Progress 05/09/12 to 05/08/17
Outputs
Progress Report 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. 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. This is the final report for this project, 2032-22000-015-00D, �Integrated Strategies for Advanced Management of Fruit, Nut, and Oak Tree Diseases� which expired in May of 2017. Please see the report for the new project, 2032-22000-016-00D, �Integrated Disease Management Strategies for Woody Perennial Species,� for further information. Substantial results were realized over the five years of the project. Cumulative progress for each of the five scientists supported on this project are presented in five sections below: 1. California produces 99 percent of the English walnuts produced in North America with nearly all production occurring on trees growing on rootstocks genetically distinct from the nut bearing part of the tree. To date, the available rootstocks have served the industry well but walnut growers and nursery operators do experience yield and tree losses due to soil borne pests and diseases. One estimate puts annual walnut orchard/ nursery losses from key soil borne pathogens such as lesion nematode, Phytophthora spp, Armillaria, and Agrobacterium tumefaciens (crown gall) at roughly 18 percent which translates into an annual loss of greater than $150 million. Currently, walnut growers have a genetically limited array of three rootstock choices. Even though these rootstock genotypes have performed well for the industry, collectively they offer little protection against crown gall, Armillaria and, with one or two exceptions, lesion nematodes and Phytophthora crown and root rot. Since there are limited post-plant options to control these soil borne diseases/pathogens, the best way to manage these diseases/pathogens is to develop rootstocks which are tolerant or resistant to them. As a first step, ARS scientists at Davis, California, exploited the walnut germplasm collection maintained by the USDA ARS National Clonal Germplasm Repository in Davis, California, which contains about 800 walnut genotypes representing about 14 Juglans species collected from around the world. Open pollinated (OP) seeds obtained from mother trees in this collection were propagated and screened for resistance to crown gall, and on a more limited initial scale, resistance to Phytophthora and lesion nematodes. The result of this work was the identification of several mother trees that produced a high incidence of OP seeds resistant to one or more of the key soil borne pathogens mentioned above. Recently we narrowed our focus to Juglans microcarpa mother trees since this species produced a higher percentage of disease resistant OP progeny than other Juglans species. In addition, given their natural range across the arid southwestern U.S., we anticipate this species may contribute useful drought tolerant traits into our rootstock breeding efforts. Several of these putative disease resistant OP Juglans microcarpa progeny have been cloned and are now in field trials to assess their commercial potential. As mentioned above, our goal is to not only identify useful disease- resistant rootstocks, but also to locate and identify the walnut genes which control disease resistance. Towards that end, we crossed the most promising Juglans microcarpa mother trees with pollen from Juglans regia to generate a family of hybrid offspring (a breeding population), which we are using to map and identify the genes for disease resistance. To facilitate the gene mapping, we sequenced the entire genome of both parents of this cross (i.e. Juglans microcarpa and Juglans regia). We now have the complete genomic DNA sequence of both parents, and understand their basic chromosomal organization. For the first time ever, in collaboration with our University of California and ARS collaborators at Davis, California, we identified the genetic regions in one or both parents that were passed along to their offspring and contributed to disease resistance in a given member of the breeding population. This level of genetic knowledge and sophistication has never before been possible in the walnut-pathogen system. Also, disease resistance screening data from evaluation of the hybrid walnut clones (breeding population) for resistance to fungal and bacterial pathogens was analyzed and used to select 20 elite clonal genotypes. Five of these �elite� disease resistant genotypes have been clonally propagated and placed in 5 large field trials where they are being examined for both horticultural and disease resistance traits across the state of California. 2. Given its limited genetic diversity, it is not understood how Phytophthora ramorum, the invasive cause of Sudden Oak Death, adapts to a changing environment and shows significant variation in colony morphology and virulence in host plants. We demonstrated that these variations were associated with originating plant host species, i.e. Phythophythora ramorum isolates from oaks and those from California bays are distinct. Furthermore, we demonstrated that when California bay isolates were inoculated and recovered from oak trunks, re-isolates displayed phenotypes associated with isolates from oaks. We subsequently demonstrated that genome rearrangement was induced inside oak trunks, which resulted in dramatic changes in Phythophythora ramorum. In order to improve our data analysis, we improved and revised the Phythophythora ramorum genome sequence. As a result, previously undetected structural variation of the genome was revealed, which enabled us to more carefully examine the mechanism of host-induced genome alteration. The above mentioned phenomenon is a previously undescribed host-pathogen interaction which may provide insight into management strategies of the disease. Recently, it came to our attention that not only in oaks but also in ornamental shrubs such as Rhododendrons, Phythophythora ramorum can convert its phenotype. We will investigate phenotypic conversions of Phythophythora ramorum on diverse host species to understand the biological implication and genetic mechanisms of the phenomenon in the new research project. 3. Potential bacterial, fungal, and oomycete contributors to Prunus replant disease (PRD) have been identified using culture-based approaches and high-throughput sequencing of key genes to identify members of each group. We carried out these analyses in both field and greenhouse-based studies of PRD. In these studies, the microbiomes of roots and root- associated soils of trees with and without symptoms of PRD were compared. Synthesis of the data across field and greenhouse studies identified microorganisms (bacteria, fungi and oomycetes) consistently associated with symptomatic trees. 4. Research on non-fumigant approaches for management of almond replant problems was continued. Third-year tree growth and nut yield evaluations were completed for our two earliest almond replant trials that were established to test anaerobic soil disinfestation (ASD) as an alternative to soil fumigation for control of Prunus replant disease. These initial almond replant trials had used rice bran as a carbon source for ASD, despite the material�s relatively high expense, due to its proven value in ASD treatments for control of soilborne strawberry diseases. Five new replicated almond orchard replant trials were established in 2017 for optimization of anaerobic soil disinfestation; the trials include evaluations of: nine different ASD carbon sources (of which several are much less expensive and much more widely available than rice bran); examinations of ASD interaction with the process of whole orchard recycling (i.e., which involves grinding up old orchards in place and using the wood residues to potentially improve soil health); factorial evaluation of each ASD component (carbon source, water, tarp); and use of commercial ASD application systems (e.g., substrate spreaders, drip lines, tarp layers). Soil measurements of temperature, redox potential, and Pythium ultimum survival were monitored during ASD treatments. The trials were planted in spring 2017, and initial growth responses and microbial monitoring in the soil and roots of the treated plots is occurring. We continue to use high-throughput sequencing technologies to examine etiology of Prunus replant disease and lethal paradox canker. During the life or our newly established research project �Integrated Disease Management Strategies for Woody Perennial Species�, we anticipate that the bioinformatic analyses of the metagenomic and metatranscriptomic data will lead to the identification of the biological agent/community which causes Prunus replant disease and lethal Paradox canker of walnut. 