybean associated with charcoal rot disease. More than 20 isolates of endophytic bacteria that show strong antimicrobial activities against the charcoal rot pathogen have been isolated. Population analysis of the bacteria in plants by Illumina sequencing is underway.</p><br /> <p>At Oklahoma State University, the genetic profiles of <em>Pythium</em> and <em>Globisporangium</em> isolates resistant and sensitive to mefenoxam were completed. Monophyletic groups with high risk of developing mefenoxam resistance were identified within <em>Globisporangium cryptoirregulare</em>, as well as low-risk phylogenetic groups. The phylogenetic relationships of four clades within <em>Globisporangium irregulare</em>, identified in 2014, were confirmed. The SSR protocol developed for <em>Fusarium proliferatum</em> was published. Strains isolated from onion in Oklahoma, transformed to express fluorescent proteins, were used to examine the infection process in onion and the patterns were compared microscopically between species. A draft of the genome of <em>Sclerotinia minor</em> was annotated and compared with the genomes of sister taxa. A bioinformatics tool for metatranscriptomic analysis for detection of soilborne fungi and oomycetes and functional analysis was developed. The population biology analysis of <em>Ophiosphaerella herpotricha</em> was conducted and a manuscript is in progress. The manuscript describing a multiplex PCR assay for discrimination of three <em>Ophiosphaerella</em> species was submitted for publication.</p><br /> <ol start="2"><br /> <li><strong><em>Examine the effect of traditional or newly developed management strategies (chemical, cultural, and biological), soil physicochemical properties, or introduced biological control agents on the microbial community and its ability to suppress soilborne pathogens.</em></strong></li><br /> </ol><br /> <p>A project established in Tennessee focused on evaluation of chemical and biorational products for controlling soilborne diseases with different application methods, intervals and reduced-rate applications in woody ornamentals. The project has resulted in development of inoculation protocols for <em>Rhizoctonia solani.</em> This evaluation (inoculation methods and inoculum level) was a necessary first step to the screening of chemical and biorational products and cultural approaches (such as biobiofumigant cover crops, solarization). Chopped potato medium, agar plugs and agar slurry inoculation methods with three inoculum levels were evaluated to determine the disease response of <em>Rhizoctonia solani</em> on Viburnum plants. Plants were evaluated 1&nbsp;month after inoculation for disease severity on root using a 1-5 ordinal scale. All inoculum levels of the agar slurry method showed significantly higher disease severity compared with the other methods. Agar slurry prepared with 7-day-old <em>R. solani </em>cultures can be used as an inoculation method which gives higher disease response in Viburnum and has the potential to be useful in future research on <em>Rhizoctonia </em>disease on other woody ornamentals as well.&nbsp;</p><br /> <p>Chemical and biorational products were evaluated for ability to control <em>Phytophthora </em>root rot in oak leaf hydrangea cvs. Queen of Hearts and Munchkin in greenhouse conditions. Treatments were RootShield WP (<em>Trichoderma harzianum </em>Rifai strain KRL-AG2), RootShield Plus (<em>T. harzianum </em>Rifai strain T-22 and <em>T. virens </em>strain G-41) and Subdue Maxx (Mefenoxam). All treatments reduced root rot disease severity compared with the inoculated, non-treated controls in both cultivars. There were no significant differences between the treatments. Root rot disease severity was significantly higher in the cultivar Queen of Hearts than cultivar Munchkin. This study shows that biopesticides, RootShield WP and RootShield Plus, should be considered to reduce the risk of <em>Phytophthora</em> root rot on container-grown oak leaf hydrangeas in the nursery.