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United States Department of Agriculture

Agricultural Research Service

Research Project: Integrated Strategies for Advanced Management of Fruit, Nut, and Oak Tree Diseases

Location: Crops Pathology and Genetics Research

2012 Annual Report


1a.Objectives (from AD-416):
1: Characterize the etiology, biology, and ecology of key phytopathogenic agents and their interactions with economically important tree and grapevine species.

Subobjective 1.1 - Determine key species of Phytophthora and Pythium contributing to root and crown rot diseases on cultivated Prunus and Juglans species in the Central Valley of California. Subobjective 1.2 - Identify member(s) of soilborne microbial communities that mediate Prunus replant disease. Subobjective 1.3 - Characterize ecology of soil-borne populations of virulent and avirulent Agrobacterium tumefaciens under nursery conditions. Subobjective 1.4 - Determine the agent responsible for grapevine necrotic union disease by characterizing the viromes in clones of Pinot noir and rootstock 110R.

2: Characterize the genetic structure and diversity of key pathogens of economically important tree and grapevine species.

Subobjective 2.1 - Characterize the genetic and phenotypic diversity of Agrobacterium tumefaciens strains occurring in CA production and nursery environments. Subobjective 2.2 - Identify the causal agent of a new leafroll-like disease spreading in California vineyards.

3: Develop cost-effective management strategies for key diseases of economically important tree and grapevine species.

Subobjective 3.1 - Evaluate genetic resistance of almond, peach and walnut rootstock germplasm to Phytophthora species. Subobjective 3.2 - Evaluate genetic resistance of almond and peach rootstock germplasm to the Prunus replant disease complex. Subobjective 3.3 - Evaluate genetic resistance of Juglans species half-sib and interspecific hybrids to Agrobacterium tumefaciens for rootstock development. Subobjective 3.4 - Develop protocols for production of crown-gall-free walnut Paradox hybrid rootstock without reliance on pre-plant soil fumigation with methyl bromide. Subobjective 3.5 - Develop interstocks to avoid walnut blackline disease caused by Cherry leafroll virus. Subobjective 3.6 - Evaluate Vitis vinifera germplasm collection maintained by USDA National Clonal Germplasm Repository and commercially available grapevine rootstocks for resistance to Grapevine leafroll associated virus 3.

4: Characterize the biology, genetics and genomics of Phytophthora ramorum and other oomycetes to facilitate identification of pathogenicity related genes.

Subobjective 4.1 - Characterize genetic and epigenetic interactions between P. ramorum and diverse host species. Subobjective 4.2 - Characterize the mode of action of phosphonate using a transcriptomics approach.


1b.Approach (from AD-416):
OBJECTIVE 1: Subobjective 1.1-Almond and walnut orchards affected by crown and/or root rot in the Central Valley of California will be located and surveyed through consultation with University of California Farm Advisors in each county where the crops are grown on thousands of acres. The number of orchards surveyed in each county will be roughly proportional to the local incidence and severity of crown and root rot losses on the crops of interest; the goal will be to sample intensively enough to detect all aggressive pathogens that are causing high incidence and severity of disease. Subobjective 1.2-Field and greenhouse bioassay experiments will be used to examine PRD-microorganism associations in multiple almond and peach replant soils. Soil in these trials will receive the following treatments: non-treated control; broad spectrum soil disinfestation; and in some cases, semi-selective chemicals. Subobjective 1.3-Examine the following A. tumefaciens population parameters under commercial nursery conditions: 1)proportion of A. tumefaciens population with the Ti-plasmid and, 2)total Agrobacterium population. Subobjective 1.4-Use biological assays to establish the presence of grapevine necrotic union (GNU) agent in the rootstock.

OBJECTIVE 2: Subobjective 2.1-characterize the genetic diversity of A. tumefaciens using three approaches; REP-PCR, fatty acid methyl ester profiles (FAME) and multilocus sequence analysis (MLSA) of 7 housekeeping genes. Subobjective 2.2-Graft transmission into test grapevines. Subobjective 3.1-Predetermined rootstocks will be used to challenge resistance of standard and prospective rootstocks for their respective hosts.

OBJECTIVE 3: Subobjective 3.2-Test standard and prospective almond and peach rootstocks for their resistance to Prunus Replant Disease under orchard and greenhouse conditions. Subobjective 3.3-Open pollinated seeds will be collected, stratified and propagated from each mother tree for evaluation of Crown Gall resistance. Subobjective 3.4-Develop a MeBr independent approach to produce crown gall free Paradox rootstocks under commercial nursery conditions. Subobjective 3.5-Construct a chimeric gene to express hairpin RNA (hRNA) corresponding to the 3'end of RNA-1 and RNA-2. Initiate agro-mediated transformation of a WIP clone 48-12 to express the hRNA. Evaluate transformed WIP clones for Cherry leafroll virus (CLRV) resistance. Subobjective 3.6-Establishment of grapevines with Grapevine leafroll associated virus 3 (GLRaV-3). Graft inoculations and evaluation for resistance/susceptibility.

