Two trials were conducted in May and June, 2011 to determine the levels of horizontal transfer (psyllid-to-psyllid spore transfer) of Ifr spores achievable with the autodisseminator (dispenser) in greenhouse tests. The results demonstrated that adult ACP transferred Ifr spores from the dispensers to nymphs developing on potted orange jasmine in a greenhouse. In the first trial, 44% of the adults and 34% of the nymphs became infected with Ifr while in the second trial 35% of the adults and 27% of the nymphs became infected. Interestingly, Ifr mycelia grew extensively as a saprophyte on molted skins, honeydew, and waxy excrement, resulting in fungal coverage of entire nymph clusters. We are currently incubating Ifr inoculated waxy excrement and molted skins to determine whether Ifr mycelium grown on this material will develop conidiospores and whether these spores, in turn, will infect nearby psyllids. A second set of tests is currently underway to compare the level of adult ACP emergence from nymph colonies that were exposed to Ifr-infected adults to that of unexposed nymph colonies. Initial testing indicates that blastospores on the dispenser surface remain viable at least 10 days after deployment in the greenhouse. Tests have just been initiated to monitor spore viability, infectiveness, and phenology for 21 days following deployment of the dispenser. These tests will determine the phenology of blastospores and condiospores on the trap coating and their relative importance in primary infection. Laboratory tests using novel behavioral assay methods to measure ACP response to combinations of visual, scent, and taste cues have showed that: 1) ACP response to particular scents is strongly influenced by scent concentration. For some scent mixtures, stronger and weaker concentrations are less stimulatory than mid-range concentrations while for other scent mixtures, response is strong to low concentrations and tapers off as concentration increases. 2) ACP can differentiate among scents composed of only three ingredients, with a synthetic mixture of terpenes from Mexican lime and orange jasmine being the most stimulatory. We will determine whether the addition of targeted compounds will enhance the attractiveness of the mixtures 3) ACP can differentiate between different shades of green and are more strongly attracted to medium-dark green objects than to pale green objects.
Native California non-target psyllid species have been collected from California’s natural habitats for host specificity tests with Tamarixia radiata. Five native psyllid colonies, Diclidophlebia fremontiae (host plant Fremontodendron californicum), Calophya nigrella (host plant Rhus ovata), Heteropsylla texana (host plant Prosopis glandulosa), Heteropsylla sp. (Host plant Acacia farnesiana) and Bactericerca cockerelli, a native pest of potatoes, tomatoes and peppers are being continuously cultured and maintained on their natural host plants in the I & Q facility at UCR. We have been unable to establish and maintain colonies of Neophyllura arctistophyli (host plant Manzanita Arctistaphylos glauca) in Quarantine. This psyllid has been dropped from the test list because of extreme difficulty in locating wild populations, an inability to maintain lab colonies and problems sourcing adequate numbers of host plants. We have been unable to produce A. glauca from seeds or cuttings (a specialist native CA plant nursery also tried and failed to produce this plant), and no California native plant nurseries produce adequate numbers of plants for this research project. Consequently, N. arctistophyli has been replaced by olive psyllid as this species is phylogenetically closely related to N. arctistophyli and ACP. A self-introduced weed biocontrol agent, the Scotch broom psyllid, Arytainilla spartiophylla, is univoltine and because of winter chilling requirements to break diapause it has not been possible to maintain a colony at UCR. These psyllids were collected from the field in northern California and shipped to Quarantine for testing. A continuous supply of healthy seedlings of host plants is very important for rearing test psyllids. Seedlings of eggplant/sweet pepper Prosopis glandulosa and Acacia farnesiana, Rhus ovata, Fremontodendron, and olive are being acquired from local nurseries or grown by CDFA at the Mt. Rubidoux facility. We have completed safety tests for three native psyllid species that occur naturally on native California host plants; Heteropsylla sp. on Acacia farnesiana, Heteropsylla texana on Prosopis glandulosa, and Calophya californica on Rhus ovata. None of these native psyllids on their respective host plants were parasitized by T. radiata. Testing of potato, scotch broom, and olive psyllids has been completed also. Scotch broom and olive psyllids were not attacked by T. radiata. A few nymphs of the pestiferous potato psyllid were attacked. We are currently running additional replications of Fremontia psyllids and Rhus psyllids for their suitability as hosts for T. radiata. Upon completion of this last round of tests the host testing of T. radiata should be concluded and the Environment Assessment report will be prepared and presented to USDA-APHIS and NAPPO for review.
