Brief Summary of Minutes of Annual Meeting

Objective 1: To identify and characterize plant microbe interactions that provide suppression of diseases caused by soilborne pathogens. California (UCR - Becker and Borneman): Worldwide, beet-cyst nematodes are major production constraints of beets and crucifers. We have investigated a field at the University of California Riverside Field Station that has been suppressive this nematode for about 25 years. Analyses of soils positively correlated with soil suppressiveness against H. schachtii have identified causal agents of the suppressiveness nature. In greenhouse trials, strains of D. oviparasitica and F. oxysporum were reintroduced into fumigated, non-suppressive soil. While F. oxysporum did not significantly decrease H. schachtii populations within two nematode generations, D. oviparasitica decreased the beet cyst nematode population to a similar level as in the suppressive soil. Moreover, the increased plant growth as a result of the pest reduction was similar to the non-infested check. This benefit of the biological control has not always been observed in the original suppressive soil. Microplot trials confirmed the greenhouse results but also indicated that Fusarium strains may require a longer time period to reduce nematode populations. California (UCR - Stanghellini): N-Serve studies: Initiate qualitative and quantitative assessment of changes in the resident bacterial, including the fluorescent pseudomonad, populations in recycled nutrient solutions amended with the chemical and determination of the biological effect of such changes on plant growth and disease control. Additionally, investigations will be initiated on the mechanism(s) associated with selective enhancement of specific bacterial populations in the chemical-amended and recycled nutrient solutions. These studies will involve the use of specific rifampicin-resistant strains of Pseudomonas spp. which have been previously associated with control of root- infecting pathogens or enhancing plant growth. Population densities of fluorescent Pseudomonas spp. increased from 103 CFU/ml to 106 CFU/ml within 48-72 hr after amending the recycled nutrient solution in a hydroponic cultural system with N-Serve. Similar results were obtained using xylene as the amendment. Pseudomonas putida, biotype A, was the dominant indigenous fluorescent Pseudomonas spp. identified following chemical amendment of the nutrient solution. In in vitro studies, significant population increases of known fluorescent Pseudomonas species (i.e., Pf-5 and P. aureofaciens 30-84) occurred following their addition to the nutrient solution in the presence of N-Serve but those populations were lower (2X) than the population increases of the indigenous fluorescent Pseudomonas spp. However, Pf-5 and P. aureofaciens 30-84 were not competitive with indigenous fluorescent Pseudomonas spp. in in vivo studies and their populations decreased significantly following their addition to the nutrient solution in the presence or absence of N-Serve. Significant disease control (using pepper plants as the host and Phytophthora capsici as the root pathogen in a model system) was achieved following amendment of the nutrient solution with various concentrations of N-Serve or xylene. Biosurfactants. Conclude studies on the efficacy of biosurfactants (Zonix and ThermX70) on the control of zoosporic pathogens in recycled nutrient solutions. Amending the nutrient solution with either a rhamnolipid or a saponin completely suppressed disease development (root rot of pepper caused by Phytophthora capsici) in both ebb-and-flow and top-irrigated cultural systems, with either an organic potting medium or rockwool. These results provide evidence that biosurfactants may be a suitable alternative to registered fungicides or synthetic surfactants in the management of diseases caused by zoosporic pathogens in cultural systems employing recycled irrigation water. New York (Cornell - Abawi, Harman, Smart): Suppressive capacity of vegetable soils under various soil management practices to soilborne pathogens: As part of a collaborative and multidisciplinary project conducted by the Cornell Soil Health Program Work Team, soil samples collected from various grower fields were assessed for their general suppressive capacity to soilborne pathogens (root health) using the soil bioassay with bean (described in detail later). In general, root health was improved with the implementation of reduced tillage, long rotations with grain crops, certain cover crops and organic amendments. Frequency distributions are being used to compare various management practices. Evaluation of selected cover crops incorporated as green manures for suppressing soilborne pathogens: Varieties of forage and grain pearl millets, canola, rapeseed, buckwheat, hairy vetch, sudangrass, oilseed radish and mustard are being evaluated in field microplots, an experimental root rot field and in the greenhouse against bean root rot pathogens, especially Rhizoctonia