2013 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):
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.
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.
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.
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.
The development of sustainable control strategies for key soil borne and foliear diseases of perennial tree and vine crops is the 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 (Juglans (walnuts) and Prunus (Almond, peach plum)) 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 were generated and screened for CG resistance. Both open pollinated progeny and progeny from directed crosses, using several Juglans species, were found resistant to CG. These genotypes were cloned via in vitro propagation. A number of these clones continue to exhibit disease 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 objective 3. A high level of CG resistance has been identified in one of the Pyrus (pear) species. ARS scientists in Davis, California, are examining the association of specific soilborne microbial communities with almond replant disorder to determine its etiology and 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 and disease problems affecting commonly grown rootstocks. Work on objective 2.2 was continued to examine etiology and characterize graft and pollen transmissible pathogens in addition to new leafroll diseases of grape. ARS scientists identified the causative agent of a new disease of grapevine known as Red Blotch Disease of Grapevine caused by a new virus, Grapevine red blotch-associated virus (GRBaV), proposed to be a member of the family Geminiviridae. 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 objective 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, California, 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.
Determine involvement of Pythium spp. in Prunus replant Disease. Prunus Replant diseases often occurs when stone fruits are grown in soil that had previously been planted with the same or similar plant species and typically leads to reductions in plant growth and crop yield. In an ARS laboratory in Davis, California, multiple pythium spp. were isolated from the root systems of trees in replant sites exhibiting Prunus replant disease. In three greenhouse trials, purified Pythium intermedium, P. irregulare, P. spinosum, and P. ultimum strains were pathogenic, could be re-isolated from their inoculated hosts but not from non-inoculated controls, and therefore were implicated as contributors to the Prunus replant diseases complex. ARS scientists also confirmed pathogenicity among isolates of four Cylindrocarpon macrodydimum strains on Nemaguard peach. Determining the cause of this disease will aid development of cost effective disease control measures.
Isolation and identification of the pathogenic agents which cause Prunus replant disease (PRD). Instances of severe PRD can kill or prevent growth in more than half of the trees in a replanted orchard. Culture-based and culture-independent samplings of bacterial, oomycete and fungal microorganisms were completed from roots of Prunus replant disease-affected and healthy rootstocks in replicated trials. Collectively, these trials revealed associations between Pythium and Cylindrocarpon species and Prunus replant disease incidence. Determining the cause of this disease and discovering resistant rootstocks will benefit the almond and stone fruit industry.
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. To date, A. tumefaciens genetic diversity appears linked to its geographic source which may facilitate forensic investigations of CG outbreaks. Understanding the diversity of A. tumefaciens will enhance our walnut Crown Gall resistance screening and control efforts.
Identification of Prunus genotypes resistant to Phytophthora. Phytophthora spp. are among the most serious soilborne pathogens among many horticultural crops in California, including Prunus spp and walnuts. A greenhouse trial was completed to evaluate genetic resistance of 21 rootstock genotypes to P. niederhauseri (Pnn), a pathogen associated with serious almond tree losses in California. Most of the 21 rootstocks are new to the almond and stone fruit industries, yet little is known about the horticultural attributes of these diverse rootstock parentages or their resistance to soilborne pathogens and all are available to growers. Rootstocks with peach and peach x almond parentage were relatively susceptible to the pathogen, whereas genotypes that included plum parentage were much more resistant. These findings will be valuable to growers as well as rootstock breeders. Developing disease resistance rootstocks allow the industry to reduce the need for ozone depleting soil fumigation.
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 have been shown to segregate for CG resistance. CG resistant individuals are being cloned using in vitro propagation for large scale field testing. These CG resistant genotypes are essential for development of CG resistant rootstocks for the walnut industry.
Characterization of the genetic and epigenetic interactions between Phytophthora ramorum (P. ramorum) and diverse host species. An ARS scientist in Davis, California, demonstrated that P. ramorum isolates originating from bay laurel artificially inoculated into boles of canyon live oak, and re-isolated 4 months later, displayed signs of phenotypic conversion. Furthermore, experimental studies on growth rate revealed re-isolates from oaks evolved significantly faster than those from bay laurels implicating participation of host-induced phenotypic diversification in adaptation. In addition, bioinformatic pipelines for transcriptome and genome analyses were constructed. This epigenetic mediated phenotypic diversification has significant implications for both the evolution and host range of P. ramorum, both of which impact regulatory and quarantine issues in the nursery trade.
Fumigant independent approaches for use in the control of crown gall. Agrobacterium tumefaciens (A. tumefaciens), causative agent of crown gall, is an excellent and long term colonist of soil particles, plant debris, and host and non-host root systems alike. Amendment of vermicompost to A. tumefaciens infested native soils effectively reduced populations of A. tumefaciens below detection limits after a 28 day exposure. As little as a 25% vermicompost mixture was as effective as 100% vermicompost. This fumigation-independent approach to controlling one of the key rootstock infecting pathogens has serious implications for both nursery and orchard production of woody perennial tree crops in the United States.
Identification of the causative agent of Red Blotch Disease of Grapevine. Red Blotch Disease causes significant vineyard losses due to reduced yields and grape quality. ARS scientists in Davis, California, discovered and characterized a new Gemini-like virus, associated with this disease, named Grapevine red blotch-associated virus (GRBaV). Epidemiology of Red Blotch Disease suggests GRBaV exhibits insect mediate transmission. Red Blotch specific DNA primers were developed to detect and quantify this virus. These DNA primers are now widely used by both diagnostic testing services and grapevine virologists around the world including USA, Australia, Canada, France, Italy, New Zealand and South Africa.
Browne, G.T., Lampinen, B.D., Holtz, B.A., Doll, D.A., Upadhyaya, S.K., Schmidt, L.S., Bhat, R.G., Udompetailkul, V., Coates, R.W., Hanson, B.D., Klonsky, K.M., Gao, S., Wang, D., Gillis, M., Gerik, J.S., Johnson, R.A. 2013. Managing the almond and stone fruit replant disease complex with less soil fumigant. California Agriculture. 67(3):128-138.
Fennimore, S.A., Serohijos, R., Samtani, J.B., Ajwa, H.A., Subbarao, K.V., Martin, F.N., Daugovish, O., Legard, D., Browne, G.T., Muramoto, J., Shennan, C., Klonsky, K. 2013. TIF film, substrates and nonfumigant soil disinfestation maintain fruit yields. California Agriculture. 67(3):139-145.