S1053: Ecological and genetic diversity of soilborne pathogens and indigenous microflora
(Multistate Research Project)
S1053: Ecological and genetic diversity of soilborne pathogens and indigenous microflora
Duration: 11/05/2012 to 09/30/2017
Statement of Issues and Justification
Although alternative chemical and non-chemical, environmentally compatible plant disease management methods have been developed, their results are still inconsistent (Keinath et al., 2000) and less effective than the previous standard methyl bromide when used individually (Gerik and Hanson, 2011; Martin, 2003). In recent years great progress has been made identifying management strategies and integrating multiple approaches for disease prevention and control. These approaches include inoculum reduction by soil fumigation and amendment with biological control agents, biopesticides and/or biofertilizers combined with modified agricultural practices (i.e. minimum tillage, drip irrigation, etc.).
One of the challenges faced by growers is the identification of effective disease management strategies for their crops in particular environmental conditions. It is well known that biological control agents will perform inconsistently in suboptimal or variable environmental conditions (Weller, 1988; Keinath et al., 2000) or in combination with other strategies (Klein et al. 2011). Hence, management methods need to be validated and evaluated in diverse cropping systems and environmental conditions. A multi-state effort to develop and validate management strategies is necessary to provide effective recommendations to growers at a regional level.
Other challenges for crop productivity are root disease complexes, where yield decline and disease symptoms are exacerbated by the combined infection of the host by two or more pathogens. Several crops suffer greater-than-normal symptoms when co-infected by Rhizoctonia, Fusarium, and/or Pythium and other soilborne pathogens, including several nematode, fungal, and bacterial species. For example, Strausbaugh et al.(2011) reported that the root rot complex of Rhizoctonia solani and the bacterium Leuconostoc mesenteroides can lead to sugar beet yield losses in the field and to additional sucrose losses due to in-storage disease infections. Nonetheless, Westphal and Xing (2011) demonstrated that the soybean disease complex of soybean cyst nematode (Heterodera glycines) and sudden death syndrome (Fusarium virguliforme) could be managed by exploiting the biological properties of nonfumigated suppressive soils.
Producers in Texas consider pod rot, frequently associated with Pythium spp. and Rhizoctonia solani, to be the most economically important disease of peanut. Management of this disease relies heavily on the use of fungicides; however, control can be sporadic. Studies evaluating different application timings and methods are needed to improve fungicide deposition and enhance control. Screening genotypes for resistance to pod rot is of major importance, as cultivar selection is the cornerstone to any disease management system. Csinos and Gaines (1986) suggested that the pod rot complex in Georgia results from a nutrient imbalance, principally calcium. However, Filonow et al. (1988) found that calcium deficiency in peanut hulls in not the primary cause of peanut pod rot. Additional research into the relationship between soil chemical properties and pod rot over time is warranted. Disease development within a field is patchy due to the distribution of R. solani and Pythium spp. The ability to characterize fields and predict where disease will occur will improve upon pod rot management and reduce fungicide expenditures for producers. A recent survey of peanut fields on the southern High Plains of Texas indicated that R. solani and Pythium spp. were found in 35 and 39% of fields, respectively (Wheeler et al. 2007). While P. myriotylum is the predominant species, other species such as P. ultimum and P. irregulare have also been identified in the complex. Furthermore, the mycoparasite, P. oligandrum, has also been isolated from symptomatic pods. Additional research is needed to better characterize fungal populations associated with pod rot.
