Reducing the amount of vegetative growth produced annually by citrus trees in Florida would reduce the opportunities for Asian citrus psyllid reproduction and thereby the spread of Hunaglongbing. This could be done in citrus without detrimental effects on yield because citrus trees in Florida produce an excess of leaves above that required to support maximum fruit yield. Excess tree growth is routinely controlled through hedging, but little research has been done to examine the effects of hedging on vegetative growth. Recent research in Florida has shown that branch re-growth can be reduced when hedging is performed in fall under Florida conditions because of the onset of cool temperatures. Additionally, research in Florida indicates that late-summer hedging may be able to synchronize a final late-season flush and thus, reduce new flush leaves present during the winter to support over-wintering psyllids. In other fruit crops, such as apple, where excessive vegetative growth can be problematic plant growth regulators (PGRs) are routinely used. However, the use of PGRs in citrus has been limited to influencing fruit development and for maintaining post-harvest quality. PGRs not only control vegetative growth, but also offer the potential to reduce insect pest populations either by reducing pest-required vegetative growth or by altering host plant metabolites or nutrition. A greenhouse trial of six different plant growth regulators and their effects on citrus tree growth and psyllid oviposition has recently been completed. While data analysis on this trial is not yet complete, early indications are that four of the six PGRs successfully reduced the growth of citrus trees and the products had varying effects on psyllid behavior, from dramatically reducing oviposition to increasing oviposition. The results of this trial are very promising and future trials are being designed to build on these findings. In addition, a wide range of PGRs are being screened in ongoing greenhouse trials for toxicity and other adverse effects. These data will be used in designing field trials during spring 2010.
This is a continuing project for which funding was released this year on 13 April. Tamarixia radiata colonies from south China, North Vietnam and Pakistan established in quarantine have now been approved by USDA-APHIS and DPI for release into the environment. Additionally, we obtained 3 more shipments of mummified nymphs of D. citri from Pakistan this quarter to refresh the Pakastani colony of T. radiata and establish an additional colony of the parasitoid Diaphorencyrtus aligarhensis at DPI ,Gainesville. For our second objective, a collaborative study with Dr. Norman Barr, USDA-ARS Mission TX, on the genetic characterization of T. radiata strains from USA and some other regions of the world was completed and is in press for publication. Using the genetic markers being developed by Dr. Barr, we hope to track the establishment and performance of different T. radiata in the field. We are consistently increasing the production of parasitoids and continuing to release and evaluate in the field (Objectives 1,3). Between March and September 2009 we produced 42,000 T. radiata wasps that were used to maintain our colonies, initiate the colony at OrangeCo, conduct experiments here and at Lake Alfred, and release in experimental, conventional, and organic groves. Twenty three percent nymphs were parasitized by T. radiata in August-September in a block of 1 m tall citrus plants at the SWFREC, Immokalee. Nymphs recovered on sentinel plants in the conventional grove were 52% parasitized by T. radiata in April but none in August and September. Diaphorencyrtus aligarhensis, an endoparasitoid of D. citri from southern China is also being reared and released throughout the state. A total of 2400, 1325, and 5050 wasps were released during 2007, 2008, and 2009, respectively, in conventional and organic citrus groves and dooryard orange jasmine (Muraya paniculata). A parasitism rate of 6-19% was calculated based on adult emergence from nymphs reared from M. paniculata in May 2008. In August-September 2009, 1500 wasps were released in dooryard Murraya and citrus and in organic groves, however, none were recovered. We are also monitoring the populations of psyllids, predatory mites, parasitoids, and other natural enemies in an organic grove in Lake Wales, FL., where we released over 0.5 million predatory mites (Amblyseius swirskii) on mature orange during bloom. Predatory mites, mostly Typhlodromalus and Euseius species averaged 3 and 5 per two tap samples per tree in March and April, respectively, declined to 1 per sample during May-June and < 1 during July-August. Psyllids per two tap samples averaged 1 during March-April, 3 in May, 1 in June, and < 1 in July and August. Incidence of parasitism is low throughout the state, presumably due to widespread use of insecticides for psyllid control; thus the need to augment populations of T. radiata. For objective 4, we assisted Orange Co. mass rear T. radiata, made several presentations and published our findings to reach the target clientele. An International Tamarixia Workshop is being organized with APHIS for Feb 2010 in McAllen TX. 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 (in press). 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.
