Growers rely on broad-spectrum agrochemicals to manage one of the most economically important fruit tree diseases, huanglongbing (HLB, citrus greening disease), presumptively caused by the gram-negative bacterium ‘Candidatus Liberibacter asiaticus’ (CLas). While genetic resistance would be an attractive alternative to chemical management, this option is not yet available for HLB. Here, we tested whether a single chain variable fragment (scFv) antibody targeting the predicted CLas outer membrane transporter NodT can inhibit CLas growth or HLB disease development when expressed in ‘Duncan’ grapefruit (Citrus x paradisi). CLas NodT is similar to TolC and could be involved in Type I secretion and virulence. The scFv antibody was expressed in grapefruit trees as a C-terminal translational fusion to the Flowering Locus T protein of Poncirus trifoliata (FT-scFv) to promote expression in the phloem. Wild type and FT-scFv-expressing transgenic lines were challenged with CLas using three inoculation approaches: psyllid-mediated inoculation, vegetative graft inoculation, and natural exposure in grove-like conditions. With the first two approaches, HLB symptom expression and CLas bacterial titers in FT-scFv grapefruit lines were not significantly different from wild-type controls. In the grove trial, one FT-scFv line exhibited a slight, but significant, reduction in canopy chlorosis compared to wild-type controls. Wild type and FT-scFv lines were similar in all other metrics. We conclude that the expression of anti-NodT FT-scFv antibody in grapefruit did not decrease HLB susceptibility. Although our approach was unsuccessful, we hope that documenting our results will be useful for those seeking to develop HLB-resistant citrus germplasm in the future.

2. Ozgur Batuman, associate professor, Plant Pathology Department, UF/IFAS Southwest Florida REC; Amit Levy, assistant professor, Plant Pathology Department, UF/IFAS Citrus REC; Mark E. Hilf, research plant pathologist (retired), USDA-ARS-USHRL; Peggy J. Sieburth, biological scientist III (retired), Bureau of Citrus Budwood Registration, FDACS DPI; Ronald H. Brlansky, professor emeritus; and William O. Dawson, professor emeritus, Plant Pathology Department, UF/IFAS Citrus REC; UF/IFAS Extension, Gainesville, FL 32611.

Vegetative grafting is a common method of transmitting and propagating ‘Candidatus Liberibacter asiaticus’, the bacterial species accepted as the causal agent of the citrus disease huanglongbing (HLB). Generally, infected tissue that is grafted to a receptor tree remains in place indefinitely to ensure transmission. In this study, individual HLB-symptomatic leaves were grafted as ‘Ca. L. asiaticus’ inoculum sources to receptor trees of six citrus types and removed after an inoculation period (IP) of 21, 51, or 81 days. The goal was to assess the effect of varying IPs on transmission of bacteria to the receptor tree and on the successful establishment of a new infection. Survival analysis of data from three trials showed a significantly reduced proportion of infected trees with an IP of 21 days compared with IPs of 51 and 81 days but that there was no significant difference in the proportion of infected trees between IPs of 51 and 81 days. In addition, the time to first detection of pathogen DNA in an infected tree was delayed significantly for the 21-day IP when compared with the 51- and 81-day IPs. Survival analysis showed that the probability of infection of sweet orange trees was significantly higher than for trees of five other citrus types throughout the experiment. There was no significant difference between the infection probabilities of these latter five citrus types. The data from this study show that successful infection by grafting is dependent upon the time of exposure to the inoculum, that shorter IPs increase the time needed to establish a systemic infection, and that citrus types vary in their overall susceptibility to infection by ‘Ca. L. asiaticus’.