5. We expanded our efforts to gain insights into the epidemiology of Grapevine red blotch virus (GRBV) which has drawn attention from wine grape producers around the country because of its adverse effect on wine quality resulting in reduced revenue. The virus was not detected in weed plant species in the vicinity of vineyards. It thus appears to be limited to the Vitis species. We continue to monitor populations of treehoppers (family Membracidae) in vineyards in collaboration with University of California researchers. The three-cornered alfalfa hopper and Tortistilus species are the most prevalent membracids in vineyards with red blotch disease. However, these two groups of insects are not present in the same vineyards and it appears three-cornered alfalfa hopper is more prevelant in the California central valley while Tortsitilus spp. occurs in the mountainous region. Their role in GRBV epidemiology is being studied in our newly established research project �Integrated Disease Management Strategies for Woody Perennial Species.� Accomplishments 01 Identification of crown gall resistant walnut rootstock genotypes. ARS researchers at Davis, California, identified walnut genotypes which exhibit a high level of resistance to Agrobacterium tumefaciens which causes crown gall disease. We crossed promising Juglans microcarpa mother trees with pollen from Juglans regia to generate a family of hybrid offspring (a breeding population) which we used to map genetic loci which mediate disease resistance. To accomplish this goal, we also sequenced the entire genome of both parents of this cross. Now that we have the complete genomic DNA sequence of both parents, and understand their basic chromosomal organization, we are ready to interpret how offspring of these parents respond to pathogens of interest. For the first time ever, we identified a region of chromosome 11 in the walnut genome which imparts crown gall resistance. 02 Characterization of the optimum carbon source for use in Anaerobic Soil Disinfestation (ASD). ARS researchers at Davis, California, screened (in vitro) approximately eighteen different carbon sources derived from agricultural wastes and identified the five most effective sources for use in ASD trials. Given the elimination of methyl bromide and increasing regulatory pressure on other fumigants, chemical-independent soil fumigation approaches are needed. ASD is one such approach showing promise across the country. The top five carbon sources were tested and found to be effective in controlling the bacterial pathogen that causes crown gall under field conditions. ASD using these carbon sources may provide a reliable and cost effective alternative to conventional soil fumigation. 03 Characterization of soil microbial communities responsive to anaerobic soil disinfestation (ASD). ARS researchers at Davis, California, are optimizing ASD for control of soil-borne pathogens in nurseries and orchards. A key factor in ASD is the carbon source, which should be easily applied by growers, readily available, inexpensive, and suppressive to a broad range of pathogens and pests. We field tested the performance of four previously identified �elite� carbon sources, which substantially reduced populations of known pathogens and sustained anaerobic conditions in in vitro ASD bioassays. Results from replicated field trials indicate different carbon sources stimulate proliferation of native bacteria with the same broad metabolic capabilities, but not always the same species. This is an important observation because microbial metabolites contribute to pathogen suppression in ASD. Functional redundancy in the communities under different carbon sources suggest ASD can be implemented with diverse carbon sources, which should aid in its local adoption in a variety of tree crops, soil types, and beyond. 04 Characterization of genome aberrations in Phytophthora ramorum. ARS researchers at Davis, California, previously discovered �host-induced genome alteration,� an undocumented plant-microbe interaction in which the genome of the pathogen is altered in the host plant. Characterizing the phenomenon has, however, been hampered by the fragmented reference genome assembled in 2006 in which DNA sequence of as much as 20 percent of the genome is not available. A combination of PacBio technology and Illumina sequencing enabled us to obtain most of the missing regions. It became clear that the missing regions were comprised of highly repetitive DNA sequences. These regions are hypervariable and probably participate in the rapid genome rearrangement in the pathogen. Such information is useful for monitoring pathogen populations and managing the disease. 05 Identification of vetchas the reproductive host of the three cornered alfalfa hopper. In the previous year, the three cornered alfalfa hopper, a minor pest of grapevines and rather obscure in vineyards, was found capable of transmitting Grapevine red blotch-associated virus (renamed recently as Grapevine red blotch virus [GRBV]) between grapevines. In an effort to understand the ecobiology of this vector, ARS scientists in Davis, California, found that vetch served as a reproductive host in the greenhouse and also in vineyards. The epidemiological studies demonstrated the significance of vetch in the spread of GRBV under vineyard conditions. This information is important in developing integrated pest management (IPM) guidelines for management of GRBV using cover crops which do not serve as reproductive hosts for the three cornered alfalfa hopper. 06 Anaerobic soil disinfestation is an effective soil treatment to reduce Prunus replant disease (PRD). Prunus replant disease is an incompletely characterized growth and yield suppression that occurs in Prunus planted after Prunus (including almond and other stone fruits). Although preplant soil fumigation can prevent the disease, human and environmental health concerns are providing incentives for development of non-fumigant approaches for management of replant problems. ARS researchers at Davis, California, determined that anaerobic soil disinfestation (ASD) based on a rice bran carbon source was as effective as optimal preplant soil fumigation for control of PRD, as measured not only by tree growth parameters, but also as measured by first-year nut yields. These were the first orchard assessments of ASD carried through examinations of early nut production for control of PRD. The findings indicate that further development of ASD for orchard replant remediation is promising and justified. 