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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]

The year 2005 marked the 25th anniversary of the establishment of the U.S. Department of Agriculture (USDA) National Plant Germplasm System (NPGS), repositories devoted to clonally propagated, horticultural fruit and nut crops. During this quarter century, facilities in Hilo, Hawaii; Mayaguez, PR,; Miami, Fla.; and Riverside, Calif. were developed to preserve collections of tropical and subtropical fruit and nut crops; facilities in Brownwood, Texas; Corvallis, Ore.; Davis, Calif. and Geneva, N.Y. preserve the temperate crops. Each of these facilities now has internationally recognized, globally diverse collections of genetic resources for their assigned genera. Germplasm of unique genotypes are maintained as growing plants, evaluated for phenotypic and genotypic traits, documented in a national public germplasm database, and freely distributed as clonal propaggules to researchers and other germplasm users around the world. Seed collections represent wild populations for some crop relatives. These 8 genebanks maintain 30,000 accessions representing 1600 species of fruit and nut crops and their wild relatives. The genebanks distribute more than 15,000 accessions annually to international researchers. Although originally conceived as working collections for crop improvement, NPGS genebanks have also become invaluable in providing the raw materials for basic plant genetic research, reservoirs tot rare or endangered species or vulnerable landraces, archives of historic cultivars, and field classrooms for educating the public. These collections preserve botanical treasures as well as the American horticultural heritage for now and for future generations. [ABSTRACT FROM AUTHOR]