The region of Araraquara has the largest insect Las+ population in summer, while Bebedouro has the smallest, during the whole year. The Asian citrus psyllid is the only known vector of the bacterium, ‘Candidatus’ Liberibacter asiaticus , that causes huanglongbing or citrus greening disease. This insect acquires CLas from an infected citrus tree while feeding as a nymph. Transmission to uninfected trees occurs when infected adults emerge and fly to, and feed on, uninfected trees. Our current understanding of the CLas-psyllid interaction suggests that adults become competent for transmission only after the bacterium moves from the insect gut into the hemolymph and eventually to the salivary glands. We hypothesize that specific molecular interactions between the bacterium and gut epithelial cell membranes are necessary to initiate the movement of the bacterium throughout the psyllid’s body. Furthermore, we hypothesize that if we could block these specific molecular interactions, we may block the bacterium from reaching the psyllid’s salivary glands and therefore block the psyllid’s ability to transmit this bacterium to healthy trees. We tested these hypotheses by first developing an assay to screen and identify psyllid gut membrane binding peptides from a combinatorial peptide library and then feeding selected peptides to psyllids and determining their ability to reduce movement of CLas from the gut to the salivary glands. A total of 7 peptides were identified that bind psyllid gut preparations that demonstrated gut-binding after oral uptake.
These peptides were detected bound to gut epithelial cells in adults that had emerged from nymphs that were fed on the peptides 4 days earlier. Using qPCR assays,ebb and flow table we demonstrated that oral delivery of a combination of three of these peptides apparently inhibited movement of the bacterium into the psyllid’s salivary glands. These results support our above stated hypotheses and demonstrate a potentially new strategy for preventing the spread of citrus greening disease. Research on development of solutions to citrus greening has been hampered by the inability to culture ‘Candidatus’ Liberibacter asiaticus , the bacterium associated with this disease. Further complicating the matter, this bacterium resides within the phloem of the plant and targeting it with bactericides requires penetration into the plant so that the bactericide can reach the bacterium. To overcome these obstacles we developed a rapid screen for both bactericidal activity against CLas and phloem mobility within the citrus phloem In this assay, single CLas+ leaves from Valencia are removed from CLas+ infected citrus, indexed for CLas titer by Q-PCR and Q-RT-PCR of petiole clippings. The leaves are then placed in liquid solutions for 6 days, after which, the CLas titer is determined in both the petiole and leaf midrib. Using this assay we have demonstrated that streptomycin and penicillin are active against the CLas bacterium but that streptomycin bactericidal activity moves more readily into the leaf phloem. We have also used this assay to identify two new antimicrobial molecules more active at reducing leaf midrib CLas than the standard antibiotics when compared at the same concentration .
Using both Q-PCR and Q-RT-PCR we demonstrated that reductions in CLas16S rRNA abundance was more pronounced than reductions in the cognate DNA sequence. Therefore analysis of 16S rRNA is a more sensitive analysis of perturbations in CLas status in the plant. This assay allows concomitant analysis of bactericide activity and plant mobility and is currently being used to screen potential antimicrobial molecules for use in fighting citrus greening. RNA interference , mediated by double-stranded RNA is a gene suppression method that triggers sequence specific mRNA degradation. In citrus, transgenic expression of antisense RNA or hairpin RNA can be used to generate plants with specific genes silenced. However, this technique involves lengthy procedures because of slow growth and long juvenility of citrus trees. Here we set out to develop a quick and convenient method to silence genes in established trees. In one test, commercially synthetized dsRNA was applied weekly to potted Hamlin sweet oranges grown in artificial rooting medium. A 170 base pair dsRNA homologous to citrus phytoene desaturase was able to significantly reduce the expression of the target transcript, at a dose of 200 µg per plant. Photo-bleaching and reduced chlorophyll content, from suppression of CiPDS, were observed in 1 tree at a dose of 200 µg and 2 trees at a dose of 2 mg per plant. In another test, citrus Terminal Flower 1 that negatively regulates flower initiation was targeted in an attempt to induce early flowering. Persian lime plants grown in soil had dsRNA applied as drench weekly, in combination with cold and drought treatment to induce flowering. After 11 treatments, flowers were observed from two individual plants treated with 200 µg dsRNA-CiTFL1, but not from the control treatment .