07 Identification of microbial communities on the roots of trees grown in non Prunus replant disease (PRD) soils. Non-fumigant treatments are being developed that may replace some of the need for soil fumigation, but a better understanding of the soil microbiology associated with PRD remediation is needed for further optimization of non-fumigant approaches. ARS scientists at Davis, California, periodically extracted total DNA from roots of almond trees in orchard replant trials and used it to create and examine diagnostic amplicon libraries from bacterial, fungal, and oomycete communities on roots of PRD-affected trees in control plots, healthy trees in fumigated plots, and healthy trees in anaerobically disinfested plots (a non-fumigant approach to PRD management). Bioinformatic examinations revealed microorganisms that may mediate PRD as well as microorganisms that respond differentially to soil fumigation and anaerobic disinfestation. It is anticipated that these results will facilitate further optimization of non-fumigant approaches to PRD control. 08 Examination of rootstock microbial communities on trees grown in Prunus replant diseased (PRD) soils. Integrated pest management strategies, which minimize unnecessary pesticide usage while achieving economical pest control, can benefit from assays that monitor target pests or pathogens. Predictive assays are needed for Prunus replant disease, a Prunus-specific growth suppression that affects successive plantings of stone fruits and nuts, such as almond, and that is prevented by soil fumigation. ARS scientists at Davis, California, conducted bioassays using a PRD-sensitive Prunus rootstock, Nemaguard, planted in fumigated, pasteurized, and non-treated portions of PRD-inducing and non-inducing replant soils from many locations in the Central Valley of California. Total DNA was extracted from the Nemaguard roots to examine amplicon libraries from bacterial, fungal, and oomycete communities in PRD- inducing and non-inducing soils. Bioinformatic examinations of the libraries revealed microorganisms associated with PRD induction, and these findings will facilitate further development of predictive assays for PRD. 09 Identification of novel walnut rootstock genotypes resistant to Phytophthora. New walnut rootstocks are needed to reduce walnut orchard tree losses due to Phytophthora crown and root rot diseases. ARS researchers at Davis, California, working in collaboration with scientists at the University of California, Davis, screened hundreds of candidate walnut rootstock hybrids and identified unique genotypes with resistance to the two most aggressive Phytophthora pathogens that affect walnut in California, Phytophthora cinnamomi and Phytophthora citricola. Elite selections from the Phytophthora resistant genotypes have been propagated and placed in five rootstock field trials in the Central Valley of California. The putatively resistant clones, some of which also offered resistance to crown gall disease, may help to reduce dependence on soil fumigation while fostering sustainability and efficiency of walnut production in the U.S.
Impacts
(N/A)
Publications
- Wallis, C.M., Sudarshana, M.R. 2016. Effects of grapevine red blotch- associated virus (GRBaV) infection on foliar metabolism of grapevines. Canadian Journal of Plant Pathology. 38:358-366.
- Strauss, S.L., Greenhut, R.F., Mcclean, A.E., Kluepfel, D.A. 2017. Effect of anaerobic soil disinfestation on the bacterial community and key soilborne phytopathogenic agents under walnut tree-crop nursery conditions. Plant and Soil. 415:493-506. doi:10.1007/s11104-016-326.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report 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. 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. ARS scientists in Davis, California, continued development of sustainable control strategies for key soil borne and foliar diseases of perennial fruit and nut tree, vine crops and oak trees (Objectives 1, 2, 3, and 4). Orchard and greenhouse trials were conducted to characterize causes of root and crown rots and Prunus Replant Disease (PRD) (objective 1). High- throughput rDNA sequencing was used to examine microbial root communities associated with PRD. Metagenomic and meta transcriptomic examinations were conducted to determine the cause of an emerging crown rot of walnut, �Paradox canker disease�. We also made substantial progress on understanding the biology and epidemiology of the newly discovered Grapevine red blotch-associated virus (GRBaV) (obj. 2). GRBaV infection is associated with poor wine quality resulting in huge economic losses to premium wine grape producers in California. In addition to Napa County, we documented virus spread in Amador and Santa Barbara counties based on the clustering pattern of diseased grapevines. GRBaV also was found in wild grapevines in riparian areas close to vineyards. Among the hemipteran insect species collected and evaluated, three cornered alfalfa hopper (Spissistilus festinus, Family: Membracidae), a minor pest of grapevines, was demonstrated to transmit the virus between grapevines under laboratory conditions. Infestation of other membracid species in vineyards affected by red blotch also has been observed. Our continued monitoring of a vineyard with limited spread of GRBaV indicated rogueing of infected grapevines is a sustainable option to manage red blotch disease. Establishment of the etiology of Grapevine necrotic union disease continues to be a challenge (Subobjective 1.4). High-throughput sequencing and bioinformatic analysis of dsRNA fractions isolated from grapevines affected by the disease, performed at three different times, has not identified any causative agents thus far. We are now moving on to evaluation of the small RNA profile from diseased grapevines. Our work on cost effective disease management strategies (objective 3.3) focuses on selection of disease-resistant rootstocks and development of fumigation-free pre-plant soil treatments for orchards and nurseries. We continue to advance our work on the identification and characterization of Crown gall- (CG) and Phytophthora-resistant host genotypes of Juglans (walnuts) and Prunus (almond, peach plum). Five elite disease resistant walnut genotypes have been commercially propagated and placed into three large-scale field trials across the state of California. To facilitate identification of genetic loci mediating CG and Phytophthora resistance, three hundred interspecific progeny from two crosses between a wild Juglans species and English walnut, (Juglans regia) were generated. The embryo was rescued from each of these 600 hybrid seeds and then used to generate 50 clonal copies of each hybrid. These plantlets have now entered both our genotype-by-sequencing and disease resistance screening pipelines. There is an urgent need to develop chemical independent �soil fumigation� approaches to control soil-borne plant pathogens and pests. We are in the second year of field trials designed to evaluate efficacy of the non- fumigant based alternative, Anaerobic Soil Disinfestation (ASD) for management of PRD and key soil borne diseases affecting walnut nursery stock (Objective 3). At a depth of 18 inches, ASD reduced populations of key soil-borne pathogens below detection limits while suppressing their populations by 90% at a depth of 30 inches. In repeated almond orchard replant trials, pre-plant ASD was confirmed to be as effective as pre- plant soil fumigation for control of PRD, and reduced-cost ASD treatments were determined provide adequate PRD control. In order to carefully examine the mechanisms which arbitrate ASD �mediated control of soil borne pathogens we developed an in vitro ASD mesocosm bioassay. This in vitro system has now been validated and used to test and identify additional and more cost effective carbon sources required for ASD. In addition, the mesocosm system is being used to examine