For the first year of research on healthy and HLB samples from one Hamlin and two Valencia harvests (15+ trees/sample), reports were given on sugar, acid, brix, titratable acidity, ratio and oil content, as well as sensory perception of flavor. The results showed that there were minimal differences between juice from healthy and HLB trees for asymptomatic fruit, but that there were differences for symptomatic fruit, both chemically and in terms of sensory perception. Now we present the data for these samples for aroma, which includes both gas-chromatography-olfactometry (GCO) and GC-mass spectroscopy (GC-MS) analyses. For GCO analysis, each sample evaluated was analyzed twice using two trained olfactory assessors. Assessors rated aroma intensity continuously throughout the chromatographic separation process using a linear potentiometer whose output was recorded and quantified using chromatography software. Retention times and verbal descriptors were recorded to permit aroma descriptors to be coupled with computerized aroma time-intensity plots. Only those compounds which were described similarly at a similar retention time and observed by half the panel considered as aroma active components. Both pasteurized and unpasteurized samples for healthy and symptomatic HLB juice were analyzed Forty-six aroma components were observed in the set of juices. Total aroma intensity from the juices were summed and averaged. Early season HLB symptomatic fruit had less overall aroma intensity than control (healthy). However, in more mature fruit there is little difference in overall intensity. On average, pasteurization increased total GCO aroma intensity. The samples were analyzed by GC-MS, primarily to identify and confirm the GCO data. Forty-two MS analyses on three sets of juice from the 2008-2009 season were analyzed. One set was Hamlin and two sets were Valencia. Within each set there were two reps of control (healthy), symptomatic and asymptomatic juices. Each set also contained paired pasteurized and unpasteurized juices of each juice type. In examining the GCO data set, it was observed that the vast majority of aroma active volatiles were very similar between HLB symptomatic and control juices. However, a few volatiles were found either exclusively or at higher aroma intensity in certain sample types. In the case of Valencia juices, 2-methylbutanol (minty, green) and decanal (citrus, fatty) were found primarily in HLB pasteurized juices. ‘-myrcene (geranium, earthy, grass) and octen-3-one, 1 (mushroom, metallic) were identified in both HLB and control unpasteurized juices, but with a higher intensities in HLB juice. An unknown minty, ocimene (tropical); 1-decanol (spicy, cilantro), farnesyl acetate (perfume); p-cymene (citrus, green) were observed only in unpasteurized control juice. Volatiles such as methional (cooked grain); dodecanal (citrus, soapy, green) and cadinene (minty, burned) were found almost exclusively in pasteurized control juice. Early season HLB symptomatic fruit had less overall aroma intensity than control (healthy). However, in more mature fruit there is little difference in overall intensity. On average, pasteurization increased total GCO aroma intensity. Since the late season Hamlin harvest was missed in 2009, samples for Hamlin were harvested last week. Analyses on these samples will be added to the final quarter first year report. The second year of research will include the establishment of thresholds for off-flavored symptomatic HLB juice in normal juice and of identified off flavored compounds in normal juice. This work has already been initiated. Also studies are underway to compare HLB juice from trees under normal cultivation and spray regimes to those undergoing nutritional and salicylic acid sprays as well as to healthy trees for chemical and sensory analyses.
For the first year of research on healthy and HLB samples from one Hamlin and two Valencia harvests (15+ trees/sample), reports were given on sugar, acid, brix, titratable acidity, ratio and oil content, as well as sensory perception of flavor. The results showed that there were minimal differences between juice from healthy and HLB trees for asymptomatic fruit, but that there were differences for symptomatic fruit, both chemically and in terms of sensory perception. One exception, chemically, that was not presented in previous reports, is that limonin and nomilin were higher in asymptmatic and especially symptomatic fruit compared to controls for January Hamlin and April Valencia, but not June Valencia, reflecting sensory results. Levels were below published taste thresholds for these compounds, however. Now we present Russ Rouseff’s data for aroma, which includes both gas-chromatography-olfactometry (GCO) and GC-mass spectroscopy (GC-MS) analyses. For GCO analysis, each sample evaluated was analyzed twice using two trained olfactory assessors. Only those compounds which were described similarly at a similar retention time and observed by half the panel considered as aroma active components. Both pasteurized and unpasteurized samples for healthy and symptomatic HLB juice were analyzed. Forty-six aroma components were observed in the set of juices. Total aroma intensity from the juices were summed and averaged. Early season HLB symptomatic fruit had less overall aroma intensity than control (healthy). However, in more mature fruit there is little difference in overall intensity. On average, pasteurization increased total GCO aroma intensity. The samples were analyzed by GC-MS, primarily to identify and confirm the GCO data. Forty-two MS analyses on three sets of juice from the 2008-2009 season were analyzed. One set was Hamlin and two sets were Valencia. Within each set there were two reps of control (healthy), symptomatic and asymptomatic juices. Each set also contained paired pasteurized and unpasteurized juices of each juice type. In examining the GCO data set, it was observed that the vast majority of aroma active volatiles were very similar between HLB symptomatic and control juices. However, a few volatiles were found either exclusively or at higher aroma intensity in certain sample types. In the case of Valencia juices, 2-methylbutanol (minty, green) and decanal (citrus, fatty) were found primarily in HLB pasteurized juices. ‘-myrcene (geranium, earthy, grass) and octen-3-one, 1 (mushroom, metallic) were identified in both HLB and control unpasteurized juices, but with a higher intensities in HLB juice. An unknown minty, ocimene (tropical); 1-decanol (spicy, cilantro), farnesyl acetate (perfume); p-cymene (citrus, green) were observed only in unpasteurized control juice. Volatiles such as methional (cooked grain); dodecanal (citrus, soapy, green) and cadinene (minty, burned) were found almost exclusively in pasteurized control juice. Early season HLB symptomatic fruit had less overall aroma intensity than control (healthy). However, in more mature fruit there is little difference in overall intensity. On average, pasteurization increased total GCO aroma intensity. Since the late season Hamlin harvest was missed in 2009, samples for Hamlin were harvested last week. Analyses on these samples will be added to the final quarter first year report, but the limonin and nomilin analyses are done and show higher levels in HLB juice, similar to the 2009 Hamlins. The second year of research (already underway) will include the establishment of thresholds for off-flavored symptomatic HLB juice in normal juice and of identified off flavored compounds in normal juice. Also studies are underway to compare HLB juice from trees under normal cultivation and spray regimes to those undergoing nutritional and salicylic acid sprays as well as healthy trees for chemical and sensory analyses.