</p><br /> <p>Chemical and biorational products (including Subdue Maxx, Empress, Segovis, OxiPhos, Terraclean, TerraGrow, MBI110, RootShield Plus and IT-1503 for <em>Phytophthora nicotianae</em> in Hydrangea; Paegant, Mural, Empress, Terraclean, TerraGrow, MBI110, RootShield Plus, Soilgard and IT-1503 for <em>Rhizoctonia solani</em> in Viburnum) were evaluated&nbsp;in field conditions. The experiment was set up at the Tennessee State University, Otis Floyd Nursery Research Center in McMinnville, TN as a randomized complete block design with four replications. Experiments are underway and plants will be evaluated for root development and disease severity on root at the end of the trial using an 1-5 ordinal scale. Also at TSU, researchers are focusing on development of improved soilborne disease management strategies based on cultural approaches for suppression of <em>Rhizoctonia</em> and (or) <em>Phytophthora</em> spp. and other soilborne pathogens. An agar slurry method was used to screen 15 biofumigant cover crops' susceptibility (belonging to the <em>Brassicaceae</em> family) to this disease under greenhouse conditions. <em>Phytophthora</em> infested rice grains were used to inoculate soil based on established protocols. Untreated biofumigant cover crops were sown in 10-cm pots containing infested soil. Non-infested sterilized soil was used as a control. Stand data and fresh weight were recorded at the end of experiments. According the analysis of germination data taken from both <em>Phytophthora</em> and <em>Rhizoctonia</em> experiments, eight cover crops having the highest germination under seedling&nbsp;disease pressure were selected. Oilseed radish, Mustard, Purple top forage turnips, Astro Arugula seeds, Mighty Mustard pacific gold, Amara Mustard, Rape and oriental Mustard were selected to use for field experiments. Cultural approaches including biofumigant cover crops, compost amendment, and solarization were evaluated under field conditions at two sites. Experiments are underway and plants will be evaluated for root development and disease severity on roots later in 2017.</p><br /> <p>During 2016, at Oregon State University &ndash; NWREC, efforts to establish guidelines for developing and implementing disease control strategies in managing Phytophthora root rot on ornamental plants continues. Ongoing trials determine the effectiveness of varying concentrations of calcium amendments, in the form of gypsum, against inoculum of two soilborne pathogens, <em>Phytophthora cinnamomi</em> and <em>P. plurivora</em>.</p><br /> <p>At Oklahoma State University, strains of <em>Fusarium</em> spp. were tested for hormetic effects at low doses of fungicides. Low-dose growth stimulation was observed suggesting that fungicides at low doses could enhance <em>Fusarium</em> spp. epidemics. New protocols for testing the effects on mycotoxin production of exposure of <em>Fusarium</em> spp. to hormetic doses of fungicides were developed. Differences in the patterns of infection of strains of <em>Fusarium oxysporum</em> and <em>Fusarium proliferatum</em> were identified by fluorescence microscopy. The protocols developed for this study will be applied to analysis of fungicide hormesis on fungal growth <em>in planta</em>.</p><br /> <p>In soybean-producing areas of Arkansas, root damage caused by southern root knot nematode (<em>Meloidogyne incognita</em>) and defoliation caused by frogeye leaf spot (a fungus, <em>Cercospora sojina</em>) are two of the most economically important soybean diseases. It is not uncommon for these diseases to be found in the same field (along with many other diseases) during the same growing season. Many times, soybean varieties are planted that are susceptible to both requiring nematicide and fungicide inputs to protect yields and minimize additional expense. Collecting data in space (yield maps, soil textural maps, and digital imagery captured by UAS or satellites) allows comparisons between multiple diseases and field characteristics. The objective of this work was to determine how two commonly occurring diseases, root damage caused by root-knot nematode (RKN) and defoliation caused by frogeye leaf spot (FLS) affected each other and overall yield using spatial data collection and analysis. Two fields, ca. 10 acres, at the Rohwer Branch of the Southeast Research and Extension Center near Rohwer, AR were split in 2014 and planted to half corn and half grain sorghum.&nbsp; In 2015, these fields were planted to Armor DK 4744 soybean and test areas were assigned 200 points (20 rows of 10 points in a row, GPS-marked) in each field with 100 in the half of the fields from corn and 100 in the half with grain sorghum in 2014. These points were untreated and served as controls. Multiple randomized treatment strips were placed between rows of points with 10 points adjacent to the untreated points in each strip (and also GPS marked). In one field, Telone II was applied in the strips between the 200 rows of points, 18 strips total with 9 strips within each set of 100 points prior to planting.