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

Outputs
OUTPUTS: We initiated four research programs to find management strategies for ARR control. Two of them, the root collar excavation and the use of Trichoderma, are long-term field experiments that are still ongoing and results are expected within the next 2 growing seasons. Two other programs, including the development and characterization of transgenic plum lines and targeted chemical treatment of trees via trunk injections, were short term projects and research results were obtained. The Gastrodia anti-fungal protein (GAFP) is a monocot mannose-binding lectin isolated from the Asiatic orchid Gastrodia elata. This lectin has provided increased disease resistance in transgenic tobacco against root diseases from different phylogenetic lineages, 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 var. Stanley) designated 4J, 4I, and 5D. These lines possessed one, two, and four copies of the GAFP-1 gene, respectively, as demonstrated by southern analysis. 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 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 lesser degree of galling than the inoculated control line. Nematode reproduction in these lines, as measured by eggs produced per gram root tissue, was not significantly different from the control line (P = 0.05). The 5D line did not demonstrate increased tolerance to either pathogen. Intravascular trunk infusion of propiconazole has been associated with beneficial effects on Armillaria root rot control in Prunus sp. but its basipetal movement has not been verified. Propiconazole, a sterol demethylation inhibitor fungicide, was more effective in inhibiting mycelial growth of Armillaria tabescens isolates in vitro (average EC50 value of 0.6 ppm) compared to fungicides from five other chemical classes (EC50 values ranging from 4.6 to >1000 ppm). The fungicide was infused into the vascular system of peach trees in the spring, summer, and fall of 2005 and 2006. Propiconazole concentration was determined using gas chromatography mass spectrometry in trunk sections above and below the infusion site and in primary roots. Over two experimental years, spring and fall infusions resulted in consistent propiconazole accumulations in primary peach roots. Spring infusions yielded propiconazole concentrations of 1.7 ppm in 2005 and 5.6 ppm in 2006, whereas the highest accumulations were detected following fall infusions with 9.2 ppm in 2005 and 6.7 ppm in 2006. Propiconazole was also consistently detected in trunk sections collected from above and below the infusion site. PARTICIPANTS: Dr. Ralph Scorza at the USDA station in Kearneysville conducted the plum transformation for us Dr. Achour Amiri is a postdoctoral scientist in my lab and conducted the injection study TARGET AUDIENCES: The entire stone fruit industry will benefit from this project. For example, Armillaria root rot is a major problem for SC and GA peach growers, MI and IL cherry growers. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The results of this study suggest that the expression of GAFP-1 in the roots of a woody plant may confer some resistance to PRR and root-knot nematode disease. This is a significant finding because rootstocks resistant to PRR are commercially not available. This genetically engineered rootstock may be the first of a line of rootstocks with resistance to ARR as well. Long term field trials will be necessary to confirm this hypothesis. Management of the disease in existing orchards is a second priority of our research. The basipetal movement of propiconazole in peach trees and its inhibitory activity against A. tabescens in vitro suggest that propiconazole infusion could be useful for targeted Armillaria root rot management. In other words, injecting propiconazole into healthy trees neighboring diseased trees may be a solution to limit the spread of the disease. This approach does not require large amounts of chemicals and is therefore environmentally much friendlier than fumigation or chemical drenches. It cannot be mechanized, however, and therefore will require some manual labor. In the future, we believe that an integrated approach of infusion, biological control, cultural method and rootstock selection will be a solution for ARR management.

Publications

• Amiri, A., Bussey, K. E., Riley, M. B., Schnabel, G. 2008. Propiconazole inhibits Armillaria tabescens in vitro and translocates into peach roots following trunk infusion. Plant Dis. 92:1293-1298.
• Nagel, A.K., Schnabel, G., Petri, C. and Scorza, R. 2008. Generation and characterization of transgenic plum lines expressing the Gastrodia antifungal protein. HortScience 43:1514-1521.

