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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 factorsmay govern the efficacy of such inoculations. Despite the rapidity at whichHLB 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 fromVolkamer 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/mg DNA), whereas ‘Valencia’ (43,000 CLas/mg DNA) and rough lemon (6700 CLas/ mg 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. Huanglongbing disease is considered the greatest threat to the U.S. citrus industry (Gottwald, 2010). In the United States, HLB is associated with CLas and its insect vector, Diaphorina citri, the ACP. Following initial infection, symptoms may take many months to become evident. As the disease progresses, trees become less vigorous, with thinning canopies and abnormal leaves contributing to yield loss and poor fruit quality (Bov e, 2006; Gottwald, 2010). Since HLB was first confirmed in Florida in 2005, it has been found in all 32 citrus producing counties in the state as well as in dooryard citrus in noncitrus producing counties (Halbert et al., 2008). It is becoming increasingly widespread in Texas, and several HLB-affected dooryard trees have now been identified in California (CDFA, 2015). Management of HLB in Florida focuses on insecticide applications for vector control in combination with a series of production practices intended to reduce stress in infected trees. These practices have markedly increased production costs per hectare, while productivity in infected Florida orchards is reported to show an estimated average reduction of 41% compared with healthy orchards (Singerman and Useche, 2015). A more sustainable approach for the management of HLB would be the cultivation of citrus cultivars which are unaffected, or less affected, by the disease rather than more susceptible types (Gottwald, 2010). Several studies indicate differences in HLB susceptibility among citrus cultivars and/or species, which may be sufficient to be economically significant (Stover and McCollum, 2011; Stover et al., 2015a, 2016). However, despite the rapid and devastating spread of HLB throughout Florida, it is remarkably difficult to quickly and efficiently screen for relative resistance and tolerance to HLB. Following graft-inoculation or exposure of germplasm to CLas-infected ACP, it often takes 8–10 months to observe a high level of CLas infection even in highly susceptible sweet orange. In discussions with many scientists studying HLB, several anecdotal observations suggested opportunities to improve inoculation efficiency. Many graft inoculations are made using buds from highly HLB-susceptible sweet orange which may have compromised health and reduced ability to transfer CLas. Therefore, this study investigates whether a graftinoculum source showing more tolerance to HLB would enhance CLas transfer and facilitate identifying differences in HLB susceptibility. Some researchers reported that transition of ACP from one genotype to another appeared to compromise ACP health and consequent transfer of CLas. Therefore, we also examined whether ACP, reared on a specific test genotype and infected with CLas, could transfer CLas to the same test genotype at a higher rate than to other genotypes. This was evaluated using a detached leaf inoculativity test that was reported to be a faster approach to assessing CLas transmission rates than using whole plants (Ammar et al., 2013). Materials and Methods All experimental trees were maintained in a greenhouse at the U.S. Horticultural HORTSCIENCE VOL. 51(7) JULY 2016 805 Research Laboratory (USHRL) in Fort Pierce, FL. Graft inoculations Six trees, each of ‘Valencia’ (Citrus sinensis, budded onto US-812, Citrus reticulata · Poncirus trifoliata), rough lemon (Citrus jambhiri, nucellar seedlings), citron (Citrus medica, cultivar Mhac Nao from rooted cuttings), and Volkamer lemon (Citrus volkamariana, nucellar seedlings) were graft inoculated on 20 Oct. 2010 with CLas using infected ‘Valencia’ budwood at three buds per tree, and these trees are hereafter referred to as source trees, because they were then used as sources of budwood for further graft inoculations. Rough lemon was shown to be more tolerant of HLB than ‘Valencia’, with sustained phloem transport even in heavily symptomatic leaves (Fan et al., 2012). Our observations indicate that Volkamer lemon and citron are also more HLB tolerant than sweet orange, and exceed rough lemon in maintaining relatively full canopies in HLB-infected trees, whereas ‘Valencia’ has substantial leaf loss and develops very thin canopies (Miles et al., 2016). The original inoculum of CLas was from an infected ‘Lisbon’ lemon (Citrus limon) from a commercial grove near Fort Pierce, FL (Albrecht and Bowman, 2008); it has been used extensively in experiments, with infected trees sustained through serial graft transmission. These source trees developed distinctive HLB mottling and CLas titer, and provided material for graft inoculations of three test tree genotypes: ‘Valencia’ (budded on rough lemon), ‘Kuharske’ (C. sinensis · P. trifoliata, from apomictic seed), and rough lemon (from apomictic seed). On 20 Mar. 2012, the three test genotypes were graft inoculated using buds from each of the four source genotypes. Two buds from the same source genotype were grafted onto each of 10 individuals of each test genotype, resulting in 10 trees each of 12 combinations (4 source genotypes · 3 target genotypes). Source buds with attached leaves were randomly assigned to test trees. Buds were cut from branches clearly showing blotchy mottle symptoms, which is a distinctive symptom of HLB, and buds with attached leaves were randomly assigned to test trees. Two source buds of the same genotype were used for each test tree. The petioles and midribs of leaves from each bud were frozen for quantitative polymerase chain reaction (qPCR) of CLas titer with complete accounting of which leaf corresponded to which bud. On 31 May 2012, any tree where both inoculum buds were dead received an additional two buds from the same initial inoculum source. At the time of bud-inoculation, test trees had a diameter of 4 to 8 mm and a height of 0.5 to 0.9 m. All trunk diameter measurements were made at 5 cm above the soil surface, which was just above the graft union on the budded trees. After budding, data were collected seven times between June 2012 and June 2013 on trunk diameter and percentage of leaves showing distinct blotchy mottle symptoms on each test tree. Mature leaves from flushes with some lignification on stems were collected on the same dates and were assessed for CLas titer using qPCR. Detached leaf assays with ACP The experiments were conducted using the detached leaf ass

Influence of Citrus Source and Test Genotypes on Inoculations with Candidatus Liberibacter asiaticus