This is a 3-year project with 3 main focal points: 1. To build a foundation of understanding of the host- Candidatus Liberibacter asiaticus (LAS) interactions that involves testing multiple genetic variants of citrus, examination of environmental and seasonal effects on symptoms and pathogen replication and movement, and understanding effects of genetic variation in the pathogen system, so that better methods of controlling the vector, detecting and monitoring the disease, and growing trees will allow better production of citrus; and, 2. To develop an understanding of how Las interacts with citrus genotypes to cause disease in sensitive varieties and to not cause disease in tolerant varieties in the hope that this information will lead to the development of approaches to produce citrus economically in the present situation with citrus greening in Florida; and, 3. To provide knowledge and resources to support and foster research in other laboratories. A substantial number of projects to be supported are based on our research and reagents (propagated healthy and HLB-infected plants, HLB inocula, nucleic acid extracts, etc.) supplied by our laboratory. We view this as one of our most important goals. We have developed a containment plant growth room to allow natural infection of citrus trees by psyllid inoculation. We are testing a series of citrus relatives for preference and inoculation by aphids. This test includes a series of different sources of poncirus. This test is at the 4 month stage. Preliminary observations suggest that psyllids are able to acquire Las from exposed plants months before PCR detection and before development of symptoms. Preliminary observations suggest that psyllids prefer to feed on older infected plants compared to younger plants with new flush, although reproduction occurs on the new flush. We also have a series of experiments testing the susceptibility of poncirus and latipes in the greenhouse. Most plants remain PCR negative after graft inoculation although the living inoculum is definitely positive. Yet an occasional plant becomes PCR positive. We continue to investigate whether this is a genetic response that can be used to produce resistant or tolerant trees. Similar experiments are being done by psyllid inoculation. A wide range of citrus relatives have been inoculated with Las and are being evalutated in the greenhouse. We are also screening a range of elite lines for the citrus improvement group and a series of transgenic trees. All of these experiments are underway. We also continue to be be a resource to supply infected trees and infected psyllids to several other research laboratories.
This is a continuing project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. With the loss of trees due to canker eradication and development and the continuing losses to greening, there is a major concern whether sufficient fruit will be produced to keep processing plants open. We are approaching this problem in two ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen to focus on antibacterial peptides because they represent one of the few choices available for this time frame. Secondly, we are developing virus vectors based on CTV to express the antibacterial peptides in trees in the field as an interim measure until transgenic trees are available. We think that this approach could be used beginning 2-3 years from now and until probably 15 years from now when resistant trees should be available. The milestones for this year’s research are: 1) Continue to screen peptides for activity against Las in citrus trees using the CTV vector. We have completed one large screening of antibacterial peptides against Las in sweet orange trees. Eighteen different peptides were tested in this experiment. The trees were infected with CTV expressing the different peptides and two months later were inoculated with HLB by grafting. We found three peptides that allow much better growth of infected trees. Some trees had no symptoms and no detectable Las, some trees had no symptoms and low levels of Las, and other trees had leaf symptoms but continued growth of the trees with normal levels of Las. This experiment also demonstrated that leader peptides for the export of the peptide from the cell from which it was produced is not needed for HLB but is needed for citrus canker. We have developed systems to test the peptides under more natural conditions. We are concerned that graft inoculation of HLB into the trunks of small trees is a too severe challenge that might cause peptides that could work in the field to be missed. We have established a containment plant growth room in which psyllids inoculate the plants expressing the peptides. An advantage is that the psyllids feed on the areas of the plants with the highest levels of expressed peptides. This experiment is at the 4 month stage. This experiment also is evaluating a series of transgenic plants for resistance or tolerance to HLB. 2) Begin high-throughput screening in tobacco against solanaceous liberbacter. (Falk, UC Davis) The subcontract has just been completed to UC Davis and the work is beginning. 3) Improve CTV-based vector to produce 2-5 peptides and to overcome cross-protection. We have built several new versions of CTV vector and have shown that two foreign genes can be expressed efficiently at the same time. We are making progress in understanding the process of cross protection to allow development of a CTV vector that will allow subsequent application of antibacterial gene products. 4) Examine survival of peptides in fruit and juice. We have developed the plants for these assays, but this project is just beginning. 5) Prepare trees for year 2.