solani and Pratylenchus penetrans. A total of 18 replicated treatments were established in the field microplot (4-ft. diam. fiberglass cylinders) test. The field soil in the microplots was additionally infested with R. solani (potato-soil inoculum preparation) prior to planting with the various cover crops. After approximately 8 weeks, the cover crops were cut and incorporated into the soil. A month later, a soil sample was collected from each microplot and bioassayed with bean in the greenhouse. Root rot severity was rated 5 weeks later on a scale of 1 (healthy root) to 9 (>75% of roots and stem tissues affected and at various stages of decay). Results obtained suggest a differential effect of the various cover crops and varieties tested, even after being incorporated for only one month. The microplots will be planted to beans in spring 2006 and root rot severity and yield will be determined. Seven cover crop treatments were established in an experimental bean root rot field (heavily infested with R. solani, Thielaviopsis bascicola, Pythium ultimum, and Fusarium solani f. sp. phaseoli) in early August after the 2005 bean crop was plowed under. Each plot was 25 x 60 ft. and the treatments were replicated 4 times. The cover crops were incorporated in early October and soil samples were collected in early November for the greenhouse soil bioassay, which is still in progress. These plots will also be planted to beans in spring of 2006 for root rot and yield assessment and evaluation. An extensive greenhouse evaluation of all the selected cover crops and varieties is in progress and is been conducted in naturally infested soil and in soil infested with P. penetrans. Oregon (Oregon State - Parke): We are investigating the ecology and epidemiology of Phytophthora ramorum in soil, potting media, and irrigation water so as to design effective cultural and biological control measures for eliminating this disease in horticultural nurseries. Infection of roots and spread in vascular tissue: Phytophthora ramorum, cause of sudden oak death and ramorum blight, is believed to be an aerial plant pathogen. We demonstrated that P. ramorum can infect roots of rhododendron grown in artificially infested potting media, spreading in the vascular tissue to stems and leaf petioles. Foliar infections also spread into stem tissue via the vascular system. Survival in soil: P. ramorum was recovered up to 20 weeks after burial of detached inoculated rhododendron leaves in potting soil and forest soil moistened to achieve a range of soil water matric potentials (-1 to -1500 kPa). Taken together, results from our experiments indicate that P. ramorum has a soil-borne phase which could be important in the epidemiology of this disease, particularly in nurseries or in landscaped settings. The significance of the soil phase of P. ramorum in natural forest settings is not yet clear. Epidemiology and control of Phytophthora species in recirculating irrigation water: We initiated studies on the detection, epidemiology, and control of Phytophthora species in recirculating irrigation systems. We constructed 32 self-contained mini-nurseries, each with its own recirculating water system, for conducting dose-response studies and for testing the efficacy of water treatment methods, including biofilters, for the elimination of Phytophthora species in irrigation water. Washington (USDA - Paulitz, Okubara, Weller, Thomashow): Suppression of Rhizoctonia bare patch with barley rotations. Rhizoctonia bare patch is caused by R. solani AG-8, and is a major problem in direct-seeded wheat and barley. Because of the wide host range of this pathogen, crop rotation had not been considered feasible as a cultural practice to manage this disease. However, recent field trials have demonstrated that wheat following barley has less disease and greater yield than continuous wheat. Because barley is also a susceptible host, the rotation effect is not due to lack of a host, but may be due to a shift in microbial communities. This phenomenon was documented in the field again this year, and a manuscript was accepted in Plant Disease. Reduction of soilborne pathogens under long-term no-till. During the conversion from conventional tillage to no-till or direct seeding, certain root diseases can become more severe. This was documented in a four-year field trial near Pullman. No-till plots were established on land that had been conventionally tilled for a long period of time. Other plots at the site were kept as conventionally-tilled, and the experiment was planted with a spring wheat-spring barley rotation. Another location, about 1 km away, had been in no-till for 12 years, and both conventional and no-till plots were established. Rhizoctonia, Pythium, and take-all were monitored over the next four years, along with plant measurements. For the first 2 years, there was no effect of stopping tillage and converting to no-till. However, in the 3rd and 4th year of the conversion, Rhizoctonia became severe, and significantly reduced