Progress 10/01/05 to 09/30/10
Outputs
OUTPUTS: Fungi classified as Rhizoctonia have evolved complex relationships with the environment, plants and microorganisms, which has ultimately contributed to their success in both space and time. To gain better insight into the factors and processes that have contributed to the ability of these soil fungi to cause plant disease and survive in absence of a host, the primary goal of this project was to provide a more comprehensive understanding of the disease ecology and population biology of this economically important pathogen of agricultural, forestry, and ornamental crops. In this project, the results of our research were disseminated to communities of interest that included the general public, growers, scientists, stakeholders, and students by numerous mechanisms. Research results were presented in a broad range of peer-reviewed scientific journals and books, and through invited oral presentations at local (North Carolina), national (California, District of Columbia, Hawaii, Kentucky, Massachusetts, Pennsylvania, and Utah) and international (Canada, Germany, New Zealand, Peru, Scotland, Switzerland, and The Netherlands) scientific conferences and workshops. The presentation and workshop that provided the greatest impact were the keynote address on Rhizoctonia disease of potato; genomics, novel biocontrol and practical disease management at the 7th World Potato Congress in Christchurch, New Zealand in 2009 and the two-day workshop on the identification of Rhizoctonia fungi conducted after the meeting. Information describing two new diseases caused by Rhizoctonia fungi not previously reported in North America was presented to growers and stakeholders at the Annual Tomato Disease Workshop in North Carolina and Rhododendron/Azalea Field Day in Mississippi. Based on our research, we provided an invited chapter for the revised edition of the Tomato Disease Compendium (published by the American Phytopathological Society) on the new Rhizoctonia disease and pathogen of tomato. To extend the research results of this project, I developed and presented a lecture (1 hr) and laboratory (7 hr) on the use of polymerase chain reaction and molecular techniques to identify Rhizoctonia fungi from plants in the USDA sponsored Ag Discovery Summer Internship Outreach Program from 2007-2010. Over the four-year period, 62 high school students participated in the program. In addition, lectures and laboratories on Rhizoctonia related fungi were presented and conducted in the Kingdom of Fungi (PP222) Plant Pathology Methods (PP 502), Global Climate Change and Agriculture (PP610) courses at North Carolina State University and the Biology of Fungi (PLSN413) course at McGill University. Over the entire lifespan of this project, these lectures and laboratories involved 125 undergraduate and 47 graduate students. In 2006, I presented information related to this project to the general public on fungi and plant diseases in the Our Land, Our Legacy College of Agriculture and Life Sciences exhibit at the North Carolina State Fair in Raleigh. PARTICIPANTS: Paulo Ceresini, Assoc. Professor, Sao Paulo University Warren Copes, Research Scientist USDA/ARS Poplarsville, MS, co-PI; David Danehower, Assoc. Professor, North Carolina State University (NCSU), co-PI; Ralph Dean, Professor, North Carolina State University (NCSU); Natalie Federova, J. Craig Venter Institute; Shuijin, Hu, Assoc. Professor, North Carolina State University (NCSU), co-PI; Kelly Ivors, Asst. Professor, North Carolina State University (NCSU); William Nierman, J. Craig Venter Institute, co-PI; Peter Ojiambo, Asst. Professor, North Carolina State University (NCSU); Tim Rinehart, Research Scientist USDA/ARS Poplarsville, MS; H. David Shew, Professor, North Carolina State University (NCSU), co-PI; Stellos Tavantzis, Professor, University of Maine, co-PI; Rytas Vilgalys, Professor, Duke University, co-PI; Cristina Pagani, post doc, NCSU; Suman Pakala, post-doc NCSU; Marianela Rodriguez-Carres, post-doc NCSU; Takeshi Toda, post-doc NCSU; Elizabeth Thomas, post-doc NCSU; Faith Bartz, graduate student, NCSU; Nikki Charlton, graduate student, NCSU; Amanda Kaye, graduate student, NCSU; Melinda