Progress 06/08/17 to 09/30/21
Outputs
Target Audience:Growers, pest control advisors, crop consultants, extension agents, graduate students Changes/Problems:Repeated efforts to culture Xylella fastidiosa from pecan petioles and grapevine leaves were not successful. Due to this difficulty, we could not perform experiment to determine host range and characterize the strains of Xylella fastidiosa. Therefore, the major changes were made to focus on survey and identification of both vectors: sharpshooters and spittlebugs. What opportunities for training and professional development has the project provided?This project has trained a postdoctoral student and four undergraduate students: Hajeewaka Mendis completed his 12-month postdoctoral fellowship on this project. undergraduates (Allay Felix,Jose Romero Gallego, Yong Zhang,Daniel Richard Collazo completed theirindependent study from 2019 to 2021, learning the research process from literature review to project planning, execution, data analysis and interpretation, and writing for publication. How have the results been disseminated to communities of interest?Project results have been disseminated to growers, pest control advisors, extension agents, industry representatives, and research community through several venues: 1) extension fact sheets on symptom identification and management; 2) symptosium; 3) farm visits, and 4) one-on-one meeting, and 5) presentation talks at several workshops, which include Southeast Ag Day and Trade show, Viticulture symptosium, and Farm and Ranch day. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
This project ascertained the causal agents and vectors of pecan leaf scorch and Pierce's Diseases: four sharpshooter species responsible for disease outbreak in pecan orchards and vineyards(blue-green: Graphocephala atropunctata, green: Draeculacephala minerva, Cuerna arida, and Hordnia sp.) and two Arizona native spittlebugs (Clastoptera lawsoni and C. arizonana). Ithas greatly advanced the science and technology in a wide range of disciplines from Xylella diagnostics to vector biology. These advancements support the hypothesis that leaf scorch can be mitigated by early detection of Xylella and management of vector populations (e.g. sharpshooters and spitllebugs). They already have enabled early adopters to implement best disease management practices for improved profitability and sustainability while reducing their environmental footprint. These advancements is highlighted by two major discoveries. First, Improved Xylella diagnostics greatly increased sensitivity and reduced the probability of false negative and false positive results. In pecan orchards and vineyards where valuable resources (i.e., water, fertilizer, and labor) have been used to raise a young seedling transplant for 3 - 4 years, and sometimes even in trees of 12 years of age and older, the tree or vine decline due to Xylella infection makes a waste of these high cost resources. The value of loss in new transplants are upwards to $2,000 per tree. It was reported that thousands of immature trees or vines were infected with Xylella fastidiosa. This is an estimated several million dollars combined loss. Early detection and adoption of effective vector management would save AZ growers hundreds of thousands of dollars. Second, high level of sharpshooter and spittle bugs were detected in two vineyards, and low vector populations were detected in pecan orchards. Monitoring vector populations could easily reduce 5 to 20% of the pesticide usage that otherwise would have been lost. Arizona pecan and grape industries are estimated to be apply pesticides annually at a direct and indirect cost of millions of dollars. This improved practice alone could save hundreds of thousands of dollars per year in Arizona.
Publications
- Type:
Other
Status:
Published
Year Published:
2018
Citation:
Hu, J. 2018. Pecan bacterial leaf scorch. The Division of Agriculture, Life and Veterinary Sciences and Cooperative Extension, University of Arizona. az1772.
|
Progress 10/01/19 to 09/30/20
Outputs
Target Audience:Growers, pest control advisors, crop consultants, extension agents, graduate students. Changes/Problems:Repeated efforts to culture Xylella fastidiosa from pecan petioles and grapevine leaves were not successful. Due to this difficulty, we could not perform experiment to determine host range and characterize the strains ofXylella fastidiosa.Therefore, the major changes were made to focus on survey and identification of both vectors: sharpshooters and spittlebugs. What opportunities for training and professional development has the project provided?Hajeewaka Mendis completed his 12-month postdoctoral fellowship on this project. Allay Felix, an undergraduate from microbiology department, completed her independent studyin 2019, learning the research process from literature review to project planning, execution, data analysis and interpretation, and writing for publication. How have the results been disseminated to communities of interest?Weprovided individual support to growers and pest control advisors to ensure that they are aware of the critical nature of Pierce's disease and pecan bacterial leaf scorch. The key to this objective is getting grower support to develop their own field monitoring programs and control strategies. This includes individual field meetings if there are questions regarding symptom identification, encouragement of tissue sampling, collecting and shipping samples to the extension plant pathology diagnostic lab at the University of Arizona, communicating results, providing information on PD and PBLS management and follow up support. Project results were also delivered through presentations at growers' meeting and extension publicastions. What do you plan to do during the next reporting period to accomplish the goals?We will focus on data analysis and writing publications. Project results will be presented at Viticulture symposium and Annual conferenceof Arizona Growers' Association.