Antagonistic microbes present in pathogen-suppressive soils are important resources for developing genetic materials for transgenic crops and biopesticides. Pathogen-suppressive soils were defined by Cook and Baker (1983) as soils in which the soil-borne pathogen does not establish or the pathogen establishes but causes little or no damage to the host plant. In agronomic ecosystems, suppressive soils have been described for many soilborne pathogens, such as Gaeumannomyces graminis var. tritici, Pythium spp., and Rhizoctonia solani (Weller et al., 2002). Bacterial species of the genera Pseudomonas, Bacillus, Enterobacter, and Streptomyces as well as species of fungal genera Trichoderma and Gliocladium are frequently identified from suppressive soils (Mazzola, 2004;Weller et al., 2002). Therefore, the suppressive soils, in which antagonistic microbes interact with the soilborne pathogens, are important pools to search for useful genetic material (Duffy et al., 2003). Mechanisms of soil suppressiveness to soilborne pathogens are complex and include antibiotic biosynthesis, resource competition, and hyperparasitism (Cook et al., 1995; Raaijmakers et al., 2002; Thomashow & Weller, 1987). One of the best-described examples is the take-all decline soil of wheat fields (Cook et al., 1995), in which fluorescent pseudomonads producing antifungal phenazines were present. Some strains of Pseudomonas have been used for biological control of plant diseases (Mavrodi et al., 2006). However, more studies on diverse disease ecosystems are needed to increase our understanding of molecular activities of these antagonistic microorganisms and provide more clues to the development of biological-based strategies for disease management (Weller et al., 2002). Moreover, by populating the endosphere and rhizosphere with beneficial plant associated microorganisms we can affect disease in all plant parts by induced systemic resistance. Although rhizosphere epiphytes seem to be variable depending on soil, plant, and environmental differences, there have been several successful efforts to populate the endosphere to achieve long-term colonization, and long term effects. Multiple microbes have been found that can support disease suppression as well as assist in supporting plant nutrient status.
Current research at the University of Arkansas has examined Rhizoctonia communities in soybean and rice fields with a history of sheath blight and aerial blight. Analysis of soil assays using a toothpick baiting method (Paulitz and Schroeder, 2005) prior to soybean planting and plant sampling at growth stage V3 revealed mostly Rhizoctonia solani AG1-IA, Rhizoctonia solani AG11, and Rhizoctonia oryzae. Binucleate Rhizoctonia were recovered in negligible amounts. The aerial blight/sheath blight pathogen made up only 2% of the Rhizoctonia population in the soil. Of greater interest for disease development is that only 33% of the Rhizoctonia isolations from plants were R. solani AG1-IA. This poses the question what roles these other Rhizoctonia are playing in the community.
Fungi antagonistic to Rhizoctonia have been identified and have been shown to have the ability to reduce the severity of disease on numerous crops. Of these, some are other nonpathogenic Rhizoctonia solani, binucleate Rhizoctonia or fungi in other genera such as Trichoderma spp. or a sterile white basidiomycete. Ichielevich-Auster (1985) showed that a nonpathogenic isolate of AG4 reduced damping off in seedlings of cotton, radish, and wheat by R. solani and Rhizoctonia zea by 76-94%. Cardoso and Echandi (1987a) reported binucleate Rhizoctonia protected bean seedlings from a virulent root rot causing isolate of AG4. At least one Trichoderma harzianum isolate also lessened disease. In another study (Cardoso and Echandi, 1987b), binucleate Rhizoctonia isolates protected snap bean seedlings from an isolate of AG4 causing root rot by what was deemed a metabolic mechanism of protection. Snap bean seedlings were exposed to the binucleate isolate and then replanted. Replanted seedlings maintained a level of suppression of the pathogen. Root exudates from the binucleate treated seedlings were also inhibitory to the pathogen in vitro. Burpee (1992) also reported disease suppression by binucleate Rhizoctonia on brown patch disease caused by R. solani AG2-2 III B on creeping bentgrass. Sumner et al. (1992) reported a significant efficacy of a binucleate Rhizoctonia CAG-2 and Trichoderma hamatum against Rhizoctonia solani AG-4, and recently Spurlock (2009) found an unidentified sterile white basidiomycete that protected zoysia grass from R. solani AG2-2(LP).
Although microbiologist and plant pathologists have been investigating the impact of soil microbial communities for several decades, we still have an incomplete picture of how microbial diversity affects crop yield, disease severity, and ecosystem function. In the case of fungi the scientific community is still left with a fragmentary understanding of fungal diversity. A modest ~7% of the estimated 1.5 million species of fungi have been described. The poor correlation between the presence of culturable fungi and bacteria or other macroscopic and microscopic structures and the full diversity of the microbiome at any sample site has shifted the focus of soil microbial ecology from culture based to molecular (DNA sequence) data, and nearly all recent attempts to characterize fungal and bacterial communities are based on sequence data (Taylor, 2008). These studies have been limited in sequence depth due to the expense and labor intensiveness of Sanger sequencing of large numbers of samples. However, recent methodological advances in the form of next generation sequencing (NGS) technologies, offer a remedy to these problems.