Spatial and Temporal Incidence of Ca. Liberibacter in Citrus and Psyllids Detected Using Real Time PCR, April 2011. In 2010, psyllid adults from our HLB negative colony on orange jasmine were caged on new shoots (10-12 adults/shoot/ per tree) that had been caged immediately after trimming and were not exposed to feral psyllid population. On the same tree an additional cage was placed on a previously uncaged shoot that was infested with feral psyllid nymphs. These adults and nymphs remained caged on the shoots for three weeks. Some adults from laboratory reproduced in the cages so when collected there were some nymphs available. Similarly cages with feral populations also had some nymphs and adults at the time of collection. Using PCR, 40 shoots tested HLB negative with average Ct value of 39.8 ‘ 0.07 and 15 shoots tested HLB positive with average Ct value of 27.4 ‘ 0.7. From lab reared adults caged on HLB negative shoots 69 were tested and all were negative. From their nymphal progeny 58 were tested and 1.7% were positive with Ct value 29.8. From the ones caged on HLB positive shoots 51 adults were tested and 12% were positive with average Ct value of 29.6 ‘ 0.7. Among 31 nymphs none were positive. Out of 88 adults that emerged from feral nymphs caged on HLB negative shoots 10% were positive with average Ct value of 28.9 ‘ 0.9. Only 5 nymphs were tested and all were negative. Among 53 adults that developed from feral nymphs caged on HLB positive shoots 9% were positive with average Ct value of 29.6 ‘ 0.9. No nymphs were tested. These colonies were re-treated on April 8, 2011 and titer values and CT values are being calculated to correlate with data from 2010. This data was also reported in April 2011 however the quantification of HLB within the psyllids is still being analyzed therefore results are pending. In April, we completed the third year of a replicated experiment in a 12-acre experiment commercial block of 8-year-old ‘Valencia’ oranges on ‘Swingle’ to test effects of micro-nutrients + systemic acquired resistance inducers, and Asian citrus psyllid (ACP) chemical control on ACP populations on Can. Libericacter asiaticus (CLas) titer, and plant yield. Psyllid populations in the insecticide and insecticide+nutritional plots reached the 0.20 threshold on 22Apr, 6May, and the insecticide+nutritional plot reached threshold on 4June. Insecticide and Insecticide+nutritional had significantly (P< 0.05) less psyllids than control or nutritional only on dates 8Apr, 22Apr, and 7May. All three treatments were significantly (P < 0.05) less than control on 21May, but no differences were found between treatments on 4Jun. Since our last report we have sprayed three applications on the treated plots: Danitol 4EC at 16oz/acre on 15 Mar11, Dibrom 8 at 16oz/acre on 28Apr, and Delegate WG at a rate of 30oz + 2gal/ac 435 oil on 12May. Three years data of sticky card sampling of adult psyllids are now being examined to analyze movement of adult psyllids between treatments. Tap and flush sampling has been modified to include bi-monthly counts of nymphs and eggs on flush to determine any correlation to tap sampling counts. In January, we collected plant samples for HLB detection, and these are still in process. Following the third harvest, fruit samples were sent to the CREC fruit quality laboratory (Apr 11) for analysis of lbs. juice per box, acid, total brix, and ratio. An initial citrus leafminer damage assessment using a modifield Horsfall Barratt scale will be done following summer flush to determine if CLM should be monitored. The objectives of this grant are ahead of schedule since grant was initiated prior to release of grant funds the first year, therefore we have three years of data as of the 2011 harvest.
