Mid Florida Citrus Foundation (MFCF) currently supports the research efforts of scientists from the University of Florida, USDA and private industry. The majority of research projects are conducted by UF scientists at the approximately 112 acre citrus grove. The MFCF supports citrus research through the employment of a full time grove manager whom works closely with researchers to ensure that their projects are handled properly and that the grove is an excellent condition. The management of this grove requires extra financial commitment as grove care cost tend to be higher than a commercial grove due to the nature of many of the research projects. Current projects being conducted at the MFCF are Asian citrus psyllid (ACP) pesticide evaluation control trials, low volume applicator trials, windbreak evaluation, HLB nutritional programs, new and existing herbicide trials, variety and rootstock evaluation trials. The MFCF is in the process of expanding grove to allow more research work to be conducted. This was made possible due to the fact that the MFCF received an EQIP grant from the NRCS. This past summer 13 acres were planted for new research trials, which included an HLB trial, multiple new herbicide trials and new variety evaluations. Additionally windbreaks were established for new and future citrus research blocks. Current plans call for the establishment of 30 additional acres of research citrus trials for the purpose of young tree psyllid control research, evaluation of new psyllid control products, economic study on rooted cuttings, evaluation of miticides and herbicides and large block planting of experimental early orange.
We are still assessing new symptoms appearance in all established experiments. Exp.1 – In the compartments 1 and 2 where citrus plants served as primary source of inoculum of CLas or CLam we are still observing a predominance of infective psyllids and diseased plants with positive conventional PCR for CLas. In average 30.4% of psyllids collected from inoculum source plants and 8.4% collected from test plants were positive for CLas and no one for CLam. In average 12.8% of test plant were positive for CLas and only 0.9% for CLam. From the three Clam citrus infected plants, two had also CLas in a mix infection. Average Ct value for leaves with CLas were 24.7 and for CLam 33.7, indicating a higher titer for CLas than for CLam. In compartment 4, where Murraya exotica served as source of inoculum, 46.0% and 9.4% of psyllids collected respectively in source plants and test plants were PCR positive for CLam, whereas in compartment 3, where Citrus sinensis served as source of inoculum, 6.35 and 4.7% of psyllids collected respectively in source plants and test plants were PCR positive for CLam. 8.2% of test plants were PCR positive for CLam when inoculum source was Murraya exotica and no positive test plant was detected when inoculum source was Citrus. All plants were in other insect-proof screenhouse and will be there for symptoms observation and leaf sampling for PCR analysis at least until December 2011. A new leaf sampling will be done in March to check the presence of both liberibacters in new test plants. A replication of this experiment was started to repeat this experiment. These results will be present in the 2nd International Research Conference on Huanglongbing in Orlando January 10-14th. Exp. 2- All test plants used to HLB-insect transmission are under evaluation for the symptom expression and the presence/concentration of the HLB bacterium by qPCR. Exp. 3 – No HLB-symptoms and infection were observed yet on inoculated trees at different sweet orange varieties and ages that were encaged under insect proof screenhouses. The disease severity (% of symptomatic canopy) of each tree from the alternative experiment under field conditions is being periodically assessed. Because of the heavy defoliation during the drought season and the recovery of tree canopy with new flushes during the spring, the average disease severity in all assessed trees seems to decrease at this time of year. New assessments after leaves maturation will be done.