Five-band Sensor Development: At Citrus Research and Education Center (CREC), a passive five-band sensor was developed and integrated as a portable, relatively inexpensive, robust, mobile sensor module that can be incorporated with the sensor platform (developed in CREC) in a gator for stress-detection applications. The sensor can measure the reflectance in five spectral bands (three visible and two infrared), and has the capability of storing the GPS coordinates and for incident light intensity correction for acquiring stable spectral reflectance. The sensor is being field tested for its ability to accurately identify stressed citrus trees in different citrus varieties. This sensor will be further tested for HLB detection. Greenhouse Experiments: The preliminary results from greenhouse experiments (CREC) for detecting HLB in early stages indicated that the spectroradiometer reflectance data could be used to identify HLB-infected trees with a classification accuracy of > 90% for Hamlin and Valencia varieties. The results are being further validated with a larger number of dataset. Aerial Hyperspectral Imaging: A new set of aerial hyperspectral imagery has been acquired in a citrus grove with the help from Dr. Yang and his colleagues (USDA ARS, Texas). The imaging site was the 20 acres Summerland grove (Consolidated Citrus Ltd.). The grove contained some dead trees (scaffold), probably from tristeza, citrus blight and HLB. Ground truthing was conducted for diseased trees. The aerial imageries along with ground data are currently being analyzed to further develop and evaluate the HLB detection algorithms. The results will be reported in next progress report. Differential mobility spectrometer sensor: In this quarter, we designed, manufactured and tested a volatile collection device holder that improved the reproducibility and eliminated false positives that occur when the collection device makes direct contact with the leaf surfaces. In addition, we expanded on our previous study of measuring volatile organic compounds (VOCs) of symptomatic and healthy sweet navel oranges. The sample collected in this quarter consisted of three groups: control plants in grove A (commercial grove, Lake Alfred, FL), healthy & asymptomatic plants in grove B (Experimental Station, Oakland, FL), and symptomatic plants in grove B. The VOCs were collected using polyacrylate solid phase microextraction (SPME) fibers and shipped to Dr. Davis’ lab for gas chromatograph/mass spectrometer analysis to classify the disease status by the VOC profile and possibly identify chemical biomarkers. As there was a large overlap between the chemical peaks among the groups, the distinction between the groups was not very clear using linear classification techniques. However, using higher order machine learning algorithms, we were able to create a model that has > 85% classification accuracy. Specifically, a couple of data analysis methods were applied to this data set. First, through a peak-by-peak comparison, we detected 11 peaks (potential biomarkers) where we could see a visible difference between the peak amplitude distributions of two groups: 5 healthy vs. 15 infected samples (p<0.15). Working on the entire chromatogram data of the samples, principal component analysis and neural network model was used for a further quantitative examination of the seperability of these two groups. Based on the leave-one-out strategy, we obtained a promising result with the specificity being 80% and the sensitivity being 87%. This diagnosis result is a promising direction for our biomarker detection and disease diagnosis research of the next step. The results of this quarter were significant because we learnt that experimental on-field conditions are very challenging because the wind mixes the VOCs and makes securing the SPME fibers difficult, and the presence of other diseases such as melanosis and leaf minor on some of the plants impact our capability of getting a clean VOC profile for HLB. We have taken these into account for our next set of studies that are currently underway.
Research results from the first season were successfully completed, the results reported at the Florida State Horticultural Society (FSHS) meetings and at Packinghouse Day and the Indian River Postharvest Workshop in Sept. 2008, a manuscript published in the FSHS proceedings (http://www.fshs.org/; Proc. Fla. State Hort. Soc. 2008. 121:322’325), and results reported in previous FCPRAC progress reports. Several experiments were conducted since the 2007-08 season. Preharvest treatments consisted of 1) foliar K (8lb K2O), 2) foliar Mg (6% solution), 3) foliar K + Mg, 4) foliar Vapor Gard’ (1% or 2%), and 5) induced tree water stress. Treatments were conducted on White grapefruit twice, Valencia oranges once, and Flame red grapefruit once. So far, we have found less promising results with foliar K alone than we did in the previous season. In one experiment, foliar K even increased peel breakdown. The reason for this is unclear. However, applying a combination of foliar K and Mg or use of an antitranspirant resulted in a significant reduction in peel breakdown during postharvest storage. Application of foliar Mg (Epsom salt) alone showed a trend in reducing peel breakdown that, while not significant, is worth repeating. We are still conducting harvests from the Flame grapefruit block that was treated in October. In general, the fruit has been very healthy with fruit harvested in early Nov. showing very little decay or peel breakdown even after over 2 months storage at ambient temperatures. We have also conducted four sets of postharvest experiments that gave similar results as the previous year. Interestingly, postharvest treatments with Imazalil sporadically reduced peel breakdown as was noted the previous year. The reason for this is unclear but worth investigating further. We are currently setting up another experiment in different Flame grapefruit block that will include a cocktail of materials, in addition to the previous treatments, to see about reducing peel breakdown. Soil water content probes are installed in a water-stress and control plot and we have arranged to have leaf and peel samples for nutrient content.
