The objective of this project is to screen citrus germplasm for resistance to the Asian citrus psyllid (ACP). Although citrus huanglongbing (HLB) is a century-old disease and the control of ACP is the key factor for HLB management, there is little information regarding citrus host resistance to ACP. Our preliminary results indicate there is ACP resistance in citrus germplasm. Historically, most (if not all) citrus resistance to HLB has been evaluated using graft inoculations, which sometimes resulted in plants becoming infected that appeared to have resistance in the field. What is needed is research to find varieties with resistance to the psyllid. Greenhouse and field evaluations of the USDA germplasm collection will be conducted. There already exists in China individual citrus plants that are thought to be HLB-resistant,but likely some of these are ACP resistant. The identification of a psyllid-resistant variety (or individual mutant) could revolutionize HLB management strategies. If we can identify ACP resistant germplasm, we can identify resistance genes for use in traditional and molecular breeding . Our intentions are to screen hundreds of sources of USDA and Chinese germplasm for resistance to ACP and to field test these to see if resistance to the psyllid negates HLB or greatly facilitates control. We expect to identify psyllid resistant citrus genotypes (or individual mutants from field-resistant collections) and/or citrus relatives within the Rutaceae that have psyllid resistance. We expect to determine traits that confer resistance and to identify traits that might be transferred to citrus varieties currently grown in order to make them resistant to the psyllid and thus less prone to contracting HLB. The ultimate return from this project would be an effective management strategy to control ACP and HLB that is less costly and friendlier to the environment and non-target organisms than the repetitive use of broad spectrum insecticides. A post doc was found and hired to conduct this research. A trip was made to China to firm up research plans with the Dr. Liu Bo and the Fujian Academy of Agricultural Sciences. Seeds representing the entire citrus/citrus relative collection at USDA-ARS-NCGR were obtained and have been planted. A field planting of many citrus genotypes and relatives was screen for psyllid infestations during July and August. Some of these genotypes were obviously highly susceptible to infestations by the psyllid while others appeared to be avoided.
Overall goal of the project was to develop and/or optimize low volume spray application technologies for controlling psyllids in Florida citrus production. Our research included both laboratory and field studies. In the laboratory, first we tested several fluorescent tracers to develop a reliable methodology for qualitative and quantitative assessment of spray deposition in low and ultralow volume applications. We used a controlled droplet applicator to generate different spray droplet sizes to investigate the effect of droplet size on the mortality of ACP. The tests included various commercially available pesticide formulations that are labeled for citrus applications. The technique involved spraying different droplet sizes on psyllid infested potted trees (Swingle) and counting the number of ACP eggs, nymphs, and adults before spraying and at 3 days and 7 days post-treatment. We tested ‘Lorsban Advanced’ ‘Danitol 2.4EC’ (synthetic pyrethroid), ‘Lorsban Advanced’ (organophosphate), and ‘Dibrom 8E’ (organophosphate), each at six droplet sizes. Our results showed that the smaller the droplet size, the greater is the percent mortality of all life stages of the ACP. For all chemistries, the results suggested up to 80 percent control of nymphs using sprays with droplet VMD of about 40 ‘ 100 ‘m. Danitol and Dibrom produced this effect by day 3, with Lorsban taking until day 7. The egg and adult stages showed similar trends, but with greater levels of control. Three other formulations screened were EverGreen (natural pyrethroid), AgriMek (abamectin), and Knack (pyriproxifen). Nonetheless, chemistries involving suspended particles could not be applied readily with our laboratory apparatus. In their present formulations, Movento (spirotetramat) and Provado (imidacloprid) are not compatible with the laboratory droplet applicator. In the field, we continued comparing various available application technologies. In one experiment, we compared high volume (dilute) spraying with low volume applications. The test involved evaluating two cold foggers and a mist blower. We also conducted several field trials with our LV-8 low volume applicator, which is becoming commonly used in the Florida citrus industry. We have applied over 4o treatments in Florida citrus groves to optimize chemistries that are labeled for the psyllid control, such as Danitol, as well as testing of unlabelled compounds. For labeled compounds, we have investigated the effect of product rate and application timing. For unlabelled compounds, we have conducted efficacy testing at standard rates to generate the needed data for label changes. Thus far, we have found no difference in efficacy between the available technologies. However, we may find differences with some of the more selective chemistries, so we are continuing this research by investigating more pesticide modes of action. We have compared the insecticide residues achieved with low volume and standard airblast sprays and have found that the residues from low volume sprayers are lower than those from conventional airblast sprayer. We have also investigated several application parameters for optimizing low volume spraying. While certain insecticides have shown similar efficacies for spraying every row versus every other row or ground speed of 5 mph versus 5-8 mph, some chemistries have shown lower efficacies when spraying every other row or at higher ground speed. In terms of the application volume, we have not found a difference between 2 and 5 gallons per acre. However, one must stay above 2 gallons per acre to remain within the boundaries of current label guidelines. We are also currently investigating whether the rate of insecticide can be reduced with low volume spraying. Given the importance of droplet size rage in label limitations, we have further refined the technique for characterizing the droplet size range of the ULV sprays used in field application.
