Progress 09/01/10 to 08/31/15
Outputs
Target Audience:The audience targeted by this project were the members of the California Leafy Greens Research Board (CLGRP), seed companies and marketers, individual growers, handlers and shippers, banks that hold the mortgage on prime production lands, and several allied industries. Stakeholders from the CLGRP, seed industries and growers played a key role in crafting the research questions throughout this project and therefore had a vested interest in the outcome. The results generated from this project are especially of interest not only to vegetable growers and seed producers, but also to regulators at the state, federal, and international levels. The recurrent introduction of novel genotypes of Verticillium dahliae from various sources, increases the likelihood of introducing strains with increased fitness, virulence or aggressiveness, all of which are detrimental to vegetable and small fruit production in coastal California and the US. The host range analyses of these novel strains supports this as well. The economic analyses of the impact of novel genotypes of this pathogen on coastal California crops both in the short and long term, further supported the conclusions drawn from our experimental work. We were in frequent contact with plant pathologists at public universities and private seed companies and shared the information from this project freely. We have close, long-standing communications with stakeholders at large. We presented our findings and received feedback twice a year at CLGRP meetings that were well attended by public and commercial crop breeders, seed company representatives, growers and shippers, extension personnel, and pest control advisors. The web platform, VertShield, facilitated sharing of new data and knowledge resulting from this project in a timely manner with specialty crop and seed industries, and other researchers and extension educators around the world. The system has also been thoroughly tested both by us and the wider scientific community. This information sharing has broadened the project impact on managing threats from V. dahliae and also helped build a global community that closely works together. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In addition to accomplishing the project objectives, one of main goals of the project was to offer training opportunities to postdoctoral researchers and developing them into independent scientists. During the reporting period, the project has had one project scientist, three postdoctoral researchers, a visiting scientist, and a visiting student in the laboratory that were instrumental in generating the information described in the previous section. A graduate student at UC Davis from the Agricultural and Resource Economics Department supported by the project adapted the simulation model developed by us to develop a theoretical and empirical model to measure the benefits and costs of policy options for controlling seedborne V. dahliae. The simulation model is now able to describe growers' profit maximizing decisions regarding which crops to plant, the timing of the plantings, and efforts to control the disease. How have the results been disseminated to communities of interest?Stakeholders from the CLGRP, seed industries and growers have played a key role in crafting the research questions being addressed in this project. The results generated from this project is therefore of special interest not only to vegetable growers and seed producers, but also regulators at the state and federal levels. Although V. dahliae is a cosmopolitan pathogen, the recurrent introduction of novel genotypes from various sources, increases the likelihood of introducing strains with increased fitness, virulence or aggressiveness, all of which may be detrimental to vegetable and small fruit production in coastal California and the US. We were in frequent contact with plant pathologists at public universities and private seed companies and will share this information freely. We have had a close, long-standing communications with stakeholders at large. We presented our findings and received feedback twice a year at CLGRP meetings that were attended by public and commercial crop breeders, growers and shippers, extension personnel, and pest control advisors (over 200 members at each meeting). In addition, the results have also been presented at professional meetings. Finally, the database Vertshield is expected to be an ongoing resource for Verticillium research community worldwide. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The major goals of the project were to: 1) analyze the genetic and phenotypic diversity of V. dahliae associated with specialty crops in central coastal California and strains from seed sources; 2) measure the migration of V. dahliae from major seed production regions in the US and abroad to specialty crop production areas in coastal California; 3) investigate whether genetic recombination occurs in V. dahliae, through cryptic sexual or parasexual recombination; 4) develop methods to mitigate or avert the effects of V. dahliae from infected seed by identifying and developing seed treatments and resistance in spinach germplasm; 5) measure the economic factors contributing to the invasion of Verticillium wilt, the economic impact of the disease on selected California's specialty crops, and the policy options available for controlling a plant disease introduced through international trade; and 6) develop a web-based platform that supports the archiving, dissemination, and utilization of the knowledge generated from this project. Every goal was accomplished conclusively. The project resulted in the 19 peer-reviewed publications, numerous talks in various fora, establishment of the VertShield as a community resource, and training of multiple postdoctoral researchers. The following are the highlights of accomplishments for each goal. Goal #1. A global collection of 1100 isolates from multiple plant hosts and countries were analyzed for genetic diversity, population structure, and demographic history. Through sophisticated population genetic analyses, seven well-differentiated genetic clusters were identified, but associations between these clusters and host/geographic origin of isolates were not apparent. These genetic clusters represented several ancient evolutionary lineages with broad geographic distribution and wide host range. Among the populations from spinach seed introduced into the lettuce production system, nearly 95% of the V. dahliae belonged to race 2 against which there is currently no resistance in lettuce. Based on these findings and the biology of V. dahliae, we concluded that anthropogenic movement has played an important role in spreading V. dahliae lineages. Goal #2. Employing population genetic analyses other than those used in goal #1, we established that V. dahliae isolates carried on spinach seed produced in northern Europe and Washington State were undifferentiated from the isolates collected from diseased lettuce. In other words, the isolates from spinach and lettuce were genetically identical. They were also cross pathogenic to each other. Furthermore, we investigated whether V. dahliae introduced into lettuce production fields by infested spinach seed is able to cause of Verticillium wilt epidemics on lettuce. In this study, amongst other ideas, the transmission of V. dahliae from infested spinach seed to lettuce was investigated. Verticillium wilt developed on lettuce following 2 or 3 plantings of Verticillium-infested spinach in independent experiments. The pathogen recovered from infected lettuce from microplots was confirmed as V. dahliae. Transmission of a GFP-tagged mutant strain of V. dahliae from spinach to lettuce following two cycles of spinach residue incorporated into the soil was also demonstrated. The study offers conclusive evidence that V. dahliae introduced via spinach seed can cause Verticillium wilt in lettuce. These findings have major implications for the development of management strategies such as quarantine measures, seed treatments, crop resistance breeding, etc. Goal #3. We explored the potential for sexual reproduction in V. dahliae. This fungus is heterothallic and that the two mating type idiomorphs required for sexual reproduction are located in different individuals. Orthologs of 88 previously characterized sex-related genes from fungal model systems in the Ascoymycota were identified in the genome of V. dahliae, out of 93 genes investigated. Experiments using both mating types revealed that 10 arbitrarily chosen sex-related genes, including MAT1-1-1 and MAT1-2-1, were constitutively expressed in V. dahliae cultures grown under