the involvement of volatile organic carbons in ASD-induced pathogen kill. This work addresses objectives 1.2 and 3.4. It is not well understood how the sudden oak death pathogen (Phytophthora ramorum) thrives and changes the California coastal ecosystems. To assess adaptability of this invasive pathogen to different host environments, we evaluated the genetic and phenotypic changes in Phytophthora ramorum isolated from different plant host species. Genome sequences of 60 representative isolates from diverse host species have been completed, and genome alterations and accompanying phenotypic changes were characterized. In order to improve the resolution of genome analysis, we re-sequenced Phytophthora ramorum using the PacBio single molecule, real-time technology and developed new assembly workflow systems with combinations of error correction protocols to produce the optimal assembly. Accomplishments 01 Three cornered alfalfa hopper mediated transmission of the Grapevine Red Blotch-associated Virus (GRBaV). Spissistilus festinus is a minor pest of grapevines that causes girdling of petioles on young shoots of grapevines. This past year ARS researchers in Davis, California, discovered that the three cornered alfalfa hopper is capable of transmitting GRBaV from infected to healthy grapevines. The discovery of GRBaV insect transmission helped describe the documented spread of red blotch disease through vineyards. Identification of the insect vector mediating GRBaV transmission was the last remaining data point needed to begin work on the development of effective red blotch disease control measures, a key concern to the wine grape industry. 02 Genome evolution in Phytophthora ramorum. ARS researchers in Davis, California, previously discovered �host-induced aneuploidy�, an undocumented plant-microbe interaction in which the genome of the pathogen is altered by the host plant species. Characterizing this phenomenon has been challenging given the fragmented Phytophthora ramorum reference genome which was assembled in 2004 used only 80% of the genome which was available at the time. By combining Pacific Biosciences� sequencing technology and Illumina sequencing, we obtained DNA sequence data of the previously missing genomic regions. With the new genome assembly, genetic variants in the pathogen are being identified at high resolution which enables examination of pathogen evolution in native settings and production nurseries. The new assembly is a useful asset which facilitates investigations into the epidemiology and genome evolution of the sudden oak death pathogen and other oomycete pathogens. 03 Fumigant independent approaches in the control of soil borne plant pathogens. Regulatory and environmental concerns are reducing and restricting the use of harsh preplant soil fumigants. ARS researchers in Davis, California, are exploring the utility of Anaerobic Soil Disinfestation (ASD) as a chemical-independent method to control soil borne pathogens. These efforts resulted in the development and validation of an in vitro ASD bioassay system to examine the molecular mechanisms which facilitate ASD-mediated control of soil borne plant pathogens. This system allows researchers to rapidly examine, in a cost effective manor, the impact of such biotic and abiotic factors as, irrigation rates, novel carbon sources and, added complex microbial communities on ASD effectiveness, etc. This in vitro ASD bioassay system facilitates development of enhanced ASD applications, which will have substantial economic and environmental implications for both nursery and orchard production of woody perennial tree crops in the United States and elsewhere. 04 Generation of interspecific walnut hybrids to facilitate mapping of genetic loci that mediate disease resistance. Mapping genetic loci, which mediate disease resistance, facilitates long-term sustainable classical breeding efforts designed to deliver disease resistant walnut root stocks. Three hundred interspecific hybrids were generated from each of three Juglans microcarpa X Juglans regia crosses. From each hybrid seed, embryos were rescued, propagated in vitro and prepared for Genotype-by-Sequencing (GBS). Approximately 50 clonal copies were generated from each of the 300 clones from each cross. ARS researchers in Davis, California, are conducting disease resistance phenotyping of these clones/plantlets which, in combination with the GBS data will facilitate mapping disease resistance genes. 05 Evidence for the recent spread of Grapevine Red Blotch-associated Virus (GRBaV) in vineyards in Amador and Santa Barbara counties. GRBaV is a recently recognized virus discovered by ARS scientists at Davis, California, in grapevines affected by red blotch disease. Previously we observed spread only in Napa county. We have now confirmed the spread of GRBaV in two other counties in California. This information benefits our stakeholders in the wine grape industry by facilitating the development of effective disease management strategies. 06 Development of a digital PCR (dPCR) for the detection of Grapevine Red Blotch-associated Virus (GRBaV). A dPCR technique has been developed and optimized for early detection of GRBaV in asymptomatic infections and potential insect vectors. This detection system offers the distinct advantage of increased sensitivity and specificity. Use of dPCR facilitated the discovery of the three corned alfalfa hopper GRBaV vector. This work also demonstrated, for the first time, the power of dPCR in the detection of low copy number targets thereby enhancing detection of early stage infections. Development and use of this detection technology enhanced identification of the insect vector, which is now saving the industry millions of dollars in potential losses to red blotch disease. 07 Detection of Grapevine Red Blotch-associated Virus (GRBaV) in wild grapevines. Wild Vitis californica x Vitis vinifera grapevines in the riparian areas close to vineyards tested positive for infection by GRBaV. This indicated a vector was potentially involved in transmitting the virus in California vineyards. This work led to the discovery of an insect vector which proved that nurseries were not solely responsible for virus spread. This finding facilitates a knowledge-based approach to the ongoing development of effective control strategies. 08 Determined cost-reduction potential for implementation of anaerobic soil disinfestation (ASD) to control Prunus replant disease (PRD). Because soil fumigation is increasingly regulated and restricted, non- fumigant methods are needed for management of replant problems. PRD causes growth and yield suppression that is induced by a soil microbial complex that accumulates in successive generations of stone fruit orchards. In previous trials, ASD was as effective as soil fumigation for control of PRD, but the cost of ASD was roughly twice that of soil fumigation. ARS scientists in Davis, California, tested modified ASD treatments that were similar in cost to soil fumigation and yet still provided acceptable levels of PRD control. The findings suggest that, with adequate optimization, ASD may become a viable alternative to soil fumigation for management of PRD.
Impacts
(N/A)
Publications
- Swiecki, T., Aram, K., Kasuga, T., Bui, M.Q., Rizzo, D., Bernhardt, E. 2016. Phytophthora ramorum causes cryptic bole cankers in Canyon line Oak. Plant Health Progress. 17:20-26.
- Kasuga, T., Bui, M.Q., Berhardt, E., Swiecki, T., Aram, K., Cano, L.M., Webber, J., Brasier, C., Press, C.M., Grunwald, N.J., Rizzo, D.M., Garbelotto, M. 2016. Host-induced aneuploidy and phenotypic diversification in the Sudden Oak Death pathogen Phytophthora ramorum. BMC Genomics. 17:385. doi: 10.1186/s12864-016-2717-z.