Sullivan, graduate student, NCSU; Doug Brown, technician, NCSU; Bryan Cody, technician, NCSU; Katie Neufeld, undergraduate and graduate student, NCSU; Juan Austarius, undergraduate student, NCSU; Lindsey Colvin, undergraduate student, NCSU; Shah Fiza, undergraduate student, NCSU; DeMonica Gentry, undergraduate student, NCSU; Lucy Liu, undergraduate student, NCSU; Nick Taylor, undergraduate student, NCSU; Sherri Thomas, undergraduate student, NCSU; Andrew Thore, undergraduate student, NCSU; Marike Boerema, high school and undergraduate student, NCSU; Moises Figueroa, high school and undergraduate student, NCSU; Amir Morgan, high school and undergraduate student, NCSU; TARGET AUDIENCES: American Phytopathological Society, General public, Mycological Society of America, North Carolina Potato Growers, North Carolina Tomato Growers, North Carolina and Mississippi Azalea Growers, North Carolina high school biology students, Rhizoctonia community, and Undergraduate students PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Field and laboratory-based research conducted for this project has contributed new fundamental knowledge to advance our understanding of the disease ecology of the soil fungus Rhizoctonia solani. Our research has revealed that R. solani is not a single species, but a diverse species complex that represents an early diverging assemblage of fungi. The results from this project have provided a conceptual framework for delineating species, examining the genetic diversity and structure of field populations, characterizing previously unreported Rhizoctonia leaf diseases found in organic tomato production fields and commercial azalea nurseries in North Carolina and Mississippi, respectively, and determining the occurrence and transmission of fungal double stranded DNA (dsRNA) viruses. Our research showed that transmission of dsRNA occurred between different genotypes of the potato pathogen R. solani AG-3 and provided the first experimental evidence for this phenomenon in this fungus. The transmission experiments were extremely challenging to design and conduct because there was no observable phenotype associated with movement of the dsRNA from the donor into the recipient fungal cells. However, we developed a method that employed a series of nuclear and mitochondrial-based genetic markers to provide evidence for horizontal transmission of the dsRNA. At the time our research was initiated, nothing was known about the genetic diversity and transmission of dsRNA in R. solani. Our research showed that a specific dsRNA element (M2) was present in a high frequency in field populations of R. solani AG-3 and at least four genetically divergent lineages of the M2 dsRNA with unique evolutionary histories of mutation and recombination. We also demonstrated that the M2 dsRNA occurred in other genera and species of related Rhizoctonia fungi. This was a significant finding since most previously described dsRNAs in filamentous fungi were assumed to be species specific. In this project, we were interested in understanding of the dynamics of fungal virus interactions and their potential for management of Rhizoctonia diseases. Although we could unequivocally demonstrate dsRNA transmission under laboratory condition, the transmitted dsRNA was unstable and eventually eliminated from the recipient fungal cells. These results suggest that the deployment of Rhizoctonia disease management strategies that rely on horizontal transmission of dsRNA elements may be challenging. Our research on the role that phenyl acetic acid (a hormone-like compound produced by R. solani that can kill plant cells and is possibly associated with the occurrence of the M2 dsRNA) plays in the disease causing activity of the fungus on potato and tomato suggests that this molecule exists as a metabolic complex consisting of at least four different methoxy and hydroxy derivatives This research is currently challenging the dogma related to the role of PAA in the disease causing activity of R. solani. Two Ph.D. students (Nikki Charlton, Senior Research Associate, Samuel Roberts Noble Foundation, and Faith Bartz (Post-doctoral Research Scientist, Emory University) were trained in this project.