The objective of this project was to investigate three questions: 1) what is the seasonal pattern of Ca. Liberibacter asiaticus (Las) 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 Las in Diaphorina citri or citrus leaves; and 3) what is the prevalence of Diaphorina citri carrying Las 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 Las 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 ACPs are feeding. This project aims to determine if there is a relationship between the frequency of disease on branches and ACPs. The backlog of plant samples has been cleared and we continue to collect additional samples from 3 groves. Two groves are in Polk County near Lake Alfred (high HLB levels) and Lake Wales (low HLB levels) and the third is in Lake County (moderate HLB levels). The Polk County groves are Hamlin and the 3rd is Early gold. We are restricted in site selection by the presence of significant populations of ACP. In each grove, two hundred trees were selected from 10 consecutive rows. One leaf/tree was randomly picked every fortnight. 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. There were 2 peaks of Las prevalence in the moderately infected grove at approx. 0.50 mid-October 2010 and April 2011, dipping to 0.20 in February 2011. Las prevalence in ACP had a similar pattern but the main peak reached 0.75 in December. In the highly infected grove, there has been a steady increase of Las prevalence in branches since July 2010 until March 2011 where a plateau of 0.75 was reached. There was the same general trend observed with ACP although the prevalence levels were 0.15 to 0.20 higher, reaching 0.90 in March 2011, but there was a large decline in ACP prevalence to 0.60 in May 2011. In the low HLB intensity grove, no Las has been detected in since the initiation of sampling in July 2010. However, very low prevalence has been detected in branches. Prevalence has hovered below 0.02 until April 2011 when it reached 0.06. The phenology data was collected at all sites since November 2010. Flush was highly synchronous in 2011 likely because of the extended cold temperatures this winter and the drought conditions. As would be expected with the very dry conditions, there is currently little new flush on the trees.
A combinatorial peptide library was synthesized by Torrey Pines Institute for Molecular Studies (TPIMS) and tested ‘in-bulk’ by addition to an artificial diet fed to the Asian citrus psyllid. This represented a collection of millions of 10-amino acid peptides (decamers). When the peptides were present in the psyllid diet, there was a clear inability to form normal salivary sheaths by the psyllids. This research is now being expanded to identify which of the peptides is responsible for the salivary sheath inhibition through a combinatorial screening protocol developed by TPIMS. Discovery of a peptide that will block the ability of psyllids to feed on citrus is being pursued in research aimed at developing novel insect control strategies based on either application of the peptides to plants or production of plants that produce this peptide as a resistance mechanism. Furthermore, continued analysis of salivary sheath composition has led to a new hypothesis on how salivary sheaths are synthesized and how their building blocks are recruited for polymerization to form the sheath.
We have completed the second year of our project. Our longterm goal is to identify and then use specific psyllid (B. cockerelli) RNA sequences to induce RNA interference (RNAi) activity in recipient psyllids. As we reported previously, we have cloned midgut sequences from B . cockerelli, and are continuing to evaluate these sequences for their ability to induce detrimental RNAi effects in psyllids. We have evaluated effector RNA sequences by intrathoracic injection (as a positive control), and by two feeding approaches. All methods have shown some potential, depending on the sequences evaluated. In vitro-generated double-stranded dsRNAs were evaluated via micro-manipulator-driven intra-thoracic injection (200nL/psyllid). This is a tedious process but is used routinely to induce whole insect RNAi effects for many different insect types. Increased mortality was observed for actin dsRNA in our experiments when compared with the control GFP dsRNA. Injection of an ATPase specific dsRNA also consistently suppressed the endogenous ATPase mRNA expression by 30% in independent experiments. We also identified specific siRNAs, hallmarks of RNAi activity in recipient insect. Taken together, these data demonstrated that psyllids are susceptible to RNAi. We developed a robust and effective in vitro feeding system for B. cockerelli. We have used this artificial diet system for screening 18 candidate sequences (as dsRNAs and siRNAs) for B. cockerelli. Four out of 18 sequences, including the homologues of actin, ATPase, Hsp70 and CLIC, caused