&nbsp; In the other field, seed treated with Ilevo was planted in 18 strips with 9 strips in each set of 100 points. Three replications of three different fungicides (Topsin XTR, Quadris Top SB, and Stratego YLD) were applied in both fields within and adjacent to Telone II and Ilevo strips for comparisons. Fields were scouted throughout the season for FLS and root galling. When FLS occurred, it was rated as a percentage within a 10-ft length of row at each point. Soil samples were taken at harvest at the 200 points assigned in each field and submitted for analysis of populations of RKN (and other nematodes) as well as soil fertility and chemistry analyses. All variables from all points were spatially interpolated and analyzed after harvest and disease differences, product efficacy, and yield compared. Points within areas of fields determined to have the greatest disease pressure were extracted using the query function in ArcMap 10.1 (ESRI, Redlands, CA) and treatment groups compared to untreated using a two-sample t-test. &nbsp;</p><br /> <p>Root-knot nematode was found in both fields along with stunt, spiral, soybean cyst, and stubby-root nematodes. Frogeye leaf spot was severe and RKN populations negatively correlated with FLS in the field where Ilevo was applied but not the field where Telone II was applied. Little galling was observed on the roots. Populations of RKN did not aggregate on the areas of each field rotated with corn, but the populations in those areas were slightly higher. In the Ilevo field, the areas with the greatest amount of disease were related to areas with the lowest soil potassium levels (<em>P</em>=0.01). Within these areas, strips of Ilevo had less overall FLS than the untreated strips. Due to the correlation between FLS and K, points below optimal levels of K were separated from points with K levels within optimal levels, above and below 130 ppm. When product efficacy was compared between K levels, the FLS percentage was significantly less for all treatments applied across areas with optimal K except Ilevo+Stratego YLD compared to the areas of below optimal K. The direct comparison of Ilevo without fungicide to the untreated checks, indicated Ilevo lowered FLS in both comparisons but was only significant in the areas of below optimal K (<em>P</em>&lt;0.0001).&nbsp;</p><br /> <p>In the Telone II field, again, the areas with the greatest amount of FLS correlated with areas of lower soil K (<em>P</em>=0.01). The addition of Telone II resulted in significantly less FLS from the untreated control in areas of the field with K levels below 130 ppm. Fungicides all performed similarly whether Telone II was applied or not when compared with the untreated control or each other. When yield within the Telone II strips was compared with that of the control, the addition of a nematicide increased yield by approximately 7 bu/acre in areas with less than 130 ppm K (<em>P</em>=0.05). Over the entire field, however, the addition of Telone II only resulted in a 2 bu/acre gain.</p><br /> <p>One of many crop management practices being adopted by cotton growers is site-specific management, which involves observing, measuring, and responding to field variables as they change across a field in order to optimize crop inputs for specific sites to maximize yield potential. Predicting and preemptive management for cotton seedling diseases could provide more uniform stand establishment. In a spatial research field trial using a range of fungicide seed treatments at the Judd Hill Plantation, stand improvement from fungicide seed treatments increased where minimal soil temperature decreased across this field in both years. Seedling disease indices increased as minimal soil temperature decreased across this field both years of this study. <em>Thilaviopsis basicola </em>soil inoculum levels and incidence on seedlings also increased as minimal soil temperature decreased across this field both years. These results show that seedling disease pressure varied spatially across this field based on predictable soil factors. This information could be used for creating prescription maps for variable rate management.</p><br /> <p>&nbsp;</p>

Publications

<p><strong><span style="text-decoration: underline;">Scientific articles</span></strong></p><br /> <p>Pradhan S, Flores F, Molineros JE, Melouk H, Walker NR, and Garzon CD. 2016. Improved assessment of mycelial growth stimulation by low doses of mefenoxam in plant pathogenic <em>Globisporangium</em> species<em>. </em>European Journal of Plant Pathology. DOI 10.1007/s10658-016-1016-5</p><br /> <p>Moncrief IR, Garzon CD, Marek SM, Stack JP, Gamliel A, Garrido P, Proa&ntilde;o MF, Gard M, Dehne H,&nbsp; Fletcher J. 2016. Development of simple sequence repeat (SSR) markers for discrimination among isolates of <em>Fusarium proliferatum</em>. Journal of Microbiological Methods. 126: 12-17.