Source: UNIVERSITY OF MAINE submitted to NRP
SUSTAINABLE STRATEGIES FOR MANAGING RHIZOCTONIA SOLANI IN POTATO
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
0193431
Grant No.
(N/A)
Cumulative Award Amt.
(N/A)
Proposal No.
(N/A)
Multistate No.
(N/A)
Project Start Date
Oct 1, 2002
Project End Date
Sep 30, 2008
Grant Year
(N/A)
Program Code
[(N/A)]- (N/A)
Recipient Organization
UNIVERSITY OF MAINE
(N/A)
ORONO,ME 04469
Performing Department
SCHOOL OF BIOLOGY & ECOLOGY
Non Technical Summary
Genomic approaches combined with sustainable agricultural practices offer solutions to difficult plant disease problems. The purpose of this project is to gain knowledge that will lead to the development of long-term, environment-friendly strategies for managing rhizoctonia disease of potatoes and several other plant species.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
21513101040100%
Knowledge Area
215 - Biological Control of Pests Affecting Plants;

Subject Of Investigation
1310 - Potato;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
The overall goal of the project is the biological control of the important soil- or tuber-borne pathogen R. solani. In the next few years, I would like to understand the genetic and biochemical basis of the M2-associated hypovirulence, determine the magnitude of this phenomenon in natural populations of the fungus, and study some of the parameters that are likely to influence decisions regarding the deployment of hypovirulent R. solani isolates in the field. The specific objectives of the proposed work are as follows: 1) Identify the genes differentially expressed in the hypovirulent Rhs 1A1 or the quinate-induced, converted-to-hypovirulent Rhs 1AP as compared to virulent Rhs 1AP. 2) Determine the behavior of M2 in field populations of R. solani: a) What is the frequency of occurrence of M2 in soil populations of R. solani, b) is it always associated with hypovirulence, and c) is M2 associated with a particular group of genotypes of the fungus. 3) Determine whether soil organic amendments containing quinate are effective in suppressing rhizoctonia disease of potato under field conditions.
Project Methods
Amplified DNA segments representing the collection of genes from the virulent genotype Rhs 1AP will be mechanically spotted at a high density on membrane filters using an x-y-z stage robotic system to create macroarrays containing the entire set of genes from this isolate. The macroarrays will be queried in hybridization assays using radiolabeled probes prepared from mRNA from Rhs 1AP and the isogenic hypovirulent Rhs 1A1(first study) or Rhs 1AP vs. quinate-induced Rhs 1AP (second study). The microarray results will be selectively confirmed by Northern blot analysis. The cDNA fragments of the selected genes will be used as probes labeled with [32P]dCTP by random primer labeling. Northern hybridization. To determine the presence or absence of the hypovirulence-associated M2 dsRNA, reverse transcription PCR (RT-PCR) experiments will be carried out using total RNA samples from a large collection (over 200 cultures) of field AG 3 isolates as templates. The M2 sequences will be compared, and virulence tests will be performed to assess the degree of pathogenicity of the respective isolates. Pilot experiments will be conducted in growth chambers to test the feasibility of using potato sprout virulence tests to determine the effect of compost type (peat vs. pine bark), age (one, four and eight weeks), and treatment (autoclaved vs. untreated) on the virulence of R. solani. Rhs 1AP will be added to the soil (with or without a compost amendment) one week prior adding six tuber-attached potato sprouts to each plastic flat and virulence assessment will be carried out. In greenhouse experiments, the same treatments and controls as those described above will be used. In field experiments, a randomized complete-block experimental design will be employed with four replications per treatment. Data to be collected will include emergence counts at 4 weeks after planting, rhizoctonia disease assessments at 6 and 10 weeks after planting, and yield and size distribution of tubers at harvest.