substantial psyllid mortality as compared with GFP dsRNA controls. Consistent and specific down-regulation of endogenous mRNAs also was revealed by qRT-PCR, and corresponding siRNAs were detected by Northern blot analysis. These latter two analyses are important and demonstrate that the orally-induced mortality is not an artifact, but due to RNAi. We also constructed an artificial chimeric sequence for actin, ATPase, Hsp70 and CLIC. Oral feeding of dsRNAs for this chimera sequence resulted in significantly higher mortality compared with the dsRNA of any of the individual sequence, demonstrating possible synergistic effects of these sequences on psyllid survival. We are also using means for more rapid evaluation of RNAi effects in plants by using plant virus-based expression systems. We have used a Tobacco mosaic virus (TMV)-based plant expression system to express B. cockerelli sequences in tomatoes. However, TMV systemic expression of insert sequences requires approximately 3-4 weeks and resulting infections are non-uniform within tomato plants. We have now evaluated different virus vectors and host plants, including TMV, Tobacco rattle virus (TRV) and Potato virus X (PVX) in different host species including tomatoes, tobacco, Datura, tomatillo and Nicotiana clevelandii, all of which are good host plants for B. cockerelli. TMV systemic infections in tomatillo develop very rapidly, within one week and the tomato psyllid readily feeds on tomatillo. TRV and PVX both also offer opportunities in other plant species. The combination of viruses and plants will allow us to rapidly test RNAi activities in plants.In summary, we illustrated the efficacy of RNAi silencing in the tomato psyllid and selected candidate sequences for RNA interference using two feeding systems, and efforts for plant virus-based and whole plant transgenic expression systems are underway.
In the previous study, imidacloprid, 0.35 g AI/plant, and thiamethoxam, 0.25 g AI/plant, applied in the nursery tree bags, before planting, were efficient to control Asian Citrus Psyllid (ACP), Diaphorina citri, until 60 days after application (DAA). The time to cause 100% of ACP mortality was between 5 to 7 days after the confinement of adults in treated plants. The first experiment was performed to determine if the systemic insecticides are effective until 90 days after application in nursery trees and if this application is effective to prevent transmission of the bacteria. In this experiment, the time to reach 100% of the adult ACP mortality ranged from 3 to 7 days for both systemic insecticides tested (imidacloprid and thiamethoxam), in the same doses of the previous experiment. The insecticides were effective up to 90 DAA. The results of PCR carried out for ACP, 15 and 30 days after application, were positive for 100% of the samples, consisting of 10 insects tested, but in the confinement held at 46 DAA, in any sample was detected the presence of the bacteria. No acquisition in this period. In bioassays performed at 75 and 90 DAA, the percentage of positive samples was from 50 to 70% and from 10 to 40%, respectively. 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 was 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%). The second experiment was conducted varying the doses of the systemic insecticides and confinement of the ACP in plants treated only 7 DAA. For thiamethoxam, the doses tested were: 1.0, 0.5, 0.1 and 0.05 g/nursery tree nursery and for imidacloprid were: 1.75, 0.9, 0.2 and 0.08 ml/nursery tree. For both insecticides, the control was more efficient in the two highest doses (mortality over 85%). The percentage of ACP positive samples ranged from 60 to 100%. As in the first experiment, there were no transmission results. In the third experiment, were tested different foliar spray insecticide to determine if they are effective to prevent the transmission and for how long. Except for etofenprox, all other foliar applied insecticides were effective in controlling adults of ACP, with variable control period. The last experiment was performed to evaluate the effect of mineral oil (MO) on the feeding behavior of ACP and its effect on the vector repellency. The results showed that up to 21 days after application, mineral oil, 1.5% and 1.0%, shows repellent effect on adults of D. citri, which prefer oil-free leave flush for oviposition. Using electrical penetration graphs (EPG) techniques showed that plants sprayed with insecticides and MO affects mainly the initial phase of probing (pathway). In the case of foliar insecticides, ACP was able to perform probes 1 DAA, but it was able to reach the phloem only in the evaluation of 15 DAA. However, on plants treated with MO around 32% of insects were able to reach the phloem 1 DAA. We still do not consider that the project is finished. To finish the project we need an extension of one year to obtain such data.
