Brief Summary of Minutes of Annual Meeting

Attendees: Craig Canaday (TN), Craig Rothrock (AR), Carla Garzon (OK), Terry Spurlock (AR, visitor), Boyd Padgett, and Shouan Zhang (University of Florida, TREC, Homestead - visitor). The meeting was hosted by University of Arkansas at Fayetteville, AR, in the Rosen Alternative Pest Control Center Conference Room beginning at 8:30 AM. Attendance was sparse due in part to restrictions in interstate travel and the difficulty in finding reasonable airline connections to the meeting site. Craig Rothrock, local arrangements host, welcomed the attendees and visitors were introduced. Craig Canaday, Chair, opened the official meeting and welcomed reports on project objectives. Carla Garzon, Secretary, recorded the presentations and comments.

Objective 1. Examine commercial and non-commercial biocontrol agents for use as seed treatments, in-furrow treatments or as potting mix amendments. The third regional trial of biocontrol agents for control of wire stem of broccoli (Rhizoctonia solani) was cancelled due to funding restrictions and changes in resource allocation. A report on the work to date is still in preparation. Shouan Zhang expressed his interest in future collaborations regarding objective 1. Dr. Zhangs research focuses on Biorational management of vegetable diseases including Phytophthora blight of squash, cause by Phytophthora capsici.

Objective 2. Examine the effect of cultural practices on soilborne pathogens and plant growth. Craig Canaday passed out reprints of the recently published report: Canaday, C. H., and Schmitthenner, A. F. 2010. Effects of chloride and ammonium salts on the incidence of Phytophthora root and stem rot of soybean. Plant Dis. 94:758-765. He presented a brief pictorial history of this work and mentioned that he was continuing to investigate the potential changes in the micro-partitioning of root calcium with chloride salts using energy-dispersive X-ray analysis. Craig Rothrock reported progress done on research on strawberry treated with green manure. The objective of the study is to determine the effect of green manure on the microbial diversity (bacteria and fungi). A metagenomics approach was used analyzing ribosomal DNA sequences by Denaturing Gradient Gel Electrophoresis (DGGE). The results presented suggest green manure treatment produced evidence of changes in the microflora of two experimental sites, over two years. A brief discussion followed about the diverse soilborne problems that affect strawberries, including Phoma, Rhizoctonia and Pythium, possible sources of inoculum, and management strategies such as soil solarization.

Objective 3. Examine the genetic diversity of Rhizoctonia solani between natural ecosystems and agricultural ecosystems. Craig Rothrocks graduate student, Terry Spurlock, presented progress reports on comparisons between two baiting protocols (toothpick and soil pellets) and selective media. He modified the toothpick protocol by using twice as many toothpicks as initially recommended in the protocol and wetting the soil. The toothpick protocol seemed the most effective to capture Rhizoctonia solani. It was especially effective to recover AG11 and AG1A. The pellet protocol was more effective to recover R. oryzae. The toothpick protocol involves saprofitic activity and requires high humidity. Ethanol-potassium nitrate medium and TS medium worked better than Ko and Horas. Contamination was a problem with the latter medium. The best baiting/selective media combination reported were toothpick baiting combined with either ethanol-potassium nitrate medium or TS medium. In the discussion which followed it was noted that the toothpick method may work best, since large amounts of soil can be assayed as compared with the pellet protocol. Craig Rothrock reported on the spatial distribution of R. solani AG1-IA in a soybean-rice rotation. Areas with higher levels of humidity had higher R. solani AG1-IA recovery rates. Isolates were recovered from asymptomatic seedlings (10/site). Epiphytic growth was observed up to 60 cm on the canopy under high humidity conditions. Aerial mycelium and abundant sclerotia were produced. The pathogen was homogenously distributed in the field, and the high incidence hides any spatial patterns. It is apparent that AG1-IA is associated with plant residue and organic matter. Periodic tilling was discussed as possible management practice but its implementation seems unlikely due to its high cost. Carla Garzon reported that DNA fingerprinting protocols (AFLP, ISSR, SSR) were standardized for analysis of Rhizoctonia solani populations. Boyd Padgett examined the Rhizoctonia diversity in Louisiana agricultural soils. Toothpick baiting was done in 8 locations (2 corn, 2 cotton, 2 grain sorghum, 2 soybean). A minimum of 31 isolates (from soybean, location 2) and a maximum of 139 isolates (from grain sorghum) of Rhizoctonia were recovered.

Plans for next year. Rhizoctonia diversity will be compared between AR fields (vegetables, cotton, rice and prairie) and LO (corn, cotton, sorghum, soybean). Craig Rothrock and Terry Spurlock will write the methods paper. Carla Garzon will compare R. solani AG1-IA genotype diversity between methods using molecular tools.

