Progress 10/01/03 to 12/31/07
Outputs
OUTPUTS: The range and severity of grape vine decline in Pennsylvania was based on data obtained from 27 vineyard sites including more than 500 acres of vines. Thirty-nine grape cultivars and 223 cultivar blocks were rated for vine decline. Vines within cultivar blocks were scored as healthy, declining, dead or missing, healthy replants, or declining replants. Percent decline was calculated as the sum of declining plants, dead or missing, plus declining replants divided by the total number of plants. Grape vine roots were examined for fungi and other potential causes of vine decline such as Tomato ringspot virus (ToRSV) and Agrobacterium vitis. Combined vine decline across all cultivars rated ranged from 0-87%. ANOVA of the vine decline data showed that vineyard location and age category of vines accounted for significant amounts of total variation in the data set. There was a vine type by age category interaction. The statistical analyses of the data were published in "Wine East",
2004. Three hundred sixty-two fungal isolates from declining vines were preserved for identification. Twenty-nine percent of all fungi identified are those listed as follows and implicated in causing Petri disease: Phaeomoniella chlamydospora, Phaeoacremonium aleophilum, P. mortoniae, P. rubrigenum, and P. viticola. Pathogenicity tests resulted in a distinct dark reddish brown streaking in the vascular tissue considered to be the initial symptoms of Perti disease. Tomato ringspot virus and crown gall are associated with vine decline. Tomato ringspot virus was detected in 23% of the 21 Native American, 37% of 81 French American hybrid, 18% of 61 vinifera cultivar blocks sampled. Crown gall was rampant on grafted vines, especially from Western nurseries. Species and genus specific primers were developed for the Petri disease fungi. The primers have been adopted by labs in South Africa and Australia for classical PCR detection systems and further exploited in developing a real-time PCR
detection method using Sybr green (Overton et al). This project also led to the "Development of a Real-Time RT-PCR SYBR Green assay for Tomato ringspot virus in grape" (Stewart et al, 2007). These highly sensitive diagnostic tools combined with tissue culture regeneration of numerous cultivars served as the core elements in our effort to promote a clean vine program for Eastern viticulture.
PARTICIPANTS: Elwin L. Stewart: Principal Investigator, provided leadership, program direction, and identified goals for all participants, and prepared the grant proposals and reports; Nancy G. Wenner: Senior Research Assistant, provided program technical support, and provided the lead role in grape meristem tip culture; Barrie E. Overton: Post doctoral fellow, provided program support and worked with project team in developing molecular diagnostic tools; Leslie Ann Long: Provided program support and completed Masters of Agriculture degree - Thesis title: Phylogenetic placement and pathogenicity of Phialophora and allied genera from declining grapevines; Xinshun Qu: Collaborator, Department of Plant Pathology Penn State, assisted with primer design for Petri disease fungi and Tomato ring spot virus, and assisted Stewart in preparation of final versions of the Stewart et al manuscript on ToRSV detection; Fred E. Gildow: Collaborator, Department of Plant Pathology Penn. State, ELISA
detection of ToRSV was conducted in the Gildow laboratory; Deborah S. Grove: Collaborator, Huck Institute, Wartik Laboratory, Penn State, provided technical advice to the team on real-time PCR; Barbara J. Christ: Collaborator, Department of Plant Pathology, Penn State, provided access to additional laboratory space and equipment and advice on fungal populations. Pennsylvania Wine Marketing and Research Board: Provided grant funds to support research; Pennsylvania Department of Agriculture: Provided substantial funding in support of the research program; and Viticulture Consortium East: provided partial financial support of the grapevine decline survey.
TARGET AUDIENCES: The results of this project have been made available to the public through print, oral, and electronic media making the information available to peoples of all nations interested in trunk diseases and grapevine decline.