During this 2nd year, data were collected from field experiment 1 (E1) until Nov/11 when all trees from experimental area were eradicated and E1 finished. The results from E1 have been compared with the results of other similar experiment (E 2) where HLB epidemics will be followed until 2012. While E1 have been carried out in a region with high external inoculum pressure because of its proximity with groves without HLB management, E2 is in a region with lower external inoculum pressure, located in the middle of a large farm with regional disease management (4 to 14 annual insecticide applications and 3 to 4 annual HLB-symptomatic trees eradication in the area up to 2 km surrounded the experimental area). The main results so far indicate that area-wild management of HLB (AWM) is essential to control the disease: i) The disease epidemics started 299 days later in area under AWM. In E1 the disease epidemics started in average about 587 days after planting, while in E2 it started later, 886 days after planting. ii) The disease epidemics were slower in area under AWM. The annual disease progress rate estimated by Gompertz model reduced 75% (1.18 for E1 and 0.30 for E2) and the cumulative incidence of HLB-symptomatic trees was reduced in 91% (E1 ‘ 76.9% and 53.1% and E2 ‘ 6.3% and 4.6% for plots without and with ACP control, respectively). iii) The local program for ACP control in E1 was efficient to reduce the number of adult psyllids captured on yellow stick traps in 81% and to reduce the number of eggs and nymphs observed on new shoots in 95%. However this local vector control reduced HLB-incidence in only 31%. iv) AWM of ACP increased the efficiency of local control measures because reduces the migrant psyllid population. The cumulative number of caught ACP adults/trap/assessment in plots without vector control was 0.46 and 0.12 for E1 and E2; and ACP population is decreased year after year even in plots without insecticide application). v) Differences on the time that a symptomatic tree remained exposed in the field before be eliminated were observed among different frequencies of local inoculum reduction. However, such differences did not result in significant differences on HLB progress rate and final HLB incidence among different frequencies of local inoculum reduction in both experiments. vi) AWM of bacterial inoculum (symptomatic trees) reduced the migrant infective psyllid population that reaches the local plots. For E1, in average, 5% of all psyllids captured on yellow stick traps of from the winter/07 to spring/10 tested positive for the presence of Candidatus Liberibacter asiaticus by conventional PCR. However, no psyllid sample was positive for CLas in E2. Infective ACPs were found both in plots with and without vector control program. This result could explain why even with similar population of adults ACP in plots with vector control (0.08 in E1 and 0.09 in E2), the incidence of HLB-symptomatic trees were much lower in vector controlled plots of E2 (53.1% in E1 and 4.6% in E2). vii) AWM reduced the control costs because with less intensive vector control program (insecticide sprays every 28 days, or even no insecticide sprays) it was possible to be more efficient than a high intensive vector control program (insecticide sprays every 14 days) applied locally within a region without regional management. viii) AWM allowed solid block replanting with the guarantee of future yield. In plots of E2 without and with vector control the yield still increasing (2 ton/ha in the 1st, 16 in the 2nd and 38 in the 3rd harvest) while in plots of E1 the yield decreased year after year (2 ton/ha in the 1st, 9 in the 2nd and 10 in the 3rd harvest for plots with insecticide application, and 2 ton/ha in the 1st, 4 in the 2nd and 3 in the 3rd harvest for plots without insecticide application).A paper with the temporal analysis of both experiments is on preparation. The results of this project were also being presented to growers to help the establishment of growers’ groups for AWM.