Recent metagenomic studies have focused on the diversity of bacteria (Will et al. 2010; Youssef et al. 2010), archaea (Leininger et al. 2006), fungi (O'Brien et al. 2005; Buee et al. 2009; Bartram et al. 2011a), viruses, or a combination of these organisms in natural systems (Bartram et al. 2011b; Fierer et al. 2007; Rousk et al. 2010) as well as the diversity of microbial communities associated with agricultural soils (Tringe et al. 2005; Delmont et al. 2011) and the rhizosphere of crops that may improve disease suppression (Inceoglu et al. 2011; Mendes et al. 2011). While these studies have shown that microbial communities are diverse at global and local spatial scales (Fierer et al. 2007), they are also quite diverse at the temporal scale (Inceoglu et al. 2011). A majority of the studies listed above focused on natural or unmanaged ecosystems. Therefore, the structure and function of microbial communities in agricultural systems still remains relatively unresolved. More importantly there is no solid grasp on how microbial communities in agricultural systems vary at the local scale (i.e. between soil types) and regional spatial scale or temporally as a result of a response to crops species; cultural, chemical and biological control strategies; and/or environmental fluctuations.
For all the power and depth provided by NGS, the technologies remain fairly complicated and may, in the absence of generally acknowledged standards, even prove counterproductive to soil microbial ecology. Various types of incompletely understood biases are introduced at different steps of the analyses, and these are often not considered during interpretation of results. Approaches to delimitation of species or operational taxonomic units (OTUs) from molecular data differ widely among users, as do ideas on how to handle abundance data, taxonomic standards, and ecological classifications. By addressing these issues within the larger context of this multi-state project, we can develop protocols and standards by which all researchers can follow to ensure our ability to compare NGS data from diverse locations and studies, which to date has remained complicated and generally incomplete due to the lack of coordination between individual investigators. This multi-state group is therefore positioned to make a valuable contribution to our understanding of soil microbial diversity across different regions and production systems.
New project members include researchers with diverse expertise that will work together to address the objectives set from different but complimentary perspectives, including basic molecular biology, genetics, population biology, metagenomics, bioinformatics, evolutionary biology, mycology, microbiology, plant disease monitoring, and chemical and biological control. The integrated effort of our research will lead to productive collaborations of relevance at the regional and national levels. The results obtained will be reported in peer reviewed scientific journals, specialized disease management journals and on-line publications, and transferred to the broader community through extension education, college and graduate level courses, informational web pages and fact sheets.
The execution of the proposed project will contribute unique information on a regional or national level and innovative protocols by characterizing selected soilborne plant pathogenic populations as impacted by cultural practices, such as rotations, soil amendments, and soil type by demonstrating the role of the microbial diversity and ecology of the pathogenic genera, as well as other organisms interacting with them, in suppressing the levels of disease that occurs in the field. The information generated will lead to management practices that take advantage of microbial interactions that reduce pathogen activity. A greater characterization of the pathogenic genera targeted in this project will provide that framework necessary to improve biological control as well as integrated disease management strategies.
The new project will encompass two main objectives:
1. Evaluate the population genetic diversity of soilborne pathogens and antagonistic microorganisms in different growing systems and regions using traditional and metagenomic approaches.
2. Examine the effect of traditional or newly developed management strategies (chemical, cultural, and biological), soil physicochemical properties, or introduced biological control agents on the microbial community and its ability to suppress soilborne pathogens.
Emphasis will put on the study of agricultural systems where Rhizoctonia and Pythium are major threats to crop productivity, but other pathosystems will be studied as well. All data will be collected in a central location, most likely by developing a password protected web site at one of the participating institutions.
Related, Current and Previous Work
Soilborne plant pathogens cause severe economic losses due to seed disease, seedling damping-off, pod and root rots. Soilborne plant pathogens can be soil inhabitants that survive in soils in the absence of host plants for prolonged periods as saprophytes (Agrios, 2005; Shurtleff and Averre, 1998) or weak soil invaders, surviving in soils for maximum 1 to 2 years in the absence of a host (Shurtleff and Averre, 1998). Although crop rotation is a common practice useful for management of many soilborne plant pathogens, soil inhabitants are recalcitrant pathogens with saprophytic behavior and very broad host ranges. Hence, crop rotation has limited effect on reducing their initial inoculum, often requiring at least 3 years out of the targeted host (Abawi and Widmer, 2000). In the past, management of soilborne plant pathogens relied heavily on soil fumigation with methyl bromide (MeBr) combined with chloropicrin (Martin, 2003). Methyl bromide phase-out has encouraged the development of alternative chemical and non-chemical management strategies, strongly grounded on a better understanding of plant pathogen diversity and the complex interactions among soil microbial communities (Martin, 2003).