USDA test 1. A new block of young, HLB’free citrus (Val on Carr) was planted on May 1, 2008 in a grove with substantial levels of HLB. Three ACP control treatments (programs) are being compared in this planting: 1) a monoculture of citrus receiving monthly insecticide applications (annual chemical cost of $198/acre); 2) citrus interplanted with jasmine with a relaxed insecticide program for the citrus (annual chemical cost of $156/acre), jasmine not treated with insecticides; and 3) citrus interplanted with jasmine with a relaxed insecticide program for the citrus and regular applications of imidacloprid to jasmine (total annual chemical cost of $213/acre). For plots with jasmine, a jasmine plant was planted between each citrus tree along some rows in each plot. Program 2 is being studied because ACP may be attracted to jasmine thus reducing numbers of ACP that go to citrus, and population levels of ACP natural enemies may be enhanced by having jasmine in the vicinity of citrus. Program 3 is being studied because ACP may be attracted to jasmine and killed, reducing numbers of ACP in citrus. HLB-infected trees are removed. None of the trees tested HLB positive just before planting in May 2008. One year later, a mean of 0.9, 0.6, and 0.6% of the trees were HLB-infected under programs 1, 2 and 3, respectively. During August 2009, a mean of 9.8, 3.9, and 4.2% of the trees were infected under programs 1, 2 and 3, respectively. The rapid increase in incidence of HLB in the plots between May and August 2009 was attributed to increases in ACP infestations during June and July, particularly in a program 1 plot that was adjacent to older citrus. In February 2010, a mean of 13%, 12% and 13% of the trees were HLB-infected under programs 1, 2 and 3. USDA test 2. Three ACP control programs are being compared for preventing HLB in a block of young, HLB-free citrus (Val on Carr): 1) citrus under a relaxed insecticide program (annual chemical cost of $173/acre); 2) citrus receiving monthly insecticide applications (annual chemical cost of $198/acre); and 3) citrus treated once every three weeks with spray oil [PureSpray Foliar (470, C27) and PureSpray Green (435, C23)] from February through November (plus a December application of Danitol) (annual chemical cost of $76/acre). There are two replications of each treatment. The experiment officially started in August 2009. In January 2010, 0%, 0.3% and 0.3% of the trees tested HLB positive under programs 1, 2 and 3, respectively. These trees were removed. UF test. The ability of systemic insecticides to protect a new planting of citrus from ACP (and consequently HLB infection) is being evaluated. 112 Hamlins were planted in two rows at 15 ft spacing on 03 Mar 2009. Half of each row was considered a replicate and divided into two main plots, treated and untreated. Treated plots were split into two subplots on 13 March, one receiving a liquid formulation of imidacloprid (Nuprid 2f @ 32 oz/acre ‘ 0.5 lbs a.i./acre) and the other receiving a solid formulation of the same (Suscon 13 @ 10 lbs/acre ‘ 0.5 lbs a.i./acre). On 11-Sep 2009, no significant treatment effects (Chi square = 3.61, P = 0.182) were found with respect to numbers of HLB trees. At that time, 10 of the 56 control (untreated) trees tested positive for HLB, 10 of the 28 trees treated with the granular slow release imidacloprid were positive, and 6 of the 28 trees treated with the liquid formulation of imidacloprid were positive. The same trees were resampled on 15 Jan, but all tested negative. PCR was run twice more on the DNA from both the Sep and Jan samples and the results were confirmed. Thus, the different result from Jan could only be attributed to effects of cold weather on titer and/or lack of ACP to reinoculate the trees.
1. Localization of Liberibacter asiaticus (Las) in the hemolymph and other tissues and organs of the Asian citrus psyllid (ACP). The following four methodologies have been tested and used with promising results. [A] Immunofluorescence confocal laser scanning microscopy: Two polyclonal and several monoclonal antibodies prepared against Las membrane proteins have been tested at various dilutions and incubation times. Some of the monoclonal antibodies produced positive labeling in the phloem area in sections of HLB- infected citrus leaves and in hemolymph smears of ACP collected from HLB-infected trees, but not in whole-mount ACP organs, suggesting that such potentially useful antibodies may be too slow to penetrate whole insect organs. We are working to resolve this issue with various permeabilization procedures or by immunofluorescent labeling of paraffin sections rather than whole organs. [B] Fluorescence In situ hybridization (FISH) using three oligonucleotide primers: Several FISH protocols have been tested on hemolymph smears and dissected organs of ACP and on leaf sections and extracts from HLB-infected plants. Green fluorescence, indicating Las, was detected in the hemolymph, filter chamber and midgut of field-collected ACP, but not in healthy controls. It was also detected in leaf sections from HLB-infected citrus and periwinkle plants but not in those from healthy plants. We are continuing to refine our FISH protocols to reduce the background fluorescence and to increase the permeability of insect tissues. [C] Quantitative RT-PCR of dissected insect organs: We tested two different RT-PCR procedures for detection of Las in dissected salivary glands, alimentary canals and other parts of individual ACP adults. In five successive experiments, Las was detected in 13-24% of the alimentary canals, 12-16% of the salivary glands, and in 16-25% of the rest of the body from psyllids collected from HLB-infected trees in Fort Pierce, FL. This is the first direct demonstration of Las (using PCR) in the alimentary canal and salivary glands of ACP. We are currently testing the relative concentration of Las in various ACP organs. [D] We are using a combination of transmission electron microscopy (TEM) and RT-PCR to compare the ultrastructure of ACP adults that have never been exposed to infected plants with those collected from HLB-infected trees and are PCR-positive for Las. This is providing a very useful library on the ultrastructure of the alimentary canal, salivary glands and other organs of ACP as well as its bacterial symbiotes, and will form the basis for future immunogold-labeling TEM studies on Las pathogen-vector interactions in ACP at the cellular and tissue levels. 2. Clarification of various acquisition and transmission parameters between ACP and Las. [A] Among ACP adults field collected from HLB-infected citrus trees, averages of 37% females and 38% males have tested positive by PCR. Significantly higher percentages of field-collected adult ACP tested positive for Las during November 2009 (38.9%) than during late March 2010 (22.1%), and CT values associated with adults collected during November (mean 24.8) were significantly lower than for adults collected during late March 2010 (mean 30.5). [B] An average of 16% individual HLB-infected adult ACP (field-collected, mean CT of 26.4) transmitted HLB to young potted citrus trees. Taken together, the data in1C and 2B above support our hypothesis that transmission barriers to Las in ACP (including the alimentary canal and salivary glands) may play an important role in HLB transmission, since much higher percentages of ACP adults were PCR-positive compared to those that were actually able to transmit HLB to citrus plants. Our studies are continuing to elucidate the roles of these and other psyllid organs/ tissues in HLB transmission by ACP.
