Brief Summary of Minutes of Annual Meeting

Objective 1. To identify and characterize new biological agents, naturally suppressive soils, cultural practices, and organic amendments that provide control of diseases caused by soilborne plant pathogens. NY: Several isolates of Rhizoctonia solani and Rhizoctonia-like fungi recovered from naturally infected vegetables were found to infect and survive on corn and other grain crops including oat, rye, sudangrass, wheat and buckwheat. Isolates belonging to AG 2-2 that were originally obtained from table beets were the most pathogenic on corn and other grains as well as beans in greenhouse tests. NY: The impact of several crop production systems on the severity and damage of root diseases was assessed using a soil bioassay with beans to determine potential root health status. A total of 305 samples from vegetable, sods/pasture, cash grain, fruit, and dairy corn (silage/grain with manure application) were analyzed in the greenhouse. Bean roots grown in soil samples from the vegetable and cash grain production systems had the poorest root health (highest root rot severity ratings), whereas those from the perennial fruit production systems had the lowest root rot severity ratings. The sods and dairy corn systems were in between and exhibited rather low root rot severity ratings. NY: A replicated trial comparing the effect of three tillage systems (ridge-till, zone-till, and plow-till) on yield and root rot severity was conducted on a 14-acre long-term soil health site at the Gates Farm, NYSAES near Geneva, NY. The zone-tilled beans had the highest yield, whereas the yield of those grown under the ridge-till and plow-till were equal. Root rot severity ratings of bean grown under the three tillage systems did not differ and was only moderate in severity. CA: Our main focus for this objective is the discovery of pathogen- and/or disease-suppressive soils, in particular those that suppress populations of plant pathogenic nematodes. For a number of years we have investigated a field suppressive to the beet cyst nematode, Heterodera schachtii. The most likely causal agents, Dactylella oviparasitica and Fusarium oxysporum were reintroduced in greenhouse and field microplot trials to fumigated, non-suppressive soil and planted to a host crop. After three weeks, the soil was infested with H. schachtii juveniles. After approximately two months, only D. oviparasitica reduced the population densities of H. schachtii to a similar degree as in the original suppressive soil. The apparent lack of efficacy of the F. oxysporum strain raised questions about strain variability. Twenty-six strains of Fusarium spp. were isolated from colonized cysts or parasitized eggs of H. schachtii. They differed substantially in colony appearance and metabolite production on solid culture media. Phylogenic analysis of their rRNA ITS nucleotide sequences revealed considerable genetic variability. Seven representative strains were individually evaluated in greenhouse tests for their potential to parasitize beet cyst nematode eggs and to cause H. schachtii population suppression. Each test strain was introduced into fumigated soil at a propagule density similar to that of F. oxysporum in suppressive soil. The suppressive soil and its fumigated equivalent served as controls. The nine treatments were arranged in a randomized complete block with four replications. A four-week-old Swiss chard seedling was transplanted into each pot that was infested with 5000 second-stage juveniles of H. schachtii. After 1180 degree days in the greenhouse, all Fusarium strains parasitized H. schachtii eggs, but at considerably different levels. While one strain parasitized less than 10% of the eggs, the most aggressive strain was found in approximately 35% of the eggs. This degree of parasitism was not significantly different from the one observed in the suppressive soil. However, in contrast to the originally suppressive soil, in none of the Fusarium-infested soils was the population of H. schachtii significantly reduced after two nematode generations. This supports our hypothesis that Fusarium spp. were not the primary cause of the population suppression. In another project we collected soil from several California locations that have been known to be inhospitable to various plant parasitic nematodes. These were assayed in greenhouse tests for suppressiveness to root-knot nematodes (Meloidogyne incognita). In one soil and with two different hosts, the population of M. incognita was consistently lower than in the fumigated, re-infested check. Aerated steam or soil fumigation eliminated the suppressive factor/s. Non-treated soil transferred the suppressiveness to fumigated, conducive soil. Fungal rRNA gene analysis of root-knot nematode egg masses collected from a series of greenhouse trials identified 11 fungal phylotypes. The phylotype containing rRNA genes with high sequence identity to Pochonia chlamydosporia exhibited the strongest negative associations. MN: We have expanded our collection of antagonistic Streptomyces and Pseudomonas isolates from native prairie