Objective 1: Survey and confirmation of HLB in seedlings from HLB-affected trees. 500 seedlings grown from seed extracted out of mild to severely HLB-affected fruit from Pineapple orange and Murcott tangor groves in Hendry Co. were assayed for HLB detection. All seedlings were negative for HLB detection by PCR in repeated assays in July and Nov. 2009. Objective 2: Thermotherapy of HLB-affected seed for the same seed sources under Objective 1 were treated at 125F, 130F and 135F to test for the effect of heat treatment on HLB detection in seedlings. Thermal treatments were lethal to Murcott, but not Pineapple seed. The emergent seedlings at 125F (142), 130F (98) and 135F (97) were PCR tested. All seedlings were negative for HLB in repeated PCR assay in July and Nov. 2009. Objective 3: In October 2009, seed source trees in two Florida nurseries were found positive for HLB by FDACS-DPI (Nursery 1) or Southern Gardens Diagnostic Lab (Nursery 2). Discovery of infected seed source trees in two Florida citrus nurseries identifies a potential (but unconfirmed) risk of outdoor seed source trees acting as a source of inoculum for introduction into nursery propagations. In Nursery 1, seed was collected from symptomatic branches of two sources of Swingle citrumelo, four sources of ‘Kuharske’ Carrizo citrange and one source of Cleopatra mandarin. In Nursery 2, seed was collected from one source of Sekwasha mandarin. From 63 to 205 seedlings from each source will have enough leaves for testing in February 2010.
Between Oct. 5th and 30th, five experiments were conducted that included Fallglo (1 time), sunburst (2 times), red grapefruit (4 times), and navel oranges (1 time). Fruit were treated on a commercial packingline (3 experiments) or on the Indian River Research and Education Center research line (2 experiments). Treatments included 1) full wash (brush bed + high-pressure wash) + waxing (carnauba), 2) full wash, 3) brush bed only, 4) brush bed with brushes rotating half normal speed, 5) high-pressure wash (HPW) only, 6) HPW for 10 seconds, 7) HPW for 5 seconds, 8) running fruit only over PVC rollers, and 9) a control (not washed or waxed). On the commercial line, fruit remained on the brush bed for ~ 1 min. 10 seconds, and on the full HPW for ~35 seconds. Normal brush rotation speed was ~100 rpm. Fruit were also evaluated for how surface dirt obscured the ability to grade the fruit for canker and other grade defects. Fruit from all treatments were degreened under simulated commercial conditions (5 ppm ethylene, 85 oF, 95% RH) and color development and weight loss measured almost daily. Fruit were subsequently stored and evaluated for the development of decay and disorders during storage under ambient conditions (~70-75 F). In general, all very early season Fallglo fruit were relatively clean and did not need washing for adequate grading. This changed somewhat by the end of October when grapefruit that received more extensive washing (i.e., full washing) was significantly easier to grade compared to unwashed fruit, and HPW producing fruit with intermediate gradability. However, even minimally washed fruit were sufficient for adequate grading. These experiments need to be repeated next season to determine variability in initial fruit cleanliness from year to year. Washing and waxing the fruit gave the greatest inhibition of degreening, almost stopping color development completely. Compared to preliminary results in 2008, results again showed that full washing of fruit on both the brush washer and HPW, or washing on the brush bed along inhibited degreening significantly more than did washing fruit only as they passed over the HPW. Fruit that were not brushed at all, but only passed over rollers experience a slight, but significant delay in color development compared to the control, but the delay was relative minor compared to the other washing treatments. As the season progresses, fruit exterior surfaces become more soiled with dirt and sooty mold that makes grading more difficult without washing. Preparations are currently underway to repeat the wash treatments of mid-season fruit to evaluate washing treatments that result in enough cleaning to allow adequate grading. The goal is to identify the minimum amount of cleaning that allows adequate grading of fresh citrus before dumping fruit on the main packingline during different times of the season. The earlier unmarketable fruit can be removed from the market chain, the less money is spent on these fruit. Furthermore, grading fruit earlier in the process could allows sales departments earlier knowledge of the amount, size, and quality of packable fruit that will be available to sell. For fruit going to export markets that tightly restrict shipment of fruit with canker symptoms, removing fruit before dumping on the main packingline offers another layer of protection against fruit with canker making it into these restrictive markets.
