Progress 10/01/06 to 09/30/12
Outputs
OUTPUTS: Tobacco blue mold, caused by the oomycete pathogen Peronospora tabacina, causes yearly epidemics in tobacco (Nicotiana tabacum) in North Carolina and other tobacco growing regions in the US and Europe. The ultimate goals of this research are to improve the detection and diagnosis of tobacco blue mold by developing new strategies to anticipate, prevent, and respond to the threat of disease. The specific objectives of the project are to: (1) collect, and preserve blue mold isolates from epidemics in NC and the US; 2) study the genetic structure of populations of the pathogen; and 3) evaluate the resistance of NC tobacco cultivars to the disease The genetic structure of P. tabacina populations in North America, Central America, the Caribbean and Europe was examined in order to understand genetic diversity, gene flow and migration within and between the regions. Nucleotide sequences from the three genes showed high genetic variability across all populations and 93 haplotypes for the Igs2 region, 93 haplotypes for the Ypt1 region and 82 haplotypes for the cox2 gene were observed. Nucleotide diversity and the mean mutation parameter per site (Watterson's theta) were higher in EULE and CCAM, populations and lower in U.S. populations. Snn Hudson's tests were performed to examine population subdivision and gene flow among populations. The U.S. and CCAM populations were pooled into a two genetically subdivided populations: North and South. An isolation-with-migration analysis (IM) indicated migration from South to North supporting the hypothesis of long-distance migration of P. tabacina from the Caribbean regions, Florida and Texas into other states further north. Within the European populations, the Hudson's test statistics were also significant and three subdivided populations: North-Central EU, Western EU and Lebanon were observed. An isolation with migration model documented migration from North Central Europe into Western Europe and Lebanon and migration from Western Europe into Lebanon. These data support migrations reported from first disease introductions into Europe. We developed a real-time PCR assay for P. tabacina that uses 5' fluorogenic exonuclease (TaqMan) chemistry to detect and quantify pathogen DNA from diseased tissue. The PtabBM TaqMan assay was optimized and performed with a final concentration of 450nM primers and 125nM probe. The real-time TaqMan assay was assessed for sensitivity and the lower detection limit was 1 fg of DNA. The assay was specific for P. tabacina. None of the DNA from other tobacco pathogens, nonpathogens, or the host were amplified. The PtabBM TaqMan assay was useful for detection of P. tabacina in field samples, artificially inoculated leaves, roots, and systemically infected tobacco seedlings. The assay was used to quantify host resistance and it was possible to detect the pathogen 4 days post inoculation in both medium resistant and susceptible tobacco varieties. The real-time PCR assay for P. tabacina will be a valuable tool for the detection of the pathogen and of use to regulatory agencies interested in preventing the spread of P. tabacina. PARTICIPANTS: Jean Ristaino, Box 7616, Dept of Plant Pathology, North Carolina State University, Raleigh, NC 27695-7616;project director. Monica Blanco Meneses, Box 7616, Dept of Plant Pathology, North Carolina State University, Raleigh, NC 27695-7616; graduate student. Kelly Ivors, Dept. of Plant Pathology, North Carolina State University, 455 Research Drive, Fletcher, NC 28732: student committee member. Aimina Mila, Box 7616, Dept of Plant Pathology, North Carolina State University, Raleigh, NC Ignazio Carbone, Center for Integrated Fungal Research, Department of Plant Pathology, Campus Box 7244 - Partners II Building, North Carolina State University, Raleigh, NC 27695-7244; Student committee member TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We ultimately plan to incorporate real -time PCR and genotype information into the forecasting system. Research and extension personnel can send samples into the clinic for rapid identification of the pathogen and the particular strains that have occurred in their region in the past. This data then can be used to track the movement of new blue mold genotypes and epidemics across the United States. Our previous work has validated a south to north migration of P. tabacina from overwintering sources in the Caribbean and in south Texas. More importantly, our findings will allow a regional evaluation of disease spread potential and could be linked to the forecasting system to give warnings and suggest options for disease management plans. This will improve disease management by ultimately enabling farmers to apply fungicides based on the forecast and thus reduce fungicide costs for growers and improve disease management.
Publications
- Trigiano, R. N., Wadl, P. A., Dean, D., Johnson, D., Scheffler, B. E., Runge,F., Telle,S., Thines, M., Ristaino, J, and Spring, O.. 2012. Twelve Polymorphic Microsatellite Loci for Peronospora tabacina, the Cause of Tobacco Blue Mold . Mycologia 104: 633-640.
- Blanco, M and Ristaino, J. B. 2011. A real-time PCR method for detection of Peronospora tabacina in tobacco. Plant Dis. 95: 673-682.