laboratory conditions. Ratios of non-synonymous (amino-acid altering) to synonymous (silent) substitutions in V. dahliae MAT1-1-1 and MAT1-2-1 sequences were indistinguishable from the ratios observed in the MAT genes of obligate sexual fungi. Patterns consistent with strong purifying selection were also observed in all 18 other arbitrarily chosen V. dahliae sex-related genes, relative to the patterns in orthologs from fungi with known sexual stages. Our findings mount further evidence for an ancestral or cryptic sexual stage in V. dahliae. Goal #4. Two treatments were identified as effective in reducing seedborne V. dahliae in spinach. A Section 24 (c) Special Local Needs registration for Topsin M 70 WP (thiophanate-methyl) was approved by the Washington State Department of Agriculture for spinach stock seed treatment. Mertect 340F (tebuconazole) was approved for a Federal spinach seed treatment registration in 2014, with a California Department of Food and Agriculture spinach seed treatment registration expected in 2015. Topsin M 70WP could not be registered federally for spinach because of the risk cup being full for that active ingredient. In addition, Rancona Spinach (ipconazole) was registered for use on spinach by Germain's Seed Technology that generated the efficacy, phytotoxicity, and residue data for this registration. Field trials in Washington State and in the Salinas Valley of California demonstrated that Mertect 340F and Topsin M 70WP could prevent seed transmission of V. dahliae. A total of 268 accessions plus nine commercial cultivars were screened against one races 1 and 2 from spinach in replicated experiments. Accessions varied in their response to V. dahliae with disease incidence ranging from 0 to 100%. The two race 2 isolates differed in their virulence against spinach genotypes. Resistant accessions were identified against both races 1 and 2. Recovery of V. dahliae from seeds plated on NP-10 medium and qPCR results were highly correlated. Some accessions identified as resistant based on disease incidence showed little seed transmission and these will be useful for spinach cultivar improvement. Goal #5. A theoretical and empirical model to measure the benefits and costs of policy options for controlling seedborne V. dahliae was developed. The simulation model is now able to describe growers' profit maximizing decisions regarding which crops to plant, the timing of the plantings, and efforts to control the disease. The economic analyses of the implications of continued immigration of virulent strains of V. dahliae on spinach seed suggested that the inoculum in the field can be reduced by fumigation and rotations with broccoli and suspending cultivation of spinach. These control options require long-term investments for future gains. Short-term growers ("renters") might not reap future benefits from these control options if they only rent the land for a short period of time. Renters therefore might not make the long-term investments needed to control Verticillium wilt. Future renters and landowners may suffer from decisions of previous renters not investing in control options. Goal #6. To further Verticillium research, we designed VertShield, a website available at http://www.verticilliumdb.org. VertShield incorporates several identification tools and other resources that were adapted from the recent literature and contain practical advice on how to identify Verticillium species. The tools include a morphological key, simplex and multiplex PCR assays, and a BLAST option for queries against a curated database of representative sequences. The General Identification Guide located under the IdentificationTools & Guide tab provides detailed steps for generating and interpreting data for species identification.
Publications
|
Progress 09/01/14 to 08/31/15
Outputs
Target Audience:The audience targeted by this project were the members of the California Leafy Greens Research Board (CLGRP), seed companies and marketers, individual growers, handlers and shippers, banks that hold the mortgage on prime production lands, and several allied industries. Stakeholders from the CLGRP, seed industries and growers played a key role in crafting the research questions throughout this project and therefore had a vested interest in the outcome. The results generated from this project are especially of interest not only to vegetable growers and seed producers, but also to regulators at the state, federal, and international levels. The recurrent introduction of novel genotypes of Verticillium dahliae from various sources, increases the likelihood of introducing strains with increased fitness, virulence or aggressiveness, all of which are detrimental to vegetable and small fruit production in coastal California and the US. The host range analyses of these novel strains supports this as well. The economic analyses of the impact of novel genotypes of this pathogen on coastal California crops both in the short and long term, further supported the conclusions drawn from our experimental work. We were in frequent contact with plant pathologists at public universities and private seed companies and shared the information from this project freely. We have close, long-standing communications with stakeholders at large. We presented our findings and received feedback twice a year at CLGRP meetings that were well attended by public and commercial crop breeders, seed company representatives, growers and shippers, extension personnel, and pest control advisors. The web platform, VertShield, facilitated sharing of new data and knowledge resulting from this project in a timely manner with specialty crop and seed industries, and other researchers and extension educators around the world. The system has also been thoroughly tested both by us and the wider scientific community. This information sharing has broadened the project impact on managing threats from V. dahliae and also helped build a global community that closely works together. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?In addition to accomplishing the project objectives, one of main goals of the project was to offer training opportunities to postdoctoral researchers and developing them into independent scientists. During the reporting period, the project has had one project scientist, three postdoctoral researchers, a visiting scientist, and a visiting student in the laboratory that were instrumental in generating the information described in the previous section. A graduate student at UC Davis from the Agricultural and Resource Economics Department supported by the project adapted the simulation model developed by us to develop a theoretical and empirical model to measure the benefits and costs of policy options for controlling seedborne V. dahliae. The simulation model is now able to describe growers' profit maximizing decisions regarding which crops to plant, the timing of the plantings, and efforts to control the disease. How have the results been disseminated to communities of interest?Stakeholders from the CLGRP, seed industries and growers have played a key role in crafting the research questions being addressed in this project. The results generated from this project is therefore of special interest not only to vegetable growers and seed producers, but also regulators at the state and federal levels. Although V. dahliae is a cosmopolitan pathogen, the recurrent introduction of novel genotypes from various sources, increases the likelihood of introducing strains with increased fitness, virulence or aggressiveness, all of which may be detrimental to vegetable and small fruit production in coastal California and the US. We were in frequent contact with plant pathologists at public universities and private seed companies and will share this information freely. We have had a close, long-standing communications with stakeholders at large. We presented our findings and received feedback twice a year at CLGRP meetings that were attended by public and commercial crop breeders, growers and shippers, extension personnel, and pest control advisors (over 200 members at each meeting). In addition, the results have also been presented at professional meetings. Finally, the database Vertshield is expected to be an ongoing resource for Verticillium research community worldwide. What do you plan to do during the next reporting period to accomplish the goals?We accomplished every goal funded by USDA-NIFA-SCRI and this being the final year, additional studies are not planned under this funded project. However, many offshoots from the current project are being pursued as part of other funding opportunities that have come our way.
Impacts
What was accomplished under these goals?