To date, three greenhouse/growth chamber studies have been completed, testing the effects of six different commercial formulations of plant growth regulators (PGRs) on the fitness (survival and oviposition) of the Asian citrus psyllid. The PGRs tested include Apogee (prohexadione calcium), Profile (paclobutrazol), Embark (mefluidide), Atrimmec (dikegulak-sodium), Sumagic (uniconazole), and Cycocel (chlormequat chloride). In both of the studies that included Atrimmec, significant phytotoxicity was observed in the form of foliage burn and dieback, thus preventing new growth and psyllid oviposition and survival. The other PGRs reduced shoot growth between 3 and 50% relative to untreated controls. Significant differences were observed in the number of eggs laid by psyllids on plants treated with different PGR’s. Oviposition was reduced by approximately 85% on plants treated with Embark and Sumagic, and by 65% on plants treated with Apogee and Profile relative to untreated controls, whereas oviposition was increased by 30% on plants treated with Cycocel Significant differences in survivorship of psyllid nymphs to adults were observed between the different PGR treatments. Survival was lowest for plants treated with Profile (7%), followed by Sumagic (36%), untreated control plants (45%) and Apogee (47%). The highest survivorship rates were observed on Cycocel (73%) and Embark (68%) treated plants. Survivorship of psyllids on plants treated with Profile was significantly lower than on plants treated with Cycocel and Embark, while all the other treatments did not differ significantly. There were significant differences in the weight of the adult psyllids reared on plants treated with the different PGRs. Adults emerged from plants treated with Sumagic weighed less than all the others with the exception of psyllids reared on Cycocel treated plants. Weight of psyllids reared on plants treated with Profile, Embark and Apogee did not differ significantly from the control. These results have been presented at the Plant Growth Regulation Society of America (Aug 2009), the Entomological Society of America (Dec 2009) and the Western Plant Growth Regulator Society (Jan 2010). It is important to note that none of the products tested to date are currently labeled for use on citrus, although Apogee and Sumagic are currently labeled for use on apples and vegetable transplants, respectively, whereas the others are only labeled for ornamental crops. Thus future greenhouse and growth chamber studies will focus on determining the plant physiological and/or metabolic changes that are occurring which are affecting psyllid behavior. Understanding these changes may allow for the selection of plants with these traits inherent or their selection in plant breeding efforts, or open avenues of research into other means of psyllid control by targeting specific biological needs of the insect. In addition, other rates and chemistries of PGRs will be tested. Field trials are planned to begin the spring of 2010.
>Work is underway in the Wang lab to understand the mechanism by which the HLB bacterial infection leads to phloem plugging and necrosis as insufficient bacteria are present in the phloem to directly plug the phloem. The procedure to obtain virulence factors from Ca. L. asiaticus was described in our last report. More than 10 potential virulence factors from the bacteria were cloned into the vector TMV30bGFP by PCR amplification and digestion with the restriction enzymes Pac1 and Xho1. The insertion of these genes in the TMV vector was confirmed by PCR amplification using gene specific primers, followed by sequencing. In vitro transcription was done using T7 RNA polymerase. The transcripts were then used to inoculate 4-5 week old Nicotiana benthamiana plants. Some plants expressed symptoms about 7 days after inoculation. The symptoms shown by the empty vector and the vector containing the potential virulence factors are being monitored. Of the 10 confirmed constructs, one construct has been shown to cause dramatic symptom changes in the Nicotiana leaves. Currently, this gene is being studied in citrus for further confirmation. Ideally these constructs should be expressed only in citrus phloem where symptom expression would more closely mimic HLB. Dr. Grosser has successfully used a construct that leads to only phloem expression of the inserted gene. This procedure will be tried for the virulence factors next year. Successful identification of the important virulence factors should help reveal the virulence mechanism of Ca. L. asiaticus, which factor(s) promote phloem disruption and possibly provide hints for management of HLB. >Transgenic approaches to disrupt HLB associated callose-phloem protein plugging and phloem dysfunction in citrus continue. Buds of the transgenic grapefruit trees that over-express 1, 3-. glucanase were grown out on Macrophylla rootstocks and challenged with HLB by bud inoculation. Only 4 of 44 plants had successful bud take and none of these show any symptoms 4 months after the inoculation attempt. The other plants will be re-inoculated with infected buds or psyllids in February after growth resumes. The citrus ‘-1,3-glucanases gene from Valencia embryogenic callus and young leaves has been cloned and multiple copies will be inserted in various citrus to see how these plants react to HLB infection. Five variations of Agrobacterium mediated or protoplast transformation using various plasmids are now perfected in the Grosser lab. Using these methods over-expressed transgenic plants are being developed and will be tested to see if production of ‘-1,3-glucan and phloem protein is blocked and to determine if these plants will show no or reduced symptoms when infected with HLB. Reaction of plants that over production these compounds will also tell us something about their role in symptom development. >Evaluation of field samples by TEM for the ratio of smooth to filamentous plugging continues. Field grown seed source trees of Swingle citrumelo were found with + PCR reactions for HLB. Leaf samples of these were fixed for examination for typical phloem plugging and necrosis symptoms. Further surveys will continue for any Carrizo seed source trees with HLB. Carrizo showed less symptoms than Swingle in greenhouse and growth chamber challenges with HLB. Based on lack of symptom development when infected, Poncirus trifoliata is another candidate for detailed phloem examination for possible tolerance to the HLB bacteria.