Progress 10/01/02 to 09/30/08

Outputs
OUTPUTS: Information generated through this project has been published in the form of refereed articles, book chapters and presentations. During the life of the project (last four years), over 30 presentations have been made at professional meetings, including regional meetings, such as the Maine Agricultural Trades Show and the Northeast Potato Technology Forum, meetings at Professional Societies, such as the American Phytopathological Society and the International Union of Microbiological Societies, and international symposia, such as the Fourth International Symposium on Rhizoctonia. 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
As discussed in previous reports, characterization of arom gene and unveiling of its expression pattern, is expected to shed light on the expression of virulence in Rhizoctonia solani. The R. solani arom gene has 5,323 base pairs (bp) including five introns as opposed to a single intron found in arom in ascomycetes. A 199-bp upstream sequence has a GC box, no TATAA box, but two GTATTAGA repeats. The largest arom transcript is 5,108 nucleotides long, excluding the poly(A) tail. It contains an open reading frame of 4857 bases, coding for a putative 1618-residue pentafunctional AROM protein. Size and sequence heterogeneity were observed at both 5'- and 3'-end of the mRNA. This work was the first report on analysis of the arom gene in a basidiomycete. We have previously shown two types of hypovirulence in R. solani: 1) The M2 dsRNA-associated hypovirulence exhibited by isolate Rhs 1A1, which has remained phenotypically stable for several years (referred to as stable hypovirulence), and 2) the quinate-induced hypovirulence (referred to as induced hypovirulence) displayed by the normally virulent Rhs 1AP. Both types of hypovirulence are associated with expression of the M2 dsRNA and the quinate pathway. To understand temporally induced hypovirulence, we set out to identify the genes induced by quinate, which is a major carbon source for soil-borne fungi and bacteria. Over 500 of these differentially expressed cDNAs were grouped into 59 contigs and 34 singletons. In addition to the putative QUT pathway genes, BLAST analysis has shown that quinate induction results in upregulation of genes involved in: a) major shifts in gene expression indicating metabolic and structural changes in the fungus; b) mitochondrial electron transport; c) membrane transport; and d) Rho signaling pathway, and e) nucleic acid binding proteins. Among other upregulated genes of potential "importance" were: transcription regulation factors, subtelomeric helicase, and ubiquitin, which in addition to its well-established role, has been associated with regulation of dynamic protein-protein interactions in the nucleus, and plant disease pathogenesis. In collaboration with Dr. Cubeta's laboratory (NCSU), 115 isolates from a potato field population of R. solani AG-3 were examined for the 3.6-kb M2 dsRNA with reverse transcription PCR (RT-PCR). M2 was detected in approximately 48% of these isolates. Phylogenetic analysis of the M2 dsRNA's suggested the occurrence of at least two genetically divergent lineages with unique evolutionary histories of mutation and recombination. To determine the efficacy of quinate-containing composts in ameliorating virulence of R. solani under commercial field conditions, a collaborative field study (Larkin and Tavantzis) was undertaken. Results showed that a conifer compost reduced Rhizoctonia disease severity significantly (as compared to untreated controls), and a combination of conifer compost and Rhs 1A1 (that is, induced hypovirulence+stable hypovirulence) results in the lowest overall disease severity and highest yield amongst different treatments that included biocontrol organisms such as Bacillus subtilis and Trichoderma spp.