Objective 1: We conducted tests with nutrient analyses of leaf samples collected from HLB-infected and healthy trees to establish relationships that could be used for (early) diagnosis of HLB. Starch accumulation in symptomatic leaves can significantly alter the interpretation of nutrient status when diagnosed on a dry weight concentration basis. When corrected for starch accumulation by using specific leaf area, Ca, Mg and B are consistently lower and K is higher in HLB-infected trees compared to healthy trees under the same fertilization program. These changes exist in both symptomatic and asymptomatic leaves from infected trees. Similarly, separation between asymptomatic healthy leaves and asymptomatic infected leaves could be achieved using the Diagnosis and Recommendation Integrated System (DRIS), particularly when assessing the Dry Mass Index (DMI) generated by the method. DMI integrates the imbalances caused by nutrient losses and starch accumulation in HLB-infected leaves, thus could indicate infection early on despite the absence of visible symptoms. We are currently sampling leaves from trees infected with other diseases (CTV, blight, phytophthora) to determine if these changes are HLB-specific and can be used as a diagnostic method. Due to stress-related abscission of HLB-symptomatic leaves from the exterior of infected canopies for much of the year, we are often forced to sample symptomatic leaves from within the canopy and asymptomatic leaves from the outside. We tested the effect of this unavoidable sampling position difference on the measured leaf nutrients analyzed in healthy trees and found no significant differences in 11 elements measured. Objective 2: Two field studies have been established to evaluate the effects of various foliar nutrient applications on the expression of HLB in infected trees by evaluating tree nutrient status, growth, yield and visual tree appearance through photographic documentation. The first of these trials is in a heavily infected mature Hamlin grove in south Florida. Since the initiation of the project the trees in this study have received four foliar applications of one of nine different treatments. Untreated trees serve as controls. The trees were harvested in December 2009. The second study is in a young (3-5 years old) commercial Valencia grove in Haines City. Treatments in this study have been underway for approximately 4 months and include fertigation in addition to foliar nutrient sprays. Our initial efforts at this site have been to demonstrate the ability to raise the levels of specific nutrients involved in plant defenses within trees. Since treatments began, B levels have been successfully raised to near toxic levels within infected and healthy trees using both foliar and fertigation applied B. This has demonstrated that good nutrient uptake can be achieved through the treatment methods in a relatively short period of time. Analysis of the large quantity of these studies have generated is still being analyzed as of the writing of this report. As soon as the analyses are complete information will be passed along to the Florida citrus community. Objective 3: A hydroponics system has been constructed in an HLB approved greenhouse at the CREC. Hamlin trees grown from rooted cuttings were purchased from a commercial nursery and are currently being established in the hydroponics system. Once the plants have made the transition from soil to hydroponics culture half of them will be inoculated with HLB by grafting material from greenhouse-grown trees with known HLB infections. All of the plants will be maintained with a complete nutrient solution until HLB symptoms develop on the inoculated trees. Previous experience with greenhouse graft inoculation indicates that this will take about 90 days. At that time, healthy and infected trees will be subjected to various changes in the nutrient solution composition. Based on the data from objective 1, the levels of Ca, Mg, and B and combinations of these three nutrients will be changed to determine plant response with and without HLB. Trees remaining in complete nutrient solution will serve as controls. This experiment will allow us to begin to separate nutrient and HLB effects on plant growth and understand the interaction of nutrition and HLB infection. Data from this project has been the basis for two Citrus Industry magazine articles in 2009. In addition, the data has been presented at Citrus Expo, the Florida State Horticultural Society conference and at six different regional grower meetings. An EDIS document about the general effects of plant nutrition on disease tolerance is being written.