yield. Population studies documented the build-up of R. solani, but not R. oryae. However, converting the no-till plots back to tillage did not result in any increase of yield or decrease in disease. Another part of the study compared long-term no-till farms to adjacent conventionally-tilled farms. No differences were found in root diseases. This indicates that Rhizoctonia becomes severe a few years after tillage is stopped, but when no-till has been practiced for 12 years or longer, disease appears to be suppressed, compared to earlier years. This phenomenon has also been documented in Australia. Future research will focus on documenting the shift in microbial communities that accompanies the transition from conventional tillage to no-till, and compare long-term no-till fields to adjacent conventionally tilled fields. Objective 2: To understand how biological and environmental factors regulate microbial populations and the expression of genes responsible for disease suppression. Arizona (U of Arizona - Pierson): Molecular analysis of negative signaling among wheat rhizobacteria. We continue to analyze the negative signal(s) produced by the wheat rhizosphere strain PU-43. The negative signal produced by this strain blocks phenazine gene expression in strain 30-84 and b-galactosidase activity in the genomic reporter strain 30-84Z. It has proven difficult to purify the negative signal. A genomic library has been constructed from strain PU-43. We identified a cosmid that contains an AHL synthase gene that rescues an AHL- strain of 30-84. A knockout mutant has been constructed that only produces the negative signal. Currently the signal can be extracted into butanol that is allowing further structural characterization. We also have identified four other cosmids from this library that result in a reduction in phenazine gene expression and may encode negative signals. One of these cosmids contains the gene oprF. OprF is a major outer membrane-associated protein in pseudomonads. In the human pathogen P. aeruginosa, OprF was shown to bind human interferon-g (IFN-g) resulting in QS-dependent induction of the lectin PA-I and the phenazine pyocyanin. Characterization of RpeB, the second part of the two component regulatory system that negatively regulates phenazine production. RpeA is a sensor kinase that appears to couple aspects of cell metabolism to secondary metabolite production. Mutations in rpeA result in higher levels of phenazine gene expression in minimal medium in comparison to the wild type strain. We have cloned and sequenced the second component, rpeB of this two compontnet system. It is similar to other known two component response regulatory genes. Of great interest is the fact that a phzR/rpeA double mutant defective in both the negative two component system and QS produces phenazines at the same level as the wild type in all medium. Thus, the loss of rpeA bypasses the need for QS in strain 30-84. We have shown that mutations in P. aeruginosa PAO1 rpeA and rpeB homologs also resulted in higher than wild type levels of pyocyanin production, suggesting this regulatory system may be universal among phenazine producers. Identification of a new regulatory gene. We have identified a cosmid from the 30-84 genomic library that when present in trans in strain 30-84Ice with a second plasmid containing the phenazine promoter fused to lacZ reduces b-galactosidase expression 100-fold (2,000 vs. 20 Miller units). Localization of the region responsible implicates psrA as the gene responsible for the observed effect. Further analysis is ongoing. New York (Cornell - Abawi, Harman, Smart): Utility of plant growth-promoting rhizobacteria for disease control of foliar pathogens: In addition to having a direct effect on soilborne pathogens, some soil-inhabiting microbes provide disease control via a process known as induced systemic resistance (ISR). The role ISR may play in control of foliar pathogens is unclear. To gain a better understanding of how ISR may lead to disease control, we are following the gene expression patterns of plants that have either been treated with plant growth-promoting Bacillus sp. or left untreated. Expression of multiple genes has been followed both pre-and post inoculation with a bacterial pathogen. For several pathogenesis related genes, we have found an increase in gene expression only visible in tomatoes treated with ISR-inducing Bacillus spp. These results support the idea that plants treated with ISR-inducing products are primed and can respond more quickly upon pathogen attack. Although the ISR-induced plants show a quicker up-regulation of defense-response genes, there was no significant disease control on these plants. Oregon (USDA - Loper): The recently-published genome of Pseudomonas fluorescens is being exploited to study patterns of gene expression by this biological control agent. A limited number of microarray slides have been spotted and tested for use in gene expression studies. Methods for isolation of RNA have been adapted for