A Southern Regional Project on soilborne plant pathogens has been active since the initial project S-26 "The Relation of Soil Microorganisms to Soilborne Plant Pathogens" was established in 1956. Throughout the series of projects it has maintained active research on important soilborne pathogens as well as examining the role of soil microflora in moderating the effects of these organisms. Other areas of emphasis during these projects have focused on the effects of the rhizosphere, the role of crop residues, amendments, and cultural practices on pathogens and other soil microflora. In 1995, the project on soilborne pathogens started an emphasis on the introduction and evaluation of biocontrol agents across crops and environments and received funding for these evaluations. In the most recent project S-1028, regional evaluation of biocontrol agents was extended to broccoli. Investigators in the project conducted research under the project on chemical and biological control of pathogens on summer squash (Seebold et al. 2008), tomato (Gwinn et al. 2010), cotton (Hu et al. 2011), snap bean (Canaday and Schmitthenner, 2010, Canaday, 2011), and soybean (Mengistu et al., 2011). Work in Tennessee characterized the endophytic nature of Beauveria bassiana. Amendments of Monarda, brassicas, or legumes as green manure for disease control were examined in cotton, tomato, and watermelon and additional amendments were examined in the regional broccoli experiments and a variety of organic amendment for Christmas trees. Microflora characterization was conducted for turf, Christmas trees, strawberries, watermelon and cotton as part of the studies (Njoroge et al. 2008). The phylogenetic diversity of Rhizoctonia solani, Sclerotinia sclerotiorum, Verticillium dahlia, Sclerotinia minor, and Phymatotrichopsis omnivora, and Pythium spp. was noted. A standardized protocol from the regional effort was developed for the recovery of Rhizoctonia using a toothpick baiting technique and selective media (Spurlock et al. 2011).
Biological seed treatments were shown to reduce the incidence of snap bean and soybean seedling diseases, increase plant stand, and increase crop yield (Canaday, 2011, Canaday, 2010b). Their efficacy was sometimes related to cultural practices and crop cultivar (Canaday, 2010a,b, Canaday and Mengistu, 2008). Use of muriate of potash (KCl), a common cultural practice, was shown to increase the incidence of seedling diseases of both soybean and snap bean (Canaday and Schmitthenner, 2010, Canaday, 2011). The observed increases in the incidence of soybean seedling diseases with application of potassium chloride (muriate of potash) appeared to be related to the presence of the chloride ion and its effects on seedling root calcium (Canaday and Schmitthenner, 2010, Canaday et al. 2011).
In the period from 2009 to 2012, 49 peer reviewed papers and reports, 28 abstracts, and four book chapters related to S-1028 project objectives were published by group members.
Evaluate the population genetic diversity of soilborne pathogens and antagonistic microorganisms in different growing systems and regions using traditional and metagenomic approaches.
Examine the effect of traditional or newly developed management strategies (chemical, cultural, and biological), soil physicochemical properties, or introduced biological control agents on the microbial community and its ability to suppress soilborne pathogens.