Impacts
Roots, trunks, and cordons from declining grape vines were collected and sectioned in the field to expose internal tissues and facilitate photographic documentation. Grapevine samples were transported to the laboratory for the fungal isolation process. Fungi were identified to genus and species using taxonomic keys and molecular methods. Three hundred sixty-two fungal isolates have been preserved for long term storage over liquid nitrogen. Twenty-nine percent of all fungi isolated and identified, were those associated with Petri disease; either Phaeomoniella chlamydospora or Phaeoacremonium species. Phaeomoniella chlamydospora was isolated from 92% of the sites sampled. Phaeoacremonium aleophilum, P. angustius, P. mortoniae, P. rubrigenum, and P. viticola were recorded collectively from 100% of the sites sampled. Other root and trunk fungi found less frequently were recorded. They include: Acremonium spp., Aureobaisdium pullulans, Botryosphaeria spp., Cadophora spp.,
Cylindrocarpon spp., Fusarium spp., Clonostachys, Lectyophora spp., Phialophora spp., Phomopsis spp., and undetermined basidiomycetes, Coelomycetes, and Hyphomycetes. This project demonstrated that grapevine decline in Pennsylvania is associated with trunk, root and graft union pathogens, tomato ring spot virus (ToRSV), and crown gall (Agrobacterium vitis). It has been estimated that grapevine decline in Pennsylvania accounts for an annual economic loss of $700,000 across 1,300 acres of wine grapes. There are doubtless many other factors involved in grapevine decline, but for the first time in Pennsylvania a science based effort was made to account for some of the causes. The cost estimates of grape vine decline and knowing some of the causes set the stage for a clean vine program for the Eastern US wine grape industry. Using morphology to identify root and trunk inhabiting microbes is slow and subject to mistaken identification so we developed and published two highly sensitive and
accurate molecular based diagnostic protocols for Perti disease fungi and ToRSV: Qualitative real-time PCR SYBR green detection of Petri disease fungi and Development of a real-time RT-PCR SYBR green assay for Tomato ring spot virus in grape. The Pennsylvania Wine Marketing and Research Board provided financial support for developing the diagnostic tools and further supported the research on developing clean vines using hot water therapy and grape meristem tip culture. The tissue culture research was published in the trade magazine, Wine East under the title: Coming clean - grape meristem tip culture. The article was intended to de-mystify the tissue culture of grape and to support a growing interest in a clean vine industry for the Northeast wine industry.
Publications
- Overton, B.E., E.L. Stewart, X. Qu, N.G. Wenner, and B.J. Christ. 2005. Real-time PCR-SYBR Green Detection of Petri Disease and ToRSV. Eastern Division Meeting of the American Enology & Viticulture Society, St. Louis, Missouri, 13-15 July 2005. Am. J. Enol. Vitic. 56(4):417-421 (Abstract).
- Long, L. A. 2004. Phylogenetic placement and pathogenicity of Phialophora and allied genera from declining grapevines. Master of Agriculture Thesis. The Pennsylvania State University, University Park, PA. 47 pp.
- Wenner, N.G. and E.L. Stewart. 2007. Coming clean--Grape meristem tip culture. Wine East May-June:10-23,56-58.
- Stewart, E.L., X. Qu, B.E. Overton, F.E. Gildow, N.G. Wenner, and D.S. Grove. 2007. Development of a real-time RT-PCR SYBR Green Assay for Tomato ring spot virus in grape. Plant Disease 91:1083-1088.
- Long, L.A., E.L. Stewart, N.G. Wenner, and B.E. Overton 2003. Molecular systematics of Phialophora and Phialophora-like fungi from declining grape vines in Pennsylvania and New York. Phytopathology 93(6):S55 (Abstract).
- Stewart, E.L., N.G. Wenner, L.A. Long, and B.E. Overton 2003. Petri and esca disease fungi in declining grapevines in Pennsylvania and New York. Phytopathology 93(6):S81 (Abstract).
- Overton, B.E., E.L. Stewart and G. Samuels. 2003. Hypocrea with effuse stromata and anamorphs in Trichoderma section Hypocreanum. Mycological Society of America/British Mycological Society, Monterey, Calif. Mycologia supplement Vol. 54(3):39 (Abstract).
- Long, L.A., E.L. Stewart, and N.G. Wenner. 2004. Molecular systematics of Phialophora-like fungi from declining grapevines. 3 rd International Workshop on Grapevine Trunk Diseases abstracts. Christchurch, New Zealand, 1-2 February 2003 Phytopathologia Mediterranea 43(1):147 (Abstract).