1) Evaluation of screens impregnated with insecticide barriers. As explained in the previous report, the experiment is being conducted on two farms located in S’o Paulo. In the farm located in S’o Manuel, the experiment was installed on 12/14/2010 and nine evaluations were performed. In the area with insecticide barrier net, no psyllids were collected. However, in the area without the barrier, the capture of adults of ACP was also low, with only two individuals collected. In Descalvado, the experiment was started on 02/08/2011 and was performed four evaluations. In this area the capture was also very low and only one psyllid was collected in the area with barrier. The low ACP capture observed in both properties, probably is related to the large amount of rainfall that occurred during the evaluations, which may have interfered in the dispersal of D. citri in citrus groves in the neighboring properties. 2) Evaluate the impact of treatment of plants with systemic insecticides on the transmission of Ca. L. asiaticus by starved psyllids. The citrus trees that will be used in this experiment have already been purchased and will be delivered to Fundecitrus in early June, in the same month, the experiment will be installed. 3) Evaluation of the effectiveness of systemic insecticides and extraction of the sap of the phloem and xylem. This will be installed in the spring (outubro/2011), favorable time for absorption and translocation of systemic insecticides on citrus plants. The equipment for this experiment are being purchased.
Exp.1 ‘ During this second year project we just monthly looked for HLB symptoms and infection by PCR analysis. In the compartments where citrus plants served as primary source of inoculum of CLas or CLam we observed a predominance of infective psyllids and diseased plants with positive conventional PCR for CLas. In average 30.4% of psyllids collected from inoculum source plants and 8.4% collected from test plants were positive for CLas and no one for CLam. In average 14.4% of test plant were positive for CLas and 1.9% for CLam. For three Clam citrus infected plants, two had also CLas in a mix infection. Average Ct value for leaves with CLas were 24.7 and for CLam 33.7, indicating a higher titer for CLas than for CLam. In compartment where Murraya exotica served as source of inoculum, 46.0% and 9.4% of psyllids collected respectively in source plants and test plants were PCR positive for CLam, whereas in compartment where Citrus sinensis served as source of inoculum, 6.35 and 4.7% of psyllids collected respectively in source plants and test plants were PCR positive for CLam. 8.2% of test plants were PCR positive for CLam when inoculum source was Murraya exotica and up to now no infected test plant was detected when citrus plants served as primary source of inoculum of CLam. For this experiment, symptoms observation and leaf sampling for PCR analysis will be done at least until Dec/11. New inoculum source Citrus and Murraya plants were prepared to repeat this experiment. Because we were not successful to obtain Murraya infected with CLas after many attempts by grafting or natural inoculations, we decided to replicate the first experiment with the same treatments; however we used higher and older source of inoculum plants and 100 test plants per compartment. The replication was started in Jan/11 with the first release of HLB-free ACPs. Exp. 2- Emerging ACP adults from nymphs reared on inoculum sources plants were used for inoculation in Mar/10. Until now no symptomatic test plant was observed. Exp. 3 ‘ During this year many problems with inoculum sources, ACP rearing, bacterial acquisition and screenhouse wind damages made impossible the re-inoculation of the three screenhouse protected sweet orange cultivars (Hamlin, Pera and Valencia) at different ages (planted in 1999, 2004 and 2006) with CLas or CLam by D. citri. Monthly assessments for symptom severity have been done, but no HLB-symptoms were observed yet on inoculated trees. Also, leaf samples have been collected to detect the presence of Liberibacter species on inoculated shoot. New inoculation of these trees is planned in Jun/11. Some symptomatic trees that appeared before the inoculation were maintained in the screenhouse and the severity progress was assessed during last year project. For Hamlin/Rangpur 99, the annual severity increased from 24.3 to 27.5% (average of 7 trees); for Valencia/Rangpur 99, from 24.5 to 38.5% (average of 5 trees); and for Hamlin/Rangpur 04, from 35.0 to 57.5% (average of 2 trees). As an alternative for this experiment, 30 trees from each five citrus blocks at different scion/rootstock combinations (Hamlin/Swingle 07, Valencia/Volkamer 07, Valencia/Swingle 07, Valencia/Swingle 99, Valencia/Swingle 95), showing very low symptoms severity, were selected in Jun/10 in farms without removal of symptomatic trees but with strong ACP control program. After one year, the disease severity increased in average from 6.2 to 7.5% for Val/Sw 95, from 4.6 to 8.5% for Val/Sw 99, from 7.9 to 15.3% for Val/Sw 07, from 8.6 to 19.3% for Val/Volk 07, and from 12.8 to 19.2% for Ham/Sw 07. As expected, the disease severity progress is faster in younger trees. Assessments of disease severity and yield (healthy versus diseased trees) will be continued for more years to allow modeling of disease severity progress.
