Source: CLEMSON UNIVERSITY submitted to NRP
PEACH TREE SHORT LIFE IN SOUTH CAROLINA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
SPECIAL GRANT
Reporting Frequency
Annual
Accession No.
0203704
Grant No.
2005-34126-15638
Cumulative Award Amt.
(N/A)
Proposal No.
2005-06137
Multistate No.
(N/A)
Project Start Date
Sep 1, 2005
Project End Date
Aug 31, 2007
Grant Year
2005
Program Code
[AC]- (N/A)
Recipient Organization
CLEMSON UNIVERSITY
(N/A)
CLEMSON,SC 29634
Performing Department
School of Agricultural, Forest, & Environmental Sciences
Non Technical Summary
The disease syndrome of peach, nectarine, and plum trees in the southeastern United States known as Peach Tree Short Life is characterized by sudden collapse of otherwise apparently healthy trees just before, during, or just after flowering from bacterial canker or cold injury. The problem, which affects more than 70% of the peach acreage in the southeastern U.S., may develop in very young orchards, but the losses generally are most common and severe in trees four to seven years of age. Severe tree mortality in the 1960s and early 1970s prompted the implementation of a "Ten-Point Program" to combat the problem. Unfortunately, effectiveness of the control program has diminished largely because the efficacy of nematode control has decreased due to the loss of chemical nematicides and the tree loss from the root rot fungus, Armillaria tabescens, has significantly increased. This research project will identify the cultural, physiological and genetic components of resistance and tolerance to peach diseases and nematodes that lead to early peach tree death in South Carolina. The project will develop nematode tolerant peach rootstocks, find molecular gene markers for nematode resistance for Rosaceae species, identify genes controlling winter dormancy in temperate fruit tree species, develop environmentally friendly, biocontrol agents (i.e., bacteria) and cultural practices for nematode control, and incorporate an Armillaria resistant gene into transgenic plums and eventually peach rootstocks.
Animal Health Component
35%
Research Effort Categories
Basic
50%
Applied
35%
Developmental
15%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2011114104030%
2121114108015%
2121114112035%
2151114112020%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 215 - Biological Control of Pests Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1114 - Peach;

