During the last three months our lab temporary stopped all PCR analysis because the building was being fixed. It delayed all the results from this period. Then, no progress was obtained in the three experiments. 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 – During January/10, ACP were reared on symptomatic inoculum sources for CLam and CLas. The emerging adults from nymphs reared on such inoculum sources were used for inoculation in March/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 were not inoculated yet, because some of screenhouse suffered some wind damages. However, until now, all encaged Pera trees did not show any HLB-symptoms, proving they are really healthy. 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.
During last three months data still have been collected from this field experiment. In average, after the symptomatic tree have been observed it was exposed in the field before be eliminated 1, 7, 29 and 67 days, respectively for frequencies of local inoculum reduction of 14, 28, 84 and 182 days. However, the shortest frequency of local inoculum reduction treatment (every 14 days) did not result in significant differences on HLB progress rate and HLB incidence 46 months after planting compared with longer frequency (every 182 days), probably because primary infections were more important and intensive than secondary ones. The tested program of ACP has been efficient to reduce the number of adult psyllids captured on yellow stick traps in 79% and to reduce the number of eggs and nymphs observed on new shoots in 94%. However this vector control reduced HLB-incidence in only 48%. The disease progress rate (estimated by Gompertz model) in plots with ACP control program was significantly reduced in 27% compared to the disease progress rate in plots without ACP control (Average Gompertz rate of 1.35 for plots without vector control and 0.99 for plots with vector control). From December/09 to March/10 the disease incidence increased from 28.9% to 35.2% in plots with ACP control program and from 61.8% to 67.1% in plots without vector control. All psyllids captured on yellow stick traps from the winter/07 to fall/09 were tested for the presence of Candidatus Liberibacter spp. by conventional PCR. PCR-positive ACP have been observed only during spring/07 (1.8% of tested samples), summer/08 (2.1%), spring/08 (4.5%), summer/09 (11.1%), and fall/09 (11.1%). Infective ACP were found both in plots with and without vector control program. Samples from spring/09 and summer/10 are being processed. 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. Because of the nature of the data (based on visual symptoms and not on infection) and because the long incubation period, new data set are being prepared to be analysed using only year based data from months were the new symptoms development is null. Also, to avoid the edge effect, only central area of each plot will be analysed. This analysis will be done at USDA lab in Fort Pierce by Gottwald’s team. The results of this experiment are being compared with the results of other similar experiment, but carried out in a region with lower external inoculum pressure. So far, it could be comments that HLB managent is much easier and economically sustainanble under low pressure of external inoculum and that only local disease management could be not enough to control HLB in a region with high external inoculum pressure. These and others results were presented in four semminars given in CREC, Florida Citrus Show, Syngentha Experimental Station, and Florida Natural’s in the last week of January/10. The assessments on this experiment will continue at least for one more year to allow more detailed temporal and spatial analysis and get final conclusions.
Several sensors and sensor aiding tools such as four-band sensor, five-band sensor (with GPS and self-incident light correction), volatile collection device holders (for volatile organic compound- VOC sampling), and moving sensor platform with retractable mast (for holding multiple sensors) were developed during the first year of this project. During the last quarter of this project period, significant progress was made in the application of mid-infrared spectroscopy (5.15-10.72 ‘m) for HLB detection. The healthy, nutrient-deficient, and HLB-infected leaf samples were analyzed using a portable mid-infrared spectrometer. The k-nearest neighbor (kNN)-based statistical algorithm yielded a healthy and diseased class average classification accuracy of 91% and 96%, respectively after data preprocessing (baseline correction, narrow waveband, principal component analysis-PCA). In addition, greenhouse experiments indicated that the visible-near infrared reflectance data (350-2500 nm) from a spectroradiometer could be used to identify HLB-infected plants with a classification accuracy of > 60% and > 90% for Hamlin and Valencia varieties, respectively (using quadratic discriminant analysis based algorithm). Aerial images were collected across 2,000 acres (Southern Gardens) during previous years to develop a spectral library. Forty-four critical wavebands (of 128 bands) were selected using image processing algorithm, which enhanced the average overall classification accuracy from 45% to 65% in 150 acres blocks. These bands also decreased the false positives by 60%. On December 9, 2009, a new set of georeferenced aerial hyperspectral imagery was acquired from a new citrus grove. Healthy and infected spectral data for ground measurement showed a clear separation in the two wavelength