Horticultural & Management

Transmission of the emerging citrus pathogen cytoplasmic citrus leprosis virus by endemic mites

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.

Coupling citrus flush management and dormant chemical spray as a strategy to control populations of Asian citrus psyllid

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.

Phloem specific responses to CLas for the identification of novel HLB resistance genes

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.

Determining best timing for Brassinosteroid (Brs) application to achieve maximum beneficial effects on citrus tree health and fruit yield and quality

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.  

Proof of concept for phage therapy in the reduction CLas titer and HLB symptoms in citrus