Cultural Practices to Prolong productive Life of HLB Infected Trees. Through the summer of 2009 trees receiving the nutritional/SAR trees were vigorous and green with only normal HLB symptoms typical of HLB infected trees. This fall of 2009 at our 100% infected trial site we have observed numerous visual HLB symptoms in multiple sectors of the trees. Although our other two sites with lower infection rates of 50% and 15-20% had increased visual symptoms of HLB this fall and winter typical of greening trees, the symptomatic leaves were not as abundant as in the more infected trees. Fruit color break from green to yellow in Valencia oranges began early (late September) at the site with 100% infected trees and was not observed at the two other sites where fruit remained green as typical in uninfected trees. Fruit drop has been observed this fall only in the 100% infected trial. Data collected show treatments with the complete Maury Boyd cocktail, with or without hydrogen peroxide, and the foliar feed macronutrients in a typical range of 2 to 5% fruit drop. The other treatments ranged from 8 to 17% fruit drop. No abnormal coloring of fruit or fruit drop has been recorded in sites with 40-50% or 15-20% HLB infection. Evaluation of Systemic Acquired Resistance Inducers Combined with Psyllid Control to Manage Greening infected Groves We are monitoring Asian citrus psyllid (ACP) populations and Can. Libericacter asiaticus (CLas) titer in plants and psyllids to evaluate main effects and interactions from a 2×2 factorial experiment with with 4 replications and 4 treatments: (1,2) (micronutrients + systemic acquired resistance inducers (Micro+SAR) with and without insecticidal control (3) insecticidal control alone, and (4) untreated. The trial is being conducted on a 12-acre commercial block of now 7-year-old ‘Valencia’ oranges on ‘Swingle’ rootstock that was held back by defoliation for canker control in 2006. Adult ACP populations have been maintained three times lower on average and up to 50 times lower on occasions in insecticide-treated plots compared to untreated plots throughout both years of the experiment. Insecticides have been applied 6 times during that period, either as a single scheduled dormant spray in winter or when adult populations exceeded a threshold of 0.5 per biweekly ‘tap’ sample made by striking a randomly selected limb while holding a laminated sheet underneath to catch fallen psyllids. ACP adults and nymphs have been collected 4 times since Nov 2008 for PCR analysis along with a leaf sample from the most symptomatic branch of every 5th tree. Insect samples were sent to our collaborators at USDA Riverside CA for analysis and plant tissue samples were processed at the PCR laboratory at SWFREC. In November 2008 and April 2009, 34% of the plants were positive for HLB over all treatments using a cutoff Ct value of 36. Incidence of HLB jumped to 85 – 100% positive in Aug. 2009. However, the two treatments employing insecticidal control had significantly higher Ct values (28.5’0.7) than the two non-insecticide treatments (26.4’0.6) indicating lower CLas titer in plants protected by insecticides. All fruit was harvested in Mar 2009 and yield was assessed by plot. Despite the high percentage of infected trees, those receiving both insecticidal and Micro+SAR produced 1.32’0.15 boxes per tree of fruit, a 30+% increase over 0.95 to 0.99 boxes per tree from the other 3 treatments. An analysis by the CREC pilot plant in Lake Alfred found no significant treatment effects on fruit or juice quality. These results could be interpreted to mean that the Micro+SAR package is capable of staving off negative impacts of HLB if CLas titer can be maintained below some threshold level through psyllid management. However, we will not begin to feel comfortable with this conclusion until we see similar encouraging results from the spring 2010 harvest.
During this reporting period Dr. Olga Minenkova of Sigma Tau pharmaceuticals (Rome, Italy) spent 2 weeks in our laboratory with Dr. Yuan and initiated scFv antibody protocols using advanced phage display technologies. This included transfer of the specialized cloning vector and E. coli strains, preparation of competent cells, purification of mRNA, construction of the cDNA library, cloning, and trouble shooting and validation of experimental protocols. As a proof of concept experiment we used Xylella fastidiosa 9a5c (citrus variegated chlorosis) mixed with psyllids to immunize mice. Psyllids were raised and provided by Drs. Brlansky and Damsteegt. Xylella fastidiosa 9a5c was added to psyllids at ~200,000,000 cells per injection and ground in extraction buffer to mimic subsequent injections with psyllids infected with ‘Ca. Liberibacter asiaticus’ from Florida. Mice were sacrificed and mRNA from spleens were used to create the phage library. The library was screened 3 times against X. fastidiosa 9a5c with excellent results. Several antibodies have been identified that react strongly against X. fastidiosa 9a5c but do not react at all against strains of X. fastidosa that cause Pierce’s disease. This is a significant ‘bonus’ result that will benefit both the citrus and grape industries, and demonstrates that our basic approach for making antibodies against ‘Ca. Liberibacter asiaticus’ will be successful. Additional screening will be done by Dr. Nandlal Choudhary in Dr. Brlansky’s lab in Florida using ELISA, western blots, tissue blots and immunogold labeling. While the phage libraries against X. fastidiosa were being screened, mice were injected 3 times over a period of three weeks with psyllid extracts containing ‘Ca. Liberibacter asiaticus’ at ~ 200,000,000 cells per insect. The mice will receive 1-2 more injections and will then be sacrificed for production of both scFv (Beltsville) and monoclonal (Agdia) antibodies. In order to prepare injections of mice, more than 500 psyllids have been individually ground in buffer and sampled by Q-PCR to quantify the amount of ‘Ca. Liberibacter asiaticus’ present in each insect. This is necessary to identify the insects with the highest titer of pathogen (~ top 2% of insects) so that each injection contains enough Ca. Liberibacter asiaticus to induce an antibody response. Symptomatic plants or fruits with the highest titers will be used to screen the phage display library for antibodies against Ca. Liberibacter asiaticus’. HLB plants in our greenhouse were individually assayed for titer of ‘Ca. Liberibacter asiaticus’ to be used in the screening of the phage display libraries. HLB symptomatic fruit have been provided by Dr. Brlansky and will be assayed individually to identify sources of extracts to be used in screening the libraries. In related work the outer membrane protein (OMP) of ‘Ca. Liberibacter asiaticus’ has been expressed and purified in the laboratory. This protein has been provided to Cocalico laboratories and they are currently raising polyclonal antibodies against this protein in rabbits. These rabbit antibodies will be useful to capture ‘Ca. Liberibacter asiaticus’ in future experiments that will use the mouse scFv or monoclonal antibodies for specific detection and quantification.