- Burns, K.N., Bokulich, N.A., Cantu, D., Greenhut, R.F., Kluepfel, D.A., O'Geen, A.T., Strauss, S.L., Steenwerth, K.L. 2016. Vineyard soil bacterial diversity and composition revealed by 16S rRNA genes: Differentiation by vineyard management. Journal of Soil Biology and Biochemistry. 103:337-348. doi:10.1016/j.soilbio.2016.09.007.
- Strauss, S.L., Kluepfel, D.A. 2015. Anaerobic soil disinfestation: a chemical-independent approach to pre-plant control of plant pathogens. Journal of Integrative Agriculture. 14(11):2309-2318. doi: 10.1016/S2095- 3119(15)61118-2.
- Bahder, B.W., Jayanth, M., Zalom, F.G., Sudarshana, M.R. 2016. Phylogeny of geminivirus coat protein sequences and digital PCR aid in identifying Spissistilus festinus (Say) as a vector of Grapevine red blotch-associated virus. Phytopathology. doi: 10.1094/PHYTO-03-16-0125-FI.
- Bahder, B.W., Zalom, F.G., Sudarshana, M.R. 2016. An evaluation of the flora adjacent to wine grape vineyards for the presence of alternative host plants of grapevine red blotch-associated virus. Plant Disease. 100:1571-1574. doi: 10.1094/PDIS-02-16-0153-RE.
- Klosterman, S.J., Rollins, J.R., Sudarshana, M.R., Vinatzer, B.A. 2016. Disease management in the genomics era - Summaries of focus issue papers. Phytopathology. 106:1068-1070.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report 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. 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. ARS scientists in Davis, California, continued development of sustainable control strategies for key soil borne and foliar diseases of perennial fruit and nut tree, vine crops and oak trees (Objectives 1, 2, 3, and 4). Orchard and greenhouse trials were conducted to characterize causes of root and crown rots and Prunus Replant Disease (PRD) (objective 1). Associated pathogenicity trials implicated several species of Pythium and Phytopythium as contributors to PRD; Phytophthora neiderhauseri was implicated as a new crown rot pathogen affecting almonds. Metagenomic examinations were conducted to determine the cause of an emerging crown rot of walnut, �Paradox canker disease�. We have made substantial progress on understanding the biology, spread and detection of the newly discovered Grapevine red blotch-associated virus (GRBaV) (obj. 2). GRBaV infection is associated with poor wine quality resulting in huge economic losses to premium wine grape producers in California. Disease frequency of GRBaV was monitored in one vineyard where disease incidence increased by 4-7%/year over a four year period. We also found that red blotch disease spread to a newly planted block but exhibited limited movement in another vineyard. Rogueing of infected grapevines holds promise to manage the disease. Our work on cost effective disease management strategies (objective 3) focuses on selection of disease-resistant rootstocks and development of fumigation-free pre-plant soil treatments for orchards and nurseries. Crown gall- (CG) and Phytophthora-resistant host genotypes of Juglans (walnuts) and Prunus (almond, peach plum) were identified, and orchard validations of resistance have been initiated. Seedlings from wild walnut species in the germplasm collection were identified with resistance/ tolerance to CG and Phytophthora. To identify genetic loci mediating CG resistance, six mother trees previously shown to produce CG and/or PHY resistant open pollinated progeny were crossed with J. regia pollen. We experienced a seed set of greater than 80%. Interspecific hybrids from these 6 crosses are being clonally propagated in vitro in preparation for disease resistance testing. Ten CG resistant F1 progeny and open pollinated progeny from last year�s crosses have been clonally propagated. These in vitro propagated putative disease resistant genotypes are being reexamined in field trials. The ability of organic amendments to modify soil-borne microbial communities and inhibit the CG pathogen under field conditions are being characterized. We established second year field trials to evaluate efficacy of the non-fumigant based alternative, Anaerobic Soil Disinfestation (ASD) for management of Prunus replant disease (PRD) and key soil-borne diseases affecting walnut nursery stock (Objective 3). At a depth of 18 inches, ASD reduced populations of key soil-borne pathogens below detection limits while suppressing their populations by 90% at a depth of 30 inches. In an almond orchard replant trial, pre-plant ASD was as effective as pre-plant soil fumigation for control of PRD. We identified several alternative carbon sources which may reduce the cost of applying ASD to the field. It is not well understood how the sudden oak death pathogen (Phytophthora ramorum) thrives and devastates Californian forests. To assess adaptability of this invasive pathogen to different host environments, we evaluated the genetic and phenotypic changes in P. ramorum isolated from different plant host species. Genome sequences of 40 representative isolates from diverse host species have been completed, and genome alterations and accompanying phenotypic changes were characterized. In order to understand the mode of action of phosphonate fungicide, we measured dosage responses of Phytophthora ramorum to phosphonate as well as to mefenoxam fungicide as a comparison in vitro. In these trials we also generated estimates of the ability of the sudden oak pathogen to develop resistance to these two fungicides while colonizing two different plant hosts. Accomplishments 01 Identification of wild walnut species/genotypes resistant to crown gall. Crown gall (CG) is a disease that debilitates young trees and facilitates development of other root-destructive diseases in walnuts. ARS scientists in Davis, California, identified open pollinated (OP) seedlings from selected walnut and wingnut (a walnut relative) mother trees which exhibit elevated levels of CG resistance over multiple dormancy periods. CG resistant hybrid progeny from directed crosses were shown to segregate for CG resistance. CG resistant individuals, both OP genotypes and interspecific hybrids, have been cloned through invitro propagation and are currently being examined in field trials. These CG resistant genotypes are essential for development of CG resistant rootstocks for the walnut industry. 02 Fumigant independent approaches in the control of crown gall disease. The causative agent of crown gall, i.e. Agrobacterium tumefaciens, is an excellent and long term colonist of soil particles and host and non- host root systems alike. ARS scientists in Davis, California, are in the second year of testing the preplant treatment of soil using Anaerobic Soil Disinfestation (ASD), an alternative to chemical-based soil fumigation which effectively reduced soil-borne populations of both fungal and bacterial pathogens below detection limits after a 28 day exposure. In addition weed species were dramatically reduced for up to 4 months post ASD application. This fumigation-independent approach to controlling key soil-borne infecting pathogens has substantial economic and environmental implications for both nursery and orchard production of woody perennial tree crops in the United States and elsewhere. 