The objective of this project is 1) to complete Las genome sequence and to conduct comparative genomics of 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 has been obtained using metagenomics approach, and published in MPMI 22:1011-1020, 2009. In collaboration with Dr. Hong Lin in USDA-ARS, Parlier, California, we have obtained approximate 1.2Mb, a nearly complete genome of Ca. L. psyllaurous with less than 20 contigs, which has ca. 34X coverage . We have also obtained the 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 both in the psyllids and host plants. Preliminary comparison revealed significant difference 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, we revealed the genetic diversity of Candidatus Liberibacter asiaticus (Las) collected from Florida, Brazil, China and Japan. The relationship between the diversity and disease phenotypes were partially correlated. A putative insect transmission determinant gene was identified. The role of this gene is being investigated. We have characterized the ATP/ADP translocase of Las, and proved its function in the heterologous E. coli system. We are currently developing a antibody-based “drug” to target this protein, aiming to disrupt the life cycle of the Las bacterium. The seed transmission of Las is tested in grapefruit, sweet orange and trifoliate orange. Relative high titer of Las detected from seed coat and inner seed coat of the seeds collected from HLB-affected citrus plants. Very low titer of Las was detected from the seedlings, ranging from 3 to 42% using nested PCR. Most, if not all the seedlings did not have typical HLB symptoms and the threshold of the bacterial titer for HLB, even in the three year old seedlings. The results indicated that the seed-transmitted Las could not cause HLB by themselves. The role(s) of these seed-transmitted Las is under investigation.
To demonstrate that Candidatus Liberibacter bacterium is the causative agent of Huanglongbing disease (HLB), or greening, KochÕs postulates need to be performed with a pure culture of Liberibacter and is still challenging various research laboratories. We want to obtain a primoculture of Candidatus Liberibacter by co-culturing the bacteria with insect cells to study Candidatus Liberibacter physiology, metabolism, virulence and its interactions with the insect vector. Indeed Liberibacter is vectored by psyllid insects and is able to proliferate inside the insect. We therefore hypothesize that insect cells could act like feeder cells, providing nutrient complements in a continuous way but also a favorable environment to Liberibacter development. We tested various insect cell lines to in vitro culture Candidatus Liberibacter asiaticus (LAS), the Asian form of HLB disease, also found in Florida. We tested Lepidoptera cell lines (Mamestra brassicae and Spodoptera frugiperda) and Diptera cell lines (Drosophila melanogaster). We inoculated this cell cultures with different inoculums (from infected Macrophylla, Lime or Periwinkle plants). We checked for the presence of LAS in inoculated cell cultures by direct and Nested PCR. Current results: -Inoculums from LAS infected citrus (Lime from Vietnam) were the best of the inoculums tested. -To this day we didnÕt detect any Las in Mamestra (hemocyte and ovarian cells) or Spodoptera (hemocyte cells) cell lines. -We detected LAS in two drosophila cell cultures by direct PCR and the signal was maintained after 6 transfers of the culture, over more than 6 weeks (and still running). It means the bacterium is multiplying. -In order to reach higher bacterial concentrations, we are currently testing complementation of the Drosophila cell culture medium with various sugars, vitamins or trace elements that could be found in citrus/periwinkle phloem. We analyzed metabolic pathways potentially encoded by the released Liberibacter genome sequences to improve growth conditions and to define limiting factors and/or growth inhibitors. We so far selected 6 complements and got positive results with 3 of them. Current work: -The detection of LAS varies over time and transfers in drosophila cell cultures; it seems to be linked to drosophila cells concentration. In order to define this link, we are setting up multiplex qPCR assays to monitor the ratio of LAS vs. drosophila DNA over culture time. -We are combining complements to the insect cell culture media and looking for new ones to improve the bacterial concentration in the insect cell cultures. -We are analyzing sugar and amino-acids variations (in particular depletion) in insect cell culture media over culture time to identify potential LAS growth limiting factors.