Business Meeting. After the research reports were finished, Craig Canaday, Chair, called the business meeting to order. C. Rothrock was nominated as secretary for 2011. The nomination was seconded and passed unanimously. C. Garzon, secretary and next years Chair, offered to host the next meeting in Oklahoma City, OK, in November 2011. The offer was accepted by all those present. The future of the S-1028 project was discussed. C. Canaday and C. Garzon will open discussion to identify new objectives for the project for the next 5 years. It was proposed to encourage team work to produce preliminary data that could be used for NIFA funded research. C. Garzon will contact Mark McLellan for guidance in the organization of a writing group for the new project. C. Canaday asked members to send him state reports before Thanksgiving. The meeting was adjourned. Members traveled home on November 7. Respectfully submitted, Carla Garzon, Secretary

Accomplishments/Outcomes (by Project Objective and State) Objective 1. Examine commercial and non-commercial biocontrol agents for use as seed treatments, in-furrow treatments or as potting mix amendments. Kentucky: A trial was conducted in the summer of 2010 to evaluate Bioten, a commercial formulation containing Trichoderma harzianum and T. viride, for suppression of Phytophthora blight, caused by Phytophthora capsici, on summer squash when applied to soil via drip irrigation, alone or in conjunction with fungicide programs. In 2009, results of the trial showed that Bioten-containing treatments reduced the severity of Phytophthora blight by 25% over the untreated control.; however, fungicide programs that contained Ridomil Gold applied pre-plant reduced incidence by over 60% compared to the untreated control. The 2010 trial was lost due to flooding of the field. It will be conducted again in 2011. South Carolina: The commercial biofungicides RootShield and Actinovate were tested for prevention of damping-off of arugula on a noncertified organic vegetable farm in Charleston County, South Carolina. Biofungicides were applied immediately after seeding over raised beds with a backpack sprayer at 32 oz/A (RootShield) or 12 oz/A (Actinovate). Water was applied as a control. Emergence and percentage damping-off did not differ significantly among the three treatments, even though Pythium spp. were isolated from 43-53% and Rhizoctonia solani was isolated from 39-78% of the plants sampled. Soil populations of R. solani (measured as percentage of organic matter colonized) were higher in the Actinovate-treated plots (51%) compared to the water control (36%). Fresh weight of arugula harvested 1 month after seeding did not differ among treatments. Tennessee: Three commercial biological seed treatment products, Kodiak Concentrate (Bacillus subtilis GB03), Subtilex (B. subtilis MBI600), and Yield Shield (B. pumilus GB34), were evaluated under a variety of environmental conditions for control of seedling diseases of snap bean and soybean in field tests conducted in soils natural infested with R. solani, Pythium spp., Macrophomina phaseolina, and Fusarium spp. GB03 or MBI600, used alone or as supplements to standard seed treatment chemicals, failed to increase snap bean stand or yield. Similarly, supplementing soybean seed treatment chemicals with GB03 or GB34 failed to increase health plant stand or yield.

Studies were initiated to address the manipulation of a biological control agent, Pasteuria nishizawae, to improve disease management of the soybean cyst nematode (SCN). In order to determine the level of P. nishizawae needed to act as an effective biological control agent, the initial objectives were (1) to determine the best method for detection of P. nishizawae levels in soil and (2) to evaluate methods for quantification of P. nishizawae in SCN cysts. Progress on both objectives was fraught with technical difficulties and more work is needed on methodology development. Project Modification: The third multi-state trial of biocontrol agents for control of wire stem of broccoli (caused by R. solani) was cancelled due to funding restrictions and changes in resource allocation. A publication on combined data from the first and second trials is being developed. Objective 2. Examine the effect of cultural practices on soilborne pathogens and plant growth. Arkansas: Research was established to characterize changes in plant pathogen and general microbial populations in soils following a summer brassica cover crop, mustard seed meal amendment, solarization or a combination of the cover crop and solarization compared to no soil treatment prior to establishing an annual strawberry crop at two different locations in Arkansas. General microbial and suspect pathogen populations from soils were quantified by plate count methods. Additional soil samples were taken after cover crop incorporation to generate denatured gradient gel electrophoresis (DGGE) profiles for bacterial and fungal populations. Roots of strawberry plants, including the initial transplants, were also analyzed for the isolation frequency of plant pathogens. Soil treatments did affect the level of bacterial, fungal and actinomycete populations in the soil at the time of treatment and at strawberry transplant. In all cases, there was a trend for higher bacterial, fungal and actinomycete populations in brassica, brassica plus solarization and mustard seed meal amended soils compared to solarized only and control soils. Total culturable, bacterial populations were significantly higher in soils that had been planted with a brassica cover crop followed by solarization and soils receiving mustard seed meal amendments at both locations at the time of strawberry transplanting. From soil samples taken at 7 and 25 days after the brassica cover crop was incorporated into the soil, DGGE produced unique profiles of bacteria and fungi compared to that of control soils. At the time of strawberry transplant, all bacterial DGGE profiles of soils from both locations receiving different treatments were still distinct and grouped separately in dendograms, while fungal DGGE profiles were not as consistently distinct among treatments. This project has successfully proven how including soil treatments such as a brassica cover crop, solarization or mustard seed meal application as a practice in annual strawberry production can enhance the soil microflora, especially the bacterial community. Since changes could be observed in both the bacterial and fungal communities throughout the sampling times, this system has the potential to produce a soil that is more diverse and possibly reduce populations or colonization of roots by soilborne pathogens. The impacts of these shifts in the soil microflora for soilborne diseases need to be compared to chemical fumigants in soil with a history of strawberry production to examine their value in developing a sustainable strawberry production system. Iowa: Biofuels are being carefully evaluated for their efficacy to replace hydrocarbons. Pyrolysis of plant materials leaves behind biochars that may impact soil cultural practices and affect soilborne organisms and plant growth. We studied corn stover and three biochars (pyrolysis products made from corn stover) and their decomposition in soil. The percentage of carbon lost from the amendments after eight weeks was greatest for corn stover (44 wt%), followed by the two less completely pyrolyzed biochars (9 and 10%), and least (4%) from the biochar made by fast pyrolysis at 500°C. Thus, all biochars contained at least some readily bioavailable carbon that could be measured as CO2 emissions over 8 weeks. Microbial availability roughly correlated inversely with the degree of pyrolysis completeness. There was an apparent shift in microbial communities from bacteria and actinomycetes to fungi with increasing pyrolysis. This work will be submitted for publication shortly.