Publications
- Bartz, F.E., Danehower, D.A., Glassbrook, N. Taylor, N., and Cubeta, M.A. 2010. Investigation of the metabolic control over phenylacetic acid production by Rhizoctonia solani AG-3, and the physiological responses of a plant host. Mycological Society Annual Meeting, Lexington, KY, Inoculum 61(4):42 (Supplement to Mycologia, Abstract). Updated
- Cubeta, M.A., Dean, R. A., Thomas, E., Pakala, S., Bayman, P., Jabaji, S., Neate, S., Schwartz, D., Zhou, S., Tavantzis, S.M., Toda, T. Vilgalys, R., Federova, N., and Nierman, W.C. 2010. Elucidating the heterokaryotic genome complexity of Rhizoctonia solani. Mycological Society Annual Meeting, Lexington, KY, Inoculum 61(4):47 (Supplement to Mycologia, Abstract). Updated
- Rodriguez-Carres, M., Vilgalys, R., Lutzoni, F., and Cubeta, M.A. 2010. Phylogeny of the Rhizoctonia species complex and closely related resupinate taxa in the Cantharelloid clade. Mycological Society Annual Meeting, Lexington, KY, Inoculum 61(4):71 (Supplement to Mycologia, Abstract). Updated.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: The soil fungus Rhizoctonia solani anastomosis group 3 (AG-3) is an important pathogen of food crops in the plant family Solanaceae that includes eggplant, pepper, potato, and tomato. In 2005 and 2006, a foliar blight of tomato was found in several fields in North Carolina and R. solani was isolated and identified from diseased leaves using hyphal anastomosis testing and sequence based analysis of the ITS rDNA region. An in planta method was developed to generate basidiospores to fulfill Koch's postulates to demonstrate the causal role of the fungus. This is the first report of foliar blight of tomato caused by R. solani AG-3 in North America. The production of the plant growth regulators phenylacetic acid (PAA) and its hydroxy and methoxy derivatives and their contribution to the infection process on tomato was investigated. The percent area of root necrosis was positively correlated with concentration of both PAA and 4-OH-PAA, though the severity and location of necrosis within the root system differed for the two compounds. Eleven field isolates of R. solani AG-3 grown in Vogel's minimal medium amended with either 25 mM quinic acid (QA) or no QA produced varying amounts of PAA and its derivatives based on gas chromatography. The production of PAA was reduced in the QA treatment, but this varied with isolate. The response of tomato to PAA and its derivative are consistent with Rhizoctonia disease symptoms and that the use of QA can reduce PAA production. Genomic DNA of R. solani AG-3 was extracted and sequenced using Sanger, 454, and Illumina methods to generate approximately 2.3 Gb of sequence data. This represents approximately 25X coverage of the 95 Mb genome. A technique was developed to generate protoplasts from mycelium of R. solani using an enzyme cocktail of cellulase, lysing enzymes from Trichoderma, driselase, and mercaptoethanol to develop an optical map to estimate the number and size chromosomes of the fungus. There are at least 23 chromosomes that range in size from 1.6 to 6.0 Mb. The size of protoplasts ranged from 2.5 to 15 microns with varying numbers of nuclei per protoplast and increased time of digestion resulted in 3.6 million protoplasts per ml. This method was effective for generating protoplasts from other isolates of R. solani representing different anastomosis groups and was subsequently modified to generate single nucleus cells for future genetic-based experiments. Based on the genome sequence data, 14 tandem repeat (microsatellite) markers were developed for population genetics studies. Additional repetitive elements (e.g., Gypsy, and Ty1/Copia transposons) were also identified in the genome. RNA was extracted from the fungus grown under seven different experimental conditions (e.g., aluminum, osmotic, pH, and temperature stress; carbon and nitrogen nutrition; and sclerotial development), and used to generate complementary DNA (cDNA) libraries to provide information on the transcriptome of R. solani AG-3. The optical map and cDNA data are currently being used to better understand gene expression and to annotate and assemble the genome sequence for use by the mycology, plant pathology and Rhizoctonia communities. PARTICIPANTS: Kelly Ivors, Asst. Professor, North Carolina State University (NCSU), co-PI William Nierman, J. Craig Venter Institute, co-PI Faith Bartz, graduate student, NCSU Nikki Charlton, graduate student, NCSU Elizabeth Thomas, post-doc NCSU Marianela Rodriguez, post-doc NCSU Bryan Cody, technician, NCSU Zhi Zhang, technician, NCSU Paulo Ceresini, ETH Zurich, Asst. Professor TARGET AUDIENCES: North Carolina Potato Growers North Carolina High school biology teachers and students International Rhizoctonia community American Phytopathological Society Mycological Society of America PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
The soil fungus Rhizoctonia solani can cause seedling diseases on many plants in agricultural and natural ecosystems. The complete DNA sequence of the fungus will reveal genes associated with its ability to cause plant disease and how the fungus survives as a saprobe in the absence of a plant host. This information will lead to better and novel ways of managing Rhizoctonia diseases that reduce economic loses to farmers and promote increased agricultural productivity and sustainability. The modification of the quinic acid (QA) content in the growth environment of the fungus can potentially reduce production of the phytotoxin PAA via metabolic regulation and an improved understanding of this mechanism may lead to more effective, environmentally sound, and novel approaches to suppress Rhizoctonia disease. In practice, composts and/or other waste products containing QA could be applied as a soil amendment to suppress Rhizoctonia disease by causing a biochemical change in fungal cell physiology that results in less production of PAA and a subsequent reduction in disease. This approach would promote soil regeneration, reduce disease pressure through restoration of soil biodiversity, enhance plant growth and could be readily integrated into sustainable/organic production systems. The fungus represents an important evolutionary link to both beneficial and disease causing fungi and this project will provide a foundation for comparative studies to better understand developmental shifts between the parasitic and saprobic stages of the life cycle.