Funds for this study were received March 18, 2009. Since these experiments take place early in the season when fruit require degreening to meet market requirements, the bulk of the funds will be used in October and November when late summer/fall rains result in small lesions on green fruit and washing the fruit may inhibit degreening. As part of another project, we recently built a research grading line that will be used for this study and allow evaluation of a number of washing treatments and other variables on the ability to effectively grade the fruit to find canker lesions and other blemishes. Modifications to our research citrus washing line are complete to evaluate different washing techniques and we’ve arranged to run fruit over a local commercial packinghouse wash line. Preliminary results from earlier experiments were presented at the FSHS meetings June 2009 and a manuscript submitted and accepted as a peer reviewed publication in the next FSHS proceedings. These results showed that inhibition of degreening was significantly less when fruit were washed for only ~15 seconds on a high-pressure washer (HPW) compared to longer (1 to 2 min) brush washing only (no HPW). On October 6th, 2009 we began our first set of experiments using Fallglo tangerines that were either washed on a commercial line (the entire brush and HPW, brush washer only, HPW only, or . or . time on the HPW), washed or washed and waxed (carnauba) on our research line, or were not washed at all but only passed over rollers on a grading table. Fruit from all these treatments were degreened under simulated commercial conditions and color development and weight loss measured almost daily. The fruit continues to be evaluated. The week of October 19 we are scheduled to conduct another, larger study using navel oranges and Ruby Red grapefruit. In all, we plan to evaluate different methods of washing on fruit coloration during and after degreening, total required degreening time to reach acceptable color, and the affect of fruit cleanliness, presence of other peel markings (i.e., windscar, melanose, etc.), speed of fruit travel, lighting conditions, roller color, grading area width, and the number of graders per unit area on the ability to detect fruit with canker lesions and other surface defects.
Research results from the first season were successfully completed, the results reported at the 2008 annual Florida State Horticultural Society (FSHS) meetings and at Packinghouse Day and the Indian River Postharvest Workshop in Sept. 2008, a manuscript published in the FSHS proceedings (http://www.fshs.org/; Proc. Fla. State Hort. Soc. 2008. 121:322’325), and results reported in previous FCPRAC progress reports. To summarize, preharvest treatments included up to three foliar potassium (K) sprays (March, October, and January) or withholding irrigation for up to two months before harvest. In one commercial block, foliar mono-potassium phosphate (MKP) was applied at 23.5 lb MKP per acre (0-52-34; 8 lb K2O per acre) with 4 lb per acre low-biuret urea (46-0-0) applied at a total volume of 125 gal per acre. In another block, the grower applied 3 gal per acre of a commercial 3-18-18 formulation at a total volume of 250 gal per acre. Postharvest treatments included holding fruit for three days at 30%, 55%, or 100% (including wetting the fruit) RH before washing and storing at 70F under ambient RH. In the first grapefruit block, preharvest foliar MKP treatments applied 2 months or more before harvest did not significantly reduce peel breakdown. However, fruit set was poor in that block producing very large and misshapen fruit. In the second block, peel breakdown was significantly reduced with foliar 3-18-18 applied 2, 3, or 4 weeks before harvest. Preventing irrigation or rainfall for 49 days before harvest increased peel breakdown, whereas wetting the fruit after harvest and maintaining high postharvest RH reduced peel breakdown compared to exposing fruit to lower RH for 3 d before washing. Funds for the second year of this study were received very late in the season on April 3, 2009. So the bulk of the funds will be used on experiments this coming season. However, we did conduct several additional experiments in 2008-09 before the project’s approval. After three sets of field trials on White grapefruit and Valencia oranges, we found less promising results with foliar K than in the previous season. In one experiment, foliar K even increased peel breakdown. The reason for this is unclear. However, applying a combination of foliar K and Mg or use of an antitranspirant resulted in a significant reduction in peel breakdown during postharvest storage. Application of foliar Mg (Epsom salt) alone showed a trend in reducing peel breakdown that, while not significant, is worth repeating. Four sets of postharvest experiments gave similar results as the previous year. Interestingly, postharvest treatments with Imazalil sporadically reduced peel breakdown as was noted the previous year. The reason for this is unclear but worth investigating further. For the current 2009-10 season we are establishing at least two field plots in two different commercial grapefruit groves to apply preharvest foliar K, antitranspirants, and Mg. We will also test combinations of these materials as well as other treatments that might be suggested by research and/or grower reports.