Pf-5. We completed studies demonstrating a role for the stationary phase sigma factor (RpoS) in stress response and rhizosphere colonization by P. fluorescens Pf-5. In this bacterium, RpoS is required for optimial stress response, including its capacity to survive desiccation in culture and in the rhizosphere of plants grown in dry soils. This work provides the first evidence for a role of RpoS in environmental fitness of a bacterium in the field. Washington (USDA - Paulitz, Okubara, Weller, Thomashow): Development of species-specific PCR primers for the detection of Pythium spp. in wheat production. In order to understand how Pythium communities vary with rotation, season, tillage and other cultural practices, we need a method for accurately following the populations. Soil dilution plating cannot distinguish among the more that 10 species of Pythium that surveys have detected. We had developed and tested 10 primer sets for quantitative capillary real-time PCR, based on ITS sequences, that are specific for the following species- P. abappressorium, P. attrantheridium ( intermedium), P. irregulare IV, P. irregulare, P. ultimum, P. rostratum (rostratifingens), P. heterothallicum, P. sylvaticum, P. paroecandrum, and an unidentified species. We have developed standard curves and extracted DNA from natural soil to quantify Pythium. Studies were conducted this year to compare the Pythium species composition in different crop rotations. Seven Pythium species were detected, but preliminary data does not show any species shift among rotations during the beginning of the season, but, in general, populations decline over the growing season. Simultaneous identification and quantification of Rhizoctonia solani and R. oryzae using real-time PCR. Rhizoctonia is also difficult to quantify in soil, and cannot be detected with soil dilution plating because of the low population density. We have developed a semi-quantitative method using toothpick baiting, but we are also need a more accurate method that not only measures hyphal activity, but also dormant inoculum. Using ITS sequences, we have developed real-time PCR assays for 2 groups of R. oryzae. We have documented the presence of R. solani AG-8, AG-10, and AG-2-1, along with a binucleate Ceratobasidium spp. R. solani AG-2-1 is highly pathogenic on canola and also causes stunting of pea, but is not virulent on wheat or barley. Interactions between wheat cultivar and bacterial isolate in rhizosphere colonization and accumulation of 2,4-diacetylphlorogucinol (DAPG) produced by Pseudomonas fluorescens. Continuing studies have shown that wheat cultivars vary in their ability to support colonization by strains of P. fluorescens. A soil-free system was developed to assay DAPG production in roots and to do microarray studies. Cultivars varied in their ability to accumulate DAPG in roots. Some host defense/stress related genes were induced in wheat roots during colonization by P. fluorescens. Populations of DAPG-producing P. fluorescens in the rhizosphere on Pythium-infected wheat. Infections of roots by soilborne pathogens can either positively or negatively affect root colonization and antibiotic production by beneficial bacteria. We tested the impact of four Pythium spp. on colonization of wheat roots by indigenous populations of 2,4-diacetylphloroglucinol (DAPG)-producing P. fluorescens in Washington State suppressive soils. Before infestation with Pythium spp., soils were cycled to wheat (cv Penawawa) to reactivate and increase indigenous populations of the DAPG producers to a density greater than 105 CFU/g fresh root. Soils were then infested with P. abappressorium, P. irregulare, P. irregulare group IV, or P. ultimum at a rate of 103 CFU/g soil. In addition, 0.5% ground rolled oats was added to infested soils. Seven days after amending the soil, pregerminated wheat seed was sown. Rhizosphere populations of DAPG-producing bacteria were assessed using the PCR-based dilution end point assay 2 weeks after sowing. Populations of DAPG-producing P. fluorescens were similar in both infested and non-infested soils. None of the Pythium spp. affected the populations of indigenous DAPG-producing P. fluorescens on the wheat roots. Objective 3: To develop and implement economic biological control systems to achieve sustainable agriculture. Alaska (U of Alaska - McBeath): Efficacy of Trichoderma atroviride in the control of Armillaria mellea on plum trees in California (This study was conducted in collaboration with Ms. Kelly McBee Walker, AmPac Biotech, Fresno California and Mr. Jay Scott, Reedly Farm, Sanger, CA). Armillaria root rot is one of the most serious diseases on fruit trees. Methyl bromide soil fumigation has been the primary strategy for disease control. International restrictions on methyl bromide use make it necessary to seek alternatives. In mid-December, 2003, an efficacy study of Trichoderma atroviride was initiated in Sanger, CA on a plum orchard heavily infested with Armillaria root rot. A section of the farm, approximately 