This is the final report of a three-component project on the Asian citrus psyllid. Sampling – Stem-tap sampling is preferred over sticky traps for making ACP density estimates and is considerably less expensive. Sampling to estimate ACP densities should be conducted throughout a block because there can be significant variation across a block of trees in numbers of ACP. For commercially-acceptable precision levels at means of 1 or more adult psyllids per sample, 28 stem-tap samples are required in a block of trees up to ten acres in size (grove edges are not sampled under this protocol). Larger numbers of samples were required at lower densities. Although no control threshold for ACP has been identified, this level would likely lower than than 1 ACP per sample, perhaps 0.2 to 0.3 ACP per sample – thus growers wanting to make insecticide decisions for ACP control should take larger sample sizes. Noted is that young trees do not lend themselves well to stem-tap sampling. Sticky traps are preferred for ACP detection, not density estimation. Three publications on sampling have been published (available on request) and one has been submitted. Future research efforts on ACP sampling should include a method of estimating densities for an entire block of trees including edges. A formal method is needed for sweep sampling. The relationship between mean number of ACP per tap sample and absolute ACP densities in trees needs to be clarified. Biological Control – Although this is the final report, research will continue under ARS funding. (a) Releases of three new biotypes of Tamarixia from Asia are being continued in southwest and east-central Florida. It is too early to gauge establishment. Research was initiated in east-central Florida on infestations of ACP and biological control of ACP by Tamarixia in urban plantings of jasmine. Releases of the new biotypes in these urban areas have not yet been made, but the old biotype of Tamarixia has been observed at some of these locations. Increasing biological control of ACP in urban settings could contribute to ACP area-wide management . (b) There is no evidence that releases in Florida of a new biotype of the parasitoid Diaphorencyrtus resulted in establishment. Some project funds supported a Ph.D. student, who is completing the degree and is in the process of preparing several manuscripts for publication. Seasonal HLB profile in adult ACP ‘ Although this is the final report, research will continue under ARS funding. From Feb 2008 through Mar 2010, a mean of 32% (SEM 3.1) ACP tested HLB positive. Percentages of ACP testing positive has varied widely among sample dates, with a mean minimum of 7.4% (SEM 3.7) during late Feb 2009 and a mean maximum of 70% (SEM 3.2) during late May 2009. An average CT value of 30.7 (SEM 0.2) was recorded among all infected ACP over the entire study period, or an average CT value of 26.1 (SEM 0.2) for ACP with CTs less than 32. For ACP with CT values below 32, mean CT values have ranged from 23.5 (SEM 0.6) in Aug 2009 to 29.4 (SEM 0.2) in late Feb 2009. An average of 77.1% (SEM 8.8) of the trees from which ACP are collected have tested HLB positive (mean CT 23.9, SEM 0.3). No trends in seasonality of HLB in ACP have yet been identified over the course of this two-year study. An average of 35.3% (SEM 4.1) ACP tested HLB positive and an average of 15.8% (SEM 4.2) infected ACP transmitted the disease, with means per sample date as low as 0% and as high as 38%. It is too early to determine if there are any seasonal trends in transmission rates.
