A number of transcriptome studies have been conducted and reported to characterize the gene expression dynamics in citrus-HLB interactions. These studies indicated that many citrus genes/pathways were modulated by HLB. The present study was conducted to gain comprehensive insight about the underlying molecular mechanisms in citrus-HLB interactions. Twenty-two publicly available citrus gene expression datasets, including 18 from HLB-susceptible and four from HLB-resistant citrus selections, were retrieved; and previously identified, differentially expressed genes were analyzed using the LIMMA and the RankProd methods. Out of a combined list of 7,412 DEGs, we identified the most significant 65 common genes and 30 R-dataset-specific DEGs. Gene Ontology analysis of these DEGs suggested that carbohydrate metabolism and transport, and stress response were the core pathways in citrus modulated by HLB. The 30 Rdataset-specific DEGs were mainly coded for LRR proteins, chitinases, CDR, miraculins, or lectins. Weighted gene co-expression network analysis of 2,499 DEGs revealed 21 modules with major hub genes. The miRNA nested network analysis suggested that csi-miR167 and csimiR396 could affect citrus transporters and defense response pathways, respectively. Collectively, these meta-analyses suggested candidate genes for further gene expression analysis,macetas de 5 litros over-expression analysis, or other genetic modification towards increased HLB resistance in citrus. Citrus Greening was first detected in Puerto Rico in 2009 affecting orange and lemon .
The causal agent was confirmed as Candidatus Liberibacter asiaticus. CG is transmitted by the Asian Citrus Psyllid Diaphorina citri Kuwayana and by human mediated grafting transmission. A survey conducted in 2010–2012 showed that the disease is widely spread in Puerto Rico. Consequently, efforts on an education program were established by the Extension Service to train agricultural agents on identification of CG and citrus orchard management practices. In order to maintain pathogen-free bud wood material, the Agricultural Experiment Station of the University of Puerto Rico, Mayagüez Campus has moved all the germplasm to insect-proof screen houses. Initially, all the germplasm was located on AES at Isabela, Puerto Rico. Currently, new collections have been moved to AES at Rio Piedras and Adjuntas, Puerto Rico. AES is the only one that has protected citrus bud wood that is certified CG free in Puerto Rico. In 2014, AES started a new institutional initiative to integrate various researchers to work with the critical citrus situation. The new initiative formed the project Production of Healthy Citrus Plants in Puerto Rico. This project combines the certification of disease free citrus plants and the development of new methodology on the production of pathogen-free citrus plants in screen-protected houses. The certification will detect citrus greening, citrus variegated chlorosis, citrus leprosis, citrus exocortis, and citrus canker. On the other hand, the new methodology will explore different media components, fertilization, container design and size, different rootstock and varieties in order to accelerate root growth and whole plant development. Current status of the screen-protected houses, current germplasm collection, and practices will be presented.In the state of Colima, Mexico, there are 20,000 hectares of Mexican lime . The Asian citrus psyllid has been present there since 2004, while the ‘Huanglongbing’ disease was detected in 2010. Seven months after the first detection, HLB positive trees were found in all producing areas of the state.