A major objective of this proposal is developing a rapid method of detection of HLB bacterium (Las) in citrus trees using non-radioactive Las specific nucleic acid probes. Towards this end, we developed specific PCR probes corresponding to the outer membrane protein (OMP), RNA polymerase beta subunit, DNA polymerase region, the r-DNA region, and the 23S and 16S ribosomal RNA inter-genic regions of the HLB pathogen. Citrus plants from the green house infected with HLB pathogen were used as the source plants for obtaining the needed tissue for the blots and to isolate nucleic acid template necessary for the amplification of Las specific DNA. In the initial tissue blot experiments we did not observe hybridization signals specific for HLB. We used healthy plants grown under similar conditions as controls. The reason probably is the low titer of pathogen and/or the non uniform distribution of the pathogen in the infected tissues. It is also possible that the PCR probes were not of sufficient specific activity so as to detect low titer of the pathogen. Therefore, the amplified regions of Las were also cloned in the transcription vector, and digoxigenin labeled strand specific RNA probes were generated by transcription. However, use of the high specific activity RNA probes did not improve hybridization of the tissue blot and occasionally non specific hybridization was also observed in healthy tissue blots. This suggested less than optimal hybridization regimen. We have subsequently focused on optimizing the hybridization conditions and use of multiple probes in an effort to increase the extent of signal. These changes have substantially improved detection of Las in citrus tissues. In addition to tissue blots of the stem sections, we have used the midrib and petiole region imprints from the leaves of infected citrus on nylon membranes. The hybridization observed with the midrib imprints showed much clear signals compared to stem imprints. In forthcoming experiments we will use imprints of the leaf midrib, petiole and stem from new flushes of infected citrus since the psyllids preferentially feed and acquire the pathogen from such tissues. We also imprint on the membrane the inside surface of the bark which contains phloem tissue in which the HLB pathogen is located. Although this procedure is not optimized, we believe this to be very useful and the distribution of Las in infected citrus tissue could be easily documented. The second area of our focus is on the detection of Las in psyllid vector by tissue blots (squash blot) on nylon membranes. At the outset, a procedure for isolating the nucleic acid from single psyllid was optimized, and we have been able to amplify Las specific amplicons from single infected psyllids using pairs of Las specific primers. Conditions of amplifications were optimized with different primer pairs and now we have been able to amplify HLB specific amplicons without non-specific bands in PCR. In dot blot hybridizations using the known amount of Las specific DNA from a psyllids we observed a linear relationship with input DNA and the degree of hybridization with probes from OMP and r-DNA. In initial studies of whole psyllid tissue blots, hybridization signal was also observed with healthy psyllids (psyllid colony from the healthy psyllid containment facility). However use of specific primer pair corresponding to the EFTU gene of Las has been promising and we will use the probe generated for this gene in tissue blots of psyllids henceforth. The observed background was probably due to the extraneous tissues of the psyllids like wings that attach to the nylon membranes during psyllid squash and very hard to exclude during membrane wash. Therefore we intend to separate the head and the abdomen regions of the psyllids in tissue blots and exclude the wings region to reduce the background signal. We will use the results of this technology to test the progression of Las spread in citrus and infection ratios in populations of psyllids in the field.
Transmission of a pure culture of ‘Candidatus Liberibacter asiaticus’ (Las) by citrus psyllids into susceptible sweet orange plants is the initial step in completing Koch’s Postulates. This project uses two mechanisms to introduce Las into the psyllids, membrane feeding and microinjection. We have done a number of experiments where Asian citrus psyllids were fed on sachets containing media with pure culture Las. The success rate of these experiments has been very low, but we report that we have successfully transmitted pure culture Las into sweet orange using psyllids. The infected trees are positive by real-time PCR, three months after inoculation, but do not show significant symptom development at this point. We are currently working to confirm the presence of Las in the inoculated trees using multiple methods including sequencing, but are reasonably confident that this represents the first success in the alternative confirmation of Koch’s postulates via transmission by vector. In addition, we have begun conducting microinjections, beginning with control experiments where healthy psyllids were injected with hemocoel from Las infected psyllids. The microinjections were successful at multiple levels, as the injected psyllids survived, tested positive for Las, and were able to transmit the Las to healthy sweet orange seedlings. We plan to move forward with micro-injection of pure cultured Las, but at this point we have great difficulty obtaining adequate pure cultures and we have begun efforts to grow our own live, pure cultures of Las using various techniques.
