We continued the work this grant focusing on the infection of various citrus and other rutaceous plants that might be alternative hosts for both the psyllid and the bacterium Candidatus Liberibacter species associated with HLB. One of the new hosts for the psyllid that we reported in the last quarter, Choisya ternata, was grafted with PCR positive budsticks of HLB infected citrus. The grafts were not successful and died in the plants. Plant materials were sent to both the USDA, ARS, Molecular Plant Pathology Lab, Beltsville Exotic Citrus Pathogen Collection and to the USDA, ARS Foreign Disease and Weed Science Research Lab, Frederick, MD for grafting and psyllid transmission of the Ca. Liberibacter americanus and the Ca. Liberibacter. africanus strains. Infectivity assays are pending. In psyllid inoculation tests with Ca. Las we were able to infect four of six Choisya ternata plants in 3 months. In field surveys for alternative hosts we sampled a group of 18 sour orange trees that were growing in a pine planting close to commercial citrus plantings. Of the 18 trees sampled one was found to be PCR positive for Ca. Liberibacter asiaticus. In other field work we sampled a swamp area close to where the initial infections of a grove were discovered. Of 35 plants samlee no infected plants were found including a number sweet orange and grapefruit seedlings. Additional sampling is planned to look for alternative hosts for HLB that could be reservoirs for infections. We continue to propagate and inoculate seedlings and grafted plants of IAPAR 73. As reported previously we were unable to infect some seedlings with HLB material. We presented these results at the Florida State Horticultural Society meeting in June and have submitted a manuscript to the proceedings. Publications: Damsteegt, V. D. E. N. Postnikova, A. L. Stone, M. Kuhlmann, A. Sechler, N. W. Schaad, R. H. Brlansky, and W. L. Schneider. 2010. The relevance of Murraya paniculata and related species as potential hosts and inoculum reservoirs of ‘Candidatus Liberibacter asiaticus’, the suspected causal agent of Huanglongbing. Plant Disease 94:528-533. (Editor’s paper of the month of May).
During the first year of this project, we evaluate the efficacy of different phytohormones in suppressing the production of new flush growth on potted citrus plants in the greenhouse. Two separate field trials were also conducted 1) to determine how new flush growth suppression by these phytohormones will relate to Asian citrus psyllid (ACP) population and 2) to elucidate the impacts of pruning date and fertilization level and timing on the population of ACP in a mature sweet orange block. In the greenhouse, potted lime trees were first pruned to stimulate flush production, and each phytohormone was immediately sprayed following the label recommended rate. Ten potted plants per treatment along with an untreated control that received tap water treatment were used. The numbers of new flush shoots on potted plants were recorded twice a week for the first two weeks and weekly during week 3 and 4. Three phytohormones, namely Apogee (0.7g/L), Sumagic PGR (100mL/L), and NAA-1-Naphthaleneacetic acid (0.5’L/L) significantly reduced the number and delayed the growth of new flush shoots produced by lime trees. The number of new flush shoots produced by plants treated with these 3 growth regulators was at least 2-fold lower than the untreated control. In field trials, rows of a mature ‘Valencia’ block were pruned and each of the 10 phytohormones was applied to a group of 2 contiguous trees. A randomized block design with 4 replications was used. Number of new flush shoots and densities of ACP were recorded weekly for 4 weeks. Out of the 10 phytohormones, only two (Apogee and NAA-1-Naphthaleneacetic acid) significantly reduced the number of new flush produced by trees, but the reduction was less than 20% relative to the untreated control. Due to the overall low numbers of psyllid recorded during the trial in October, no meaningful statistical inference could be made. In the second field trial two hedging dates (one early in February and one late in April) and two nitrogen fertilization regimes (one application of 100 lb/ac in February and two applications of 50 lb/ac each in February and in June) were tested in a factorial design in a mature sweet orange block. Weekly counts of new flush shoot growth and ACP densities were made. Both hedging dates and application of nitrogen significantly affected ACP infestation and densities. Hedging significantly altered the phenology and intensity of new flush shoot production on trees. Both the early and late hedging dates of trees stimulated profuse flush shoot production 2 to 3 weeks post-hedging. Significantly higher ACP infestation levels and densities were recorded in the late hedging date compared to the other treatments from May to October. Although more new flush shoots were produced in the early hedging treatment relative to the non-hedged treatment, ACP populations were comparable in these two treatments. These results clearly demonstrate that early hedging (February) should be encouraged as it prevents severe outbreaks of ACP populations, while providing the intended physiological benefit of the practice. By contrast, late pruning in spring will likely lead to ACP outbreaks in citrus orchards. Application of nitrogen also affected the abundance of new flush growth. Although no alteration of flush cycles resulted from N application, densities of new flush growth were higher in fertilized plots than in the non-fertilized control blocks. The effect was more dramatic in blocks where N was applied in a single dose. In the one-time N application treatment, significantly more new flush shoots were produced which resulted in higher densities of ACP eggs and nymphs for most of the sampling dates. ACP densities in the split application and non-fertilized control were similar throughout the sampling period. In summary, N management, and in particular, split N fertilization and early pruning of trees were associated with lower ACP population densities on sweet orange trees. These cultural practices can be used to manipulate the phenology and abundance of citrus flush shoots and consequently the population densities of ACP.