The major goals of the project were to: 1) analyze the genetic and phenotypic diversity of V. dahliae associated with specialty crops in central coastal California and strains from seed sources; 2) measure the migration of V. dahliae from major seed production regions in the US and abroad to specialty crop production areas in coastal California; 3) investigate whether genetic recombination occurs in V. dahliae, through cryptic sexual or parasexual recombination; 4) develop methods to mitigate or avert the effects of V. dahliae from infected seed by identifying and developing seed treatments and resistance in spinach germplasm; 5) measure the economic factors contributing to the invasion of Verticillium wilt, the economic impact of the disease on selected California's specialty crops, and the policy options available for controlling a plant disease introduced through international trade; and 6) develop a web-based platform that supports the archiving, dissemination, and utilization of the knowledge generated from this project. Every goal was accomplished conclusively. The project resulted in the 19 peer-reviewed publications, numerous talks in various fora, establishment of the VertShield as a community resource, and training of multiple postdoctoral researchers. The following are the highlights of accomplishments for each goal. Goal #1. A global collection of 1100 isolates from multiple plant hosts and countries were analyzed for genetic diversity, population structure, and demographic history. Through sophisticated population genetic analyses, seven well-differentiated genetic clusters were identified, but associations between these clusters and host/geographic origin of isolates were not apparent. These genetic clusters represented several ancient evolutionary lineages with broad geographic distribution and wide host range. Among the populations from spinach seed introduced into the lettuce production system, nearly 95% of the V. dahliae belonged to race 2 against which there is currently no resistance in lettuce. Based on these findings and the biology of V. dahliae, we concluded that anthropogenic movement has played an important role in spreading V. dahliae lineages. Goal #2. Employing population genetic analyses other than those used in goal #1, we established that V. dahliae isolates carried on spinach seed produced in northern Europe and Washington State were undifferentiated from the isolates collected from diseased lettuce. In other words, the isolates from spinach and lettuce were genetically identical. They were also cross pathogenic to each other. Furthermore, we investigated whether V. dahliae introduced into lettuce production fields by infested spinach seed is able to cause of Verticillium wilt epidemics on lettuce. In this study, amongst other ideas, the transmission of V. dahliae from infested spinach seed to lettuce was investigated. Verticillium wilt developed on lettuce following 2 or 3 plantings of Verticillium-infested spinach in independent experiments. The pathogen recovered from infected lettuce from microplots was confirmed as V. dahliae. Transmission of a GFP-tagged mutant strain of V. dahliae from spinach to lettuce following two cycles of spinach residue incorporated into the soil was also demonstrated. The study offers conclusive evidence that V. dahliae introduced via spinach seed can cause Verticillium wilt in lettuce. These findings have major implications for the development of management strategies such as quarantine measures, seed treatments, crop resistance breeding, etc. Goal #3. We explored the potential for sexual reproduction in V. dahliae. This fungus is heterothallic and that the two mating type idiomorphs required for sexual reproduction are located in different individuals. Orthologs of 88 previously characterized sex-related genes from fungal model systems in the Ascoymycota were identified in the genome of V. dahliae, out of 93 genes investigated. Experiments using both mating types revealed that 10 arbitrarily chosen sex-related genes, including MAT1-1-1 and MAT1-2-1, were constitutively expressed in V. dahliae cultures grown under laboratory conditions. Ratios of non-synonymous (amino-acid altering) to synonymous (silent) substitutions in V. dahliae MAT1-1-1 and MAT1-2-1 sequences were indistinguishable from the ratios observed in the MAT genes of obligate sexual fungi. Patterns consistent with strong purifying selection were also observed in all 18 other arbitrarily chosen V. dahliae sex-related genes, relative to the patterns in orthologs from fungi with known sexual stages. Our findings mount further evidence for an ancestral or cryptic sexual stage in V. dahliae. Goal #4. Two treatments were identified as effective in reducing seedborne V. dahliae in spinach. A Section 24 (c) Special Local Needs registration for Topsin M 70 WP (thiophanate-methyl) was approved by the Washington State Department of Agriculture for spinach stock seed treatment. Mertect 340F (tebuconazole) was approved for a Federal spinach seed treatment registration in 2014, with a California Department of Food and Agriculture spinach seed treatment registration expected in 2015. Topsin M 70WP could not be registered federally for spinach because of the risk cup being full for that active ingredient. In addition, Rancona Spinach (ipconazole) was registered for use on spinach by Germain's Seed Technology that generated the efficacy, phytotoxicity, and residue data for this registration. Field trials in Washington State and in the Salinas Valley of California demonstrated that Mertect 340F and Topsin M 70WP could prevent seed transmission of V. dahliae. A total of 268 accessions plus nine commercial cultivars were screened against one races 1 and 2 from spinach in replicated experiments. Disease incidence, severity, and seed transmission through plating on NP-10 medium and qPCR were assessed. Accessions varied in their response to V. dahliae with disease incidence ranging from 0 to 100%. The two race 2 isolates differed in their virulence against spinach genotypes. Resistant accessions were identified against both races 1 and 2. Recovery of V. dahliae from seeds plated on NP-10 medium and qPCR results were highly correlated. Some accessions identified as resistant based on disease incidence showed little seed transmission and these will be useful for spinach cultivar improvement. Goal #5. A theoretical and empirical model to measure the benefits and costs of policy options for controlling seedborne V. dahliae was developed. The simulation model is now able to describe growers' profit maximizing decisions regarding which crops to plant, the timing of the plantings, and efforts to control the disease. The economic analyses of the implications of continued immigration of virulent strains of V. dahliae on spinach seed suggested that the inoculum in the field can be reduced by fumigation and rotations with broccoli and suspending cultivation of spinach. These control options require long-term investments for future gains. Short-term growers ("renters") might not reap future benefits from these control options if they only rent the land for a short period of time. Renters therefore might not make the long-term investments needed to control Verticillium wilt. Future renters and landowners may suffer from decisions of previous renters not investing in control options. Goal #6. The database Vertshield currently contains procedures for long-term storage of isolates, assays to enumerate microsclerotia, and an ability to identify strains to species based on the sequences of five key genes. Vertshield also archives commonly used lab protocols (e.g., storage of Verticillium strains, pathogenicity tests) and images of typical Verticillium wilt symptoms in various crop plants.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Short, D. P. G., S. Gurung, P. Gladieux, P. Inderbitzin, Z. K. Atallah, F. Nigro, G. Li, S. Benlioglu, and K. V. Subbarao. Globally invading populations of the fungal plant pathogen Verticillium dahliae are dominated by multiple divergent lineages. Environmental Microbiology 16: doi: 10.1111/1462-2920.12789.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Short, D. P. G., Sandoya, G., Vallad, G. E., Koike, S. T., Xiao, C. L., Wu, B. M., Gurung, S., Hayes, R. J., and Subbarao, K. V. Dynamics of Verticillium dahliae microsclerotia in field soils in response to fumigation, cropping patterns, and flooding. Phytopathology 105:638-645.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Gurung, S., Short, D. P. G., Hu, X., Sandoya, G. V., Hayes, R. J., Koike, S. T., and Subbarao, K. V. Host range of Verticillium isaacii and Verticillium klebahnii from artichoke, lettuce and spinach. Plant Dis. 99:933-938.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Novakazi, F., P. Inderbitzin, G. Sandoya, R. J. Hayes, A. von Tiedemann, and K. V. Subbarao. The three lineages of the diploid hybrid Verticillium longisporum differ in virulence and pathogenicity. Phytopathology 105:662-673.