Publications

  • Tavantzis, S. M. and Manmathan, H. 2008. Global gene expression induced by quinic acid in the presence of host, Solanum tuberosum, in the plant pathogenic basidiomycete Rhizoctonia solani. Fourth International Symposium on Rhizoctonia. Berlin, Germany.
  • 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.
  • Tavantzis, S. M. 2008. Partitiviruses of Fungi. In Encyclopedia of Virology, 3rd Edition, Vol. 4, pp. 63-68. B. W. V. Mahy, M. H. V. Van Regenmortel, Eds. Elsevier, Ltd. & Academic Press, San Diego, CA., U. S. A.
  • Charlton, N. D., Tavantzis, S. M., and Cubeta, M. A. 2007. Detection of Double-stranded RNA Elements in Plant Pathogenic Fungi. In Plant Pathology Techniques and Procedures, Chapter 14, pp 171-182. Burns R., Ed. Humana Press, 2nd Edition, Tocawa, NJ, USA.
  • Erich, S., Larkin, R.P., Alyokhin, A., Bernard, E., Gross, S. Tavantzis, S. 2009. A Systems Approach to Soil Fertility, Plant Health and Disease Suppression: Soil Properties and Fertility. Maine Agricultural Trades Show, January 13-15. Augusta, ME.
  • Larkin, R.P., Erich, S., Alyokhin, A., Bernard, E., Gross, S. Tavantzis, S. 2009. A Systems Approach to Soil Fertility, Plant Health and Disease Suppression:Soil-borne Diseases and Yield. Maine Agricultural Trades Show, Jauary 13-15. Augusta, ME.
  • Gross, S., Alyokhin, A., Larkin, R.P., Erich, S., Bernard, E., Tavantzis, S. 2009. A Systems Approach to Soil Fertility, Plant Health and Disease Suppression: Effects on Insect Pest Densities. Maine Agricultural Trades Show, Jauary 13-15. Augusta, ME.
  • Bernard, E., Larkin, R.P., Tavantzis, S., Erich, S., Alyokhin, A., Gross, S. 2008. Compost and Biological Amendments in Potato Systems: Effects on Soil Microbial Communities. Northeast Potato Technology Forum, Fredericton, New Brunswick, Canada (Abstract). Tavantzis, S., Larkin, R.P., Alyokhin, A., Erich, S., Bernard, E., Gross, S. 2008. Compost and Biological Amendments in Potato Systems: Effects on Soilborne Diseases and Yield. Northeast Potato Technology Forum, Fredericton, New Brunswick, Canada (Abstract).
  • Larkin, R.P., Alyokhin, A., Erich, S., Bernard, E., Gross, S.Tavantzis, S. 2008. Compost and biological amendment effects on soilborne diseases and yield in potato. . 67th Annual Maine Agricultural Trades Show, Augusta, ME.
  • M. Susan Erich, S. Tavantzis, R. Larkin, S. Gross and A. Alyokhin. 2008. Effect of Compost Amendment on Soil Properties in Low and High Organic Matter Soils. Joint Annual Meeting of GSA, SSSA, ASA, CSSA, GCAGS, HGS in Houston, TX. Joint Annual Meeting Program, p. 349.
  • Erich, S., Tavantzis, S., Larkin, R.P., Alyokhin, A., Gross, S. 2008. Compost and Biological Amendments in Potato Systems: Effects on Soil Properties and Fertility. Northeast Potato Technology Forum, Fredericton, New Brunswick, Canada (Abstract).
  • Gross, S., A. Alyokhin, R. Larkin, S. Erich and S. Tavantzis. 2008. Reduced pest insect densities following compost application in organic and conventional systems. Northeast Potato Technology Forum, Fredericton, New Brunswick, Canada (Abstract).
  • Alyokhin, A., Gross, S., R. Larkin, S. Erich and S. Tavantzis. 2008. Compost effects on insect pests of potato. 67th Annual Maine Agricultural Trades Show, Augusta, ME.
  • Erich, S., Tavantzis, S., Larkin, R.P., Alyokhin, A., Gross, S. 2008. Compost effects on soil properties and fertility. 67th Annual Maine Agricultural Trades Show, Augusta, ME.
  • Larkin, R.P., Tavantzis, S., Bernard, E., Alyokhin, A., Erich, S., Gross, S. 2008. Compost and Biological Amendment Effects on Soilborne Disease and Soil Microbial Communities. Annual Meeting of the American Phytopathological Society. Phytopathology 98:586 (Abstract).
  • R. P. Larkin, A. Alyokhin, M. S. Erich, E. Bernard, S. Gross, and S. Tavantzis. 2008. A Systems Approach for Enhancing Soil Functionality and Plant Health to Suppress Plant Diseases and Pests. IOP PDs Meeting and SARE Conference, Kansas City.
  • Bartz, Faith E., Danehower, David A., Tavantzis, Stellos and Cubeta, Marc A. 2008. Quinic acid catabolism and production of the plant growth regulator phenylacetic acid by Rhizoctonia solani AG-3. Inoculum Supplement to Mycologia Vol. 59(4), p.19 - 2008


Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: We have continued our field studies on complementing stable hypovirulence in Rhizoctonia solani (isolate Rhs 1A1 containing the M2 dsRNA) and transient hypovirulence induced by quinic acid found in compost. Our previous data on the synergistic effects of the two types of hypovirulence in a conventional potato production site were confirmed. Current work on an exemplary organic farm showed that the hypovirulent isolate Rhs 1A1 significantly reduced the total incidence and severity of all tuber diseases whereas compost significantly increased tuber yields, especially the production of large tubers (32% increase), and also increased plant emergence. We have sequenced more than seven hundred cDNAs specifically associated with quinate-induced hypovirulence and have reported previously on some of the gene groups expressed under induced hypovirulence conditions in the presence of the potato host. We have used EST-specific primers in genome walking experiments to locate potential clusters of quinate-utilization pathway genes. After sequencing a total of 4,389 base pairs of a genomic region, we determined the sequence of the activator of the QUT pathway, qutA gene (sum of exxons 2,238 bp), and the respective QUTA protein (746 amino acids). The amino acid similarities of the R. solani QUTA protein with those of some other fungi are as follows: Coprinopsis cinerea (basidiomycete) 56.5%, Ustilago maydis (basidiomycete) 32.4%, Aspergillus terreus (ascomycete) 32.4%, Neurospora crassa (ascomycete) 30.0%. These similarities are significant considering that the similarities between corresponding QUT proteins within the same genus in Neurospora or Aspergillus are less than 30%. In regards to the qutD gene (quinate transporter), we sequenced 7,730 bp upstream and downstream of the respective EST/cDNA sequence by genome walking. The sum of the exxons is 1,821 bp, and the protein (QUTD) is comprised of 607 amino acids. The similarities (at the amino acid level) with other fungal QUTD's are 38.3% for U. maydis, 33.5% for N. crassa, and 32.7% for A. terreus, respectively. PARTICIPANTS: Tavantzis, S. M. (PI) (UMaine, School of Biology and Ecology); Larkin, R.P. (co-PI)(New England Plant, Soil and Water Lab., USDA-ARS); Alyokhin, A. (co-PI)(UMaine, School of Biology and Ecology); Erich, M. S. (co-PI)(UMaine, Dept. of Plant, Soil and Env. Sciences); Cubeta, Marc A. (Collaborator) (NCSU, Dept. of Plant Pathology). Five graduate students, and several undergraduate students at UMaine are being trained through this project). TARGET AUDIENCES: Conventional potato growers,organic potato growers, conventional vegetable growers, organic vegetable growers.

Impacts
Knowledge obtained from this research project will lead to the implementation of the novel concept of complementing quinate-induced hypovirulence activated by quinate-rich compost amendments with stable hypovirulence (attributed to the M2 dsRNA), which will be most useful when quinate becomes unavailable due to dry soil conditions or complete decomposition of compost.

Publications

  • Larkin, R.P., Manmathan, H., Tavantzis, S. 2007. Effects of Compost and Biocontrol Amendments on Stem Canker, Black Scurf, and Common Scab of Potato, 2006. Plant Disease Management Reports. vol 1: p. v065.
  • Charlton, N.D., Tavantzis, S., Carbone, I., Cubeta, Marc A. 2007. Evolutionary history and population dynamics of the M2 double-stranded RNA of the soil fungus Rhizoctonia solani. International Meeting on "Population and Evolutionary Biology of Fungal Symbionts", Ascona, Switzerland.
  • Bartz, E. F., S. M. Tavantzis and M. A. Cubeta. 2007. Inluence of quinic acid catabolism on the growth and aggressiveness of Rhizoctonia solani. Joint Meeting of the American Phytopathological Society, and the Society of Nematologists in San Diego, CA.


Progress 10/01/05 to 09/30/06

Outputs
Transmission of the hypovirulence-associated M2 dsRNA (stable hypovirulence) under field conditions requires transmission of M2 to several R. solani isolates representing the somatic incompatibility groups occurring in a particular field. In contrast, quinate-induced hypovirulence involves amending the soil with lignin-rich plant composts and circumvents somatic incompatibility problems. Lignin degradation gives rise to quinic acid, which induces synthesis of full-length transcript of M2 and its corresponding polypeptide (pA). Moreover, plant composts promote soil regeneration, reduction of disease pressure through restoration of soil biodiversity, systemic induced resistance, and enhancement of plant growth. To determine the efficacy of quinate-containing composts in ameliorating virulence of R. solani under commercial field conditions, a three-year collaborative field study (Larkin and Tavantzis) was undertaken. Results show that lignin-rich conifer compost reduced Rhizoctonia disease severity significantly (as compared to untreated controls). The hypovirulent, M2-containing, strain Rhs 1A1 alone or a combination of conifer compost and the Rhs 1A1 (e. g., induced hypovirulence + stable hypovirulence) resulted in the lowest overall disease severity both in terms of black scurf and stem canker. Conifer compost brought about significantly higher tuber yields (total weight and marketable weight, respectively).

Impacts
Knowledge obtained from this research project will lead to the implementation of the novel concept of complementing quinate-induced hypovirulence activated by quinate-rich compost amendments with stable hypovirulence (attributed to the M2 dsRNA), which will be most useful when quinate becomes unavailable due to dry soil conditions or complete decomposition of compost.