This progress report summarized research activities from October 15, 2009 to January 15, 2010. During this period, we continued working on (1) ‘Identification of additional Simple Sequence Repeat (SSR) loci and design and validation of new Las SSR markers’ and (2), ‘Analyses of genetic diversity of HLB Las populations. Followings are the summary of research activities and accomplishments during this period. Previously, we reported that we had designed and experimentally evaluated 40 new Las SSR primers. Among them, 25 primers showed clean and single amplified bands with expected amplicon sizes while the rest of 15 primers showed multiple bands when PCR evaluations were conducted. We then re-evaluated 15 primers by adjusting annealing temperature and Mg++ concentrations. PCR experiments were conducted with annealing temperatures 55oC, 57oC and 59oC along with 1.0 mM 1.5 mM and 2.0 mM Mg++ concentrations using an ABI Veriti thermal-cycler with a temperature gradient heating block (ABI, Foster City, CA). The results showed that PCR amplification conditions with 57oC and 1.0 Mg++ improved the specificity of PCR amplification. 8 primers that showed clean and single bands under this condition were therefore selected. Those primers that passed quality test were then subjected to discrimination power test. Total 33 primers (8 primers and 25 primers) were amplified with six asiaticus-infected DNA samples, 2 from Florida, 2 from India and 2 from China, respectively. Amplified PCR products were then separated by the high resolution of 5% polyacrylamide gels. Gels were visualized by silver staining. 16 out of 33 primers showed polymorphism with alleles ranging from 150bp to 320 bp per locus. Seven of SSR primers were synthesized with 5’-labeled fluorescent dyes (FAM, NET, VIC) and assayed by an ABI 3130 Genetic Analyzer. All rest of primer evaluation will be completed in next three months. We conducted global genetic assessment of asiaticus populations. A total of 166 HLB isolates representing four populations from US Florida, Brazil, India and China were analyzed using multilocus SSR markers. Based allele frequencies of SSR marker loci across four populations, a data matrix that consisted of 71 alleles and 166 samples was generated. The genetic distance analyses were then performed using Neil’s coefficient (Neil, M.,1972) with 1,000 bootstrap and 95% confidential intervals. A pair wise population genetic analysis showed that the genetic distance of the populations between Brazil and China, or Brazil and Florida or Brazil and India were 0.8261, 0.9598 and 0.9330, respectively, while genetic distances between Florida and China populations, or Florida and India populations were 0.6016 and 1.3327, respectively. The results indicated that the genetic distances among the populations are not always reflected to their geographic relationships. For example, the genetic distance between Florida Las population and China Las population was closer (0.6016) than that of any other populations compared while the Las populations between China and India are most genetically distant (1.3327) even though both are in Asia. Since Brazil Las population has more or less equal distance to other three populations, it brings up a question of why both populations are closely related or what is the origin of Florida HLB populations. High numbers of commonly-shared alleles of Las populations in Florida and in China suggest both populations are genetically related. In contrast, the genetic distance of Las populations between India and China is 1.7212, the largest distance as compared with other populations indicating that both populations have been differentiated for certain time and there is limited gene flow between the populations. Reference: Neil, M. 1972. Genetic distance between populations. Amer. Natur. 106:238-292.
Significant progress with funds released in June 2009 is described below by objective number. 1) We developed a rapid and easy to use tap sample method to monitor psyllid adults and other insect pests and beneficials in citrus trees (Qureshi and Stansly, 2007). A tap sample consists of 3 consecutive strikes on a randomly chosen branch using the hand or a PVC pipe, and counting fallen insects on a laminated white sheet of paper held 1 ft below the foliage. Studies were undertaken in response to FCPRAC review panel suggesting comparison of different sampling methods in addition to the proposed objectives. Four sampling methods, the tap, Yellow Corn Rootworm Trap (YCRW), ACP Trap, and sweep net, were compared at low and high density psyllid populations in insecticide treated and untreated blocks of mature citrus. At low density, adult psyllids on YCRW traps and ACP traps hung within the canopies of citrus trees in a commercial grove for two weeks averaged 1.8 ‘ 0.3 and 1.1 ‘ 0.02, respectively, compared to 0.08 ‘ 0.04 per tap sample made once per tree at the end of the two week period. Averages of 9.3 ‘ 1.2, 4.8 ‘ 0.6, and 0.5 ‘ 0.1 were obtained for YCRW trap, ACP trap, and tap sample, respectively, at the high density. Sticky traps have a high cost (approx. $ 1 per trap), only provide data after at least a 1-week delay, and require an average 7 minutes per trap to deploy and collect data compared to an average of 3 seconds for a tap sample that provides instant information. Therefore, many samples can be taken in the time one sticky trap can be processed, and at less cost. Sampling conducted at the experimental grove of Southwest Florida Research and Extension Center compared two tap samples and two net sweeps on the same tree. Significantly more adults were detected using the tap sample compared to the sweep net at low density with means of 0.1 ‘ 0.02 and 0.06 ‘ 0.01 per tap or sweep sample per tree, respectively. Means of 0.3 ‘ 0.1 per tap and 0.4 ‘ 0.1 per sweep per tree were obtained at high density and were not significantly different. Time to conduct one sweep (3.1 ‘ 0.04 s) was significantly greater than one tap sample (2.9 ‘ 0.04 s) at low density but did not differ between the two methods at high density and averaged 3.4 ‘ 0.1 s. However, sweep samples are more tiring, require more equipment, knock off fruit and can spread canker, especially when foliage is wet. 2) Regression analysis based on bootstrapping of the first set of low density data indicated that to obtain an SEM:Mean ratio of 0.1, considered necessary for research purposes, numbers of YCRW traps, ACP traps, and tap samples needed would be 130, 219, and 1929, respectively, compared to 75, 74, and 66, respectively, for the high density data. For a ratio of 0.25, considered necessary for routine monitoring, required numbers of YCRW traps, ACP traps, and tap samples were 16, 43, and 267, respectively, at low density, and 12, 13, and 8, respectively, at high density. For the second data set at low density, the required numbers of tap or sweep net samples were 554 and 1281, respectively, for a SEM:Mean ratio of 0.1 and 98 and 158, respectively, for a ratio of 0.25. Therefore, the sweep net would take more time to provide the same data and more work and difficulty in counting at high densities. These results have been prepared for submission to a refereed journal in an article entitled: “Efficiency of sampling methods for the adult Asian citrus psyllid, Diaphorina citri (Hemiptera: Psyllidae) at low and high population densities”. 3) We continue to obtain satisfactory results after 4 years of using the tap sample for our many field studies in young and mature citrus at experimental and commercial groves. The method has also gained wide acceptance by growers and consultants in SW Florida. A recent survey of grove managers covering 106,148 acres of citrus reported all 95% conducting sampling use tap method, along with shoot examination (76%), sticky traps (14%), and sweep nets (9%). We made 4 presentations specifically on monitoring, conducted 4 workshops at SWFREC with a total of over 200 attendants and put up details of sampling procedures on our website at http://www.imok.ufl.edu/entlab/.