This is the final report of a three-component project on the Asian citrus psyllid. Sampling – Stem-tap sampling is preferred over sticky traps for making ACP density estimates and is considerably less expensive. Sampling to estimate ACP densities should be conducted throughout a block because there can be significant variation across a block of trees in numbers of ACP. For commercially-acceptable precision levels at means of 1 or more adult psyllids per sample, 28 stem-tap samples are required in a block of trees up to ten acres in size (grove edges are not sampled under this protocol). Larger numbers of samples were required at lower densities. Although no control threshold for ACP has been identified, this level would likely lower than than 1 ACP per sample, perhaps 0.2 to 0.3 ACP per sample – thus growers wanting to make insecticide decisions for ACP control should take larger sample sizes. Noted is that young trees do not lend themselves well to stem-tap sampling. Sticky traps are preferred for ACP detection, not density estimation. Three publications on sampling have been published (available on request) and one has been submitted. Future research efforts on ACP sampling should include a method of estimating densities for an entire block of trees including edges. A formal method is needed for sweep sampling. The relationship between mean number of ACP per tap sample and absolute ACP densities in trees needs to be clarified. Biological Control – Although this is the final report, research will continue under ARS funding. (a) Releases of three new biotypes of Tamarixia from Asia are being continued in southwest and east-central Florida. It is too early to gauge establishment. Research was initiated in east-central Florida on infestations of ACP and biological control of ACP by Tamarixia in urban plantings of jasmine. Releases of the new biotypes in these urban areas have not yet been made, but the old biotype of Tamarixia has been observed at some of these locations. Increasing biological control of ACP in urban settings could contribute to ACP area-wide management . (b) There is no evidence that releases in Florida of a new biotype of the parasitoid Diaphorencyrtus resulted in establishment. Some project funds supported a Ph.D. student, who is completing the degree and is in the process of preparing several manuscripts for publication. Seasonal HLB profile in adult ACP ‘ Although this is the final report, research will continue under ARS funding. From Feb 2008 through Mar 2010, a mean of 32% (SEM 3.1) ACP tested HLB positive. Percentages of ACP testing positive has varied widely among sample dates, with a mean minimum of 7.4% (SEM 3.7) during late Feb 2009 and a mean maximum of 70% (SEM 3.2) during late May 2009. An average CT value of 30.7 (SEM 0.2) was recorded among all infected ACP over the entire study period, or an average CT value of 26.1 (SEM 0.2) for ACP with CTs less than 32. For ACP with CT values below 32, mean CT values have ranged from 23.5 (SEM 0.6) in Aug 2009 to 29.4 (SEM 0.2) in late Feb 2009. An average of 77.1% (SEM 8.8) of the trees from which ACP are collected have tested HLB positive (mean CT 23.9, SEM 0.3). No trends in seasonality of HLB in ACP have yet been identified over the course of this two-year study. An average of 35.3% (SEM 4.1) ACP tested HLB positive and an average of 15.8% (SEM 4.2) infected ACP transmitted the disease, with means per sample date as low as 0% and as high as 38%. It is too early to determine if there are any seasonal trends in transmission rates.
The results of this project over the last year have shed new light on pathogen transmission by ACP, some of which is radically different from the outcomes of other previously published studies. We have demonstrated in both laboratory and field studies that psyllids acquire the Las pathogen more readily as nymphs than as adults when feeding on infected plants. Furthermore, just because a psyllid tests positive for the Las pathogen does not mean that it can successfully inoculate a healthy plant. In pathogen transmission experiments, a very low rate of successful pathogen inoculation occurred from feeding by single psyllids. Much higher numbers of Las carrying psyllids were required to successfully inoculate at an average rate of 70%. We have also demonstrated that low rates of transovarial passage (mother to egg) of Las does indeed occur. Thus, Las+ nymphs collected from plants not showing symptoms of HLB may not necessarily indicate that the plant is already infected, but could be the result of oviposition by a Las+ female. In studies where psyllids reared on Las+ plants were continually transferred to Las- plants over time, the percentage of psyllids retaining the Las pathogen decreased to as low as 20% after 24 days on Las- plants. This is evidence that Las may not replicate within the psyllid as previously suggested; psyllids may need continual access to Las+ plants for acquisition to be capable of vectoring the pathogen over extended periods of time. In experiments designed to determine the effects of Las presence on psyllid biology, there was a significantly higher rate of oviposition by Las+ female psyllids compared to female psyllids not carrying the pathogen. However, longevity of adult Las+ female psyllids was significantly reduced when compared with Las- females, perhaps as a consequence of increased energy expended for oviposition by Las+ females. We have completed two years worth of surveys determining the incidence of Las+ psyllids in commercial groves. The objective of the repeated sampling of ACP populations on a monthly basis was to determine if there was sufficient variation in the incidence of Las+ psyllids to adjust pesticide applications to only those times of the year when the potential for pathogen transmission was increased. While we observed variation in the incidence of Las+ psyllids throughout the year, the months with highest incidence of Las+ psyllids was not consistent year to year. While not accounted for in this study, we suspect that flushing patterns of HLB infected trees (which vary year to year) are the primary factor responsible for the fluctuations in Las+ psyllids. The sampling of citrus groves under different HLB/ACP management regimes also showed that there was a much higher incidence of Las+ psyllid in groves were little or no HLB management programs were in place thus providing some indirect evidence that HLB management programs are likely to be beneficial in terms of reducing rate of pathogen spread. In studies using an Electrical Penetration Graph (EPG) monitor to examine psyllid feeding behavior on insecticide treated versus untreated plants, we have found that the feeding behaviors responsible for pathogen transmission (inoculation) can be significantly disrupted or prevented for certain insecticides. Soil-applications of imidacloprid greatly reduced psyllid feeding behaviors including phloem salivation indicating that imidacloprid can reduce the likelihood of successful inoculation of uninfected plants with Las. However, soil-applications of aldicarb did not have any effect on psyllid feeding behaviors in the first 18 hours of feeding suggesting aldicarb will provide little protection from inoculation by Las+ psyllid migrating from surrounding areas. Thus growers should not rely solely on aldicarb for psyllid control but instead should incorporate foliar applications with the aldicarb which provides extended systemic control of developing nymphs. Foliar applications of fenpropathrin prevented phloem feeding (inoculation) behaviors due to the rapid mortality of adult psyllids exposed to residues.