Publications
- Bartz, F.E., Danehower, D.A., Taylor, N., and Cubeta, M.A. 2009. Investigation of the metabolic control over phenylacetic acid production by Rhizoctonia solani AG-3, and the physiological responses of a plant host. Mycological Society Annual Meeting, Snowbird, UT, Inoculum 60:10 (Supplement to Mycologia, Abstract).
- Cubeta, M.A., and Bartz, F.E. 2009. The role of the phenylacetic acid metabolic complex in the parasitic activity of Rhizoctonia solani AG-3. Soilborne Diseases of Potato Workshop, Proceedings of the World Potato Congress, Christchurch, New Zealand (Abstract).
- Ivors, K., Bartz, F.E., Toda, T., Naito, S., and Cubeta, M.A. 2009. First occurrence of a foliar blight of tomato caused by Rhizoctonia solani AG-3 in North America. 101th American Phytopathological Society Annual Meeting, Portland, OR Phytopathology 99:S57 (Abstract).
- Kanetis, L., Wang, X., Wadl, P.A., Neufeld, K., Holmes, G.A., Ojiambo, P., Cubeta, M.A., and Trigiano, R.T. 2009. Microsatellite loci in the downey mildew pathogen, Pseudoperonospora cubensis. Molecular Ecology Resources (In Press).
- Rodriguez-Carres, M., Vilgalys, R., Lutzoni, F., and Cubeta, M.A. 2009. Phylogeny of the Rhizoctonia species complex and closely related taxa. Mycological Society Annual Meeting, Snowbird, UT, Inoculum 60:37-38 (Supplement to Mycologia, Abstract).
- Sullivan, M. J., Parks, E. J., Cubeta, M. A., Gallup, C. A., Moyer, J.W., and Shew, H.D. 2009. Assessment of genetic diversity from a field population of Phytophthora nicotianae with a changing race structure. Plant Disease 92: (In Press).
- Thomas, E. and Cubeta, M.A. 2009. The world after Novozyme 234: a method for preparing protoplasts of Rhizoctonia solani. Mycological Society Annual Meeting, Snowbird, UT Inoculum 60:44 (Supplement to Mycologia, Abstract).
- Cubeta, M.A., Nolte, P., Tavantzis. S.M., and Larkin, R. 2009. Fungal virus-based management of Rhizoctonia disease of potato. Proceedings of the World Potato Congress, Christchurch, New Zealand (Abstract).
- Ivors, K., Bartz. F.E., and Cubeta, M.A. 2009. Rhizoctonia foliar blight of tomato. In Compendium of Tomato Diseases, 2nd Ed., eds. S. Miller and J. Jones, APS Press, St. Paul, MN (In Press).
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The fungus Rhizoctonia solani anastomosis group 3 (AG-3) is an important pathogen of food crops in the plant family Solanaceae. In this project, closely related strains of R. solani AG-3 and other members of the R. solani species complex were examined for the presence the M2 double stranded RNA (dsRNA) virus-like element. The M2 dsRNA has been hypothesized to regulate biochemical pathways associated with the production of plant growth regulator phenylacetic acid (PAA) and catabolism of the carbon source quinic acid (QA). Experiments were conducted to better understand the role that PAA and QA play in the parasitic and saprobic activities of the fungus and to complement the current genome sequencing project on R. solani AG-3 isolate Rhs1AP. A conserved 1000 nucleotide region of the dsRNA was amplified with reverse transcription PCR and the M2 dsRNA was detected in representative isolates belonging to R. solani (AG-1-IA, AG-4, and AG-6, teleomorph=Thanatephorus) and four AGs of binucleate Rhizoctonia (AG-A, AG-F, AG-R, and AG-U; teleomorph=Ceratobasidium). Amplified PCR products from the 3 prime region of the M2 dsRNA from a representative sample of 12 isolates from eight different AGs were sequenced and subjected to parsimony analysis and coalescent simulations to infer ancestral lineages and to reconstruct the ancestral history of haplotypes. Seven dsRNA haplotypes were inferred from the sample of 12 isolates. One haplotype was composed of only isolates of Ceratobasidium belonging to different AGs. The rooted gene genealogies from coalescent simulations suggested that the ancestral M2 dsRNA haplotype most likely evolved in R. solani AG-1-IA and has recently been acquired by isolates of Ceratobasidium. Reconstruction of the ancestral history of haplotypes with a parsimony-based approach that assumes both mutation and recombination suggested that four haplotypes recombined before coalescing to their most recent common ancestor, while