Repeated low volume (LV) applications of horticultural mineral oil (HMO) were initiated this year on 14 April although funding for this project was released on 27 April. The replicated experiment is being conducted in the same 65 acre block as last year, although we have increased the rate from 1 to 2 gal/ac pure FL-435 oil and are comparing two truck-mounted LV applicators: a modified London Fogger model 18-20 provided by Chemical Containers and the Proptec rotary atomizer P400D. Applications continued through fall flush at 2-4 week intervals depending on pest pressure. Significant differences in the number of Asian citrus psyllid (ACP) adults between the Proptec P400D (0.06’0.06 ACP adults/tap) and the control (0.23’0.08 ACP adults/tap) were observed on 15 June. A similar trend has continued to be observed, although there were no statistically significant treatment effects due to low psyllid populations. Leaf extractions have been made as a first step in the process of quantifying mineral oil deposition on leaf surfaces of citrus following LV ground applications. Results from these studies support those obtained last year indicating the effectiveness of frequent LV applications of HMO, but we still need higher populations to feel confident with this tactic. We also conducted a separate study on a highly infested, 38-acre block of ‘pineapple’ orange in Glades County to compare the London Fogger ‘ applying 2 gal/ac to bed tops only (typical application) with an airblast sprayer treating tops and swales at 116 gal/ac. Delegate WG (spinetoram) @ 4 oz/ac. and Dimethoate 4EC (24 oz/ac.) were both applied with 2 gal/ac. oil on 15 June. Insecticides sprayed with the airblast showed significantly fewer adults compared to the control from week 1 to week 4. LV sprays showed significantly lower populations than the control only for weeks 3 and 4. This experiment demonstrated that LV application to bed tops with these two insecticides did not provide adequate control. Growers should evaluate such applications carefully. In a third experiment, we evaluated the effectiveness of aerial versus ground application of two selective insecticides: Delegate WG (spinetoram) @ 4 oz/ac, and Movento (spirotetramat) @ 10oz./ac., and a broad-spectrum insecticide Imidan 70W phosmet (Imidan) @ 1Lb/ac, all with 2gal/ac of 435 mineral oil. The trial was conducted on thirty 24-acre blocks of mature orange trees. Imidan by ground (0.1’0.1 ACP/tap) was the only treatment that significantly reduced ACP populations compared to the untreated control (2.6’0.5 ACP/tap) 2 weeks after application. At week 4, ground applications resulted in lower ACP numbers when compared with aerial applications. We also saw fewer psyllids on trees treated with Movento (by air: 0.7’0.1 ACP/tap, by ground: 1.1’0.3 ACP/tap), and Imidan by ground (0.2’0.2 ACP/tap) compared with the control (2.1’0.5 ACP/tap). The effect of these treatments on rust mites was evaluated on 6 August by counting the number in 2 lens fields (14x) per fruit, 4 fruits per tree, 10 trees per plot . Ground applications of Delegate (0.13’0.03 mites/fruit), Movento (0.12’0.02 mites/fruit), and Imidan by ground (0.28’0.08 mites/fruit) and by air (0.16’0.07 mites/fruit) had significant lower populations when compared with the untreated control (0.42’0.13 mites/fruit) and oil only (0.40’0.08 mites/fruit). Results from these trials have been presented at the Florida State Horticultural Society 2009 annual meeting and will be published in their proceedings, and also be presented at the Entomological Society of America December 2009. In an area wide spray during the 2008/09 dormant season, 80,000 acres were treated by air and more by ground. A field survey coordinated by SWFREC-Entomology and DPI-CHRP found that 5 months after the application groves treated by air (0.1’0.02 ACP/tap) and by ground (0.3’0.1 ACP/tap) had significantly fewer ACP than the untreated which had 2.8 ACP/tap, a 2400% increase. We are working with Golf Citrus Growers Association to organize two area wide sprays this season.