As a preliminary step to understand and characterize what metabolites are responsible for the bitter off-favor of Huanglongbing infected fruit, the thresholds of limonin, nomilin, and their combination in a sugar and acid matrix, as well as in healthy ‘Valencia’ orange juice were determined by taste panels. Food grade limonin and nomilin were added alone or in combination to a simple (sucrose and citric acid) or complex (sucrose, glucose, fructose, citric and malic acid) matrix, or were added directly into orange juice. Thresholds were determined by taste panels, composed of 16 to 23 trained panelists, using a three-alternative forced choice (3-AFC) method (ASTM: E-679). In the simple matrix, the threshold of limonin was lower than nomilin. The synergetic effect of limonin and nomilin was significant in decreasing their individual thresholds. Interestingly, the thresholds of limonin and nomilin were lower in orange juice compared to the thresholds measured in the complex matrix. Our current results show that the threshold concentrations of limonin and nomilin when added to healthy ‘Valencia’ orange juice are higher than the concentrations of those compounds measured in juice made with symptomatic HLB fruit, which was perceived bitter by a taste panel. Possibly, the lower sugar and higher acid content of HLB fruit decreased the threshold of those bitter compounds. Moreover, different concentrations of ‘Valencia’ and ‘Hamlin’ HLB infected juice were blended into healthy juice to determine the detection and recognition thresholds. Panelists were able to detect the symptomatic HLB juice at different levels depending on the variety. For both Hamlin and Valencia juices, however, panelists could detect a difference when blending normal juice with 25% HLB symptomatic juice and could describe the difference (bitter, metallic) when normal juice was blended with 50% HLB symptomatic juice. Nomilin was discovered to have a lingering metallic taste that was different from limonin, which was found to be just bitter. This study looked at flavor compounds in juice made from fruit harvested from 15+ trees symptomatic for HLB compared to healthy trees grown in the same area for multiple harvests of Hamlin (December/January, 2009) and Valencia (April/June, 2009). Fruit from HLB symptomatic trees were separated into asymptomatic (normal looking, HLBAS) and symptomatic (small, green and lopsided, HLBS) fruit for comparison to healthy (H) fruit prior to juicing using a JBT extractor/pasteurizer. For Valencia, there were no differences in Brix, but HLB juices tended to have higher titratable acidity (TA), lower ratio (April) and higher oil (June). For Hamlin, Brix was higher in H juice (December), TA higher in HLBS (December), ratio lower in HLBS and oil higher for HLBS juice (December). Healthy juice tended to have higher levels of sucrose and fructose, whereas glucose was variable, especially for HLBS juice. HLB juice had higher levels of citric acid (December Hamlin/April Valencia) but there were no differences for the other two harvests/cultivars. Malic acid tended to be lower in HLB juices. Limonin and nomilin were higher in HLB juices, especially HLBS, but were below reported thresholds. Gas chromatography-olfactometry (GC-O) research did not show differences between H and HLBAS juice, but observed some volatiles that were found either exclusively or at higher aroma intensity in either H or HLBS juices. There were more :green’/ ‘fatty’ aromas in HLBS while there were more ‘sweet’/’fruity’ components in H juices with respect to each other.