Funding obtained from this grant has allowed us to develop a comprehensive management program that includes: sampling plans for monitoring psyllid populations and preemptive dormant sprays of broad-spectrum insecticides targeting adult psyllids that has been expanded to a cooperative area wide effort among the growers in the region. We also developed a wide range of alternatives including: (1) recurrent low volume application of horticultural oil, (2) selective and broad-spectrum insecticides for the growing season, and (3) strategies for conservation and augmentation of biological control. A survey of local managers of 106,148 commercial citrus acres in SW Florida indicated that there has been widespread adoption of these management strategies as indicated below under specific objectives of the project. (1) Assist growers to plan and implement effective strategies for psyillid control. All growers surveyed used dormant sprays during the 2008-2009 dormant season in cooperation with a voluntarily area wide program initiated by ourselves and promoted by Gulf Citrus Growers Association. Pilots reported 103,431 acres sprayed by air with an additional estimated 42,000 sprayed by ground, virtually all the commercial acreage in SW Florida. Field counts obtained from FDOACS-DPI and grower personnel trained by us indicated that up to 4 months later in May, treated populations were within 50-85% of winter numbers compared to a 28-fold increase in untreated blocks. Participation is expected to be even better this year and 73,500 acres were already sprayed aerially by 14 Jan. (2) Assist in development of efficient monitoring methods. The “tap” sampling method we developed is an effective and efficient tool for monitoring ACP adults, other pests as well as beneficial insects. We integrated this and a technique for assessing immature stages into a rapid and easy to use system promoted in Citrus Industry Magazine and fully explained on our website www.imok.ufl.edu. (3) Accelerate testing of new chemical tools for psyllid suppression. We completed 4 field evaluations of insecticides for ACP control in 2009 and submitted reports for publication in the ESA journal Arthropod Management Tests. These join 17 other published reports on insecticidal control of ACP available on the website. This year we found that foliar application of spirotetramat, and fenproximate with 435 Oil or NIS (spirotetramat) or 435 Oil alone during bloom suppressed psyllids for 2 months. (4) Evaluate the economic feasibility of psyllid management strategies. The cost of two aerial dormant sprays is less than $25/ac each and could be as low as $15/acre. The cost of monitoring psyllids using the tap is about $25 per block up to 100 acres so could be done 20 times per year for as little as $5/ac, and would avoid unnecessary sprays during the growing season that could cost up to $50/ac and cause secondary pest outbreaks of mites, scales and leafminers that are already occurring according to 70% of survey respondents. (5) Provide an information bridge between growers and researchers: Experimental results and recommended management programs have been presented at 18 extension meetings held across the State including two Production Managers meetings and two CCA trainings, as well as the Entomological Society of America (ESA), the Florida State Horticultural Society (FSHS), the Florida Entomological Society (FES), Citrus Huanglongbing (HLB) & Potato Zebra Chip (ZC) Conference in Mc Allen, TX, and the XIII Simposio Internacional de Citricultura. CD Victoria, Mexico. Ninety one percent of surveyed growers professed to be satisfied at some level by their present management programs with 85% of respondents monitoring ACP more than 3 times per year. We are working on emphasizing the use of this information to make informed decisions to spray insecticides only when needed. Stansly, P. A., J.A. Qureshi, and H.A. Arevalo. 2009. Why, when and how to monitor and manage Asian citrus psyllid. Citrus Industry 90(3):24-26. Stansly, P.A., H.A Arevalo, M. Zekri, and R. Hamel. 2009. Cooperative dormant spray program against Asian citrus psyllid in SW Florida. Citrus Industry 90(10):14-15
Funding obtained from this grant has allowed us to develop a comprehensive management program that includes: sampling plans for monitoring psyllid populations and preemptive dormant sprays of broad-spectrum insecticides targeting adult psyllids that has been expanded to a cooperative area wide effort among the growers in the region. We also developed a wide range of alternatives including: (1) recurrent low volume application of horticultural oil, (2) selective and broad-spectrum insecticides for the growing season, and (3) strategies for conservation and augmentation of biological control. A survey of local managers of 106,148 commercial citrus acres in SW Florida indicated that there has been widespread adoption of these management strategies as indicated below under specific objectives of the project. (1) Assist growers to plan and implement effective strategies for psyillid control. All growers surveyed used dormant sprays during the 2008-2009 dormant season in cooperation with a voluntarily area wide program initiated by ourselves and promoted by Gulf Citrus Growers Association. Pilots reported 103,431 acres sprayed by air with an additional estimated 42,000 sprayed by ground, virtually all the commercial acreage in SW Florida. Field counts obtained from FDOACS-DPI and grower personnel trained by us indicated that up to 4 months later in May, treated populations were within 50-85% of winter numbers compared to a 28-fold increase in untreated blocks. Participation is expected to be even better this year and 73,500 acres were already sprayed aerially by 14 Jan. (2) Assist in development of efficient monitoring methods. The “tap” sampling method we developed is an effective and efficient tool for monitoring ACP adults, other pests as well as beneficial insects. We integrated this and a technique for assessing immature stages into a rapid and easy to use system promoted in Citrus Industry Magazine and fully explained on our website www.imok.ufl.edu. (3) Accelerate testing of new chemical tools for psyllid suppression. We completed 4 field evaluations of insecticides for ACP control in 2009 and submitted reports for publication in the ESA journal Arthropod Management Tests. These join 17 other published reports on insecticidal control of ACP available on the website. This year we found that foliar application of spirotetramat, and fenproximate with 435 Oil or NIS (spirotetramat) or 435 Oil alone during bloom suppressed psyllids for 2 months. (4) Evaluate the economic feasibility of psyllid management strategies. The cost of two aerial dormant sprays is less than $25/ac each and could be as low as $15/acre. The cost of monitoring psyllids using the tap is about $25 per block up to 100 acres so could be done 20 times per year for as little as $5/ac, and would avoid unnecessary sprays during the growing season that could cost up to $50/ac and cause secondary pest outbreaks of mites, scales and leafminers that are already occurring according to 70% of survey respondents. (5) Provide an information bridge between growers and researchers: Experimental results and recommended management programs have been presented at 18 extension meetings held across the State including two Production Managers meetings and two CCA trainings, as well as the Entomological Society of America (ESA), the Florida State Horticultural Society (FSHS), the Florida Entomological Society (FES), Citrus Huanglongbing (HLB) & Potato Zebra Chip (ZC) Conference in Mc Allen, TX, and the XIII Simposio Internacional de Citricultura. CD Victoria, Mexico. Ninety one percent of surveyed growers professed to be satisfied at some level by their present management programs with 85% of respondents monitoring ACP more than 3 times per year. We are working on emphasizing the use of this information to make informed decisions to spray insecticides only when needed. Stansly, P. A., J.A. Qureshi, and H.A. Arevalo. 2009. Why, when and how to monitor and manage Asian citrus psyllid. Citrus Industry 90(3):24-26. Stansly, P.A., H.A Arevalo, M. Zekri, and R. Hamel. 2009. Cooperative dormant spray program against Asian citrus psyllid in SW Florida. Citrus Industry 90(10):14-15