The objective of this project is to investigate three questions: 1) whether HLB symptoms or boron/zinc deficiencies alone affect how ACP responds to citrus; 2) whether feeding patterns by adults, length and location of feeding, are altered by HLB infection or boron/zinc deficiencies; and 3) whether different strains of Ca. Liberibacter asiaticus (Las) differentially affect the response of ACP to citrus. In other pathogen/host/vectors systems, such as that with Ca. Phytoplasma mali and Cacopsylla picta (the apple psyllid), the pathogen manipulates the plant host metabolism so that diseased plants become more attractive to the psyllid vector, thereby spreading the pathogen more rapidly than if no plant host manipulation occurred. Since nutrient deficiencies are often associated with HLB in citrus, we wished to confirm that the reported attraction of Diaphorina citri to HLB symptomatic plants over uninfected plants was due to changes in host metabolism by the pathogen rather than physiological changes due to poor nutrition. The production of greenhouse-grown citrus trees with nutrient deficiency symptoms for both zinc and boron continues. Deficiency symptoms are expected to take a few months to develop. This is especially the case for zinc because of the high mobility of zinc within the plant allowing redistribution of existing zinc to new flush, delaying the development of symptoms. Multiplication of our stocks of HLB infected citrus is also underway to provide a constant source of symptomatic tissue for experimentation once the nutrient deficiency symptoms have developed. We are also interested to determine if strains of Las will have any effect on the attractiveness of trees to D. citri. It has been reported that Las strains have varying levels of virulence and symptomatology (Tsai et al. 2008). We have analyzed DNA samples from HLB positive trees from Polk and Highlands counties as well as the ‘Smoak Grove’ CREC greenhouse strain by PCR and sequencing. Three putative strains of Ca. Liberibacter asiaticus (Las) were found with 5 (CREC greenhouse isolate), 13, and 15 tandem repeats of DNA in the LAPGP locus described by Chen et al. 2010 and have identified sources of budwood. We are currently using the identified budwood sources to acquire greenhouse isolates of the three strains. Cloning and sequencing of loci including the b-operon, OMP (outer membrane protein) gene and phage DNA polymerase to support the differentiation of the three strains is underway (Bastianel et al. 2005; Lin et al. 2008; Okuda et al. 2005; Tomimura et al. 2009).
The objective of this project was to investigate three questions: 1) what is the seasonal pattern of Ca. Liberibacter asiaticus prevalence in leaf tissue on a grove scale; 2) what are the flushing patterns of citrus and whether these flushing patterns affect the prevalence of Ca. Liberibacter asiaticus in Diaphorina citri or citrus leaves; and 3) what is the prevalence of Diaphorina citri carrying Ca. Liberibacter asiaticus on a grove scale and how does it compare the results from the citrus trees in the same grove. In 2008 and 2009 Ebert and Rogers demonstrated that the prevalence of Ca. Liberibacter asiaticus in the Asian citrus psyllid varied seasonally but the pattern between seasons was not consistent. It was suggested that perhaps the reason for the differences between the years related to the flushing patterns of citrus and the prevalence of the bacterium in the leaves where psyllids are feeding. This project aims to determine if there is a relationship between the frequency of disease on branches and psyllids. The postdoc has been hired for this project and is currently processing the backlog of samples but we have some preliminary results from 1 grove. An ‘Earlygold’ grove in Orange county Florida was selected to