three haplotypes coalesced without recombination in the recent past. There was no unique association of haplotype within a specific AG of either Ceratobasidium or Thanatephorus to support co-evolution of the M2 dsRNA within the fungal host. To our knowledge, this is the first report of a dsRNA occurring in Ceratobasidium that is also present in Thanatephorus. Experiments were conducted to quantify the effects of QA on mycelial growth and PAA production of R. solani AG-3. Biomass was quantified by measuring the mycelial dry weight of each isolate after 3 wk incubation in Vogel's minimal medium amended with concentrations of QA ranging from 0-250 mM. All isolates used QA as a carbohydrate source, with more biomass produced in the QA treatments than in the control. Isolates varied in their growth rates and in the shape of their growth curve in response to increasing concentrations of QA. Gas chromatography was used to quantify PAA and its hydroxy and methoxy derivatives produced in the 0 and 25 mM QA treatments for each isolate. Isolates varied in the quantity and type of PAA produced. There was a significant reduction in the production of PAA and or its derivatives for two isolates when grown with QA. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Double stranded RNA (dsRNA) virus-like elements commonly occur in most anastomosis groups (AG) of the soil fungus Rhizoctonia solani and have been the subject of investigation for more than 40 years because of their observed effect on the pathogenic activity of the fungus and potential for biological control of Rhizoctonia disease of plants. Because of the prevalence of these elements in field populations of R. solani, it is possible that they play a pivotal role in the biology and ecology of the fungus. Through a better understanding of the evolutionary history and function of these elements, it may be possible to develop more effective, environmentally sound and novel strategies for managing this important pathogen, which has often been difficult to manage with traditional approaches such as breeding crops for resistance and crop rotation. This project will contribute significantly to our understanding of the role that derivatives of PAA play in the parasitism and infection process and whether this process can be manipulated with quinic acid (QA) utilization by the fungus. In practice, composts and/or other waste products containing QA could be potentially applied as a soil amendment to suppress Rhizoctonia disease by causing a biochemical change in fungal cell physiology that results in less production of PAA and a subsequent reduction in disease. This approach would promote soil regeneration, reduce disease pressure through restoration of soil biodiversity, enhance plant growth and could be readily integrated into sustainable/organic production systems.
Publications
- Bartz, F.E., Danehower, D.A., Tavantzis, S.M., and Cubeta, M.A. 2008. Carbon metabolism and plant growth regulation: the influence of quinic acid on phenylacetic acid production and pathogenic activity of Rhizoctonia solani AG-3. Proc. 4th International Rhizoctonia Symposium, Berlin, Germany (Abstract).
- Bartz, F.E., Tavantzis, S.M., Danehower, D.A., and Cubeta, M.A. 2008. Influence of quinic acid catabolism on the production of the plant growth regulator phenylacetic acid by Rhizoctonia solani AG-3. Mycological Society Annual Meeting, State College, PA, Inoculum 59:19 (Supplement to Mycologia, Abstract).
- Bartz, F.E., Tavantzis, S.M., Danehower, D.A., and Cubeta, M.A. 2008. Influence of quinic acid catabolism on the production of the plant growth regulator phenylacetic acid by Rhizoctonia solani AG-3. 100th American Phytopathological Society Annual Meeting, Minneapolis, MN, Phytopathology 98:S18 (Abstract).
- Charlton, N.D., Carbone, I., Tavantzis, S.M., and M.A. Cubeta. 2008. Phylogenetic relatedness of the M2 double stranded RNA in Rhizoctonia fungi. Mycologia 100:555-564.
- Charlton, N.D., Tavantzis, S.M. and Cubeta, M.A. 2008. Detection of double stranded RNA viruses in the soil fungus Rhizoctonia solani, In Plant Pathology Techniques and Procedures, Chapter 14, pp. 171-182. Eds. R. Burns, Humana Press, 2nd Edition, Tocawa, NJ.