USDA test 1. A new block of young, HLB’free citrus (Val on Carr) was planted on May 1, 2008 in a grove with substantial levels of HLB. Three ACP control treatments (programs) are being compared in this planting: 1) a monoculture of citrus receiving monthly insecticide applications (annual chemical cost of $198/acre); 2) citrus interplanted with jasmine with a relaxed insecticide program for the citrus (annual chemical cost of $156/acre), jasmine not treated with insecticides; and 3) citrus interplanted with jasmine with a relaxed insecticide program for the citrus and regular applications of imidacloprid to jasmine (total annual chemical cost of $213/acre). For plots with jasmine, a jasmine plant was planted between each citrus tree along some rows in each plot. Program 2 is being studied because ACP may be attracted to jasmine thus reducing numbers of ACP that go to citrus, and population levels of ACP natural enemies may be enhanced by having jasmine in the vicinity of citrus. Program 3 is being studied because ACP may be attracted to jasmine and killed, reducing numbers of ACP in citrus. HLB-infected trees are removed. None of the trees tested HLB positive just before planting in May 2008. One year later, a mean of 0.9, 0.6, and 0.6% of the trees were HLB-infected under programs 1, 2 and 3, respectively. During August 2009, a mean of 9.8, 3.9, and 4.2% of the trees were infected under programs 1, 2 and 3, respectively. The rapid increase in incidence of HLB in the plots between May and August 2009 was attributed to increases in ACP infestations during June and July, particularly in one program 1 plot that was adjacent to older citrus. In February 2010, a mean of 13%, 12% and 13% of the trees were HLB-infected under programs 1, 2 and 3. USDA test 2. Three ACP control programs are being compared for preventing HLB in a block of young, HLB-free citrus (Val on Carr): 1) citrus under a relaxed insecticide program (annual chemical cost of $173/acre); 2) citrus receiving monthly insecticide applications (annual chemical cost of $198/acre); and 3) citrus treated once every three weeks with spray oil [PureSpray Foliar (470, C27) and PureSpray Green (435, C23)] from February through November (plus a December application of Danitol) (annual chemical cost of $76/acre). There are two replications of each treatment. The experiment officially started in August 2009. In January 2010, 0%, 0.3% and 0.3% of the trees tested HLB positive under programs 1, 2 and 3, respectively. These trees were removed. UF test. The ability of systemic insecticides to protect a new planting of citrus from ACP (and consequently HLB infection) is being evaluated. 112 Hamlins were planted in two rows at 15 ft spacing on 03 Mar 2009. Half of each row was considered a replicate and divided into two main plots, treated and untreated. Treated plots were split into two subplots on 13 March, one receiving a liquid formulation of imidacloprid (Nuprid 2f @ 32 oz/acre ‘ 0.5 lbs a.i./acre) and the other receiving a solid formulation of the same (Suscon 13 @ 10 lbs/acre ‘ 0.5 lbs a.i./acre). On 11-Sep 2009, no significant treatment effects (Chi square = 3.61, P = 0.182) were found with respect to numbers of HLB trees. At that time, 10 of the 56 control (untreated) trees tested positive for HLB, 10 of the 28 trees treated with the granular slow release imidacloprid were positive, and 6 of the 28 trees treated with the liquid formulation of imidacloprid were positive. The same trees were resampled on 15 Jan, but all tested negative. PCR was run twice more on the DNA from both the Sep and Jan samples and the results were confirmed. Thus, the different result from Jan could only be attributed to effects of cold weather on titer and/or lack of ACP to reinoculate the trees.