Impacts
What was accomplished under these goals?
Significant achievement has been made on this project objectives and goal: 1) We deployed about 1,000 yellow sticky traps in pecan orchards and vineyards; many species of sharpshooters and skpittlebugs have been identified and their seasonal fluctuation patterns were recorded in both vineyards and pecan orchards; 2) Vineyards with high incidence of PD was found and the causes of vine decline were determined. ELISA and PCR tests were performed on more than 300 plant specimens for Xylella fastidiosa. Vines and trees positive for XF were marked for removal to reduce further spread; and 3) several attempts to culture Xylella fastidiosa using PD3 mediawas not successful.
Publications
- Type:
Other
Status:
Published
Year Published:
2020
Citation:
Hu, J. Pierces disease of grape. The Division of Agriculture, Life and Veterinary Sciences and Cooperative Extension, University of Arizona. az1861.
|
Progress 10/01/18 to 09/30/19
Outputs
Target Audience:Growers, pest control advisors, and extention educators in pecan and wine grape industry. Consultants, Extension specialists,Agents, Plant disease diagnosticians, regulatory personnel, Undergraduate and graduate students, postdoctoral associates Educators, bacteriologists, and researchers, Government policy makers Changes/Problems:Several attempts were made to isolate X. fastidiosa strains from pecan leaves and branches, none of them were successful. This is due to the slow growing of the Xf strain associated with pecan bacterial leaf scorch. To date, we did not obtain pure culture of Xf. Consequently, we were not able to perform strain characterization and host range study. Instead, we focus our work on improving Xylella diagnostics and expanding our survey efforts to cover both symptomatic and nonsymptomatic trees. What opportunities for training and professional development has the project provided?Jin Huang completed her 12-month visiting scholarship on epidemiology and management of bacterial pathogens.Ally Felix and Shizhe Li, two undergraduate students at U of A, completed their independent study in 2019, learning the research process from literature review to project planning, execution, data analysis and interpretation. Hongsheng Yang, a visiting scholar, is learning field survey methodology for monitoring Xf and its vectors. How have the results been disseminated to communities of interest?Presentation at Grower Meetings and other professional meetings: "Disease Issues in Arizona Pecans". The 2019 Annual Meeting of Arizona Pecan Growers Association, Tucson, Arizona "Step-by-Step Guide to Vineyard Diagnostics". 2018. Arizona Grape Growers Symposium, Benson, Arizona "Arizona Tree Disease Update". 2018. The Shade Conference, Arizona Nursery Association, Phoenix, Arizona" "Recognizing and Managing Diseases on Landscape Plants". 2018. The 27th Annual Desert Horticulture Conference, Tucson, Arizona "Xylella Leaf Scorch". 2017. The Annual Conference & Workshop, Arizona Community Tree Council, Prescott, Arizona What do you plan to do during the next reporting period to accomplish the goals?In addition to planned activities, we plan to deploy more insect traps to monitor vector populations in pecan orchard. Survey on PD in Az Vineyards is planned. We will repeat and expand our survey of pecan orchards. Focus will be on relationship between symptomology and tree age and cultivars.
Impacts
What was accomplished under these goals?