03 Use of anaerobic soil disinfestation (ASD) to prevent Prunus replant disease (PRD). Because soil fumigation is increasingly regulated and restricted, non-fumigant methods are needed for the management of replant problems affecting almond and stone fruits. This includes the most widespread replant malady, PRD (growth and yield suppression induced by a soil microbial complex in successive generations of almond and stone fruit orchards). In almond orchard replant trials near Parlier, California, ARS researchers based in Davis, California, determined that pre-plant ASD was as effective as pre-plant soil fumigation in reducing soil populations of Pythium ultimum (a PRD contributor) and in preventing PRD. The results have led to support for optimization and commercial testing of ASD for management of PRD and other replant problems. 04 Development and application of a greenhouse-based soil bioassay to detect the potential of orchard soils to express Prunus replant disease (PRD). PRD is a widespread malady that can suppress growth and yields in successive plantings of almond and stone fruit orchards yet it is difficult to predict, and its incidence and severity vary significantly among different soils. PRD can be reliably prevented with preplant soil fumigation, but treatments are costly and burdensome. ARS scientists in Davis, California, collected soil samples from 25 orchard plots throughout the San Joaquin and Sacramento Valleys and evaluated each soil�s potential to induce PRD using a greenhouse bioassay with Nemaguard peach seedlings. Based on the greenhouse bioassay, some soils showed no benefit from preplant fumigation while others exhibited marked stimulation of peach seedling growth by preplant soil fumigation. This greenhouse-based soil bioassay, along with orchard validations will facilitate PRD risk assessments and reduced fumigant use. 05 Characterization of host-induced genome alterations in Phytophthora ramorum. Phytophthora ramorum causes sudden oak death disease and also infects many other plant species (i.e. foliar hosts) without causing major symptoms. ARS researchers in Davis, California, discovered major genome alterations such as formation of partial trisomy and monosomy in isolates from oaks infected by Phytophthora ramorum but not in those from the foliar hosts Rhododendron and California bay. Although, generation of chromosomal abnormalities has been investigated in pathogenic yeasts treated with antifungal drugs, this work shows for the first time that chromosomal aberrations are triggered not by drug treatment but by the physical and chemical environment of the plant host. Compounds that cause lethality in eukaryotic cells with an abnormal number of chromosomes may be exploited to potentially help the host plant control/eradicate the pathogen. 06 Development of broad-spectrum primers and probes for molecular detection of three grapevine viruses. For Grapevine leafroll- associated virus 1 (GLRaV-1) and GLRaV-3, the DNA probes designed for sensitive molecular assays, nearly a decade ago, were found inefficient because of the existence of genetically divergent strains. For Grapevine red blotch-associated virus (GRBaV), such probes had not been developed. ARS scientists in Davis, California, assembled the complete genome sequences of all available isolates of GLRaV-1, GLRaV-3 and GRBaV, the three most important grapevine viruses of concern to wine grape production in the U.S. Using this nucleic acid sequence data, effective, and sensitive molecular tools for the detection of these key viral pathogens have been developed. These detection tools will facilitate production of virus-free nursery stock. 07 Documentation of evidence for the spread of Grapevine red blotch- associated virus (GRBaV) in vineyards in Napa County. GRBaV is a recently recognized virus discovered by ARS scientists at Davis, California. It infects grapevines causing a disease known as red blotch disease. For the establishment of management guidelines, it is essential to know if the virus spreads in and between vineyards. ARS scientists have documented evidence for the increase of disease incidence over several years and horizontal spread of the virus. Identification of vineyards where spread has occurred will help focus efforts to identify the vectors of GRBaV and evaluate risk factors that contribute to virus spread. This information benefits the growers and nurseries that produce grapevines for planting. 08 Development of a clearing technique for the identification of mealybugs and soft-bodied insects. Mealybugs, vectors of grapevine leafroll- associated viruses and vitiviruses, are difficult to prepare for slide mounts and taxonomic identification because of their waxy coating. In a collaborative effort, ARS scientists in Davis, California, and entomologists at University of California, Davis, optimized a wax and body content clearing technique for mealybugs using proteinase treatment and heating. This allows extraction of DNA for genetic characterization and preparation of slide mounts for morphological examination of individual insects. This was an offshoot of efforts to identify the vector of GRBaV in California vineyards. This accomplishment benefits entomologists and insect identifiers and is an improvement over the currently used protocol for mealybugs and soft insects at the USDA insect identification laboratories.
Impacts
(N/A)
Publications
- Al Rwahnih, M., Rowhani, A., Golino, D., Islas, C., Preece, J.E., Sudarshana, M.R. 2015. Detection and genetic diversity of Grapevine red blotch-associated virus isolates in table grape accessions in the National Clonal Germplasm Repository in California. Canadian Journal of Plant Pathology. 37:130-135.
- Bag, S., Al Rwahnih, M., Li, A., Gonzalez, A., Rowhani, A., Uyemoto, J.K., Sudarshana, M.R. 2015. Detection of a new luteovirus in imported nectarine trees: A case study to propose adoption of metagenomics in post-entry quarantine. Phytopathology. 105:840-846.
- Sudarshana, M.R., Perry, K.L., Fuchs, M. 2015. Grapevine red blotch- associated virus, an emerging threat to the grapevine industry. Phytopathology. doi: org/10.1094/PHYTO-12-14-0369-FI.
- Szewczyk, E., Kasuga, T., Fan, Z. 2014. A self-excising beta-recombinase/ six cassette for repetitive gene deletion and homokaryon purification in Neurospora crassa. Journal of Microbiological Methods. 91(100):17-23.
- Bahder, B.W., Bollinger, M., Sudarshana, M.R., Zalom, F.G. 2015. Preparation of mealybugs (Hemiptera: Pseudococcidae) for genetic characterization and morphological examination. Journal of Insect Science. doi: 10.1093/jisesa/iev086.
- Knipfer, T., Brodersen, C., Zedan, A., Kluepfel, D.A., Mcelrone, A.J. 2015. Patterns of drought-induced embolism formation and spread in living walnut saplings visualized using x-ray microtomography. Tree Physiology. doi: 10.1093/treephys/tpv040.