- Blanco, M., Carbone I., Ivors, K. and Ristaino, J. B. 2008. Genetic structure of populations of the tobacco blue mold pathogen, Peronospora tabacina in North America, Central America, the Carribean and Europe. Phytopathology S:23.
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: Tobacco blue mold, caused by the oomycete pathogen Peronospora tabacina, causes yearly epidemics in tobacco (Nicotiana tabacum) in North Carolina and other tobacco growing regions in the US and Europe. The genetic structure of P. tabacina populations in North America, Central America, the Caribbean and Europe was examined in order to understand genetic diversity, gene flow and migration within and between the regions. Several nuclear Igs2,Ypt1) and mitochondrial genes (cox2)were amplified, cloned and sequenced from fifty four isolates from the U.S., Central America-Caribbean-Mexico (CCAM) and Europe and the Middle East (EULE). Nucleotide diversity and the mean mutation parameter per site (Watterson's theta) were higher in EULE and CCAM, populations and lower in U.S. populations. Neutrality tests were significant for all populations and the equilibrium model of neutral evolution was rejected, indicating an excess of recent mutations or rare alleles. The U.S. and CCAM populations were pooled into a two genetically subdivided populations: North and South. An isolation-with-migration analysis (IM) indicated migration from South to North supporting the hypothesis of long-distance migration of P. tabacina from the Caribbean regions, Florida and Texas into other states further north. Within the European populations, the Hudson's test statistics were also significant and three subdivided populations: North-Central EU, Western EU and Lebanon were observed. An isolation with migration model documented migration from North Central Europe into Western Europe and Lebanon and migration from Western Europe into Lebanon. These data support migrations reported from first disease introductions into Europe. P. tabacina could be considered a high evolutionary risk plant pathogen due to its large effective population size and mechanism of dispersal. Intraspore genetic diversity, genome instability due to transposable elements, gene conversion, mitotic and/or sexual recombination may play a role in the high levels of nucleotide variability observed in this pathogen. We developed a real-time PCR assay for P. tabacina that uses 5' fluorogenic exonuclease (TaqMan) chemistry to detect and quantify pathogen DNA from diseased tissue. The primers and probe were designed using 5.8S rDNA sequences from 12 fungal and Oomycete tobacco pathogens and 24 Peronospora species. The PtabBM TaqMan assay was optimized and performed with a final concentration of 450nM primers and 125nM probe. The real-time TaqMan assay was assessed for sensitivity and the lower detection limit was 1 fg of DNA. The assay was specific for P. tabacina. The PtabBM TaqMan assay was useful for detection of P. tabacina in field samples, artificially inoculated leaves, roots, and systemically infected tobacco seedlings. The assay was used to quantify host resistance and it was possible to detect the pathogen 4 days post inoculation in both medium resistant and susceptible tobacco varieties. The real-time PCR assay for P. tabacina will be a valuable tool for the detection of the pathogen and of use to regulatory agencies interested in preventing the spread of P. tabacina. PARTICIPANTS: Dr Jean Beagle Ristaino, North Carolina State University, Raleigh NC Monica Blanco, Former Graduate Assistant, North Carolina State University, Raleigh NC, and now Assistant porfessor, Agronomy Dept. Universidad de Costa Rica, San Jose Costa Rica Tobacco Foundation, North Carolina State University Tobacco Commission, North Carolina State University Phillip Morris Undergradutae Research Internship - Kari Peeden TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
We ultimately plan to incorporate real -time PCR and genotype information into the forecasting system. Research and extension personnel can send samples into the clinic for rapid identification of the pathogen and the particular strains have occurred in their region in the past. This data then can be used to track the movement of new blue mold genotypes and epidemics across the United States. Our previous work has validated a south to north migration of P. tabacina from overwintering sources in the Caribbean and in south Texas. More importantly, our findings will allow a regional evaluation of disease spread potential and will be linked to the forecasting system give warnings and suggest options for disease management plans. This will improve disease management by ultimately enabling farmers to apply fungicides based on the forecast and thus reduce fungicide costs for growers and improve disease management.
Publications
- Blanco, M., Carbone I., Ivors, K. and Ristaino, J. B. 2008. Genetic structure of populations of the tobacco blue mold pathogen, Peronospora tabacina in North America, Central America, the Carribean and Europe. Phytopathology S:23.
- Blanco-Meneses, M, Carbone, I, and Ristaino, J. B. 2007. Genetic structure of populations of the tobacco blue mold pathogen, Peronospora tabacina in North America, Central America, the Caribbean and Europe. Phytopathology 97: S 11.