In June 2013, a systematic sampling was done in 299 orchards checking 7500 trees, resulting that 100% of the orchards and 100% of the sampled trees had HLB symptoms, which had a canopy portion affected by the disease ranging from 25 to 75%. During 2010, 2011, and 2012, the number of fruits per square meter of canopy in different orchards was quantified. In these 3 years, the results were similar; it was observed that trees with HLB symptoms in over 75% of the canopy tend to reduce their production of fruit between 40 and 60%. Also, it was evident that asymptomatic sectors in HLB-affected trees present good production and good fruit size. And contrary to that, symptomatic sectors have chlorotic foliage, a reduced yield, and a fruit size slightly smaller in comparison with fruits from healthy trees. Until nowadays, in Mexican lime, it has not been detected misshapen fruit, inverted ripening, or an increased number of aborted seeds related to HLB. During 2013, an average yield of 14.4 t/ha was recorded, representing a reduction of 23.4 and 39.7% compared to that recorded in 2010 and 2012, respectively. This partly reflects the effect of HLB on Mexican lime production in Colima. It has been observed that an integrated crop management, with special emphasis on nutrition, although with a lower yield, allows HLB-affected trees to continue producing fruit. This represents an option for producers while HLB tolerant varieties are generated.Severe pruning has been suggested as a strategy to rejuvenate citrus trees which have been adversely affected by HLB. Pruning rebalances a tree’s root to canopy ratio and thus allows an infected tree to increase its capacity to set fruit. An experiment was initiated at the UF/IFAS Southwest REC in February 2010 to measure the effects from severely pruning HLB-infected trees. Four years of data including shoot growth, canopy development, and fruit yield were collected from 2010 through 2013. Fifteen-year-old Valencia orange on Swingle citrumelo root stock grown on two-row beds typical of citrus in the Florida flat woods were selected, pruning one row and leaving the other row unpruned.
Three foliar nutritional treatments plus a standard grower fertilizer program were applied to both pruned and unpruned trees.The harvestable yield from pruned trees was minimal in 2010, the year of pruning, and constituted the largest financial penalty when attempting to rejuvenate HLB-infected trees through severe pruning. Production from pruned trees recovered during the second season and was statistically equal to production from unpruned trees. In 2012 and 2013, production from pruned trees surpassed the production from unpruned trees. Juice quality data generally showed no significant differences between pruned and unpruned trees. Cost effectiveness of pruning, however, depended on the enhanced foliar nutritional program. Pruned trees on two of the three nutritional programs produced nearly a box more fruit than unpruned trees receiving the same nutritional program. The increase in net returns was estimated to nearly $4 per tree. The cost of severe pruning plus the value of yield loss in the first year after pruning was estimated to be less than $2.30 per tree. Foliar nutritional programs have been adopted by nearly all Florida citrus growers as a strategy to maintain production from HLB-infected trees. Important questions arise, however, as to the relative importance and necessary quantity of micro- and macro-nutrients. Five years of production and cost data were collected from a trial on a commercial block of Valencia on Swingle on which various combinations of nutritional supplements were applied three times a year corresponding to the spring, summer, and fall flushes. Nine foliar treatments were designed and replicated five times over a 30-acre block. All treatments received a uniform ground fertilizer application and a uniform psyllid control spray program. The foliar treatments represented various combinations of micro- and macro-nutrients. Annual fruit yield and juice quality were recorded for each treatment and analyzed both by individual year and cumulatively across the 5-year period. Fruit revenue was estimated using average fruit prices reported in the Annual Citrus Summary . The costs of the individual nutritional treatments were itemized using 2013 retail fertilizer prices. Significant yield differences between all foliar nutritional programs and a standard were noted starting in the second year of the trial.