MethodsOBJECTIVE 1. Evaluate the population genetic diversity of soilborne pathogens and antagonistic microorganisms in different growing systems and regions using traditional and metagenomic approaches. Rhizoctonia solani is an important pathogen on many crops grown in the southern U.S. and throughout much of the growing regions of North America. The Rhizoctonia population is very diverse, with several of the new AGs that have been discovered recently in the southern region. This survey will increase the awareness of the importance of R. solani on these crops and in the southern U.S. Crops to be examined will include peanut, rice, soybean, corn, cotton, switchgrass, and potato (Appendix Table 1). In addition, Rhizoctonia populations will be sampled from native areas to examine the diversity and presence of important AGs. Site information will include GPS location, cropping history, cultivar, and cultural practices. The survey will emphasize the occurrence of Rhizoctonia spp. on crops early in the season. Seedlings should be dug between 3 and 5 weeks after planting. Seedlings (a minimum of 50 seedlings per test site) will be selected arbitrarily to represent the crop in that field. Seedlings will be evaluated for growth by recording the number of nodes or leaves from five arbitrarily selected seedlings. Pythium (Oomycota) spp. are ubiquitous soilborne plant pathogens that cause seed and seedling diseases, as well as root and crown rots in established plants. Most Pythium species have broad host ranges, and many produce abundant waterborne zoospores. Pathogenicity varies significantly among and within species (Csinos and Hendrix, 1978; Csinos, 1979; Abad et al. 1994), and disease severity will depend on the aggressiveness of strains as well as on the developmental stage and intrinsic susceptibility of the hosts. Pythium species diversity can vary extensively among soil types and geographic locations (Broders et al. 2009). Little is known about the ecological interactions of Pythium species with other microorganisms and their effects on disease incidence and severity. Pythium spp. have been associated with disease complexes in apple, wheat, and cotton (Mazzola, 1998; Tunali et al., 2008; Mazzola et al. 2009). Antagonistic bacteria and fungi have been reported to suppress Pythium spp. (Mao et al. 1997; Benhamou et al. 2000; Mazzola et al. 2007). A better understanding of the interactions of Pythium with other microbial communities and their role in diseases suppression will help to identify effective antagonistic agents, soil amendments and, overall, integrated disease management strategies. Crops that will be sampled include cotton, soybean, switchgrass, and ornamentals (Appendix Table 1). The genus Phytophthora is comprised primarily of plant pathogens, many of which are soilborne and problematic to agriculture in the United States (Lamour and Kamoun, 2009). The vegetable pathogen Phytophthora capsici is of particular interest as it is an exotic that is now resident at many locations nationwide (Lamour et al., 2011). Once introduced, it is very difficult, if not impossible, to eradicate and can cause up to 100% loss of peppers, pumpkins, melons, and other cucurbits and significant losses to tomatoes and snap and lima beans (Hausbeck and Lamour, 2004; Gevens et al., 2008). Phytophthora capsici populations are genetically very diverse as the pathogen is outcrossing and survives many years as dormant thick-walled oospores. Crop rotation and heavy fungicide use has not provided adequate control and understanding how P. capsici is spread and is responding to novel (e.g. resistant plants) selection pressures is crucial. Isolation of Rhizoctonia and Pythium species Rhizoctonia. Seedlings will be rinsed for 20 min in running tap water. Seedlings will be rated for hypocotyl or coleoptile disease severity and root system discoloration. Seedlings will be scanned for a permanent record. Fifty seedlings from the arbitrary sample will be surface disinfested by immersion for 1.5 min in 0.5% NaClO, blotted dry in a paper towel, and plated on water agar (1.3%) amended with 10 mg and 250 mg of the antibiotics rifampicin and ampicillin, respectively, and 0.5 µl of the miticide Danitol (Valent Chemical Co.) per liter. Resulting colonies will be transferred to PDA and Rhizoctonia isolates will be labeled and stored for later identification of AG groups. Composite soil samples will be collected from the top 6 inches (15 cm) of soil. At least 15 individual soil cores will be collected to represent the field or area of the field being sampled. Soil will be assayed for Rhizoctonia populations using the toothpick method. Soil samples will be uniformly mixed and large pieces of organic debris will be removed. For each sample, three 10-cm-diameter plastic, square greenhouse pots 7.5 cm deep with soil will be filled. Soil will be watered to saturation and allowed to drain for 24 h. Nine white birch toothpicks (Diamond round) will be inserted vertically in soil to a depth of 5 cm (1 cm remaining above the soil line) about 2.5 cm apart in a 3 x 3 grid in soil in each pot. Pots will be incubated in the dark at 22±1 C. After 48 h, toothpicks will be removed and placed on the selective TS medium, 3 toothpicks per plate. After 24 to 72 h, the number of colonies of Rhizoctonia growing from the toothpicks will be recorded. Physical and chemical properties of the soils will be determined by a single analytical laboratory. Closely situated natural ecosystems (i.e., ecosystems that represent what was present prior to agriculture or are being reverted back to a natural ecosystem