A major objective of this project is to develop an understanding of how the HLB bacterium (Las) interacts with citrus genotypes to cause disease. After finding that different citrus genotypes respond differently to Las from extremely sensitive (sweet orange and grapefruit) to tolerance with minor symptoms, we have focused on the one citrus genotype that is most resistant to citrus. Las is restricted to very low levels in Poncirus trifoliata. Most plants remain PCR negative, but a few have barely detectable levels of Las. We are determining whether this is due to plant genetics, Las variation, or randomness. Some Poncirus hybrids are more susceptible than others suggesting that resistance to Las is segregating. We are beginning experiments to map citrus genes that provide Las resistance. Las also appears to have difficulty spreading in Poncirus. We are examining the value of using Poncirus rootstocks and interstocks to reduce or prevent spread of the disease in sweet orange or grapefruit. We have developed a containment plant growth room to examine natural infection of citrus trees by psyllid inoculation. We already have made several significant observations: First, we have found that the time period between when plants first become exposed to infected psyllids and the time that new psyllids can acquire Las for those plants can be as little as 6 weeks. We are examining this process in more detail now. Second, when we allowed the infected psyllids a choice of different citrus genotypes, there was a large difference in the time and number of plants that were inoculated by the psyllids: (Citrus macrophylla >> Swingle citrumelo >> Volkamer lemon = Duncan grapefruit > Madam Vinous sweet orange >> Carrizo citrange). Most of the Citrus macrophylla plants became infected with only 2 months of exposure in the epidemic room, whereas only a few of the sweet orange and grapefruit became infected after 4 months. Since there was such a clear preference, we are now investigating its cause ‘ whether the preference is related to genotype, growth habit, flushing, or other possible differences. It is clear that psyllids reproduce on new flush, but feed on older leaves. We are examining whether and how well the psyllid can transmit the disease in the absence of flush. Third, these results have led to the development of methods to greatly speed up results of field tests for transgenic or other citrus trees or trees being protected by the CTV vector plus antibacterial or antipsyllid genes. In order to interpret results of a field test, most control trees need to become diseased. Under natural field pressure in areas in which USDA APHIS will allow field tests, this level of infection could take 2-3 years. By allowing the trees to become adequately inoculated by infected psyllids in a containment facility, we can create the level of inoculation that would naturally occur in the field within 2-3 years in 2-5 months in the containment room, after which the trees are moved to the field test site. Another objective is to provide knowledge and resources to support and foster research in other laboratories. A substantial number of funded projects in other labs are based on our research and reagents. We supply infected psyllids to Mike Davis’s lab for culturing of Las and Kirsten Pelz-Stelinski’s lab for psyllid transmission experiments. Among the plants being screened for resistance or tolerance to HLB for other labs are: 1) a series of elite lines for the citrus improvement group; 2) a series of transgenic plants designed to examine the relationship of pectin production to disease development for Jude Grosser, Gene Albrigo, and Nian Wang; 3) we are testing a series of transgenic plants that we made in collaboration with Zhonglin Mou to have increased disease resistance. The trees, which have high resistance to citrus canker, are presently being tested against HLB; and, 4) a series of lemonine trees reported to be resistant to HLB for Gene Albrigo.
This is a continuing project to find an interim control measure to allow the citrus industry to survive until resistant or tolerant trees are available. We are approaching this problem in two ways. First, we are attempting to find products that will control the greening bacterium in citrus trees. We have chosen initially to focus on antibacterial peptides because they represent one of the few choices available for this time frame. We also are testing some possible anti-psyllid genes. Second, we are developing virus vectors based on CTV to effectively express the antibacterial genes in trees in the field as an interim measure until transgenic trees are available. We think that this approach could be used beginning 2-3 years from now and until probably 15 years from now when resistant trees should be available. With effective antibacterial or antipsyllid genes, this will allow protection of young trees for perhaps the first ten years with only pre-HLB control measures. Third, we are examining the possibility of using the CTV vector to express antibacterial peptides to treat trees in the field that are already infected with HLB. With effective anti-Las genes, the vector should be able to prevent further multiplication and spread of the bacterium in infected trees and allow them to recover. We have completed several large screenings of antibacterial peptides against Las in sweet orange trees. About 40 different antibacterial peptides have been tested in trees. We initially found three peptides that allow much better growth of trees that were grafted with HLB-infected buds. Some trees had no symptoms and no detectable Las, some trees had no symptoms and low levels of Las, and other trees had leaf symptoms but continued growth of the trees with normal levels of Las. Another result is that we found that leader peptides for the export of the peptide from the CTV-infected cell is not needed for HLB but is needed for citrus canker. Because we were concerned that graft inoculation of HLB into the trunks of small trees is a too severe challenge that might cause peptides that could work in the field to be missed, we developed a system that only allows inoculation by infected psyllids. We have established a containment plant growth room in which psyllids inoculate the plants expressing the peptides. These experiments are on-going, but we appear to have several more peptides that are protecting sweet orange. To speed up the search for effective anti-HLB genes, Falk (UC Davis) has developed a tobacco-tomato psyllid/liberibacter model to screen for effective genes against the similar bacterium. This system is working and screening is on-going. We also are improving the CTV-based vector to be able to produce 2-5 peptides at the same time. This will allow expression of genes against both HLB and canker or multiple of genes against HLB. We have developed a vector that can be re-added to trees if the anti-Las gene is lost or a better gene becomes available. A major objective that we are pursuing is to make a vector that cannot be transmitted by aphids. Another major goal is to do a field test of the CTV vector with antibacterial peptides, which is an initial step in obtaining EPA and FDA approval for use in the field. We have received permission from USDA APHIS for the field test, but were delayed by EPA. We are now submitting a revised application to USDA APHIS to include EPA requirements and are expecting to establish the field test this spring. In addition, we are screening a series of transgenic sweet orange and grapefruit expressing antibacterial genes for Erik Mirkov of Texas A&M and Mike Irey of Southern Gardens.