ranges: 500-800 nm and 1500-2000 nm. Studies on VOC profiling for HLB detection were performed in the greenhouse. These experiments were conducted to identify volatile biomarkers. Volatile samples were collected from two varieties (Valencia and Hamlin) of healthy and HLB-infected samples. After data pre-processing (baseline correction, computation of auto-regression coefficients), PCA and PC regression with leave-one-out validation were applied for HLB diagnosis. The corresponding systematical accuracy, false positive rate and false negative rate are: 80.7%, 10.0%, and 36.4% for Valencia; 76.0%, 16.7%, and 30.1% for Hamlin; and 70.0%, 18.8%, 45.8% for the varietal independent case. With these results, the next step will be to eliminate background variations between sampling batches and varietals. In regard to the application of fluorescence-based HLB detection, a subcontract was signed between University of Florida and Embrapa Agricultural Instrumentation to allow partnership. However, the process could not be completed due to some complications resulting from the change in rules by the Brazilian Government. Embrapa is making efforts to resolve this issue to receive funding for this project. For HLB detection, a sensor prototype PHOTONCITRUS was developed. In the laboratory, the accuracy for detecting HLB-infected citrus leaves and healthy leaves using the calibrated system increased to 95% and 80%, respectively. The sensor will be further evaluated to detect HLB-infected trees under field conditions. The work done in Brazil will be joint and partially moved to Florida to avoid funding transfer problems. In summary, progress has been made in the developing sampling protocols, establishing experimental protocols, evaluating multiple sensors under laboratory and field conditions, as well as developing data analysis algorithms and improving their performances for accurately detecting HLB-infected citrus trees. In the second year, we will further expand our research to validate the current findings on a larger dataset under field experiments and further refine the sensing techniques and classification accuracies.
Two years funding for this project was recommended by NAS panel. Significant progress was made during the first year for all 3 objectives. 1) Evaluation and refinement of the tap sample method developed by Qureshi and Stansly (2007) to monitor psyllid adults. We compared tap sample, the Yellow Corn Rootworm Trap (YCRW), the ACP Trap, and a sweep net, at low and high density psyllid populations in insecticide treated and untreated blocks of mature citrus at two locations. 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 1.04 ‘ 0.2, 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. One sticky trap cost approximately $1, only provides data after at least a 1-week delay, and requires an average 7 minutes to deploy and collect data compared to an average of 3 seconds for a tap sample that provides instant data with no more supplies than a laminated sheet of letter size paper or a smooth white surface such as a clipboard, and a one-foot piece of PVC tube. Therefore, many tap samples can be taken in the time one sticky trap can be processed and cost much less. Comparison of the two sampling methods in mature citrus at Southwest Florida Research and Extension Center showed that 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 at high density 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 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) Test the influence of adult density and shoot infestation rates on precision of estimated means and distribution of population within blocks. Regression analysis based on bootstrapping of the data from the first trial indicated that the precision targets of 0.25 and 0.01 SEM:mean were reached at high psyllid density with 8 and 66 tap-samples, respectively, compared to 12 and 75 YCRW traps and 13 and 74 ACP traps. At low psyllid density, 267 and 1929 tap-samples, 16 and 130 YCRW traps, and 43 and 219 ACP traps were needed to attain these precision ratios, respectively. In the second trial at low psyllid density, 98 and 554 tap-samples and 158 and 1281 sweep net samples were needed at ratios of 0.25 and 0.1, respectively, compared to 35 and 196 tap samples and 37 and 198 sweep net samples at high density. Therefore, the sweep net would take more time to provide the same data and more work and difficulty in counting at high density. We are conducting more sampling and integrating shoot density and infestation data into overall sampling for psyllid. 3) Evaluate and integrate methods for assessing psyllid density, shoot density, and infestation rates into a user friendly system accessible to consultants and managers. A rapid system that includes tap samples, flush inspections and natural enemy evaluations along with forms and spreadsheets is available on our website swfrec.ifas.ufl.edu/entlab/. We made 5 presentations specifically on monitoring psyllids and conducted 5 workshops at SWFREC with a total of over 250 attendants. The tap sampling method has gained wide acceptance and is adopted by growers and consultants in SW Florida and our cooperators in California and Mexico. A recent survey of grove managers by SWFREC and IFAS extension covered 106,148 acres of commercial citrus and reported that all 95% conducting sampling use the tap method, along with shoot examination (76%), sticky traps (14%), and sweep nets (9%). Findings from the above studies have been submitted for publication in citrus industry and Florida Entomologist.