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Progress 01/01/06 to 12/31/06
Outputs
Roots, trunks, and cordons from declining vines were collected and sectioned in the field to expose internal tissues and facilitate photographic documentation. Grapevine samples were transported to the laboratory for the fungal isolation process. Fungi were identified to genus and species using taxonomic keys and molecular methods. 362 fungal isolates have been preserved for long term storage over liquid nitrogen Twenty-nine percent of all fungi isolated and identified, are those associated with Petri disease. The Petri disease fungi are assigned to two genera named: Phaeomoniella and Phaeoacremonium. Phaeomoniella chlamydospora was isolated from 92% of the sites sampled and represent 12% of all fungi isolated. Phaeoacremonium aleophilum, P. angustius, P. mortoniae, P. cf. rubrigenum, and P viticola were recorded from declining vines. Collectively, the five Phaeoacremonium spp. were isolated from 100% of the sites sampled and represent 17% of all the fungi isolated.
Phialophora-like fungi were isolated from 100% of the sites and represent 24% of all fungi isolated. Cylindrocarpon destructans and undetermined Cylindrocarpon spp. were isolated from 62% of the sampled sites and accounted for 6% of all fungi isolated. Phomopsis was recovered frequently from nearly all of the sampled sites, and was often observed causing shoot dieback, leaf distortion with necrotic lesions, and fruit cluster damage. Field observations indicate that mechanical pruning could leave shoots heavily infected with Phomopsis deep within the canopy. Canes riddled with Phomopsis fruiting bodies, if left within the canopy produce the inoculum for re-infection and can significantly contribute to vine dieback and decline. Numerous other fungi were also isolated and identified including basidiomycetes, Fusarium spp., Clonostachys and Botryosphaeria dothidea. Manganese (Mn) oxidation was tested on Petri disease fungi by using potato dextrose agar amended with Mn sulfate.
Phaeomoniella chlamydospora showed reduced growth on PDA amended with 3000 ppm Mn sulfate and exhibited no growth at higher concentrations. Phaeoacremonium spp. evaluated grew on PDA amended with as much as 6000 ppm Mn sulfate. Stereum hirsutum and Fomitiporia punctata, known white rot basidiomycetes, were used as controls. Stereum hirsutum oxidized Mn sulfate in vitro and grew on PDA amended with 6000 ppm Mn sulfate. Fomitiporia punctata had reduced growth at 300 ppm and no growth at higher concentrations of Mn sulfate with no indication of Mn oxidation. Morphological studies and phylogenetic analyses of DNA sequences from three genomic regions were used to investigate the systematics of Hypocrea citrina and related species. A neotype was designated for H. citrina. Historical information and results from this study provided the data needed for selection of a lectotype specimen from Fries herbarium for H. lactea. Hypocrea pulvinata contain geographical subgroupings between European
and North American collections. Hypocrea citrina var. americana was elevated to species status, H. americana. Hypocrea eucorticioides, H. victoriensis, H. parmastoii, and H. alcalifuscescens were published as new names.
Impacts
The goal of this project has been to develop a functional model system for a "clean vine program" to serve the North Eastern grape and wine industry. Fungi, viruses and bacteria are associated in high numbers with grapevines that decline early in their expected life span which represents a significant economic loss to the industry in what is being termed replacement viticulture in the East. The economic loss to the wine sales industry is also significant because the wine makers lack access to the old vine grape juice from which the premium priced wines are produced. The most frequently isolated fungi, Tomato ringspot virus, and Agrobacterium vitis associated with young and old vine decline in Pennsylvania have now been identified Molecular detection protocols developed in this project provide the means of detecting pathogens in the field and in vines produced from tissue culture. The methods to develop cleaner vines are not complicated and can be adopted by the
nursery industry as economics demand. When this transition occurs the entire wine grape industry will benefit economically. The Hypocrealean fungi contain many economically and ecologically important species. Prior to the efforts of a number of systematics the taxonomy was confused and incomplete. Overton et al. stabilized the identity of H. citrina and established its closest relatives. In addition, several Hypocreanum anamorphs are redescribed and described as new names to science.
Publications
- Overton, B. E., Stewart, E. L. and Wenner, N. G. 2006. Manganese oxidation in Petri disease fungi as a novel taxonomic character. Phytopathologia Mediterranea 45: S131-S134.