examine seasonal prevalence of HLB over a 3-year period. Two hundred trees were selected from 10 consecutive rows. One leaf/tree was randomly picked every fortnight. Sample collection began at the end of June 2010 with collections ongoing. For qPCR detection of Las, the midribs of five random leaves were pooled to obtain 40 samples/date. An estimated Las prevalence in the branches was generated from the pools with PooledInfRate v3. Simultaneously, ACPs were collected from the same location to compare the seasonal prevalence of Las in citrus branches and ACPs. Las prevalence increased (from 0.14, 0.20, 0.21 to 0.27) from June to September. The data from ACPs are still being processed. Comparing the data from ACPs and citrus trees could provide more information on seasonal variation in Las prevalence in both the vector and the host, and thereby enhancing our management strategies by identifying the essential periods for ACP control to prevent HLB transmission.
Under Objective 1 (define rates and formulations of copper sprays for more effective control) one trial with Red grapefruit is on-going in Ft. Pierce, FL and likewise one trial with Hamlin orange in Hardee County, each with 14 treatments of formulations, rates and combinations with other chemical treatments of interest. This season, trials have low to moderate disease epidemics as a result of a periodically wet spring, moderately wet summer and an absence of tropical storm events. Evaluations of fruit infection in these trials will begin in November 2010. Under Objective 2 (establish the period of fruit susceptibility, residual activity and phytotoxicity of copper) These results have been summarized for publication in Florida State Hort. Society (FSHS) Proceedings 2010. Major findings reported are: 1) Copper formulations containing copper hydroxide or basic copper sulfate (metallic rates of 0.67 to 1.12 kg/ha), varied from low to moderately effective for canker control depending on late-season, wind-blown rains. 2) Copper pentahydrate, with a lower metallic copper rate/ha per application, provided equivalent control to other copper formulations. 3) Streptomycin, alone or with a reduced rate of Kocide 3000, in July and early August gave equivalent control to Kocide 3000 alone. 4) Copper phytotoxicity risk coincided with accelerated fruit growth after summer rains. 5) Greater canker susceptibility of fruit in later season is likely because of increased opening of stomates for bacterial infection coincident with more numerous windblown rain events. 6) The only factor that led to significant reductions in copper residue per fruit surface area over time was the Kocide 3000 concentration. PDS had no effect on the residue. Under Objective 3 ( evaluate the use of streptomycin [Firewall]) Two trials, one non-bearing and one bearing, in grapefruit are underway to further evaluate FireWall for canker control. The package to obtain an EPA Section 18 Emergency Exemption for FireWall has been submitted to FFVA based on data from Florida trials conducted in 2008-2009. Under Objective 4 (To define risk for development of bacterial resistance to copper and streptomycin in FL citrus groves) a number of factors favorable for the development of copper resistance in Xcc were identified, but further investigation is necessary to fully assess the risk for streptomycin resistance. Previously reported findings are being summarized for publication. Under Objective 5 (rapid transfer improved canker management technology to the Florida citrus industry), 2010-11 canker management recommendations have been published in the Florida Citrus Pest Management Guide and Citrus Industry Magazine. Oral presentations have been delivered to the Florida Citrus Production Managers, at county extension meetings, and at the FSHS annual meeting.