Publications

  • Lakshman, D. K, Liu, C., Mishra, P. and Tavantzis, S. M. 2006. Characterization of the arom gene in Rhizoctonia solani, and transcription patterns under stable and induced hypovirulence conditions. Current Genetics 49: 166-177.
  • Charlton, N. D., Carbone, I., Tavantzis, S. M., and Cubeta, M. A. 2006. Analysis of genetic diversity and evolutionary history of the M2 dsRNA of Rhizoctonia solani AG-3. Joint Annual Meeting of Mycol. Soc. of America, American Phytopath. Society, and Canadian Phytopathol. Society.
  • Charlton, N.D., Tavantzis, S.M., Carbone, I., and M.A. Cubeta. 2006. Analysis of the genetic diversity and evolutionary history of the M2 dsRNA of Rhizoctonia solani AG-3. Inoculum 57:23. (Supplement to Mycologia).


Progress 10/01/04 to 09/30/05

Outputs
We have shown previously that quinic acid (QA) induces hypovirulence in the virulent isolate Rhs 1AP of R. solani. We have sequenced 431 QA-induced cDNAs, which have been grouped into 52 contigs and 33 singletons. One of our primary goals is to identify cDNAs of the quinate utilization (QUT) pathway genes in R. solani (Rhs 1AP). Nine contigs and singletons were found to have a significant sequence similarity with four different QUT genes (QUTA-activator, QUTD, QUTH, and QUTR-suppressor) from the ascomycetous genera Aspegillus and Neurospora. In addition to the putative QUT pathway genes, BLAST analysis showed quinate induction results in up-regulation of genes involved in a) major shifts in gene expression indicating metabolic and structural changes in the fungus; b) saprophytism; c) mitochondrial electron transport; d) membrane transport; e) Rho signaling pathway. Among other upregulated genes of potential importance were: transcription regulation factors, subtelomeric helicase, and ubiquitin, which in addition to its well-established role, has been associated with regulation of dynamic protein-protein interactions in the nucleus. Interestingly, one of the upregulated cDNAs in the above experiment is a Tn10 transposase, which is highly concerved in a wide variety of eukaryotes (plants, birds, mammals).

Impacts
Knowledge gained from this work will set the stage for dissecting stable hypovirulence, induced hypovirulence, and virulence in R. solani, and proceeding with the design of directed and novel approaches to plant disease management.

Publications

  • Lakshman, D. K, Liu, C., Mishra, P. and Tavantzis, S. M. 2005. Characterization of the arom gene in Rhizoctonia solani, and transcription patterns under stable and induced hypovirulence conditions. Current Genetics (In Press).
  • Tavantzis, S. M. and Manmathan, H. K. 2005. Functional Genomics of induced hypovirulence in Rhizoctonia solani. Abstracts of the XI International Congress of Mycology. Pp. 22
  • Lakshman, D.K., Liu, C., Mishra, P.K., Tavantzis, S.M. 2005. Characterization And Substrate Induced Transcriptional Regulation Of The Pentafunctional Arom Gene Of Rhizoctonia Solani. Phytopathology 95:S56.
  • Charlton, N., Tavantzis, S., Cubeta, M. 2005. Genetic diversity of the M2 dsRNA mycovirus in a population of the soil fungus Rhizoctonia solani. Abstracts of the XI International Congress of Mycology. Pp. 40
  • Charlton, N., Carbone, I., Tavantzis, S., Cubeta, M. 2005. Genetic diversity of the M2 dsRNA mycovirus in a population of the soil fungus Rhizoctonia solani. Phytopathology 95:S18


Progress 10/01/03 to 09/30/04

Outputs
We constructed a quinate induced Rhs 1AP subtracted cDNA library using the BD Clontech PCR-Select cDNA Subtraction Kit (BD Clontech). cDNA from quinate-induced Rhs 1AP was subtracted by cDNA from untreated Rhs 1AP both grown in the absence of the potato plant host. Since we have provided biochemical evidence concerning the quinate-mediated induction of quinate genes and the M2 dsRNA in Rhs 1AP, the above subtraction will result in the identification of the quinate pathway genes. This experimental approach will allow us to (a) study their expression patterns in Rhs 1A1, Rhs 1AP and quinate-induced Rhs 1AP, and (b) identify potential sequence similarities between the M2-encoded polypeptide A (pA) and the putative suppressor of the quinate pathway, and (c) examine potential protein-protein interactions between pA and the activator of this pathway using the yeast dihybrid assay. Results regarding two constitutive genes indicated that subtraction was efficient and as expected from the Invitrogen protocol. In experiments aimed at studying the expression of the arom gene, the PCR product band intensities generated from quinate-induced Rhs 1AP cDNA were greater than those from uninduced cDNA at the different step cycles. These results were in agreement with northern blot hybridization analysis data and published reports regarding elevated amounts of the AROM protein in A. nidulans cultures with a dysfunctional suppressor (QUTR) or over-expression of one or more quinate pathway genes. In the absence of environmental quinate, these conditions lead to leakage of the two shared metabolites (3-dehydroquinate and dehydroshikimate) from the AROM enzyme. Interestingly, the fungus elevates the concentration of the AROM protein to compensate (starvation effect) for the leakage of these metabolites from the AROM enzyme. The higher level of arom mRNA we detected in quinate-induced Rhs 1AP may be analogous to the starvation mechanism observed in A. nidulans.