Field Of Science
1040 - Molecular biology; 1120 - Nematology; 1080 - Genetics;
Goals / Objectives
There are 8 objectives which are as follows: (1) We will evaluate of longevity and productivity of peach BY520-9 (GuardianTM Brand BY520-9) rootstocks on Peach Tree Short Life (PTSL) sites in the Southeast and replant sites throughout North America. (2) We will develop molecular markers to identify (i.e., fingerprint) each BY520-9 selection under field-testing, and to find markers (MAS) to identify nematode or PTSL tolerant and susceptible genotypes in F1 and F2 seedling populations from crosses of field tested tolerant and susceptible genotypes. (3) We will try to identify and characterize the evergrowing gene and its function in peach dormancy behavior to determine if it may be a factor in predisposing peach trees on susceptible rootstocks to cold damage and subsequent PTSL. (4) Our lab will maintain, monitor and evaluate bacterial populations, nematode reproduction, tree growth, foliar nutrient status, root growth and physiology, and incidence of PTSL in the on-going preplant solarization/BG33R inoculation field trials. A new solarization field trial will be inoculated with a bacterial cocktail that also includes the bench mark strain P. synxantha BG33R. (5) We will continue the development of a Pseudomonas synxantha BG33R interactive website for rapid dissemination of scientific data to the scientific community, and to encourage a timely real-time interaction between biocontrol and bacterial genetics research groups involved in this important area of research. (6) We will initiate a global-gene-expression study of the bacterial genes involved in the communication, interaction, and subsequent colonization of the peach root by the bacterium Pseudomonas synxantha BG33R. This also will include genes expressed during growth in both egg-kill factor inducing and noninducing conditions. (7) We will determine the usefulness of the gastrodianin gene to create disease tolerant rootstocks. (8) We will investigate root collar excavation, various fumigants, and trunk injections of fungicides for Armillaria root rot control.
Project Methods
The development of a new commercial rootstock from the Guardian Brand BY520-9 line will continue. A seed orchard of 1200 virus-indexed trees (6 genotypes) produces about 1,500,000 seed for distribution to the commercial nursery industry per year. Data from a 2001 NC-140 peach rootstock trial show that a BY520-9 selection is one of the most vigorous rootstocks across all sites. This rootstock was also one of the most productive rootstocks from the 1994 NC-140 peach trial. New selections will be rigorously tested in grower field trials. We have completed sequencing a 70.5 kb region of the wild type peach genome putatively containing the EVG locus. Analysis of the sequenced locus predicted several candidates for the EVG mutation. The region we have sequenced has at least six similar copies of the MIKC structural class of MADS-box genes. We will explore the functions of these genes in relation ot winter cold hardiness. Application of the ring nematode ovicidal bacterium Pseudomonas synxantha BG33R through the drip irrigation system will continue in the solarized and non-solarized experimental peach orchard plots at the USDA-ARS Research Station in Byron, GA. These biocontrol applications will be made throughout the year. Sampling of soil for bacteria and nematodes also will continue throughout the year. Work is continuing on the genetics and functional genomics of the plant/microbe interaction of Pseudomonas. Current work has focused on the identification of bacterial genes involved in colonization and communication between the bacterium and the plant roots. These studies, which include the use of high-density, full genome Pseudomonas microarrays and plant-modulated promoter probe mutant libraries of P. synxantha BG33R, have targeted numerous genes that display altered expression patterns in the rhizosphere. We will investigate the function of these genes. We also engineered transgenic tobacco and plum plants containing the GAFP gene under the control of the 35S promoter so that we can study the potential of GAFP as a resistance determinant for soil borne diseases in agricultural systems. Plum was used as a woody plant model system because a transformation system was already in place, plum is susceptible to all major root pathogens, and plum can be used as rootstock for many stone fruit crops. We analyzed GAFP gene expression in transgenic tobacco lines with real-time PCR and normalized the data with the expression of 18S ribosomal DNA. Initial results have indicated a seven-fold difference in relative expression between low and high expressors and the majority of lines expressed the gene 2-3 fold higher than the lowest expressor. We will try to elucidate the function and/or effect of these amplified expressed genes in Armillaria infection and colonization of tobacco roots and ultimately transgenic plum roots.

Progress 09/01/05 to 08/31/07

Outputs
OUTPUTS: Four F2 populations of Guardian and Nemaguard rootstock crosses were created, cloned and then evaluated on a severe peach tree short life (PTSL) site. DNA was collected from each genotype in all 4 families before planting. Markers in the lab were then developed based on the genotypes observed PTSL tolerance or susceptibility in the field test. We have completed the sequencing of the genomic region responsible for the evergrowing non-dormancy trait. Additionally, we have evaluated seasonal gene expression trends for the identified candidate genes. We also designed RNAi vectors that specifically knock-down individual candidate genes for functional testing by transgenic approaches. Field trials to examine cultural and biological methods to control Armillaria root rot were established in 2005 and 2006. Each trial was replicated twice, but it is still too early to make recommendations to manage this disease. The Gastrodia anti-fungal protein (GAFP), a lectin isolated from the Asiatic orchid Gastrodia elata, was inserted via Agrobacterium tumefaciens-mediated transformation, where we obtained 4 transgenic plum (Prunus domestica) lines of which three lines (4J, 4I and 5D) were used in our Armillaria research. The biocontrol bacterial research provides insights into the mechanisms underlying the biocontrol of nematodes by the BG33R bacterium and other organisms, which has facilitated the development of more effective biocontrol strategies such as pre-plant solarization to improve agriculture production. This information was disseminated via technical and scientific presentations at local, regional, national and international scientific and grower meetings as well as grower field days and workshops. Additional work on all areas of this grant continue and results have been shared with both the stakeholders and scientific community via publications, reports, meetings, and sharing of molecular probes, techniques and genetic maps. PARTICIPANTS: P.I. Gregory Reighard, Dept. of Horticulture, Clemson University Collaborators: Dr. Guido Schnabel, Dept. of Entomology, Soils and Plant Scienes, Clemson University Dr. Desmond Layne, Dept. of Horticulture, Clemson University Dr. Douglas Bielenberg, Dept. of Horticulture, Clemson University Dr. Christina Wells, Dept. of Horticulture, Clemson University Dr. Daniel Kluepfel, USDA, Davis, California Dr. Andrew Nyczepir, USDA, Byron, Georgia Post Doctoral and Research Associates: Dr. Pat Wechter, USDA, Charleston, South California Dr. Greg Fang, Dept. of Entomology, Soils and Plant Scienes, Clemson University Dr. Sergio Tarado, Zaragoza, Spain Graduate Students: Mr. Cory Tanner, Dept. of Horticulture, Clemson University Ms. Xiaoyu Liu, Dept. of Horticulture, Clemson University Mr. Antonio Weibel, Dept. of Horticulture, Clemson University Undergraduate Stud; Dr. Achour Amiri, post doctoral scientist; Clemson University; Karen Bussey, graduate student, Clemson University; Alexis Nagel, graduate student, Clemson University; Daniel Toms, undergraduate student, Clemson University; Andrew Phillips, undergraduate student. Clemson University. TARGET AUDIENCES: The target audience were fruit growers nationwide and scientist peers worldwide. This project provided cultural information for fruit tree growers in the United States via extension activities at local, regional, and national meetings. Scientific advances from this project were discussed at regional, national and international meetings and disseminated via journal publications.