The Citrus Greening Bibliographical Database [ http://swfrec.ifas.ufl.edu/hlb/database/ ] is managed by the Entomology group at the University of Florida-IFAS in Immokalee, in collaboration with the Center for Library Automation at the University of Florida in Gainesville. The database was constructed from the ground up, and after less than a year contains 1,770 references, 880 of which are linked to original sources. With the help of our users, we are continually searching for new information, cross-referencing existing data for accuracy, and updating the database regularly. Ninety percent of the entries are in English, with the remaining 10% in Spanish, Portuguese, Afrikaans, Japanese, Chinese, French, German, Vietnamese, Dutch, Farsi, Arabic, Czech and Hebrew. The database includes references from refereed and non-refereed research and extension publications, presentations, websites, proceedings, grant reports, periodicals, dissertations, book chapters and complete books. Entries are the product research worldwide on the various aspects of Huanglongbing (HLB): the associated bacteria (Candidatus Liberibacter spp.), vectors [Diaphorina citri Kuwayama and Trioza erytrea (Del Guercio)], plant and vector effects of the disease, and management tactics. The database was designed as a tool for growers, researchers, and students to access HLB related information. It is also readily accessible to the general public through a user-friendly interface. This project has been promoted in presentations at several national and international meetings in the U.S and Mexico to invite researchers to use the database and to contribute to it by submitting their publications. We have received a positive response from many researchers attending these meetings, and several have sent us their presentations and publications to be included in the database. During 2009, the database received close to 5,000 visits. The majority of those connecting to the Internet Protocol (IP) addresses are from Florida and California. However, we have also received visits from IPs located in several other countries such as China, Mexico, Colombia, and Brazil. These results indicate that the database is becoming an integral source of information for a growing number of researchers, growers, and the citrus community. Pending continued funding, unfortunately not requested in the original proposal, our goal for the second year of the project is to continue providing this service by including the most current information as it becomes available, increasing the number of linked documents, and creating an interactive forum in which researchers and growers can exchange HLB related information. Presentations related to the citrus greening database database: – Arevalo, H. A., A. B. Fraulo, and P. A. Stansly. 2009. Update on the Citrus Greening Bibliographical Database. (Poster). HLB/ ZC meeting, McAllen Texas – Arevalo. H. A. and P. A. Stansly. 2009. Monitoreando el Psillydis Asiatico de los Citricos en el Campo y el Internet (Monitoring the Asian Citrus Psyllid in the field and the Internet). XIII Simposio Internacional de Citricultura. CD Victoria, Tamaulipas, Mexico. – Arevalo, H. A., A. B. Fraulo, and P. A. Stansly. 2009. Update on the Citrus Greening Bibliographical Database. (Poster). Florida Entomological Society Annual Meeting. Ft. Myers, FL July 2009 – Arevalo, H. A., G. Snyder, and P. A. Stansly. 2008. The citrus greening Bibliographical Database, a New Tool for Researchers, Students and Growers. (Poster). International Research Conference on Huanglongbing. Orlando, FL December 1-5, 2008
The Citrus Greening Bibliographical Database [ http://swfrec.ifas.ufl.edu/hlb/database/ ] is managed by the Entomology group at the University of Florida-IFAS in Immokalee, in collaboration with the Center for Library Automation at the University of Florida in Gainesville. The database was constructed from the ground up, and after less than a year contains 1,770 references, 880 of which are linked to original sources. With the help of our users, we are continually searching for new information, cross-referencing existing data for accuracy, and updating the database regularly. Ninety percent of the entries are in English, with the remaining 10% in Spanish, Portuguese, Afrikaans, Japanese, Chinese, French, German, Vietnamese, Dutch, Farsi, Arabic, Czech and Hebrew. The database includes references from refereed and non-refereed research and extension publications, presentations, websites, proceedings, grant reports, periodicals, dissertations, book chapters and complete books. Entries are the product research worldwide on the various aspects of Huanglongbing (HLB): the associated bacteria (Candidatus Liberibacter spp.), vectors [Diaphorina citri Kuwayama and Trioza erytrea (Del Guercio)], plant and vector effects of the disease, and management tactics. The database was designed as a tool for growers, researchers, and students to access HLB related information. It is also readily accessible to the general public through a user-friendly interface. This project has been promoted in presentations at several national and international meetings in the U.S and Mexico to invite researchers to use the database and to contribute to it by submitting their publications. We have received a positive response from many researchers attending these meetings, and several have sent us their presentations and publications to be included in the database. During 2009, the database received close to 5,000 visits. The majority of those connecting to the Internet Protocol (IP) addresses are from Florida and California. However, we have also received visits from IPs located in several other countries such as China, Mexico, Colombia, and Brazil. These results indicate that the database is becoming an integral source of information for a growing number of researchers, growers, and the citrus community. Pending continued funding, unfortunately not requested in the original proposal, our goal for the second year of the project is to continue providing this service by including the most current information as it becomes available, increasing the number of linked documents, and creating an interactive forum in which researchers and growers can exchange HLB related information. Presentations related to the citrus greening database database: – Arevalo, H. A., A. B. Fraulo, and P. A. Stansly. 2009. Update on the Citrus Greening Bibliographical Database. (Poster). HLB/ ZC meeting, McAllen Texas – Arevalo. H. A. and P. A. Stansly. 2009. Monitoreando el Psillydis Asiatico de los Citricos en el Campo y el Internet (Monitoring the Asian Citrus Psyllid in the field and the Internet). XIII Simposio Internacional de Citricultura. CD Victoria, Tamaulipas, Mexico. – Arevalo, H. A., A. B. Fraulo, and P. A. Stansly. 2009. Update on the Citrus Greening Bibliographical Database. (Poster). Florida Entomological Society Annual Meeting. Ft. Myers, FL July 2009 – Arevalo, H. A., G. Snyder, and P. A. Stansly. 2008. The citrus greening Bibliographical Database, a New Tool for Researchers, Students and Growers. (Poster). International Research Conference on Huanglongbing. Orlando, FL December 1-5, 2008
