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Huanglongbing (HLB), also known as citrus greening, is an insidious disease in citrus and has become a threat to the sustainability of the citrus industry worldwide. In the U.S., Candidatus Liberibacter asiaticus (CLas) is the pathogen that is associated with HLB, an unculturable, phloem-limited bacteria, vectored by the Asian Citrus Psyllid (ACP, Diaphorina citri). There is no known cure nor treatment to effectively control HLB, and current control methods are primarily based on the use of insecticides and antibiotics, where effectiveness is limited and may have negative impacts on beneficial and non-target organisms. Thus, there is an urgent need for the development of effective and sustainable treatment options to reduce or eliminate CLas from infected trees. In the present study, we screened citrus-derived endophytes, their cell-free culture supernatants (CFCS), and crude plant extracts for antimicrobial activity against two culturable surrogates of CLas, Sinorhizobium meliloti and Liberibacter crescens. Candidates considered high-potential antimicrobial agents were assessed directly against CLas in vitro, using a propidium monoazide–based assay. As compared to the negative controls, statistically significant reductions of viable CLas cells were observed for each of the five bacterial CFCS. Subsequent 16S rRNA gene sequencing revealed that each of the five bacterial isolates were most closely related to Bacillus amyloliquefaciens, a species dominating the market of biological control products. As such, the aboveground endosphere of asymptomatic survivor citrus trees, grown in an organic orchard, were found to host bacterial endophytes capable of effectively disrupting CLas cell membranes. These results concur with the theory that native members of the citrus microbiome play a role in the development of HLB. Here, we identify five strains of Bacillus amyloliquefaciens demonstrating notable potential to be used as sources of novel antimicrobials for the sustainable management of HLB. [ABSTRACT FROM AUTHOR]

We adopted a systems-based approach to determine the role of two Candidatus Liberibacter asiaticus (C Las) proteins, LasP 235 and Effector 3, in Huanglongbing (HLB) pathogenesis. While a published work suggests the involvement of these C Las proteins HLB pathogenesis, the exact structure-based mechanism of their action has not been elucidated. We conducted the following experiments to determine the structure-based mechanisms of action. First, we immunoprecipitated the interacting citrus protein partners of LasP 235 and Effector 3 from the healthy and C Las-infected Hamlin extracts and identified them by Liquid Chromatography with tandem mass spectrometry (LC–MS/MS). Second, we performed a split green fluorescent protein (GFP) assay in tobacco to validate that the interactions observed in vitro are also retained in planta. The notable in planta citrus targets of LasP 235 and Effector 3 include citrus innate immune proteins. Third, in vitro and in planta studies were performed to show that LasP 235 and Effector 3 interact with and inhibit the functions of multiple citrus proteins belonging to the innate immune pathways. These inhibitory interactions led to a high level of reactive oxygen species, blocking of bactericidal lipid transfer protein (LTP), and induction of premature programed cell death (PCD), all of which are beneficial to C Las lifecycle and HLB pathogenesis. Finally, we performed molecular dynamics simulations to visualize the interactions of LasP 235 and Effector 3, respectively, with LTP and Kunitz protease inhibitor. This led to the design of an LTP mimic, which sequestered and blocked LasP 235 and rescued the bactericidal activity of LTP thereby proving that LasP 235 , indeed, participates in HLB pathogenesis. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB), or citrus greening, is the most devastating disease in citrus worldwide. Commercial citrus varieties including sweet orange (Citrus sinensis) are highly susceptible to HLB, and trifoliate orange (Poncirus trifoliata , a close Citrus relative) is widely considered resistant or highly tolerant to HLB. In this study, an intergeneric F1 population of sweet orange and trifoliate orange was genotyped by Genotyping-by-Sequencing, and high-density SNP-based genetic maps were constructed separately for trifoliate orange and sweet orange. The two genetic maps exhibited high synteny and high coverage of the citrus genome. Progenies of the F1 population and their parents were planted in a replicated field trial, exposed to intense