Progress 08/01/06 to 07/31/07

Outputs
We have established several field trials to investigate root collar excavation and biocontrol agents for Armillaria root rot (ARR) control. Each trial is repeated twice at different locations with different soil-types. At each location, the experiments were conducted with at least 30 single tree replicates per treatment. First results are expected in 2008. Intravascular trunk infusion of propiconazole was investigated for ARR control. Propiconazole, a demethylation inhibitor (DMI), was more effective in inhibiting Armillaria tabescens isolates growth in vitro with an average EC50 value of 0.6 ug/ml compared to fungicides from five other chemical classes (with EC50 values ranging from 4.6 to >1000 ug/ml). The fungicide was infused into the vascular system of peach trees in the spring, summer, and fall of 2005 and 2006. Propiconazole concentration was determined using gas chromatography mass spectrometry (GC-MS) in sections above and below the infusion site and in primary roots. Trunk sections and roots were harvested 48 h after treatment and stored at -20 oC until analyzed. Results from two consecutive years showed consistent and significant propiconazole accumulation in roots after fall infusions (9.2 and 6.7 ug/g tissue in 2005 and 2006, respectively). Propiconazole was also consistently detected in trunk sections collected from both above and below the infusion site. Another aspect for ARR control is the creation of transgenic, disease resistant rootstocks. Using Agrobacterium tumefaciens-mediated transformation, we obtained four transgenic plum (Prunus domestica) lines expressing gastrodia antifungal protein (GAFP); three of which (lines 4J, 4I, and 5D) were used for further analysis. All transgenic lines produced GAFP in root and leaf tissue, 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 but not 5D exhibited increased resistance to Phytophthora root rot, caused by P. cinnamomi, and the root-knot nematode, Meloidogyne incognita. All transgenic lines were susceptible to white rot caused by Sclerotium rolfsii and the ring nematode Criconemoides xenoplax.

Impacts
An integrated strategy to control ARR is necessary to sustain peach production in southeastern peach orchards. We are investigating cultural, chemical, biological and biotechnology-based methods that in the future will be the pillars of an integrated pest management strategy. The documentation of basipetal movement of propiconazole following fall injection in peach trees along with its inhibitory activity against A. tabescens in vitro constitute a strong basis for the future potential application of propiconazole infusion for targeted ARR management. Also, we document the first evidence that a plant lectin such as GAFP has the potential to confer increased levels of resistance to agriculturally important plant pathogens in a tree species. First results from our field trials are expected in 2008.

Publications

• Nagel, Dominy and Schnabel 2007. Understanding GAFP: A Unique Defense Lectin with Broad-Spectrum Inhibitory Activity.
• 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. 2006. Management options for Armillaria root rot control in the southeastern United States. Mitt. Biol. Bundesanst. Land- Forstwirtsch. 400: p. 130.

Progress 08/01/05 to 07/31/06

Outputs
At two grower sites a novel cultural system called the Above-Surface Root Collar system is being investigated for Armillaria root rot control. One-year-old trees were planted in an orchard where trees had recently declined from Armillaria root rot. The trees were planted 18 inches above the soil line in open bottom 27 x 18 inches pots made out of black fabric (SMART POT). Treatments included (i) the gower standard, (ii) trees permanently planted in smartpots, and (iii) trees temporarily planted in smartpots. Soil of treatment 3 will be removed from the root collar after one year of establishment. A research trial was also established on a commercial peach replant site with a history of Armillaria root rot 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. By 2 years after planting, 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. Both Guardian and Halford rootstocks had performance superior to Lovell. There was no benefit to preplant root dipping with MycorTree. Experimental results were not influenced by the location of trees on the site. The efficacies of nine fungicides from six chemical classes were evaluated in vitro against two Armillaria tabescens isolates to select an effective fungicide for injection experiments. Propiconazole was the strongest inhibitor 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 at three times during the growing season. Distribution of propiconazole two days post-infusion was determined by GC-MS analysis. Following spring and summer applications, propiconazole was found up to 5 cm below the infusion sites, and there was strong evidence of propiconazole transport to the root system in the fall. Research on the improvement of transformation systems for hypocotyls slices, immature cotyledons and peach leaves is being undertaken with the aim of transferring the Gastrodia Anti-fungal Protein (GAFP) gene into peach. In order to improve the regeneration system different basal media, gelling agents, growth regulators, dark induction timing, ethylene inhibitors [Silver thiosulphate (STS), AVG] and different polyamines are being tested. Preliminary results show that QL salts increased regeneration rate compare with MS salts. Also STS increased regeneration reaching 33.4 and 34 % from hypocotyls slices of cv. Lovell and cv. Bounty, respectively. We are also developing new vectors to improve the efficacy of GAFP protein. We have put the GAFP gene under control of the Ubiquitin 1 promoter. This plant promoter seems to be an inducible promoter and is stronger expressed in roots than in other plant tissues.

Impacts
Armillaria root rot has developed to the most important cause of peach tree decline in the southeastern United States. Virtually all growers are hit by this disease every year due to the widespread occurrence of this pathogen and its persistence in the soil. Trees flush out normally in the spring and suddenly collapse when the fungus girdles the crown and cuts off the water supply. Management options are currently non-existent. This project will develop a much needed integrated management approach for Armillaria root rot in commercial peach orchards to ensure sustainability of peach production in the southeastern United States.

Publications

• 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.