The objective is to evaluate soil-applied neo-nicotinoids and other SAR inducers on HLB disease progress in newly planted citrus trees subjected to psyllid-mediated infection or graft-inoculation. One yr-old Hamlin trees were planted in May 2009 and treated as follows: 1) non-treated check (UTC), 2) foliar insecticide to control psyllids, 3) soil-applied imidacloprid/thiamethoxam (IMID/THIA) to induce SAR, 4) soil-applied IMID/THIA plus foliar insecticides, 5) graft-inoculated UTC, 6) graft-inoculated with IMID/THIA. There were 40 trees per treatment (5 blocks of 8 trees). In 2009, the effect of SAR inducers on HLB infection progress was inconclusive perhaps attributable to the interaction of IMID/THIA with psyllid control which may have an uncontrolled effect on psyllid transmission. In 2010, the SAR inducer acibenzolar-S-methyl (ASM, Actigard 50WP) which does not control psyllids was substituted in treatments 3, 4 and 6. At 15 months after treatments began, 56 trees were PCR+ (23%) in the trial. Higher number of PCR+ occurred in the UTC (14), the UTC with graft inoculation (13), and the IMID/THIA/ASM with graft-inoculation (13). Lower number of PCR+ trees occurred without graft inoculation in treatments with SAR inducers (7), foliar insecticides (6), and foliar insecticide plus SAR inducers (3). An HLB-SAR experiment has set up in Parana, Brazil. The trial utilizes soil applied ASM (Bion) for the SAR treatments and is proximal to tree in a farm with HLB. The first tree to show HLB symptoms after six months is a tree treated with ASM. While effect of SAR on HLB disease progress is still inconclusive, thus far these trials and other evidences indicate lack of promise for SAR in HLB management.
Objective 1: Potential for soil application of the neonicotinoids, Admire (imidacloprid, IMID)and Platinum (thiamethoxam, THIA), and Actigard (acibenzolar-S-methyl, ASM), to provide long-lived SAR control of canker was evaluated. In the field trial of 3- and 4-year-old ‘Ray Ruby’ grapefruit trees in Ft. Pierce, Florida, soil drenches of IMID, THIA, and ASM were compared with contact activity of Kocide 3000 (copper hydroxide, CH) and/or Firewall (streptomycin, STREP) applied as a foliar spray at 21-day interval for canker control on foliage. Canker on each set of vegetative flushes was assessed as the percentage of leaves with lesions. In 2008, despite above average rainfall and a tropical storm event, all treatments significantly reduced incidence of foliar canker. Spray of CH was the most effective treatment. As a group, soil drenches of SAR inducers reduced foliar disease incidence depending on rate and frequency of application. In 2009, all treatments significantly reduced incidence of foliar canker compared to the untreated check. IMID, THIA, and ASM as soil drenches were each effective for sustained control of canker on young trees under epidemic conditions. Control with SAR inducers was highest for four applications of ASM in 2008 and 2009 seasons which demonstrated the value for maintenance of SAR with repeated soil applications. Objective 2 Integration of soil applied IMID with foliar applications of copper sprays for control of canker. IMID applied once at the beginning of the season followed by 11 CH sprays gave the best control in the 2009 trial. This suggests that SAR and copper could be used in an integrated program for augmenting canker control for young fruiting trees. In 2010, a trial integrating ASM at different frequency of soil application with THIA and CH sprays at 21 day interval was set up on 1-yr-old Vernia sweet orange in Ashland, FL. Incidence of newly infected trees is just starting to increase in the various treatments this summer. This highest incidence of disease trees and leaves is in the nontreated check. Objective 3 is to evaluate of the complementary use of ASM, THIA and IMID soil applications to increase and/or extend canker control in 2-yr-old grapefruit trees. Canker was first observed in the trial area in September 2009 after a very high rainfall period in August (17 inches). The pattern of disease spread was across the trial area from southwest to northeast. Incidence of trees with canker was 56% in the non-treated check trees, whereas in most of the SAR treatment combinations the incidence was less than 10%. Incidence of canker in this trial continues to increase in 2010. A field trial with soil applied neonicotinoids in Parana, Brazil was evaluated. IMID(Confidor) as a soil drench and IMID (Winner) applied to trunk gave comparable in disease control activity on 2-yr old Valencia orange trees, as well as, the other neonicotinoids tested, THIA (Actara) and Clothianidin (Poncho). In Brazil a greenhouse evaluation, soil drenches of these neonicotinoids and ASM were effective for reducing lesion and Xcc population development in leaves. ASM was the best treatment among those evaluated. Thus, SAR not only prevented infection but also acted post-infection to reduce the level of bacterial populations in lesions. This confirms the best activity that the inducers of SAR have for reducing canker incidence and epidemic development of disease on leaves and fruit.
Objective 1 is to conduct a field evaluation nutritional sprays for control of HLB and HLB symptom expression and yield. The field study was set up May 2010 in Southern Grove, Hendry Co., FL. Six treatments were set in 4 plots of 150 trees per treatment (interior 10 trees in each block were identified for PCR, leaf nutrition sampling, tree health and yield evaluation). Treatments were 1) non-treated check; 2) Nutri-Phite sprayed 3times bimonthly; 3) N-Sure sprayed bimonthly; 4) Agra Sol Mn/Zn/Fe plus Nutri-Phite plus triazone urea sprayed bimonthly; 5) Keyplex 1400 DP plus Nutriphite plus triazone urea sprayed bimonthly; 6) Wettable powder nutrients (Diamond R #2) plus Nutri-Phite P+K sprayed bimonthly. The materials were applied to both sides of the tree in 125 gallons per acre with an airblast sprayer driven at 2 mph to obtain thorough coverage. Three applications have been completed as of October 2010. Ratings of tree health, leaf nutritional status and PCR status will commence in November 2010. Objective 2 is to determine the mechanism of HLB symptom suppression by foliar nutritional application. Hamlin sweet orange trees have been inoculated and are being treated bimonthly with the nutritional sprays. Infection rate and progress are being monitored by qPCR monthly. One month after inoculation, the new flush on a few of graft inoculated trees was weakly positive for Las. Two months after inoculation, new flush on some trees is strongly positive for Las; however, too few of the trees are positive to determine any treatment effects. Three month post inoculation samples are currently being processed. Sampling and fixing of plant material for microscopy of phloem and leaf blade tissue began at two months and continues monthly. Fixed samples will be observed by microscopy once strongly symptomatic tissue is available for comparison.