Objective 1 was to understand the cause of leaf chlorosis. In the leaf, starch packing of chloroplasts occurred upstream after phloem blockage and often after some necrosis was detected. Subsequently chloroplast grana were disorganized and disappeared and chlorosis of these affected cells in the leaf tissue occurred. Phloem plugging was elucidated as smooth (callose formation) and fibrillar (phloem protein 2 (PP2) ligand formation) materials in sieve elements accompanied by some phloem necrosis. The combination leads to phloem blockage and starch accumulation above blocked phloem. The cascade of blockage down the phloem system leads to root starvation and tree decline. Plugging by callose was about 3 times more prevalent than PP2 plugging in HLB affected trees in both the greenhouse and field. Objective 2 was to attempt to alter the primary plugging cause (callose formation) and determine how important callose plugging was to the overall disease symptomology of HLB. This was accomplished by over expressing the citrus beta-1, 3 glucanase gene in citrus plant phloem. The goal was to either accelerate breakdown of the callose ligand or block its breakdown depending on how metabolism reacted to the over expression. Plants were produced and are now under greenhouse screening to compare their development to non-transformed plants both exposed to the HLB causing bacteria C. Las and without the disease. Objective 3 was to determine if any virulent genes from C. Las could directly cause any of the symptoms described for HLB infected citrus plants. The known bacterial genes were evaluated for overexpression in HLB affected citrus and 44 putative virulent factors were identified. Two of the hypothetical genes that were overexpressed in planta were screened in Nicotiana benthamiana, and the tobacco plants showed disease symptoms, primarily general necrosis. Transgenic citrus plants (Duncan) expressing these two genes were constructed at the Citrus Transformation Facility. Another potential virulence factor overexpressed in planta was hemolysin. Results indicated that C. Liberibacter encodes a functional hemolysin protein which might be involved in pathogen and host interaction. Two papers and 2 abstracts were published related to Objective 1 and 2.
Transmission tests using endemic healthy Brevivalpus phoenicis mites from Florida were done in quarantine at the USDA, ARS, Foreign Disease and Weed Science Research Unit, Ft. Detrick, MD. Cytoplasmic citrus leprosis infected samples were sent under permit from Colombia to the USDA, APHIS, PPQ quarantine facility at Beltsville, MD and then shipped under permit to the Ft. Detrick lab. The leprosis materials arrived in excellent condition as compared with previous samples that were detained in customs. Mite transmission tests were also done at the same time by our cooperator in Colombia. Results of both transmission tests are pending. The manuscript describing the new cytoplasmic citrus leprosis virus from Colombia (“A Novel Virus of the Genus Cilevirus Causing Symptoms Similar to Citrus Leprosis) was accepted for publication in Phytopathology) Contacts have been made with Mexico where another type of citrus leprosis has been detected.
Cytoplasmic citrus leprosis infected samples were sent from Colombia to quarantine facilities at the USDA, APHIS, PPQ, CPHST, Beltsville, MD. Again they were negative in PCR and antibody tests for cytoplasmic citrus leprosis virus (CiLV-C). Samples were from different cultivars of citrus in order to see if the samples differed in the virus contained. Samples again were prepared for electron microscopy to verify the type of viral particle and particles similar to those previously published for cytoplasmic citrus leprosis were seen. Sequencing (funded on another project) was completed and the bioinformatics confirmed a new cytoplasmic citrus leprosis virus. New primers were produced for detection and samples were sent from Colombia for detection of both types of cytoplasmic citrus leprosis virus. Mr.Leon in Colombia as earlier reported continues to do successful mite transmission experiments with PCR negative isolates from Colombia. Samples were sent under permit to the USDA and using primers to the the new cytoplasmic citrus leprosis virus (CilV-C2) we have found that he has transmitted this new virus with Brevipalpus mites from Colombia. A manuscript is in preparation.
Healthy endemic Brevivalpus phoenicis mites from Florida were reared and acquired from Dr. Jorge Pena, University of Florida, Tropical Research and Education Center, Homestead, FL. The mites were shipped under permit to the USDA, ARS, Foreign Disease and Weed Science Research Unit, Ft. Detrick, MD. Cytoplasmic citrus leprosis infected samples were sent under permit from Colombia to the USDA, APHIS, PPQ quarantine facility at Beltsville, MD and then shipped under permit to the Ft. Detrick lab. Healthy citrus plants were shipped at the same time for transmission tests. Transmission tests were performed however mite survival was less than expected. We are awaiting results. Mite transmission tests were also done by Guillermo Leon in Colombia and the mites were shipped in alcohol for comparison with the Florida mites. The Florida mites and a sample of the mites were prepared for low temperature SEM by Dr. Ronald Ochoa and Dr. Gary Baucham of the USDA, ARS, Beltsville, MD. They reported and produced photos of the mites. The mites from Colombia and the Florida mites were both found to be B. phoenicis type II. All transmission experiments will be with the same mite species and type.