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Subbarao, K. V., Sundin, G. W., and Klosterman, S. J. Focus issue articles on emerging and re-emerging plant diseases. Phytopathology 105:852-854.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2015
Citation:
Gurung, S., D. P. G. Short, X. Hu, G. V. Sandoya, R. J. Hayes, and K. V. Subbarao. Screening of wild and cultivated Capsicum germplasm reveals new sources of Verticillium wilt resistance. Plant Dis. 100:
- Type:
Journal Articles
Status:
Accepted
Year Published:
2015
Citation:
Hu, X., S. Gurung, D. P. G. Short, W. Shang, G. V. Sandoya, and K. V. Subbarao. 2015. Verticillium dahliae non-defoliating and defoliating strains from cotton conform to race 1 and race 2 from tomato and lettuce. Plant Disease 100:
|
Progress 09/01/13 to 08/31/14
Outputs
Target Audience: The audience targeted by this project are the members of the California Leafy Greens Research Board (CLGRP), individual growers, handlers and shippers, banks that hold the mortgage on prime production lands, and several allied industries. Stakeholders from the CLGRP, seed industries and growers have played a key role in crafting the research questions being addressed in this project and therefore have a vested interest in the outcome. The results generated from this project will be of special interest not only to vegetable growers and seed producers, but also regulators at the state and federal levels. Although V. dahliae is a cosmopolitan pathogen, the recurrent introduction of novel genotypes from various sources, increases the likelihood of introducing strains with increased fitness, virulence or aggressiveness, all of which may be detrimental to vegetable and small fruit production in coastal California and the US. We are in frequent contact with plant pathologists at public universities and private seed companies and are sharing the information from this project freely. We have close, long-standing communications with stakeholders at large. We present our findings and receive feedback twice a year at CLGRP meetings that are well attended by public and commercial crop breeders, growers and shippers, extension personnel, and pest control advisors. The web platform,VertShield, facilitates sharing of new data and knowledge resulting from this project in a timely manner with specialty crop and seed industries, and other researchers and extension educators around the world. This information sharing broadens the project impact on managing threats from V. dahliae and also helps build a global community that closely works together in the long run. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? In addition to accomplishing the project objectives, one of main goals of the project is to offer training opportunities to postdoctoral researchers and developing them into independent scientists. During the reporting period, the project has had one project scientist, three postdoctoral researchers, a visiting scientist, and a visiting student in the laboratory that were instrumental in generating the information described in the previous section. A graduate student at UC Davis from the Agricultural and Resource Economics Department supported by the project adapted the simulation model developed by us to develop a theoretical and empirical model to measure the benefits and costs of policy options for controlling seedborne V. dahliae. The simulation model is now able to describe growers' profit maximizing decisions regarding which crops to plant, the timing of the plantings, and efforts to control the disease. How have the results been disseminated to communities of interest? Stakeholders from the CLGRP, seed industries and growers have played a key role in crafting the research questions being addressed in this project. The results generated from this project is therefore of special interest not only to vegetable growers and seed producers, but also regulators at the state and federal levels. Although V. dahliae is a cosmopolitan pathogen, the recurrent introduction of novel genotypes from various sources, increases the likelihood of introducing strains with increased fitness, virulence or aggressiveness, all of which may be detrimental to vegetable and small fruit production in coastal California and the US. We are in frequent contact with plant pathologists at public universities and private seed companies and will share this information freely. We have close, long-standing communications with stakeholders at large. We present our findings and receive feedback twice a year at CLGRP meetings that are well attended by public and commercial crop breeders, growers and shippers, extension personnel, and pest control advisors. In addition, the results have also been presented at professional meetings. Finally, the database Vertshield is expected to be an ongoing resource for all researchers on Verticillium spp. What do you plan to do during the next reporting period to accomplish the goals? We will finalize our population analyses of the V. dahliae isolates from the global collection to establish migratory patterns, determine the relationships between mating types and virulence, develop seed treatments for spinach seedlots, release sources of spinach germplasm with resistance to Verticillium wilt, and complete the economic analyses of the continued migration of the pathogen through seed trade and alternate policy options available.
Impacts
What was accomplished under these goals?
The spread of aggressive fungal pathogens into previously non-endemic regions is a major threat to plant health and food security. We elucidated the genetic diversity, population structure, and demographic history of worldwide invasion of Verticillium dahliae, a soil-borne pathogen, using a global collection of 1100 isolates from multiple plant hosts and countries. Seven well-differentiated genetic clusters were revealed through discriminant analysis of principal components (DAPC), but no strong associations between these clusters and host/geographic origin of isolates were found. These genetic clusters represented several ancient evolutionary lineages with broad geographic distribution and wide host range. Comparison of different scenarios of demographic history using Approximate Bayesian Computations revealed the branching order among the different genetic clusters and lineages. Based on these findings and the biology of V. dahliae, we conclude that anthropogenic movement has played an important role in spreading V. dahliae lineages. Our findings have implications for the development of management strategies such as quarantine measures and crop resistance breeding. We explored the potential for sexual reproduction in V. dahliae. Only 1% of the isolates in a global collection of 1120 isolates contained the MAT1-1 idiomorph, whereas 99% contained MAT1-2. Nine unique multilocus microsatellite types comprised isolates of both mating types, eight of which were collected from the same substrate at the same time. Orthologs of 88 previously characterized sex-related genes from fungal model systems in the Ascoymycota were identified in the genome of V. dahliae, out of 93 genes investigated. Experiments using both mating types revealed that 10 arbitrarily chosen sex-related genes, including MAT1-1-1 and MAT1-2-1, were constitutively expressed in V. dahliae cultures grown under laboratory conditions. Ratios of non-synonymous (amino-acid altering) to synonymous (silent) substitutions in V. dahliae MAT1-1-1 and MAT1-2-1 sequences were indistinguishable from the ratios observed in the MAT genes of obligate sexual fungi. Patterns consistent with strong purifying selection were also observed in all 18 other arbitrarily chosen V. dahliae sex-related genes, relative to the patterns in orthologs from fungi with known sexual stages. Our findings mount further evidence for an ancestral or cryptic sexual stage in V. dahliae. Whether V. dahliae introduced into lettuce production fields by infested spinach seed is able to cause of Verticillium wilt epidemics on lettuce was also completed. In this study, amongst other ideas, the transmission of V. dahliae from infested spinach seed to lettuce was investigated. Verticillium