Data still have been collected from this field experiment. From June/09 to September/09 the disease incidence increased from 15.4 to 20.3% in plots with psyllid control program and from 34.1 to 44.9% 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). Until now, there was no significant relationship between the frequency of local inoculum reduction and all assessed variables. Even the local vector control program significantly reduced the adult psyllid population in 79% and the psyllid eggs and nymphs in 95% in treated trees; psyllid control significantly reduced the cumulative number of HLB-symptomatic trees in 55%. No significant difference was observed on the delay of the beginning of epidemics and on disease progress rates. This preliminary result shows that the tested vector control program did not completely avoid HLB contamination from external source of inoculum (20.3% of HLB-symptomatic trees 40 months after planting). After the first harvest in September/09, the average yield on plots without vector control (40.4 box/acre) was 56% smaller than the average yield on plots with vector control (91.6 box/acre). Additionally, the yield per plant was 38% less in non-treated trees (0.33 box/plant) than in insecticide treated trees (0.54 box/plant). Trees without systematic insecticide sprays had higher attack of other pest such as citrus leafminer and aphids that reduced the normal vegetative flux development. Until September/09, US$ 7.79 per plant was spent on psyllid vector control program. Additional data for economic analysis have been collected. Annual maps of HLB-symptomatic trees are being prepared for spatial analysis using stochastic models (MCMC) to verify the effects of each treatment on primary and secondary spread of HLB. Also, all psyllids captured on yellow stick traps since the winter/07 are being prepared to be tested for the presence of Candidatus Liberibacter spp. by PCR. The assessments on this experiment will continue to allow more detailed temporal and spatial analysis and get better conclusions.
Funds for this project were released on 9 June 2009. Objectives and corresponding results are listed below. 1) Evaluate and refine the use of the tap sample to monitor adult psyllids and methods of assessing flush density, and infestation rates in blocks of growing and dormant citrus trees of different ages and varieties planted at experimental and commercial groves. Tap sampling, sticky traps, and sweep nets were compared for sampling low and high density populations of psyllid adults as maintained in several insecticide treated and untreated blocks. A tap sample consists of 3 consecutive strikes on a randomly chosen branch using the hand or a PVC pipe, and counting fallen psyllids on a laminated white sheet of paper held 1 ft below the foliage. At low density populations, adult psyllids per trap per tree on ‘Yellow Corn Rootworm Trap’ (YCRW), and ‘ACP Trap’ hung within the canopies of citrus trees in a commercial grove over a two week period 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, using the above procedure at the high density. Sticky traps have a high cost (approx. $ 1 per trap), provide data after at least a 1-week delay and require an average 7 minutes to deploy and collect data from one trap compared to an average of 3 sec for a tap sample that provide instant information. Sampling conducted at the experimental grove of Southwest Florida Research and Extension Center used two tap samples and two sweeps on the same tree. Significantly more adults were detected using tap sample compared to the sweep net at low density with means of 0.1 ‘ 0.02 and 0.06 ‘ 0.01 per one tap or sweep sample per tree, respectively. Means of 0.3 ‘ 0.1 per tap and 0.4 ‘ 0.1 per sweep sample per tree were obtained at high density and were not significantly different. Time to conduct one sweep (3.1 ‘ 0.04 sec) was significantly more than to conduct one tap sample (2.9 ‘ 0.04 sec) at low density but did not differ between the two methods at high density and averaged 3.4 ‘ 0.1 sec. 2) Test the influence of these parameters on precision of estimated means and the distribution of the population within blocks using the appropriate aggregation and regression models 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 could take less time to provide the same data but could also involve more work and difficulty in counting at very high densities and could spread citrus canker. 3) Evaluate and integrate methods for assessing these parameters into a user friendly system accessible to consultants and managers. We made several presentations and published to reach consultants and managers. Several of our presentations, publications, and details of tap sampling procedure are available at our website: http://www.imok.ufl.edu/entlab/. A survey of 27 groves conducted by UF-IFAS and Multi-County Cooperative Extension reported 95% growers use tap sample along with flush observations (76%), sticky traps (14%), and sweep net (9%), with 85% sampling > 3 times a year.