Objective 1: Sprays of copper (Cu) formulations, containing copper hydroxide (Kocide, Champ, Kentan, Badge) or copper sulfate (Cuprofix), were moderately to highly effective for control of canker on fruit of susceptible Ruby red grapefruit and Hamlin orange in the lack of high disease pressure due to early and late season wind-blown rain storms in 2009. A chelated Cu (Magna-Bon, copper pentahydrate) at a 50% lower rate of Cu per application than standard Cu formulations performed as well for reducing fruit disease incidence for grapefruit or canker-induced fruit drop for Hamlin. Early season infection and fruit drop of grapefruit and Hamlin was minimal because April was relatively dry and Cu treatments were initiated before significant rainfall occurred in May. Cumulative fruit drop due to early season infection of untreated Hamlin amounted to about 5%. Five sprays of Cu formulations reduced the incidence fruit drop due to canker by about 40-50% to 2.5% cumulative fruit loss. Objective 2: In Marsh grapefruit, canker control increased with number of Cu sprays from 3 to 11 (April to October), canker infection and copper burn occurred after rains commenced in July. In August, fruit were growing most rapidly which would produce a thinning of fruit cuticle and an increase in the rate that new stomates open due to more rapid expansion of the fruit surface. Season-long copper spray also gave the best control of late season scab and melanose on fruit. In Hamlin, sprays beyond mid-July provided additional canker control of fruit drop confirming that late season lesions do not cause fruit drop like early season lesions. Objective 3: In two grapefruit trials, Firewall (streptomycin[Sm]) applied alone or in combination with a reduced rate of Kocide 3000 in July and early August gave equivalent control on grapefruit to Kocide alone. The adjuvant, Polymer Delivery System (PDS) did not increase the residual activity of Cu on grapefruit or control efficacy of Cu. The residual activity of Cu on fruit was not affected by Kocide rate but decreased with time after application due to increase in fruit surface area over 21 days. This result supports the recommendation for use of 21 day interval Cu sprays for adequate canker control and explains the reduced efficacy of 28-day interval sprays. Objective 4: The Cu resistance gene was identified as CopL on a plasmid from a resistant Xanthomonas citri subsp. citri (Xcc) strain from Argentina that was exposed long-term to Cu for canker control. The identical resistance gene sequence was found in Xanthomonas spp. causing bacterial spot in tomato and pepper. Primers constructed based on the gene sequence were used to screen the remaining Cu resistant strains of Xcc from Argentina and Cu resistant strains of X. alfalfae. pv citrumelonis from Florida citrus nurseries with citrus bacterial spot. All strains screened thus far contain the CopL resistance gene. In addition, a non-pathogenic strain of Xanthomonas isolated from a citrus grove was found to be Cu resistant and may represent a pre-existing source of risk in citrus groves for horizontal transfer of CopL into Xcc. Cu and Sm resistance were monitored in Xcc and epiphytic bacterial populations on grapefruit trees sprayed with Cu or Sm every 21 days for two growing seasons (22 sprays total). Each season Cu and Sm sprays increased the ratio of epiphytic bacterial population with tolerance to these chemicals. Overall, the Sm resistant bacterial populations were proportionally lower than Cu tolerant bacterial population. No resistance to either Cu or Sm was verified in Xcc or epiphytic populations after two years of season long sprays. Objective 5: In 2009, canker management talks were given at county extension and other meetings. Updated 2010 canker management recommendations have been published in the Florida Citrus Pest Management Guide and Citrus Industry Magazine. Oral presentations have been scheduled for the Florida Citrus Production Managers and Florida Citrus Show.