All objectives for the first year of this project have been completed or exceeded. Objective 1: Compare aerial and conventional ground application of insecticides for psyllid control. Two trials were conducted for this objective. Low-volume (LV) aerial applications at 10 GPA were compared with ground airblast applications at125 GPA during the summers of 2008 and 2009. Results in 2008 indicated that broad spectrum insecticides work well by air and ground but that the selective insecticides, spinetoram and imidacloprid, provided much better control when applied by ground. During 2009, we followed up with two selective insecticides (spinetoram and spirotetramat) compared with the broad spectrum phosmet, all applied at the same volumes as above by air and ground in replicated (N=4) 48 or 24-acre plots respectively of mature orange trees. In this second trial, all materials were more effective by ground, however differences between broad spectrum and selective insecticides were less clear, phosmet providing best control with both methods. Nevertheless, the ‘Cooperative Dormant Spray’ program we initiated with Gulf Citrus Growers Association resulting in over 70,000 total acres sprayed by air the last two winters demonstrates that aerial applications are efficient and effective during the “dormant” season (see report on FCPRAC #000). A second year of ‘Cooperative Dormant Sprays’ is presently being evaluated . We also compared LV applications using a modified London Fogger 18-20 by Chemical Containers @ 2GPA applied to bed tops only with conventional airblast sprayer that treated both tops and swales @ 116GPA on a highly infested plot of ‘Pineapple’ oranges. Applying only tops weighted the odds against the LV, but the objective was to test this typical LV application. Spinetoram (4oz/ac) and dimethoate (24oz/ac.) were both applied with 2GPA of horticultural mineral oil (HMO). Conventional ground applications resulted in fewer ACP for the duration of the one-month trial compared with the control, whereas LV applications resulted in statistically fewer ACP for only the last two weeks. So again, conventional application proved superior in terms of control with the products and under the conditions tested. Objective 2: Assess the effects of frequent nocturnal LV applications of horticultural mineral oil (HMO) on psyllid populations. In a preliminary replicated trial testing LV @1GPA of only HMO every two weeks for 4 months in summer of 2008, we found 14.1’3.8% of the flush was infested in untreated plots compared with 2.1’1.2% for the HMO-treated plots. In 2009, we compared the modified London Fogger and the Proptec P400D @ 2GPA, applied every 2 to 4 weeks depending on ACP populations, and monitored ACP every two weeks. High frequency LV applications of HMO with the Proptec maintained lower populations (1.4 ‘ 0.75 ACP adults x days) compared with the London Fogger (4.4 ‘ 1.9), and the untreated control (6.3 ‘ 3.9). In 2010, we are conducting experiments to evaluate frequent LV applications of HMO alone or tank mixed with micronutrients as a complete ACP/HLB management package. We are also collaborating with CREC to assess the deposition of oil on citrus foliage by LV application with protocols we are developing using gas chromatography to quantify residues eluted from leaves of treated trees. Objective 3: Extend results to the citrus industry. Results from these experiments have been presented at 18 extension meetings across the state in 2009 including two production managers meetings and two CCA trainings, the Entomological Society of America (ESA), and the Florida State Horticultural Society (FSHS). – Stansly, PA, HA Arevalo, M Zekri, and R Hamel. 2009. Cooperative dormant spray program against Asian citrus psyllid in SW Florida. Citrus Industry 90(10):14-15 – Arevalo, H. A. and P. A. Stansly. 2009. Comparison of Ground and Aerial Applications for Control of Adult Asian Citrus Psyllid, Diaphorina citri Kuwayama. Proc. Fla. State Hort. Soc. 122: (186-188)
Significant progress was made in rearing, release, and evaluation of parasitism rates to enhance biological control of ACP in Florida as described under objective 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, 23,571 (S. China), 18,783 (N. Vietnam), and 11,294 (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 1,325, and 5,750 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 from our 3 new colonies, as well as from Florida and the Caribbean 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 the previously established strain of T. radiata which we continue to release and evaluate in the field. The colony produced 88,000 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 release blocks compared to 7-18% in blocks where parasitoids were not released. Parasitism on sentinel plants placed in a conventional grove averaged 60%, 26%, and 27% in Apr 09, Nov 09 and Jan 10, respectively, and 36% and 22% in Nov 09 and Jan 10, respectively, at SWFREC. 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, and monthly average for psyllid adults was < 1 per tap sample for the year except May when 1.5 per tap sample were observed. 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" in Feb 2010 at McAllen TX, with support from sponsors including Florida Citrus Mutual. The objective was to improve the technology by sharing practical information. 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 ACP Diaphorina citri (Hemiptera: Psyllidae) populations in Florida citrus. Biological Control 50: 129'136.