Obj. 1. Carry out DNA bar coding to establish the identity and diversity (if it exists) in south Florida (Stansly, Brown). PCR using mtCOI primers amplify a 850 bp fragment from colony psyllid adults; DNA sequencing confirmed. FL field collections from citrus and other spp. obtained from cooperator collections in previous years; 2010 spring and summer FL (and elsewhere) field collections are planned when psyllid dispersal is underway. Obj. 2. Employ qPCR to detect Ca. Liberibacter presence (or absence) in the psyllid colony cohorts over different AAPs, for immatures and adults (Roberts, SWFREC, HLB diagnostics lab). The FL psyllid rearing system is fully functional and Ca. Liberibacter-infected and bacterium-free colonies are being maintained. Climatic factors affect growth of colony host plants (Citrus, Murraya) and seem to influence bacterial titer in plants and in the psyllid vector instars. Using qPCR (DNA isolation) we are monitoring over time the detectable bacterial titer in psyllid colonies and the host plant, toward quantitative detection in fresh flush plant tissue and in whole insect bodies. Obj. 3a. Carried out exploratory analysis of psyllid organs (guts) for HLB detection using qPCR (consulted methods of: Li et al. 2006 for plant material; Manjaunth et al. 2008 for psyllids). Obj. 3. Using the colonies (analyzed above by qPCR), localize Ca. Liberibacter in whole psyllids and dissected organs to develop a gross anatomical road map of Ca. Liberibacter accumulation in organs, tissues, and cells (adult and 5th instar psyllids) (Cicero, Brown). [In our hands, D. citri does not render a signal when processed with the standard protocol proven for whiteflies (Gottlieb et al 2006)]. We have observed positive fluorescence by in-situ hybridization (FISH) with a Cy5-HLB 16S rDNA probe in variously prepared samples. We have observed a robust signal in infected adult psyllids cut in half to improve infiltration. These results represent extensive experimentation to develop and optimize fixation, dehydration, decolorization and hybridization procedures for this species. After replicated experimentation and further validation of this and several other probes, probes can then be applied to thick sections, which in turn can be compared to ultrathin TEM sections (gold probes) (Obj. 4) for precisely localizing interactions between HLB and the organs involved in transmission. Obj. 4. Using the resultant thick section road map, elucidate at the TEM level, specific organs, tissues, and cells where Ca. Liberibacter accumulates (Cicero, Brown). Using TEM we observed bacterial cells on the outside of the esophagus and of the PSGs. By SEM, bacteria were observed on the outside and inside of the midgut epithelial cells. This suggests that Ca. Liberibacter bacterial colonies exist in multiple locations, perhaps adhering to the upper alimentary canal and midgut, and elsewhere. We also have observed by TEM groups of phage-like particles, intercellularly in Liberibacter-infected adult psyllids (unknown significance). We have compiled an anatomical, serial, ultrathin-section library of infected, 5th instar D. citri larvae. It is apparent that our focus must include both adult and late immature instars based on recent results of transmission studies. Therefore we are developing procedures for adult and immature instars to extirpate and SEM-stage digestive organs, followed by TEM. We also have carried out initial SEM exploration of extirpated guts from adults and 5th immature instars.
This work will determine if certain alternative plant species are better hosts for the suspected HLB bacterial pathogens (Ca. Liberibacter asiaticus (Ca. Las), Ca. Liberibacter americanus (Ca. Lam) and Ca. Liberibacter africanus (Ca. Laf)) and can serve as a reservoir hosts for infection to citrus. During this quarter work was again was performed at all four investigator locations. At the University of Florida, CREC Lake Alfred, quantitative real time PCR (qPCR) was done on Severinia buxifolia (orange boxwood) and showed that it was a good host for the Las bacterium. HLB healthy Asian citrus psyllids were allowed to feed on HLB infected S. buxifolia and 20-30% of them were found PCR positive. Initial PCR tests of sweet orange plants that psyllid transmissions were previously done from the infected Severinia produced some positive plants. Additional PCR testing will be done after further incubation. This research was done by Hao Hu a graduate student studying with Dr. Brlansky. Real time PCR results on rough lemon (C. jambhiri) showed infection of both symptomatic as well as asymptomatic shoots and leaves with similar PCR values for all shoots. Rough lemon continues to grow even when infected with Ca. Las. Rootstocks from field experiments are being tested to determine infection. Various sweet orange cultivars were inoculated with Florida Ca. Las and one cultivar tested PCR negative in multiple tests. At the Texas A&M Citrus Center, Weslaco psyllid feeding tests continued on the rutaceous plants that are established there. More replications of psyllid feeding experiments were done with Esenbeckia berlandieri (jopoy), Amyris madrensis (torchwood), Choisya ternata and C. arizonica. All are feeding hosts for the psyllid. As previously reported egg laying was found on torchwood but the psyllids did not complete development. Egg laying and nymphal development to adults were found on C. ternata. This work is part graduate student Jose Sandoval’s research with Dr. da Graca. At the USDA, ARS, Beltsville quarantine greenhouse. Graft inoculations were done to Murraya paniculata with exotic Ca. Ca. Lam and Ca. Laf isolates and will be tested soon. No symptoms are apparent. Work with dodder as an alternative host was completed studying the plant infection process and for its use to transmit all Liberibacters to plants that are not graft compatible with citrus. A manuscript was submitted for publication in Phytopathology by Drs. Hartung and Brlansky. In this publication dodder became infected and phloem necrosis occurred similar to that in citrus. In addition we found that the Liberibacter exists in two morphological forms in the dodder similar to that seen in citrus and periwinkle. In addition we found the intermediate between the two forms which proves that both forms are the same bacterium. At the USDA, ARS, FDWSRU, Ft. Detrick, MD a manuscript entitled ‘The relevance of Murraya paniculata and related species as potential hosts and inoculum reservoirs of ‘Candidatus Liberibacter asiaticus’, causal agent of Huanglongbing (HLB)’ was completed and accepted for publication. Authors from the grant include Drs. Damsteegt, Brlansky and Schneider. The details of this work were reported in earlier reports. Psyllid transmissions from HLB (Ca. Laf and Ca. Las (Thailand strain)) infected sweet orange to Severinia buxifolia are underway.