HLB pressure for 3 years, and then evaluated for susceptibility to HLB over 2 years. The F1 population exhibited a wide range in severity of HLB foliar symptom and canopy damage. Genome-wide QTL analysis based on the phenotypic data of foliar symptom and canopy damage in 2 years identified three clusters of repeatable QTLs in trifoliate orange linkage groups LG-t6, LG-t8 and LG-t9. Co-localization of QTLs for two traits was observed within all three regions. Additionally, one cluster of QTLs in sweet orange (linkage group LG-s7) was also detected. The majority of the identified QTLs each explained 18–30% of the phenotypic variation, indicating their major role in determining HLB responses. These results show, for the first time, a quantitative genetic nature yet the presence of major loci for the HLB tolerance in trifoliate orange. The results suggest that sweet orange also contains useful genetic factor(s) for improving HLB tolerance in commercial citrus varieties. Findings from this study should be very valuable and timely to researchers worldwide as they are hastily searching for genetic solutions to the devastating HLB crisis through breeding, genetic engineering, or genome editing. [ABSTRACT FROM AUTHOR]

Citrus Huanglongbing (HLB) associated with ‘Candidatus Liberibacter asiaticus’ (Las) and citrus canker disease incited by Xanthomonas citri are the most devastating citrus diseases worldwide. To control citrus HLB and canker disease, we previously screened over forty antimicrobial peptides (AMPs) in vitro for their potential application in genetic engineering. D2A21 was one of the most active AMPs against X. citri, Agrobacterium tumefaciens and Sinorhizobium meliloti with low hemolysis activity. Therefore, we conducted this work to assess transgenic expression of D2A21 peptide to achieve citrus resistant to canker and HLB. We generated a construct expressing D2A21 and initially transformed tobacco as a model plant. Transgenic tobacco expressing D2A21 was obtained by Agrobacterium-mediated transformation. Successful transformation and D2A21 expression was confirmed by molecular analysis. We evaluated disease development incited by Pseudomonas syringae pv. tabaci in transgenic tobacco. Transgenic tobacco plants expressing D2A21 showed remarkable disease resistance compared to control plants. Therefore, we performed citrus transformations with the same construct and obtained transgenic Carrizo citrange. Gene integration and gene expression in transgenic plants were determined by PCR and RT-qPCR. Transgenic Carrizo expressing D2A21 showed significant canker resistance while the control plants showed clear canker symptoms following both leaf infiltration and spray inoculation with X. citri 3213. Transgenic Carrizo plants were challenged for HLB evaluation by grafting with Las infected rough lemon buds. Las titer was determined by qPCR in the leaves and roots of transgenic and control plants. However, our results showed that transgenic plants expressing D2A21 did not significantly reduce Las titer compared to control plants. We demonstrated that transgenic expression of D2A21 conferred resistance to diseases incited by P. syringae pv. tabaci and X. citri but not Las. Our results underscore the difficulty in controlling HLB compared to other bacterial diseases. [ABSTRACT FROM AUTHOR]

The U.S. Department of Agriculture citrus scion breeding program is urgently working on developing huanglongbing (HLB; pathogen Candidatus Liberibacter asiaticus)-tolerant cultivars with excellent fruit quality and productivity when HLB-affected. The slow process of assessing new citrus hybrids is a major impediment to delivery of these much-needed cultivars. We generate thousands of hybrids each year, germinate the seedlings, grow them for 2 years in the greenhouse, plant them at high density in a field where the disease HLB is abundant, grow them for 5 to 10 years, and make selections based on tree performance and fruit quality of these HLB-affected trees. Based on promising reports of accelerated citrus growth when grown in a metallized reflective mulch (MRM) system, we tested the hypothesis that the MRM system may accelerate growth and selection of new hybrid seedlings compared with conventional soil culture (CSC). In the MRM system, tree rows are covered with a layer of metallized plastic film and drip irrigation is installed beneath the plastic. In 2 years of analysis, tree canopy volume was significantly greater with MRM in 2020 (27% greater than CSC) but not in 2021, and MRM tree height was greater in 2021 (7% greater than CSC). Mortality was significantly greater with MRM in both 2020 and 2021(in 2021: 32% vs. 17% under CSC), and MRM trees had more chlorotic leaves. Because of staff limitations, plant debris and soil were not routinely cleared from MRM, thus diminishing any benefit from the reflective surface. Better maintenance might have resulted in more sustained evidence of MRM growth benefits. With the current resource availability, the MRM system does not appear to accelerate the assessment of hybrid seedling trees. [ABSTRACT FROM AUTHOR]