or situated close to agriculture) will be sampled close to selected agricultural sites. Rhizoctonia strains will be isolated as described above. Results from surveys and population characterization in Objective 1 and microbial interactions studies will be used to develop and validate new integrated pest management strategies for root rot complexes and examine the value of integrated management systems for limiting Rhizoctonia on selected crops. The value of cover crops or crop rotations as well as physical properties such as soil texture will be selected in sites found to increase nonpathogenic populations of Rhizoctonia spp. that result in population shifts and minimize disease incidence and severity. Aspects that will be examined include frequency of the susceptible crop and cover crop biomass and timing of incorporation. Populations of Rhizoctonia spp. will be examined as well as disease incidence and severity. Pythium. Isolates will be collected from soils and potting mixes in diverse agricultural systems including cotton, soybean, peanut, ornamental crops, and from the natural ecosystem of the Tallgrass Prairie Preserve, Oklahoma. Soil (four samples per site, 500 g each) and root samples will be collected. Conventional baiting methods to detect species of Pythium will be used (Ferguson and Jeffers, 1999; Moorman et al., 2002; Pettitt et.al. 2002). Clean cultures bearing oospores will be grown in water agar, cut into pieces and put in vials with sterile water for long term storage. Liquid cultures in potato dextrose broth 5 days old will be processed for DNA extraction with Qiagen DNeasy Plant Mini Kits (Qiagen, Valencia CA) (Garzon et al. 2005) Characterization of Rhizoctonia and Pythium species Species identification. Rhizoctonia isolates will be identified to species and anastomosis group by nuclear staining and testing against known AG-tester and molecular techniques. Anastomosis testing will use the technique of Kronland and Stanghellini (1988) in accordance with the reaction types described by Carling (MacNish et al., 1993; Carling, 1996). Pythium isolate identification to species will be conducted based on morphology (Van der Plaats-Niterink) and verified using nuclear and mitochondrial gene sequences (Martin 2000; Moorman et al. 2002; Garzon et al. 2007). Molecular characterization. Species identification will be validated by sequence analysis of ribosomal DNA and beta-tubulin genes of Rhizoctonia (Gonzalez et al., 2001, 2006) and the internal transcribed spacers of the ribosomal DNA and cytochrome oxidase I-II of Pythium (Moorman et al. 2002; Martin 2000; Garzon et al. 2007). Comparisons of the genetic diversity of isolates among crops and between agricultural and natural soils will be conducted by assessing the occurrence of anastomosis groups, haplotype diversity, and species diversity per sample. Furthermore, phylogenetic relationships among species from the soils studied and intraspecific and intra-anastomosis group population structure will be examined. Phylogenetic relationships will be inferred by maximum parsimony and maximum likelihood analyses, and the robustness of tree topologies will be assessed by bootstrap analyses. Species and anastomosis group diversity. Soil samples will be characterized by metagenomic analyses of the ITS region or the b-tubulin gene using 454 sequencing, modified protocols (Budge et al. 2009; Gonzalez et al. 2001), and de novo developed markers. Population structure. The population structure of the predominant Rhizoctonia and Pythium species and anastomosis groups will be examined using standard population genetic analyses (AMOVA, GST, Nm, PCO) on microsatellite (Zala et al. 2008) and ISSR and AFLP dominant markers (Zietkiewicz et al. 1994; Vos et al. 2004). Characterization of Phytophthora capsici Populations of P. capsici at key locations in the U.S. and worldwide will be sampled and the isolates characterized with a battery of single nucleotide polymorphism (SNP) markers that span the genome. In addition to selectively neutral markers, a subset of the markers will reside in key genes and gene families that encode proteins thought to play an important role in pathogenesis and virulence (e.g. RxLR effectors). SNP discovery and genotyping will be accomplished using a variety of tools including next generation re-sequencing of field isolates and a moderately high throughput approach known as DNA melting analysis (DMA) (Gobena et al., 2012). Data will be analyzed using both a phylogenetic and population genetic approach to determine the relatedness of isolates on a local, regional, and national scale. All data will be made publically available through peer-reviewed publication. OBJECTIVE 2. Examine the effect of traditional or newly developed management strategies (chemical, cultural, and biological), soil physicochemical properties, or introduced biological control agents on the microbial community and its ability to suppress soilborne pathogens. This objective seeks to improve our understanding of microbial interactions (ecologic, metabolic, genomic) in the rhizosphere, as well as soil chemistry and composition, that can exacerbate root diseases in multiple cropping systems across the southern region (Appendix Table 2). Information on the Rhizoctonia community will be examined using common Rhizoctonia isolates from selected sites. The role of nonpathogenic isolates or groups on pathogens will be examined by infesting soils with more than one group. Initially this will be done by infesting soil with an isolate of Rhizoctonia solani pathogenic to a specific crop and other AGs of R. solani