OBJECTIVE 4: Subobjective 4.1-Study of host-pathogen genetic interactions underlying the broad host range and evaluate host-induced epigenetic alterations. Subobjective 4.2-Determine phosphonate mode of action and drug target mutant screening.


3.Progress Report:
This new project was implemented on May 09, 2012 and replaces former project 5306-22000-014-00D). The development of sustainable control strategies for key soil borne diseases of perennial tree and vine crops is a major focus of our research program, addressing Objectives 1, 2, 3, and 4. Work contributing to objectives 1 and 3 include identification of crown gall (CG) and Phytophthora-resistant host genotypes under both greenhouse and field conditions. Wild walnut species were identified with resistance to CG and Phytophthora crown rot. To identify genetic loci mediating CG resistance, F1 progeny generated from crosses between CG resistant female parents and CG susceptible pollen donors are being screened for CG resistance. A genetically diverse collection of Prunus rootstocks is being examined in 3 geographically distant field trials for resistance to CG and Prunus replant disease as described in obj. 3. ARS scientists in Davis, CA, are examining the association of specific soilborne microbial communities with almond replant disorder to determine its etiology and.
2)characterized soilborne microbial communities associated with the inhibition of CG under field conditions. ARS scientists established new trials to evaluate efficacy of fumigant and non-fumigant based alternatives to pre-plant soil fumigation with methyl bromide for management of orchard replant problems. Work on obj. 2.2 was continued to examine etiology and characterize graft and pollen transmissible pathogens in addition to new leafroll diseases of grape. The sudden oak death pathogen Phytophthora ramorum propagates clonally, yet displays diverse phenotypes due to host-induced epigenetic alteration. Compared with P. ramorum isolates originating from bay laurel, those from oak display irregular colony morphology, reduced virulence, and diverse global gene/transposon expression profiles. This modification of fungal gene expression and transposon activation, as a function of host colonization, is being characterized using deep sequencing as described in obj. 4. Mode of action of phosphonate fungicide, which is effective in slowing infection and canker expansion on Phytophthora-induced diseases, is uncharacterized. ARS scientists in Davis, CA, are examining the molecular mode of action of phosphonate fungicide using a genetically tractable host and pathogen with well-annotated genomes to understand molecular microbial interactions in this system.


4.Accomplishments
1. Determining the involvement of Pythium spp. in Prunus Replant Disease. Prunus Replant diseases often occur when stone fruits are grown in soil that had previously been planted with the same or similar plant species and typically lead to reductions in plant growth, crop yield and production duration. In an ARS laboratory in Davis, CA, pythium spp. were isolated from orchard replant sites using a peach seedling bioassay and direct isolation from root systems of orchard trees affected by Prunus replant disease. rDNA sequences were used to identify many of the isolates, which will be tested for pathogenicity. Determining the cause of this disease will aid development of cost effective control measures for use by the grower.

2. Isolation and identification of the pathogenic agents which cause Prunus replant disease (PRD). PRD causes severe growth suppression in almond and stone fruit orchards planted after one another. Instances of severe PRD can kill or prevent growth in more than half of the trees in a replanted orchard. Culture-based isolations and rDNA-based identifications were completed for organisms associated with Prunus replant disease in a trial evaluating resistance of rootstocks to the complex. Determining the cause of this disease and discovering resistant rootstocks will benefit the almond and stone fruit industry.

3. Establishing a representative collection of wild-type Agrobacterium tumefaciens virulent on walnut. Each year the walnut industry of California suffers significant yield and tree losses due to crown gall caused by the soil-borne bacterium Agrobacterium tumefaciens. ARS scientists at Davis, California, continue to expand and characterize the diversity of the collection of virulent A. tumefaciens isolates from the walnut production regions of California. Understanding the diversity of A. tumefaciens will enhance our walnut Crown Gall resistance screening efforts.

4. Identification of walnut genotypes resistant to Phytophthora. Phytophthora spp. are among the most serious soilborne pathogens among many horticultural crops in California, including walnuts. Three experiments evaluating the resistance of walnut (Juglans microcarpa) clones to Phytophthora cinnamomi and P. citricola were completed. Developing disease resistance rootstocks allow the industry to reduce the need for ozone depleting soil fumigation.

5. Identification of walnut genotypes resistant to crown gall (CG). Crown gall is a disease that saps young trees and triggers root-destructive diseases in walnuts. ARS scientists in Davis, California, collected "open pollinated nuts" from selected walnut mother trees which were then germinated and cultivated in the greenhouse and screened for CG resistance. Directed crosses have been made with selected walnut mother trees with pollen from English cv Serr. Progeny from these crosses will be harvested this fall and tested for crown gall resistance. This work is essential to the development of CG resistant rootstocks for the walnut industry.


Last Modified: 8/22/2014
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