soils in the upper Midwestern United States, and from diverse natural habitats around the globe, adding approximately 300 new isolates. These isolates are being characterized with respect to their pathogen inhibitory activity. WA: Brassicaceous seed meals (BSMs) were evaluated for the potential to control the biologically complex phenomenon termed apple replant disease. Pathogen control via incorporation of BSMs was previously shown to involve a biological or chemical mechanism, which varied dependent upon the target pathogen and with time. BSM-induced suppression of root infection by Rhizoctonia solani is derived in part through amplification of resident Streptomyces populations. BSMs exhibited small differences in capacity to increase Streptomyces numbers, and the ability to control R. solani (Mazzola et al., 2007a). More recently, it was determined that there was no association between antagonistic potential of individual resident Streptomyces spp. isolates and control of Rhizoctonia root rot, that induction of plant defense responses does have a role in the capacity of certain Streptomyces isolates to elicit disease suppression, that composition of the resulting Streptomyces populations was dramatically altered in a BSM-specific manner, and that certain components of the resulting community while not functioning as apple root pathogens, could amplify R. solani root infection. A BSM that yielded a more uniform Streptomyces community exhibited a reduced capacity to control R. solani. One of the long-term goals of this research is to develop new and more sustainable strategies to manage soil-borne plant pathogens. To accomplish this, we are endeavoring to understand soils that naturally inhibit plant pests and disease, termed suppressive soils. Key steps in realizing the potential of these soils are to identify the causal organisms and then understand the agronomic and environmental factors that enable them to function. Armed with such knowledge, it should be possible to develop effective and sustainable pest management strategies through the application of the organisms and through agronomic practices that influence their populations. Towards this goal, the first Specific Objective of this research is to elucidate the interactions among the beneficial microorganisms, pathogens and crops that lead to the development and stability of the pathogen suppressiveness. To date, we have identified several fungi involved in suppressing sugarbeet cysts nematodes (Dactylella oviparasitica and Fusarium oxysporum) and root-knot nematodes (Pochonia chlamydosporium and a Tetracladium sp.). Current research is to elucidate the interactions among the beneficial microorganisms, nematode and crops that lead to the development and stability of the suppressiveness. Another long-term goal of this research is to develop new and more sustainable strategies for managing replant suppression. Replant suppression is a disease or disorder that often occurs when crops are "replanted" in soil that had previously supported the same or similar plant species. It typically leads to reductions in plant growth, crop yields and production duration. As the causal agent(s) remain ill-defined, the second Specific Objective of this research is to identify the microorganisms that cause Prunus replant suppression. Identifying the causal agents will represent a crucial step in the development of new management strategies, as this will facilitate targeted breeding programs and or the selection of targeted biological, chemical or integrated pest management strategies, thereby eliminating the need for soil fumigants such as methyl bromide. To date, we have obtained strong evidence that two stramenopile species (a diatom and Pythium ultimum) are the causal agents of Prunus replant disease in a peach orchard soil. OH: We characterized the effects of different farm management strategies on native populations of microbes and plant health. The use of compost for building fertility in transitional organic cropping systems led to improvements in soil fertility and soil structure leading to dramatic increases in yield potential. And, regardless of fertility level, mixed hay cropping provided durable soilborne pathogen suppression. These results suggest that mixed hay can be used as a useful cover crop or mixed-species green manure to provide vegetable growers with naturally disease suppressive soils. NM: Seed treatment with biofungicides (Micro108 and Kodiak), and transplant treatment with biofungicides (Mycostop Mix and Actinovate AG) and botanical extracts (garlic extract and Capsicum oleoresins) were tested for effectiveness against Phytophthora blight (Phytophthora capsici) on chile pepper. Seeds treated with a chemical fungicide (Apron XL LS) and seeds not treated (control) were included in the experiments. Although seed treatment did not significantly afford plants with protection against P. capsici after seedlings were transplanted in soil infested with P. capsici, results show that plant protection could be achieved with treatment of transplants with botanical extracts. Pungent Capsicum