wilt developed on lettuce following 2 or 3 plantings of Verticillium-infested spinach in independent experiments. The pathogen recovered from infected lettuce from microplots was confirmed as V. dahliae. Transmission of a GFP-tagged mutant strain of V. dahliae from spinach to lettuce following two cycles of spinach residue incorporation into the soil was also demonstrated. The study offers conclusive evidence that V. dahliae introduced via spinach seed can cause Verticillium wilt in lettuce. A theoretical and empirical model to measure the benefits and costs of policy options for controlling seedborne V. dahliae was developed. The simulation model is now able to describe growers' profit maximizing decisions regarding which crops to plant, the timing of the plantings, and efforts to control the disease. The economic analyses of the implications of continued immigration of virulent strains of V. dahliae on spinach seed suggested that the inoculum in the field can be reduced by fumigation and rotations with broccoli and suspending cultivation of spinach. These control options require long-term investments for future gains. Short-term growers ("renters") might not reap future benefits from these control options if they only rent the land for a short period of time. Renters therefore might not make the long-term investments needed to control Verticillium wilt. Future renters and landowners may suffer from decisions of previous renters not investing in control options. Finally, the database Vertshield was significantly improved over the past year. The site currently contains procedures for long-term storage of isolates, assays to enumerate microsclerotia, and an ability to identify strains to species based on the sequences of five key genes.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Short, D. P. G., S. Gurung, S. T. Koike, S. J. Klosterman, and K. V. Subbarao. Frequency of Verticillium species in commercial spinach fields and transmission of V. dahliae from spinach to subsequent lettuce crops. Phytopathology 104:80-90.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Robbertse, B., C. L. Schoch, V. Robert, V. Duong, G. Cardinali, L. Irinyi, W. Meyer, R. H. Nilsson, K. Hughes, A. N. Miller, P. M. Kirk, K. Abarenkov, M. C. Aime, H. A Ariyawansa, M. Bidartondo, T. Boekhout, B. Buyck, Q. Cai, L. J. Chen, P. W. Crous, U. Damm, W. Z. De Beer, B. T. M. Dentinger, P. K. Divakar, M. Due�as, N. Feau, K. Fliegerova, M. A. Garc�a, G. W. Griffith, J. Z. Groenewald, M. Groenewald, M. Gryzenhout, L. Guo, S. Hambleton, R. Hamelin, K. Hansen, V. Hofstetter, J. Houbraken, K. Hyde, P. Inderbitzin, P. R. Johnston, S. C. Karunarathna, U. K�ljalg, G. M. Kov�cs, E. Kraichak, K. Krizsan, K.-H. Larssen, S. Leavitt, P. M. Letcher, K. Liimatainen, J-K. Liu, J. D. Lodge, Janet Jennifer Luangsaard, H. Thorsten Lumbsch, Sajeewa S.N. Maharachchikumbura, D. Manamgoda, M. P. Mart�n, A. Minnis, G. Mul�, K. K Nakasone, T. Niskanen, Ibai Olariaga, Tam�s Papp, Tam�s Petkovits, Raquel Pino-Bodas, Martha J. Powell, H. A. Raja, D. Redecker, K. A. Seifert, B. Shrestha, S. Stenroos, B. Stielow, S-O. Suh, K. Tanaka, L. Tedersoo, M. T. Telleria, D. Udayanga, W. A. Untereiner, J. D. Uribeondo, K. V. Subbarao, C. V�gv�lgyi, C. Visagie, D. M. Walker, B. S. Weir, M. Wei�, Z. Wen-Ying, N. N. Wijayawardene, M. J. Wingfield, J. P. Xu, Z. L. Yang, N. Zhang, S. Federhen. Finding needles in haystacks: linking scientific names, reference specimens and molecular data for fungi. DATABASE 2014:1-21.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Short, D. P. G., S. Gurung, X. Hu, P. Inderbitzin, and K. V. Subbarao. Maintenance of sex-related genes and the co-occurrence of both mating types in Verticillium dahliae. PLoS One 9: e112145.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2015
Citation:
Novakazi, F., P. Inderbitzin, G. Sandoya, R. J. Hayes, A. von Tiedemann, and K. V. Subbarao. The three lineages of the diploid hybrid Verticillium longisporum differ in virulence and pathogenicity. Phytopathology 105:
- Type:
Journal Articles
Status:
Accepted
Year Published:
2015
Citation:
Short, D. P. G., Sandoya, G., Vallad, G. E., Koike, S. T., Xiao, C. L., Wu, B. M., Gurung, S., Hayes, R. J., and Subbarao, K. V. Dynamics of Verticillium dahliae microsclerotia in field soils in response to fumigation, cropping patterns, and flooding. Phytopathology 105:
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Progress 09/01/12 to 08/31/13
Outputs
Target Audience: Leafy greens producers, packers, and consumers as also those involved in crop production such as the PCAs, pest management and produce harvest companies, etc. Seed industry (spinach and lettuce) personnel, spinach and lettuce growers. The VertShield facilitates sharing of new data and knowledge resulting from this project in a timely manner with specialty crop and seed industries, and other researchers and extension educators around the world. This information sharing broadens the project impact on managing threats from V. dahliae and also helps build a global community that closely works together in the long run. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Collaborators from across the globe have contributed isolates for this project. Two project scientists (Maruthachalam and Inderbitzin), two postdoctoral scholars (Short and Gurung) and a student (Christine Carroll) have worked on this project at the University of California at Davis. The California Leafy Greens Board has provided matching funds and a number of grower collaborators have allowed access to their fields. We received seeds from the USDA spinach germplasm collection in Ames, Iowa for these experiments. A half-time student was hired to help with spinach germplasm-screening experiments including planting, inoculation, transplanting, watering, fertilization, disease and insect control, evaluation and data collection, harvesting, and seed cleaning. Dr. Karunakaran Maruthachalam tested seed infection % by using NP-10 plate assay. Dr. Steve Klosterman determined pathogen copy numbers in qPCR tests. Ocean Mist Farms was the grower-cooperator in Salinas, CA in 2011, and Christensen & Giannini was the grower-cooperator for the 2012 CA trial. TriCal did the soil fumigation for both trials at no charge. Holaday Seeds, Germains Technology Group USA, and Schafer Ag Services provided assistance with the CA spinach-lettuce field trials (labor, lab resources, and some travel expenses covered). The Puget Sound Seed Growers’ Association in WA is also supporting the research financially and in-kind. Two Ph.D students in the Bioinformatics and Genomics program at Penn State, Bongsoo Park and Venky Moktali, built the VertShield platform, in collaboration with PD’s laboratory. CoPD Kang supervised these students and guided the development and optimization of VertShield. How have the results been disseminated to communities of interest? Research resuts have been presented to the California leafy greens producers, packers, and consumers as also those involved in the production such as PCAs, produce harvest companies, etc. Seed industry (spinach and lettuce), spinach and lettuce growers. The VertShield facilitates sharing of new data and knowledge resulting from this project in a timely manner with specialty crop and seed industries, and other researchers and extension educators around the world. This information sharing broadens the project impact on managing threats from V. dahliae and also helps build a global community that closely works together in the long run. What do you plan to do during the next reporting period to accomplish the goals? We plan to complete the analyses of the global collection of isolates to determine global migratory patterns, their diversity, mating type distribution, identification of potential recombinant, begin a breeding project to develop resistance in spinach against Verticillium wilt from germplasm identified during the past three years, pursue registration of Mertect 340F for seed treatment, complete the bioeconomic modeling of measures to control seedborne V. dahliae, and release the fully functional website to the public.
Impacts
What was accomplished under these goals?