A soil biology educational website containing video of important soil organisms including fungi, ectomycorrhizae, endomycorrhizae, and their activities in the soil food web is maintained at (http://www.agron.iastate.edu/~loynachan/mov/). Two presentations were given in October at Hoover High School in Des Moines on living organisms in the soil. Louisiana: Foliar-applied biological fungicides were evaluated. Fungicide field studies evaluating Ballad Plus (Bacillus pumilus (Strain QST 2808)) for managing fungal diseases of wheat, corn, and soybean were conducted during 2010. Foliar applications were made when each crop reached a selected growth stage. Diseases were monitored if present and quantified in each plot during the growing season. Yields and test weights were collected when possible. Ballad Plus was applied at rates of 0.5 and 1.0 qt/A on wheat during growth stage F8 (flag leaf emergence). These products were applied alone or in combination with Headline fungicide (pyraclostrobin) (3.0 and 6.0 fl oz/A). Leaf rust (Puccinia triticina) severity (except for one rating), test weights, or grain yield did not differ among the non-treated wheat and solo applications of Ballad Plus. The addition of Headline fungicide resulted in less disease severity and higher yields than the non-treated. Ballad Plus applied alone at 0.5 or 1.0 qt/A or in combination with Headline at 3.0 or 6.0 fl oz/A was evaluated in field corn. Foliar applications were made to corn during the R1 growth stage (tasseling). Diseases did not develop to damaging levels in this test. Yields did not differ among treatments. Ballad Plus applied alone at 0.5 or 1.0 qt/A or in combination with Headline at 3.0 or 6.0 fl oz/A was evaluated in soybean. Foliar applications were made when soybean was in the R3 growth stage (pod initiation). Diseases did not develop to damaging levels and did not differ among treatments. Test weights and yield did not differ among any treatments. Mississippi: Mississippi is the third largest sweetpotato producer in the United States after California and North Carolina (USDA National Agricultural Statistics Service, 2009). Sweetpotato storage rots in Mississippi have increased annually since 2005 and are occurring earlier each year. Although increasing in incidence, the rot seldom damages enough of the crop to cause serious economic loss. Until 2008, the most common rots were Rhizopus and several Fusarium rots, circular spot, charcoal rot, punky rot, and Java black rot. However, during 2008-2009 growing season, an unknown rot complex dramatically impacted stored roots and coupled with other rots caused substantial economic losses. These losses would represent 1,334,313 boxes of sweetpotatoes or ca. $16,775,000 for the 2008 production year.

Repeated random isolations from sweetpotatoes symptomatic of the new rot and drawn from different bins of multiple growers, failed to obtain a consistent pathogen, usually resulting in one of eight different Fusarium spp., many of which are common soil inhabitants and M. phaseolina. These findings illuminated the need for baseline data of the associate root microorganisms of sweetpotato roots that could support determination of a causal agent(s), or be used to systematically assess biologically based management schemes or evaluate crop rotations. Therefore, the objectives of the initiated studies were: (1) to utilize selective media to determine the diversity and densities of fungi and bacteria associates from sweetpotato tissues, including roots and stems from key production stages in the field and storage; (2) to co-sample the same tissues and utilize molecular cloning and sequence data to compare organism(s) diversity to results from objective (1); and (3) to conduct pathogenicity screening of the putatively important bacterial and fungal isolates (ca. 100 to 150 total species expected) in sweetpotato tissues to confirm parasitism using Kochs postulates.

Two fields with a history of sweetpotato rot, with 1-2 years of continuous sweetpotato production were selected. Both fields were planted with Beauregard varieties. The cultural practices from bedding to harvesting followed the recommendations of Mississippi State University Extension Services and Thompson et al. (2002). Tissue sampling was divided into eight periods during sweetpotato production. The sampling periods were: (1) sampling of seed stock roots prior to bedding to include four pieces each from eyes at the distal and proximal ends of the potato, as well as eight samples drawn from the internal storage parenchyma obtained by slicing the root horizontally down the middle, (2) at six weeks after seed bed establishment [roots and attached slips were dug and tissues processed as in period 1 except tissue at the proximal end were sampled instead of the proximal eyes]; (3) 1 cm long replicate tissue pieces removed from the base, central node, and top of each slip [sampling as in period 2]; (4) roots dug prior to herbicides application(s) ca. 45 days after slip transplanting [sampling as in period 2]; (5) roots were dug immediately prior to mechanical vine removal [sampling as in period 2]; (6) except roots were dug immediately after vine removal or during harvest [sampling as in period 2]; (7) at ca. 60 days after harvest [sampling as in period 1 except mother roots were selected from four replicate storage bins per location for tissue assay]; (8) at ca. 90 days after harvest [sampling as in period 7]. Four isolation media were used to culture bacteria and fungi from the tissues sampled in each of the 8 sample periods. Nutrient glucose agar (NGA) and Kings B agar (KB) media were used for bacteria and potato dextrose agar (PDA) and water agar (WA) for fungi. Identification of bacteria and fungi used fatty acid methyl esters analyses for bacteria and morphological features for fungi. Molecular techniques will be used to confirm identification. Representative tissues from each sample are currently stored in -80°C for further processing (i.e., DNA extraction). Approximately 2400 isolates of fungi and 2500 isolates of bacteria were collected from sampling period 1-6. Samples from period 7 are currently being processed. Identification of bacteria and fungi is currently under way. Several fungal genera which tentatively have been identified included Fusarium (several species), Candida, Curvularia, Cercosporella, Phoma-like, Aspergillus, and Macrophomina. Following identification, representative isolates of each taxon (fungi and bacteria) will be stored in -80°C as sweetpotato end/tip rot study culture collections.

