Luo, Weiqi

Abstract Candidatus Liberibacter asiaticus (Las) is one of the causal agents of huanglongbing (HLB), the most devastating disease of citrus worldwide. Due to the intracellular lifestyle and significant genome reduction, culturing Las in vitro has proven to be extremely challenging. In this study, we optimized growth conditions and developed a semi-selective medium based on the results of nutritional and antibiotic screening assays. Using these optimized conditions, we were able to grow Las in the LG liquid medium with ca.100- to 1000-fold increase, which peaked after 4 to 6 weeks and were estimated to contain 106 to 107 cells/ml. The cultured Las bacteria remained in a dynamic state of growth for over 20 months and displayed limited growth in subcultures. The survival and growth of Las was confirmed by fluorescence in situ hybridization with Las-specific probes and expression of its metabolic genes. Growth of Las in the optimized medium relied on the presence of a helper bacterium, Stenotrophomonas maltophilia FLMAT-1 that is multi-drug resistant and dominant in the Las co-culture system. To recapitulate the disease, the co-cultured Las was inoculated back to citrus seedlings via psyllid feeding. Although the Las-positive rate of the fed psyllids and inoculated plants were relatively low, this is the first demonstration of partial fulfillment of Koch’s postulates with significant growth of Las in vitro and a successful inoculation of cultured Las back to psyllids and citrus plants that resulted in HLB symptoms. These results provide new insights into Las growth in vitro and a system for improvement towards axenic culture and anti-Las compound screening.

‘Candidatus Liberibacter asiaticus’ (Las) is the prominent species of Liberibacter associated with huanglongbing, a devastating disease of citrus worldwide. In this study, we report the identification of an ∼8.3-kb DNA region of the Las genome containing eight putative open reading frames flanked by two inverted repeats, which was not present in the Las str. psy62 genome. Comparisons with other genome sequences established this region as a unique genetic element associated with genome plasticity/instability. Primers specific for both the presence (Las wild type) and absence (Las mutant) of this region were designed to study the population dynamics and host adaptation of the two strains. Las populations with and/or without the wild-type strain were detected and differentiated in >2,300 samples that included psyllids, periwinkle, and several species of citrus. In psyllids, although a mixed population of the wild type and mutant was observed in most samples (88%), the wild-type Las was detected alone at a rate of 11%. In contrast, none of the infected citrus plants were positive for the wild type alone, which harbored either the mutant strain alone (8%) or a mixed population of the mutant and wild type (92%). Furthermore, the dynamics of these two major Las populations varied with different citrus hosts, whereas an in-depth study on grapefruit that did not rapidly succumb to disease revealed that the population of mutant alone increased with time, indicating that the absence of this genetic element is associated with the fitness of Las in planta under the selection pressure of its host.

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’.

‘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) 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.