We have now developed a series of flexible stochastic models to predict the temporal increase and spatial spread of diseases. The models were initially characterized for citrus canker spreading in plantation and urban (backyard) environments. They have subsequently been extended to HLB in this project. The models can be used in a number of ways: to predict spread and to analyze the effectiveness of control strategies. Most attention has been given to spread within plantations, including allowance for proliferation of infection along boundaries in response to vector behavior. The models can readily be extended to consider spread at larger scales including spread through heterogeneous environments up to State-wide scales. We have also considered the effects of uncertainties in the distribution of host crops for example the effects of small areas of crop that may not be recorded but which can act as ‘bridges’ in transmitting disease. The effects of uncertainty in parameter estimates for dispersal parameters and transmission rates have also been included. Additional computer-friendly formulations of the models have also been developed to aid in education of stake-holders to illustrate the effects of uncertainty in predicting future disease spread and the effectiveness of alternative methods of control. Estimation of parameters for dispersal of HLB poses considerable statistical challenges, especially where trees may become infectious before they are symptomatic/detected. Here we use an SEIDR model (Susceptible, Exposed (latently infected but not yet infectious), Infectious but not yet symptomatic/detected, Detected and infectious and Removed trees). Using MCMC methods, and extensive data from Southern Gardens for successive snapshots of the occurrence of symptomatic/detected trees in known populations of susceptible trees, we are able to estimate model parameters for the transmission rates and dispersal kernel for the disease. Current work is focused on the differential effects of host age on epidemiological parameters as well as variability across the plantation. From these it is possible to allow for uncertainty in the parameters as well as variability over time and through space. The uncertainty is then incorporated in models to predict spread and to allow for uncertainty in the efficiency and comparison of control methods.
Methods and models for the control of HLB disease of citrus. Citrus huanglongbing (HLB) is the most serious disease of citrus worldwide and presently for the very existence of citrus industry of Florida. The approach is two-fold: First was the examination of the effect of various control strategies on HLB, in control plots, established in 2007. I this phase of the study, five treatments were examined: Minimal control, Insecticide vector control, Roguing, Roguing via PCR+, and Comprehensive. Results indicated that although treatments were significantly different, there was no benefit of any control treatment over another. Small differences were due to plot location, not treatment effects. The tests are being repeated, however, the results point to a need for regional control strategy and that small plantings that can’t control neighbors cannot control the disease. Our estimates indicated that for each tree with visual symptoms, there were an average of 13 (range 2-52) that were infected but asymptomatic, i.e., infections that have occurred over the duration of the epidemic but that have not yet expressed symptoms. New plots have been established at the USDA, ARS , in Fort Pierce, Florida and the data collection is under way. Psyllids are also being trapped (in each plot) to estimate populations and correlate with disease progress in each plot. To date 3 HLB+ trees have been identified within the plots, but no statistical difference among plots is yet apparent. Differences should become noticeable in 2010. The second approach is to develop epidemiological models of HLB disease dynamics which improve the understanding of vector-driven disease transmission and analyze disease control policies aimed at disrupting vector population dynamics. In previous and current work we have developed a model for citrus canker. This is the basis upon which we have built a preliminary model for HLB. The HLB simulation model is stochastic based on biological, epidemiological, and meteorological parameters using Markov Chain Monte-Carlo simulation methods and SIR modeling protocols. The model is fit to the HLB data collected in various observed epidemics from Florida and SE Asia by thousands of simulations. Linked-differential equations are used that describe the temporal increase in HLB infected trees and explicitly characterize the population dynamics of the vector. The effectiveness of different disease control measures such as intercropping with guava, roguing and insecticide use will eventually be analyzed via this model and a suite of mathematical tools to identify the most effective strategies. HLB data sets will be correlated with various disease mitigation strategies/events from our epidemiology trials in Florida. The stochastic models allow testing of multiple disease management strategies in thousands of simulated epidemics to determine which will have the optimum effect and in what combination these methods can best be deployed for maximal disease control. The HLB model will continue to be augmented and improved over the next 1.5 years but are well near completion. Data continue to be generated for multiple test plots to parametrize the models. Models are being validated against actual data to ensure correct estimation of disease dynamics. New data sets have been acquired from south Florida and will be used for validation and further model improvement during the first part of 2010. We are also setting up an extensive field experiment at Fort Piers in 2010 to examine the latency period between transmission and infection. This has never been documented with precision in field trees and is a key component in he model.