The main focus thus far has been to improve the current media to increase growth of the group of bacteria known as Candidatus Liberibacter. Our main focus for improving the growth has focused on understanding the phloem chemistry of citrus. The completed phloem and genomic metabolic analysis indicated several key growth factors to be tested. The nitrogen sources ethanolamine and micronutrient zinc have shown improve growth. Further improvement of the media is ongoing with quantification now possible using microscopy. With the improved media, we are also quantifying differences between different DNA extraction methods using phase contrast and light field microscopy. In collaboration with Ft. Pierce, FL, the testing of Koch’s postulates under field conditions has continued this quarter. Symptoms have not appeared, but are not expected for six to nine months (March-June). RT-PCR assays have not found Ca. L. asiaticus beyond the inoculation point and more tests are planned for the coming quarter. The second focus has been sequencing the genomes of the three Liberibacter species from pure culture. In cooperation with the National Center for Genome Resources (NCGR), we used Illumina sequencing to sequence the strains used in the published pathogenicity trials (China1 Ca. L. asiaticus). The current genome is in the alignment and annotation. The second strain used in the pathogenicity trials (Brazil1 Ca. L. americanus) is also in the alignment and annotation phase at NCGR. Currently we are ensuring the quality and purity of the representative strain of Ca. L. africanus (Africa2) which will be shipped by the end of January.
Incidence of psyllid parasitism has decreased globally in recent years, presumably due to intensified insecticidal control; thus the need to augment beneficial populations. Progress in 2009 is described here by objective number. 1) Tamarixia radiata colonies from south China, North Vietnam and Pakistan were established in DPI quarantine and parasitoid releases initiated in October after approval by USDA-APHIS and DPI. So far, 11130 (S. China), 11266 (N. Vietnam), and 6459 (Pakistan) wasps have been released in Zolfo springs, Lake Wales, and Immokalee, respectively. Psyllid populations were low at release sites in Zolfo springs and Lake Wales, and high in Immokalee particularly in a block of 1 m tall citrus plants at the SWFREC. During Oct-Nov, parasitism averaged 10, 2, and 60% at Zolfo springs, Lake Wales, and Immokalee, respectively. We also established an additional colony of the parasitoid Diaphorencyrtus aligarhensis at DPI ,Gainesville, and released 1325, and 5750 wasps during 2008 and 2009, respectively, in conventional and organic citrus groves and dooryard Muraya paniculata. A parasitism rate of 6-19% was observed from nymphs reared from M. paniculata in May 2008, although none were recovered in 2009. 2) A collaborative study with Dr. Norman Barr, USDA-ARS Mission TX, and others on the genetic characterization of T. radiata strains from USA and some other regions of the world was completed and published. We will continue development of genetic markers for T. radiata to track establishment and performance of different strains in the field. 3) We are consistently improving rearing methods and increasing the production of previously established strain of T. radiata which we continue to release and evaluate in the field. The colony produced 71,000 wasps between March and December 2009 that were used to initiate and help maintain the colony at OrangeCo, conduct laboratory experiments at SWFREC, Immokalee and CREC, Lake Alfred, and release in experimental, conventional, and organic groves. During Oct-Nov, parasitism averaged 8-50% in the release blocks compared to 7-18% in the blocks where parasitoids were not released. We also released over 0.5 million predatory mites (Amblyseius swirskii) on mature orange during bloom in an organic grove in Lake Wales, FL. Predatory mites, averaged 3 and 5 individuals per two tap samples per tree in March and April, respectively, with psyllid numbers per two tap samples averaging only 1 mite/tap during March-April, 3 in May, 1 in June, comparable to many sprayed groves. 4) We assisted Orange Co. and DPI to establish their mass rearing facilities, made several statewide and national presentations and published our findings to reach the target clientele. Our efforts have generated much interest in the US and Latin America in mass release of T. radiata and the number of projects has mushroomed. For this reason, we organized with the USDA-APHIS and the California Department of Agriculture, the “International Tamarixia Workshop” to take place this February in McAllen TX, with support from sponsors including Florida Citrus Mutual. The objective is to improve the technology by sharing practical information and the workshop is full. 1. Barr, N.B., D.G. Hall, A. Weathersbee, R. Nguyen, P. A. Stansly, J. A. Qureshi, and D. Flores. 2009. Comparison of laboratory colonies and field populations of Tamarixia radiata, an ecto-parasitoid of the Asian Citrus Psyllid, using ITS and COI DNA sequences. Journal of Economic Entomology. 102: 2325-2332. 2. Qureshi, J. A., M. E. Rogers, D. G. Hall, and P. A. Stansly. 2009. Incidence of invasive Diaphorina citri (Hemiptera: Psyllidae) and its introduced parasitoid Tamarixia radiata (Hymenoptera: Eulophidae) in Florida citrus. Journal of Economic Entomology. 102: 247-256. 3. Qureshi, J.A., and Stansly P.A. 2009. Exclusion techniques reveal significant biotic mortality suffered by Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae) populations in Florida citrus. Biological Control 50: 129’136.