Citrus tristeza virus (CTV) is a major cause of the decline and eventual death of trees on sour orange rootstocks. This 2-page fact sheet is part of the 2018–2019 Florida Citrus Production Guide. Written by O. Batuman, A. Levy, M. E. Hilf, P. J. Sieburth, W. O. Dawson, and R. H. Brlansky, and published by the Plant Pathology Department, January 2019. PP-181/CG039: 2022–2023 Florida Citrus Production Guide: Tristeza Decline (ufl.edu)

Citrus tristeza virus (CTV) is a major cause of the decline and eventual death of trees on sour orange rootstocks. This 2-page fact sheet is part of the 2019–2020 Florida Citrus Production Guide. Written by O. Batuman, A. Levy, M. E. Hilf, P. J. Sieburth, W. O. Dawson, and R. H. Brlansky, and published by the Plant Pathology Department, March 2019. PP-181/CG039: 2022–2023 Florida Citrus Production Guide: Tristeza Decline (ufl.edu)

‘Candidatus Liberibacter asiaticus’ is a phloem-colonizing intracellular bacterial pathogen of citrus associated with the disease huanglongbing. A study of patterns of colonization and bacterial population growth in new growth of different citrus types was conducted by pruning infected citron, sweet orange, sour orange, mandarin, citrange, and Citrus macrophylla trees to force the growth of axillary and adventitious shoots. The first three leaves on newly emerged shoots were collected at 30, 60, and 90 days to assess colonization and population growth of ‘Ca. L. asiaticus’ using real time PCR (qPCR). Single trials were conducted with mandarin and citron, two trials each for citrange, sour orange and sweet orange, and four trials for C. macrophylla. In citron the proportion of colonized leaves increased significantly over time, with 67, 85, and 96% of leaves colonized at 30, 60, and 90 days, respectively. For the other citrus types, the exact proportion of colonized leaves differed, but colonization exceeded 60% in mandarin, sour orange, and citrange, and exceeded 80% at 30 days in two trials with sweet orange and three trials with C. macrophylla, but there was no significant increase in the proportion of colonized leaves at 60 and 90 days. Bacteria were readily detected by 30 days in new leaves of all citrus types. Differences in the growth of the bacterial population between citrus types and at different times of the year were noted, but common trends were apparent. In general, bacterial titers peaked at 60 days, except in leaves of C. macrophylla where bacterial titers peaked at 30 days. The early and consistently high proportion of leaf colonization observed for new growth of sweet orange during two trials and for C. macrophylla during three trials indicates a near synchronous colonization of new leaves by 30 days.

Huanglongbing (HLB) is a chronic, progressive decline disease in citrus associated with a systemic infection by the bacterium 'Candidatus Liberibacter asiaticus'. Transmission of the bacterium in the field is by the Asian citrus psyllid, Diaphorina citri Kuwayama. Experimental propagation of 'Ca. L. asiaticus' is done primarily by grafting pieces of bud wood from an infected plant. To produce a small-scale model system for investigation of pathogen biology, we investigated grafting single leaves from infected citrus plants as sources of inoculum for propagation of the bacterium. In total, 162 plants ranging in age from 3 to 18 months were grafted. Grafting with intact asymptomatic and HLB-symptomatic leaves resulted in 61 of 78 (78%) and 35 of 41 (85%) of the plants infected with 'Ca. L. asiaticus', respectively. Inoculum consisting of the leaf petiole only or only an inoculum tissue remnant under the bark of the receptor tree resulted in 6 of 12 (50%) and 7 of 31 (23%) infected trees, respectively. Real-time polymerase chain reaction (qPCR) assays verified the infection in plants, a majority of which developed the foliar blotchy mottle symptom considered diagnostic for HLB, while some plants also displayed the stunted, chlorotic shoots for which the disease is named. The qPCR data together with the symptoms displayed demonstrated that individual leaves from infected trees can serve as effective inoculum sources for transmission and propagation of 'Ca. L. asiaticus' via grafting.