The objective is to test the effect of soil applied neonicotinoid insecticds used for systemic psyllid control as inducers of SAR to determine if possible the direct effect of SAR on HLB disease progress in newly planted citrus trees subjected to psyllid mediated infection or budwood-inoculated infection. Hamlin trees were planted in May 2009 at the USDA-ARS, Picos Farm in Ft. Pierce FL and treated as follows: 1) untreated check, 2) foliar insecticide to control psyllids, 3) soil applied imidacloprid/thiamethoxam to induce SAR, 4) soil applied imidacloprid/thiamethoxam plus the foliar insecticide to induce SAR and control psyllids, 5) HLB bud graft-inoculated untreated check, 6) HLB bud graft-inoculated with soil applied imidacloprid/thiamethoxam to induce SAR. The experimental design is 50 trees per treatment in a completely randomized block design (5 blocks of 10 trees per block). Tree and insecticide treatments commenced in May and HLB budwood graft inoculations occurred in July. In December 2009 (6 months post-HLB inoculum challenge), 34 of 300 trees in the experiment are positive (11%). Most of the infected trees are located on the west side of the trial area which is proximal to the infected citrus, but few infected trees occur on the east side bordered by pine flatwoods. Based on the incidence of pysllid transmission versus graft-transmission, the gradient of infected trees from west to east is unlikely to persist given the preponderance of HLB-infected citrus and pysllids in the site. The highest number of HLB positives trees (8) is in the untreated checks with or without graft inoculation (treatments 1 and 5), followed by 6 positives in the graft-inoculated soil applied imidacloprid/thiamethoxam (Treatment 6), 5 positives in the foliar insecticide and soil applied imidacloprid/thiamethoxam (treatments 2 and 3), and 2 positives in foliar insecticide plus soil applied treatment (treatment 4). The effect of SAR induction on HLB infection progress is inconclusive because the soil applied imidacloprid/thiamethoxam control of the psyllid is causing an uncontrolled interaction with psyllid transmission. Although the best treatment to control psyllids, foliar insecticide plus soil applied (Treatment 4) has the lowest incidence of infection, this treatment is still not completely preventing pysllid infection. Hence even the most stringent psyllid control (foliar insecticdes applied every two weeks plus systemic activity) has not protected trees from transmission by infected psyllids. In a separate project on SAR-Canker control (see annual report for contract no. 72642) we have confirmed that soil applied acibenzolar-s-methyl (Actigard, Syngenta) induces SAR in the field and provides similar levels of canker control as the imidacloprid/thiamethoxam neonicotinoid insecticides but not interact with psyllid control. Henceforth, the trial will utilize soil-applied Actigard instead of imidacloprid/thiamethoxam in treatments 3,4 and 6. Two HLB-SAR experiments of similar design have been set up in Parana, Brazil in late 2009 (spring for southern hemisphere). One is HLB graft-inoculated and other proximal to an unmanaged farm with HLB. Each of these trials will utilize soil applied Actigard for the SAR treatments.
The objective is to test the effect of soil applied neonicotinoid insecticds used for systemic psyllid control as inducers of SAR to determine if possible the direct effect of SAR on HLB disease progress in newly planted citrus trees subjected to psyllid mediated infection or budwood-inoculated infection. Hamlin trees were planted in May 2009 at the USDA-ARS, Picos Farm in Ft. Pierce FL and treated as follows: 1) untreated check, 2) foliar insecticide to control psyllids, 3) soil applied imidacloprid/thiamethoxam to induce SAR, 4) soil applied imidacloprid/thiamethoxam plus the foliar insecticide to induce SAR and control psyllids, 5) HLB bud graft-inoculated untreated check, 6) HLB bud graft-inoculated with soil applied imidacloprid/thiamethoxam to induce SAR. The experimental design is 50 trees per treatment in a completely randomized block design (5 blocks of 10 trees per block). Tree and insecticide treatments commenced in May and HLB budwood graft inoculations occurred in July. In December 2009 (6 months post-HLB inoculum challenge), 34 of 300 trees in the experiment are positive (11%). Most of the infected trees are located on the west side of the trial area which is proximal to the infected citrus, but few infected trees occur on the east side bordered by pine flatwoods. Based on the incidence of pysllid transmission versus graft-transmission, the gradient of infected trees from west to east is unlikely to persist given the preponderance of HLB-infected citrus and pysllids in the site. The highest number of HLB positives trees (8) is in the untreated checks with or without graft inoculation (treatments 1 and 5), followed by 6 positives in the graft-inoculated soil applied imidacloprid/thiamethoxam (Treatment 6), 5 positives in the foliar insecticide and soil applied imidacloprid/thiamethoxam (treatments 2 and 3), and 2 positives in foliar insecticide plus soil applied treatment (treatment 4). The effect of SAR induction on HLB infection progress is inconclusive because the soil applied imidacloprid/thiamethoxam control of the psyllid is causing an uncontrolled interaction with psyllid transmission. Although the best treatment to control psyllids, foliar insecticide plus soil applied (Treatment 4) has the lowest incidence of infection, this treatment is still not completely preventing pysllid infection. Hence even the most stringent psyllid control (foliar insecticdes applied every two weeks plus systemic activity) has not protected trees from transmission by infected psyllids. In a separate project on SAR-Canker control (see annual report for contract no. 72642) we have confirmed that soil applied acibenzolar-s-methyl (Actigard, Syngenta) induces SAR in the field and provides similar levels of canker control as the imidacloprid/thiamethoxam neonicotinoid insecticides but not interact with psyllid control. Henceforth, the trial will utilize soil-applied Actigard instead of imidacloprid/thiamethoxam in treatments 3,4 and 6. Two HLB-SAR experiments of similar design have been set up in Parana, Brazil in late 2009 (spring for southern hemisphere). One is HLB graft-inoculated and other proximal to an unmanaged farm with HLB. Each of these trials will utilize soil applied Actigard for the SAR treatments.