Impacts
The release of an improved Guardian rootstock selection that increases orchard survival and productivity by a few percent will increase profitability for the $60 million southeastern peach industry by several million dollars per year. This is magnified by the increased longevity of trees on an improved rootstock and thus collectively has an impact much greater than $1 million/year. Several molecular markers have been found that distinguish PTSL susceptible rootstock seedlings and thus may reduce the time of selecting superior rootstocks for PTSL resistance. The candidate dormancy genes appear to be photoperiod regulated with distinct peaks in expression at different points of physiological interest during the year, such as approximate date of bud formation or solstice. These expression data correlated well with the non-dormant phenotype of mutant trees which do not respond to inducing conditions. Controlled environment studies demonstrated that these genes had strong responses to photoperiod in the absence of changes in temperature which may explain the majority of the expression patterns seen under field conditions. Preliminary data indicate that trees planted in Smartpots grew less vigorously compared to control trees. Also, they were more drought stressed during the dry 2007 season. Root suckering of excavated Guardian trees was significantly increased compared to Guardian control trees, whereas excavated Lovell trees did not sucker more than Lovell control trees. All transgenic plum lines produced GAFP-1 in root tissues, and Southern blot analysis revealed that lines 4J, 4I, and 5D possessed one, two, and four copies of the GAFP-1 gene, respectively. Lines 4J and 4I were not phenotypically different from the non-transformed control line, but line 5D showed significant divergence with regard to its leaf morphology and growth habit. Compared to the inoculated control, lines 4J and 4I exhibited increased tolerance to Phytophthora root rot, caused by P. cinnamomi, and the root-knot nematode, Meloidogyne incognita. This study documented the first evidence that a plant lectin such as GAFP has the potential to confer increased tolerance to agriculturally important plant pathogens in a tree species. Our understanding of the biocontrol microorganism Pseudomonas synxantha BG33R can be summarized in the following three areas:(a)for biocontrol activity we identified an unusual broad-spectrum of antagonistic activity of the BG33R that its biocontrol activity spans a highly diverse taxa, including nematodes, fungi, oomycetes, and insect. To our knowledge, such broad effectiveness has never been reported from biocontrol microorganisms so far identified;(b)for the biochemical characterization we discovered that the nematocidal and anti-oomycetes activity are closely associated with the biosurfactant-type compounds while distinct compound(s) contributes to the insecticidal activity and (c)for the molecular characterization the GacS/GacA two-component regulatory system is involved in the modulation of the antagonistic activities that suppress nematodes, oomycetes and fungi. However, the insecticidal activity appeared to be independent from the system.