Huanglongbing (HLB) disease is the most serious threat to citrus production worldwide and, in the last decade, has devastated the Florida citrus industry. In the United States, HLB is associated with the phloem-limited α-proteobacterium ‘Candidatus Liberibacter asiaticus’ and its insect vector, the Asian citrus psyllid (ACP; Diaphorina citri). Significant effort is being put forth to develop novel citrus germplasm that has a lower propensity to succumb to HLB than do currently available varieties. Effective methods of screening citrus germplasm for susceptibility to HLB are essential. In this study, we exposed small, grafted trees of 16 citrus types to free-ranging ACP vectors and ‘Ca. L. asiaticus’ inoculum in the greenhouse. During 45 weeks of exposure to ACP, the cumulative incidence of ‘Ca. L. asiaticus’ infection was 70%. Trees of Citrus macrophylla and C. medica were most susceptible to ‘Ca. L. asiaticus’, with 100% infection by the end of the test period in three trials, while the complex genetic hybrids ‘US 1-4-59’ and ‘Fallglo’ consistently were least susceptible, with approximately 30% infection. Results obtained in this greenhouse experiment showed good agreement with trends observed in the orchard, supporting the validity of our approach for screening citrus germplasm for susceptibility to HLB.

'Candidatus Liberibacter asiaticus' (CLas), the bacterial pathogen associated with citrus greening disease, is transmitted by Diaphorina citri, the Asian citrus psyllid. Interactions among D. citri and its microbial endosymbionts, including 'Candidatus Profftella armatura', are likely to impact transmission of CLas. We used quantitative mass spectrometry to compare the proteomes of CLas(+) and CLas(-) populations of D. citri, and found that proteins involved in polyketide biosynthesis by the endosymbiont Profftella were up-regulated in CLas(+) insects. Mass spectrometry analysis of the Profftella polyketide diaphorin in D. citri metabolite extracts revealed the presence of a novel diaphorin-related polyketide and the ratio of these two polyketides was changed in CLas(+) insects. Insect proteins differentially expressed between CLas(+) and CLas(-) D. citri included defense and immunity proteins, proteins involved in energy storage and utilization, and proteins involved in endocytosis, cellular adhesion, and cytoskeletal remodeling which are associated with microbial invasion of host cells. Insight into the metabolic interdependence between the insect vector, its endosymbionts, and the citrus greening pathogen reveals novel opportunities for control of this disease, which is currently having a devastating impact on citrus production worldwide.

A survey of seven citrus cultivars (Citrus sinensis, C. paradisi, ‘Temple’ tangor, ‘Minneola’ and ‘Orlando’ tangelos, and ‘Fallglo’ and ‘Sunburst’ mandarin hybrids) growing in commercial citrus orchards in Florida revealed a strong correlation between HLB incidence and severity and Candidatus Liberibacter asiaticus (CLas) titer (Stover and McCollum, 2011). Temple tangor and grapefruit consistently exhibited the least severe HLB symptoms and lowest CLas titers, followed in increasing order of HLB symptoms by ‘Fallglo’ and C. sinensis.In the orchard, Candidatus Liberibacter asiaticus (CLas) is transmitted to citrus hosts via the Asian citrus psyllid (ACP); however, in most greenhouse studies CLas inoculation of citrus has been conducted by grafting. In an attempt to more closely mimic the natural process of CLas transmission by ACP to citrus we conducted a greenhouse study that included CLas-infected citron (Citrus medica) to serve as a source of inoculum, free-ranging ACP to vector the pathogen, and 16 citrus genotypes (C. medica, C. reticulata, C. grandis, C. sinensis, C. paradisi, Poncirus trifoliata, and nine citrus hybrids) as hosts. Our objectives were to determine the incidence of CLas infection and titer among the 16 genotypes over time. The experiment was conducted three times. Leaf samples were collected at regular intervals over a period of ca. 300 days, and each sample was assayed for the presence of CLas. In each experiment, CLas titer remained at less than 101 copies 16S rDNA g-1 fwt until 150 to 175 days after placing CLas negative trees into the greenhouse, and thereafter increased steadily for the remainder of the experiments. After 300 to 350 days in the greenhouse grand means for CLas titer ranged from 103 to 105 g-1 fwt, although HLB symptoms were not apparent. Significant differences in CLas titer among the cultivars were first detected at approximately 125 days and throughout the remainder of the experiment. Our results have significant implications for studies involving transmission of CLas by ACP and subsequent disease development.