1. Localization of Liberibacter asiaticus (Las) in the hemolymph and other tissues and organs of the Asian citrus psyllid (ACP). [A]. Fluorescence In situ hybridization (FISH) studies are continuing using three oligonucleotide primers (including a newly designed one) based on the following sequences of Las: Primer 1 (HLBr): TCG AGC GCG TAT GCA ATA CG, Primer 2 (LL-REV): TCC CTA TAA AGT ACC CAA CAT CTA GGT AAA; Primer 3 (Cr-RDR-1): TAT ATT GTT CAC ATG AGG GAG CAT TTA ACC. Several FISH protocols have been tested on hemolymph smears and dissected organs of ACP and on leaf sections and extracts from HLB-diseased plants. Green fluorescence, indicating Las, was detected in the hemolymph, filter chamber and midgut of field-collected ACP, but not in healthy controls. It was also detected in leaf sections from HLB-diseased citrus and periwinkle plants but not in those from healthy plants. We are continuing to refine our FISH protocols to reduce the background fluorescence and to increase the permeabilization of insect tissues. [B]. Quantitative RT-PCR of dissected insect organs: We tested two different RT-PCR procedures for detection of Las in dissected salivary glands, alimentary canals and other parts of individual ACP adults. In five successive experiments, Las was detected in an average of 13-24% of the alimentary canals, 12-16% of the salivary glands, and in 16-25% of the rest of the body from psyllids collected from HLB-infected trees in Fort Pierce, FL. To our knowledge, this is the first direct demonstration of Las (using PCR) in the alimentary canal and salivary glands of ACP. Additionally, percentage of infection did not differ significantly between the three insect parts in males but was significantly lower in the salivary glands than in the alimentary canals of females suggesting that in the latter the salivary glands may act as transmission barriers. [C]. Transmission electron microscopy (TEM): We are continuing to use a combination of TEM and RT-PCR, comparing the ultrastructure of psyllid adults that have never been exposed to infected plants with those collected from HLB-infected trees and tested positive for Las with RT-PCR. This is providing a very useful library on the ultrastructure of the alimentary canal, salivary glands and other organs of ACP as well as its bacterial symbiotes, and will form the basis for future immunogold-labeling TEM studies on Las pathogen-vector interactions in ACP at the cellular, tissue and organ levels. 2. Clarification of various acquisition and transmission parameters between ACP and Las. [A] Among ACP adults field collected from HLB-infected citrus trees, averages of 37% females and 38% males have tested positive by PCR (not significantly different). Significantly higher percentages of field-collected adult ACP tested positive for Las during November 2009 (38.9%) than during late March 2010 (22.1%), and CT values associated with adults collected during November (mean 24.8) were significantly lower than for adults collected during late March 2010 (mean 30.5). [B] An average of 16% individual HLB-infected adult ACP (field-collected, mean CT of 26.4) transmitted HLB to young potted citrus trees. Taken together, the data in 2A and 2B, combined with those in1B above, support our hypothesis that transmission barriers to Las in ACP (including the alimentary canal and salivary glands) may play an important role in HLB transmission, since much higher percentages of ACP adults proved PCR-positive compared to those that were actually able to transmit the disease to citrus plants. Our studies are continuing to elucidate the roles of these and other psyllid organs in HLB transmission by its vector.
March, 2010 – 15 McPhail traps were established in locations where Mustang had been sprayed for Asian Citrus Psyllid control, 15 McPhail traps were set where Malathion had been sprayed for Asian Citrus Psyllid control and 15 McPhail traps were set in the control area. GPS locations were recorded and a helper was trained. The servicing schedule was set up and the results of each service date were recorded.
The first year of a 2-year research proposal (FDACS Contract Number 58-1920-9-925 #40) has been completed. Our research objectives were: (1) Devise and perform alternative methods (microinjection and membrane uptake) to complete Koch’s postulates using a pure culture of bacteria isolated and cultured in our laboratory and healthy psyllids as a transmission tool; and, (2) following successful inoculation or loading of the psyllids, we will complete Koch’s postulates. The cultures of two strains of ‘Candidatus Liberibacter asiaticus’ (Las) from China (China1) and Taiwan (B239) were produced in our and a collaborator’s laboratory and determined to be Las by PCR and sequencing. We prepared stretched parafilm membrane sachets containing sucrose solutions in 1XTE buffer in which the cultured bacteria were suspended. Sucrose solutions from 5-20% were tested, however, most experiments were done with 10% sucrose. The titer of the bacteria in sucrose was roughly assessed using real time PCR (Ct values ranged from 20 to 38). Membrane sachets were suspended over 15 ml or 50 ml conical tubes within which 15-20 healthy adult psyllids and 5th instar nymphs were allowed to probe the membranes. After 48 – 72 hr the feeding psyllids were transferred onto sweet orange seedlings and the remaining culture fluid in the sachets was assayed by real time PCR. Psyllids and nymphs were allowed to feed on the sweet orange seedlings for 14 days after which they were removed, and assayed for Las by real time PCR. From more than 30 membranes attempted, nine test membranes, each containing 250 ‘ls of culture in 0.25 ‘ 0.5 ml sucrose solution, were successful in uptake of bacterial culture by the psyllids. The orange seedlings were observed for symptom development and assayed three months post-inoculation by real time PCR. Although no symptoms were observed at 3 months, Madam Vinous sweet orange seedlings inoculated with infectious culture had Ct values ranging from 19.5 to 43.8. Typical HLB leaf symptoms were observed by six months. Test plants are being monitored for additional symptoms and plans are to re-isolate the bacteria from positive seedlings, assay for Las titer, sequence the bacteria to validate the strain originally inoculated, and grow the re-isolated bacteria in culture media. This would complete Koch’s Postulates for Las as the causal agent of Huanglongbing. A second approach used direct microinjection of the culture fluid into the hemolymph of adult and 5th instar nymphs of Diaphorina citri. Psyllids were immobilized with a low velocity stream of C02 and approximately 0.01’l of bacterial culture (Ct value of the bacterial culture suspensions ranged from 32.9 to 38.3) was injected into the abdomen of each adult psyllid. Microinjection of 5th instar nymphs was attempted but mortality was very high. Psyllids were allowed to recover and then placed on young sweet orange seedlings for recovery feeding for 14 days. Surviving psyllids were captured, assayed by real time PCR for the presence of Las, and plants were observed for 3 months for symptom development. While this does not serve as additional confirmation of Koch’s postulates for Las, it does provide important insights into the interaction of Las with the vector. Continued refinement of the microinjection technique is underway. We have developed a usable membrane uptake mechanism of transferring cultured bacteria of strains of Las. Our primary objective for year 1 was to complete Koch’s postulates on a pure culture of Las and we are on the threshold of completing that objective. This work is not only demonstrating the causal relationship of Las to HLB, it is providing important insights about the nature of the pathogen-vector interaction.