Publications

  • Reighard, G., R. Andersen, J. Anderson, W. Autio, T. Beckman, T. Baker, R. Belding, G. Brown, P. Byers, W. Cowgill, D. Deyton, E. Durner, A. Erb, D. Ferree, A. Gaus, R. Godin, R. Hayden, P. Hirst, S. Kadir, M. Kaps, H. Larsen, T. Lindstrom, N. Miles, F. Morrison, S. Myers, D. Ouellette, C. Rom, W. Shane, B. Taylor, K. Taylor, C. Walsh, and M. Warmund. 2007. Growth and yield of Redhaven peach on 19 rootstocks at 20 Locations in North America. Acta Hort. 732: 271-278.
  • Reighard, G.L., D. R. Ouellette, and K. H. Brock. 2007. Survival, growth and yield for Carogem peach on an interstem and two dwarfing rootstocks. Acta Hort. 732:303-306.
  • Reighard, G., T. Beckman, R. Belding, B. Black, J. Cline, W. Cowgill, R. Godin, M. Kaps, T. Lindstrom. D. Ouellette, L. Stein, K. Taylor, C. Walsh, M. Whiting and T. Robinson. 2006. Performance of Prunus rootstocks in the 2001 NC-140 peach trial. Sodininkyste Ir Darzininkyste 25(3): 327-333.
  • Blenda, A.V., I. Verde, L.L. Georgi, G.L. Reighard, S.D. Forrest, M. Munoz-Torres, W.V. Baird, and A.G. Abbott. 2007. Construction of a genetic linkage map and identification of molecular markers in peach rootstocks for response to peach tree short life syndrome. Tree Genetics and Genomes DOI 10.1007/s11295-006-0074-9, 10pp.
  • Bielenberg, D.G., S. Fan, G.L. Reighard and A.G. Abbott. 2007. Sequencing and annotation of the peach Evergrowing locus from wild-type and mutant genomes reveals several candidate genes for the control of terminal bud formation in response to dormancy inducing conditions. Acta Hort. 738: 559-565.
  • Zhebentyayeva, T., D.A. Lalli, D. Jiwan, J.H. Jun, J.. Duncan, D. Main, G. L. Reighard, R. Scorza and A. G. Abbott. 2007. From BAC to trait and back: Exploiting structural and functional genomics databases for gene identification in peach. Acta Hort. 738: 711-717.
  • Nagel, A.K., Nyczepir A.P., Scorza, R. and Schnabel, G. 2007. The Gastrodia Anti-Fungal Protein confers increased resistance to Phytophthora root rot and the root-knot nematode in a fruit tree species. Phytopathology 97:S82.
  • Schnabel, G. A. Nagel, and D. R. Layne 2007. Development of transgenic rootstocks with resistance to oak root rot. South Carolina Peach Council 2006 Research Reports, 6:14-15.
  • Schnabel, G. 2007. Investigation of a Cultural Method to Manage Armillaria Root Rot Disease of Peach. Proceedings of the 82nd Cumberland-Shenandoah Fruit Workers Conference, Winchester, VA, 112-113.
  • G. C. Fang, V. C. Waldrop, W. P. Wechter, and D. A. Kluepfel. 2007. A broad-spectrum antagonistic activity of the biocontrol agent Pseudomonas synxantha BG33R. Phytopathology 97:S34
  • A. P. Nyczepir, and D. A. Kluepfel. 2007. Impact of solarization, rootstock and Pseudomonas synxantha on Criconemoides xenoplax populations and tree growth in a peach tree short life site. Journal of Nematology 39(1):75