Disease control of citrus huanglongbing (HLB) by interplanting with guava. HLB is the most devastating disease of citrus worldwide and presently threatens the existence of the citrus industry in Florida. In Vietnam guava has been shown to be an effective deterrent to HLB. For all plots and experiments, Guava trees, (Vietnamese white cultivar) were propagated and grown to appropriate size requiring about one year. Guava vs no guava nurseries: Two nursery sites, a guava protected citrus nursery versus and unprotected nursery, have been established. Disease free, PCR-negative citrus trees (2 sweet orange and 1 grapefruit cultivars) were located in the protected and unprotected plots in June 2009. The guava were established over a year ago and grown to appropriate size as indicated in Vietnam. Trees are assayed for HLB every 60 days, and are in their second assay. Psyllid populations are also being monitored continuously every two weeks within plots to document any repulsion of the vector due to guava. To date no HLB+ plants have been identified in the nursery plantings after multiple assays. Citrus/guava interplantings: 2 commercial plantings with multiple replications each have been established. This has taken considerable time. Guava trees were propagated and grown to transplant size. These were then out planted and grown for a year per Vietnam protocols. One trial was established in a commercial orchard with collaborators in Southern Gardens Citrus. A second trial planting was established at the USHRL Picos Farm in Fort Pierce. To date no HLB+ plants have been identified in the USHRL plantings after multiple assays. A severe frost last winter affected both the USHRL and the Southern Gardens plots causing a delay in the experiment. Damage was extensive in both plots. The damage to the guava was overcome by pruning and replanting of damaged guava trees. Renovation of the USHRL plot was less, and the guava have now been interplanted with the citrus as of August 2009 in the USHRL Picos Farm plot. To date no HLB+ plants have been identified in the USHRL plantings after multiple assays. In the Southern garden plots, damage was more severe and the guava have now been renovated sufficiently that the Souther Gardens plot will be interplanted with citrus. The guava have now been interplanted with the citrus as of November 2009 in the USHRL Picos Farm plot. To date no HLB+ plants have been identified in the SG plantings after multiple assays. Both nursery and field citrus trees are assayed for HLB every 60 days, and are in their second assay. Psyllid populations are also being monitored continuously every two weeks within interplanted plots to document any repulsion of the vector due to guava. Data collection continues and is currently ongoing.
Cultural Practices to Prolong productive Life of HLB Infected Trees. Through the summer of 2009 trees receiving the nutritional/SAR trees were vigorous and green with only normal HLB symptoms typical of HLB infected trees. This fall of 2009 at our 100% infected trial site we have observed numerous visual HLB symptoms in multiple sectors of the trees. Although our other two sites with lower infection rates of 50% and 15-20% had increased visual symptoms of HLB this fall and winter typical of greening trees, the symptomatic leaves were not as abundant as in the more infected trees. Fruit color break from green to yellow in Valencia oranges began early (late September) at the site with 100% infected trees and was not observed at the two other sites where fruit remained green as typical in uninfected trees. Fruit drop has been observed this fall only in the 100% infected trial. Data collected show treatments with the complete Maury Boyd cocktail, with or without hydrogen peroxide, and the foliar feed macronutrients in a typical range of 2 to 5% fruit drop. The other treatments ranged from 8 to 17% fruit drop. No abnormal coloring of fruit or fruit drop has been recorded in sites with 40-50% or 15-20% HLB infection. Evaluation of Systemic Acquired Resistance Inducers Combined with Psyllid Control to Manage Greening infected Groves We are monitoring Asian citrus psyllid (ACP) populations and Can. Libericacter asiaticus (CLas) titer in plants and psyllids, and impacts in a young citrus block as the result of the main effects and interaction of two treatments: 1) micronutrients + systemic acquired resistance inducers (Micro+SAR), and 2) Psyllid chemical control applications based on scouting. This trial was designed as a replicated (n=4) complete block for a 2×2 factorial experiment on a 12-acre commercial block of now 7-years-old ‘Valencia’ oranges on ‘Swingle’ rootstock that was held back by defoliation for canker control in 2006. Adult ACP populations have been maintained three times higher on the average, and up to 50 times higher on occasion in insecticide-treated plots compared to treated plots throughout both years of the experiment. Insecticides have been applied 6 times during that period, either as a single scheduled dormant spray per year or when adult populations exceeded a threshold of 0.5 per biweekly ‘tap’ sample made by striking a limb while holding a laminated sheet underneath to catch falling psyllids. The Micro+SAR treatment has had no effect on ACP populations. ACP adults and nymphs have been collected 4 times since Nov 2008 for PCR analysis along with a leaf sample from the most symptomatic branch from every 5th tree in the block. Insect samples were sent to our collaborators at the USDA in Riverside, CA for analysis and plant tissue samples were processed at the PCR laboratory at SWFREC. In November 2008 and April 2009, 34% of the plants were positive for HLB over all treatments using a cutoff CT value of 36. This jumped to from 86 to 100% of the plants positive in Aug. 2009 with no significant differences among treatments by ANOVA. However, the two treatments employing insecticidal control had significantly higher Ct values (avg. 28.5) than the two non-insecticide treatments (avg. 25.5) indicating lower CLas titer in plants protected by insecticides. Yield was assessed on the entire block, plot by plot in March 2009. Despite the high percentage of infected trees, we observed that trees receiving both insecticidal and nutritional treatments produced 1.32’0.15 boxes per tree of fruit, a 30+% increase over 0.95-0.99 boxes per tree from the other 3 treatments. Fruit was analyzed by the CREC pilot plant in Lake Alfred and no significant treatment effects on fruit or juice quality were observed. These results could be interpreted to mean that the Micro+SAR package is capable of staving off negative impacts of HLB if CLas titer can be maintained below some threshold level through psyllid management. However, we will not feel comfortable with this conclusion until we see similar results from the March/April 2010 harvest.
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