Plants inoculated with the huanglongbing (HLB)-associated bacterium, Candidatus Liberibacter asiaticus (CLas) typically must be monitored for 8-10 months to identify differences in susceptibility between genotypes. Continuous light is reported to accelerate development of HLB symptoms and field observations suggest that trees girdled by tags or tree ties showed greater symptoms. Therefore, an experiment was conducted assessing HLB susceptibility as influenced by light/dark periods of 12 hours: 12 hours and 24 hours: 0 hours, in combination with scoring tree trunks to disrupt phloem. Sixty trees of each of three citrus genotypes ('Kuharske', previously shown to be HLB resistant; rough lemon, previously shown to be HLB tolerant; and 'Valencia', highly HLB susceptible) were bud grafted using two CLas-infected buds (rough lemon and citron) per tree on 26 Mar. 2012, and were placed in controlled growth rooms (one 12 hour light: 12 hour dark and one constant light) on 4 June 2012. Ten trees of each genotype in each growth room were scored 10 cm above the soil (cutting through the bark but not the wood) with a knife on 18 July 2012 and the scoring was repeated at the same scoring wounds on 30 Aug. 2012. Trees were removed from growth rooms on 12 Dec. 2012 and subsequently maintained in a greenhouse. At two to three month intervals between June 2012 and May 2013, HLB symptoms and stem diameter at 5 cm above the soil were assessed, and three leaves per tree were collected for quantitative polymerase chain reaction (qPCR) determination of CLas titer. Six months after inoculation and 3 months following imposition of treatments, the 'Valencia' scored in the 12 hour light: 12 hour dark regime, the 'Valencia' non scored trees in 24 hours of light and the 'Kuharske' scored trees in 24 hours of light displayed higher CLas titers than most other trees. After an additional two months, both scored and non-scored trees of all three genotypes in 24 hours of light had significantly elevated CLas titers compared with trees in 12 hour light: 12 hour dark regime, but within most treatments all three genotypes had titers which were not statistically different from each other. Growth of 'Kuharske' and rough lemon was enhanced; whereas 'Valencia' growth was reduced when graft-inoculated plants were maintained in continuous light. Scoring enhanced early CLas development in 'Kuharske' when combined with continuous light, had no effect in rough lemon, and showed inconsistent effects in 'Valencia'. Although continuous lighting enhanced disease progression, it did not reveal differences in HLB susceptibility. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB or citrus greening disease) caused by Candidatus Liberibacter asiaticus (Las) is a great threat to the US citrus industry. There are no proven strategies to eliminate HLB disease and no cultivar has been identified with strong HLB resistance. Citrus canker is also an economically important disease associated with a bacterial pathogen (Xanthomonas citri). In this study, we characterized endogenous citrus thionins and investigated their expression in different citrus tissues. Since no HLB-resistant citrus cultivars have been identified, we attempted to develop citrus resistant to both HLB and citrus canker through overexpression of a modified plant thionin. To improve effectiveness for disease resistance, we modified and synthesized the sequence encoding a plant thionin and cloned into the binary vector pBinPlus/ARS. The construct was then introduced into Agrobacterium strain EHA105 for citrus transformation. Transgenic Carrizo plants expressing the modified plant thionin were generated by Agrobacterium-mediated transformation. Successful transformation and transgene gene expression was confirmed by molecular analysis. Transgenic Carrizo plants expressing the modified thionin gene were challenged with X. citri 3213 at a range of concentrations, and a significant reduction in canker symptoms and a decrease in bacterial growth were demonstrated compared to nontransgenic plants. Furthermore, the transgenic citrus plants were challenged with HLB via graft inoculation. Our results showed significant Las titer reduction in roots of transgenic Carrizo compared with control plants and reduced scion Las titer 12 months after graft inoculation. These data provide promise for engineering citrus disease resistance against HLB and canker. [ABSTRACT FROM AUTHOR]

