An oil treatment program for Asian citrus psyllid is proposed for situations such as organic blocks where conventional insecticides can not be used. The objective of the project is to determine the effects of 0, 6 (fall) and 12 (spring and fall) applications of PureSpray green 0.25% oil applied in 250 gpa using an airblast sprayer with outside coverage, on tree health, fruit production and fruit quality of California navels. The experiment is being conducted at Lindcove Research and Extension Center in Exeter CA. The plots (5 rows by 8 trees) were assigned treatments based on pretreatment evaluations of fruit yield and size prior to treatment. Treatments were applied to three replicated plots during spring (6 applications 2 weeks apart) and fall (6 applications 2 weeks apart) during 2010. Trees were evaluated for leaf drop and no significant differences were found between treated and nontreated trees. In November 2010, color was evaluated in the four quadrants of 6 trees per plot. The percentage of fruit with 75-100% color and juice weight were significantly higher in the two oil treatments compared to the nontreated control, indicating the oil treatments affect fruit maturation. Fruit quality measurements (brix, California Standard, rind thickness, juice weight, fruit length and width) are currently underway. Harvest of individual trees will be conducted in February 2011 and we will evaluate yield, fruit size and fruit grade. This experiment will determine if repeated low rate, low volume oil applications affect citrus production or fruit quality under California conditions. This information is critical as we prepare for the spread of Asian citrus psyllid into commercial citrus orchards.
Evaluation of the passage and mortality Diaphorina citri on screens impregnated with insecticide The experiment was conducted in two farms located in S’o Paulo State. The first farm is located in the municipality of S’o Manuel, where a block was selected that had a strong record of psyllids capturing in its surround. The barrier installed, has a length of 100 meters, width of 2.7 m and a distance to the ground about 60 cm. This was positioned next to the orchard at a distance of 5 m. The green sticky traps were equally divided between areas with and without barrier, 15 days after the first assessment was conducted, but none of the areas the psyllid was collected. In the second farm, the experiment was similar to the first one; however in this case an additional protective barrier 80 m was added to the side of the block. On this farm the first evaluation has not been realized. A first selection to determine which screens have the potential to be used as barriers was carried out. D. citri could go through all the screens evaluated (75%). But it was observed that after contact with the screen, about 85% of the insects have died before having contact with citrus trees, except the screen which showed a COD 196 60% mortality. For the field trials was selected to screen COD205. This was sent by the supplier, but with a higher dose (4g deltamethrin/kg of net). A new test was performed, and observed a mortality of 98.3%. Evaluation of screens impregnated with insecticide barriers The experiment was conducted in two farms located in S’o Paulo State. The first farm is located in the municipality of S’o Manuel, where a block was selected that had a strong record of psyllids capturing in its surround. The barrier installed, has a length of 100 meters, width of 2.7 m and a distance to the ground about 60 cm. This was positioned next to the orchard at a distance of 5 m. The green sticky traps were equally divided between areas with and without barrier, 15 days after the first assessment was conducted, but none of the areas the psyllid was collected. In the second farm, the experiment was similar to the first one; however in this case an additional protective barrier 80 m was added to the side of the block. On this farm the first evaluation has not been realized.