Verticillium wilt caused by the fungus Verticillium dahliae is an emerging disease that threatens to devastate lettuce and other leafy greens production in the Western U.S. V. dahliae is seedborne, making it critical to assess migration patterns and develop management strategies to curb severe epidemics on specialty crops. To address the patterns of migration of V. dahliae, we analyzed over 1,400 isolates from a worldwide collection involving many sources and crops. These analyses revealed that the net migration of V. dahliae into the United States occurs only through contaminated spinach seed. We therefore focused our attention on understanding the diversity of isolates from spinach seed production sources. Of the five spinach seed production sources evaluated, we determined that the population from Denmark had the highest measures of gene diversity and allelic richness suggesting that this may be a source population. Furthermore, the current population in coastal California is primarily composed on a single mating type. The isolates from Denmark, however, belonged to both mating types, and thus, migration of these isolates into coastal California can facilitate sexual reproduction in V. dahliae leading to even greater diversity in the V. dahliae. This scenario is likely to compromise all known sources of resistance in lettuce and other specialty crops and become a significant threat to many of these crops. We also documented that in addition to V. dahliae, the most well-known species that causes economic damage to crops, two other species of Verticillium, V. isaacii and V. klebahnii are also carried on contaminated spinach seed and are being introduced into coastal California. Although most isolates from these two species are non-pathogenic, a few isolates are capable of causing significant damage to crops such as artichoke, strawberries, and lettuce. We have developed both singleplex and multiplex PCR methods to identify the different Verticillium species either singly or together rapidly. We also have developed PCR methods to identify the two races of V. dahliae from lettuce and other crops. Both techniques have been made available widely to private laboratories and scientific peers. Since the best option to manage diseases caused by Verticillium species is host resistance, we screened 273 accessions of USDA spinach germplasm for resistance to both Races 1 and 2 of V. dahliae and identified ten accessions that had no symptoms on plants, low percentage of seed infection, and low pathogen copy numbers in qPCR tests. These represent the best sources of resistance against the Races 1 and 2 of V. dahliae. To reduce the levels of inoculum carried on spinach seed, we also tested three conventional and organic treatments each identified as effective in previous studies in fields in WA and CA. Syngenta is pursuing a federal spinach seed treatment registration for Mertect 340F that will be approved in spring 2014. We built an online platform entitled VertShield (http://www.verticilliumdb.org/) to archive and disseminate a large amount of molecular data and practical knowledge on Verticillium wilt from this project. It currently archives sequences of seven phylogenetically informative loci generated from 288 Verticillum isolates representing 11 species. The BLAST function is available to enable sequence-based identification of a newly isolated strain. A user guide for sequence-based identification was also built. We also developed a theoretical and empirical model to measure the benefits and costs of policy options for controlling seedborne V. dahliae. We developed a simulation model to describe growers' profit maximizing decisions regarding which crops to plant, the timing of the plantings, and efforts to control the disease. The results of our ongoing work has been discussed with the members of the advisory committee for the project, stakeholders, growers, pest control advisors, seed industry representatives, etc. in formal and informal meetings. The formal meetings organized twice yearly by the California Leafy Greens Advisory Board and the annual reports prepared have been a major avenue of dissemination of the research outcomes.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
: Maruthachalam, K., Klosterman, S. J., Anchieta, A., Mou, B., and Subbarao, K. V. 2013. Colonization of spinach by Verticillium dahliae and effects of pathogen localization on the efficacy of seed treatments. Phytopathology 103:268-280. Previously reported as in press. Inderbitzin, P., R. M. Davis, R. M. Bostock, and K. V. Subbarao. 2013. Identification and differentiation of Verticillium species and V. longisporum lineages by singleplex and multiplex PCR assays. PLoS One 8:e65990.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2014
Citation:
Kasson, M.T., ONeal, E.S., Short, D.P.G., Subbarao, K. V., and Davis, D.D. 2014. Verticillium wilt of the invasive Ailanthus altissima caused by Verticillium nonalfalfae: phylogenetics, efficacy, and intra-specific resistance screening. Phytopathology 103: in press.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2014
Citation:
Short, D.P.G., S. Gurung, K. Maruthachalam, K. V. Subbarao. 2014. Verticillium dahliae race 2- specific PCR reveals a high frequency of race 2 strains in commercial spinach seed lots and delineates race structure. Phytopathology 103: in press.
- Type:
Journal Articles
Status:
Submitted
Year Published:
2014
Citation:
Gurung, S., D. P. G. Short, P. Inderbitzin, Z. K. Atallah, F. Nigro, S. Benlioglu, G. Li, and K. V. Subbarao. Population genetic analyses of the fungal plant pathogen Verticillium dahliae indicate a Northern European center of origin and a potential for sexual reproduction. Molecular Ecology
- Type:
Journal Articles
Status:
Submitted
Year Published:
2014
Citation:
Wu, B. M., and K. V. Subbarao. A model for multi-seasonal spread of Verticillium wilt of lettuce. Phytopathology 104
- Type:
Journal Articles
Status:
Submitted
Year Published:
2014
Citation:
Inderbitzin, P. and K. V. Subbarao. Verticillium systematics and evolution: Implications of information confusion on Verticillium wilt management and potential solutions. Phytopathology 104
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2013
Citation:
Carroll, C., C. A. Carter, R. Goodhue, C-Y. C. Lin, and K. V. Subbarao. 2013. Bioeconomic modeling of an imported disease in California lettuce. Proceedings of the Agriculture and Applied Economic Association. Washington D. C.