Assessments of the resistance of citrus germplasm to huanglongbing (HLB) can be expedited by inoculating plants under laboratory or greenhouse settings with the HLB bacterium, Candidatus Liberibacter asiaticus (CLas). Consistent rapid screening is critical to efficiently assess disease resistance among plant materials; however, a number of factors may govern the efficacy of such inoculations. Despite the rapidity at which HLB can spread in a grove, it often takes 8 to 10 months for high levels of CLas and HLB symptoms to develop even in highly susceptible sweet orange. Therefore, two experiments were conducted to assess factors that might influence efficiency in screening for HLB resistance. In one experiment, three test citrus genotypes ('Kuharske', previously shown to be HLB resistant; rough lemon, previously shown to be HLB tolerant; and 'Valencia', HLB susceptible) were bud grafted using CLas-infected buds from four different source genotypes. All bud source genotypes had similar levels of CLas titer, but citron, rough lemon, and Volkamer lemon were hypothesized to be better bud inoculum sources as they are more tolerant of HLB than 'Valencia'. Among the three test genotypes over all sources of infected buds, inoculations of 'Kuharske' resulted in lower CLas titers and fewer HLB symptoms than inoculations of rough lemon or 'Valencia'. Inoculations of rough lemon resulted in higher CLas titers and more pronounced HLB symptoms when it was inoculated using infected buds from rough lemon or 'Valencia'. Grafting 'Valencia' with infected buds from Volkamer lemon resulted in less disease than when 'Valencia' was grafted with infected citron, rough lemon, or 'Valencia' buds. Overall, these results suggest that the source of CLas-infected buds used to graftinoculate some genotypes will influence disease development. Trunk cross-sectional area increase for the year following infection was 33 higher in 'Kuharske' and rough lemon, compared with 'Valencia'. 'Kuharske' had very low levels of CLas (30 CLas/µg DNA), whereas 'Valencia' (43,000 CLas/µg DNA) and rough lemon (6700 CLas/ µg DNA) had relatively high levels. As an alternative to graft-inoculating plants with CLas-infected buds, plants can be subjected to infestations of CLas-infected Asian citrus psyllid (ACP) as occurs naturally. Of interest is if transmission rates of CLas and the development of HLB in a genotype are greater when the ACP have been feeding on the same host genotype. An experiment was therefore conducted to assess transmission of CLas by ACP reared on CLas-infected rough lemon to five different genotypes ('Carrizo', 'Flame' grapefruit, rough lemon, 'Temple', and 'Valencia'). These assessments were made using a detached leaf assay recognized as a faster method of gauging transmission rates of CLas than using whole plants. Higher percentages of ACP died when they were transferred from infected rough lemon to healthy 'Carrizo', and lower percentages died when they were transferred to rough lemon or 'Flame'. However, CLas transmission by infected ACP occurred to at least some leaves of each genotype in each of the five different assays, with 70% or more leaves of each genotype becoming infected in at least one assay. Over all assays, there was relatively little variation among genotypes in the percentage of leaves becoming CLas infected and in the titer of CLas developing in infected leaves. However, there were relatively large differences in transmission rates among individual assays unrelated to differences among test genotypes. Because of the rapidity of the detached leaf assay, efforts are merited to improve consistency of this inoculation method. [ABSTRACT FROM AUTHOR]