- Sudarshana, M.R., Fuchs, M. 2015. Grapevine red blotch. In: Wilcox, W.F., Gubler, W.D., Uyemoto, J.K., editors. Compendium of Grape Diseases. 2nd edition. St. Paul, MN: American Phytopathological Society Press. p. 122- 123.
- Strauss, S.L., Stover, D.A., Kluepfel, D.A. 2014. Impact of biological amendments on Agrobacterium tumefaciens soil survival. Applied Soil Ecology. 87:39-48.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report 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. 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. The development of sustainable control strategies for key soil borne and foliar diseases of perennial fruit and nut tree, vine crops and oak trees is the focus of our research program (Objectives 1, 2, 3, and 4). Work contributing to objectives 1 and 3 include identification of crown gall- (CG) and Phytophthora-resistant host genotypes of Juglans (walnuts) and Prunus (almond, peach plum). This work is now into its third year of full- scale resistance evaluations. Seedlings from wild walnut species in the germplasm collection were identified with resistance/tolerance to CG and Phytophthora. To identify genetic loci mediating CG resistance, again this year, 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 were found resistant/tolerant to CG and/or Phytophthora. These genotypes were cloned at a commercial nursery. We established a repeat field trial to evaluate the resistance of 21 almond and stone fruit rootstock genotypes to Prunus replant disease (PRD). As shown previously, peach parentages were susceptible to PRD while peach-almond hybrids and some plum parentage exhibited moderate to good resistance. ARS scientists in Davis, California, are examining the associations of specific soilborne microbial communities and their members with almond PRD. Microbial communities associated with the CG inhibition under field conditions are also being characterized. ARS scientists in Davis, California, established new trials to evaluate efficacy of non-fumigant based alternatives such as Anaerobic Soil Disinfestation for management of PRD and key soil-borne diseases affecting walnut nursery stock (Objective 3). Work on objective 2.2 was continued to examine etiology and characterize graft and pollen transmissible pathogens in addition to new leaf roll- like diseases of grape. ARS scientists in Davis, California, identified the causative agent of a new disease of grapevine known as Red blotch disease of grapevines caused by a new virus, Grapevine red blotch- associated virus, proposed to be a member of the family Geminiviridae. We examined the gene activity of Rhododendron roots infected with sudden oak death fungus and found host marker genes associated with Phytophthora ramorum infection in asymptomatic and uninfected leaf tissues. Such host markers or gene �signatures� will be of value for disease diagnostics. It is not well understood how the sudden oak death fungus thrives and devastates Californian forests. In order to assess adaptability of the pathogen to different hosts, we evaluated the genetic and phenotypic changes in Phytophthora ramorum isolated from different plant host species. Genome sequences of 18 representative isolates have been completed and gene expression analyses are underway. Bioinformatic tools for raw genomic data processing and mutant identification have been established. In order to understand the mode of action of phosphonate fungicide, we have measured dosage responses of Phytophthora ramorum to phosphonate as well as to mefenoxam fungicide as a comparison. In addition, we are using the almond-Phytophthora citricola interaction as a model pathosystem to elucidate the mode of action of phosphonate. Accomplishments 01 Characterization of the virus which causes grapevine red blotch disease. The virus is widely distributed in the major grape growing regions across the country and causes grapes to develop with low sugar content which results in poor wine quality. Detection of the virus prior to symptom development is required for development of effective control measures. ARS researchers at Davis, California, have developed a new set of DNA primers targeted to a highly conserved region of the Grapevine red blotch virus for the detection of the virus using PCR. These primers are now widely used by commercial testing service labs for the detection of the red blotch virus which facilitates effective control measures at participating nurseries. 02 Discovery of a new luteovirus in nectarine lines showing stem pitting. Nectarine trees, recently identified as exhibiting novel disease symptoms, were shown to be negative for all known viruses. ARS researchers at Davis, California, conducted metagenomic analysis on nectarine samples showing the stem disease and found a new luteovirus. An RT-PCR test has been developed for the detection of this new virus and the information was provided to regulatory agencies. This important research will be helpful for the detection of this virus during the importation of plant germplasm from other countries. 03 Recognition of infection of blackberry (Rubus discolor) bramble by Grapevine red blotch-associated virus. The virus is widely distributed in grapevines in major grape growing regions in the country yet nothing is known of its epidemiology. ARS researchers at Davis, California, have detected the presence of this virus in blackberry. This is an important find as blackberry is an invasive plant species commonly found in/near the vineyards. Recognition of this alternate host will have a significant impact on the design of effective disease management strategies in all grape growing regions of the country. 04 Identification of wild species walnut genotypes resistant to crown gall. Crown gall (CG) is a disease that debilitates young trees and facilitates other root-destructive diseases in walnuts. ARS scientists in Davis, California, identified seedlings from selected walnut and wingnut (a walnut relative) mother trees which continued to exhibit elevated levels of CG resistance over multiple dormancy periods. Progeny from directed crosses were shown to segregate for CG resistance. CG resistant individuals are being cloned for large scale field testing using in vitro propagation. These CG resistant genotypes are essential for development of CG resistant rootstocks for the walnut industry. 05 Fumigant independent approaches in the control of crown gall disease. The causative agent of crown gall, i.e. Agrobacterium tumefaciens, is an excellent and long term colonist of soil particles and host and non- host root systems alike. ARS scientists in Davis, California, conducted research on pre plant treatment of soil using Anaerobic Soil Disinfestation (ASD), an alternative to chemical-based soil fumigation, which effectively reduced soil-borne populations of both fungal and bacterial pathogens below detection limits after a 28 day exposure. In addition weed species were dramatically reduced for up to 4 months post ASD application. This fumigation-independent approach to controlling key rootstock infecting pathogens has serious economic and environmental implications for both nursery and orchard production of woody perennial tree crops in the United States. 06 Gene expression of Rhododendron hosts as function of infection by the sudden oak death (SOD) pathogen. There is a pressing need to identify plant host genes which respond to invasion by the SOD pathogen which could be used to rogue out infected plant prior to symptom expression. An ARS scientist in Davis, California, conducted gene expression analysis that showed gene activity of non-infected and asymptomatic infected Rhododendron leaves was significantly different. Genes whose expression is altered during the course of Phytophthora ramorum root infection were identified. This information can potentially be used to develop methods for the detection of latent infection in nursery setting dramatically reducing costs and limiting legal liabilities for the nursery operator. 