Huanglongbing (HLB) is a disease of citrus associated with a systemic infection by the α-proteobacterium ‘Candidatus Liberibacter asiaticus’. Infection of an individual tree can occur via psyllids (Diaphorina citri Kuwayama) carrying the bacterium or if the tree is propagated from infected budwood. Seed transmission is another possible mode of dissemination of the pathogen. Rootstock varieties are propagated from seed so we assessed the seed transmission among eighteen rootstock varieties using seeds from mature fruit collected in late winter and immature fruit collected in late summer. In dissected seeds real-time PCR detected pathogen DNA in seed coats at an incidence of 0-100%, whereas no pathogen DNA was detected in cotyledons or embryos from any variety. Seeds collected in late winter were germinated in a greenhouse and no pathogen DNA was detected in extracts of shoots of 425 seedlings harvested at 7-10 days post-germination whereas a small amount of pathogen DNA was detected in extracts from 6 of 425 roots. All six positive samples were the same rootstock variety, which had 100% colonization of seed coats. The positive samples likely are a result of remnant seed coat, which was not completely removed from cotyledons of the harvested seedlings. The data from this study suggest the pathogen can colonize the seed coat but it does not colonize embryos, which makes seed transmission unlikely.

Huanglongbing, or citrus greening disease, is associated with infection by the phloem-limited bacterium 'Candidatus Liberibacter asiaticus'. Infection with 'Ca. L. asiaticus' is incurable; therefore, knowledge regarding 'Ca. L. asiaticus' biology and pathogenesis is essential to develop a treatment. However, 'Ca. L. asiaticus' cannot currently be successfully cultured, limiting its study. To gain insight into the conditions conducive for growth of 'Ca. L. asiaticus' in vitro, 'Ca. L. asiaticus' inoculum obtained from seed of fruit from infected pomelo trees (Citrus maxima 'Mato Buntan') was added to different media, and cell viability was monitored for up to 2 months using quantitative polymerase chain reaction in conjunction with ethidium monoazide. Media tested included one-third King's B (K), K with 50% juice from the infected fruit, K with 50% commercially available grapefruit juice, and 100% commercially available grapefruit juice. Results show that juice-containing media dramatically prolong viability compared with K in experiments reproduced during 2 years using different juice sources. Furthermore, biofilm formed at the air-liquid interface of juice cultures contained 'Ca. L. asiaticus' cells, though next-generation sequencing indicated that other bacterial genera were predominant. Chemical characterization of the media was conducted to discuss possible factors sustaining 'Ca. L. asiaticus' viability in vitro, which will contribute to future development of a culture medium for 'Ca. L. asiaticus'.

We determined the frequency of Ct values for 20,000 HLB diagnostic samples collected by field scouts in Florida. The Ct frequency distribution revealed that the vast majority of samples produced no amplicon (Ct values > 40) and were therefore considered negative for CLas, indicating that scouts were commonly sampling leaves that exhibiting symptoms that mimicked HLB, but were not actually HLB. However, these samples were collected fairly early in the Florida HLB epidemic when symptoms were more ambiguous than is the current situation. Of the samples that did produce amplicons, Ct = 22 was by far the most frequent value and was the peak of a perfectly symmetrical bell shaped curve with a leading edge of Ct=28 and a tailing edge of Ct=18. Because scouts collect samples based on visual HLB symptoms we can conclude that Ct values between 28 and 18 represent the range of Ct values most typical of symptomatic leaves. Conversion of Ct values to CLas genomes∙g-1 fwt based on a standard curve reveals that: 1) the majority of samples were CLas-negative (Ct > than 38); 2) CLas-infected, asymptomatic leaves have CLas titers of 102 to 104 genomes g∙-1 fwt (Ct 38-30); 3) the titer of CLas in HLB-symptomatic fwt is 106 to 107 genomes∙ g-1 fwt (Ct 24-19) and 4) no samples exceed 108 CLas genomes g∙-1 fwt (Ct < 18). These results provide insights into the relationship between Ct values, CLas titers and HLB symptoms.