the project goals were to establish issue culture cell lines derived from the citrus greening vectoring psyllid, D. citri and we have over a dozen actively growing insect cell lines. the two major cell types in the lines are heterogenous adherant cells and more homogenous suspension cells. Most lines display an asymmetric growth pattern in which cell populations appear to grow slowly, then rapidly, then slowly again. We have also developed antibiotic free cell lines for use in culturing the Liberibacter organism. Project goals that have been met include determining the optimal conditions for passage of the cell lines, measuring the stability of the cell lines and storage/preservation of the cell lines (in progress). A new project for funded for the 2010 fiscal year (Application of Asian Citrus psyllid, Diaphorina citri, tissue culture cell lines) will seek to disseminate the cell line for various purposes. current groups that have requested and recieved cell lines/materials include Dr. Davis (Lake Alfred Citrus Research Center) and Dr. J. Brown (University of Arizona, Dept. of Plant Sciences). It is anticipated that the major project goals will be complete within the 2010 funding period.
Objective 1. Develop baseline susceptibility information for commonly used insecticides for ACP control. The baseline susceptibility data (LD50) of laboratory colony of Asian citrus psyllid (ACP) have been developed for 14 insecticides that are labeled for use in Florida citrus. Also, the baseline sensitivity data of acetylcholinesterase (AChE) to inhibition by selected OP and carbamate insecticides have been generated using biochemical assays. All goals for Objective 1 have been completed. Objective 2. Compare the insecticide susceptibility levels of field ACP populations in different parts of the state to monitor resistance development. The susceptibility of field collected ACP from 5 FL counties has been determined for 14 insecticides from Objective 1 using topical bioassays. all five ACP populations collected from five Counties showed decreased susceptibility to all the tested compounds (especially imidacloprid) when compared with laboratory strain. Also, the baseline sensitivity data of AChE to inhibition by OP and carbamate insecticides was determined for these insecticides.AChE of all five ACP populations collected from five Counties showed decreased sensitivity to inhibition to all the tested insecticides compared with laboratory strain with few exceptions. These data corroborate with insecticide susceptibility data obtained from bioassays and will help monitor changes in resistance levels in field populations both at physiological level for all insecticides and at the target site level for OP and carbamate insecticides. Objective 3. Select ACP for imidacloprid (other insecticide) resistance and determine the mechanisms of resistance. For this objective, two ACP field colonies have been established in a greenhouse and are being subjected to imidacloprid or chlorpyrifos selection pressure in every generation for developing resistant colonies. Thus far, we have selected for up to 50 fold resistance to imidacloprid and 25 fold resistance to chlorpyrifos in the laboratory. Further selection of future generations and determining the mechanisms of resistance by toxicological and biochemical studies are in progress. These colonies will be used for further studies on determining the rate of resistance and cross-resistance development potential in ACP and mechanisms of resistance. Objective 4. Determine the cross-resistance potential in imidacloprid (other insecticide)-selected ACP strain. This objective will be started in the 2nd year once the highly resistant ACP colonies for imidacloprid and chlorpyrifos insecticides are selected in the greenhouse, which is currently in progress as described in objective 3. In the 2nd year of this project, we will continue screening more field ACP populations collected from 4-5 different locations in Florida for determining the changes in insecticide resistance levels both at physiological and target site which will be helpful in monitoring insecticide resistance levels. We will also continue selection of two field colonies for imidacloprid and chlorpyrifos resistance in the greenhouse. The information from the objectives 2, 3 and 4 should help monitor the onset and levels of resistance in ACP to different insecticides. Also, it is helpful in determining the rate of resistance development and cross resistance potential to various insecticides. This will enable us to design, develop and implement an effective insecticide resistance management program for ACP.