Recent research at Mississippi State University has focused on understanding the mechanisms of antifungal bacteria present in soil-borne disease systems. More than 50 bacteria obtained from the local soils of Mississippi exhibited significant antifungal activities against pathogenic fungi. Strain MS14 possesses a very broad range of antifungal activities against economically important fungi, including plant and animal pathogens. It was revealed that a set of nonribosomal peptide synthetase genes, which are harbored in a 56-kb gene cluster in the MS14 genome, are required for the antifungal activities and the antifungal compound was an oligopeptide (Gu et al. Biochem. Biophys. Res. Commun. 380:328-332). Two key regulatory genes ambR1 and ambR2 positively regulate expression of antifungal activities of strain MS14 (Gu et al. FEMS Microbiol. Lett. 297:54-60). The genes involved in modification and secretion of the oligopeptide have been characterized from the right and left boards of the gene cluster. In collaboration with Dr. James L. Smith, the chemical structure of the oligopeptide (named occidiofungin) was characterized as a glycopeptide containing eight amino aids and may target glucan biosynthesis of fungal cell walls (Lu et al. Biochemistry, 48:8812-8321). Analyses of the antifungal spectrum of activity and the minimum inhibitory concentration revealed occidiofungin is of great potential in both plant disease management and animal disease treatment. North Carolina: Wood-based mulches are used in avocado production and are being tested on Fraser fir for reduction of Phytophthora root rot, caused by Phytophthora cinnamomi Rands. Research with avocado has suggested a role of microbial cellulase enzymes in pathogen suppression, through effects on the cellulosic cell walls of Phytophthora. This work was conducted to determine whether cellulase activity could account for disease suppression in these systems. A standard curve was developed to correlate cellulase activity in mulches with concentrations of a cellulase product. Based on this curve, cellulase activity in mulch samples was equivalent to a cellulase enzyme concentration of 25 U/ml or greater of product. Sustained exposure of P. cinnamomi to cellulase at 10-50 U/ml significantly reduced sporangia production, but biomass was only reduced with concentrations over 100 U/ml. In a lupine bioassay, cellulase was applied to infested soil at 100 or 1000 U/ml with three timings. Activity diminished by 47% between one and 15 days after application. Cellulase applied at 100 U/ml two weeks before planting yielded activity of 20.08 µmol glucose equivalents per gram soil water (GE/g aq) at planting, a level equivalent to mulch samples. Activity at planting ranged from 3.35 to 48.67 µmol GE/g aq, but no treatment significantly affected disease progress.

Phytophthora root rot of Fraser fir, caused by several Phytophthora spp., is a severe problem in Christmas tree production. Since fungicides are not economically viable for disease management in field plantings and host resistance is not available, cultural control methods were investigated. Mulches, dairy compost, and soil pH adjustment were tested at five field sites in North Carolina. Treatments included wood chips, wood chips plus compost, or pine bark as raised beds, and compost or sulfur tilled into soil. Soil and mulch microbial populations were characterized by dilution plating and calculation of a log series diversity index, and by enzyme analyses at 5, 12, 17, and 24 months after planting. Bacterial and fungal counts, microbial activity, and cellulase activity were higher in mulch than in soil at all sites and times (P<0.01), and generally did not differ among mulch types or among soils. Treatments significantly affected disease ratings and tree survival at three of five sites, with one or more mulch treatments yielding lower disease ratings and greater survival than controls. Tree mortality at each time point varied significantly with cellulase activity in the upper root zone (P=0.005). Other biological variables did not show significant relationships with disease ratings or mortality. Ohio: A biochemical assay was developed to detect Phytophthora sojae infestation in soil. The approach was based on profiling total cellular fatty acid methyl esters (FAME) of P. sojae in pure culture. A total of 12 fatty acids (14:0, 16:0, 18:0, 16:1 É7, 18:1 É9, 18:2 É6, 18:3 É6, 20:1 É9, 20:3 É6, 20:4 É6, 22:1 É9 and 24:1 É9) were identified in the FAME profiles of P. sojae (races 1, 4 and 7) pure cultures. The predominant fatty acids in the FAME profiles are the unsaturated 18C fatty acids (18:1É9 and 18:2 É6) followed by the saturated and unsaturated 16C fatty acids (16:0 and 16:1 É7). The ratio of some of the individual fatty acids significantly changed due to growth conditions, and to a lesser extent due to pathogen race. Zoospores of P. sojae additionally contained the long-chain saturated fatty acids (20:0 and 22:0), which were not detected in the mycelium of this organism. The potential of using FAME profiles of P. sojae for detecting the pathogen in soil was evaluated by adding a known number of zoospores of P. sojae to soil. The results showed that fatty acids such as 18:1w9, 18:2w6, 20:1w9, 20:4w6 and 22:1w9 could be detected and quantified against the background levels of fatty acids present in soil. This outcome is significant because it offers the potential for a simple and rapid method for determining P. sojae infestations in soil.