Various symptoms of leaf scorching, yellowing, twig dieback and defoliation were noted on 490 trees of 5-year-old pecan in several pecan orchards in southeast Arizona. The disease survey indicated that 68.6% of marked trees (n=490) had over 50% of their leaves with scorching symptom. Symptoms of leaf scorching, yellowing, twig dieback and defoliation appeared to occur together on many pecan trees with advanced symptoms. The expression of yellowing along with leaf scorching and defoliation does not match the typical symptom of pecan bacterial leaf scorch caused by Xylella fastidiosa (namely XF). Surprisingly, XF was not detected in any of 318 pecan sap samples that were taken in September and October. 180 yellow sticky trap deployed from August to October did not catch any sharpshooters or spittlebugs.Research data translation and dissemination to end users has emerged as a central theme of our project activities this past year. The most significant accomplishments included: (i) severalwell-attended extension meeting on PBLSand PD, and (ii) publication of a comprehensive extension publications on bacterial leaf scorch of pecan.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Jiahuai Hu.2019. Disease Issues in Arizona Pecans. The 2019 Annual Meeting of Arizona Pecan Growers Association, Tucson, Arizona
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2018
Citation:
Jiahuai Hu. 2018. Step-by-Step Guide to Vineyard Diagnostics. Arizona Grape Growers Symposium. Benson, Arizona
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Jiahuai Hu. 2017. Xylella Leaf Scorch. The Annual Conference & Workshop, Arizona Community Tree Council, Prescott, Arizona
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Jiahuai Hu. 2017. Bacterial Leaf Scorch by Xylella fastidiosa. The Shade Conference, Arizona Nursery Association, Phoenix, Arizona
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2017
Citation:
Jiahuai Hu. 2017. Controlling Diseases of Fruit and Nut Trees and Grapes. The 26th Annual Desert Horticulture Conference, Tucson, Arizona
|
Progress 10/01/17 to 09/30/18
Outputs
Target Audience:Pecan growers, farm managers, crop consultants, pest control agents, and university cooperative extension cooperators. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One undergraduate student and one visiting scientist scholarship. How have the results been disseminated to communities of interest?Results of this research have been published in the Extension Bulletin, they have been presented at many grower meetings in Arizona. What do you plan to do during the next reporting period to accomplish the goals?We plan to Improve Xylella isolation techniques as follows: Twigs from 12 different trees with PBLS symptom were cut into pieces. 8 to 10 cm long (0.5-0.8 cm diameter), washed under tap water, surface-sterilized in 2% sodium hypochlorite for 2 min, soaked in 70% ethanol for 2 min and rinsed three times in sterile water. Each piece was cut in half and squeezed at one end with a plier, while the other end was gently pressed on a buffered charcoal yeast extract (BCYE) growth medium37 to make 2-3 imprints. For each sample three BCYE plates were spotted (ca. 20-30 spots per plate), incubated for 3-4 weeks at 28 °C and weekly inspected for the growth of Xylella colonies. Identification of individual colonies as X. fastidiosa was based on in vitro fastidious growth, coloration and PCR using RST31/ RST33 primers. We plan to use this mehtod to isolate X. fastidiosa trains from pecan trees. If successful, These Xylella strains will be used to conduct host range and pathogenicity test.
Impacts
What was accomplished under these goals?
Objective 1: disease surveys were conducted to determine the incidence of X. fastidiosa infestations in Arizona vineyards and pecan orchards. The surveys investigated the occurrence, host range and presence of X. fastidiosa in asymptomatic trees and vines. Approach: Comprehensive sampling of grapevines and pecan trees were carried out in several Arizona vineyards and pecan orchards across a broad range of soil types and varieties. Pecan orchards and vineyards in Cochise county were subjected to random and systematic collection of samples from individual vines/trees. In 2017, the survey for leaf scorch symptoms in pecan cultivars present in Arizona orchards was conducted from July through October. Orchards were selected to represent the major areas of managed pecan production. Four pecan orchards in Cochise and Pima Counties were selected for survey study of PBLS incidence. The selected orchards are located close to riparian areas and had varying degrees of management intensity ranging from being well managed to poorly managed. The age of the trees assayed for X. fastidiosa ranged from 5 to over 50 years. Thirty trees were sampled at each location. Eight leaves with or without scorch symptoms were taken from each tree and pooled together in a plastic bag. Leaf samples were placed in an ice cooler, transported back to Laboratory, and kept overnight at 4 to 7°C. Serological assay for presence of the bacterium was conducted the next day using a commercial ELISA kit for X. fastidiosa (Agdia, Inc., Elkhart, IN). Leaflets were discarded and a section approximately 4 cm long was cut from each rachis and homogenized