Significant progress was made in rearing, release, and evaluation of parasitism rates to enhance biological control of ACP in Florida as described under objective 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, 23,571 (S. China), 18,783 (N. Vietnam), and 11,294 (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 1,325, and 5,750 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 from our 3 new colonies, as well as from Florida and the Caribbean 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 the previously established strain of T. radiata which we continue to release and evaluate in the field. The colony produced 88,000 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 release blocks compared to 7-18% in blocks where parasitoids were not released. Parasitism on sentinel plants placed in a conventional grove averaged 60%, 26%, and 27% in Apr 09, Nov 09 and Jan 10, respectively, and 36% and 22% in Nov 09 and Jan 10, respectively, at SWFREC. 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, and monthly average for psyllid adults was < 1 per tap sample for the year except May when 1.5 per tap sample were observed. 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" in Feb 2010 at McAllen TX, with support from sponsors including Florida Citrus Mutual. The objective was to improve the technology by sharing practical information. 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 ACP Diaphorina citri (Hemiptera: Psyllidae) populations in Florida citrus. Biological Control 50: 129'136.
In the fourth quarter, we have primarily focused on selecting T1 Arabidopsis transformants expressing citrus SA homologs and testing some T2 transgenic plants in disease resistance. So far, preliminary data obtained from Arabidopsis expressing ctNDR1 look promising. The Arabidopsis ndr1-1 mutant was shown previously to lack a hypersensitive response (HR) when challenged with the Pseudomonas syringae avrRpt2 strain. We transformed ndr1-1 with ctNDR1 and obtained T2 seeds. The T2 plants were infected with the P. syringae avrRpt2 strain at OD=0.1 for a HR test. We found that T2 plants from 10 independently transformed lines were segregated into HR+ and HR-, consistent with the fact that the T2 transgenic plants are heterozygous in the transgene. In the control experiments, all T2 plants showed no HR when challenged with a virulent isogenic P. syringae strain. In addition, when we infected the T2 ndr1-1 plants expressing ctNDR1 with P. syringae avrRpt2 strain at OD=0.0001 for a disease resistance test, we observed a segregation of resistance and susceptibility among the T2 lines. As controls, WT plants were resistance and ndr1-1 plants were more susceptible to this strain. These data suggest that the ndr1-1 mutant is likely complemented by the ctNDR gene. We will isolate homozygotes of the transgenic plants and test them again for defense response in the next generation. In the meantime, the ctNDR1/pBINplusARS construct will be placed in the pipeline for citrus transformation. Summary for additional transgenic plants are in the following: 1. CtNPR1/pBINplusARS to Col or the npr1-1 mutant: we infected T2 transgenic npr1-1 plants expressing ctNPR1. So far we did not observe a drastic change of disease resistance in the T2 plants. We will isolate homozygous lines and plant them side by side with WT and the npr1-1 mutant for a disease resistance test in the next generation. 2. CtPAD4/pBINplusARS to Col or the pad4-1 mutant: We obtained T2 seeds from Col and the pad4-1 mutant expressing ctPAD4 and will infect the T2 plants soon with P. syringae. 3. CtEDS5/pBINplusARS to Col or the eds5-1 mutant: We screen T0 seeds for transgenic plants expressing ctEDS5 but were not able to obtain any lines. This is possibly due to the toxicity of overexpressing ctEDS5 and/or the failure of transformation. We are now repeating the transformation process. In the meantime, we will transiently express ctEDS5 in tobacco to see if overexpression of this gene could cause cell death or other damages to tobacco cells. In addition, we continue to clone additional SA genes from citrus. We are currently working on cloning several additional SA homologs from citrus, including ctEDS1, ctSID2, ctALD1, and ctWIN3. All these genes were previously shown to be involved in either SA biosynthesis or regulating SA levels in Arabidopsis. We already obtained 3′ end of ctEDS1 and in the process of cloning other genes.