- Charlton, N.D., Carbone, I., Tavantzis, S.M., and Cubeta, M.A. 2008. Evolutionary history and population genetics of the M2 double-stranded RNA of Rhizoctonia solani anastomosis group 3. Proc. 4th International Rhizoctonia Symposium, Berlin, Germany (Abstract).
- Copes, W.E., Rinehart, T. A., Toda, T., and Cubeta, M.A. 2008. Rhizoctonia species associated with bark media and plant strata of container-grown azalea. Proc. 4th International Rhizoctonia Symposium, Berlin, Germany (Abstract).
- Cubeta, M.A., Dean, R. A., Bayman, P., Jabaji, S., Neate, S., Nolte, P., Tavantzis, S.M., Toda, T. Vilgalys, R., Federova, N., and Nierman, W.C. 2008. Whole genome sequencing of the soil fungus Rhizoctonia solani AG-3. Mycological Society Annual Meeting, State College, PA, Inoculum 59:21 (Supplement to Mycologia, Abstract).
- Ferrucho, R.L., Zala, M., Zhang, Z., Cubeta, M.A., Garcia-Dominguez, C., and Ceresini, P.C. 2008. Highly polymorphic in silico-derived microsatellite loci in the potato-infecting fungal pathogen Rhizoctonia solani anastomosis group 3 from the Colombian Andes. Molecular Ecology Resources (In Press).
- Kaye, A., Parks, L., Kennedy, G., Shew, B.B., Carbone, I., Cubeta, M.A., and Moyer, J.W. 2008. Population genetics of Tomato Spotted Wilt Virus on peanut in North Carolina and Virginia. 100th American Phytopathological Society Annual Meeting, Minneapolis, MN, Phytopathology 98:S79 (Abstract).
- Toda, T., Upchurch, R.G., and Cubeta, M.A. 2008. Development of an Agrobacterium-based transformation system for Rhizoctonia solani. Proc. 4th International Rhizoctonia Symposium, Berlin, Germany (Abstract).
- Toda, T., Strausbaugh, C., Vilgalys, R., and Cubeta, M.A. 2008. Characterization of a basidomycete fungus from sugarbeet. Mycological Society Annual Meeting, State College, PA, Inoculum 59:59 (Supplement to Mycologia, Abstract).
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Progress 10/01/06 to 09/30/07
Outputs
The M2 double-stranded RNA (dsRNA) from the soil fungus Rhizoctonia solani anastomosis group 3 (AG-3), an economically important pathogen of cultivated plants in the family Solanaceae, has been hypothesized to regulate metabolic pathways associated with the parasitic and saprobic activity of the fungus. Phylogenetic analyses of a field population sample of 59 isolates of R. solani AG-3 identified distinct evolutionary lineages and unique histories of mutation and recombination among clades of the M2 dsRNA. The reconstructed genealogies and analysis of the ratio of nonsynonymous to synonymous nucleotide substitutions AG-3 suggest that differential selective forces of mutation and recombination have contributed to the evolution of the M2 dsRNA genome. The M2 dsRNA was also detected in representative isolates belonging to three anastomosis groups (AG) of R. solani (AG-1-IA, AG-4, and AG-6; teleomorph=Thanatephorus) and four AG of binucleate Rhizoctonia (AG-A, AG-F, AG-R,
and AG-U; teleomorph=Ceratobasidium). Phylogenetic analysis of M2 dsRNA sequence data resulted in seven inferred haplotypes and there was no unique association with AG to support co-evolution of the M2 dsRNA haplotype within the fungal host. Based on the rooted gene genealogy inferred from coalescent analyses, the ancestral M2 dsRNA haplotype most likely evolved in R. solani AG-1-IA and has recently been acquired by isolates of Ceratobasidium. To our knowledge, this is the first report of a dsRNA occurring in isolates other than R. solani AG-3. Horizontal transmission of the M2 dsRNA between mycelia of eight somatically incompatible donor isolates of R. solani AG-3 containing the M2 dsRNA and three recipient isolates where the M2 dsRNA was absent. Reverse-transcription PCR (RT-PCR) was used to detect horizontal transmission of the M2 dsRNA via hyphal anastomosis from donor to recipient isolates by examining hyphal explants taken 3-cm from the hyphal interaction zone. PCR-RFLP
genetic-based markers of two nuclear loci and one mitochondrial locus were used to confirm identity and transmission between donor and recipient isolates of R. solani AG-3. The frequency of transmission observed between 72 pairings of the eight donor and three recipient isolates was approximately 4% of the total pairings and differences in the phenotype of the recipient isolates after acquisition of the M2 dsRNA via horizontal transmission were observed. To our knowledge, this represents the first demonstration of transmission of dsRNA between genetically different individuals of R. solani confirmed with nuclear and mitochondrial markers. These results suggest that transmission can occur between somatically incompatible isolates of R. solani AG-3, but that maintenance of the dsRNA in the recipient isolates was not stable following repeated subculturing on nutrient medium.