with 4 to 5 ml of the extraction buffer with a mortar and pestle. Results for the presence of the bacterium were recorded based on color reaction and the absorbance at 490 nm. An absorbance ≥2.5 times the extract buffer and a distinct color reaction were recorded positive for X. fastidiosa. During collection of leaves with scorch, leaf samples were taken from trees not displaying symptoms to determine whether asymptomatic tree harbors the Xylella bacterium. In 2018, We surveyed another pecan orchard in Santa Cruz County. A total of 16 pecan trees were selected for sampling and eight petioles will be taken from each plant. Both symptomatic and asymptomatic foliage were collected. The petiole samples were placed in individual plastic bags and transported back to the laboratory on ice and kept at -80oC until further analysis. The Xf detection assay were performed as described above. Results: PBLS-like symptoms were frequently be observed in all four orchards in Arizona. All seven cultivars were identified as having Symptoms similar to PBLS. Three cultivars, including cv. Waco, Western, and Wichita, were positive for infection with X. fastidiosa. Sixty percent (48/80) of the trees with PBLS symptoms were positive for X. fastidiosa. All cultivars that had PBLS-like symptoms were positive for the bacterium. Disease incidence within cultivars varied from 20 to 80% between orchards. The severity of symptoms varied from tree to tree within a cultivar in an orchard. Severity was not quantified, but most cultivars generally had similar but mild symptoms when it was present in the same orchard. Some, however, did exhibit a higher level of scorch in some orchards. Forty trees without symptoms selected from four orchards were assayed for the bacterium. Fourteen of these asymptomatic trees gave positive reactions for X. fastidiosa. Objective 2: Isolation of X. fastidiosa from infected pecan trees. Approach: Method 1: pecan leaf samples positive for X. fastidiosa were used to isolate the bacterium. Leaves were surface sterilized by submerging in 70% ethanol for 2 min, followed by submerging the leaf in 2% sodium hypochlorite for 1.5 min. The leaves were rinsed in sterile distilled water twice. Leaf sections from ten separate leaves, consisting of mainly the petiole and main veins, were finely chopped on sterile filter paper and in mesh bag containing 5 ml of succinate-citrate-phosphate buffer (1 g/L disodium succinate, 1 g/L trisodium citrate, 1.5 g/L K2HPO4, 1 g/L KH2PO4, pH 7). The homogenate was incubated for 20-min at room temperature and then was made into serial dilution up to 10-4 by serial dilution method. 50µl of the dilution from 10-1, 10-2, 10-3, and 10-4 were placed onto PWG medium. The PWG plates were then incubated at 28oC and monitored for colony development for 6 weeks. Result: Forty eight isolation plates were inoculated with symptomatic pecan leaf petioles. Twenty days after inoculation, numerous colonies were developed on PWG medium. Small white colonies were transferred to new PWG media. Genomic DNA templates were isolated from purer culture using Omega genomic DNA isolation kit. However, X. fastidiosa was not detected using RST31 and RST33 primers. Several Bacillus spp. were confirmed by direct sequencing of amplicons produced from 16S nrRNA using primers 8F and 1492R. Method 2: 2 to 3 cm long pieces of pecan petioles and midribs from plants exhibiting PBLS symptoms were transferred to sterile plastic bags, surface sterilized by soaking for 3 min in 1% sodium hypochlorite and 4 min in 70% ethanol, and rinsed three times each for 3 min in sterile distilled water. The tissues were transferred to new plastic bags in 3 mL sterile succinate-citrate-phosphate buffer. After a 20-min incubation period at room temperature, 50 μL of the liquid were streaked on the media PD3 (Davis et al., 1980), PW (Schaad et al., 2001), X. fastidiosa-D1 (Almeida et al., 2004) and nutrient agar (Schaad et al., 2001). Alternatively the petioles and midribs were crushed in the plastic bags using a pair of sterile pliers and 30 μL of extract were streaked on culture medium. The plates were incubated at 28°C for 4 weeks and examined with a stereomicroscope at weekly intervals for the presence of Xylella-like colonies (Wells et al., 1987; Chen et al., 2007). Result: a total of 120 isolation plates were inoculated with symptomatic pecan leaf petioles. Three weeks after plating on PD3 and PW media, small white colonies and yellow smooth colonies, and other wrinkled colonies were observed on the isolation plates. Small white colonies were re-streaked three times onto PW medium to ensure the purity of the strains. Twenty four subcultures of bacterial cell were established on PW medium. Bacterial cells were Gram stained and observed at 1000× magnification using phase contrast microscopy. Confirmation of X. fastidiosa was made using RST31 and RST33 primers. X. fastidiosa was not detected in any of 24 bacterial isolates recovered from symptomatic pecan leaf petioles. Results: X. fastidiosa was not cultured from infected pecan trees using PWG media. Twenty days after plating on PWG medium, small white colonies were visible. These colonies were confirmed as Bacillus spp. by direct sequencing of amplicons produced from 16S nrRNA using primers 8F and 1492R.