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. A phloem analysis of sweet orange was completed by UC Davis MetaCore facility using GC/MS. Along with the genomic metabolic analysis of the available Ca. L. asiaticus genome, several key growth factors were indicated for further testing. Several nitrogen sources were tested as additions to the published media; ethanolamine has shown to improve initial growth. The seven micronutrients were tested with only the addition of zinc showing any improved growth. None of the amino acids or carbohydrates tested thus far have shown any improved growth, but several remain to be added to the media. Our current focus is to combine the different metabolites that show improved growth to optimize the media. In addition to these metabolites, sterilized fresh juice from uninfected plants was added to media in place of the citrus vein extract. We found that bacteria isolated from sour plants (lemons, limes) grew better on plates with sour juice compared to sweet juice and vice versa. From this information, we plan to do phloem analyses on multiple species and cultivars of citrus to identify commonalities and differences in the phloem chemistries. In collaboration with Ft. Pierce, FL, the testing of Koch’s postulates under field conditions began this quarter. A Florida-derived culture of Ca. L. asiaticus was initiated at Ft. Pierce last quarter. Inoculations using original isolations and transfers occurred this quarter. Symptoms are not expected for several months, but we will use RT-PCR assays to determine if Ca. L. asiaticus is present in the next 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 strain used in the published pathogenicity trials (China1 Ca. L. asiaticus). The current genome is in the alignment phase and we plan to release the sequence in the third quarter. The second strain used in the pathogenicity trials (Brazil1 Ca. L. americanus) is ready for shipment to the NCGR and sequencing will begin once the alignment of China1 is complete. Currently we are ensuring the quality and purity of the representative strain of Ca. L. africanus (Africa2).
This work will determine if certain alternative hosts are better hosts for the suspected HLB bacterial pathogens (Ca. Liberibacter asiaticus (Ca. Las), Ca. Liberibacter americanus (Ca. Lam) and Ca. Liberibacter africanus (Ca. Laf)) and can serve as a reservoir hosts for infection to citrus. During this quarter work was again was performed at all four investigator locations. At the University of Florida, CREC Lake Alfred HLB infection once again was achieved by graft transmission from HLB (Ca. Las) infected citrus to Severinia buxifolia (orange boxwood) and to C. jambhiri. Quantitative real time PCR (qPCR) was done on both hosts and showed that they were very good hosts for the Las bacterium. Psyllid transmission tests from Severinia to sweet orange were repeated this quarter and will be PCR tested after the appropriate incubation period. Additional infections were achieved with rootstocks Carrizo citrange, Swingle citrumelo and Kinkojii. At the Texas A&M Citrus Center, Weslaco psyllid feeding tests continued on the rutaceous plants that are established there. Esenbeckia berlandieri (jopoy), Amyris madrensis (torchwood), Choisya ternata and C. arizonica all were found to be feeding hosts for the psyllid. Egg laying was found on torchwood and egg laying and nymphal development were found on C. ternata. This work is part graduate student Jose Sandoval’s researchwith Dr. da Graca. Plant materials of Severinia buxifolia and Murraya paniculata were established at the USDA, ARS, Beltsville quarantine greenhouse and inoculations are pending with exotic Ca. Las, Ca. Lam and Ca. Laf isolates. Work with dodder as an alternative host to study the plant infection process and for its use with plants that are not graft compatible with citrus was concluded and a manuscript was submitted for publication. Dodder becomes infected and phloem necrosis occurs similar to that found in citrus. At the USDA, ARS, FDWSRU, Ft. Detrick, MD a manuscript was completed and submitted for publication on the work of infection of Murraya paniculata and the psyllid transmission to sweet orange. A presentation will be made at the HLB-Zebra Chip workshop in Weslaco in November.
The goal of the proposed research is to understand how Candidatus Liberibacter asiaticus causes Huanglongbing (HLB) disease on citrus. Citrus HLB is the most devastating disease on citrus. There are very few options for management of the disease due to the lack of understanding of the pathogen and citrus interaction. Understanding the citrus and citrus HLB pathogen interaction is needed in order to provide knowledge to develop sustainable and economically viable control measures. We proposed to expand our current research to different citrus varieties which are either susceptible or tolerant to HLB and a more facile model system tobacco (Nicotiana tabacum Xanthi). The specific objectives of this proposal are: transcriptional and microscopic analyses of citrus varieties which are either susceptible or tolerant to Ca. L. asiaticus infection at different infection stages in greenhouse and citrus grove; and transcriptional and microscopic analyses of host response to Ca. L. asiaticus infection with a model system tobacco. Microarray analysis and/or suppressive subtractive hybridization libraries approaches will be used to study the host response to the HLB pathogen infection followed by confirmation with Northern blot or quantitative reverse transcriptional PCR. Anatomical study will be performed with light microscopy using different staining methods. The following objectives related to this project were pursued: (A) An initial microarray analysis of host response of sweet orange to Las infection in greenhouse and in field; and (B) A preliminary study of the effects of HLB on phloem at the microscopic level and phloem transport of sweet orange. The results of these experiments are detailed below. Major achievements: 1. Microarray analysis of host response of sweet orange to Las infection in greenhouse. The results have been published in the following paper: Kim, J., Sagaram, U.S., Burns, J. K., and Wang N*. 2009 Response of sweet orange (Citrus sinensis) to Candidatus Liberibacter asiaticus infection: microscopy and microarray analyses. Phytopathology 2009 99:50-7. 2. To further expand our current understanding of Las-host interaction, we are currently comparing two susceptible and two resistant hybrid/cultivars in greenhouse. Currently, we have collected samples for more than 4 months for the two susceptible varieties including grapefruit and Madam Venus. The two resistant/tolerant varieties have not been inoculated successfully. 3. Leaf samples from different varieties including grapefruit, Murcott, and Hamlin were collected from Florida citrus groves. Both healthy and infected trees were sampled. It is expected gene expression of those trees in the citrus grove will reveal more information when compare with the gene expression profile in greenhouse. Those samples will be used for gene expression analysis using microarray or SSH approaches. 4. Gene expression of Valencia in citrus grove have been done. The data is under analysis.