Those treatments that combined both micro- and macro-nutrients yielded the greatest yield benefits. While the treatment with the greatest complement of microand macro-nutrients resulted in the largest numerical yield gain, the yield differences among most of the foliar nutritional programs were not significantly different. The costs of nutritional products, however, ranged significantly from $100 to $550 per acre.Huanglongbing or citrus greening, caused by the phloem-limited bacterium Liberibacter asiaticus , is threatening the viability of the citrus industry in the United States. The search for an effective treatment of HLB is imperative. Using the closest culturable relative to Las, Liberibacter crescens ,macetas cultivo as a model organism, we have quickly and inexpensively screened a variety of likely phloem mobile compounds for effectiveness prior to field experiments. An existing model describes the optimal chemical characteristics of phloem mobile xenobiotics as: an octanol/water partition coefficient between -1.5 and 2.5, a molar volume ≤300 cm3mol- 1 , and ionizability between 2 and 15 . Following these assumptions, we selected and screened 145 compounds for Lc sensitivity using a high-throughput 96-well assay method. The percent inhibition was evaluated for multiple concentrations of each compound and the MIC90 and MIC50 was noted. Of the 145 screened compounds, 50 showed ≥90% inhibition at one or more concentrations. Of those, 12 were penicillins, 16 were cephalsporins, 2 were carbapenems, 7 were tetracyclines, 6 were aromatic hydrocarbons, 3 were nitro compounds, and 4 were organic chemicals . The 27 quinolines and 20 sulfones tested showed little impact on Lc growth. Our next step is to test the phytotoxicity of those antimicrobials that are phloem mobile and inhibit Lc. Oxytetracycline fits these criteria and showed no phytotoxicity after repeated foliar sprays at 200 ppm. Using this selection and assay method, we can efficiently sift through multiple treatment options and only select the most suitable for more laborious field-testing, and ideally provide a short-term treatment option for commercial citrus growers. Eventually, these results could help guide the production of effective novel compounds that will provide additional treatment options.Current strategies for managing Huanglongbing include area–wide psyllid vector control, inoculum removal, use of clean planting stock, and foliar nutritional supplements to sustain productivity of groves with infected trees. Foliar nutritional supplementation has had mixed results, in part, because the basis for such supplementation to suppress HLB has either not been established or is useful in correcting specific nutrient deficiencies observed in trees. The possibility that other mechanisms such as toxicity and other nutrient interactions could be interfering with tree metabolism has not been addressed. In this multi-year field/lab study , the impact of HLB infection on leaf mineral contents of adequately-fertilized grapefruit and sweet orange trees was investigated in Texas. Symptomatic and non-symptomatic leaves from known HLB-infected trees and leaves from non-infected trees, selected based on visual observations and qPCR tests, were analyzed for mineral composition .
Leaves from HLB-infected trees had significantly higher TNC levels than leaves from healthy trees. HLB-infected trees also exhibited significant decreases in leaf nitrogen , phosphorus , magnesium , calcium and zinc , and significant increases in sodium , copper , and boron concentrations of symptomatic leaves compared to healthy trees, whereas asymptomatic leaves from HLB-infected trees had intermediate values. Significant correlations were obtained between leaf nutrient concentration and CT values. The present observations shed further light on the physiological and biochemical changes associated with HLB disease development. Since these groves were well fertilized, the observed differences seem to arise, at least in part, from an imbalance in uptake of soil minerals, suggesting that the decline in leaf/tree physiological function may be due to toxicity associated with elevated tissue concentrations of Na and other trace elements.In 2011, we have demonstrated that feeding dsRNA targeting specific genes within the insect are toxic through RNA silencing mechanisms. This approach opened the door to use psyllid specific dsRNAs that high amounts are by citrus tristeza virus in the phloem, the site of the viral replication and D. citri site of feeding. Some of the dsRNA molecules can move in the citrus once expressed in the phloem into the xylem a minor site of psyllids feeding. Two important targets were selected: a) important gut digestive enzyme and b) enzymes that control synthesis and metabolism of juvenile hormone, an important hormone that controls metamorphism, egg development, and behavior. D. citri infected with Liberibacter asiaticus were allowed to feed on citrus trees that were transfected with CTV synthesizing dsRNA targeting digestive and juvenile hormone synthesis and metabolic enzymes. Adult D. citri were assayed initially by PCR for L. asiaticus and allowed to lay eggs on the leaves and the newly emerged adults were assayed for number of surviving adults and for L. asiaticus titers using PCR. A marked decrease in the number of the surviving adults was noted when compared with controls citrus trees that did not express dsRNA. The surviving adults when assayed by PCR were negative for L. asiaticus indicating that expressing specific dsRNA molecules in citrus can be highly effective against the spread of L. asiaticus by D. citri. A road map to control psyllid infestations and L. asiaticus spread will be discussed. Only two therapies have been shown to be effective for treating trees infected with “Candidatus Liberibacter asiaticus” : thermal therapy and bactericide therapy. There have been reports of several bactericides reducing CLas titer and alleviating HLB symptoms.