Transmission tests using endemic healthy Brevivalpus yothersii (syn. phoenicis) mites from Florida continued in quarantine/containment at the USDA, ARS, Foreign Disease and Weed Science Research Unit, Ft. Detrick, MD. Two tests were completed during this quarter. Cytoplasmic citrus leprosis infected samples were sent under permit from Colombia to the USDA, ARS, Foreign Disease and Weed Science Research Unit, Ft. Detrick, MD. Mite transmission tests were also done at the same time by Guillermo Leon our cooperator in Colombia who is also working with the mite transmission of cytoplasmic citrus leprosis virus. Positive transmission tests were done multiple times in Colombia. In containment at Ft. Detrick all tests have been negative with the endemic Florida mites. The Florida mites and the Colombian mites were identified by an expert on mite taxonomy, Dr. Ron Ochoa, as being the same species. A manuscript describing the new cytoplasmic citrus leprosis virus from Colombia (“A Novel Virus of the Genus Cilevirus Causing Symptoms Similar to Citrus Leprosis) was published in Phytopathology, May 2013). Contacts have been made with Mexico where another type of citrus leprosis (CiLV-N) has been detected and we are working with collaborators on characterization and transmission of this virus.
For the second year of this project, we evaluated the impact of an insecticide spray application in combination to the two main objectives i.e., effect of harvest date and fertilization regimes on the population densities of the Asian citrus psyllid. The two evaluated hedging dates in 2010 were hedging during the dormant season before the first flush of the year in January and hedging in April after the first flush but before the second major flush. Three nitrogen fertilization regimes (no fertilization, one full application of 100 N lb/acre in February, and a split application of 50 N lb/ac each in mid-February and in mid-June, respectively) were tested in a factorial design in a mature ‘Marrs’ sweet orange block. These blocks were subsequently split in two, and half the block received insecticide spray applications at the beginning of each major flush cycle. All three main factors (hedging, fertilization and spraying) and their interactions significantly affected D. citri adult populations. Similarly, D. citri densities varied with sampling date and this variation correlated with flush cycle. Although late hedging in mid-April produced a spike in D. citri population in mid-May due the new flush growth on these hedged trees, its overall impact on total psyllid counts throughout the year was not different from the non-hedged treatment because the traditional June flush cycle in Texas was very light to absent on the late hedging treatment in contrast to the no-hedging treatment. The early hedging had the lowest overall psyllid count mainly due to the fact that trees that were hedged in January had profuse flush shoot production in February when psyllid populations were very low. Consequently, mean psyllid numbers in the no-hedging and late hedging treatments were twice as high as the one recorded in the early hedging during the dormant season. Based on these observations, we can recommend early hedging when possible, as a strategy to reduce D. citri populations. D. citri numbers were affected with nitrogen application with significantly more psyllids recorded in the one-time application of 100 lb of N per acre. Consequently, all plots receiving this single dose harbored more psyllids than the split and fertilization treatments that were not different. Application of nitrogen as single dose is a common practice in Texas, but the present results showed that this practice has the potential to significantly increase psyllid population in groves. As expected the sub-plots that received insecticide sprays had lower psyllid counts than the unsprayed ones within each block. However, there were significant interactions between spray application and hedging and fertilization. Our study demonstrated that that growth care practices including nitrogen fertilization and tree canopy management can significantly affect D. citri densities in citrus groves. To ensure that these practices do not negatively affect our psyllid control efforts with insecticides, late hedging (in April or May) and one single rate application of 100 lb N/ac should be avoided at least be followed with a spray application at the beginning of the subsequent flush cycle.
Our project is examining phloem gene expression changes in response to CLas infection in HLB-susceptible sweet orange and HLB-resistant Poncirus and Carrizo (a sweet orange – Poncirus cross). We are using a recently developed methodology for woody crops that allows gene expression profiling of phloem tissues. The method leverages a translating ribosome affinity purification strategy (called TRAP) to isolate and characterize translating mRNAs from phloem specific tissues. Our approach is unlike other gene expression profiling methods in that it only samples gene transcripts that are actively being transcribed into proteins and is thus a better representation of active cellular processes than total cellular mRNA. Sweet orange, and HLB-resistant Poncirus and Carrizo (sweet orange x Poncirus) will be transformed to express the tagged ribosomal proteins under the control of characterized phloem-specific promoters; tagged ribosomal proteins under control of the nearly ubiquitous CaMV 35S promoter will be used as a control. Transgenic plants will be exposed to CLas+ or CLas- ACP and leaves sampled 1, 2, 4, 8, and 12 weeks later. Ribosome-associated mRNA will be sequenced and analyzed to identify differentially regulated genes at each time point and between each citrus cultivar. Comparisons of susceptible and resistant phloem cell responses to CLas will identify those genes that are differentially regulated during these host responses. Identified genes will represent unique phloem specific targets for CRISPR knockout or overexpression, permitting the generation of HLB-resistant variants of major citrus cultivars.