Under Objective 1 (define rates and formulations of copper sprays for more effective control) one trial with Red grapefruit was conducted in Ft. Pierce, FL and 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 had 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 were made in November 2010 (grapefruit) and January 2011 (Hamlin). Copper formulations containing copper hydroxide or basic copper sulfate (metallic rates of 0.67 to 1.12 kg/ha), varied from low (Hamlin) to moderately effective (grapefruit) for canker control of fruit disease incidence. Copper pentahydrate, at a lower metallic copper rate/ha per application, provided equivalent control to film-forming copper formulations. Greater canker susceptibility of fruit occurred in later season and was likely because of more prolonged opening of stomates in cooler weather and enhanced bacterial entry, coincident with more numerous windblown rain events. Although Hamlin fruit disease incidence was higher, copper protection against early season fruit infection was effective for prevention of premature fruit drop. Under Objective 2 (establish the period of fruit susceptibility, residual activity and phytotoxicity of copper). In 2010, we compared the copper fruit residues from different copper-containing products up to 28 days after application to grapefruit. The copper product used and the number of days after application significantly affected the amount of copper residue. These effects were consistent whether the data were analyzed as copper/fruit or copper/surface area. The rate of copper residue decrease was different for the various products tested. Copper products forming films, Kocide 3000 (copper hydroxide), Cuprofix Ultra 40 Disperss (copper sulfate), and Badge X2 (copper hydroxide and oxycloride) decreased at a similar rate, whereas the non-film forming Magna-Bon CS2005 (copper sulfate pentahydrate) decreased at a higher rate and left about 75% less fruit residue. This was expected as Magna-Bon is applied at approximately 15% of the metallic contained in film-forming coppers. Since Magna-Bon performed as well as film-forming copper products for control of canker on grapefruit the last two seasons, we have hypothesized that the Magna-Bon copper may be locally systemic in fruit rind tissues. This coming season, we will attempt to assay the activity of copper in the rind against Xcc to determine whether the copper is located internally as well as externally. Under Objective 3 ( evaluate the use of streptomycin [Firewall]) As in the past three seasons, applications of Firewall in July and August, were effective for canker control on grapefruit either in combination with a reduced rate of copper or when substituted for copper in the spray program. Based on Firewall’s efficacy and ability to lower the risk of copper phytotoxicity, an application has been submitted by FFVA and FDACS to gain EPA Section 18 emergency registration for use of Firewall against canker on fresh grapefruit. 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. Findings are being prepared for publication. Under Objective 5 (rapid transfer improved canker management technology to the Florida citrus industry), 2011 canker management recommendations have been submitted for publication in the Florida Citrus Pest Management Guide and to Citrus Industry Magazine. Oral presentations have been delivered at the Florida Citrus Show and at a multi-county extension meeting. Results of the Hamlin trials will be presented at the 2011 FSHS annual meeting.
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 4 times 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. Two disease ratings have been taken so far and no significant change in tree health has been observed yet in treated or control, including symptomatic trees. There were no significant treatment differences in yield at the first harvest, after the initiation of treatments in April. 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 1, 2, 3, and 5 from objective 1. Infection rate and progress are being monitored by qPCR monthly. Two months after inoculation, new flush on some trees was strongly positive for Las; however, too few of the trees were positive to determine any treatment effects. Samples from 3 and 4 months post inoculation continue to show an increase in trees detected positive. No significant differences have been observed, although treatment 5 has consistently had the highest number of positive trees each month. Five month qPCR samples showed a leveling off of incidence as most of the trees test positive and the number of trees still testing negative agrees with known graft inoculation efficiencies. Routinely, the first positive sample on a tree is in the newest flush and older leaves remain negative or only weakly positive. No significant differences in Las titer have been observed between treatments to date, although the highest Las titers each month have been in treatment 3 and 5. Initial positives were detected in new flush near the graft inoculation site. 5 months post inoculation Las appears to be moving systemically through the canopy with detections occurring in old leaves, recently hardened off leaves, and topmost new flush depending on the plant. There is a trend towards high incidence of detection in the topmost flush of treatment 5, which, combined with higher titers, could have implications for vector transmission in disease management. 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. At five months, symptoms began to develop on tissues that had been Las positive the longest according to qPCR. This suggests that it takes 2-3 months after Las invades leaf tissue for symptoms to develop. No treatment differences in symptom development have been observed, except that symptom development first began in the trees from treatment 3 and 5 that were the first to test positive 2 months after inoculation. Currently, no differences in tree vigor have been observed, but this is not expected until strong symptoms develop. As soon as symptoms develop in control plants, analysis of fixed samples will begin, which is expected at month 6 or 7. Sampling for 6 months post inoculation is underway at the time of this report.
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 50 trees per treatment (5 blocks of 10 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 17 months after treatments began, 65 trees were PCR+ (22%) 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 (18). Lower number of PCR+ trees occurred without graft inoculation in treatments with SAR inducers (6), foliar insecticides (8), and foliar insecticide plus SAR inducers (6). At this time, the effect of SAR on HLB disease progress is minimal, which indicates a lack of promise for use of SAR inducers in HLB management.