Impacts
The relatively high frequency of occurrence of the M2 double stranded RNA (dsRNA) in field populations of R. solani suggests that it may provide important functions in the fungus. It is possible that M2 dsRNA may regulate the expression of genes associated with the shikimic (SA) and quinic acid metabolic pathways that are thought to be associated with the parasitic and saprophytic activity of R. solani AG-3. This evidence is based on the observations that: a) phenyl acetic acid, a phytotoxic compound produced in the SA pathway, is needed by the fungus to cause damage to plant cell that results in disease, and b) the utilization of quinic acid by R. solani reduces production of phenyl acetic acid and decreases the ability of fungus to cause disease. With a better understanding of how the M2 dsRNA influences the metabolism of the fungus, we will be able to develop more effective, environmentally sound and novel strategies for managing this important pathogen, which has
often been difficult to manage with traditional approaches such as breeding crops for resistance and crop rotation. In practice, quinic acid based plant composts could be applied to soil to induce a biochemical change in the fungus rendering it unable to cause disease for a 2-3 weeks period after which time the plants would be less susceptible to infection. The use of quinic acid-based, plant composts would promote soil regeneration, reduce disease pressure through restoration of soil biodiversity, enhance plant growth and could be readily integrated into sustainable/organic production systems.
Publications
- Mozley-Standridge, S.E., Porter, D, and Cubeta, M.A. 2007. Concepts:Zoosporic fungi. In Plant Pathology Laboratory Exercises and Concepts. CRC Press, eds. Trigiano and Windam, (In Press, revised from 2003 with concept examples)
- Charlton, N.D., Tavantzis, S.M. and Cubeta, M.A. 2007. Detection of double stranded RNA viruses in the soil fungus Rhizoctonia solani, In Plant Pathology Techniques and Procedures, Eds. R. Burns, Humana Press, 2nd Edition, Tocawa, NJ (In Press)
- Keinath, A.P., Cubeta, M.A., and Langston, D. 2007. Cabbage Disease, Ecology and Control. In Encyclopedia of Pest Management, ed., David Pimentel, Markel-Dekker, (In Press)
- Charlton, N.D. and Cubeta, M.A. 2007. Transmission of the M2 double-stranded RNA virus in Rhizoctonia solani anastomosis group 3 (AG-3). Mycologia 99:(In Press)
- Charlton, N.D., Tavantzis, S.M., Carbone, I., and M.A. Cubeta. 2007. The occurrence of the M2 double stranded RNA element in Rhizoctonia fungi. Mycologia 99: (Accepted)
- Rinehart, T., Copes, W., Toda, T. and Cubeta, M.A. 2007. Genetic characterization of binucleate Rhizoctonia species causing web blight on azalea in Mississippi and Alabama. Plant Dis. 91:616-623.
- Smith, D.L., Cubeta, M.A., Toda, T., and Shew, B.B. 2007. PCR-based detection of Sclerotinia minor. Phytopathology 97:S109.
- Bartz, F.E., Tavantzis, S.M., and Cubeta, M.A. 2007 Influence of quinic acid catabolism on the growth and aggressiveness of Rhizoctonia solani AG-3. Phytopathology 97:S8.
- Ceresini, P.C., Shew, H.D. Vilgalys, R., T.Y. James, and Cubeta, M.A. 2007. Molecular diversity and phylogeography of Rhizoctonia solani AG-3 based on sequence analysis of two nuclear loci. BMC Evolutionary Biology 7:163-184. doi:10.1186/1471-2148-7-163.
- Cubeta, M.A., Mozley-Standridge, S.E., and Porter, D. 2007. Laboratory Exercises with Zoosporic Fungi. In Plant Pathology Laboratory Exercises and Concepts. CRC Press, eds. Trigiano and Windam, (In Press, revised from 2003)
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