Objective 1: Potential for soil application of the neonicotinoids, imidacloprid (Admire) and thiamethoxam (Platinum), and acibenzolar-s-methyl (Actigard), to provide long-lived SAR control of canker was evaluated in a trial of 4-yr old grapefruit trees in Ft. Pierce, FL. Despite above average rainfall in May, the disease on the spring flush that emerged in March when it was dry was free of canker. In contrast, spring-summer flushes evaluated in September had 62% incidence of canker diseased leaves. Several of the SAR treatments significantly controlled disease but not as well as with Kocide 3000 and Firewall (streptomycin) sprays at a 21 day interval. SAR treatments that failed to significantly reduce foliar disease were the two applications of Actigard, Platinum split rate and isonicotinic acid treatments. Incidence of canker on the spring- summer-fall flushes evaluated at the end of the season in November had a slightly higher canker incidence than on the earlier set of flushes. By this time all the SAR treatments significantly reduced disease compared to the non-treated check. The most effective treatment was 4 applications of Actigard at 2 oz and least effective treatment was 2 applications of Actigard at 1.0 oz. The best treatment matched the control on the flushes attained by the 11 sprays of Kocide 3000 and/or Firewall. The 4-yr old trees produced enough fruit to evaluate canker incidence. Effectiveness of treatment on incidence of canker fruit varied from moderate for the 4 applications of Actigard at 2 oz, Platinum and Admire to ineffective for isonicotinic acid and Actigard at lesser rates and applications. Kocide 3000 and Firewall were significantly more effective than SAR treatments for reducing fruit disease. Harvestable fruit was reduced by canker’induced premature fruit drop. The non-treated check had the lowest fruit harvested per plot while the 4 applications of Actigard at 2 oz had the highest number of fruit. The number of fruit harvested was significantly negatively correlated with the incidence of disease on the spring summer-fall flush disease, but was not correlated with fruit disease incidence. Control of canker on the leaves apparently reduced the inoculum available for early season infection of fruit. Based on the trial results, Syngenta the manufacturers of Actigard are supportive of seeking a label for soil application of Actigard for SAR control of canker on non-bearing citrus. Objective 2 Integration of soil applied SAR inducers with foliar applications of copper sprays for control of canker on grapefruit was evaluated in the 4 yr-old grapefruit trial above, the best control was Admire applied once at the beginning of the season followed by 11 Kocide 3000 sprays. A trial in 4 yr-old fruiting Hamlin trees was set up in Arcadia, FL to compare trunk and soil applications of Admire at 3x the label rate to account for effect of the larger tree volume. SAR control of canker on foliage and fruit was equal to that of six 21 day sprays of Kocide 3000 starting in March. This suggests that SAR and copper could be used in an integrated program for augmenting canker control for young fruiting trees. However, it is unlikely that either neonicotinoid will be approved for use on bearing trees due to potential risks of increasing rates of soil application leaching into groundwater and residues in the flowers. Because the best SAR treatment for fruit disease control in 4-yr -old tree trial was the 4 applications of the 2 oz rate of Actigard, trials of this SAR treatment for young bearing trees are planned. Objective 3 is to evaluate of the complementation of the use of Actigard and neonicotinoids Platinum and Admire to increase and/or extend canker control in 1-yr-old grapefruit trees. Canker was first observed in the trial area in September 2009 after a very high rainfall period in August (17 inches). The pattern of disease spread was across the trial area from southwest to northeast. Incidence of trees with canker was 56% in the non-treated check trees, whereas in most of the SAR treatment combinations the incidence was less than 10%. This trial will continue in 2010.
Objective 1: Survey and confirmation of HLB in seedlings from HLB-affected trees. 500 seedlings grown from seed extracted out of mild to severely HLB-affected fruit from Pineapple orange and Murcott tangor groves in Hendry Co. were assayed for HLB detection. All seedlings were negative for HLB detection by PCR in repeated assays in July and Nov. 2009. Objective 2: Thermotherapy of HLB-affected seed for the same seed sources under Objective 1 were treated at 125F, 130F and 135F to test for the effect of heat treatment on HLB detection in seedlings. Thermal treatments were lethal to Murcott, but not Pineapple seed. The emergent seedlings at 125F (142), 130F (98) and 135F (97) were PCR tested. All seedlings were negative for HLB in repeated PCR assay in July and Nov. 2009. Objective 3: In October 2009, seed source trees in two Florida nurseries were found positive for HLB by FDACS-DPI (Nursery 1) or Southern Gardens Diagnostic Lab (Nursery 2). Discovery of infected seed source trees in two Florida citrus nurseries identifies a potential (but unconfirmed) risk of outdoor seed source trees acting as a source of inoculum for introduction into nursery propagations. In Nursery 1, seed was collected from symptomatic branches of two sources of Swingle citrumelo, four sources of ‘Kuharske’ Carrizo citrange and one source of Cleopatra mandarin. In Nursery 2, seed was collected from one source of Sekwasha mandarin. From 63 to 205 seedlings from each source will have enough leaves for testing in February 2010.
