Progress 09/01/04 to 08/31/06
Outputs
We improved Guardian rootstock, which is used by 90 percent of the commercial peach growers in South Carolina. We found genetic markers for tolerance to ring nematode in Prunus. We completed the sequencing of a 132 kilobase region of the peach genome assembled from three overlapping BACs that encompass the complete evergrowing (EVG) gene region. This region had six highly similar MIKC-type MADS-box transcription factors that are candidates for the EVG gene. In wild-type tissues, the six MADS genes are expressed in leaves and roots but two are not expressed in stem tissues. We identified a broad spectrum of hosts for the BG33R bacterium that extends from our original report on the control of the ring nematode (C. xenoplax) to phytopathogenic fungi and insects. To our knowledge, there is no other biological control microorganism that has ever been reported to exhibit such wide-span target range. This makes BG33R a high potential, biocontrol agent. A total of 11 bacteria
mutants were identified from a transposon integration library generated by Tn5::xylE random integrations. These mutants were disruptive for zoosporicidal activity in that they were no longer able to disintegrate the zoospores of Phytophthora nicotianae, a major plant pathogen with highly broad host range. We challenged Gastrodianin gene containing transgenic tobacco lines with several root disease pathogens from several higher-order eukaryotic lineages. Transgenic tobacco lines had reduced symptom development and improved plant vigor compared to non-transformed and empty vector control lines when challenged with various pathogens. These lines also exhibited reduced root galling when challenged by M. incognita. Preplant fumigation with Enzone to prevent Armillaria root rot resulted in tree mortality and stunting but Methyl bromide or Telone II enhanced tree survival and growth. Guardian and Halford rootstocks performed superior to Lovell after 2 years. Preplant root dipping with
mycorrhizal fungi provided no apparent benefit for tree growth or survival 2 years following planting. The DMI fungicide propiconazole was found to be the strongest inhibitor of A. tabescens isolates out of seven chemical classes tested with EC50 values ranging from 0.49-0.86 mg/L. Both pressurized injection and non-pressurized infusion systems were tested on peach, but only the non-pressurized infusion system was suitable for chemical delivery. It was used to apply formulated propiconazole in three seasonal application timings. We found that propiconazole can move into the primary roots, however, translocation was dependent on application timing and weather conditions. U. S. Provisional Patent Application, G. Schnabel, R. Scorza and D. Layne. Increased resistance of plants to oomycetes, nematodes and fungi. Date of Deposit: April 2005. Docket No. CXU-464-P.
Impacts
This research has identified some cultural, physiological and genetic components of resistance and tolerance to peach diseases and nematodes that predispose peach trees to early death (Peach Tree Short Life) in South Carolina. This project developed a nematode tolerant peach rootstock (Guardian), found molecular gene markers for ring nematode resistance for peach, identified genes (6 MADS box) influencing winter dormancy in peach, developed environmentally safe biocontrol agents (BG33R) for ring nematode and fungal (Phytophthora) control, cultural practices for Armillaria root rot control, and incorporated an Armillaria resistant gene into transgenic plums. Results from the study have provided new and novel, environmentally friendly methods to control the Peach Tree Short Life syndrome, which causes millions of dollars of losses annually in peach orchards in South Carolina, Georgia, Alabama, North Carolina, California, and New Jersey.
Publications
- 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.
- Reighard, G.L., D. R. Ouellette, and K. H. Brock. 2006. Growth and survival of 20 peach rootstocks and selections in South Carolina. Acta Hort. 713: 269-274.
- Bielenberg, D.G., Y. Wang, G.L. Reighard and A.G. Abbott. 2006. Sequencing and analysis of the peach EVG locus. Acta Hort. 713: 73-82.
- Nyczepir, A.P., T.G. Beckman and G.L. Reighard. 2006. Field evaluation of Guardian peach rootstock to different root-knot nematode species. Acta Hort. 713: 303-310.
- Tanner, S.C., Wells, C.E., and G.L. Reighard. 2006. Soil treatments differentially affect peach tree root development and demography in a replant site. Acta Hort. 713: 381-390.