oleoresins were shown to have the potential to reduce Phytophthora blight on chile pepper. OR: We are conducting studies in horticultural nurseries to identify sources of Phytophthora contamination in potting media and soil and developing recommendations for specific cultural practices that will be effective in eliminating those sources of contamination. We are also developing an online course to better educate nursery personnel about Phytophthora diseases in their nurseries. CA: We are assessing the significance of insects in the acquisition and dissemination of root-infecting pathogens belonging to the Phyla Oomycota (Pythium and Phytophthora spp). We are searching for chemical substrates which selectively enhance resident populations of fluorescent pseudomonads in amended recycled irrigation water. We are continuing studies on the identification of the soil microbes and other factors that induce germination of ascospores of Monosporascus cannonballus. We are looking at the association of Olpidium bornovanus association with vine decline of melon. WA: Novel phenazine producers are predominant in dryland wheat soils. Phenazine produced by fluorescent pseudomonads was initially hypothesized to be involved in take-all suppression in the late 1970s. This work was mostly done in irrigated plots to favor take-all. Later work with molecular tools in the mid 1990s implicated phloroglucinol producers as being responsible for take-all suppression. However, this work was also done in irrigated fields and in the high rainfall areas of eastern Washington. Recent sampling work at a dryland plot in which we identified possible Rhizoctonia suppression has recovered high numbers of phenazine producing bacteria, both Pseudomonas and Burkholderia. Several important discoveries have been made. High levels of phenazine have been detected from the rhizosphere of wheat and barley growing on this site, using mass spectroscopy. This group of phenazine producers is widespread across the low rainfall dryland region in wheat. Based on sequences of the biosynthesis genes for phenazine and 16s rDNA genes, these strains are unique from previous strains, and are closest to Pseudomonas orientalis. Some of the strains are active against Rhizoctonia in vitro. Irrigation completely shifts the population toward phloroglucinol producers. Phenazine producing strains may be involved in Rhizoctonia suppression and patch disappearance at this site. WA: Composition of Rhizoctonia and Pythium spp. in cereal cropping systems is influenced by cropping systems and climatic zones.We are continuing our survey of eastern Washington, using real-time PCR to quantify 10 Pythium spp. and 6 Rhizoctonia groups. We have picked up some broad trends. R. solani AG-8 is found in high populations in low rainfall areas with sandy loam, areas with patch symptoms under direct seeding. In higher rainfall areas of the Palouse, AG-8 is detectable but not quantifiable. R. oryzae is found in annually cropped areas. R. solani AG 2-1 is associated with grain legume rotations. P. ultimum and P. irregulare Group 1 are found in high rainfall areas with grain legume rotations, but not in low rainfall areas with summer fallow. However, P. irregulare group 4 is widely distributed across all zones. We also found that most Pythium species, except P. abappressorium and P. irregulare Group 4, were almost absent from fallow treatments. WA: Timing of herbicide sprays can greatly affect greenbridge carryover of inoculum from volunteer to planted crop.A second year of field trials were conducted. All seedling measurements were affected by greenbridge treatments. The longer the greenbridge period with green volunteer/weeds before planting, the lower the crop performance. The longer the period of time the weeds and volunteer are killed before planting, the better the crop performance. The critical threshold period was between 2 and 4 weeks before planting. In other words, if the crop is sprayed out less than 2-4 weeks before planting, the crop suffered. The response seemed to be asymptotic after this 2-4 week threshold- i.e. longer periods free of the greenbridge after this threshold period did not always result in additional improvement to the crop. WA: Phialophora isolates are more tolerant to phloroglucinol We hypothesized that Ggt isolates from long-term take-all decline may develop resistance to the antifungal compound phloroglucinol (DAPG), which is responsible for natural suppression. We tested a large collection for in-vitro sensitivity to DAPG and virulence in the greenhouse. Most isolates were sensitive to DAPG, with ED90s ranging from 3 to 11 ppm. There was no correlation between sensitivity to DAPG and virulence. However, we also found a population of weakly pathogenic Phialophora (the asexual state of Ggt) strains that are more tolerant of DAPG (5 times more), which were more predominate in long-term no-till, and may be selected for in long-term take-all decline. Objective 2. To understand how microbial populations and their gene expression are regulated by the biological (plants and microbes) and physical environment