The citrus disease huanglongbing (HLB) has become endemic in Florida, with estimates that greater than 80% of citrus trees are currently infected. Although there are no commercial citrus varieties with strong HLB resistance, some field tolerance has been observed in trees exposed to the disease after they were mature. There is great urgency to identify citrus which may permit economic citrus production where HLB is endemic. Therefore, the objective of this study was to assess field tolerance to HLB. To expedite the trial due to urgency, nursery trees were purchased on rootstock varieties as available. The trial included the following unbalanced scion/rootstock combinations: 'Hamlin/ Kinkoji', 'Hamlin/Cleopatra', 'Temple/Cleopatra', 'Fallglo/Kinkoji', 'Sugar Belle/Sour Orange', 'Tango/Kuharske', and 'Ruby Red/Kinkoji', with most comparisons based solely on scion/rootstock combinations. A randomized complete block experiment was established at Fort Pierce, FL, in Sept. 2010. All trees exhibited symptoms of HLB and tested positive for the Candidatus Liberibacter asiaticus (CLas) bacterium by Oct. 2012, with similar titers [directly assessed as cycle threshold (Ct) using quantitative polymerase chain reaction (qPCR)] measured for all scion/rootstocks at most sample dates, but early titer development in 'Ruby Red/Kinkoji' was significantly lower than several other scion/rootstocks. Across all time-points, 'Fallglo/Kinkoji' had the lowest rating of distinctive HLB mottling and 'Ruby Red/Kinkoji' had the highest rating, but 'Sugar-Belle/Sour Orange' had the highest percentage of leaves affected. After 5 years, 'SugarBelle/Sour Orange' and 'Tango/Kuharske' had the greatest overall increase in trunk diameter, and were among the healthiest in overall appearance. In Oct. 2015, 'SugarBelle/Sour Orange' and 'Tango/Kuharske' trees had significantly greater fruit load (80-88 fruit/tree) followed by 'Temple/Cleopatra' and 'Fallglo/Kinkoji' (31-35 fruit/tree) while 'Hamlin/Kinkoji', 'Hamlin/Cleopatra', and 'Ruby Red/Kinkoji' produced less than 20 fruit per tree. Despite becoming infected by CLas in less than 2 years after planting, the trees continued to grow and all scion/rootstocks displayed increasing fruit production, although very low in 'Hamlin/Kinkoji', 'Hamlin/Cleopatra', and 'Ruby Red/Kinkoji'. Growth and fruit production in the highest performing scion/ rootstocks were likely less than would be expected for healthy trees, but these results are promising, with markedly better response of some scion/rootstocks with mandarin hybrid scions compared with trees with sweet orange or grapefruit scions. Larger fully replicated trials are underway. It is noteworthy that the most pronounced HLB symptoms and higher early pathogen titer, which are the two criteria most widely used in assessing HLB resistance, were not associated with the lowest growth and cropping, and focus on early symptomatic traits when screening for resistance may obscure important disease tolerance. [ABSTRACT FROM AUTHOR]

Background: Citrus Huanglongbing (HLB) is a bacterial disease with high economic significance. The associated agent Candidatus Liberibacter asiaticus is a fastidious, phloem-limited, intracellular bacterium that is transmitted by an insect vector the Asian citrus psyllid (ACP). The genome of Ca. L. asiaticus contains protein secretion machinery that suggests host cell modulation capacity of this bacterium. Results: A total of 28 candidate effectors, an important class of secreted proteins, were predicted from the Ca. L. asiaticus genome. Sequence specific primers were designed for reverse transcription (RT) and quantitative PCR (qPCR), and expression was validated for 20 of the effector candidates in infected citrus with multiple genetic background. Using detached leaf inoculation, the mRNA of effectors was detected from 6 h to 7 days post ACP exposure. It was observed that higher bacterial titers were associated with a larger number of effectors showing amplification across all samples. The effectors' expression were compared in citrus hosts with various levels of HLB tolerance, including susceptible Duncan grapefruit and Washington navel orange, tolerant citron and Cleopatra mandarin, and resistant Pomeroy trifoliate and Carrizo citrange. Across all genotypes relatively high expression was observed for CLIBASIA_03695, CLIBASIA_00460, CLIBASIA_00420, CLIBASIA_04580, CLIBASIA_05320, CLIBASIA_04425, CLIBASIA_00525 and CLIBASIA_05315 in either a host-specific or -nonspecific manners. The two genotypes in each HLB-response group also show effector-expression profiles that seem to be different. In a companion study, the expression of effectors was compared between leaves and roots of own-rooted citrus that had been Ca. L. asiaticus-infected for more than a year. Results indicated relatively high expression of CLIBASIA_03875, CLIBASIA_04800 and CLIBASIA_05640 in all leaf and some root tissues of citron, Duncan and Cleopatra. Conclusion: This temporal and spatial expression analysis of Ca. L. asiaticus effectors identified candidates possibly critical for early bacterial colonization, host tolerance suppression and long-term survival which are all worthy of further investigation. [ABSTRACT FROM AUTHOR]