Progress 09/01/05 to 08/31/06

Outputs
In the first year of this grant, different genotypes of Armillaria tabescens were found and characterized and a fast and reliable method was developed to identify A. tabescens from other Armillaria species. A. tabescens was found to cause Armillaria root rot on Pindo Palm and other ornamental crops in South Carolina. The plant lectin gastrodianin was shown to provide tolerance to nematodes, fungi and stramilopiles in transgenic tobacco plants. A new open-source software application (RootFly) for the analysis of fine root images was developed. We demonstrated the efficacy of soil solarization as an alternative to methyl bromide fumigation of replanted peach orchards and isolated and identified cyanide-producing rhizobacteria from replanted peach orchards. An F2 population of rootstocks that have exhibited increased tolerance to ring nematodes and segregate for a marker for susceptibility began dying in 2005 thereby allowing for scoring of PTSL segregation in the population. We made the initial identification of the nature of the lesion responsible for the 'Evergrowing' trait in the mutant genome and identified putative candidate genes for the control of the 'Evergrowing' trait in peach and performed the initial analysis to demonstrate that putative candidate genes are expressed in wild type peach germplasm. Large-scale field-testing of both the delivery techniques and effectiveness of Pseudomonas synxantha under commercial orchard conditions was initiated.

Impacts
The release of a new Guardian rootstock selection that can increase orchard survival and productivity by a few percent will also increase profitability for the $60 million southeastern peach industry by several million dollars per year. This is magnified by the increased longevity of trees on an improved rootstock and thus collectively have an impact much greater than $1 million/year. The soil bacteria and solarization research has the potential to be used commercially within as little as 2-3 years in some commercial orchards. Identification of bacterial strains and the nematode egg-kill factor (EKF) produced by these strains will eventually lead to a new natural nematocide, which might interest private industry and could also become a new tool for organic growers. From the Armillaria research, gastrodianin gene insertion into commercial peach rootstocks may become possible. In addition, localized soil removal and tree injection of fungicides could be an effective tool to prevent Armillaria infection in peach orchards. The outcome of this research will be an environmentally friendly, integrated peach tree short life (PTSL) management system that will successfully control PTSL in peach trees.

Publications

  • Bielenberg D.G., Li Z., and Reighard G.L. 2006. The peach evergrowing locus contains a cluster of MIKC-type MADS-box genes with tissue specific expression patterns. Poster abstract no. 03011. Annual Meeting, Boston, MA, August 5-9, 2006. American Society of Plant Biologists.
  • Blenda, A.V., W.P. Wechter, G.L. Reighard, W.V. Baird and A.G. Abbott. 2006. Development and characterization of diagnostic AFLP markers in Prunus persica for its response to peach tree short life syndrome. J. Horticultural Sci. & Biotechnology 81(2):281-288.
  • Blenda, A.V., G.L. Reighard, W.V. Baird and A.G. Abbott. 2006. Simple sequence repeat markers for detecting sources of tolerance to PTSL Syndrome in Prunus persica rootstocks. Euphytica 147:287-295.
  • Cox, K. D., D. R. Layne, R. Scorza, and G. Schnabel. 2006. Gastrodia Anti-Fungal Protein from the orchid Gastrodia elata confers disease resistance to root pathogens in transgenic tobacco. Planta DOI 10.1007/s00425-006-0322-0.
  • Layne, D.R. and G. Schnabel. 2006. Preplant practices to manage Armillaria root rot disease and other soil pathogens on a commercial peach replant site. HortScience 41(4):1028.
  • Layne, D. R., G. Schnabel, K. D. Cox, R. Scorza, and K. E. Bussey. 2005. Armillaria root rot of peach: a multipronged management approach. HortScience 40(4):1026.
  • Schnabel, G., J. S. Ash, and P. K. Bryson. 2005. Identification and characterization of Armillaria tabescens from the southeastern United States. Mycol. Res. 109:1208-1222.
  • Schnabel, G., K. E. Bussey, and P. K. Bryson. 2005. First Report of Armillaria gallica causing Armillaria root rot in daylily in South Carolina. Plant Dis. 89:683, 2005.
  • Schnabel, G., P. K. Bryson, and M. A. Williamson. 2006. First Report of Armillaria tabescens causing Armillaria root rot of Pindo Palm in South Carolina. Plant Dis. 90:1106.