Citrus greening (Huanglongbing, HLB) is one of the most destructive diseases of citrus worldwide. In South Asia HLB has been known for more than a century, while in Americas the disease was found relatively recently. HLB is associated with three species of 'Candidatus Liberibacter' among which 'Ca. Liberibacter asiaticus' (CLas) has most wide distribution. Recently, a number of studies identified different regions in the CLas genome with variable number of tandem repeats (VNTRs) that could be used for examination of CLas diversity. One of the objectives of the work presented here was to further validate the VNTR analysis-based approach by assessing the stability of these repeats upon multiplication of the pathogen in a host over an extended period of time and upon its passaging from a host to a host using CLas populations from Florida. Our results showed that the numbers of tandem repeats in the four loci tested display very distinguishable "signature profiles" for the two Florida-type CLas haplotype groups. Remarkably, the profiles do not change upon passage of the pathogen in citrus and psyllid hosts as well as after its presence within a host over a period of five years, suggesting that VNTR analysis-based approach represents a valid methodology for examination of the pathogen populations in various geographical regions. Interestingly, an extended analysis of CLas populations in different locations throughout Florida and in several countries in the Caribbean and Central America regions and in Mexico where the pathogen has been introduced recently demonstrated the dispersion of the same haplotypes of CLas. On the other hand, these CLas populations appeared to differ significantly from those obtained from locations where the disease has been present for a much longer time.

Citrus tristeza virus (CTV) is the most destructive viral pathogen of citrus and has been an important concern for the citrus industry in the Dominican Republic. Earlier studies documented widespread distribution of mild isolates of the T30 genotype, which caused no disease in the infected trees, and a low incidence of isolates of the VT and T3 genotypes, which were associated with economically damaging decline and stem-pitting symptoms in sweet orange and Persian lime, the two major citrus varieties grown in the Dominican Republic. In light of the dramatic increase in the number of severely diseased citrus trees throughout the country over the last decade, suggesting that field populations of CTV have changed, we examined the CTV pathosystem in the Dominican Republic to assess the dynamics of virus populations. In this work, we characterized the molecular composition of 163 CTV isolates from different citrus-growing regions. Our data demonstrate a dramatic change in CTV populations, with the VT genotype now widely disseminated throughout the different regions and with the presence of two new virus genotypes, T36 and RB. Multiple infections of trees resulted in development of complex virus populations composed of different genotypes.

'Candidatus Liberibacter asiaticus' is the bacterium implicated as a causal agent of the economically damaging disease of citrus called huanglongbing (HLB). Vertical transmission of the organism through seed to the seedling has not been demonstrated. Previous studies using real-time polymerase chain reaction assays indicated abundant bacterial 16S rRNA sequences in seed coats of citrus seed but the presence of intact bacterial cells was not demonstrated. We used microscopy to verify that intact bacterial cells were present in citrus seed coats. Bacterial cells with the morphology and physical dimensions appropriate for 'Ca. L. asiaticus' were seen in phloem sieve elements in the vascular bundle of grapefruit seed coats using transmission electron microscopy (TEM). Fluorescence in situ hybridization (FISH) analyses utilizing probes complementary to the 'Ca. L. asiaticus' 16S rRNA gene revealed bacterial cells in the vascular tissue of intact seed coats of grapefruit and pummelo and in fragmented vascular bundles excised from grapefruit seed coats. The physical measurements and the morphology of individual bacterial cells were consistent with those ascribed in the literature to 'Ca. L. asiaticus'. No bacterial cells were observed in preparations of seed from fruit from noninfected trees. A small library of clones amplified from seed coats from a noninfected tree using degenerate primers targeting prokaryote 16S rRNA gene sequences contained no 'Ca. L. asiaticus' sequences, whereas 95% of the sequences in a similar library from DNA from seed coats from an infected tree were identified as 'Ca. L. asiaticus', providing molecular genetic corroboration that the bacterial cells observed by TEM and FISH in seed coats from infected trees were 'Ca. L. asiaticus'.