or other Rhizoctonia species found associated with the pathogen from surveys. Soil will be planted to the crops of interest and disease assessments will be done on that crop as changes in symptoms severity or crop development. Additional experiments will examine competitive colonization of the crop by pathogenic and nonpathogenic isolates. Field experiments will be conducted in a similar manner by infesting soils with isolates of Rhizoctonia in the planting furrow. To determine whether the seedling diseases of snap bean and soybean receiving muriate of potash may be reduced with an increase in seedling root calcium, sources of supplementary calcium will be added to soil naturally infested with several soil-borne pathogens and their effects on the incidence and severity of seedling diseases and root pruning determined in a series of laboratory and greenhouse experiments. Identified sources of supplementary calcium will be evaluated for their effects on seedling root calcium levels using energy-dispersive X-ray analyses. Finally, the effects of these calcium supplements will be evaluated in field tests both with and without muriate of potash fertilization for their effects on snap bean and soybean seedling diseases, root pruning, plant growth, and crop yield. To obtain antagonistic bacteria from soybean and cotton rhizospheres, the soils showing disease suppressiveness will be collected from the root systems associated with different soilborne diseases. Bacteria will be isolated using various selective culture media (Schaad et al., 2001). Bacteria antagonistic to major soilborne pathogens will be selected using plate assays (Lu et al., 2002). Initial identification of bacterial genera will be performed as described by Schaad et al. (2001). The isolated antagonistic bacteria will be grouped and identified using the Microbial Identification System (MIDI, Microbial ID, Newark, DE) and based on the production of various antibiotics using antibiotic-specific PCR primers (Raaijmakers et al., 2002) or designed during this study. The species, which might have different antagonistic mechanisms to plant pathogens will be further identified and characterized using biochemical and molecular approaches, such as MicroSeq 16S rRNA Gene Kit (PE Applied Biosystems). To characterize the genes dedicated to antagonism, mutagenesis of the antagonistic isolates of interest will be performed using an EZ::TN transposon system as described previously (Gu et al. 2009). Plasmid rescue techniques will be used to clone the transposon-targeted genes from the resulting mutants deficient in antifungal activities as described by the manufacturer. Partial genes associated with its antifungal activity will be sequenced. To clone the intact genes of interest, a fosmid library will be constructed using the CopyControl Fosmid Library Production Kit (Epicentre, Madison, WI) according to the kit manual. Screening the resultant library using the DNA fragments of the targeted genes as probes will identify the fosmids that carry the intact genes. Complementation of mutant strains will be performed with the identified intact gene carried by compatible expression vectors (Lu et al. 2002; Gu et al. 2009; Cardona and Valvano, 2005). Sequence analysis of the targeted genes will provide insights for predicting the possible products contributing to antagonism. More genes will be identified and characterized using this procedure from the novel antagonistic bacteria identified from Objective 1. Success of cloning the partial genes dedicated to antifungal activities of isolate MS14 demonstrates that the procedures described above worked efficiently for obtaining the genes of interest from bacteria. Efficacy of plant associated microbes that produce plant growth promotion, disease suppression and/or nutrient sufficiency improvements when colonized plants will be compared to non-colonized plants. Colonization will be determined at multiple dates, and in directly treated and non-treated plant parts. Disease will be assessed using both objective and subjective methods. Plant growth and yield will be determined when appropriate. The effect of subinhibitory doses of fungicides on radial growth in vitro and pathogenicity of soilborne oomycetes and fungi will be examined. Recently standardized methods for assessment of hormesis in plant pathogenic fungal pathogens will be used (Garzon et al., 2011; Flores, 2010).
Measurement of Progress and Results
- Understanding of the differences and similarities among particular agricultural and natural ecosystems regarding the genetic diversity of Rhizoctonia spp., Pythium spp, and Phytophthora capsici.
- Improved control of seedling diseases of soybean and snap bean through increases in seedling root calcium levels.
- Deeper understanding of the interactions between fungi and bacteria present in soil and plant microbial populations.
- Understanding of the usefulness and feasability of inoculation of plant endophytes for management of soilborne diseases.
- Optimized disease management protocols, using targeted management of field soilborne diseases according to spatial distribution of inocula.
- Output 6 Understanding of the impact of exposure to subinhibitory fungicides on the pathogenicity of soilborne fungal plant pathogens. Output 7 Genetic and physical resources for the analysis of the population structure of Phytophthora capsici, including a reference genome, a large set of single nucleotide polymorphism (SNP) markers, and a dense genetic linkage map. Output 8 Improved protocols for collection, detection and diagnosis of soilborne fungal plant pathogens.