Objective 1: Soil applications of inducers of systemic acquired resistance (SAR), imidacloprid, thiamethoxam or acibenzolar-S-methyl, at various rates and application frequencies were evaluated for control of citrus canker caused by Xanthomonas citri subsp. citri in a field trial of 3- and 4-year-old ‘Ray Ruby’ grapefruit trees in southeastern Florida. Canker control on foliage produced by one, two or four soil applications of imidacloprid, thiamethoxam, and acibenzolar-S-methyl was compared with 11 foliar sprays of copper hydroxide and streptomycin applied at 21-day intervals. In 2008 and 2009 crop seasons, canker incidence on each set of vegetative flushes was assessed as the percentage of the total leaves with lesions. In 2008, despite above average rainfall and a tropical storm event, all treatments significantly reduced foliar incidence of citrus canker. Sprays of copper hydroxide was the most effective treatment for reducing canker disease incidence compared to the untreated control. Compared to the untreated control, soil applications of SAR inducers reduced foliar disease depending on rate and frequency of application. In 2009, all treatments significantly reduced the incidence of foliar canker compared to the untreated control. Four applications of acibenzolar-S-methyl at 0.2 g a.i. per tree was the most effective SAR treatment for reducing foliar disease incidence in both 2008 and 2009, but soil application of all the inducers was effective for reducing foliar incidence of canker on young trees under epidemic conditions. In Brazil and FL greenhouse evaluations, 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 systemic activity that the inducers of SAR have for reducing canker incidence and epidemic development of disease on leaves and fruit. 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. 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. In 2010, trials integrating ASM at different frequency of soil application with THIA and CH sprays at 21 day interval was conducted in 2 yr-old Ray Ruby grapefruit and 1-yr-old Vernia sweet orange. The highest incidence of disease trees and/or leaves is in the non-treated check in each trial compared with a very low incidence of canker in the integrated SAR treatments. 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).
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. Strong characteristic deficiency symptoms have yet to develop. Deficiency symptoms are expected to take several months to develop, which is exacerbated by the slow growth due to cold weather. 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. As soon as strong deficiency symptoms develop, then psyllid testing for objective 1 and 2 will commence and is expected to move rapidly. 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 and bulk up isolates of the three strains in the greenhouse. 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). Current analysis suggests strong support for at least two strains and we are currently trying to determine if the third strain is an independent strain or a subpopulation of one of the other two. To insure that the isolates are CTV free they must be passaged through Carrizo, which will take at least 3-6 months due to the latent period.
Experiments using electrical penetration graph (EPG) showed that in plants treated with imidacloprid and thiamethoxam, the phloem sap ingestion (waveform E2) by D. citri is statistically reduced, and after the first feeding on phloem, the adults do not do more probing. Despite the acquisition of the bacterium by ACP, evidenced by the positive results of PCR in insects, no plants have been detected the presence of the bacterium Ca. L. asiaticus. No transmission results yet. In the 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, 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.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). Mineral oil reduced the ratio of psyllids that reached the phloem, but these insects were able to sustain ingestion for 1 to 2 h. Mineral oil has a moderate interference on probing behavior of ACP. We started the EPG experiment using pymetrozine.
A series of greenhouse research experiments have been completed that show that a number of commercial formulations of plant growth regulators (PGRs) (GA biosynthesis inhibitors) reduce Asian citrus psyllid fitness. Specifically, we found that two of the PGRs tested (prohexadione calcium and mefluidide) reduced ACP fecundity and survivorship while two others, uniconazole and paclobutrazol, reduced fecundity or survivorship, respectively. These results have been summarized in a manuscript that has been submitted to the Entomological Society of America for publication in a peer reviewed journal. A follow-up study to determine the duration of efficacy of these treatments has recently been completed using one of the PGRs (prohexadione calcium). Unfortunately, we had very poor egg laying in all treatments, including untreated control trees, and the experiment was unsuccessful. We are trying to determine why this happened and repeat the experiment. An experiment has been completed to begin to determine what changes are occurring within plants that elicit the observed changes in psyllid fitness. Large container grown trees were treated with prohexadione calcium and tissue samples were harvested at 3 day intervals. The samples have been analyzed for nutrients and free amino acid content. We are still working on analyzing phenolic and flavonoid contents as well. We anticipate having the analyses and preliminary data summary completed during the next quarter. Results of this study will be used to develop further studies to determine the basis for the observed effects. New work is being developed to determine whether the effects of PGRs on psyllid fitness correspond with changes in either acquisition or transmission of Candidatus Liberibacter asiaticus (Las). The first step in preparing for that line of research is to develop a population of Las-infected trees that can be used in acquisition studies. Greenhouse grown Las-infected trees used as infected budwood source material has been confirmed Las+ by PCR . Budwood from these trees has been harvested and used to graft inoculate a population of citrus trees. Acquisition studies will proceed as soon as the new plant material has been successfully infected. We anticipate that occurring early in 2011. In the interim, a population of ACP is being maintained and increased for use as soon as the plant material is ready. Field trials are planned for 2011 to test the efficacy of the PGRs against field populations of ACP. This trial was attempted in 2010, but we were unable to develop a sufficient ACP population in our field plots due to area wide ACP management. If this is the situation again this season, we will cage psyllids on treated trees to make the assessments.