Between Oct. 5th and 30th, five experiments were conducted that included Fallglo (1 time), sunburst (2 times), red grapefruit (4 times), and navel oranges (1 time). Fruit were treated on a commercial packingline (3 experiments) or on the Indian River Research and Education Center research line (2 experiments). Treatments included 1) full wash (brush bed + high-pressure wash) + waxing (carnauba), 2) full wash, 3) brush bed only, 4) brush bed with brushes rotating half normal speed, 5) high-pressure wash (HPW) only, 6) HPW for 10 seconds, 7) HPW for 5 seconds, 8) running fruit only over PVC rollers, and 9) a control (not washed or waxed). On the commercial line, fruit remained on the brush bed for ~ 1 min. 10 seconds, and on the full HPW for ~35 seconds. Normal brush rotation speed was ~100 rpm. Fruit were also evaluated for how surface dirt obscured the ability to grade the fruit for canker and other grade defects. Fruit from all treatments were degreened under simulated commercial conditions (5 ppm ethylene, 85 oF, 95% RH) and color development and weight loss measured almost daily. Fruit were subsequently stored and evaluated for the development of decay and disorders during storage under ambient conditions (~70-75 F). In general, all very early season Fallglo fruit were relatively clean and did not need washing for adequate grading. This changed somewhat by the end of October when grapefruit that received more extensive washing (i.e., full washing) was significantly easier to grade compared to unwashed fruit, and HPW producing fruit with intermediate gradability. However, even minimally washed fruit were sufficient for adequate grading. These experiments need to be repeated next season to determine variability in initial fruit cleanliness from year to year. Washing and waxing the fruit gave the greatest inhibition of degreening, almost stopping color development completely. Compared to preliminary results in 2008, results again showed that full washing of fruit on both the brush washer and HPW, or washing on the brush bed along inhibited degreening significantly more than did washing fruit only as they passed over the HPW. Fruit that were not brushed at all, but only passed over rollers experience a slight, but significant delay in color development compared to the control, but the delay was relative minor compared to the other washing treatments. As the season progresses, fruit exterior surfaces become more soiled with dirt and sooty mold that makes grading more difficult without washing. Preparations are currently underway to repeat the wash treatments of mid-season fruit to evaluate washing treatments that result in enough cleaning to allow adequate grading. The goal is to identify the minimum amount of cleaning that allows adequate grading of fresh citrus before dumping fruit on the main packingline during different times of the season. The earlier unmarketable fruit can be removed from the market chain, the less money is spent on these fruit. Furthermore, grading fruit earlier in the process could allows sales departments earlier knowledge of the amount, size, and quality of packable fruit that will be available to sell. For fruit going to export markets that tightly restrict shipment of fruit with canker symptoms, removing fruit before dumping on the main packingline offers another layer of protection against fruit with canker making it into these restrictive markets.
To date, three greenhouse/growth chamber studies have been completed, testing the effects of six different commercial formulations of plant growth regulators (PGRs) on the fitness (survival and oviposition) of the Asian citrus psyllid. The PGRs tested include Apogee (prohexadione calcium), Profile (paclobutrazol), Embark (mefluidide), Atrimmec (dikegulak-sodium), Sumagic (uniconazole), and Cycocel (chlormequat chloride). In both of the studies that included Atrimmec, significant phytotoxicity was observed in the form of foliage burn and dieback, thus preventing new growth and psyllid oviposition and survival. The other PGRs reduced shoot growth between 3 and 50% relative to untreated controls. Significant differences were observed in the number of eggs laid by psyllids on plants treated with different PGR’s. Oviposition was reduced by approximately 85% on plants treated with Embark and Sumagic, and by 65% on plants treated with Apogee and Profile relative to untreated controls, whereas oviposition was increased by 30% on plants treated with Cycocel Significant differences in survivorship of psyllid nymphs to adults were observed between the different PGR treatments. Survival was lowest for plants treated with Profile (7%), followed by Sumagic (36%), untreated control plants (45%) and Apogee (47%). The highest survivorship rates were observed on Cycocel (73%) and Embark (68%) treated plants. Survivorship of psyllids on plants treated with Profile was significantly lower than on plants treated with Cycocel and Embark, while all the other treatments did not differ significantly. There were significant differences in the weight of the adult psyllids reared on plants treated with the different PGRs. Adults emerged from plants treated with Sumagic weighed less than all the others with the exception of psyllids reared on Cycocel treated plants. Weight of psyllids reared on plants treated with Profile, Embark and Apogee did not differ significantly from the control. These results have been presented at the Plant Growth Regulation Society of America (Aug 2009), the Entomological Society of America (Dec 2009) and the Western Plant Growth Regulator Society (Jan 2010). It is important to note that none of the products tested to date are currently labeled for use on citrus, although Apogee and Sumagic are currently labeled for use on apples and vegetable transplants, respectively, whereas the others are only labeled for ornamental crops. Thus future greenhouse and growth chamber studies will focus on determining the plant physiological and/or metabolic changes that are occurring which are affecting psyllid behavior. Understanding these changes may allow for the selection of plants with these traits inherent or their selection in plant breeding efforts, or open avenues of research into other means of psyllid control by targeting specific biological needs of the insect. In addition, other rates and chemistries of PGRs will be tested. Field trials are planned to begin the spring of 2010.