</p><br /> <p>Abd-Elmagid A, Hunger R, Garzon CD, Jub H, Payton M, and Melouk H. 2015. Effect of osmotic and matric potentials on <em>Sclerotinia minor</em> and <em>Sclerotinia sclerotiorum</em> virulence on peanut. Int. J. Phytopathol. 04: 147-158.</p><br /> <p>Spurlock, T. N., Rupe, J. C. 2016. Aerial blight. Compendium of Soybean Diseases and Pests, Fifth Edition.&nbsp; APS Press, St. Paul, MN.</p><br /> <p>Spurlock, T. N., Rothrock, C. S., Monfort, W. S., Griffin, T. W. 2016. The distribution and colonization of soybean by <em>Rhizoctonia solani</em> AG11 in fields rotated with rice.&nbsp; 2016. Soil Biology &amp; Biochemistry 94:29-36.</p><br /> <p>Allen, T., Bluhm, B., Conner, K., Doyle, V., Price, T., Sikora, E., Singh, R., Spurlock, T., Tomaso-Peterson, M., Wilkerson, T. 2016. First report of taproot decline, a previously undescribed soybean disease, in the southern United States.&nbsp; Plant Health Progress. (submitted)</p><br /> <p>Spurlock, T. N. 2016. Using spatial data layers to understand soybean diseases.&nbsp; Crop &amp; Soil 49:12-15.</p><br /> <p>Tomaso-Peterson, M., Allen, T., Singh, R., Spurlock, T., Price, P. 2016. Characterization of taproot decline in southern soybean. Proceedings of the 43^rd Annual Southern Soybean Disease Workers, Pensacola, FL.</p><br /> <p>Wilson, K., Rothrock, C., and Spurlock, T. 2016. Spatial examination of soil factors on cotton seedling disease pressure. Page 593 in: Proceedings of the 2016 Beltwide Cotton Conferences.</p><br /> <p><strong><span style="text-decoration: underline;">Extension publications:</span></strong></p><br /> <p>Espindola AS, Garzon CD, Marek SM, Melouk H, Schneider W. 2016. Genome (DNA) Sequencing of <em>Sclerotinia minor</em>.&nbsp; Partners in Progress - Peanuts. Peanut Research. Oklahoma State University, P-1047. P. 19-22.</p><br /> <p>Baysal-Gurel, F., Liyanapathiranage, P., Blalock, A. 2015. Southern blight management for woody ornamentals. ANR-PATH-02-2015. TSU-16-0191(A)-15-61065.</p><br /> <p>Baysal-Gurel, F., Kabir, Md N., Blalock, A. 2016. Root diseases of hydrangeas. ANR-PATH-4-2016. TSU-16-0237(A)-15-61065.</p><br /> <p><strong><span style="text-decoration: underline;">Abstracts/Posters</span></strong></p><br /> <p>Iturralde JF, Flores FJ, Koch AR, Garz&oacute;n CD, Walker NR. 2016. A Multiplex end-point PCR assay for the detection and identification of three species of <em>Ophiosphaerella</em> that cause spring dead spot of bermudagrass. 5th European Turfgrass Society, Jun. 5-8, Albufeira, Portugal.</p><br /> <p><strong><span style="text-decoration: underline;">Presentations / Professional Meetings</span></strong></p><br /> <p>Proa&ntilde;o MF, Ayala C, Chiriboga A, Daughtrey M, Garzon CD. 2016. Characterization and identification of <em>Pythium</em> and <em>Globisporangium</em> species present in ornamental greenhouses from Long Island, New York. APS Annual Meeting. Jul. 30- Aug. 3, Tampa, FL. 60-O</p><br /> <p>Kabir, Md N., Liyanapathiranage, P., Simmons, T., Baysal-Gurel, F. 2016. Effect of biopesticides on <em>Phytophthora</em> root rot disease of oakleaf hydrangea. APS Annual Meeting. Jul. 30- Aug. 3, Tampa, FL.</p><br /> <p>Kabir, Md N., Simmons, T., Liyanapathiranage, P., Curry, C., Alexander, L., Baysal-Gurel, F. 2016. Evaluation of stem inoculation technique for assessing resistance to <em>Phytophthora nicotianae </em>in hydrangea cultivars. APS Annual Meeting. Jul. 30- Aug. 3, Tampa, FL.</p><br /> <p>Spurlock, T., Rothrock, C., Urrea-Romero, K., Tolbert, A. 2016. Spatial distribution of taproot decline of soybean. Phytopathology 106:S2.14.</p><br /> <p>Urrea, K., Rothrock, C., and Winters, S. 2016. Virulence of <em>Rhizoctonia solani</em> anastomosis groups on cotton, corn, rice, and soybean. Phytopathology 106:S4.30</p><br /> <p>Stetina, T., Rothrock, C., Spurlock, T., and Tzanetakis, I. 2016. The virosome of <em>Rhizoctonia solani </em>and its effect in fungal pathogenicity. Phytopathology 106:S4.43.</p><br /> <p><strong><span style="text-decoration: underline;">Theses</span></strong></p><br /> <p>Espindola, AS. 2016.&nbsp; Eukaryotic Plant Pathogen Detection through High Throughput DNA/RNA Sequencing Data Analysis. Doctor of Philosophy in Plant Pathology, Oklahoma State University.</p><br /> <p>Proa&ntilde;o, MF. 2016. Temporal and Host Related Variation of <em>Pythium</em> and <em>Globisporangium</em> Species in Floricultural Crops. Master of Science in Entomology and Plant Pathology, Oklahoma State University.</p><br /> <p>&nbsp;</p>

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1. INFEWS N/P/H2OA: Impact of soil water fluctuation on multi-scale soil nitrogen and phosphorus chemical processes (Williams, Virginia Tech, $472,326, NSF)

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