Work was also conducted to investigate extracellular proteins belonging to class-I elicitins in Phytophthora. It is known that elicitins are involved in sterol acquisition, but very little is known about the relationship between sterols and elicitin gene expression. The objective was to determine the pattern of class-I elicitin gene expression in P. sojae, when its growth medium contains different types of sterol (fungal, plant or animal). It was discovered that the growth of P. sojae was stimulated by nanomolar concentrations of all the sterols tested. This also resulted in a differential regulation of class-I elicitin gene expression compared to controls when monitored over-time using real time Reverse Transcription Polymerase Chain Reaction (RT-PCR). Generally, class-I elicitin genes became down-regulated over time which also coincided with a reduction in elicitin biosynthesis when any of the sterols was present in the growth medium. However, kinetics of down-regulation varied as a function of sterol structure, which may be related to binding efficiencies for sterols with elicitins. Also, using Elemental Analysis-Isotopic Ratio Mass Spectrometry (EA-IRMS), it was discovered that P. sojae rapidly assimilated 15N-labeled extracellular proteins (which predominantly constitute class-I elicitins) into its mycelium. This happened when stigmasterol was added to the growth medium, suggesting that elicitins are involved in sterol sequestration by this organism. This study is the first to show that sterols regulate the expression of class-I elicitin genes in Phytophthora and provides strong evidence for the involvement of elicitins in sterol acquisition. Pennsylvania: The ecology and diversity of endophytic and soilborne fungi and bacteria are poorly understood. Verticillium dahliae causes wilt diseases in hundreds of economically important crops worldwide. Populations of this pathogen are highly structured and composed of Vegetative Compatibility Groups (VCGs) that define clonal lineages. Over sixty isolates, representing all VCGs (1A, 1B, 2A, 2B, 4A, 4B, and 6), numerous hosts and geographic origins were typed based on IGS sequences and six anonymous genomic regions containing single nucleotide polymorphisms. Results showed that IGS sequences display a very complex structure with numerous large indels with varying copy numbers between isolates. Maximum parsimony analysis indicated that certain subgroups (e.g., 1A and 1B) are indeed closely related and placed in the same clade; however, other subgroups (e.g., 4A) are related more closely to members of a different VCG (2B) than to subgroups of the same VCG (4B). MP analysis that VCG2B is polyphyletic and comprises at least three distinct phylogenetic lineages. Endospore-forming bacterial endophytes were isolated from Theobroma cacao. Cacao leaves, pods, branches, and flower cushions were removed from cacao trees escaping disease and tissue was surface sterilized, heat treated (75°C for 15 min), and plated. 69 endophytic endospore-forming bacteria were isolated and the 16S rRNA gene was sequenced to determine species identity. Isolates were further classified for characteristics of biological control activity. Sixteen isolates were chitinolytic and in antagonism studies against cacao pathogens, 42% were antagonistic to Moniliophthora roreri, 33% to M. perniciosa, and 49% to Phytophthora capsici. 25% of isolates inhibited the growth of both Moniliophthora spp., while 22% of isolates inhibited the growth of all three pathogens. Isolates that were chitinolytic and tested negative on Bacillus cereus (Bc) agar were tested in planta. All 14 isolates were capable of endophytic colonization; while 8 isolates significantly inhibited P. capsici lesion formation in detached leaf assays. ARISA was used to estimate the diversity of total bacteria and bacilli within cacao leaves. Inundative application of a single bacterial species to cacao leaves did not impact native bacterial communities. Bc is a common inhabitant of soil as well as the endophytic portions of plant tissue. Although common in the environment, some isolates cause foodborne illness through the production of enterotoxins. A collection of endophytic Bc isolates were obtained from several crops including apple, cacao, tomato, and potato. The presence of several genes for enterotoxins was tested by PCR. Of the 35 endophytic Bc isolates, the majority had one or more of the genes required for enterotoxin production and 14% of isolates tested lacked any enterotoxin genes. The endophytic host had no impact on the presence of enterotoxin genes, as they were detected in isolates from all tested plants. The enterotoxin genes from endophytic isolates were sequenced and compared to Bc isolates known to cause foodborne illness. While Maria del Mar Jimenez-Gasco and Paul A. Backman are the principal investigators of this project, other participants included Anissa Poleatewich (former graduate student), Rachel L. Melnick (former graduate student), and Glenna Malcolm (postdoctoral researcher). South Carolina: The rootstocks Emphasis, Macis, and WMXP 3945 (Lagenaria siceraria), Shintosa Camel and Strong Tosa (Cucurbita moschata x Cucurbita maxima), and Ojakkyo (Citrullus lanatus var. citroides) were evaluated in a field experiment. The rootstocks were grafted with seedless watermelon Tri-X 313 (Citrullus lanatus var. lanatus); Tri-X 313 not grafted or grafted to itself served as the controls. Plants were transplanted to a field in which the transplanting holes had been infested with a 1:1 mixture of races 1 and 2 of Fusarium oxysporum f. sp. niveum, the causal agent of Fusarium wilt of watermelon. Wilt incidence and plant death was highest in the self-grafted Tri-X 313. Ojakkyo citron had more wilted plants than plants grafted to Lagenaria rootstocks. Yield (weight and number of fruit) were greater with Lagenaria rootstocks than with Tri-X 313 (mean of not grafted and self-grafted) or with Cucurbita rootstocks.