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Progress 09/01/11 to 08/31/12
Outputs
OUTPUTS: Verticillium wilt caused by the fungus Verticillium dahliae is an emerging disease that threatens to devastate lettuce and other leafy greens production in the Western U.S. V. dahliae is seedborne, making it critical to assess migration patterns and develop management strategies to curb severe epidemics on several specialty crops. Over the past year, we investigated the genetic diversity of 1,400 isolates from several countries using 12 Simple Sequence Repeat (SSR) molecular markers and tested for differentiation among strains of V. dahliae to test the hypothesis of global gene flow. Nearly 593 unique microsatellite genotypes were discovered among the 1113 isolates. Major clonal complexes were detected at the single locus variant level, which included a few genotypes isolated with high frequency representing founding genotypes. Genetic differentiation varied among populations from different countries. Populations from China and Turkey were genetically most distinct but the U.S. population showed low differentiation from populations from several other countries, indicating transcontinental migration of V. dahliae. Five genetic clusters were detected in the dataset, which appear to be distributed in various proportions among the 10 countries. The population from Denmark had a low clonal fraction and the highest measures of gene diversity and allelic richness suggesting that this may be a source population. Estimates of historical migration between continents were highest between Europe and the U.S. We screened the 273 accessions of USDA spinach germplasm for resistance to Race 1 and 2 of V. dahliae. Disease severity, wilting time of leaves were noted for each accession. Three conventional and organic treatments each identified as effective in previous studies were evaluated in fields in WA and CA during 2011 and 2012. Spinach seed treated with each treatment were planted into fumigated and non-fumigated soils to determine the effects of the treatments on seedborne vs. soilborne V. dahliae. The 2012 trial is still in progress, the lettuce crop will be harvested in October and residue incorporated soon thereafter, and soil samples collected from each plot five weeks later for assay of V. dahliae microsclerotia. We built an online platform entitled VertShield (http://www.verticilliumdb.org/) to archive and disseminate a large amount of molecular data and practical knowledge on Verticillium wilt from this project. It currently archives sequences of seven phylogenetically informative loci generated from 288 Verticillum isolates representing 11 species. The BLAST function is available to enable sequence-based identification of a newly isolated strain. A user guide for sequence-based identification was also built. The results of our ongoing work has been discussed with the members of the advisory committee for the project, stakeholders, growers, pest control advisors, seed industry representatives, etc. in formal and informal meetings. The formal meetings organized twice yearly by the California Leafy Greens Advisory Board and the annual reports prepared have been a major avenue of dissemination of the research outcomes. PARTICIPANTS: Collaborators from across the globe have contributed isolates for this project. Two project scientists (Maruthachalam and Inderbitzin), two postdoctoral scholars (Short and Gurung) and a student (Christine Carroll) have worked on this project at the University of California at Davis. The California Leafy Greens Board has provided matching funds and a number of grower collaborators have allowed access to their fields. We received seeds from the USDA spinach germplasm collection in Ames, Iowa for these experiments. A half-time student was hired to help with spinach germplasm-screening experiments including planting, inoculation, transplanting, watering, fertilization, disease and insect control, evaluation and data collection, harvesting, and seed cleaning. Dr. Karunakaran Maruthachalam tested seed infection % by using NP-10 plate assay. Dr. Steve Klosterman determined pathogen copy numbers in qPCR tests. Ocean Mist Farms was the grower-cooperator in Salinas, CA in 2011, and Christensen & Giannini was the grower-cooperator for the 2012 CA trial. TriCal did the soil fumigation for both trials at no charge. Holaday Seeds, Germains Technology Group USA, and Schafer Ag Services provided assistance with the CA spinach-lettuce field trials (labor, lab resources, and some travel expenses covered). The Puget Sound Seed Growers' Association in WA is also supporting the research financially and in-kind. Two Ph.D students in the Bioinformatics and Genomics program at Penn State, Bongsoo Park and Venky Moktali, built the VertShield platform, in collaboration with PD's laboratory. CoPD Kang supervised these students and guided the development and optimization of VertShield. TARGET AUDIENCES: California leafy greens producers, packers, and consumers as also those involved in the production such as PCAs, produce harvest companies, etc. Seed industry (spinach and lettuce), spinach and lettuce growers. The VertShield facilitates sharing of new data and knowledge resulting from this project in a timely manner with specialty crop and seed industries, and other researchers and extension educators around the world. This information sharing broadens the project impact on managing threats from V. dahliae and also helps build a global community that closely works together in the long run. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Three globally distributed genetic populations were identified, indicating sustained migration among these distinct geographic regions with multiple spinach crops produced each year and repeated every year in coastal California. The population structure of V. dahliae from coastal California lettuce plants was heavily influenced by migration from spinach seed imported from Denmark and Washington. In a study of major significance, we also redefined the genus Verticillium that now includes 10 species, five of which are new to science. At least two new species are also seedborne, one of which is causing extensive losses on lettuce in Japan and may arrive in California eventually. Two other new species described are represented among the Verticillium species recovered from spinach seed. We are currently assessing the threat posed by these two species of Verticillium on coastal California crops. Knowledge of pathogen localization in seed is critical to develop methods to reduce seedborne inoculum. Pathogen colonization studies with spinach inoculated with with a green fluorescent protein (GFP)-tagged strain of V. dahliae revealed the maximum concentration of the pathogen in the seed coat. Infection of V. dahliae in spinach seed was systemic and transmissible to developing seedlings. Additional analyses indicated that two fungicidal seed treatments reduced detectable levels of the pathogen, but did not eliminate the pathogen from the seed. Genotypes were variable for disease incidence and severity. About ten accessions had no symptoms on plants, low percentage of seed infection, and low pathogen copy numbers in qPCR tests. These represent the best sources of resistance against the Races 1 and 2 of V. dahliae. These accessions can be used in breeding programs to develop cultivars with resistance to Verticillium wilt that may eventually reduce the inoculum brought by spinach seeds. Results have been disseminated to seed companies through the biennial meetings of California Leafy Greens Research Program. Seed treatments reduced spinach seed transmission of V. dahliae. In 2011 in WA, V. dahliae averaged <4 CFU/g in soil planted with untreated spinach seed, <3 CFU/g in soil with Apron + Thiram-treated seed, and <0.5 CFU/g in soil with other treatments and in no spinach control. No Verticillium wilt symptoms were observed on lettuce following a single crop of spinach. Soil sampled 5 wk post-lettuce residue incorporation in 2011 in CA had no V. dahliae, and in 2011 in WA ranged from 0 to 0.2 CFU/g soil with no treatment differences. Syngenta is pursuing a federal spinach seed treatment registration for Mertect 340F that will be approved in spring 2014. The VertShield was built to support the preservation of the genotypic and phenotypic data and educational materials resulting from this project in a format that is readily accessible and searchable by members of the global Verticillium research community. Via systematically archiving datasets from multiple areas of research on Verticillium, the VertShield will help efficiently leverage new knowledge based on the existing knowledge and support problem solving. The economic analyses are in progress.
Publications
- Atallah, Z. K., and K. V. Subbarao. 2012 Population biology of fungal plant pathogens. Bolton, M. and Thomma, B.P.H. J. (Eds.). Pages 333-363 in: Plant Fungal Pathogens: Methods and Protocols. Methods in Molecular Biology 835. Humana Press, New York, NY.
- Klosterman, S. J., K. V. Subbarao, S. K. Kang, P. Veronese, S. E. Gold, et al. 2011. Comparative genomics of plant vascular wilt fungal pathogens, Verticillium dahliae and Verticllium albo-atrum, with Fusarium oxysporum. PLoS Pathogens 7: e1002137.
- Inderbitzen, P., R. M. Bostock, R. M. Davis, T. Usami, H. W. Platt, and K. V. Subbarao. 2011. Phylogenetics and taxonomy of the fungal vascular wilt pathogen Verticillium, with the descriptions of five new species. PLoS One 6: e18260.
- Hayes, R. J., K. Maruthachalam, G. E. Vallad, S. J. Klosterman, and K. V. Subbarao. 2011. Selection for resistance to Verticillium wilt caused by race 2 isolates of Verticillium dahliae in accessions of lettuce (Lactuca sativa L.). Hort Science 46:201-206.
- Klosterman, S. J., A. Anchieta, M. D. Garcia-Pedrajas, K. Maruthachalam, R. J. Hayes and K. V. Subbarao. 2011. SSH reveals a linkage between a senescence-associated protease and Verticillium wilt symptom development in lettuce (Lactuca sativa). Physiological and Molecular Plant Pathology 76:48-58.