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 (ACP) 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 working on this project 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 in citrus branches increased from 0.15 in June to a peak of 0.47 in mid-October 2010. It then declined to between 0.20-0.30 in mid-December. The ACP Las prevalence in stayed between 0.10-0.15 from July to September and rose to 0.30 by October. There was a positive correlation between the Las prevalence in citrus branches and ACP (Pearson r=0.62, p=0.05). From a subset of samples of the other two sampling locations, the trend for citrus prevalence is similar. The samples from these sites are currently being processed to give a more complete picture. At this time there does not seem to be a relationship to environmental variables, ambient temperature and rainfall. Sampling is continuing for ACP, citrus and phenology at all 3 sites. Results of this project were presented at the International Research Conference on Huanglongbing in Orlando January 2011.
Based on the analysis of more than 78,000 psyllids collected monthly from commercial citrus groves in Florida, we have identified certain times of the year when psyllids are more likely to be carrying the Las pathogen with Oct-Dec having the highest likelihood of Las+ psyllids being present. However, our results also suggest that risk of pathogen transmission can be present at any time of the year which we can not yet predict. Further work is ongoing to correlate plant related factors with psyllid transmission of the pathogen for more reliable prediction of risk and need for increased psyllid control efforts. The effects of insecticide applications on the ability of psyllids to successfully transmit Las were investigated using Electrical penetration graph (EPG) monitors. Certain groups of insecticides (particularly Pyrethroids and certain OPs) were able to prevent pathogen transmission by disrupting psyllid feeding behavior while some of the more selective insecticide groups had no discernible effect on pathogen transmission. The effects of three soil-applied neonicotinoid insecticides were also examined and despite some differences, all three were shown to provide benefit in preventing or reducing pathogen transmission. EPG investigations are continuing with additional products as well as looking at the extended duration of protection provided by these insecticides. Development of new approaches to managing pathogen spread requires a better understanding of the factors responsible for pathogen survival in the vector. For this reason, psyllid populations from various locations in Florida, Texas, Hawaii, South America, and Asia have been collected for analysis of endosymbiont populations. To determine the associations between psyllid populations and the frequency of their associated bacterial endosymbionts, 16S clone libraries obtained from these samples have been sequenced and are being subjected to DNA homology searches. PCR results from these samples indicated that Wolbachia infection differs considerably among psyllid populations within and outside of Florida. In addition, laboratory experiments indicated that there is no apparent effect of temperature on ACP endosymbiont populations. As a follow-up to this study, we analyzed additional psyllid samples to determine whether specific endosymbionts are associated with changes in Las- infection rates within ACP populations over time. Finally, we have successfully developed a psyllid microinjection system for inoculating psyllids with antibiotics. Initial injections with rifampicin were unsuccessful, as subsequent PCR analysis with 16S primers failed to identify aposymbiotic psyllids. Injections with tetracycline are currently being conducted to obtain axenic psyllids for studies of the biological functions of psyllid endosymbionts. Experiments evaluating the effect of temperature on Las acquisition and inoculation were completed. Peak acquisition (approximately 50%) occurred at 25oC. Significantly fewer ACP (< 10%) acquired Las below 22.5oC and above 30oC. We have begun assessing plants used in inoculation studies initiated five months ago. Thus far, none of these plants have tested positive for Las. Previously we reported that psyllid survival decreases when the insects are infected with Las. In experiments to determine the effect of Las on psyllid fitness, we have determined that nymphs reared on Las-infected plants develop faster than nymphs reared on healthy plants and that there are no differences in fertility among infected and healthy psyllids. Severinia buxifolia (box orange) was shown to be a non citrus rutaceous host for Ca. L. asiaticus. Psyllid transmissions from S. buxifolia to citrus were successful with a transmission rate of 46% and to S. buxifolia at a rate of 54%. A newly devised qPCR method that will determine live versus dead bacteria is being utilized for studies with inoculative psyllids.