Incidence of psyllid parasitism has decreased globally in recent years, presumably due to intensified insecticidal control; thus the need to augment beneficial populations. Progress in 2009 is described here by objective number. 1) Tamarixia radiata colonies from south China, North Vietnam and Pakistan were established in DPI quarantine and parasitoid releases initiated in October after approval by USDA-APHIS and DPI. So far, 11130 (S. China), 11266 (N. Vietnam), and 6459 (Pakistan) wasps have been released in Zolfo springs, Lake Wales, and Immokalee, respectively. Psyllid populations were low at release sites in Zolfo springs and Lake Wales, and high in Immokalee particularly in a block of 1 m tall citrus plants at the SWFREC. During Oct-Nov, parasitism averaged 10, 2, and 60% at Zolfo springs, Lake Wales, and Immokalee, respectively. We also established an additional colony of the parasitoid Diaphorencyrtus aligarhensis at DPI ,Gainesville, and released 1325, and 5750 wasps during 2008 and 2009, respectively, in conventional and organic citrus groves and dooryard Muraya paniculata. A parasitism rate of 6-19% was observed from nymphs reared from M. paniculata in May 2008, although none were recovered in 2009. 2) A collaborative study with Dr. Norman Barr, USDA-ARS Mission TX, and others on the genetic characterization of T. radiata strains from USA and some other regions of the world was completed and published. We will continue development of genetic markers for T. radiata to track establishment and performance of different strains in the field. 3) We are consistently improving rearing methods and increasing the production of previously established strain of T. radiata which we continue to release and evaluate in the field. The colony produced 71,000 wasps between March and December 2009 that were used to initiate and help maintain the colony at OrangeCo, conduct laboratory experiments at SWFREC, Immokalee and CREC, Lake Alfred, and release in experimental, conventional, and organic groves. During Oct-Nov, parasitism averaged 8-50% in the release blocks compared to 7-18% in the blocks where parasitoids were not released. We also released over 0.5 million predatory mites (Amblyseius swirskii) on mature orange during bloom in an organic grove in Lake Wales, FL. Predatory mites, averaged 3 and 5 individuals per two tap samples per tree in March and April, respectively, with psyllid numbers per two tap samples averaging only 1 adult/tap during March-April, 3 in May, 1 in June, comparable to many sprayed groves. 4) We assisted Orange Co. and DPI to establish their mass rearing facilities, made several statewide and national presentations and published our findings to reach the target clientele. Our efforts have generated much interest in the US and Latin America in mass release of T. radiata and the number of projects has mushroomed. For this reason, we organized with the USDA-APHIS and the California Department of Agriculture, the “International Tamarixia Workshop” to take place this February in McAllen TX, with support from sponsors including Florida Citrus Mutual. The objective is to improve the technology by sharing practical information and the workshop is full. 1. Barr, N.B., D.G. Hall, A. Weathersbee, R. Nguyen, P. A. Stansly, J. A. Qureshi, and D. Flores. 2009. Comparison of laboratory colonies and field populations of Tamarixia radiata, an ecto-parasitoid of the Asian Citrus Psyllid, using ITS and COI DNA sequences. Journal of Economic Entomology. 102: 2325-2332. 2. Qureshi, J. A., M. E. Rogers, D. G. Hall, and P. A. Stansly. 2009. Incidence of invasive Diaphorina citri (Hemiptera: Psyllidae) and its introduced parasitoid Tamarixia radiata (Hymenoptera: Eulophidae) in Florida citrus. Journal of Economic Entomology. 102: 247-256. 3. Qureshi, J.A., and Stansly P.A. 2009. Exclusion techniques reveal significant biotic mortality suffered by Asian citrus psyllid Diaphorina citri (Hemiptera: Psyllidae) populations in Florida citrus. Biological Control 50: 129’136.
