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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]

In a Fort Pierce, FL, field planting, plant growth, and Huanglongbing (HLB) severity were assessed as indicators of HLB tolerance on progenies of 83 seed-source accessions of Citrus and Citrus relatives mainly from the Riverside, CA, genebank. The HLB-associated pathogen [Candidatus Liberibacter asiaticus (CLas)] and vector [asian citrus psyllid (ACP), Diaphorina citri] were abundant, and trees were naturally challenged for 6 years before metrics (leaf mottle, percent canopy mottle, overall health, canopy density, canopy width, canopy height, and trunk diameter) were collected in Oct. and Nov. 2015. The healthiest trees with low or no HLB symptoms were distant citrus relatives: Balsamocitrus dawei, Bergera koenigii, Casimiroa edulis, Clausena excavata, Murraya paniculata, and one accession of Severinia buxifolia. Within Citrus, most of the healthiest trees with densest canopies, little leaf loss, and greater growth were those with pedigrees that included Citrus medica (citron). These included progenies of Citrus hybrid ('Limon Real'), Citrus limetta, Citrus limettioides, Citrus limonia, C. medica, Citrus volkameriana, and some Citrus limon accessions. Trees in this category exhibited distinct leaf-mottle characteristic of HLB and substantial pathogen titers, but maintained dense canopies and exhibited good growth. Trees from seed-source accessions in the genus Citrus without citron in their background were generally among the least healthy overall with less dense canopies. The exceptions were progenies of two Citrus aurantium accessions, which were markedly healthier than progenies of other Citrus seed-source accessions not derived from citron. Linear regression analysis, between metrics collected and pedigree of seed parent, indicated that percentage of citron in the pedigree significantly correlated with measures of tolerance. Although no commercial Citrus genotypes yielded progenies with strong HLB resistance, in this field experiment several progenies maintained dense canopies and good growth, and may be useful for breeding HLB tolerant cultivars. [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]

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]