During the first year of our CRDF funded project, we have demonstrated that we currently have the tools necessary to greatly reduce the likelihood of young trees from becoming infected with HLB; the focus now is to determine the most cost-effective (and practical) manner for Florida citrus growers to achieve this goal in order to maintain the long-term viability of the Florida citrus industry. During 2010, we began by evaluating the most effective way to protect young trees from psyllids using soil-applied systemic insecticides. In these evaluations we used three different active ingredients, imidacloprid (Admire Pro), thiamethoxam (Platinum) and clothianidin (Belay). While most growers have had extensive experience with imidacloprid products, thiamethoxam was labeled for use in citrus in 2009 and clothianidin was registered for use on non-bearing citrus most recently in 2011. Due to the differences in the water solubility of these products (differences in availability and rate of uptake by the citrus root system), and our past success using these products via different application methods, we examined three different application methods for the most effective use of these products; soil drench (standard), soil band spray, and trunk application. The use of trunk applications was examined as a result of reports from South Africa touting trunk applications of imidacloprid (Confidor) as providing several MONTHS of residual control of the African citrus psyllid. In our trial work, we obtained the South African formulation (Confidor) and compared the efficacy of that product, using rates typical to those applied in S.A., to the products available for use in the U.S. For all three active ingredients and application methods evaluated, soil drench and trunk applications were most effective and consistent. Regardless of the particular product used, our results suggest that Florida citrus growers using soil-band applications of any of these three neonicotinoids will likely experience significant variation in psyllid control (failures) on a tree-by-tree basis and thus should not use this application method until further work on application volumes and subsequent irrigation practices can be investigated to make this method of application more reliable. While Florida citrus growers now have 3 “neonics” that can provide nearly season-long control of psyllids, additional control methods are needed to prevent HLB infection as neonic levels decline between applications and also to minimize the likelihood of resistance to this important group of pesticides that can be used for young tree protection. [Ongoing work in our CRDF funded project Huanglongbing: Understanding the vector-pathogen interaction… has shown that all 3 of these neonics can disrupt psyllid feeding behavior such that pathogen transmission is unlikley to occur.] As part of the overall goal of the current project, we are investigating the use of products that could potentially deter psyllid feeding in addition to that provided by systemic neonic products, particularly between neonic applications. The results of choice and no-choice experiments in the laboratory have demonstrated that the use of our primary psyllid repellent of choice can provide relatively extended and efficient reduction in psyllid feeding. These results have been further confirmed using EPG monitors to demonstrate a reduction in psyllid (phloem) feeding behaviors responsible for successful transmission of the HLB pathogen. This work is being continued in 2011 in field studies investigating these methods and the benefit of supplemental foliar sprays of different modes of action for protection young trees from HLB infection prior to reaching fruit-bearing age.
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. Data analysis of the amino acids has not progressed as quickly as we had anticipated, but we will have them complete by the end of the project year. Results of this study will be used to develop further studies to determine the basis for the observed effects to be conducted in both field and greenhouse studies this summer. 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. We are sampling these trees periodically for confirmation of Las transmission by PCR. Psyllid acquisition studies will proceed as soon as the new plant material has been successfully infected. We anticipate that occurring during the next quarter. 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. This trial will begin in mid-June or at the onset of the rainy season and summer flush development.
Oviposition and nymphal development experiments were conducted that included topical applications of dsRNA targeting specific psyllid genes. Elevated psyllid nymphal mortality was observed in plants on which dsRNA targeting distinct psyllid genes was applied to the soil of small potted citrus plants. This mortality was seen in the early instars but not later in nymphal development. Ultimately, adult psyllids did emerge from the surviving nymphs, but the number of emerging adults was reduced significantly (greater than 60%) as compared to plants either treated with water or dsRNA with a sequence that did not match any known psyllid gene. These experiments are being repeated. Continued bioinformatic analysis of psyllid transcriptome response to dsRNA feeding shows the major perturbations in the digestive tract gene regulation are in the pathways related to the genes being targeted. Observed changes include the apparent up-regulation of genes in the same pathway as the targeted gene showing how the cells are trying to compensate for the loss of function of this pathway due to dsRNA inhibition. Finally, field experiments have been initiated to test the effectiveness of topically applied dsRNA as a psyllid control strategy. Trees have been treated and performance is being evaluated.