Spatial and Temporal Incidence of Ca. Liberibacter in Citrus and Psyllids Detected Using Real Time PCR Funds for this project were released on 19 Mar 09. Objective 1. Assess seasonal patterns of pathogen incidence in citrus trees and psyllids in regions of high HLB incidence. This objective includes establishing field plots, which has been done, and sampling plant and psyllid for the HLB pathogen at the different locations over time. This objective has been established and sampling has been initiated and it ongoing. Included in the sampling objectives was to validate our PCR test at SWFREC for the psyllids by comparing field to ‘hot’ colonies and to results from similar sampling for USDA-Riverside. A 12 acre block of ‘Valencia’ orange trees was selected at a commercial grove in 2008 and a block at SWFREC was established for these experiments. Monthly samples of 100-200 psyllids collected from SWFREC and commercial groves have been collected since November 2008 and are under analysis at the SWFREC-HLB and USDA-Riverside. Plant and psyllid samples for commercial experiment conducted in December 2009 using shoots infested with natural populations and lab reared psyllids adults are also being analyzed. A sample of psyllid adults collected in the block in November 2008 and analyzed at USDA-ARS Riverside labs contained 28% HLB positive psyllids. Subsequent sampling in spring and fall in 2009 during the months of April, May, and August detected 5-10% positive in April and 10% positive in May of adult psyllids from the blocks. Psyllids from ‘hot’ colonies tested 18% positive on grafted plants by the USDA-Riverside lab and the psyllids from the same test site tested 0-27% positive in duplicated runs at SWFREC lab. The ‘cold’ colonies tested 11% positive by USDA-Riverside lab and 5% at SWFREC in August 2009. Hot colony on plants with field psyllids tested 9% positive by USDA-Riverside in August 2009 and a similar colony tested by SWFREC in September and October was less than 10% positive. Similarly, plants infested with natural populations or lab reared psyllids were tested throughout the year in commercial and SWFREC test blocks. The percentage of positive samples from trees in the commercial grove were 16, 44, 45, 10, 35 and 42% for two samples dates each in June, Aug and September respectively. The 10% sample was collected on August 7, 2009. Plants grafted from HLB positive source and plants inoculated with field psyllids were followed for percentage of HLB from April through August. Initial samples were negative but 75% were positive by PCR within 6 weeks of inoculation. The percentage of positives dropped slightly in June, probably due to sampling issues, but otherwise has been fairly consistently above 90% positive in subsequent testing indicating that a positive source of HLB has been established for future research. Sampling of the block receiving two levels of micronutrients+SAR, insecticide treatments, or left as control continues. Plant and psyllid samples are collected at four months intervals to test for Candidatus Liberibacter asiaticus using PCR. Insecticide applications significantly suppressed psyllids compared to control. Other objectives are pending completion of objective 1.
This first section of the report covers the entire period that Huanglongbing Diagnostic Laboratory has been in service since February 2008 because one of the objectives for the funding was to continue to provide continuous, seamless service for as long as the growers deemed it necessary. This has been achieved as detailed in the report below. The HLB Diagnostic Laboratory located at the University of Florida Institute of Food and Agricultural Science Southwest Florida Research and Education Center (UF-IFAS-SWFREC), was establish with funds appropriated by the Florida legislature to serve grower and researcher HLB diagnostic needs in citrus. The lab has been in operation since February 2008. In addition to providing diagnostic services to the growers, the lab continued to develop and apply techniques, protocols and efficiency. Samples The lab has been in operation for nearly 2 years, and has received to date more than 16,000 grower samples, approximately 5,700 research samples, and 2,100 screen house samples from the Budwood facility located on the SWFREC property. Approximately 2600 of the sample total were submitted between October through December 2009. Additionally, of the 5,700 total samples have been processed for research, of which nearly 700 were within that same time frame. These totals represent a several-fold increase in number of sample submissions when compared to the year’s previous two quarters, reflecting seasonal fluctuation in sample volume. The lab has received samples from growers throughout Florida, with the highest number of samples received from growers in Collier, Highlands, and Hendry Counties. There is a slight seasonality to the sample submission volume with respect to harvesting and new growth (flushing) events. Techniques, Protocols and Research The new DNA isolation kit implemented last quarter into routine operation of the lab has provided us with exceptionally fast and efficient turnaround time for growers as well as research samples. This new kit employs magnetic bead technology to effectively and efficiently isolate whole genomic DNA from plant tissue including DNA of the HLB pathogen. We are using liquid nitrogen to grind all samples as this provides the most effective maceration. We continue to use the magnetic particle based system, which has proved both reliable and fast. We have developed and are in the beginning stages of implementing a protocol for the detection of HLB in Asian Citrus Psyllid (ACP) with the primary goal of serving research projects within the Entomology and Plant Pathology departments with compensation through their programs. To improve sensitivity to detect HLB, we are investigating other probe/primer combinations in different areas of Candidatus Liberibacter asiaticus gene and have the ability to detect other greening organisms such as Candidatus Liberibacter africanus and Candidatus Liberibacter americanus. We have attempt to culture the organism Candidatus Liberibacter asiaticus either in co-culture or isolate it in pure culture using reports cited in literature and will use the pure culture to calibrate the sensitivity of our lab protocols to determine the minimum number of cells of Liberibacter needed for detection.