100 acres, was prepared as the study site. Plant Helperâ (flowable formulation), containing 107 cfu/g of T. atroviride spores, was sprayed on the root of plum stocks, at the application rate of one pound per acre, before being transplanted. Two rows of the transplants adjacent to the road were kept as an untreated control. Observations were made on the health of the trees. At the end of the second year, ten trees were randomly selected from the treated plot and untreated control and measured. The circumference of the tree trunk was taken at 12 inches above the crown. Data on leaf samples, ten leaves randomly harvested per tree, from the control and treated sample trees, were also collected and analyzed (using the Student t-test). The Plant Helperâ treatment continued to produce favorable results. The trunk of the treated trees, with a mean circumference of 32.88 cm was found significantly thicker than the untreated control (27.0 cm). Leaves from the treated trees were found to be significantly longer (14.35 cm) and broader (3.73 cm) than the untreated control (12.91 x 3.16 cm). Furthermore, the color of the leaves of the treated trees was greener as the leaves of the untreated control were in various stages of senescence. Growth and yield responses of potatoes to Trichoderma atroviride seed piece treatment in Alaska. Trichoderma atroviride is an extremely versatile microorganism. It has been found to parasitize and destroy a wide range of economically important plant pathogens, and sequestrate heavy metals. In 2005, results of an experiment conducted in a commercial potato field near North Pole, AK, illustrated the ability of T. atroviride to strongly enhance the growth and yield of plants. Plant Helperâ (dust formulation), containing 103 cfu/g of T. atroviride spores, was applied on cut potato seed pieces, at a rate of 15 kg of Plant Helperâ/400 kg of seed pieces/acre. A strip of the field was planted with untreated potato seed pieces as control. Potatoes were planted in early June. The first sampling taken in mid July, showed that the potato plants grown from treated seed pieces were more advanced in their development than the untreated control. In mid-August, ten plants each were randomly harvested for evaluation. Data collected were: numbers of main shoots, numbers of side shoots, plant weight (fresh and dry), root weight (fresh, dry), numbers of stolon, numbers and total weight of progeny potato tubers, numbers of marketable potatoes (>0.2 kg). Progeny tubers were also examined for any signs of scab, dry rot, or black scurf. Data was analyzed using the Student t-test. No lesion or sign of diseases was found on any of the progeny tubers examined. Significant difference was found between T. atroviride treated potatoes and untreated control on fresh plant weigh, dry plant weigh, fresh root weigh, dry root weigh, numbers of main shoots, numbers of side shoots, total weigh of progeny potato tubers, total numbers and weigh of marketable potatoes. No significant difference between T. atroviride treated potato and control on the numbers of stolon and progeny potato tubers/plant. However, treated potatoes had more main shoots, side shoots, biomass, root mass, larger potatoes and more marketable potato tubers. This experiment illustrated that T. atroviride promotes the growth and development of potato plants and increases the yield. T. atroviride seed piece treatment will enable farmers to harvest their potatoes one or two weeks earlier. In Alaska and northern countries where the growing season is short and weather capricious, T. atroviride treatment of seed pieces can be most beneficial to potato farmers. New York (Cornell - Abawi, Harman, Smart): Second year evaluation of plant activators and a biological nematicide for the control of soilborne pathogens on table beets, onions and cabbage. In 2004, foliar applications of Actigard (acibenzolar-S-methyl) showed promising results in controlling foliar infections caused by Rhizoctonia solani on table beets and the root-knot nematode (Meloidogyne hapla) on onion. However, Actigard applications in 2005 did not reduce the incidence of R. solani infections on beets or significantly affect marketable beet yield. Actigard applications to onions slightly reduced the root-galling severity and root-knot nematode numbers in roots and soil, but it was not significantly different from the control. Also, Actigard and the other control products included in the test had no significant effect on onion yield, probably due to the extreme dry and hot weather conditions that prevailed during the season. The inclusion of a biological nematicide (STAN, a product of Syngenta) as a component of a seed treatment package was effective in reducing the number of P. penetrans in roots and soil of cabbage seedlings in a growth chamber and also in field trials (data not shown). Screening of selected bean germplasm for resistance to root rot pathogens. In 2004 and 2005 replicated trials were conducted to evaluate the field reaction and yield