We completed experiments evaluating the effect of temperature on Las acquisition. Peak acquisition (approximately 50%) occurred at 25oC. Significantly fewer ACP (< 10%) acquired Las below 22.5oC and above 30oC. Experiments to determine the effect of temperature on Las inoculation were also completed and we will begin assessing plants used in these experiments for Las infection later this month. Analysis of endosymbionts from different psyllid populations is underway. Thus far we have amassed a 16S library containing 1000 sequences from psyllid populations collected from seven counties in Florida as well as India, Argentina, Mexico, Texas, and Hawaii. PCR results from these samples indicated that Wolbachia infection differs considerably among psyllid populations within and outside of Florida. In addition, laboratory experiments indicated that there is no apparent effect of temperature on ACP endosymbiont populations. Further experiments are underway to evaluate the role of endosymbionts in Las infection differences observed in Florida psyllids. In experiments to determine the effect of Las on psyllid fitness, we have assessed the fecundity, fertility, development time and adult survival of Las-infected ACP. Results from these experiments suggest that nymphs reared on Las-infected plants develop faster than nymphs reared on healthy plants and that there are no differences in fertility among infected and healthy psyllids. Additional investigations revealed an increase in the fecundity of infected psyllids. Infected psyllids produced nearly 200 eggs per female over a 25 day period, compared to only 100 eggs per healthy female. Vertical transmission of Las by ACP, determined in our previous experiments (Pelz-Stelinski et al. 2010), and increased fecundity of infected psyllids suggest that Las is well-adapted to psyllids. The practical implication of this result is that greater egg production in Las-infected ACP may increase disease spread. Finally, infected adult psyllids exhibited reduced longevity compared to Las-free ACP, suggesting a possible fitness trade-off between reproduction and longevity when infected with Las.
Field data collected mostly in Lake Alfred commercial groves over two growing seasons were used to generate a model and produce recommendations for general optimal field conditions that ensures maximum effectiveness of low volume insecticide applications. Data from the nearby FAWN weather station was used to compare relevant meteorological data from application times against the day 3 (knock-down) and day 28 (residual) percent reduction of Asian citrus psyllid (ACP) nymphs and adults vs. 2 sets of untreated control plots. Applications of test insecticides were made using the Curtis Dyna-Fog’ ‘LV-8’ sprayer at 6 gpa/10 mph in 0.1 acre/3 middles plots of Valencia grove. The volume median diameter of spray droplets was about135 microns. Sprays were made in 4 replicates during conditions of thermal inversions, i.e. ~11 pm onwards and when wind speeds were less than 3-5 mph. Still nights were avoided to ensure label compliance. Products tested were for evaluations of novel active ingredients (AI), existing chemicals requiring LV registration, formulation types as well as experimental investigations into spray adjuvants. Efficacy evaluations were conducted on days 3, 7, 14 and 28 using: a) tap counts, beating randomly selected branches in each plot and enumerating the ACP adults disturbed onto a white sheet, b) yellow sticky traps, deployed 4 per plot and the adults counted on the card, and c) flush rating, 10 flush terminals per plot were collected and the nymphs surveyed in the lab by rankings. All of these data were then analyzed using ANOVA versus the untreated control. Overall the results from the experimental applications were that the LV-8 is an effective method of controlling ACP in a mature citrus grove. The wide scale analysis highlighted the differences in the active ingredients. The insect growth regulators showed lower rates of knockdown, but were effective at the end of the sampling period, whereas the organophosphates showed very effective knockdown, with levels of control tailing off at the end. The newer products that combine two different classes of AI gave good overall control for both periods. Use of the same AI and different classes of adjuvant showed differences in levels of control which prompts further research. Our applications followed the label and best practices defined by the applicators license, so we did not experience suboptimal performance from the products, the use of meteorological data was more akin to fine tuning of best practice. When the micro-meteorological data were incorporated into the rankings, we noticed that the wind speeds during LV applications affected the efficacies. The lowest efficacies were recorded for the higher wind speed; about 3.5 mph. Low humidity during the applications also reduced the efficacy in the long term applications. Conclusions- For the products tested, the LV-8 was an effective application method; products consisting of mixtures of different insecticide classes show good knockdown and longer term control; and that lower, steady wind conditions with higher humidity gave the best control.