Outcomes or Projected Impacts
- Awareness of the diversity of soilborne plant pathogens will allow effective disease management by taking into account the differences in sensitivity of each pathogenic species to particular chemical and biological treatments.
- Changes in seed treatment and fertilization recommendations to growers will reduce plant losses, increase yields, and increase grower profits.
- Understanding of plant pathogen interactions with soil and plant microbial commutities will result in identification of new biological control agents and management protocols.
- The new physical and genetic resources for the study of population structure of Phytophthora capsici will benefit the plant pathology scientific community in general, and those studing P. capsici in particular, by providing a unique model system with resources for large scale population genetic studies unprecedented so far.
- Understanding the effects of subinhibitory effects of fungicides on diverse fungal soilborne plant pathogens will create awareness of the risks involved in the misuse of chemical control.
- Outcome/Impact 6 Improved protocols for detection and diagnosis of soilborne plant pathogens will result in more effective management practices and higher crop productivity.
Milestones(2013): - Collection, isolation and identification of fungal and microbial strains interacting in each of the systems under study - Treatments found to increase calcium levels will be verified and improved control of soybean and snap bean seedling diseases documented - Changes to extension publications will be recommended and manuscripts submitted to refereed journals. - Assessment of effects of subinhibitory fungicides on multiple fungal plant pathogens - Generation of a draft genome for P. capsici - Evaluation of current collection, detection and diagnosis protocols
(2014): - Identification of microbial communities suppressive to soilborne plant pathogens - Evaluation of modified collection, detection and diagnostic protocols - Assessment of responses to subinhibitory fungicides in fungal plant pathogen populations - Data analyses, publication of scientific reports and outreach materials - Progress reports at APS national and regional meetings as well as in the annual project meeting - Draft P. capsici SNP database
(2015): Identification of microbial species with potential for biological control of soilborne plant pathogens - Development of new collection, detection and diagnostic protocols - Assessment of responses to subinhibitory fungicides in fungal plant pathogen populations - Updated draft of P. capsici SNP database - Data analyses, publication of scientific reports and outreach materials - Progress reports at APS national and regional meetings as well as in the annual project meeting
(2016): Evaluation of microbial species with potential for biological control of soilborne plant pathogens - Validation of new collection, detection and diagnostic protocols - Assessment of responses to subinhibitory fungicides in fungal plant pathogen populations - Population genetic analysis of P. capsici using SNP database - Data analyses, publication of scientific reports and outreach materials - Progress reports at APS national and regional meetings as well as in the annual project meeting - Development and delivery of extension education and outreach materials
(2017): - Completion of data analyses, publication of scientific reports and outreach materials - Extension education and outreach presentations and delivery of educational materials - New project / objectives writing
Projected ParticipationView Appendix E: Participation
The effects of treatments on seedling diseases of soybean and snap bean, seedling root calcium levels, crop growth, and yield will be published in a combination of extension publications, non-refereed but peer reviewed reports, and refereed journal articles to ensure access to research results by growers, the agricultural industry, other scientists, and interested parties. The results obtained and protocols developed will be reported in peer reviewed scientific journals, specialized disease management journals and on-line publications, and transferred to the broader community through extension education, college and graduate level courses, informational web pages and fact sheets.
This project has a very simplified organization. There are two officers: the meeting chair and the meeting secretary. The meeting secretary becomes the meeting chair at the subsequent annual meeting. Thus, there is only one 'elected' position each year. The meeting secretary records the minutes of the annual meeting of the project membership and submits the minutes to the chairman within 30 days of the close of the meeting. The chairman (1) obtains approval for the annual meeting from the project administrative advisor (PAA), (2) prepares the annual meeting agenda, (3) notifies the membership and interested parties of the date, location, and time of the annual meeting at least 30 days in advance, (4) presides over the annual meeting, and (5) submits the annual report to the PAA within 60 days of the meeting.
There is one annual committee with one or more members - the local arrangements committee. Once the site (i.e., city and state) and date for the next annual meeting has been identified by the project membership attending the current meeting, this committee (1) identifies the specific location for the annual meeting (building, room, time, etc.), (2) obtains approval for its use from the appropriate local authorities, (3) identifies local accommodations and reserves (if possible) a block of rooms for meeting attendees, and (4) identifies appropriate locations for meals, etc.
Other committees may be formed as identified by the needs of the membership, e.g. to facilitate publication of project research results or to identify or formulate standard research procedures.
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