In summary, initial results indicated root application of dsRNA is capable of triggering gene silencing in citrus. We hypothesize that the magnitude and consistency of the silencing effect can be further improved through optimized application protocols. With appropriate targets identified, we hope that RNAi may be used in citrus plantings to alter physiological/developmental plant responses to improve tolerance to Candidatus Liberibacter, or mitigate negative effects caused by infection.Pathogen-associated molecular patterns -triggered immunity is an important component of the inducible defense system of plants. Citrus species possess varying levels of responsiveness to PAMP flg22 derived from the flagellum of Xanthomonas citri ssp. citri , the bacterial causal agent of citrus canker. Higher sensitivity to Xcc-flg22 is mediated by robust expression of the plant cell surface receptor gene FLS2 and confers an increased level of canker resistance. In this study, we explored the induction of PTI by chitin, another PAMP that is a structural component of insect stylets and exoskeleton, and studied its effect on the feeding behavior of Asian citrus psyllid , the vector of Huanglongbing. The results showed that infiltration of chitin solution into ‘Sun Chu Sha’ mandarin leaves induced expression after 1h of defense genes including WRKY22, GST1, SGT1, RAR1, PAL1, NDR1 and EDS1, a set of genes previously shown to be induced by Xcc-flg22 in the canker-resistant ‘Sun Chu Sha’, but not in canker-susceptible varieties. However, the chitin-induced gene expression was not detected at 18h after infiltration. ACP feeding behavior was monitored using electrical penetration graph to study how induction of PTI by chitin modifies the number and duration of xylem and phloem feeding bouts. When adult ACP started feeding at 1h after infiltration, reduced xylem and phloem feeding were observed in chitin-treated leaves compared with untreated controls. However, this difference was smaller when psyllids started feeding at 18h after infiltration. In summary, our study shows that chitin treatment induced a transient PTI that can affect ACP phloem ingestion,flood table and this inducible defense will be further studied for its utilization on selection and generation of resistant citrus materials. Citrus production has plummeted in Florida by more than 50% in the last 10 years, although many Florida growers still maintain yields in spite of huanglongbing by increasing inputs of insecticides and nutrients. Consequently, focus is shifting to health and profitability of the next generation of trees. Area wide Asian citrus psyllid management and new planting systems are needed to maintain tree health and bring new blocks into rapid production. Systemic insecticides applied to the soil supplemented by foliar sprays provide some protection against ACP but have proven insufficient to adequately control HLB infection during critical the first 3 years after planting. Planting into a bed covered with metalized polyethylene film offers an additional layer of protection by warding off incoming ACP adults through flight disruption. Lack of similar effects with white mulch reflecting only visible light indicates that reflected light in the UV range is required. Use of plastic mulch is a novel practice in citrus production requiring delivery of water, nutrients and systemic pesticides by drip irrigation. Therefore, adoption of this practice to commercial production is necessarily a gradual process. Nevertheless, some large scale trials have been conducted on commercial farms and interest in growing as results come in and technology improves. The metalized mulch must be of sufficient thickness and protected with a clear coat to last 3 years.
Ground must be well prepared and film mechanically installed as a tight 125-145 cm wide strip, higher in the center to shed water and sprays. Drip irrigation can be provided by one or two relatively inexpensive drip tubes placed under the mulch or punch-in emitters at the base of each tree. Trees are typically planted through 15 cm holes cut in the plastic using a post-hole digger. Oil containing sprays should be avoided to protect the polyethylene from undo degradation. Metalized mulch consistently reduces ACP populations and HLB incidence compared to whiteface mulch or bare ground. In addition, the mulch plus drip irrigation system increases soil moisture, reduces weed pressure, and accelerates growth. Chemigation has proved more efficient than soil drenches for delivering systemic insecticide once the root system is established. Yield after 3.5 years in a large scale replicated experiment was improved by 44% from trees planted on UV reflective mulch and treated with insecticides compared to insecticides alone and brix improved by 5%. Thus, metalized mulch can improve growing conditions and augment current insecticide based ACP control measures for young trees. The potential to more than compensate for additional costs by savings of water, fertilizer and herbicides, shortening time to crop profitability and increasing yields has inspired interest in using this technology to protect young citrus plantings threatened by HLB. Huanglongbing is severely impacting Florida citrus. The sweet oranges and grapefruit dominating Florida production appear to be among the most HLB-susceptible citrus cultivars. Transition to resistant or tolerant cultivars will be essential to sustain the Florida citrus industry, with solutions needed in the short and long term. Evidence mounts that useful resistance/tolerance to HLB is present in cultivated citrus and greater resistance may be drawn from more distant members of the gene pool, and both approaches are targeted by the USDA citrus breeding program. Potentially useful HLB- tolerance is apparent in several mandarin hybrids.After six years of growth following almost immediate infection, ‘SugarBelle/Sour Orange’ and ‘Tango/Kuharske’ had the largest trunk diameters, good canopy density, and were progressing into fairly normal bearing. In a planting of seedlings from 85 diverse gene bank accessions there is a strong association between tolerance and pedigrees including citron. Hybridization is ongoing to combine diverse sources of tolerance. There is evidence of resistance to HLB in several Citrus crosscompatible genera. In a study of diverse scions on both unifoliate and trifoliate root stocks, root stock had little effect but scions with Poncirus in their pedigrees had significantly lower populations of the HLB bacterium. Strong resistance and even immunity should be possible using genetic engineering and USDA efforts are also underway to produce HLB-resistant scions using biotechnology. Programmed cell death or apoptosis is a plant defense response that can be triggered upon pathogen infection, and some triggers, such as Reactive Oxygen Species , can activate or prime plant defenses over long distances. There are also some anti-apoptosis proteins in plants which dampen plant defense responses. By suppressing expression of these anti-apoptosis proteins, PCD should occur in a more rapid manner and with augmented defense responses upon pathogen infection. Huanglongbing , caused by Ca. Liberibacter asiaticus , threatens citrus production worldwide. Las grows strictly inside living citrus cells. Citrus cells detect Las, but fail to trigger adequate native defense systems or PCD until the bacteria have multiplied to very high levels. To help accomplish this defense suppression, Las secretes both a functional peroxiredoxin and a peroxidase directly into the citrus cell cytoplasm that significantly dampen both the ROS response and signaling pathway. We have developed a citrus gene silencing strategy to suppress a brake on the natural citrus defense reaction to Las. Five silencing constructs were used to transform Carrizo root stocks. Average silencing efficiencies of the transformed root stocks ranged from 77.30% to 82.35%. Using a Las flagellin protein fragment applied at 10 µM as a proxy for Las infection, expression levels of three citrus defense response genes NDR1, PR1 and EDS1 were significantly higher in silenced lines than in NT controls. Challenge inoculations with Las were then performed by approach grafting selected transgenic Carrizo lines representing all 5 silencing constructs to mature Las-infected nontransgenic citrus scions.