Transmission of Candidatus Liberibacter asiaticus by the Asian citrus psyllid (ACP), Diaphorina citri, probably involves complex psyllid-pathogen interactions, which should be understood in order to improve management strategies to control HLB. The goal of this research is to determine factors that influence the risks of acquisition or inoculation of the pathogen (Candidatus Liberibacter asiaticus – CLas) by D. citri, e.g vector developmental stage, feeding periods, leaf age and symptom expression/bacterial population in disease plants, in order to optimize strategies to avoid or reduce disease spread within and between citrus groves. We already set up the first four of five proposed studies and the project is progressing as planned originally. Partial results of previous studies showed that bacterial acquisition can occur when ACP adults feed on asymptomatic infected plants, although acquisition efficiency is higher on citrus plants with higher bacterial titers, usually symptomatic. We also have reported that acquisition efficiency by psyllid adults is markedly affected by citrus leaf age and duration of acquisition access periods (AAP). Efficient acquisition depends on the availability of young leaves in infected plants and long AAPs (>2 days). In this quarterly report, we present details of the first study, concluded in September/2010, which was carried out to determine how acquisition efficiency of CLas varies in relation to ACP developmental stages. Health insects of known age were obtained from a lab colony maintained on Murraya paniculata. In each experiment trial, groups of 40 adults with 3-5 days post-emergence and newly-molted nymphs of all development stadia (1st, 2nd, 3rd, 4th and 5th instars) were confined on young leaves of a same symptomatic branch of an infected plant, during a 48-h AAP. Psyllids of each treatment (developmental stage) were confined on a separate leaf. After the AAP, the insects were maintained on healthy sweet orange seedlings for 21 days (healthy seedlings were replaced after the first 14 days). Surviving psyllids were then fixed in 100% ethanol and individually tested for the presence of CLas by real-time quantitative PCR (qPCR). As a negative control, a group of 40 psyllids of the same rearing batch was transferred directly to the healthy seedlings (without previous AAP on infected plants). The experiment was repeated three times using distinct pathogen source plants. Acquisition efficiencies by 1st, 2nd, 3rd, 4th and 5th instars, as measured by the proportion of infective psyllids by qPCR, were 68.4, 60, 66.6, 84 and 65.3%, respectively. In contrast, only 42.9% individuals were infective when acquisition took place during the adult stage. Interestingly, a higher concentration of CLas was observed in psyllids that acquired the bacterium during the 4th or 5th instar. These results show that all developmental stages of ACP are able to acquire the bacterium with moderate to high efficiencies during a 48-h AAP on infected plants, but nymphs are more efficient than adults. We are now concluding PCR assays for detection of CLas in the test seedlings exposed to the psyllids of each treatment, to check if the higher acquisition rates by nymphs result in higher transmission rates of this pathogen.
Imidacloprid (Confidor 700 GrDA), 0.35 g AI/plant and thiamethoxam (Actara 250 WG) 0.25 g AI/plant, applied in the nursery tree bags, before planting, was efficient to control ACP until 60 days after application. The time to cause 100% of ACP mortality was between 5 to 7 days after the confinement of adults in treated plants. However, researches using electrical penetration graph (EPG) showed that in plants treated with imidacloprid and thiamethoxam, after the first feeding on phloem, the adults do not do more probing. We carried out the first PCR of the plants in this experiment and the results were negative, no plants have been detected the presence of the bacterium L. Ca asiaticus. No transmission results yet. We finish the second experiment that was performed to determine if the systemic insecticides are effective until 90 days after application and its effect on transmission of the bacteria. In this experiment, the time to reach 100% of mortality ranged from 3 to 7 days for both systemic insecticides tested (imidacloprid and thiamethoxam). The insecticides were effective up to 90 days after application. The results of PCR carried out for the ACP, in some periods, were positive for 100% of the samples, consisting of 10 insects tested, but in the confinement held at 46 days after application, in any sample was detected the presence of the bacteria. No acquisition in this period. In bioassays performed at 75 and 90 days after application, the percentage of positive samples was 50 to 70% and 10 to 40%, respectively. We finish the experiment 2, the difference from the experiment 1 is the application of varying doses of the systemic insecticides and confinement of the ACP in plants treated only 7 days after application. To thiamethoxam (Actara 250 WG), the doses tested were: 1, 0.5, 0.1 and 0.05 g/nursery tree and imidacloprid (Provado 200 SC) were: 1.75, 0.9, 0.2 and 0.08 mL/nursery tree. We also finish the experiment 3, using different insecticide spraying to determine if they prevent the transmission and for how long. In both experiments the systemic insecticides were effective in control of ACP, but have no result of transmission. Plants treated with insecticide, the proportion of insects reaching the phloem was similar between plants treated with imidacloprid (0.35 g AI/tree), thiamethoxam (0.25 g AI/tree) and control (untreated plants), being respectively 74, 72 and 76%. The time to perform the first ACP salivation was also similar between treatments, 118.4, 103.2, and 112.6 minutes, respectively. However, the time of phloem ingestion is drastically reduced compared to untreated plants. Apparently, ACP can only distinguish between plants with and without treatment from the moment that start ingesting the phloem sap. In this case, it was observed that after ingestion of sap with insecticide, the ACP removes the stylet from the plant and rarely returns to start a new probe on the same plant. With systemic insecticides, the main interference on probing behavior occurred during the phloem phase; phloem sap ingestion, as measured by duration of waveform E2, is significantly reduced (approximately 91%), and the insect subsequently withdrawals the stylets from the plant and rarely restarts a new probe. We finish the experiment to evaluate the effect of oil on the feeding behavior of ACP and its effect on repellency of the vector. The results showed that up to 21 days after application, mineral oil, 1.5% and 1.0%, shows repellency to adults of D. citri. Using electrical penetration graphs (EPG) techniques, we are studying the probing behavior of ACP in plants that were applied mineral oil. Plants sprayed with insecticides and M.O. affect mainly the initial phase of probing (pathway). In the case of insecticides, D. citri was able to perform probes 1 DAA, but it was able to reach the phloem only in the evaluation of 15 DAA. However, on plants treated with M.O. around 32% of insects were able to reach the phloem 1 DAA. We started the EPG experiment using pymetrozine.