- Overton, B.E., Stewart, E. L., Geiser, D. M., and Jaklitsch, W. M. 2006. Systematics of Hypocrea citrina and related taxa. Studies in Mycology 56:1-38.
- Overton, B. E., Stewart, E. L., and Geiser, D. M. 2006. Taxonomy and phylogenetic relationships of nine species of Hypocrea with anamorphs assignable to Trichoderma section Hypocreanum. Studies in Mycology 56:39-65.
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Progress 01/01/05 to 12/31/05
Outputs
Tomato ring spot virus (ToRSV) has been shown to be associated with vine decline in commercial vineyards in the Northeast. The primer pair Torsv2f/2r was designed for one-step real-time reverse transcription (RT)- PCR amplification of ToRSV based on sequences in Genbank from several hosts, including grape. Cuttings from grapevines naturally infected with ToRSV were grown in the greenhouse and tested using real-time RT-PCR and enzyme-linked immunosorbent assay (ELISA). Vines testing positive based on ELISA were also positive based on real-time RT-PCR. The sequences of the PCR products suggested that a unique, highly divergent, Northeastern strain of ToRSV was present in grape from the Northeast. ToRSV from grape in the Pacific Northwest, often referred to as Grape yellow vein virus (GYV), has been shown to have a divergent coat protein gene sequence. The Northeast strain identified in this study differed substantially from GYV. The results of this study established a
one-step, RT-PCR method that does not require a RNA quantification step. A 1:25 dilution of a total RNA preparation was adequate for detection of ToRSV and was more sensitive than ELISA. Two primer-pairs were designed and evaluated in this study. Although both amplified ToRSV, the primer pair ToRSV2f/2R (330-bp) was most efficient and was used for comparison with results obtained from ELISA. Amplification curves for the 330-bp product crossed the cycle threshold setting early (Ct < 24 cycles). The primer-dimer for the 330-bp product was minimal occurring after 36 cycles and easily distinguished from the true product using the Cepheid Smart Cycler melt curve analysis program. The RT-PCR product from ToRSV1f/1r primer pair yielded a 130-bp product, but amplification curves did not cross the cycle threshold until later in the amplification cycle (Ct > 33 cycles). In addition, the melt derivative of the 130-bp product was closer to the melt derivative of the primer-dimer than for the
330-bp product. This is important because the Cepheid Smart Cycler software allows the operator to set the optics to take fluorescence readings in a fourth PCR step above the melt derivative of the primer-dimer, eliminating the spurious fluorescence readings that a primer-dimer will cause in a SYBR Green assay. The closer the melt derivative of the primer-dimer to that of the actual PCR product, as in the case of the 130-bp product (Table 1), the less effective this method is in eliminating false positives caused by dimers. Both primer pairs failed to amplify an isolate of PRMV, a non-target nepovirus. Comparison of test results between ELISA and real-time, RT-PCR with SYBR Green from greenhouse grown vines shown that RT-PCR is more sensitive in early detection of ToRSV. Real-time RT-PCR was able to detect all ELISA positive samples, but ELISA failed to detect all real-time RT-PCR positive samples in 2004. However, ELISA eventually detected ToRSV in sample 16 in 2005.
Impacts
This study provides: (A) a cost effective, one-step, real time, RT-PCR detection assay for ToRSV in grape, (B) RT-PCR products can be sequenced to characterize variation in ToRSV isolates, and (C) evidence establishing divergent strains of ToRSV from grape; all of which are paramount for establishing a virus control program in the Northeast region for ToRSV.
Publications
- Chamberlain, H. L., Rossman A. Y., Stewart, E. L., Ulvinen, T. and Samuels, G. J. 2004. The stipitate species of Hypocrea (Hypocreales, Hypocreaceae) including Podostroma. Karstenia 44:1-24.
- Morton, L., Wenner, N.G., Stewart, E.L. and Overton, B.E. 2005. Fungi isolated from failing graft unions with necrotic lesions. 4th International Workshop on Grapevine Trunk Diseases. Abstracts. Stellenbosch, SA. 20-21 January 2005. P. 24.