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Progress 01/01/05 to 12/31/05
Outputs
The development of a commercial rootstock from the GuardianTM Brand BY520-9 line continues and a rootstock seed orchard produced 1,600,000 seed for the commercial nursery industry in 2005. Data from a 2001 NC-140 peach rootstock trial show that a BY520-9 selection is one of the most vigorous and productive rootstocks at 10 North American locations. 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. Analysis of the sequences information from all three BACs confirms that we have identified all of the flanking markers used to fine map the position of the locus and therefore should have delimited the extent of the candidate genes in the region. The region we have sequenced has at least six similar copies of the MIKC structural class of MADS-box genes. Propiconazole was injected in the trunk of peach trees and its mobility was
determined in bioassays and HPLC analysis of roots. Results indicate that the compound is mobile and therefore may be used for injection of peach trees. We also propagated transgenic plum plants. Preliminary results on the mobility of GAFP in plum indicate no protein movement across the graft union. In addition, we set up two more root collar excavation experiments in grower orchards. We established a trial on a farm with a history of Armillaria disease. Trees were either planted on raised beds or not. Prior to planting, trees were either dipped with mycorrhizal fungi or not. One year after planting, half of the trees on raised beds will have a partial excavation of the crown to expose primary roots to sunlight and UV. We continue to use belowground video technology to monitor root behavior in response to both solarization and BG33R inoculation. We have partnered with computer engineers to develop an image processing software package (Rootfly) that speeds up root data collection by an
order of magnitude. The unique root detection algorithms developed through this project have resulted in software that is distributed free of charge to interested researchers in the plant science community. We are currently utilizing full-genome microarrays to perform gene expression studies of Pseudomonas putida KT2440 in the rhizosphere of Arabidopsis thaliana. We identified 26 bacterial genes that were induced and 149 genes that were repressed in the rhizosphere at a level of 2-fold or greater. In addition, 195 bacterial genes were modulated as a function of growth in the rhizosphere. Thirty-one percent of the genes up-regulated after 48 hr returned to basal expression levels 72 hr later while 85 unique genes were up-regulated only at 120 hr. We have generated site directed P. putida mutants in two of the genes that have been identified as being upregulated during root colonization; Methionine gamma-lyase and the araC regulatory gene. Both of these mutants now have been examined in
root colonization bioassays and have been found to be debilitated in their ability to colonize the plant rhizosphere.
Impacts
This research 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 and cultural practices for nematode control, and incorporate an Armillaria resistant gene into transgenic plums and eventually peach rootstocks.
Publications
- Horn, R., A.C. Lecouls, A. Callahan, A.Dandekar, L. Garay, P. McCord, W. Howad, H. Chan, I. Verde, K. Ramaswamy, D. Main, S. Jung, L. Georgi, S. Forrest, J. Mook, T. Zhebentyayeva, Y. Yu, H.R. Kim, C. Jesudurai, B. Sosinski, P. Arus, W.V. Baird, D. Parfitt, G. Reighard, R. Scorza, J. Tomkins, R. Wing, and A.G. Abbott. 2005. Candidate gene database and transcript map for peach, a model species for fruit trees. Theor. Appl. Genet. 110:1419-1428.
- Schnabel, 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.
- Bielenberg DG, Wang Y, Reighard GL, and Abbott AG. 2005. New gene candidates for the regulation of winter dormancy in perennial plants. HortScience 40:1109.
- Wells, CE and DR Layne. 2005. Fine root dynamics of peach (Prunus persica) under 2 irrigation regimes. HortScience 40:1038.
- Cox, K. Layne, D. and Schnabel, G. 2005. A lectin gene from a Chinese orchid, Gastrodia elata, confers tolerance to root diseases in transgenic tobacco. Phytopathology 95:S22.
- Layne, D.R., G. Schnabel, K. Cox, R. Scorza, and K. Bussey. 2005. Armillaria root rot of peach: a multipronged approach. HortScience 40(4):1026.
- Tanner, SC, Wells, CE and GL Reighard. 2005. Soil solarization and methyl bromide fumigation alter fine root dynamics at a peach replant site. HortScience 40:1038-1039.
- 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.
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Progress 01/01/04 to 12/31/04
Outputs
We found in addition to six MADS-box genes in the sequenced Evergrowing (evg) gene region, a Ca-binding protein (CaBP). The fragment hybridizing to CaBP was missing in the evg mutant genome when compared to the wild-type genome, suggesting that a large deletion spans this region. The region affected by the apparent deletion also contained two other gene sequences. One was a copia-like retrotransposon, which is not predicted to play a functional role in the dormancy behavior of perennial trees. Another was a large, hypothetical protein that was also predicted in the region. We generated a small-insert genomic library by hydroshearing P. synxantha BG33R genomic DNA into 2000 bp fragments and cloning these into a high-copy vector. This library, which represents a 16X coverage of the BG33R genome, consists of over 50,000 clones and has been stored in 384-well microtiter plates. A library of 4000 transcriptional fusion mutants of BG33R which contain a promotorless xylE Tn5
cassette were screened in a root-exudate study in which root exudates were incorporated in a minimal salts medium. Eleven xylE Tn5 mutants were identified which displayed an altered gene expression level when grown in the presence of root exudates when compared with the same mutant grown in a non-amended medium. In previously engineered transgenic tobacco and plum plants expressing the GAFP gene under the control of the 35S promoter, the expression and translation of GAFP in tobacco and plum transplants was verified using real-time PCR and dot blot analysis. A preliminary in vitro screening was conducted to determine the sensitivity of regional isolates of Armillaria tabescens to propiconazole (Orbit). We found that propiconazole had a strong inhibitory effect on the mycelial growth of A. tabscens. EC50 values for A. tabescens ranged from 0.45 to 0.49 ppm, which is comparable to the current label rate for brown rot control on peach. These results support the application of
reduced-risk fungicides using an intravascular injection method.
Impacts
This research 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 and cultural practices for nematode control, and incorporate an Armillaria resistant gene into transgenic plums and eventually peach rootstocks.
Publications
- No publications reported this period
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