This is the first year, 3nd quarter progress report; our grant started December 1, 2018. In the last three months, the post-doctoral researcher, Tami Collum, has started optimizing nucleic acid extraction protocols for citrus. For objective 6 (Additional Approach: Phloem limited citrus tristeza virus vectors will be used to express the His-FLAG-tagged ribosomal protein in healthy and CLas infected citrus) Dr. Dawson’s lab has all necessary constructs and has moved many of them into citrus. CTV-infected plants will soon be ready for shipment to Maryland. Again, the majority of our efforts in the 3nd quarter were focused on objective 2 (production of transgenic citrus lines). The Stover lab has performed Agrobacterium-mediated transformation of seedling epicotyls from all three citrus genotypes indicated in the grant (Carrizo, Poncirus and Hamlin sweet orange) with the His-FLAG tagged RPL18 (ribosomal protein L18) under the 35S promoter and all three phloem promoters pSUC2, pSUL and p396ss. Carrizo transgenic plants with three promoters are already acclimatized in the greenhouse: p35S::HF-RPL18 (12 plants), pSUL::HF-RPL18 (21 plants), and p396ss::HF-RPL18 (30 plants), with many plants >25 cm and suitable for taking cuttings for replication. Seven plants transformed with each promoter were evaluated for presence (PCR) and expression (RT-qPCR) of the HF-RPL18 gene, and 100% of the plants are expressing the gene. The newly transformed Carrizo with the pSUC2 promoter has been transferred to greenhouse and will be evaluated soon. Putative transgenic plants of Poncirus harboring the 35S::HF-RPL18 (12 plants) and pSUL::HF-RPL18 (10 plants) were moved to the soil. Poncirus plants with constructions p396ss::HF-RPL18 and pSUC2::HF-RPL18 are still in rooting medium (16 and 49 plantlets, respectively). Hamlin transformation was intensified in this quarter and many shoots have being transferred to rooting media, and one plant to soil. Since Hamlin has a much lower transformation efficiency, some transformations were repeated and also cotyledons have being used as a new transformation target explant for this genotype. Carrizo plants expressing the HF-RPL18 gene will be replicated and transferred to Ft. Detrick in the next quarter.
1. Please state project objectives and what work was done this quarter to address them: The objectives of this project are: 1, To study the effect of Brs on priming immunity on young, newly planted trees. This will allow to know for how long immune response will last after Br application, so we can adjust timing (number of applications).After 1 year (with a monthly application), some trees (around 20%) are still HLB-negative. Denser foliage has consolidated in a denser canopy with less leaf drop in winter. Spring flush in Br treated trees has already started in early February and is highly syncronized, following the trend we observed during Summer and Fall flushes. This confirms our previous observations that flushing occurred at the same time in Br-treated trees, and will allow better planification of insecticide sprays. 2, To determine the best time of application (frequency) to achieve maximum protection against pests and disease in newly planted trees. We have started to get data showing that even though immunity is maximum by 30 days, it is still significantly high 60 days after treatment as compared to controls. 3, To determine the effect of Br application on advancing fruit maturation in both Valencia and Hamlin. Treatments started in September on Hamlin. We performed two different sets of treatments: biweekly treatment and only once treatment. Last season, there were no differences between biweekly treatments and only once treatment in terms of yield and quality. This season, we wanted to confirm this. We have been following quality every 15 days, including internal quality and external color development. We started to see a significant increase in Brix with Br treatment performed in mid-November for Hamlin, just like last year. In this case, we had 9.2 Brix in Br treated fruit as compared to 8.4 in controls at harvest. Ratio was again greatly increased,15 in Br-treated as compared to 11.8 in controls. External color is also increased. Together this is encouraging, as we are confirming results from last year in Hamlin. Interestingly, we had also a mild 25% increase in total yield at harvest. These trees were harvested in January 12. We started treatments in Valencia on January. Outreach: -OJ BREAK Jan 17, Lake Alfred. IPC’s and Brassinosteroids to Prolong Health and Improve Fruit Yield and Quality in Newly Planted Trees Under HLB 2. Please state what work is anticipated for next quarter: We will continue treatments in Valencia until harvest. We will perform juice analisis as we did last year in both Valencia and Hamlin to determine the composition of sugars and acids. 3. Please state budget status (underspend or overspend, and why): Spending continues on track.
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