Irrigation and fertilizer practices in the Advanced Citrus Production System flatwoods site at SWFREC indicate potential for improved plant nutrition of three year old trees. Hamlin and Valencia trees were planted in 2006 at the flatwoods site at 151, 198 and 545 trees per acre on rootstocks Cleopatra mandarin (Hamlin) or Volkamer lemon (Valencia), Swingle citrumelo, and Flying Dragon; respectively. Irrigation/fertigation practices were daily drip fertigation (Drip OHS), daily irrigation and weekly fertigation with a strip shaped microsprinkler (Microsprinkler OHS) and a Grower Microsprinkler control with periodic irrigation based on soil depletion and monthly fertigation. Adequate tree nutrient status, root density in the irrigated zone and water conservation were three factors investigated in 2009. Leaf N , P and K content were similar for all treatments at the beginning of 2009 (January and March), however samples taken in June and September indicate that the Drip OHS and Microsprinkler OHS maintained leaf concentrations greater that those for the Grower Microsprinkler irrigation treatment. The exception to this is P, where no significant differences were found among treatment at any sample date. All treatments maintained average leaf nutrient concentrations in the optimum or high range. Root length density (RLD, root diameter <1mm) varied as a function of irrigation/fertilizer treatment (p<0.05), decreasing with depth (p<0.0001) and distance from the tree (p<0.0001). RLD was consistently higher for Drip OHS than the other two treatments. Prior to the start of the summer rainy season, Drip OHS significantly increased RLD by 13-90% for roots <3mm in diameter in both the irrigated and non-irrigated zones when compared with the conventional practice. After the rainy season, RLD in the Drip OHS plots were 19% and 112% higher than conventional practice in the non-irrigated and irrigated zones, respectively. These data indicate that RLD in the drip irrigation zone may not be greatly influenced by the rainy season as first speculated. Thus, irrigation methods such as drip which apply water and fertilizer frequently and in small pulses within a limited root zone offer a viable option for increasing root water and nutrient uptake efficiency compared with the microsprinkler based systems when the trees are small. Total water used was greatest for the Grower Microsprinkler treatments plots and increased with planting density ranging between 10 and 18 inches. Water use was lowest for Drip OHS irrigation treatments at low (5 in yr-1) and moderate (6 in yr-1) densities compared with the Grower Microsprinkler treatments at the same densities (10 and 14 in yr-1, respectively). Thus, overall water use efficiency for the Drip OHS and Grower Microsprinkler treatments were 0.03 and 0.07 inches per tree, respectively. However, the highest water user was the high density planting with drip irrigation (19 in.). The reason water use increased about 3 to 4 fold for the drip treatment at high tree density is that each tree had the same number of drippers and thus water use was a function of tree density (i.e. 545 trees at high density, 151 and 198 at low and moderate densities). The relationship of increased water use with increased tree density existed for the microsprinkler treatments but not at the same ratio as tree density because the emitter output and pattern sizes were selected to give similar application rates on a gal. per area basis with little overlap.
QUARTERLY RESEARCH REPORT (Jan. 08, 2010) In the past 3 months (from Oct. 2009 to Dec. 2009), the research was focused on the on-going evaluation of the new candidate compounds for HLB control using the optimized regeneration system in periwinkle, the effectiveness of the compounds that killed Liberacter in periwinkle to control HLB in citrus and the effects of PS on microorganism diversity in rhizosphere soil. RESULTS 1. On-going evaluation of the new compounds MDL, OPP and SA were selected and evaluated against the Las bacteria and for their phytotoxiticy using the optimized regeneration system in periwinkle. MDL is an antibiotic drug used against anaerobic bacteria. The results showed MDL is effective in eliminating Las bacteria in the Las-infected periwinkle cuttings. The Las can’t be detected in the treated, regenerated plants. MDL is currently being tested in the citrus grove at the Pico Farm, USDA. OPP and SA were not effective. High concentrations of OPP and SA inhibited regeneration rates in Las-infected periwinkle. The regenerated plants from OPP- or SA-treated cuttings kept high titers of Las bacteria. The average Ct value in the regenerated plants was less than 30. 2. Effects of the screened compounds on HLB-affected citrus HLB-affected container-grown citrus was treated with PS, one of the screened effective compounds against Las bacteria. The HLB-affected citrus seedlings were soaked in PS three times. Citrus samples were taken once each month for Liberibacter analysis. The Las bacteria could not be detected in the container-grown citrus two months post treatment, by Q-PCR. The PS-treated citrus grew better than citrus receiving control treatments. When PS combined with other PGRs was injected into HLB-affected, container-grown citrus, PS can also eliminate the Las bacteria in the greenhouse. Thus, PS can control Las in container-grown citrus when applied either as a root drench or by injection. In August 2009, PS at three different rates (2.5g. 5g and 10g per tree) was injected into HLB-affected field citrus trees with 10~15 cm in diameters. Leaf samples were randomly taken and tested in Oct and Dec, 2009. The average Ct value in the PS-treated citrus grove increased from 26 in the pre-treatment to 32 in the post-treatment. All treatment rates were effective. Some other compounds, such as MDL and KESU are also being evaluated in the field. 3. Effect of PS on microorganism diversity in rhizosphere soil Rhizosphere soil samples were taken from the PS-treated citrus pot. Soil DNA was extracted using a Soil DNA Extraction Kit (Mio-Bio). Diversity of microorganisms in the soil was evaluated by RFLP. The results showed that treatment of field citrus with PS had no effect on the diversity of soil microorganisms. The residues of PS in the citrus fruit will be tested later. CONCLUSIONS 1. PS was effective against Las bacteria in periwinkle or in citrus. 2. MDL is also effective compound against Las, but MDL is not easily dissolved in water. Improving the water solubility of MDL and would be was necessary for MDL application in HLB control. 3. OPP or SA was not effective in control of Las bacteria.