We have proposed to identify and assess gene sequences for their negative effects on sap-sucking Hemipteran insects via RNAi using both in vitro and in planta dsRNA feeding assays. Objective 1 of our proposal intended to evaluate candidate genes for dsRNA-induced lethality of Diaphorina citri and our model organism, Myzus persicae, using artificial feeding assays. To date, we have cloned sequences at least 400 bp in length from nine Intellectual Property-Free (IP-Free) homologous D. citri and M. persicae transcripts. In addition, we have carried out artificial feeding assays on M. persicae using dsRNA derived from the salivary gland-specific Coo2, midgut-specific glutathione-S-transferase S1 and constitutively expressed S4e ribosomal protein from M. persicae, as well a control derived from green fluorescent protein sequence. We are currently repeating our feeding assay experiment to confirm our initial results, which indicated that dsRNA had a negative effect on both the lifespan of the insects and the number of offspring generated. Since our last report we have also made considerable headway with regards to objective 2: to evaluate the RNAi strategy in planta for its effects against our model insect, M. persicae. Since recent evidence suggests that RNAi in sap-sucking insects may operate more effectively in planta than in vitro, this approach may prove to be critical to the success of this study. This research requires the use of Gateway-based vectors that express the selected insect dsRNA either constitutively (35S promoter) or in a phloem-specific manner. We previously cloned and confirmed the phloem-specificity of both the AtSUS1 and AtSUC2 promoters from Arabidopsis thaliana, and also cloned the putative promoter regions (approximately 1.5 kb upstream of the translational start site) of SUS1 (CsSUS1-1 and 2) and SUC2 (CsSUT1) homologues from Citrus sinensis cv. valencia. We have since confirmed the phloem-specificity of two separate alleles of the CsSUS1 promoter and have determined that the CsSUT1 promoter confers flower-specific expression in A. thaliana. Expression driven by the CsSUS1-1 and CsSUS1-2 promoters occurs at very high levels, bearing a closer resemblance to the robust AtSUC2 promoter than the weaker AtSUS1 promoter. To test the ability of our citrus promoters to drive phloem-specific expression in a wide range of species, both CsSUS1::GUS and CsSUT1::GUS cassettes have also been transformed into a variety of other plants, including Nicotiana tabacum (tobacco), Prunus domestica (plum) and Malus domestica (apple). GUS assays will be carried out on leaf tissues of potted plants in the coming weeks. To develop an IP-free system, we have successfully replaced the 35S promoter contained within our original Gateway vector with a multiple cloning site that will allow the introduction of our IP-Free efficient phloem-specific promoter to drive the expression of our insect-derived IP-Free dsRNAs for our in planta RNAi assays. In summary, we have cloned a number of transcripts from both D. citri and our model organism, M. persicae, and are repeating our analysis of a subset of derived IP-Free dsRNAs to test their effect on M. persicae using in vitro assays (objective 1). We have also cloned several IP-Free phloem-specific promoters from various organisms, including Citrus sinensis, and are in the process of evaluating their expression patterns in a number of plant systems. Finally, we are developing new IP-Free Gateway-derived vectors bearing a constitutive promoter and phloem-specific promoters (AtSUC2 and CsSUS1), respectively, for use in RNAi against sap-sucking insects in planta (objective 2).
This report covers the period October 1, 2009 through December 31, 2009. This project was funded July 1, 2009. Five coordinating meetings were held during this period. Seven meetings were held with field personnel to arrange, in particular, intensive trapping experiments. This program coordinated with the aerial application program through Southeastern Air and the Indian River Citrus League. Pesticide applications were mapped for the Indian River and St. Lucie counties for subsequent for time and space analyses of Asian citrus psyllid populations. Coordination with citrus growers was increased during this period in order to understand the relationship between grower programs to suppress Asian citrus psyllid populations with pesticide applications by ground and the areawide management program by aerial pesticide application. Trapping was adjusted by blocks to obtain more control(non-sprayed) areas within the area covered by the areawide aerial program. Traps set by county were: Indian River, 205; St. Lucie, 225; Martin, 72. Total traps set and retrieved during this period were: 7,070. Trees surveyed during this period were: Indian River, 2,870; St. Lucie, 3,150; Martin, 1,050. Total Asian citrus psyllids caught were: Indian River 3,413; St. Lucie, 2,510; Martin, 2,020. A new intensive trapping program was begun in Indian River County(the Marsh) and in St. Lucie and Okeechobee Counties(McArthur Farms) to provide baseline psyllid population data prior to applying pesticides by air for the areawide program. Citrus growers were contacted, new blocks for sampling were established by GPS and mapping and traps were set in these new areas. Data sets were now transferred weekly for statistical and other analyses. It is noted that 29 Caribbean fruit flies were caught on the yellow sticky traps in this areawide program during this period. This period established field procedures through experience and correction and provided a large data set for statistical analyses.