Oilseed radish (Raphanus sativus), mustard (Brassica juncea cv. Pacific Gold), and winter rapeseed (B. napus cv. Dwarf Essex) were grown in the spring for seven weeks before incorporation as biofumigation cover crops. Plots were covered with black virtually impenetrable film (VIF) after incorporation in late May. Control treatments were fallow with and without VIF. Five weeks after incorporation, populations of Pythium did not differ among any treatments. Populations of R. solani were higher in fallow without VIF than in all mulched treatments, indicating that the VIF increased soil temperatures enough to reduce populations of R. solani. Pepper plants transplanted into the plots were stunted due to Pythium infection of the roots in all plots but particularly in the fallow plots without VIF. Yields of pepper over 10 weeks were greater in the winter rapeseed, mustard, and fallow plus VIF treatments than in the fallow plots without VIF. The lack of an effect of the cover crops may be due to the relatively low biomass obtained with spring-planted brassica cover crops in coastal South Carolina. Tennessee: Interactions between two fertilizers (muriate of potash and sulfate of potash), two biological control agents (GB03 and MBI600), and seed treatment fungicides were evaluated for control of snap bean seedling diseases in an April-planted field test. Fertilizers were applied at 135 kg K2O/ha. The biological agents were evaluated both with and without supplemental seed treatment fungicides. Over 4 cm of rain were received as the snap bean seedlings were beginning to emerge. Another 36 cm of rain were received during the next 10 days. Under these very disease-conducive conditions, GB03 and MBI600 failed to increase snap bean stand or yield without the use of seed treatment fungicides. MBI600 reduced plant stand and yield when combined with captan + metalaxyl and increased plant stand and yield when combined with fludioxonil + mefenoxam. Highest plant stands and yield were achieved with the use of trifloxystrobin + metalaxyl seed treatment fungicides without a supplemental biological agent. Use of sulfate of potash instead of muriate of potash increased plant stands by over 12% and increased snap bean yield by over 50%.

Potash fertilizer form, fertilizer application timing, and seed treatment materials were evaluated for control of charcoal rot of soybean (caused by M. phaseolina) in both May-planted and June-planted field tests. Potash fertilizer form (sulfate of potash versus muriate of potash) and application timing (March or at planting) had no effect on soybean stand or yield in the 2010 tests. Adding biological agents (MBI600 or GB34) to a soybean seed treatment of fludioxonil + mefenoxam had no effect on plant stand or yield.