Impacts
Addressing the objectives described above will help us determine a) how similar the phenomena of permanent (Rhs 1A1) and induced hypovirulence (quinate-treated Rhs 1AP) are, and b) whether they can be used in a complementary manner to manage Rhizoctonia-caused plant diseases.

Publications

  • Charlton, N.D., Tavantzis, S.M., and Cubeta, M.A. 2004. Genetic diversity of double stranded RNA mycoviruses in a population of Rhizoctonia solani anastomosis group 3 (AG-3). Inoculum 55:11. (Supplement to Mycologia). (Abstr.)


Progress 10/01/02 to 09/30/03

Outputs
The goal of the project in its current phase is to understand the interactions between Rhizoctonia solani and the potato plant at the molecular level, and use this knowledge to effectively manage numerous plant diseases caused by this pathogen using environmentally sensible strategies. The specific objectives are to: 1) Detect genes in R. solani that are specifically associated with the following conditions: a) virulence in culture, b) virulence in the presence of the plant host (potato), c) hypovirulence in culture, d) hypo-virulence in the presence of the plant host, e) induced hypovirulence (quinate-treated Rhs 1AP), and f) induced hypovirulence in the presence of the plant host. This will be accomplished by use of macroarrays that allow detection of differentially activated genes. 2) Identify genes co-regulated in virulence (Rhs 1AP), stable hypovirulence (Rhs 1A1), and quinate-induced hypovirulence in the presence or absence of the host plant. This will be achieved by applying a clustering method to the macroarray data collected under the conditions of the above treatments. A novel clustering algorithm known as adaptive double self-organizing map (ADSOM) will be used for this purpose. ADSOM has unique features in that it 1) has a flexible topology that performs clustering and cluster visualization simultaneously, and 2) allows automatic detection of number of gene clusters. The algorithm will be applied to cluster the gene expression profiles obtained from the macroarrays generated by objective #1. The goal is to identify genes that are active under different conditions that up- or down-regulate virulence. Suppression subtractive hybridization (SSH) will be used to compare mRNA (cDNA) populations in Rhs 1AP and Rhs 1A1 under the conditions described in objective #1. SSH is an amplification-based cDNA subtraction method designed to detect genes that are over expressed or exclusively expressed in one tissue compared to another. The substrates for subtraction are total cDNA (tester) containing differentially expressed cDNAs (target molecules) and an excess of cDNA (driver) used for comparison (deprived of target molecules). This step achieves a greater than 1000-fold enrichment of differentially expressed cDNAs. We have carried out successfully all stages of SSH using the Clontech PCR-Select Subtraction Kit and its satellite kits. Two house-keeping genes have been used in these experiments: glyceraldehyde 3-phosphate dehydrogenase (G3PDH), and beta-tubulin. We used primers from published sequences of the respective genes previously cloned from anastomosis group 3 (AG 3), the AG to which Rhs 1AP and Rhs 1A1 belong. In both cases (G3PDH and beta-tubulin), there was a dramatic reduction of abundance in quinate-induced Rhs 1A1 cDNA subtracted by Rhs 1AP cDNA.

Impacts
Addressing the objectives described above will help us determine a) how similar the phenomena of permanent (Rhs 1A1) and induced hypovirulence (quinate-treated Rhs 1AP) are, and b) whether they can be used in a complementary manner to manage Rhizoctonia-caused plant diseases.

Publications

  • Liu, C., Lakshman, D. K, Tavantzis S. M. 2003 Quinic acid induces hypovirulence, and expression of a hypovirulence-associated double-stranded RNA in Rhizoctonia solani. Current Genetics 43:103-111.
  • Liu C., Lakshman D. K., Tavantzis S. M. 2003. Expression of a hypovirulence-causing double-stranded RNA (dsRNA) is associated with up-regulation of quinic acid pathway, and down-regulation of shikimic acid pathway in Rhizoctonia solani. Current Genetics 42:284-291.