The literature shows that transmission efficiency of Candidatus Liberibacter asiaticus (CLas) by the Asian citrus psyllid (ACP), Diaphorina citri, can be quite variable (1-100%). We are conducting a series of studies to determine the impact of various factors on the efficiency of acquisition of CLas by ACP), such as vector development stadia and duration of acquisition access period (AAP) (study 1), as well as leaf age (study 2), pathogen titer and symptom expression in infected citrus (study 3). We are also investigating how transmission efficiency varies depending on the duration of the inoculation access period (study 4), and if a systemic insecticide can prevent transmission (study 5). We finished study 1 and are close to conclusion of studies 2 and 3. Studies 4 and 5 are being set up and will require another year for completion. In this quarterly report we show results of experiments related to study 1 (see previous reports for partial results of studies 2 and 3). In study 1, we compared acquisition efficiency of CLas by different ACP nymphal stadia (1st, 2nd, 3rd, 4th and 5th instars) and adults (1 wk old). Insects of each age group were allowed a 2-day acquisition access period (AAP) on CLas-infected plantas, then kept on citrus healthy plants for 19 days and tested by real-time (RT) PCR. The experiment was repeated three times with different source plants. Mean acquisition efficiencies by 1st, 2nd, 3rd, 4th and 5th instars, as measured by the proportion of infective psyllids by RT-PCR, 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. In a second experiment, we compared CLas acquisition efficiency by 3rd-instar nymphs and adults in relation to duration of the AAP (1.5, 6, 12, 24, 48 or 96 h). Almost 30% of the nymphs and adults were PCR positive for CLas when submitted to the shortest AAP tested, suggesting that the minimum AAP required for acquisition is <1.5 h. Acquisition efficiency by nymphs increased at a logarithmic rate in relation to AAP duration, reaching an average of 91% with 24-h AAP and stabilizing around this level with longer AAPs. In contrast, acquisition efficiency of CLas by adults increased linearly with AAP duration, reaching the maximum level (93%) only after an AAP of 96 h. Overall, the results show that both nymphs and adults can efficiently acquire the pathogen if allowed to feed for 1 and 4 days, respectively, on young shoots of infected plants. In a third experiment, we used the Electrical Penetration Graph (EPG) technique to determine stylet penetration activities and probing periods required for pathogen acquisition and inoculation in citrus. Healthy ACP adults were connected to the EPG system and placed on CLas-infected plants for different probing periods. Probes were artificially terminated by pulling the insects out of the plant after the following stylet penetration periods (treatments; n=30 insects per treatment): I) 20 min in waveform C (pathway phase through epidermis and parenchyma); II) waveform C + 30 s in waveform D (first contact with phloem tissue); III) waveforms C+D + 90 s in waveform E1 (penetration and possibly salivation in the phloem sieve elements); and IV) waveforms C+D+E1+ 1 h in E2 (phloem sap ingestion). Following each probing treatment, the insects were kept on healthy seedlings for 21 days and then tested by RT-PCR for CLas infectivity. We observed infective psyllids (5 out of 30) only when waveform E2 was performed (treat. IV), indicating that CLas acquisition by ACP occurs only after its stylets reach the phloem tissue, and sap ingestion (represented by E2) takes place. We hope to obtain additional replicates per treatment to confirm these results. This knowledge is important to analyze the efficacy of some control tatics (e.g. insecticides, host plant resistance and feeding deterrents) aimed to prevent pathogen acquisition by ACP.
Progress has been made toward developing a GC-EAD method that should be useful for determining which specific chemicals may serve as psyllid attractants. This method records specific impulses from psyllid antennae in response to blends of chemicals allowing precise identification of which specific chemicals the insect antenna is responsive to. Software and hardware-related obstacles to recording antennal signals from the Asian citrus psyllid were overcome during 2010, resulting in the first successful recordings from a psyllid antenna. Citrus volatiles were collected from citrus flush and analyzed by gas chromatograph-coupled electroantennography (GC-EAD). GC-EAD identified several peaks that reliably elicited antennal response. These peaks were subsequently identified by GC-mass spectometry (GC-MS) and will now be confirmed by testing individual compounds in behavioral bioassays. Varying concentrations of ‘pure’ purchased odor compounds will be ‘puffed’ over antennae to generate dose-response curves. In addition to identifying active plant odors, our primary focus will be to use our recording system to search for pheromone compounds that may be present in odors produced by male and/or female psyllids. Currently, we have identified seven compound of particular interest, which are being pursued in behavioral testing. Also, we have been analyzing cuticular extracts of pysllids and have identified several differences between males and females. We are investigating these chemicals that differ between males and females in an effort to identify the specific behaviorally active pheromone components.
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