Work on Objective 1 was initiated this season. The purpose of this objective was to determine how best to apply systemic insecticides to young trees to gain the longest lasting duration of psyllid control possible. Three neonicotinoid insecticides were evaluated in replicated field plots on trees approximately two years of age. These three products were imidacloprid (Admire), thiamethoxam (Platinum) and clothianidin (a product expected to be registered for use in citrus in the coming year). Each of these products were evaluated using three different application methods; soil drench, soil band spray and trunk application with multiple application rates for each method tested. For the trunk applications with imidacloprid, in addition to evaluating Admire, trunk applications were also made using Confidor 200SL which is the formulation reportedly used in the past for trunk applications in South Africa. Using the same material and rate as used in South Africa where several months of control are touted, we will be able to determine if we can obtain the same level of control reported from South Africa and also determine if by altering application method can we use our currently labeled products to obtain a level of control similar to that reported from S. Africa under Florida growing conditions. Currently, this trial is still ongoing with weekly evaluations of psyllid adults and nymphs being made. We are also collecting data on control of citrus leafminer to ensure that changing application methods does not have any negative effects on control of this pest as well. Results should be reported in the next quarterly progress report.
Previously we reported on results from this project indicating that Cleopatra mandarin is an unsuitable host for the Asian citrus psyllid with little or no reproduction occurring on this citrus species (Citrus reshni Hort. ex Tan.). These results were recently published: Tsagkarakis, A.E. and M.E. Rogers. 2010. Suitability of ‘Cleopatra’ mandarin as a host plant for Diaphorina citri (Hemiptera: Psyllidae). Florida Entomologist 93 (3): 451-453. As a followup to this work, we conducted additional studies with Cleopatra mandarin hybrids to determine if there would be differences among these hybrids in susceptibility to psyllids that might be used in future psyllid management approaches. For these experiments, 30 field grown mature Cleopatra mandarin hybrids were selected for evaluation. For each of these hybrids selected, young flush was available for psyllid oviposition at the time experiments were conducted. The initial evaluation was conducted by caging adult psyllids on the new flushes of each of the 30 hybrids which were selected at random. The flushes remained caged for the next 21 days to allow for psyllid oviposition, development of nymphs and subsequent emergence to the adult stage to ensure that psyllids were able to complete their lifecycle on these hybrids. In this initial evaluation, there was successful psyllid reproduction on 9 of the 30 hybrids. It appeared that there was some gradient of suitability between these plants however with some plants having only 1 or 2 surviving offspring whereas other hybrids produced up to 30 offspring per flushing terminal. This experiment was repeated a second time using all the plants for which some psyllid reproduction was found and an equal number of plants for which no psyllid reproduction was previously found. In this second round of evaluations, we again found psyllids were able to reproduce and complete development successfully on all of the Cleopatra hybrids for which there previously had been relatively high levels of reproductive success (10-30 offspring per branch). However, we did not see successful development on some of the hybrids which previously had low levels of psyllid development. We also did not see any reproduction on the hybrids for which there previously was no reproduction recorded. Thus, based on the outcome of these two evaluations, we have identified three groups of Cleopatra mandarin hybrids with varying levels of susceptibility to psyllids; no reproduction, little or no reproduction, and moderate to high reproductive success. The next step in this work will be to evaluate plants from these three susceptibility groups to determine if there are physical or chemical differences that are responsible for the suitability of the host plant for psyllid development.