The objectives of the first year’s research project are focusing on: 1. Conduct genome-wide sequence analysis to identify Simple Sequence Repeat (SSR) loci from genomic sequences of ‘Candidatus Liberibacter asiaticus’ (Las). Design and develop PCR-based multi-locus SSR molecular diagnosis assay. 2. Analyzing Las population structures, assessing the genetic diversity of Las in Florida populations. We have conducted genome wide sequence analysis to identify (SSR) loci in Las. This resulted in the identification of ~175 total sequencing loci in Las containing mono, di-, tri-, tetra-, penta-, hex- type of perfects, in-perfect and compound types of simple repeats. Based on the criteria for primer design and BLAST analysis against available microbe sequences in the NCBI databases, 112 SSR loci were selected for primer design. We then designed and experimentally evaluated Las SSR primers. These evaluations were based on PCR experiments using 2-3 Las isolates collected from US Florida, India, China and Brazil, respectively. These efforts led to the successful validation and development of 10 SSR primers. While we continue working on more SSR marker development, these 10 SSR markers that showed to be useful for unambiguous detection and discrimination of Las genotypes were used for Las population genetic analysis. To adapt high through-put sample analysis platform, these SSR markers were labeled with fluorescent dyes (FAM, NET, VIC). These labeled SSR markers can be multiplexed and analyzed by an automatic ABI 3130 Genetic Analyzer. This Las genetic analysis system can process 4 x 96 samples in 2.5 hours. We assessed Florida Las population and compared it with a global genetic diversity of Las populations using a multi-locus SSR marker system. A total of 166 HLB isolates representing four major citrus production regions from US Florida, Brazil, India and China were analyzed using 5 SSR primers. Two of them (India and China) represent populations in Asian continent where HLB has been for centuries while another two (US Florida and Brazil) represent recently emerging populations in American continent. Based on allele frequencies of SSR loci among the four populations, a genetic similarity matrix that consisted of 71 alleles and 166 samples was generated. The genetic distance analyses were performed using Neil’s coefficient with 1,000 bootstrap and 95% confidential intervals. A pair wise population genetic analysis indicated that among four Las populations, the genetic distance between Florida vs China Las populations is 0.61, while the genetic distance between Florida vs India is 1.35, Florida vs Brazil is 0.96, China vs Brazil is 0.84, India vs Brazil is 0.95 and China vs India is 1.72, respectively. The results indicated that the genetic distance between Florida and China have the closest distance (0.61) as compared with other populations. High percentage of commonly-shared allele types detected in both populations suggests that Las populations in Florida and China are more related. This leads to a hypothesis that Las populations in Florida could be possibly derived from China. It is not clear if the introduction was from single or multiple times and/or from multiple sources as well. Our results demonstrate the useful of multi-locus SSR marker system for genetic analysis of Las. Bacterium has very compact genome. Sequencing variations of SSR loci located within or near the coding and/or gene regulatory regions could have profound effect on gene expression and functions. We have mapped SSR loci genome containing genes of interest. Our next step is to examine the functionality of the candidate genes using a standard in vitro heterologous expression system. This type of study will facilitate the link of DNA-based genotyping to phenotyping of Las. We have accomplished objective 1 and 2 during the first year of the project. Research had been summarized and reported in 2009 and 2010 American Phytopathological Society meetings.
The objective of this project is 1) to complete the Las genome sequence and conduct comparative genomics studies on the Liberibacter species; 2) to explore the potential role of the microbial community and genetic diversity of Las bacteria in HLB development; 3) to confirm if Las bacteria are seed-transmissible and their role in HLB development. A complete circular genome of Candidatus Liberibacter asiaticus was obtained using a metagenomics approach and published in MPMI 22:1011-1020, 2009. In collaboration with Dr. Hong Lin at the USDA-ARS in Parlier, California, we have obtained approximate 1.25Mb of sequence from Ca. L. psyllaurous. This nearly complete genome contains less than 10 contigs and has ca. 34X coverage . We have also obtained a draft genome (approximately 70%) of Ca. L. americanus using multiple displacement amplification and 454 pyrosequencing technologies. We are currently confirming the sequence of these contigs in both psyllids and host plants. A preliminary comparison revealed significant differences between Ca. L. asiaticus and Ca. L. americanus. The information from our genome sequence allowed us to design new primers and probes that target various regions of the bacterial genome. Using these new primers and probes, genetic diversity of Candidatus Liberibacter asiaticus (Las) samples collected from Florida, Brazil, China, Philippines, Thailand, India and Japan can be found. The relationship between the diversity and disease phenotypes were partially correlated. A putative insect-transmission determinant gene was identified and the role of this gene is under investigation. We have characterized the ATP translocase from Las and proved its function using a heterologous E. coli system. This data was published in J. Bacteriol. 192:834-840, 2010. We are currently developing an antibody-based “drug” to target this protein, aimed at disrupting ATP import, which may be important for its survival. We have also characterized the individual genes of two putative zinc operons in Las, with an overall aim of interfering with the ability of Las to regulate zinc uptake. Seed transmission of Las was tested in grapefruit, sweet orange, and trifoliate orange. Relatively high titers of Las were detected from both seed coats and inner seed coats collected from HLB-affected citrus plants. A very low titer of Las was detected from the embryos and seedlings using nested PCR and real-time PCR. Most, if not all the seedlings did not show typical HLB symptoms and contained a relatively low Las bacterial titer for HLB, even in the three to four year old seedlings. The results indicated that the seed-transmitted Las could not cause typical HLB disease by themselves, which suggested “Detection of Candidatus Liberibacter asiaticus was NOT necessarily equal to the presence of “HLB disease” in plants.” A super sensitive qPCR detection technology has been developed, which increased the sensitivity of detection by 100-2000 fold, thereby eliminating the need for DNA isolation and increasing the throughput of the detection method. The cost savings can be up to 500%. Because the detection is based on HLB bacterium-specific primers, the detection data further confirmed our results on seed transmission and HLB disease phenotypes with low bacterial titers. The role(s) of the seed-transmitted Las is under investigation.