- Trigiano, R. N., Wadl, P. A., Dean, D., Johnson, D., Scheffler, B. E., Runge,F., Telle,S., Thines, M., Ristaino, J, and Spring, O. 2012. Twelve Polymorphic Microsatellite Loci for Peronospora tabacina, the Cause of Tobacco Blue Mold . Mycologia 104 : doi:10.3852/11-288 .
- Blanco, M and Ristaino, J. B. 2011. A real-time PCR method for detection of Peronospora tabacina in tobacco. Plant Dis. 95: 673-682.
- Blanco, M., Ivors, K., Carbone, I. and Ristaino, J. 2010. Tracking regional migrations of the tobacco blue mold pathogen, Peronospora tabacina in North America, Central America, the Carribean and Europe using gene genealogies. Mol. Ecol. submitted.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Peronospora tabacina Adam. is the causal agent of blue mold or downy mildew of tobacco. The pathogen is a fungus-like organism and is a member of the Oomycota. P. tabacina is an obligate parasite restricted to species in the genus Nicotiana. The disease has been responsible for multi-million dollar losses to growers and resistance to the commonly used fungicide metalaxyl has made management increasingly difficult. Blue mold can be extremely severe in tobacco transplant production houses and movement of infected plants to the field can lead to widespread disease occurrence. The spread of the pathogen occurs through aerial long distance dispersal of inoculum and severe epidemics occur yearly in tobacco growing areas of the world. Blue mold spores may also be transported in the atmosphere across states, threatening tobacco production hundreds to thousands of miles away from their point of origin. In previously work funded by the NC Tobacco Commission and Foundation, we examined the genetic structure of P. tabacina populations in North America, Central America, the Caribbean (CCAM) and Europe (EU) and determined the direction of migration of the pathogen. The intergenic spacer Igs2 region of the nuclear ribosomal DNA (rDNA) and the Ras-related protein (Ypt1) gene, and the mitochondrial cytochrome c oxidase subunit 2 (cox2 gene) were sequenced. Populations of P. tabacina were characterized by high nuclear diversity and low population division. Subpopulations from CCAM had the highest estimates for nucleotide diversity and mean mutation rate. Large population size, the mechanism of dispersal, the parameters of mutation rate and genetic diversity found for the whole population indicate that this pathogen is a high evolutionary risk plant pathogen. An isolation with Migration (IM) model was used to study genetic diversity in the U.S./Central America and the Caribbean (CCAM). Two subpopulations referred to North and South were found. The data support migration of P. tabacina from the CCAM region, Florida and Texas into northern states in the U.S. including North Carolina. These data validate previous meteorological based migration reports of the pathogen by the TBMWS at NCSU. We have also developed both conventional and a quantitative real-time PCR assay for P. tabacina. The real time assay uses a TaqMan probe and an ABI Prism 7300 real time PCR machine. The PtabBM TaqMan assay was optimized to perform on a final concentration of 450nM primers and 125nM probe, half of what it was recommended by the manufacturer reducing cost of the assay. The PtabBM TaqMan assay was tested for sensitivity and specificity. The minimum level of detections was 1 fg of DNA. Tests for specificity showed that none of 12 other tobacco pathogens, other Oomycete organisms, or DNA from tobacco were amplified by this assay. The PtabBM TaqMan assay was capable of detecting the presence of P. tabacina in field samples, artificially inoculated leaves, seedlings and roots. It was also a useful tool for the detection of the pathogen in asymptomatic systemic infected tobacco seedlings. PARTICIPANTS: Jean Ristaino, Box 7616, Dept of Plant Pathology, North Carolina State University, Raleigh, NC 27695-7616;project director. Monica Blanco Meneses, Box 7616, Dept of Plant Pathology, North Carolina State University, Raleigh, NC 27695-7616; graduate student. Kelly Ivors, Dept. of Plant Pathology, North Carolina State University, 455 Research Drive, Fletcher, NC 28732: student committee member. Aimina Mila, Box 7616, Dept of Plant Pathology, North Carolina State University, Raleigh, NC Ignazio Carbone, Center for Integrated Fungal Research, Department of Plant Pathology, Campus Box 7244 - Partners II Building, North Carolina State University, Raleigh, NC 27695-7244; Student committee member TARGET AUDIENCES: Tobacco Growers Oomycete Molecular Genetics Researchers PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Yearly, epidemics reach most tobacco growing areas in the US from Florida to Canada and significant crop loss can occur. We have clearly documented gene flow between tobacco fields in Central America and the Caribbean and NC suggesting long distance pathogen spread. Results from our work will improve the management of tobacco blue mold in the US. We have already developed improved methods for detection (real time PCR) of the pathogen. We are now in a position to deploy these methods to improve the accuracy of detection in grower fields.