Publications
- Type:
Other
Status:
Published
Year Published:
2018
Citation:
Hu, J. 2018. Pecan Bacterial Leaf Scorch. University of Arizona Cooperative Extension az1772
|
Progress 06/08/17 to 09/30/17
Outputs
Target Audience:Fruit nut growers, Crop Advisor, Pest Control Advisor, Extension Agents, Research and extension plant pathologists, Plant disease diagnosticians, Agricultural and horticultural extension agents, and Policy-makers. Changes/Problems:Several attempts to culture Xylella strain from pecan petiole samples were not successful. Xylella isolation from pecan is difficult and requires repeated attempts and tissue samples with high bacterial titers. We are working to improve isolation methods. What opportunities for training and professional development has the project provided?One postdoctoral associates (Utpal Handique) and one graduate student (Leo) worked on this project at University of Arizona and developed expertise and practical experience in testing and isolation of Xylella research and education programming. This project also provided a unique opportunity for them to interact with farmers and extension plant pathologists. Additional undergraduate student workers (Alice and Brianna) were trained to process biological samples. How have the results been disseminated to communities of interest?The implications of these data and recommendations on pecan bacterial leaf scorch disease management have been communicated with growers and pest control advisors via numerous local and regional extension venues including presentations, on-site farm visits, phone calls, E-mail, and disease diagnostic reports. A fact sheet incorporating the final results from the recent field trials is being developed for regional distribution. What do you plan to do during the next reporting period to accomplish the goals?Improve isolating and culturing protocol for Xylella fastidiosa in pecan. PCR genotyping of Xylella strains from pecan trees. Provide pecan producers with research based information for managing pecan bacterial leaf scorch, through publications, presentations, and individual contacts.
Impacts
What was accomplished under these goals?
The survey for leaf scorch symptoms in pecan cultivars present in Arizona orchards was conducted from July through October in 2017. Orchards were selected to represent the major areas of managed pecan production. Four pecan orchards in St David, and green valley were selected for survey study of PBLS incidence. The selected orchards are located close to riparian areas and had varying degrees of management intensity ranging from being well managed to poorly managed. The age of the trees assayed for X. fastidiosa ranged from 5 to over 50 years. Thirty trees were sampled at each location. Eight leaves with or without scorch symptoms were taken from each tree and pooled together in a plastic bag. Leaf samples were placed in an ice cooler, transported back to Laboratory, and kept overnight at 4 to 7°C. Serological assay for presence of the bacterium was conducted the next day using a commercial ELISA kit for X. fastidiosa (Agdia, Inc., Elkhart, IN). Leaflets were discarded and a section approximately 4 cm long was cut from each rachis and homogenized with 4 to 5 ml of the extraction buffer with a mortar and pestle. Results for the presence of the bacterium were recorded based on color reaction and the absorbance at 490 nm. An absorbance ≥2.5 times the extract buffer and a distinct color reaction were recorded positive for X. fastidiosa. During collection of leaves with scorch, leaf samples were taken from trees not displaying symptoms to determine whether asymptomatic tree harbors the Xylella bacterium. PBLS-like symptoms were frequently observed in all four orchards in Arizona. All seven cultivars were identified as having Symptoms similar to PBLS. Three cultivars, including cv. Waco, Western, and Wichita, were positive for infection with X. fastidiosa. Sixty percent (48/80) of the trees with PBLS symptoms were positive for X. fastidiosa. All cultivars that had PBLS-like symptoms were positive for the bacterium. Disease incidence within cultivars varied from 20 to 80% between orchards. The severity of symptoms varied from tree to tree within a cultivar in an orchard. Severity was not quantified, but most cultivars generally had similar but mild symptoms when it was present in the same orchard. Some, however, did exhibit a higher level of scorch in some orchards. Forty trees without symptoms selected from four orchards were assayed for the bacterium. Fourteen of these asymptomatic trees gave positive reactions for X. fastidiosa. Pecan leaf samples positive for X. fastidiosa were used to isolate the bacterium. Leaves were surface sterilized by submerging in 70% ethanol for 2 min, followed by submerging the leaf in 40% bleach (2% sodium hypochlorite) for 1.5 min. The leaves were rinsed in sterile distilled water twice. Leaf sections from ten separate leaves, consisting of mainly the petiole and main veins, were finely chopped on sterile filter paper and in mesh bag containing 5 ml of succinate-citrate-phosphate buffer (1 g/L disodium succinate, 1 g/L trisodium citrate, 1.5 g/L K2HPO4, 1 g/L KH2PO4, pH 7). The homogenate was incubated for 20-min at room temperature and then was made into serial dilution up to 10-4 by serial dilution method. 50µl of the dilution from 10-1, 10-2, 10-3, and 10-4 were placed onto PWG medium. The PWG plates were then incubated at 28oC and monitored for colony development for 6 weeks.X. fastidiosa was not cultured from infected pecan trees using PWG media. Twenty days after plating on PWG medium, small white colonies were visible. These colonies were confrmed to be Bacillus spp. rather than X. fastidiosa by direct sequencing of amplicons produced from PCR reactions. We are working to improve isolation techniques.
Publications
- Type:
Other
Status:
Accepted
Year Published:
2018
Citation:
Hu, J. 2018 Pecan bacterial leaf scorch. University of Arizona Extension Publication
|
|