We have accomplished the first objective to duplicate at other grove sites the positive response in HLB infected trees by applying nutrient/SAR foliar sprays obtained by citrus grower Maury Boyd in his Orange Hammock grove in Felda. The second objective was to identify the important components in the foliar spray cocktail. This was approached by arranging the cocktail components into groups of micronutrients (Mg, Zn, Mn, Mo), SARs (Salicylic acid, Serenade Max WP), Phosphite, Hydrogen peroxide, and macronutrients. We have determined that the nutrients are giving the strongest results in rejuvenating and maintaining HLB infected trees. These nutrients include macros and micros, and phosphite. The role of the SARs is undetermined but a synergistic affect may be involved when added to the mix. In 2010 we will separate SAR products and evaluate their contribution (see budget justification). Leaf samples were collected and analyzed at selected intervals since summer 2008. Four treatments (i.e. 4, 5, 6, and 8) do not contain the micronutrient component of the complete HLB cocktail. Trees were consistently and significantly lower in manganese and zinc but not magnesium when compared with trees receiving micronutrients. Micronutrient deficiencies were only observed in treatments not containing micronutrients. Boron was found to be significantly lower after June 2009, presumably after reserves had been exhausted. Boron has been added to the mix. In 2010 we will compare the contribution of the micronutrients in both sulfate and phosphite forms (see budget justification). Yield in 2009 from the commercial grove was higher when trees received phosphite with the nutrients. Based on input prices prevalent during the latter part of 2009, the cost of the foliar nutrient program has been estimated to increase production costs by $200/acre/year over the ‘standard’ HLB management strategy that requires tree inspections and removal of symptomatic trees. A 15-year net present value (NPV) model determined that for a delivered-in price of $1.25 per p.s., annual HLB tree losses had to be greater than 3% before the foliar nutrient program would return a higher 15-year NPV. The cost and future value of resets will be included in this analysis. A first draft of the NPV model has been posted on the SWFREC website to allow growers to determine a HLB tree loss threshold based on their production cost data. Asian citrus psyllid (ACP) populations and Can. Libericacter asiaticus (CLas) titer in plants and psyllids, are being monitored. Two treatments: 1) micronutrients + systemic acquired resistance inducers (Micro+SAR), and 2) Psyllid chemical control applications are being evaluated. Adult ACP populations have been maintained 3 times lower, and up to 50 times lower in insecticide-treated plots compared to insecticide-free plots. The Micro+SAR treatment has had no effect on ACP populations. Overall PCR positive trees increased from 29% in November 2008 to 83% in January 2010. Treatments with insecticidal control had significantly higher Ct values (avg. 25.4) than non-insecticide treatments (avg. 24.1) indicating lower CLas titer in plants protected by insecticides. Despite the high percentage of infected trees, yield collected in March 2009 from trees receiving both insecticidal and nutritional treatments produced 1.32’0.15 boxes/tree, a 30%+ increase over 0.95-0.99 boxes/tree other treatments. 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 reducing negative effects of HLB if CLas titer can be maintained below some threshold level through psyllid management. We will not feel comfortable with this conclusion until we see similar results from the 2009 crop harvested in spring 2010.