and how they influence disease. MN: We have developed methods to study the effects of agricultural nitrogen applications (urea) on pathogen-antagonist interactions, and specifically whether urea application reduces biocontrol efficacy. AZ: We found previously that quorum sensing (QS) is important in biofilm initiation in Pseudomonas. We have identified additional roles for phenazines in the biology of P. chlororaphis 30-84. We have also characterized the role of the RpeA/RpeB negative two component regulatory system in strains 30-84 and P. aeruginosa PAO1. NY: In the Smart lab, we are studying the effect of multiple plant activators on plants in the field to determine if induced systemic resistance (ISR)-inducing biocontrol agents are compatible with other systemic acquired resistance (SAR)-inducing compounds. Additionally, a DNA-based macroarray was developed which can detect over 40 fungal and oomycete pathogens simultaneously. This array is effective on soil and plant tissue samples, and is currently being tested on irrigation water. Finally, the development of detection and identification systems for beneficial soil-inhabiting microbes will, together with the pathogen detection arrays, enable growers to know both the pathogen and beneficial content of their soils. WA: Previously, the zoosporicidal activity and control of Pythium root rot of flower bulbs by Pseudomonas fluorescens SS101 was attributed, in part, to the production of the cyclic lipopeptide biosurfactant massetolide A. However, when he capacity of strain SS101 and its surfactant-deficient massA mutant 10.24 to suppress populations and root infection by complex Pythium spp., both strains effectively suppressed resident Pythium populations to an equivalent level and ultimately suppressed Pythium root infection to the same degree on all host plants. Pythium spp. populations indigenous to the two soils employed in these studies were composed primarily of P. irregulare, P. sylvaticum, and P. ultimum var. ultimum. These Pythium spp. either do not or rarely produce zoospores, which could account for the observation that both SS101 and mutant 10.24 were equally effective in disease control. Collectively, the results showed that (i) Pseudomonas fluorescens SS101 is very effective in controlling diverse Pythium populations on different crops grown in different soils and (ii) production of the cyclic lipopeptide massetolide A does not play a significant role in disease suppression. Other, as yet undefined mechanisms appear to play a significant role in the interaction between P. fluorescens SS101 and soilborne Pythium spp. communities (Mazzola et al., 2007b) OH: We determined that DAPG and phlD+ Pseudomonas can alter crop root architecture. Specifically, terminal necrosis and secondary root generation were observed. This phenomonenon occurred at concentrations similar to those reported for in situ measurements of DAPG and the induction of host resistance pathways in some plant species. Such changes appeared to affect both tomato and corn, but the greater affects were observed on the dicot species. Preliminary data also indicate that such changes might also affect nutrient uptake in corn. WA: In collaboration with Scot Hulbert, we have been using pyrosquencing (454) to look at total bacterial microbial communities in soil based on 16s ribosomal genes, to see how tillage and crop rotation affect community structures. We have conducted two years of sampling from a long-term site near Manhattan, KS, comparing 4 treatments in a 2 X 2 factorial experiment (two rotations- wheat-wheat or wheat-soybean) and 2 tillage treatments (conventional or no-till). Over two years, we characterized over 15,000 sequences, which were classified into bacterial groups. Acidobacteria was the most predominate group, representing almost 20% of the sequences. Gemmatimonads and Actinomycetales were also common. Other common groups were Burkholdariales, Rhizobiales, and Sphingobacteriales. Surprisingly, groups of Acidobacteria were influenced by the cropping practice. For example, Group 2 was more predominate in no-till, while Group 4 was more numerous in the soybean rotation. This new method is a powerful tool to look at total non-culturable microbial communities, and will be used next year in comparing long-term no-till plots to conventional tilled plots in eastern Washington. Objective 3. To develop and implement biological control in agriculture. NY: A replicated trial comparing the effect of three tillage systems (ridge-till, zone-till, and plow-till) on yield and root rot severity was conducted on a 14-acre long-term soil health site at the Gates Farm, NYSAES near Geneva, NY. The zone-tilled beans had the highest yield, whereas the yield of those grown under the ridge-till and plow-till were equal. Root rot severity ratings of bean grown under the three tillage systems did not differ and was only moderate in severity. The capacity of Brassicaceae seed meals to provide replant disease control, both in greenhouse and field trials, was found to vary in both a seed meal and apple rootstock-dependent manner. When