07 Evaluation of the genetic and phenotypic changes in the same Phytophthora ramorum (p. ramorum) strain isolated from different host species. P. ramorum is the oomycete plant pathogen known to cause the disease Sudden oak death (SOD). The disease kills oak and other species of tree and has had devastating effects on the oak populations in California and Oregon as well as also being present in Europe. ARS researchers in Davis, California, discovered major genome alterations in P. ramorum isolates recovered from oak hosts while only minor genomic changes were detected in isolates from artificially infected Rhododendron, California bay laurel, and Viburnum tinus. This ability of the pathogen to rapidly alter its genome may make P. ramorum a devastating invader. Understanding the ability of P. ramorum to modify and adapt its genome depending on which host it is colonizing is critical to developing effective control strategies and science based regulatory restrictions. 08 Understanding the mode of action of phosphonate fungicides. Phytophthora citricola (P. citricola) is an important fungal pathogen of walnut and almond in California, but little is known about its genetics. Understanding both genome composition and gene expression in this fungus is essential if we are to fully exploit the use of phosphonate as a fungicide. ARS scientists in Davis, California, have determined the complete genome sequence of a virulent Californian isolate of P. citricola Initial gene identification has also been accomplished. This information will help us understand P. citricola�s sensitivity to phosphonate which will result in development of cost effective control strategies. 09 Pythium helicoides identified as an almond and stone fruit pathogen. Phytophthora and Pythium species attack a wide range of woody ornamentals, including species and cultivars of almond and walnut. Our previous research revealed novel contributions of Phytophthora and Pythium species to root and crown diseases of almond and walnut trees, but knowledge of these diseases is incomplete. ARS researchers in Davis, California, determined that Pythium helicoides, which was not previously known as a fruit or nut tree pathogen, can cause root and crown rot and replant growth suppression in almond and stone fruit orchards in the San Joaquin Valley. The identification of Pythium helicoides as an important source of economic loss in these crops will facilitate development of appropriate diagnostics and disease management strategies. 10 Evaluation of disease resistance among wild species of walnut genotypes. Phytophthora cinnamomi and Phytophthora citriocla are serious soilborne fungal pathogens of walnut worldwide. ARS Scientists in Davis, California, conducted greenhouse trials to evaluate genetic resistance of clonal hybrid selections and seedlings from 32 mother trees for resistance to Phytophthora cinnamomi. Among the clonal hybrids, numerous selections expressed high resistance to both Phytophthora cinnamomi and Phytophthora citricola. Among the seedlings, disease resistance differed significantly according to mother tree source, which helped identify mother trees with superior breeding value for development of resistance to Phytophthora. These and previous evaluations have helped provide promising releases of new candidate rootstocks for testing in the California walnut industry. 11 Assessing resistance of selected almond and stone fruit rootstock genotypes to the Prunus replant disease (PRD) complex. Successive plantings of almond and stone fruit orchards on a given site typically sustain poor tree growth and yield loss. ARS Scientists in Davis, California, evaluated the resistance of 21 almond and stone fruit rootstock genotypes to PRD in a repeat field trial. Most rootstocks with only peach parentage exhibited little resistance to PRD, while peach � almond hybrid rootstocks were relatively resistant. Rootstocks with plum parentage varied in response. The PRD resistance profiles generated by our trials are facilitating rootstock breeding efforts, informing rootstock selections made by growers and nurseries, and helping to reduce reliance on soil fumigation. 12 New primers developed for the detection of the virus that causes grapevine red blotch disease. The virus is widely distributed in the major grape growing regions across the country and causes grapes to develop with low sugar content which results in poor wine quality. Detection of the virus prior to symptom development is required for development of effective control measures. ARS researchers at Davis, California, have developed a new set of DNA primers targeted to a highly conserved region of the Grapevine red blotch virus for the detection of the virus using PCR. These primers are have been made available to commercial testing service labs to facilitate elimination of virus-infected material by participating growers and nurseries.
Impacts
(N/A)
Publications
- Al Rwahnih, M., Daubert, S.D., Sudarshana, M.R., Rowhani, A. 2013. Gene from a novel plant virus satellite from grapevine identifies a viral satellite lineage. Virus Genes. 47:114-118.
- Al Rwahnih, M., Dave, A., Anderson, M.M., Rowhani, A., Uyemoto, J.K., Sudarshana, M.R. 2013. Association of a DNA virus with grapevines affected by red blotch disease in northern California. Phytopathology. 103:1069- 1076.
- Kasuga, T., Gijzen, M. 2013. Epigenetic mechanisms and the evolution of virulence. Trends in Microbiology. 21:575-582.
- Yakabe, L.E., Parker, S.R., Kluepfel, D.A. 2014. Incidence of Agrobacterium tumefaciens biovar 1 in and on �Paradox� (Juglans hindsii x Juglans regia) walnut seed collected from commercial nurseries. Plant Disease. 98:766-770.
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. 02 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. 03 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. 04 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. 05 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. 06 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. 07 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. 08 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.
Impacts
(N/A)
Publications
- 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.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report 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. 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. 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. Accomplishments 01 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. wer 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 isolat which will be tested for pathogenicity. Determining the cause of this disease will aid development of cost effective control measures for use the grower. 02 Isolation and identification of the pathogenic agents which cause Prunus replant disease (PRD). PRD causes severe growth suppression in almond a 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 tria evaluating resistance of rootstocks to the complex. Determining the caus of this disease and discovering resistant rootstocks will benefit the almond and stone fruit industry. 03 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 tumefaciens will enhance our walnut Crown Gall resistance screening efforts. 04 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 th need for ozone depleting soil fumigation. 05 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 "ope pollinated nuts" from selected walnut mother trees which were then germinated and cultivated in the greenhouse and screened for CG resistan Directed crosses have been made with selected walnut mother trees with pollen from English cv Serr. Progeny from these crosses will be harveste this fall and tested for crown gall resistance. This work is essential t the development of CG resistant rootstocks for the walnut industry.
Impacts
(N/A)
Publications
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