A laboratory study on the effects of chloride salts on the micro-partitioning of root Ca was initiated using energy-dispersive analysis of x-rays. After 136 separate EDX analyses of root tissues from six different plants, detectable Ca levels in the outer cells of taproots were notably lower when plants were grown in soil treated with MgCl2 than when grown in untreated soil. There were no detectable treatment effects on lateral roots. Objective 3. Examine the genetic diversity of Rhizoctonia solani between natural ecosystems and agricultural ecosystems. Arkansas: R. solani populations were examined by the protocol developed by the regional project which included two selective media, ethanol-potassium nitrate and Ko and Hora, and two sampling techniques, multiple-pellet soil sampler and toothpick method. In 2010, soils used in evaluations in Arkansas were one soybean field in rotation with rice and a prairie soil in Prairie County, three cotton fields in Ashley and Poinsett Counties, and a vegetable production field in Sebastian County. The diversity of Rhizoctonia species in these cropping systems included R .solani AGs 11, 1-1A, 4 and 2-1, R. oryzae and bi-nucleate Rhizoctonia species. Ethanol or Ko and Hora media both allowed the isolation of R. solani. However, Ko and Hora medium had more problems with fungal contaminants. Anastomosis groups detected were similar for the two selective media, two techniques, and seedlings. The toothpick method was an effective method for the recovery of R. solani from soils with diverse cropping histories because no special tools and few units were needed to detect the population. By comparing the soil pellet weight assayed to toothpick isolation frequency, the volume of soil assayed by the toothpick method can be estimated. Kentucky: Samples were collected from three locations (histories of vegetable, tobacco, and soybean production) in May and June, 2010 and were assayed using the toothpick method chosen by the S1028 group. Isolates have been recovered and stored for analysis. They will be sent to C. Rothrock for complete identification. Louisiana: A study was conducted at the Macon Ridge Research Station near Winnsboro, LA to assess the diversity of Rhizoctonia sp. in corn, cotton, grain sorghum, and soybean fields. A standardized protocol developed by the S1028 group was used. Soil samples were taken from fields (two per crop) and placed in plastic containers. Moisture was supplemented to soil to saturation and toothpicks were placed in soil and left for a predetermined time according to protocol. Toothpicks were plated onto ethanol-potassium nitrate medium. Plates were monitored for growth of Rhizoctonia at 24, 48 and 72 hours. Fungal growth was minimal at 24 and 48 hours; however, colonies were counted 72 hours after toothpicks were placed on plates. There were 142, 158, 233, and 113 colonies counted for corn, cotton, grain sorghum, and soybean, respectively. Oklahoma: DNA fingerprinting protocols (AFLP, ISSR, SSR) were standardized for analysis of R. solani populations. Peanut populations of the Sclerotinia minor from Oklahoma were characterized using AFLP and ISSR. Multiple strains of Pythium irregulare species complex from diverse locations and hosts were characterized. Phylogenetic analyses of this clade showed evidence that multiple cryptic species exist. The genetic fingerprinting of multiple isolates of Phymatotrichopsis omnivora from Oklahoma, New Mexico, Texas and Arizona was completed. The genetic fingerprinting was completed and population genetic analyses performed of multiple isolates of Fusarium oxysporum f. sp. palmarum in collaboration with Monica Elliot, University of Florida. It was demonstrated that sublethal doses of two fungicides, Cyazofamid and Propamocarb, as well as ethanol induce stimulation in Pythium aphanidermatum and that hormesis is involved in the stimulation processes. It was also demonstrated that low doses of ethanol induce stimulation in Rhizoctonia zeae and that hormesis is involved in the stimulation process, while Propiconazole at sub-lethal doses do not have hormetic effects on either R. zeae or on R. solani. Diagnostic primers were designed for multiple Pythium, Rhizoctonia, and Sclerotinia spp. Tennessee: Previous studies had shown that populations of Rhizoctonia solani were greatest in soil with a current crop of fall-planted broccoli, followed by fallow soil with a previous spring broccoli crop, and lowest in non-agricultural soil; detection was based on incubation of a toothpick bait in soil, and then plating onto Ko and Hora culture medium. To support the results of these studies, and to determine if populations of Rhizoctonia detected were pathogenic, disease assays with broccoli seedlings as the bait plant, were conducted. In the disease assays, the incidence of damping-off of broccoli seedlings was 0% in the wooded non-agricultural soil, 40% in soil with a previous crop of spring broccoli, and 56% in the soil that was currently cropped to fall broccoli. The rates of damping-off were consistent with the population levels of R. solani detected in the three soils. Rhizoctonia solani was re-isolated from all damped-off broccoli seedlings. This confirmed previous studies on detection methods and microbiological culture media for identification of pathogenic R. solani from soil. Grant Funding Relevant to Objectives Canaday, C. H. Elemental Analysis of Tap Root Tissues Using Energy-Dispersive X-ray (EDX) Analysis. University of Tennessee Professional Development Award, $4,955. Cox, M. and Rothrock, C. S. Microbial changes associated with use of brassica cover crops compared to traditional production systems for strawberry. Graduate student Project, Southern Region SARE Program, $9,971. Donald, P., Biggerstaff, J., and Canaday, C. Advisory Program for Management of Soybean Cyst Nematode. Tennessee Soybean Promotion Board, $46,198. Ivors, K. and Benson, D.M. Evaluating integrated strategies for management of Phytophthora root rot on Fraser fir Christmas trees in North Carolina. NC Agricultural Foundation. $21,073. Mengistu, A., Arelli, P., Canaday, C. Screening of soybean varieties, breeding lines for charcoal rot, frogeye leaf spot and Phomopsis seed decay resistance, Tennessee Soybean Promotion Board, $30,000. Seebold, K.W. and Ji, P.J. 2010. Managing Phytophthora capsici on Pepper and Summer Squash with Combinations of Bioten and Conventional Fungicides. IR-4 Biopesticide Program, $20,000.