Asiatic huanglongbing (HLB) is a devastating disease of citrus associated in North America with the bacterium 'Candidatus Liberibacter asiaticus' (LAS) vectored by the Asian citrus psyllid (ACP), Diaphorina citri Kuwayama. ACP management is considered a vital component of a program aimed at reducing the incidence and spread of HLB. Considerable research has been published comparing the efficacy of different insecticides for ACP control as well as on seasonal strategies for applying ACP insecticides. However, published information was largely lacking for even the most intense insecticide programs on their effectiveness for keeping HLB out of a new citrus planting in Florida citrus. We therefore conducted two replicated (individual plots 0.2 to 0.4 ha in size) experiments on protecting young citrus from HLB using different ACP management programs. An intensive insecticide program was evaluated in each experiment: eight annual calendar applications of traditional insecticides (hereafter referred to as the "complete" program). In one experiment, citrus was either planted alone and subjected to the complete program or citrus was interplanted with orange jasmine, Murraya exotica L. (a favored ACP host plant) and subjected to a reduced insecticide program (four calendar sprays of traditional insecticides). There was one set of plots in which both jasmine and citrus were treated with insecticides and one set in which jasmine was not treated at all. In the second experiment, citrus was either subjected to the complete program or to one of two other programs: a reduced insecticide program consisting of five calendar applications of traditional insecticides or a mineral oil program (oil applications every 3 weeks plus one dormant insecticide spray). The results of the two experiments were similar. Relatively good ACP control was achieved under each ACP management program during the first year but, as the experiments progressed and trees increased in size, ACP outbreaks occurred regardless of the psyllid management program. Little HLB developed under any ACP management program during the first year, but thereafter HLB increased and large percentages of the trees in each experiment became LAS-infected in less than two to three years. The combined results of the experiments indicated that up to eight monthly pesticide treatments per year applied on a calendar schedule were ineffective for preventing young citrus from becoming diseased. Of important significance is that the orchard within which the two experiments were conducted was subjected to a minimal psyllid management program and contained many older trees known to be infected by the HLB pathogen, a challenging situation for getting young trees into production without contracting the disease. Thus, the ACP management programs we evaluated might have been more effective if ACP in the surrounding areas had been more aggressively controlled and diseased trees in the surrounding areas removed to reduce inoculum loads. Also, the programs might have been more effective for slowing the spread of HLB if our research plots had been larger. [ABSTRACT FROM AUTHOR]

Huanglongbing (HLB) is one of the most destructive diseases of citrus worldwide. The three known causal agents of HLB are species of α-proteobacteria: 'Candidatus Liberibacter asiaticus', 'Ca. L. africanus', and 'Ca. L. americanus'. Previous studies have found distinct variations in temperature sensitivity and tolerance among these species. Here, we describe the use of controlled heat treatments to cure HLB caused by 'Ca. L. asiaticus', the most prevalent and heat-tolerant species. Using temperature-controlled growth chambers, we evaluated the time duration and temperature required to suppress or eliminate the 'Ca. L. asiaticus' bacterium in citrus, using various temperature treatments for time periods ranging from 2 days to 4 months. Results of quantitative polymerase chain reaction (qPCR) after treatment illustrate significant decreases in the 'Ca. L. asiaticus' bacterial titer, combined with healthy vigorous growth by all surviving trees. Repeated qPCR testing confirmed that previously infected, heat-treated plants showed no detectable levels of 'Ca. L. asiaticus', while untreated control plants remained highly infected. Continuous thermal exposure to 40 to 42°C for a minimum of 48 h was sufficient to significantly reduce titer or eliminate 'Ca. L. asiaticus' bacteria entirely in HLB-affected citrus seedlings. This method may be useful for the control of 'Ca. Liberibacter'-infected plants in nursery and greenhouse settings. [ABSTRACT FROM AUTHOR]