In 2003, a replicated long-term research trial was established on a commercial peach replant site with a history of Armillaria root rot and other soilborne diseases. The objectives of the trial were to determine the short- and long-term effects of preplant fumigation, rootstock, and preplant root dipping with mycorrhizal fungi and beneficial bacteria on tree growth, productivity, and survival. Preplant fumigants included none (control), methyl bromide, Telone II, or Enzone. Rootstocks tested included Guardian, Lovell, and Halford. Root dipping (or not) was with MycorTree. The scion cultivar was Big Red. There were a total of 24 experimental treatment combinations and the trial site comprised more than 1500 trees on 11.5 acres. By 2 years after planting, fumigation with Enzone was disadvantageous when compared with no treatment at all. Enzone-treated blocks had higher tree mortality or were significantly reduced in growth compared to other treatments. Preplant fumigation with Telone II or methyl bromide, however, resulted in reduced tree stunting and phytotoxicity and increased tree growth when compared to the untreated control. After 2 years, 10% of the total trees planted were dead. Half of these were from the Enzone treatment. Enzone does not appear to be a viable preplant fumigation product for South Carolina peach growers, based on this preliminary data. Both Guardian and Halford rootstocks had performance superior to Lovell during the first 2 years. Although Guardian trees were smaller than Halford at the time of planting, by the end of the second growing season, their TCA was not significantly different. There was no benefit to preplant root dipping with MycorTree. Experimental results were not influenced by the location of trees on the site.
Desmond R. Layne and Guido Schnabel
Alexis K. Nagel, Hetal Kalariya and Guido Schnabel
The Gastrodia antifungal protein (GAFP-1) is a monocot mannose-binding lectin found in the Asiatic orchid Gastrodia elata. Transgenic plum (Prunus domestica var. ‘Stanley’) lines (4J and 4I) expressing GAFP-1 exhibit enhanced disease resistance to the stramenopile pathogen Phytophthora cinnamomi and the root-knot nematode Meloidogyne incognita. Rootstocks created from such transgenic lines might be more readily accepted by consumers if it can be shown that foreign gene products are not migrating into a grafted, nontransgenic scion on which fruit is produced. In this study, wild-type (WT) plum tissue was budded onto transgenic plum lines 4J and 4I to create chimeric-grafted trees. Tissues from chimeric-grafted trees were analyzed for gafp-1 transcripts (leaf and root) and protein (leaf, soft shoot, and root) by reverse transcription–polymerase chain reaction and immunodetection, respectively. Transcripts of gafp-1 were detected consistently in the root tissues but not within the leaves of the grafted, WT scions. Similarly, the GAFP-1 lectin was identified within the roots, but not in the soft shoot or leaf tissues of the grafted, WT scions. These results suggest that gafp-1 mRNA and protein are not moving into the WT scion tissues of chimeric-grafted plum trees.
Hetal M. Kalariya, Guido Schnabel, Cesar Petri and Ralph Scorza
The Gastrodia antifungal protein (GAFP-1) is a mannose-binding lectin that can confer increased disease resistance in transgenic tobacco and plum. In all previously generated, transgenic lines, the gene was under the control of the 35SCaMV promoter. In this study, transgenic plum lines were created from seeds derived from open pollination of the cultivar Bluebyrd (BB-OP) with gafp-1 under the control of the polyubiquitin promoter bul409 and evaluated for Phytophthora root rot (PRR) and Root knot nematode (RKN) susceptibility. One of nine transgenic lines synthesizing GAFP-1 exhibited increased tolerance to PRR caused by P. cinnamomi. The same line (BB-OP-1) was also significantly more tolerant to RKN infection caused by Meloidogyne incognita. BB-OP-1 was more resistant to PRR and equally resistant to RKN compared with the cultivar Stanley-derived 4J line, which expresses gafp-1 under the control of the 35SCaMV promoter. GAFP-1 synthesis in BB-OP-1 was not elevated by pathogen infection, suggesting that the bul409 promoter is not inducible in the plum/GAFP-1 system. This study confirms the usefulness of the gafp-1 gene in various cultivars of transgenic plum and establishes that the bul409 promoter is at least equal in effectiveness to the 35SCaMV promoter for gafp-1 expression in transgenic lines of woody plants.