The data pertaining to low volume field trials of the 2009 and 2010 seasons were analyzed to establish the relationship between the weather parameters and efficacy of the Asian citrus psyllid (ACP) control. Throughout the trials, application time wind speed varied from 0.9 ‘ 5.9 m/s and relative humidity from 87.6 to 91.3%. Mortality of the adult ACP at day-3 was not correlated with the humidity, but was significantly correlated with humidity at day-28, i.e., higher humidity resulted in better residual ACP control. Considering both the day-3 and day-28 efficacy results, wind speed did not appear to have a meaningful correlation with ACP control. Furthermore, the control of ACP nymphs was not affected significantly by the weather parameters. Air temperature at the time of application did not have a significant effect on mortality of either nymph or adult ACP. For the five top ranked treatments, the lowest relative humidity was 91.5% and the highest wind speed 1.8 m/s. The most effective application against both life stages occurred during meteorological conditions of 94.5% relative humidity and 0.9 m/s ambient wind speed. Data analysis will be continued to investigate the effect of post-treatment weather conditions on residual control of psyllids.
This project was designed to examine the potential disease control of citrus huanglongbing (HLB) by interplanting citrus with guava. In Vietnam guava has been shown to be an effective deterrent to HLB, slowing the disease and keeping plantings alive for up to 15 years that normally succumb in 2-3 year. For all plots and experiments, Guava trees, (Vietnamese white cultivar) were propagated and grown to appropriate size requiring ~1 year. Both nursery and field citrus trees are assayed for HLB every 60 days, and have been assayed multiple times. Psyllid populations are monitored continuously every 2 weeks to document repulsion of the vector. Results: Guava vs no guava nurseries: Two nursery sites, a guava protected citrus nursery versus an unprotected nursery, have were established with disease free, PCR-negative citrus trees (2 sweet orange and 1 grapefruit cultivars) in June 2009 and were located in the protected and unprotected plots. The guava trees were grown to appropriate size as indicated in Vietnam prior to outplanintg. To date HLB appears to be progressing more slowly in nursery plots interplanted with guava than in non interplanted plots. The freezes discussed below did not adversely affect these nursery plots. Citrus/guava interplantings: 2 commercial plantings with multiple replications were established but due to freezes and property sales these plantings are no longer viable. A third trial planting was established at the USHRL Picos Farm in Fort Pierce. The Picos plot was interplanted with citrus in August 2009. Severe frosts during 2008/2009 and again during 2009/2010 winters affected the USHRL plots and caused a delay in the experiment. A final hard freeze during the 2010/2011 season killed most of the guava trees. Data analysis to date indicates no differences were observed among treatments, i.e., guava interplanted vs. non-interplanted plots prior to the final demise of the plots. Our interpretation is that Florida is actually a subtropical environment, prone to intermittent freezes and cool or cold temperatures. Whereas, Vietnam and Indonesia, where the guava effect seems to work, are truly tropical without such broad temperature swings. After freezes it takes a considerable time to either replant guava or for the freeze damage guava to recover. Even during cool weather guava trees are very sensitive and do not continue to flourish and grow. It is the new flush of guava which appears to be the best at producing ACP repellent volatiles. Cool or freezing temperatures inhibits volatile production and thus the citrus crop is left unprotected from ACP. While guava not be a viable deterrent as an intercrop, it still may be possible to identify individual volatiles from guava that might be useful under field applications as chemical applications. We are now switching direction slightly to investigate other more temperate Myrtaceous plant species that are more cold tolerant and might be useful as intercrops. Research continues using a Y-tube olfactometer to continue to investigate guava volatiles as repellents of the psyllid as well as to investigate the feasibility of other Myrtaceous plants.
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