A paper resulting from this funded project was recently published: Pelz-Stelinski, K.S., Brlansky, R.H., Ebert, T.A. and M.E. Rogers. 2010. Transmission parameters for Candidatus Liberibacter asiaticus by Asian Citrus Psyllid (Hemiptera: Psyllidae). Journal of Economic Entomology 103 (5): 1531-1541. ABSTRACT The purpose of this investigation was to evaluate acquisition and inoculation efficiency of Candidatus Liberibacter asiaticus (Las), the pathogen associated with huanglongbing (HLB) or citrus greening disease, by the Asian citrus psyllid, Diaphorina citri (Kuwayama). In laboratory studies, nymphs reared on Las-infected plants were more likely to acquire the bacterium than adults. Acquisition by nymphs ranged from 60-100%, whereas acquisition by adults only reached 40% after 5 wk of feeding on Ca. Las infected plants. Similar rates of pathogen acquisition by psyllids following nymphal and adult feeding were observed in the field. Transmission of Ca. Las from parent to offspring (transovarial) occurred at a rate of 2-6%. One year after psyllid inoculations, successful transmission by individual D. citri ranged from 4-10%, while groups of 100 or more D. citri transmitted the pathogen at a rate of approximately 88%. In addition, the proportion of Ca. Las-positive adult psyllids, determined using quantitative real-time PCR, decreased over time when held on healthy plants. Due to the low rate of pathogen acquisition and long period required for successful inoculation by adult D. citri, experiments designed to determine the latent period required for replication and successful inoculation of Ca. Las by D. citri did not result in Las infected plants after more than one year of incubation following inoculation. Collectively, these results indicate that adult D. citri which acquire the HLB pathogen as adults are poor vectors of the pathogen compared to adults which acquired the pathogen as nymphs. END ABSTRACT. In other ongoing related work…experiments to evaluate the effect of temperature on acquisition are nearly finished. Preliminary data indicate that peak acquisition occurs between 25-30C, with approximately 50% of D. citri becoming infected after two weeks of feeding on infected plants. Only 4% of psyllids acquired Las at 20C. In addition, Las acquisition decreased at temperatures above 30C, although acquisition was not as low as that observed at 20C. Collectively, these data suggest that little acquisition of Las occurs during winter months when temperatures are low. Currently, experiments are underway to evaluate the effect of temperature on transmission. Studies to determine whether there are fluctuations in the seasonality of pathogen transmission by psyllids continues. Since this study began in 2008, by analyzing more than 80,000 psyllids collected from 6 studies sites across the state we have documented significant grove-to-grove, year-to-year and month-to-month variability in the number of Las+ psyllids. However, there has been no consistent pattern in the month-to-month change in number of Las+ psyllids within each grove. The changes in abundance of Las+ psyllids is most likely a cyclical pattern associated more with plant related factors than weather per se. The one pattern most evident from this work however is the overall abundance of Las+ psyllids from year-to-year. In 2008, a low level of Las+ psyllids were collected from most sites followed by a dramatic increase in the abundance of Las+ psyllids at all sites in 2009. In 2010, to date, the overall abundance of Las+ psyllids has been much lower, possibly as a result of enhanced psyllid control programs being conducted.
All experiments are already established and we are now waiting for symptoms appearance. Exp.1 – It was observed the predominance of diseased plants with positive conventional PCR for CLas in the two compartments where citrus plants served as primary source of inoculum of CLas and CLam. In compartment 1, CLas infected 9.6% of test plants (15 of 156) and in compartment 2, CLas infected 13.5% (21 of 156) and Clam infected 1.9% of plants (3 of 156). The first CLas and CLam citrus infected plant were detected respectively 6 and 27 months after the beginning of the experiment. From the three Clam citrus infected plants, two had also CLas in a mix infection. There were 12 infected citrus plants with CLam in compartment 4, where Murraya exotica served as source of inoculum and no plant with positive for CLam in compartment 3, where citrus served as inoculum source. Since the ACP population was much higher in the last three months of 2009, all plants were moved to other insect-proof screenhouse and will be there for symptoms observation and leaf sampling for PCR analysis at least until December 2011. A new leaf sampling was done at the beginning of October to check the presence of both liberibacters in new test plants. A replication of this experiment was started in October 2009 to repeat this experiment. Exp. 2- A factorial designed experiment with two HLB bacterium species (CLam and CLas) and two sources of inoculum (symptomatic and asymptomatic) were established. Five to ten adults of Diaphorina citri had the access to infected sweet orange plants (symptomatic and asymptomatic) during 2-3 weeks. Both those adults and the adults emerged from nymphs developed in those infected sweet orange plants were transferred to young flushes of health sweet orange plants (test plants) to evaluate the proportion of transmission of the bacterium species. The adults used in the tentatively transmission of liberibacters were processed by PCR. Also, during 12-18 months all test plants used to HLB-insect transmission will be evaluated about the symptom expression and the presence/concentration of the HLB bacterium by qPCR. We are waiting for the PCR results. Exp. 3 – No HLB-symptoms and infection were observed yet on inoculated trees at different sweet orange varieties and ages that were encaged under insect proof screenhouses. As an alternative experiment, even without control of number and time of infections, 30 trees with initial symptoms of HLB were selected in different citrus blocks (B1 – Hamlin/Swingle planted in 2007; B2 – Valencia/Volkamer planted in 2007; B3 – Valencia/Swingle planted in 2007; B4 -Valencia/Swingle planted in 1999, and B5 – Valencia/Swingle planted in 1995) in a farm without removal of symptomatic trees but with strong ACP control program. In that farm, last inspection and eradication of HLB-symptomatic trees was done in April 2010. The symptomatic tree selection was done in the end of June 2010, so the symptoms on selected trees were quite new (less than 2 months). The average initial severity was 10.8%, 8.6%, 7.9%, 4.6%, and 6.2% respectively for B1 to B5. The disease severity (% of symptomatic canopy) of each tree is being periodically assessed. A new group of new symptomatic trees in the same blocks will be selected in December/10.
We continued the assessments of two experiments under high and low external inoculum pressure (E1 and E2, respectively). At the end of September 2010, HLB incidence in plots without vector control was 76.8% and 6.2% in E1 and E2, respectively. In plots with vector control program, the disease incidence was 53.1% for E1 (insecticide sprays every 14 days) and 4.6% and 6.3% for E2 (every 14 days and 28 days, respectively). This results indicates that with regional management of HLB, with less intensive vector control program (insecticide sprays every 28 days, or even no insecticide sprays) it is possible to be more efficient than a high intensive vector control program (insecticide sprays every 14 days) applied locally within a region without regional management. It has been observed that cumulative ACP adult population caught per yellow stick trap in plots without vector control program in E1 was high and quite stable year after year (5.0 in 2007, 30.5 in 2008, 10.8 in 2009 and 13.9 in 2010), while the cumulative population in plots without vector control program in E2 has decreased year after year (11.3 in 2007, 2.4 in 2008, 2.2 in 2009 and 0.1 in 2010). It is an indication that the local population of ACP do not stay in the plots without vector control during all the year (probably after some period or year season all adults move to other place), and the yearly population with be dependent of the influx of ACP from outside citrus blocks or alternative hosts. With the implementation of regional HLB management around E2, the influx of migrant ACP population has decreased yearly causing the reduction of the initial ACP population on plots without vector control. No significant differences on HLB progress rate and HLB incidence among different frequencies of local inoculum reduction in each experiment was observed yet. However in a regional scale, the 3-4 inspection/eradication of symptomatic HLB trees around E2, were enough to reduce the average incidence of HLB in E2 compared with E1 surrounded by citrus without any eradication of HLB infected trees. PCR-positive ACPs have been observed both in plots with and without vector control program for E1 and no psyllid sample was positive for Candidatus Liberibacter spp. in Exp.2. This result could explain why even with similar population of adults ACP in plots with vector control, the incidence of HLB-symptomatic trees were much lower in vector controlled plots of E2. Spatial analysis of annual maps of HLB-symptomatic trees were done at USDA lab in Fort Pierce by Gottwald’s team by stochastic models (MCMC) to verify the effects of each treatment on primary and secondary spread of HLB. Now we are interpreting the outcome results. The assessments on both experiments will continue to allow more detailed temporal and spatial analysis, as well economical analysis, and get final conclusions. These and others preliminary results were recently presented in two workshops in Mexico (2nd International Workshop on Huanglongbing and Asian Citrus Psyllid – July/10, and the 2nd International Citrus Research Workshop, July/10), and two meetings in Brazil (the Annual Meeting of Brazilian Society of Phytopathology, Aug/10, and in the Brazilian Workshop of Plant Disease Epidemiology, Oct/10). The results of this project are also being presented to growers to help the establishment of growers’ groups for regional management of HLB in S’o Paulo, Brazil.
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