response of a large number of bean germplasm lines and varieties grown under severe root rot pressure in an established root rot field at NYSAES in Geneva, NY. The germplasm lines included in these trials differed greatly in the percent of emergence, number of productive plants at harvest, root rot severity ratings and seed or pod yield. Field trials of plant growth-promoting rhizobacteria for the control of a bacterial pathogen of tomato. We have conducted a preliminary field experiment investigating the efficacy of systemic acquired resistance (SAR) and induced systemic resistance (ISR) inducers to control bacterial speck in the field in New York. The results of this experiment reveal that the SAR-inducing compound (acibenzolar-S-methyl) controlled the pathogen to the same level as the standard copper-based spray program. These results are similar to those observed in other states. However, the ISR-inducing Bacillus spp. was ineffective in disease control. The ISR-inducing bacteria did increase yield as measured by both fruit number and weight. Interestingly, when the SAR- and ISR-inducing products were combined, disease control was observed but there was no increase in yield. Montana (Montana State - Callan): Stolon Decay of Peppermint. Fall planting of peppermint in the Flathead Valley of Montana typically results in poor emergence the following spring. The resulting loss of stand leads to lower oil production and a greater requirement for weed control measures. Rhizoctonia, Fusarium, Sclerotinia, and Pythium have been isolated from stolon decay lesions. Research at Montana State Universitys Northwestern Agricultural Research Center (NWARC) has demonstrated the effectiveness of fungicide application at fall planting of mint stolons. Replicated plots were established at the NWARC on October 27, 2004, to evaluate fungicides and biocontrol agents for control of mint stolon decay. Plots consisted of four rows 4.6 m long and 0.3 m apart, with four replications. Stolon segments (1.4 kg, 10-15 cm long) were coated with 300 ml of the designated treatment and laid in furrows 4-5 cm deep. The viability of biological inocula was confirmed by dilution plating. Only the fungicides Gem and Prevail increased emerged plant stand in the spring of 2005 over the water-only control, and Gem, Tilt and Tops MZ increased dry matter production over the control. Oil yield was not significantly influenced by treatment, but was highly correlated with dry matter (r2 = 0.89, P = <0.0001). The relationship between stand and dry matter (r2 = 0.25, P = 0.0807) and stand and oil yield (r2 = 0.29, P = 0.0565) was weaker due to the ease of spread of the mint plant from stolons. Gem and Prevail, fungicides that were most effective in improving plant stand, are primarily labeled for control of Rhizoctonia diseases. Tops MZ and Tilt are also labeled for this pathogen. Tops MZ and Prevail increased dry matter and/or oil yield in previous studies of stolon decay in Montana. We can speculate that Rhizoctonia had a greater impact in this planting than did Pythium, Fusarium, and Sclerotinia, other genera of pathogenic fungi that have been isolated from decayed mint stolons. The biocontrol agents Trichoderma atroviride, Trichoderma harzianum, and Bacillus pumilus, did not increase stand and yield of peppermint under these conditions. Trichodeerma harzianum is labeled for Rhizoctonia diseases and B. pumilus has activity against Rhizoctonia solani (Kanjanamaneesathian, World J Microbiol Technol 16:523, 2000). Trichoderma atroviride is also effective against Rhizoctonia (McBeath, personal communication). V. Collaboration with Other W-147 Members: The USDA Pullman group will be initiating a collaboration with Borneman on how cultural methods affect microbial populations in no-till cereal production, and how a suppressive microflora may develop during long-term no-till. Our unit has a long collaboration with S. Pierson at Univ. of Arizona in studying phenazine biosynthetic pathways. Our unit has also collaborated with N. Grünwald on the development of AFLP methods to study populations of Rhizoctonia. Borneman and Stanghellini have a collaboration to identify microorganisms involved in the germination of Monosporascus cannonballus ascospores. Borneman and Becker have a collaboration to identify microorganisms involved in (i) Meloidogyne suppressiveness and (ii) replant suppression of walnut and peach. Determine the amount of Dactylella oviparasitica needed to create Heterodera schachtii suppressiveness and how crop type influences this suppressiveness. Loper and Pierson collaborate on studies evaluating gene expression of Pf-5 using microarrays. McBeath collaborates with Callan and Miller of Montana State on the control of decline of peppermint and snow mold of turfgrasses using Trichoderma. McBeath also collaborates with Mr. Jay Scott, Reedly Farm, Sanger, CA and Ms. K. Walker, AmPac Biotech Corporation, Fresno CA. Dr. Bowen McBeath, Portland State examining the control of Armillaria root rot on plum.