Andrew P. Nyczepir, Alexis K. Nagel and Guido Schnabel
The expression of gastrodianin antifungal protein (GAFP) in a form of its VNF isoform increases tolerance to Phytophthora root rot (Phytophthora cinnamomi) and the root-knot nematode (Meloidogyne incognita) in transgenic plum lines. However, nothing is known about the potential of the GAFP lectin to confer disease resistance to the ring nematode, Mesocriconema xenoplax, in plum. Three transgenic plum lines (4I, 4J, and 5D) expressing gafp-1 under the control of CaMV 35S promoter sequence were evaluated for their response to M. xenoplax in the greenhouse. All plum lines were rated as hosts of M. xenoplax. Among the individual plum lines tested, the number of M. xenoplax per gram of dry roots was lowest in the rhizosphere of transgenic line 5D, intermediate in that of the nontransformed control line, and greatest in line 4J. The results of this study indicate that the comparisons of the final soil densities (Pf) of adult and juvenile M. xenoplax expressed as nematodes per gram of dry roots provide a better measure of the nematode carrying capacity by the tested lines than Pf values referred to as number of M. xenoplax/100 cm3 soil.
Alexis K. Nagel, Guido Schnabel, Cesar Petri and Ralph Scorza
The Gastrodia antifungal protein (GAFP) is a monocot mannose-binding lectin isolated from the Asiatic orchid Gastrodia elata. This lectin has previously been shown to provide increased resistance in transgenic Nicotiana tabacum against taxonomically unrelated root pathogens Phytophthora nicotianae, Rhizoctonia solani, and Meloidogyne incognita, but its potential to confer disease resistance in tree species is not known. Agrobacterium tumefaciens-mediated transformation yielded three gafp-1 expressing plum lines (Prunus domestica) designated 4J, 4I, and 5D. These lines possessed one, two, and four copies of the gafp-1 gene, respectively, as demonstrated by DNA blotting. Lines 4J and 4I were not phenotypically different from the nontransformed control line, but line 5D showed significant divergence in leaf morphology and growth habit. Compared with the inoculated control line, lines 4J and 4I exhibited increased tolerance to Phytophthora root rot (PRR) caused by P. cinnamomi. When inoculated with the root-knot nematode, Meloidogyne incognita, the 4J and 4I lines showed a significantly lower degree of root galling than the inoculated control line. Nematode reproduction, as measured by the presence of egg masses and the number of eggs produced per gram fresh root, was significantly reduced in line 4J compared with the inoculated control line. The results of this study suggest that the expression of gafp-1 in the roots of a woody plant may confer some level of resistance to PRR and root-knot nematode. Long-term field trials will be necessary to confirm this hypothesis.
Desmond R. Layne, Guido Schnabel, Kerik Cox, Ralph Scorza and Karen Bussey
Armillaria root rot (ARR) of peach caused by the soil-borne basidiomycete fungus Armillaria tabescens is causing premature decline and mortality of peach trees on most southeastern U.S. peach farms. Soil inoculum may be present both in former peach orchard sites and on sites that were once in hardwood forest. The fungus is protected under the bark of dead root pieces and may survive up to 100 years at various depths in the soil profile. No commercially available rootstocks are resistant to ARR. Since 2002, we have embarked on a multipronged strategy to develop control options to combat ARR. First, we have two replicated trials on commercial grower replant sites with a history of ARR. Trial 1 compares four preplant fumigation treatments (none, Telone II, methyl bromide, and Enzone), three rootstocks (Lovell, Halford, and Guardian) and preplant root dips with endomycorrhizal fungi. Trial 2 compares the use of raised beds, root collar excavation and preplant root dips. Both trials examine long-term productivity and tree survival. Second, we are examining the use of systemic fungicide injection into infected trees to protect trees around infection foci. Third, we are trying to develop a genetically modified ARR-resistant rootstock. We have inserted the gene encoding the gastrodia antifungal protein (GAFP—a low molecular weight lectin that binds mannose and chitin) from a Chinese orchid into tobacco (model herbaceous system) and plum (model Prunus system). GAFP has antifungal activity against several basidiomycete root rot pathogens. Pathogenicity tests with transformed tobacco plants show enhanced tolerance to several root rot pathogens when compared to nontransformed plants. Transformed plums are being multiplied for pathogenicity tests.