During the past year, 4 main objectives are being pursued with the goal of better understanding the psyllid/host plant interaction to improve psyllid management using alternative control tactics to broad-spectrum insecticides. 1) Psyllid fitness on various host plants – investigations were initiated to examine the fitness of the Asian citrus psyllid when reared on 8 different citrus varieties. Experiments conducted in both the greenhouse and growth chambers examined psyllid longevity, number of eggs produced, immature survival and development rate for each of these eight varieties examined. During the first set of evaluations, it was found that psyllids were not able to complete development on one of the root stock varieties evaluated, Cleopatra mandarin. Because of the potential significance of this finding, this objective was temporarily set aside to focus more in depth and confirm that Cleopatra mandarin was indeed not a suitable host for the psyllid (see objective 2 below). Work has now resumed on this first objective. Results to date suggest that not all citrus varieties are equally suitable for psyllid development. An electrical penetration graph (EPG) monitor is being used to record psyllid feeding behavior on these 8 host plants to determine if there is a difference in psyllid feeding behavior that can be correlated with difference in psyllid fitness on certain host plants. Preference experiments using an olfactometer are being used to determine if plant volatiles may also be responsible in part for psyllid preference of certain varieties under field conditions. 2) Survival of psyllid on Cleopatra mandarin – based on the preliminary results from objective 1, we conducted a series of replicated experiments to confirm our findings that Cleopatra mandarin is an unsuitable host plant for ACP. Survival of ACP on Cleopatra mandarin was less than 5% compared to greater than 60% survival on sour orange. Female ACP also laid significantly fewer eggs on Cleopatra compared to other host plants. The factors affecting the “unsuitability” of Cleopatra mandarin as a host for psyllid are being further investigated. Working with the CREC plant improvement program (Fred Gmitter), we are conducting evaluations of ACP survival on numerous Cleopatra hybrids in the CREC field collection to determine if any differences in suitability for ACP exiists between these hybrids. Any differences in suitability for ACP will be further examined to determine the responsible factors. Such factors could be exploited in breeding programs for psyllid resistant plants. 3) Effects of host plant nutrition on ACP fitness – the effects of fertilization practices on psyllid population dynamics was examined in greenhouse studies. High levels of nitrogen fertilization resulted in more rapid psyllid development, whereas high levels of potassium significantly slowed down psyllid development. There was also a similar effect for psyllid body mass. Ongoing studies are examining psyllid fecundity and longevity under the varying fertilization regimes. Field trials are planned for 2010 to confirm these findings under typical commercial growing conditions. The goal of this work is to determine if adjusting nutrient applications (within acceptable limits for commercial groves) can be used to help minimize psyllid populations. 4) Effects of boron application on psyllid populations – Based on initial findings from Objective 3 (above), investigations were undertaken to determine the effects of Boron application on ACP populations, particularly adult ACP mortality. Evaluations of differing rates of Boron in both greenhouse and growth chamber experiments demonstrated that Boron alone caused significant mortality of ACP. Filed experiments are underway currently to investigate the use of Boron as an alternative to traditional pesticides for ACP control.
The first year of a 2-year research proposal (FDACS Contract Number 58-1920-9-925 40) has been completed. Our research objectives were: (1) Devise and perform alternative methods (microinjection and membrane uptake) to complete Koch’s postulates using a pure culture of bacteria isolated and cultured in our laboratory and healthy psyllids as a transmission tool; and, (2) following successful inoculation or loading of the psyllids, we will complete Koch’s postulates. The cultures of two strains of ‘Candidatus Liberibacter asiaticus’ (Las) from China (China1) and Taiwan (B239) were produced in our and a collaborator’s laboratory and determined to be Las by PCR and sequencing. We prepared stretched parafilm membrane sachets containing sucrose solutions in 1XTE buffer in which the cultured bacteria were suspended. Sucrose solutions from 5-20% were tested, however, most experiments were done with 10% sucrose. The titer of the bacteria in sucrose was roughly assessed using real time PCR (Ct values ranged from 20 to 38). Membrane sachets were suspended over 15 ml or 50 ml conical tubes within which 15-20 healthy adult psyllids and 5th instar nymphs were allowed to probe the membranes. After 48 – 72 hr the feeding psyllids were transferred onto sweet orange seedlings and the remaining culture fluid in the sachets was assayed by real time PCR. Psyllids and nymphs were allowed to feed on the sweet orange seedlings for 14 days after which they were removed, and assayed for Las by real time PCR. From more than 30 membranes attempted, nine test membranes, each containing 250 ‘ls of culture in 0.25 ‘ 0.5 ml sucrose solution, were successful in uptake of bacterial culture by the psyllids. The orange seedlings were observed for symptom development and assayed three months post-inoculation by real time PCR. Although no symptoms were observed at 3 months, Madam Vinous sweet orange seedlings inoculated with infectious culture had Ct values ranging from 19.5 to 43.8. Typical HLB leaf symptoms were observed by six months. Test plants are being monitored for additional symptoms and plans are to re-isolate the bacteria from positive seedlings, assay for Las titer, sequence the bacteria to validate the strain originally inoculated, and grow the re-isolated bacteria in culture media. This would complete Koch’s Postulates for Las as the causal agent of Huanglongbing. A second approach used direct microinjection of the culture fluid into the hemolymph of adult and 5th instar nymphs of Diaphorina citri. Psyllids were immobilized with a low velocity stream of C02 and approximately 0.01’l of bacterial culture (Ct value of the bacterial culture suspensions ranged from 32.9 to 38.3) was injected into the abdomen of each adult psyllid. Microinjection of 5th instar nymphs was attempted but mortality was very high. Psyllids were allowed to recover and then placed on young sweet orange seedlings for recovery feeding for 14 days. Surviving psyllids were captured, assayed by real time PCR for the presence of Las, and plants were observed for 3 months for symptom development. While this does not serve as additional confirmation of Koch’s postulates for Las, it does provide important insights into the interaction of Las with the vector. Continued refinement of the microinjection technique is underway. We have developed a usable membrane uptake mechanism of transferring cultured bacteria of strains of Las. Our primary objective for year 1 was to complete Koch’s postulates on a pure culture of Las and we are on the threshold of completing that objective. This work is not only demonstrating the causal relationship of Las to HLB, it is providing important insights about the nature of the pathogen-vector interaction.
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