The main focus thus far has been to improve the current media to increase growth of the group of bacteria known as Candidatus Liberibacter. To quantify differences between media, microscopy is being used to calculate the number of cells/colonies present on the different media. We are also quantifying differences between different DNA extraction methods using phase contrast and light field microscopy from culture material. This methodology will also establish a correlation between cell/colony count and the Ct value of RT-PCR assays. In collaboration with Ft. Pierce, FL, the testing of Koch’s postulates under field conditions has continued this quarter. Symptoms have not appeared, and a second inoculation will be started this quarter. The second focus has been sequencing the genomes of the three Liberibacter species from pure culture. Using amplified DNA from cultured cells and Illumina Solexa second generation sequencing technology, we sequenced L. asiaticus strain China1 previously shown to be pathogenic. A collection of 21M trimmed paired-end 90mer reads obtained from this sequencing was aligned to all published bacterial genomes at NCBI using GSnap. Notably only 2,220 reads aligned to the current reference for L. asiaticus CP001677.2, primarily to regions identified as rDNA. Some alignment to chromosome 1 of Ralstonia pickettii and Ralstonia megaplasmid was observed. Reads also demonstrated alignment to the family Rhizobiaceae. Over 90 percent of reads were novel, not aligning to any bacterial genome at NCBI. The read set was used to construct a draft Phase 1 assembly (contigs unordered and unoriented) resulting in 481 contigs, indicating a genome size of 3.8M bp. These contigs demonstrate local similarity to bacterial sequences at NCBI, but contain much novel material and in many cases ORFs similar to known genes or conserved domains. Sequence-derived evidence was used to confirm the presence or absence of these sequence contigs in 8 culture samples and 34 diseased tissue samples. The presence of Rhizobiaceae-like sequence in all samples confirmed the successful sequencing of the genome from cultured cells of Liberibacter. Ralstonia plasmid sequence was found in 25% of Asian samples, but not in North and South American samples. Unique sequences showed two patterns; sequence present in all samples suggesting bacterial chromosomal DNA and sequence present in a percentage of the samples suggesting plasmid DNA. The genomes of L. asiaticus and L. americanus are currently being assembled and annotated. The genome of L. africanus is still in the sequencing phase.
This work on this project 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. This work is a collaborative effort of five researchers located at four locations since part of the work involves exotic HLB associated pathogens. At the University of Florida, CREC Lake Alfred, quantitative real time PCR (qPCR) work focused on Severinia buxifolia, rough lemon, Calomondin and Murraya paniculata. Graft inoculations to Severinia buxifolia (orange boxwood) and showed that it was an excellent host for the Las bacterium. Asian citrus psyllids were allowed to feed on HLB infected S. buxifolia and 20-30% were found PCR positive. Transmission tests from S. buxifolia to sweet orange was done and the transmission rate was found to be high. This research was done by Hao Hu a graduate student studying with Dr. Brlansky on this project. Studies to determine the absolute quantification of live bacteria in the tissues has been done using two compounds to separate dead from live bacteria. We feel that this is necessary since previous results have shown high numbers of Las in infected tissues but microscopy has revealed few bacteria. 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. Other commercial rootstocks have been tested and results show that many of them continue to grow even when infected with Las. In field experiments rootstocks from clipped HLB positive trees have shown that rootstock sprouts are positive for Las and maybe a source for further spread. Various commercial sweet orange cultivars were inoculated with Florida Ca. Las and one cultivar tested PCR negative in multiple graft inoculation tests. We have expanded tests on this cultivar and plan to report these results at the Florida State Horticultural Society meetings in June. Experiments on the existence of alternative hosts where edge effects next to non- citrus plantings has begun. Zanthozylum species have been found but PCR tests are negative. At Texas A&M Citrus Center, psyllid feeding tests on eight indigeous rutaceous plants. Esenbeckia berlandieri (jopoy), Amyris madrensis (torchwood), Choisya ternata and C. arizonica and Zanthoxylum fragara, Casimiroa terameria were found to be feeding hosts for the psyllid. 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 and C. arizonica. Feeding and egg laying on 3 of the others species but nymphs died. At the USDA, ARS, Beltsville quarantine greenhouse graft inoculations were done to M. paniculata with exotic Ca. Ca. Lam and Ca. Laf isolates and are being tested. 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. Dodder became infected and phloem necrosis occurred similar to that in citrus. We showed that Liberibacter exists in two morphological forms in the dodder similar to that seen in citrus and periwinkle and an intermediate between the two forms was discovered. 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 have been done and testing will begin after incubation.
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