Citrus Huanglongbing (HLB) is one of the most destructive diseases on citrus. Very little is known about the causal pathogen and the microbiome associated with the infected trees. The goal of the proposed research is to characterize the bacteria (endophytes and plant pathogens) associated with HLB positive citrus, the Asian citrus psyllid, dodder, and periwinkle. The specific objectives of this proposal are: (1) Comparison of the microbiomes in leaf midribs and roots of HLB pathogen positive and free citrus of different varieties; (2) Characterization of the microbiomes that can be transferred among citrus, psyllid, dodder, and periwinkle; and (3) Understanding how Candidatus Liberibacter asiaticus colonizes and moves inside the phloem. Comparison of the microbiomes associated with HLB pathogen positive and negative citrus will illuminate the causal agent of citrus greening. Potential beneficial endophytic microorganisms could be identified from escape plants which survived in heavily infected citrus groove with HLB. Beneficial microorganisms have been shown in previous studies to have the capacity to control plant diseases by accelerating seedling emergence, promoting plant growth and development, and preventing the invasion of plant pathogens. The investigation of the microbiomes associated with different hosts will help understand the transmission of microorganisms between different hosts. Major achievements: 1. A comprehensive study of the bacterial diversity associated with healthy and HLB diseased citrus indicated that Candidatus Liberibacter asiaticus as the pathogen responsible for HLB disease in Florida. Phytoplasma was not found in any of the samples collected from Florida. The result has been published in the following publication: Uma Shankar Sagaram, Kristen M. DeAngelis, Pankaj Trivedi, Gary L. Andersen, Shien Lu, and Nian Wang 2009 Bacterial diversity analysis of Huanglongbing pathogen-infected citrus using PhyloChips and 16S rDNA clone library sequencing. Applied and Environmental Microbiology 2009 75: 1566-1574. 2. Samples (leaf and root) from different varieties including grapefruit, Murcott, and Hamlin were collected from Florida. Both healthy and infected trees were sampled. Bacteria associated with the root system will be studied comprehensively. 3. Samples (leaf and psyllid) from three different locations were collected. The bacterial diversity study of the HLB infected trees and the psyllids in those groves will reveal potential competitive microorganisms for the control purpose. 4. Isolation of bacteria with the potential of plant growth promoting and biological control potential might reveal innovative ways controlling the HLB disease. Fifty-four morphologically distinct isolates were obtained from surface sterilized roots of symptomatic and asymptomatic (potential escape trees) citrus plants from a citrus grove with a HLB infection rate of more than 60% and an infection history of approximate five years. Qualitative screening showed that for all of these criteria, asymptomatic plants harbor a significant greater diversity of potentially beneficial bacterial strains. 5. Development of a proper in vitro screening system that provides repeatable and reliable results in shorter periods of time is an important step for isolation of efficient bacterial antagonists. We have developed a method to screen antagonistic bacteria against unculturable HLB pathogen. The method uses the discrimination of live-dead cells by EMA and speed and sensitivity of QPCR. Six bacterial isolates were found to reduce the number of viable Las cells.
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