Publications
- Blanco, M., Carbone I., Ivors, K. and Ristaino, J. B. 2008. Genetic structure of populations of the tobacco blue mold pathogen, Peronospora tabacina in North America, Central America, the Caribbean and Europe. Phytopathology S:23.
- Blanco, M. and Ristaino, J. B. 2009. Genetic structure of populations of the tobacco blue mold pathogen, Peronospora tabacina, in North America, Central America and the Caribbean and Europe. Oomycete Molecular Genetics Network, March 15-16, Asilomar, CA.
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: Blue mold is a devastating downy mildew disease of tobacco caused by the Oomycete pathogen Peronospora tabacina Adam. Sporangiospores of Peronospora tabacina do not produce zoospores, and infection occurs via direct germination. The asexual sporangiospores of the pathogen can be dispersed thousands of kilometers and are the primary source of inoculum for epidemics. Sporangiospores may be spread yearly from the southeastern United States on wild tobacco in Texas or may originate in Caribbean countries and move northward and infect tobacco as it is planted in fields. Blue mold can be extremely severe in tobacco transplant production houses and movement of infected plants to the field can lead to widespread disease occurrence. We have developed methods to sequence and genotype trains of P. tabacina. We have sequenced several mitochondrial and nuclear loci and established methodologies to name haplotypes in populations of P. tabacina. Eight frequent haplotypes and 116 less frequent or rare haplotypes were found across populations using the IGS region of DNA. Shared haplotypes were found between Texas, the putative center of origin of inoculum for North American epidemics and site of overwintering and sexual reproduction of the pathogen, and North Carolina burley and flue cured tobacco indicating a potential migration from TX to NC. Shared haplotypes were also found in other Carribean and central American countries including the Dominican Republic and NC indicating a potential migration from this source to NC fields. We have clearly documented gene flow between tobacco fields in Central America and the Caribbean and NC suggesting long distance pathogen spread. We have also developed both conventional and a quantitative real-time PCR assay for P. tabacina. The real time assay uses a TaqMan probe and an ABI Prism 7300 real time PCR machine. The PtabBM TaqMan assay was optimized to perform on a final concentration of 450nM primers and 125nM probe, half of what it was recommended by the manufacturer reducing cost of the assay. The PtabBM TaqMan assay was tested for sensitivity and specificity. The minimum level of detections was 1 fg of DNA. Tests for specificity showed that none of 12 other tobacco pathogens, other Oomycete organisms, or DNA from tobacco were amplified by this assay. The PtabBM TaqMan assay was capable of detecting the presence of P. tabacina in field samples, artificially inoculated leaves, seedlings and roots. It was also a useful tool for the detection of the pathogen in asymptomatic systemic infected tobacco seedlings. PARTICIPANTS: Jean Ristaino, Box 7616, Dept of Plant Pathology, North Carolina State University, Raleigh, NC 27695-7616, project director; Monica Balnco Meneses, Box 7616, Dept of Plant Pathology, North Carolina State University, Raleigh, NC 27695-7616, graduate student; Kelly Ivors, Dept. of Plant Pathology, North Carolina State University, 455 Research Drive, Fletcher, NC 28732, student committee member; Ignazio Carbone, Center for Integrated Fungal Research, Department of Plant Pathology, Campus Box 7244 - Partners II Building, North Carolina State University, Raleigh, NC 27695-7244, Student committee member. TARGET AUDIENCES: Scientific community, growers, PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Yearly, epidemics reach most tobacco growing areas in the US from Florida to Canada and significant crop loss can occur. We have clearly documented gene flow between tobacco fields in Central America and the Caribbean and NC suggesting long distance pathogen spread. Results from our work will improve the management of tobacco blue mold in the US. We have already developed improved methods for detection (real time PCR) of the pathogen. We are now in a position to deploy these methods to improve the accuracy of detection in grower fields.
Publications
- Ristaino, J. and Blanco, M. 2008. A real-time PCR method for detection of Peronospora tabacina in tobacco. Proceedings of the 43rd Tobacco Workers Conference, 2008, Savannah, GA, Jan 2008.
- Blanco, M., Ristaino, J., and Carbone I. 2008. Tracking regional migrations of the tobacco blue mold pathogen Peronospora tabacina using gene genealogies. Proceedings of the 43rd Tobacco Workers Conference, Savannah, GA, Jan 2008.
- Blanco, M., Carbone I., Ivors, K. and Ristaino, J. B. 2008. Genetic structure of populations of the tobacco blue mold pathogen, Peronospora tabacina in North America, Central America, the Carribean and Europe. Phytopathology S:23.
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