- Atallah, Z. K., R. J. Hayes, and K. V. Subbarao. 2011. Fifteen years of Verticillium Wilt of lettuce in America's Salad Bowl: A tale of immigration, subjugation and abatement. Plant Disease 95:784-792.
- de Jonge, R., H. P. van Esse, K. Maruthachalam, P. Santhanam, T. Usami, K. V. Subbarao, and B. P.H.J. Thomma. 2012. Tomato immune receptor Ve1 recognizes effector of multiple fungal wilt pathogens uncovered by genome and RNA sequencing. PNAS 109: 5110-5115.
- Duressa, D., G. Rauscher, S. T. Koike, B. Mou, R. J. Hayes, K. Maruthachalam, K. V. Subbarao, S. J. Klosterman. 2012. Quantification of Verticillium dahliae in spinach germplasm and seed lots by real-time PCR. Phytopathology 102:443-451.
- Atallah, Z. K., K. Maruthachalam, and K. V. Subbarao. 2012. Sources of Verticillium dahliae populations affecting lettuce. Phytopathology 102:1071-1078.
- Maruthachalam, K., Klosterman, S. J., Anchieta, A., Mou, B., and Subbarao, K. V. 2012. Colonization of spinach by Verticillium dahliae and effects of pathogen localization on the efficacy of seed treatments. Phytopathology 102: in press.
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: Verticillium wilt caused by the fungus Verticillium dahliae is an emerging disease that threatens to devastate lettuce and other leafy greens production in the Western U.S. This Project quantifies the pathogen migration into this economically vital region and its long-term impact on affected crops. V. dahliae is seedborne, making it critical to assess migration patterns of the pathogen on seed and propagative material, and develop management strategies to curb severe epidemics on several specialty crops and stealthy introduction of exotic strains. Over the past year, we assembled over 1500 isolates of V. dahliae from many important crops from around the world for analyses during the course of this study. The isolates are being curated and DNA extracted currently. Analyses of these isolates to determine potential migratory patterns using the microsatellite markers will begin soon. Efforts are also underway to determine the factors involved in pathogen evolution. There are no sources of spinach germplasm with resistance to Verticillium wilt. Seed of germplasm that already harbors V. dahliae is unlikely to be a source of resistance, and thus, pre-screening of seed has facilitated the selection of germplasm for detailed plant screening. A collection of 273 USDA germplasm accessions and 9 control cultivars were screened against a race 1 isolate of V. dahliae in replicated trials and results confirmed also by quantifying the pathogen in seed using quantitative PCR tests. Data collection for economic analyses of the impact of seed trade in the dissemination of V. dahliae is also in progress. Archiving all of this information in a web-based platform is being delayed until some data from the above studies are available. A field trial was completed in CA to evaluate the effects of spinach seed treatments, as well as soil fumigation, on seed transmission of Verticillium from planting infected spinach seed as a 'baby leaf' crop, on soil infestation by the pathogen, and on development of Verticillium wilt in a head lettuce crop planted after the spinach crop. The trial will be completed in January 2012 when the last of the soil assays for Verticillium is completed. The results of our ongoing work has been discussed with the members of the advisory committee for the project, stakeholders, growers, pest control advisors, seed industry representatives, etc. in formal and informal meetings. The formal meetings organized twice yearly by the California Leafy Greens Advisory Board have been a major avenue of dissemination of the research outcomes from this project. We have identified 19 putative resistant accessions that had low or no disease incidence/symptoms, seed infection percentage from NP-10 plate assay, and pathogen copy numbers in quantitative PCR tests. For the next reporting period, we will retest the putative resistant varieties identified in the preliminary tests with more replications and controls to confirm the results. We will also test them against a Race 2 isolate of the pathogen. We have requested and received seeds from the USDA spinach germplasm collection in Ames, Iowa for these experiments. PARTICIPANTS: Collaborators from across the globe have contributed isolates for this project. Two project scientists, two postdoctoral scholars and a student have worked on this project at the University of California at Davis. The California Leafy Greens Board has provided matching funds and a number of grower collaborators have allowed access to their fields. We received seeds from the USDA spinach germplasm collection in Ames, Iowa for these experiments. Ocean Mist Farms was the grower-cooperator in Salinas, CA. TriCal did the soil fumigation for this trial at no charge. Holaday Seeds, Germains Technology Group USA, and Schafer Ag Services provided assistance with the CA spinach-lettuce field trial (labor, lab resources, and some travel expenses covered). TARGET AUDIENCES: California leafy greens producers, packers, and consumers as also those involved in the production such as PCAs, produce harvest companies, etc. Seed industry (spinach and lettuce), spinach and lettuce growers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
In a study of major significance, we have redefined the genus Verticillium that now includes 10 species, five of which are new to science. At least two of these new species are also seedborne and have been causing extensive losses on lettuce in Japan and Washington State and may arrive in California eventually. Two other new species described are represented among the Verticillium species recovered from spinach seed. This novel information has been enthusiastically welcomed by the California Leafy Greens Advisory Board. A worldwide collection of V. dahliae isolates from a variety of hosts has been assembled for migration analyses. Their potential threat to California specialty crops therefore remains to be assessed. We have identified 19 putative resistant accessions that had low or no disease incidence/symptoms, percent seed infection from NP-10 plate assay, and pathogen copy numbers in quantitative PCR tests. We will retest these putative resistant varieties with more replications and controls to confirm resistance. We will also test them against a Race 2 isolate of the pathogen. Results of the field trial will be analyzed statistically in January after the final soil assays have been completed. Residue analyses of spinach leaves harvested from plots planted with spinach seed that had been treated with Farmore D300+Mertect 340F showed <10 ppb thiabendazole, the active ingredient in Mertect 340F. These results are being used by Syngenta Crop Protection to pursue a federal seed treatment registration for Mertect 340F on spinach.
Publications
- Inderbitzen, P., R. M. Bostock, R. M. Davis, T. Usami, H. W. Platt, and K. V. Subbarao. 2012. Phylogenetics and taxonomy of the fungal vascular wilt pathogen Verticillium, with the descriptions of five new species. PLoS One: in press.
- Atallah, Z. K., R. J. Hayes, and K. V. Subbarao. 2011. Fifteen years of Verticillium Wilt of lettuce in America's Salad Bowl: A tale of immigration, subjugation and abatement. Plant Disease 95:784-792.
- Maruthachalam, K., S. J. Klosterman, S. Kang, R. J. Hayes, and K. V. Subbarao. 2011. Insertional mutagenesis through Agrobacterium tumefaciens-mediated transformation facilitate identification of pathogenicity genes in Verticillium dahliae. Molecular Biotechnology 49:209-221.
- Hayes, R. J., M. J. Truco, G. E. Vallad, L. K. McHale, O. E. Ochoa, R. W. Michelmore, S. J. Klosterman, K. Maruthachalam, and K. V. Subbarao. 2011. The inheritance of resistance to race 1 isolates of Verticillium dahliae in the lettuce cultivar La Brillante. Theoretical and Applied Genetics 123:509-517.
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