Refereed. Bartz, F. E., M. A. Cubeta, T. Toda, S. Naito, and K. Ivors, 2010. An in planta method for assessing the role of basidiospores in Rhizoctonia foliar disease of tomato. Plant Disease 94:515-520. Berbegal, M., A. Ortega, M. M. Jimenez-Gasco, C. Olivares-Garcia, R. M. Jimenez-Diaz and J. Armengol. 2010. Genetic diversity and host range of Verticillium dahliae isolates from artichoke and other vegetable crops in eastern-central Spain. Plant Disease 94: 396-404. Canaday, C.H. and A.F. Schmitthenner, 2010. Effects of chloride and ammonium salts on the incidence of Phytophthora root and stem rot of soybean. Plant Dis. 94:758-765. Gwinn, K.D., B.H. Ownley, S.E. Greene, M.M. Clark, C.L. Taylor, T.N. Springfield, D.J. Trently, J.F. Green, A. Reed, and S.L. Hamilton. 2010. Role of essential oils in control of Rhizoctonia damping-off in tomato with bioactive Monarda herbage. Phytopathology 100:493-501. Gu, G., L. Smith, N. Wang, H. Wang, and S.-E. Lu., 2009. Biosynthesis of an antifungal oligopeptide in Burkholderia contaminans strain MS14. Biochemical and Biophysical Research Communications. 380: 328-332. Gu, G., N. Wang, N. Cahney, L. Smith, and S.-E. Lu. 2009. AmbR1 is a key transcriptional regulator for antifungal activity of Burkholderia contaminans strain MS14. FEMS Microbiology Letter. 297:54-60. Jangid, K., M. A. Williams, A. J. Franzluebbers, J. M. Blair, D. C. Coleman, and W. B. Whitman, 2010. Development of soil microbial communities during tallgrass prairie restoration. Soil Biology & Biochemistry 42:302-312 Kaye, A.C., Moyer, J.W., Parks, E.J., Carbone, I., and Cubeta, M.A., 2011. Population genetic analysis of Tomato spotted wilt virus on peanut in North Carolina and Virginia. Phytopathology 101:147-153. Keinath, A. P., Hassell, R. L., Everts, K. L., and Zhou, X.-G. 2010. Cover crops of hybrid common vetch reduce Fusarium wilt of seedless watermelon in the eastern United States. Online. Plant Health Progress doi:10.1094/PHP-2010-0914-01-RS. Kim, D.-G, T. M. Isenhart, T. B. Parkin, R. C. Schultz, and T. E. Loynachan. 2010. Methane flux in cropland and adjacent riparian buffers with different vegetation covers. J. Environ. Qual. 39:97-105. Lu, S.-E., J. Novak, F. W. Austin, G. Gu, D. Ellis, M. Kirk, S. Wilson-Stanford, M. Tonelli, and J. L. Smith. 2009. Occidiofungin, a unique antifungal glycopeptide produced by a Strain of Burkholderia contaminans. Biochemistry 48: 8312-8321. Njoroge, S. M. C., Toler, J. E. and Keinath, A. P. 2010. Soil solarization on populations of Pythium spp., fluorescent Pseudomonas, and damping-off of broccoli and cucumber. Int. J. Veg. Sci. 16:15-31. Sullivan, M. J., Parks, E. J., Cubeta, M. A., Gallup, C. A., Moyer, J.W., and Shew, H.D. 2010. An assessment of genetic diversity from a field population of Phytophthora nicotianae with a changing race structure. Plant Disease 94:455-460. Abstracts Berbegal, M., C. D. Garzon, A. Ortega, J. Armengol, R. M. Jimenez-Diaz and M. M. Jimenez-Gasco. 2009. Development and application of new molecular markers for the analysis of genetic diversity in Verticillium dahliae populations. In 10th International Verticillium Symposium Book of Abstracts, November 16-20, 2009, Corfu, Greece. p. 29. Jimenez-Gasco, M. M., M. Berbegal, G. M. Malcolm, J. Armengol and R. M. Jimenez-Diaz. 2009. New insights on the phylogenetic relationships of vegetative compatibility groups in Verticillium dahliae. In 10th International Verticillium Symposium Book of Abstracts, November 16-20, 2009, Corfu, Greece. p. 28. Spurlock, T. N., Rothrock, C. S., and Monfort, W. S. 2010. Evaluation of selective media and selective chemicals on the isolation of Rhizotonia spp. from soil. (Abstr.) Phytopathology 100(6) Supplement S121. Book Chapters. Benson, D.M., and B.H. Ownley. 2010. Exercícios de laboratório com fitopatógenos do solo. Pages 255-270 in: Fitopatologia: Conceitos e Exercícios de Laboratório. 2a Edição. R. Trigiano, M. Windham, and A. Windham, eds., Taylor and Francis, CRC Press, Boca Rotan, FL. (Translation in Portuguese). Ownley, B.H., and D.M. Benson. 2010. Fitopatógenos do solo. Pages 237-254 in: Fitopatologia: Conceitos e Exercícios de Laboratório. 2a Edição. R. Trigiano, M. Windham, and A. Windham, eds., Taylor and Francis, CRC Press, Boca Rotan, FL. (Translation in Portuguese). Ownley, B.H., and M. Windham. 2010. Controle biológico de fitopatógenos (Revised). Pages 447-460 in: Fitopatologia: Conceitos e Exercícios de Laboratório. 2a Edição. R. Trigiano, M. Windham, and A. Windham, eds., Taylor and Francis, CRC Press, Boca Rotan, FL. (Translation in Portuguese). Ownley, B.H., K.D. Gwinn, and F.E. Vega. 2010. Fungal entomopathogens with activity against plant pathogens: ecology and evolution. Pages 113-128 in: The Ecology of Fungal Entomopathogens. H.E. Roy, F.E. Vega, D. Chandler, M.S. Goettel, J. Pell, and E. Wajnberg, eds. Springer-Verlag, NY. Other Publications. Canaday, C.H., 2010, Effects of seed treatments, potash formulation and application time on plant height, stand loss, and yield, 2009. Plant Disease Management Reports (online), Report 4:ST012. doi:10.1094/PDMR04. Canaday, C.H., 2010, Evaluation of seed treatments, in-furrow sprays, and biological agents for control of seedling diseases of snap bean, 2009. Plant Disease Management Reports (online), Report 4:V084. doi:10.1094/PDMR04. Francis, R., and Keinath, A. 2010. Biofungicides and chemicals for managing diseases in organic vegetable production. Clemson Cooperative Extension Information Leaflet 88. Malcolm, G. M., and M. M. Jimenez-Gasco. 2010. Associations of Verticillium dahliae with host and non-host plants in agricultural fields. In 95th Ecological Society of America Annual Meeting, August 1-6, 2010, Pittsburgh, PA. p COS4. Yousef, L.F. 2010. Class-I elicitins in relation to sterol acquisition and lipid profiling of Phytophthora sojae. PhD Dissertation. The Ohio State University, Columbus, OH.