- Overton, B. E., Stewart, E. L., Qu, X., Wenner, N. G. and Christ, B. 2004. Qualitative Real-time PCR SYBR Green detection of Petri disease fungi. Phytopathologia Mediterranea. 43:403-410.
- Overton, B.E., Stewart, E. L., Qu, X., N. G. Wenner, N. G., Christ, B.J. and Gildow F.E. Gildow. 2005. Real-time PCR-SYBR Green detection of grapevine decline pathogens. 4th International Workshop on Grapevine Trunk Diseases. Abstracts. Stellenbosch, SA. 20-21 January 2005. P.14.
- Overton, B.E., Stewart, E. L. and N.G. Wenner. 2005. Molecular phylogenetics of grapevine decline fungi from Pennsylvania and New York. 4th International Workshop on Grapevine Trunk Diseases. Abstracts. Stellenbosch, SA. 20-21 January 2005. P.27.
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Progress 01/01/04 to 12/31/04
Outputs
Grapevine decline has gained the attention of grape growers and scientists in the major wine and processing grape regions of the world. Grapevine decline may be caused by numerous biotic and abiotic agents. This project focus is to quantify grapevine decline associated with fungal, viral (tomato ringspot virus), and bacterial (Agrobacterium vitis) pathogens. Twenty-seven vineyard sites including more than 500 acres of vines have been examined. Thirty- nine grape cultivars and 223 cultivar blocks were rated for vine decline. Combined vine decline across all cultivars ranged from 0-87%. An ANOVA if the decline data demonstrated that vineyard location and age category of vines accounted for a significant amount of the total variation. There was also a vine type (vinifera, French American hybrid, Native American) by age category interaction. A means separation test showed significant differences (0.05) between vine types and between own-rooted and grafted vines. The mean
percentage dead or declining vines for vinifera, French American hybrid, and Native American cultivars were 22.615%, 20.488%, and 11.528% respectively. The mean percentage dead or declining for own-rooted and grafted vines was 16.810% and 22.687%, respectively. There was no vine type, root interaction effect on vine decline. Vine decline in general was observed to increase as the vines aged. However, percentage dead and declining vinifera decreased, although, not significantly, between the age categories 0-5, 6-10 and 11-20 years. With vinifera, there was a significant increase from the 11-20 to 21-30 year old age category. The percentage dead and declining French American hybrids increased significantly between the 6-10 and 11-20 year old age category. There was no significant increase in dead and declining vines among the Native American cultivars from the 11-20 to the 50+ year age category. More than 500 fungal isolates from declining vines are being identified using morphology and
molecular techniques. Tomato ring spot virus was detected in 23% of 21 Native American, 37% of 81 French American hybrid, and 18% of 61 vinifera cultivar blocks sampled. Crown gall (Agrobacterium vitis) was observed in high frequency during the survey but was not quantified due to the production of galls below ground and the lack of weed control in many vineyards. Root and trunk fungi, tomato ringspot virus, and crown gall are associated with and likely contribute to vine decline. Real-time PCR probes are being developed to detect fungi, viruses, and bacteria most frequently associated with grapevine decline.
Impacts
This project has produced the first attempt to quantify vine decline in Pennsylvania and New York states due to Petri disease, tomato ringspot virus, and crown gall. Growers more fully understand vine decline, the causes, and cultural practices needed to avoid compromising vine vigor such as: controlling broad leaf weeds, irrigating plantings for a minimum of two to ten years, avoiding over-cropping, balance pruning, avoid pruning cuts more than one inch in diameter, removing prunings from the vineyard, managing foliar pathogens, insects, protecting young graft unions in the winter, maintaining vine nutrition, using acceptable planting methods to avoid J-rooting, and planting vines that are known to be free of internal pathogens. This project is the basis of a 'clean vine' program initiative in the Mid-Atlantic States.
Publications
- Stewart, E.L. and Wenner, N.G. 2004. grapevine decline in Pennsylvania and New York. Wine East (July-August). 12-52.
- Overton, B.E., Stewart, E.L., Qu, Xinshun, Wenner, N.G. and Christ, B.J. 2004. Real-time PCR-SYBR Green detection of Phaeomoniella chlamydospora and Phaeoacremonium spp. Abstract. North East Division, American Phytopathological Society Meeting, State College, PA. October 6-8, 2004. pp. 37.
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