>Work is underway in the Wang lab to understand the mechanism by which the HLB bacterial infection leads to phloem plugging and necrosis as insufficient bacteria are present in the phloem to directly plug the phloem. The procedure to obtain virulence factors from Ca. L. asiaticus was described in our last report. More than 10 potential virulence factors from the bacteria were cloned into the vector TMV30bGFP by PCR amplification and digestion with the restriction enzymes Pac1 and Xho1. The insertion of these genes in the TMV vector was confirmed by PCR amplification using gene specific primers, followed by sequencing. In vitro transcription was done using T7 RNA polymerase. The transcripts were then used to inoculate 4-5 week old Nicotiana benthamiana plants. Some plants expressed symptoms about 7 days after inoculation. The symptoms shown by the empty vector and the vector containing the potential virulence factors are being monitored. Of the 10 confirmed constructs, one construct has been shown to cause dramatic symptom changes in the Nicotiana leaves. Currently, this gene is being studied in citrus for further confirmation. Ideally these constructs should be expressed only in citrus phloem where symptom expression would more closely mimic HLB. Dr. Grosser has successfully used a construct that leads to only phloem expression of the inserted gene. This procedure will be tried for the virulence factors next year. Successful identification of the important virulence factors should help reveal the virulence mechanism of Ca. L. asiaticus, which factor(s) promote phloem disruption and possibly provide hints for management of HLB. >Transgenic approaches to disrupt HLB associated callose-phloem protein plugging and phloem dysfunction in citrus continue. Buds of the transgenic grapefruit trees that over-express 1, 3-. glucanase were grown out on Macrophylla rootstocks and challenged with HLB by bud inoculation. Only 4 of 44 plants had successful bud take and none of these show any symptoms 4 months after the inoculation attempt. The other plants will be re-inoculated with infected buds or psyllids in February after growth resumes. The citrus ‘-1,3-glucanases gene from Valencia embryogenic callus and young leaves has been cloned and multiple copies will be inserted in various citrus to see how these plants react to HLB infection. Five variations of Agrobacterium mediated or protoplast transformation using various plasmids are now perfected in the Grosser lab. Using these methods over-expressed transgenic plants are being developed and will be tested to see if production of ‘-1,3-glucan and phloem protein is blocked and to determine if these plants will show no or reduced symptoms when infected with HLB. Reaction of plants that over production these compounds will also tell us something about their role in symptom development. >Evaluation of field samples by TEM for the ratio of smooth to filamentous plugging continues. Field grown seed source trees of Swingle citrumelo were found with + PCR reactions for HLB. Leaf samples of these were fixed for examination for typical phloem plugging and necrosis symptoms. Further surveys will continue for any Carrizo seed source trees with HLB. Carrizo showed less symptoms than Swingle in greenhouse and growth chamber challenges with HLB. Based on lack of symptom development when infected, Poncirus trifoliata is another candidate for detailed phloem examination for possible tolerance to the HLB bacteria.
This progress report summarized research activities from October 15, 2009 to January 15, 2010. During this period, we continued working on (1) ‘Identification of additional Simple Sequence Repeat (SSR) loci and design and validation of new Las SSR markers’ and (2), ‘Analyses of genetic diversity of HLB Las populations. Followings are the summary of research activities and accomplishments during this period. Previously, we reported that we had designed and experimentally evaluated 40 new Las SSR primers. Among them, 25 primers showed clean and single amplified bands with expected amplicon sizes while the rest of 15 primers showed multiple bands when PCR evaluations were conducted. We then re-evaluated 15 primers by adjusting annealing temperature and Mg++ concentrations. PCR experiments were conducted with annealing temperatures 55oC, 57oC and 59oC along with 1.0 mM 1.5 mM and 2.0 mM Mg++ concentrations using an ABI Veriti thermal-cycler with a temperature gradient heating block (ABI, Foster City, CA). The results showed that PCR amplification conditions with 57oC and 1.0 Mg++ improved the specificity of PCR amplification. 8 primers that showed clean and single bands under this condition were therefore selected. Those primers that passed quality test were then subjected to discrimination power test. Total 33 primers (8 primers and 25 primers) were amplified with six asiaticus-infected DNA samples, 2 from Florida, 2 from India and 2 from China, respectively. Amplified PCR products were then separated by the high resolution of 5% polyacrylamide gels. Gels were visualized by silver staining. 16 out of 33 primers showed polymorphism with alleles ranging from 150bp to 320 bp per locus. Seven of SSR primers were synthesized with 5’-labeled fluorescent dyes (FAM, NET, VIC) and assayed by an ABI 3130 Genetic Analyzer. All rest of primer evaluation will be completed in next three months. We conducted global genetic assessment of asiaticus populations. A total of 166 HLB isolates representing four populations from US Florida, Brazil, India and China were analyzed using multilocus SSR markers. Based allele frequencies of SSR marker loci across four populations, a data matrix that consisted of 71 alleles and 166 samples was generated. The genetic distance analyses were then performed using Neil’s coefficient (Neil, M.,1972) with 1,000 bootstrap and 95% confidential intervals. A pair wise population genetic analysis showed that the genetic distance of the populations between Brazil and China, or Brazil and Florida or Brazil and India were 0.8261, 0.9598 and 0.9330, respectively, while genetic distances between Florida and China populations, or Florida and India populations were 0.6016 and 1.3327, respectively. The results indicated that the genetic distances among the populations are not always reflected to their geographic relationships. For example, the genetic distance between Florida Las population and China Las population was closer (0.6016) than that of any other populations compared while the Las populations between China and India are most genetically distant (1.3327) even though both are in Asia. Since Brazil Las population has more or less equal distance to other three populations, it brings up a question of why both populations are closely related or what is the origin of Florida HLB populations. High numbers of commonly-shared alleles of Las populations in Florida and in China suggest both populations are genetically related. In contrast, the genetic distance of Las populations between India and China is 1.7212, the largest distance as compared with other populations indicating that both populations have been differentiated for certain time and there is limited gene flow between the populations. Reference: Neil, M. 1972. Genetic distance between populations. Amer. Natur. 106:238-292.
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