grown in replant orchard soils amended with BSM in the field, apple rootstocks differed in the capacity to support resident Streptomyces populations, with Geneva series rootstocks typically superior to Malling-series rootstocks in this attribute. These same Geneva rootstocks exhibited a greater growth response in the BSM amended soils, typically to a level equivalent to that attained through soil pasteurization or fumigation. Among those evaluated, Brassica juncea seed meal provided superior control of Pythium spp. and Pratylenchus penetrans, whereas lesion nematode control in response to Brassica napus seed meal was only attained on rootstocks (Geneva series) that maintained an inherently lower P. penetrans populations (Mazzola et al., 2009). WA: Evaluation of limestone amendments for control of Fusarium wilt in a spinach seed crop, 2006 Lindsey du Toit, at the WSU research station in Mt. Vernon, has conducted field trials, to investigate whether limestone amendments can suppress Fusarium wilt of spinach, a major seed crop in NW Washington. If a field becomes infected, it cannot be used seed production for at least 10 years. Fields were planted with the male, a susceptible female and a moderately susceptible female line. In 2006, rates from 0 to 4.2 tons/acre were tested, and rates of 0 to 8 tons in 2007. In 2006, there was a significant interaction between rates and plant lines- with the greatest effects seen in the most susceptible lines. In 2007, rates from 2, 4, 6, and 8 tons reduced wilt, with no interaction between rates and plant lines. All treatments increased soil pH by 0.5 to 1 unit by the end of the season. Future research will focus on the microbial basis of this suppression. Objective 4. Provide outreach, education, extension and technology transfer to our clients and stakeholders- growers, biocontrol industry, graduate and undergraduate students, K-12 students and other scientists. AZ: P. chlororaphis 30-84 is used as a model genetic system in microbial genetics laboratory course. In spring 2008 there were 88 total students enrolled in 2 sections of the laboratory course. Each pair of students performed transposon mutagenesis of strain 30-84 and screened for unusual phenotypes. The students then isolated genomic DNA and attempted to clone out the mutated region of the chromosome. MN: We are training 4 Ph.D. students in our laboratory. OH: The PI facilitated an open discussion session at the annual meeting of the American Phytopathological Society on use and adoption of biological controls in organic agriculture and he also presented a workshop on transitioning to organic agriculture presented by the Ohio Ecological Food and Farming Association. Consultation with industry on biopesticide discovery, testing, and product formulation was also conducted. For organic growers, we developed and distributed the first Extension FactSheet that described the utility of commercially-available microbial biopesticides for plant disease control. WA: Dr. Lindsey du Toit has an extension appointment, and devotes much of her time to extension talks and training. ARS scientists at Pullman, WA lead a science outreach and engagement program, with participation by USDA, NRCS, Washington State University Cooperative Extension, WSU Plant Pathology Department, and Bellevue Community College, to The Confederated Tribes of the Colville Reservation and the region surrounding Omak, WA. This is one of the most remote, poorest and underserved regions in Washington State. The program entitled "Pumping-Up the Math and Science Pipeline: Grade School to College" has four components: i) development of energy independence on the Colville Reservation through the production of biofuels; ii) hands-on science education in reservation schools by ARS research scientists, WSU faculty and BCC instructors; iii) development of and participation in on-reservation summer science camps; and iv) employing Native American, Latino and rural high school summer science interns in ARS laboratories. Examples of these activities include: 1) field tests of spring and winter canola varieties and biodiesel production on Colville Reservation land; 2) monthly visits to Nespelem Elementary School and Pascal Sherman Indian School on the Colville Reservation to present science modules, October 2007 to May, 2008) sponsorship of and participation in the Second Annual Skwant Life Science Camp at the Pascal Sherman Indian School (Skwant is translated as 'Waterfalls'), June 2007 (two 1-week sessions for 5th-6th and 7th-8th graders and a total of 70 campers); and 4) molecular biology training for 6 summer high school interns (July 2008) in ARS labs at Pullman. This award-winning program is promoting economic development on the Colville Reservation, enhancing science awareness among Native American, Latino and rural youth, and training the next generation of scientists. Native American reservations and rural communities throughout the Pacific Northwest are requesting expansion of this ARS program to their communities. Numerous extension talks were given by members of the group, listed under Publications.