Experiment 1 – Since the last report, no new symptomatic plant were observed in any compartment. Leaf samples of all the plants were collected with the objective to detect the presence and to identify the bacterial species in each plant in November/09 (total of 1268 samples collected since the beginning of the experiment in April/08). Psyllids were collected in November and December/09 to evaluate their infectivity. The samples were submitted to conventional PCR and real-time PCR. These analyses are in progress. In December/09 we stopped to release new ACP and killed all psyllids present in the compartments. All plants were moved to other insect-proof screenhouse and will be there for symptoms observation and leaf sampling for PCR analysis. New inoculum source Citrus and Murraya plants are being prepared to repeat this experiment after March/10. At this time we want to include sources of M. paniculata infected with CLas. Experiment 2 – The delay to start this experiment was caused because took so long to obtain inoculum source plants for CLam, but this problem was already solved. During January/10, ACP will be reared on symptomatic inoculum sources for CLam and CLas. The emerging adults from nymphs reared on such inoculum sources will be used for inoculation in February/10. After the inoculation, adults of ACP free of liberibacters will be periodically feed on these inoculated plants to detect the moment when they can acquire the bacteria from inoculated plants. Experiment 3 – Nine insect-proof screenhouses were built in a commercial citrus farm to protect Hamlin, Pera and Valencia sweet oranges with three different age. Plants from Hamlin and Valencia were already inoculated at the beginning of July/09 with infected adults of ACP and plants from Pera will be inoculated in January/10. Monthly assessments for symptom severity have been done, but no HLB-symptoms were observed yet. Also, leaf samples have been collected to detect the presence of Liberibacter species on inoculated shoot. PCR analyses are in progress, no result yet.
Data still have been collected from this field experiment. The longer time of exposition of symptomatic trees in plots with longer frequencies of local inoculum reduction treatments did not result in significant differences on HLB progress rate and HLB incidence 42 months after planting. The tested program of ACP has been efficient to reduce the number of adult psyllids captured on yellow stick traps in 78.6% and to reduce the number of eggs and nymphs observed on new shoots in 94.6%. The disease progress rate (estimated by Gompertz model) in plots with ACP control program was significantly reduced in 21% compared to the disease progress rate in plots without ACP control, reducing the incidence of HLB-symptomatic trees in 53.2% during these 42 months. No significant difference was observed on the delay of the beginning of epidemics. From October/09 to December/09 the disease incidence increased from 20.3 to 28.9% in plots with ACP control program and from 44.9 to 61.8% in plots without vector control. Like observed in past years, the seasonality of HLB symptoms was repeated this year. The incidence of symptomatic trees in each assessment started to increase after February (end of summer) reaching high values in April to June (autumn), and started to decrease after July, reaching low values in September (beginning of spring). However, in 2009 there was a second increase of symptomatic trees detection from October to December probably associated to abnormal amount of rain during the winter. All psyllids captured on yellow stick traps since the winter/07 were tested for the presence of Candidatus Liberibacter spp. by conventional PCR. Higher frequencies of PCR-positive ACP have been observed during spring and summer. Until the first harvest in September/09, US$ 7.37 per plant was spent on psyllid vector control program. The cumulative costs for scouting symptomatic trees were respectively US$ 1.56 and 0.12 per tree for 14 and 182 days of inoculum reduction frequencies. US$ 0.79 was the average cost to remove and eliminate each symptomatic tree. Considering the average cost of HLB management, the cost of eliminated trees and the income by first fruit harvest (US$ 4.12per box paid to citrus growers in Sao Paulo in 2009), no treatment was economically sustainable so far. Additional data for economic analysis have been collected. Annual maps of HLB-symptomatic trees were prepared for spatial analysis using stochastic models (MCMC) to verify the effects of each treatment on primary and secondary spread of HLB. This analysis will be done at USDA lab in Fort Pierce by Gottwald’s team. The assessments on this experiment will continue at least for one more year to allow more detailed temporal and spatial analysis and get better conclusions. According to the estimated disease progress rates, the HLB incidence will be 62.8 and 83.4% respectively for plot with and without vector control at the end of 2010. These results will be presented in the next Florida Citrus Show (January 27th).
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