McCollum, Greg

Prophages/phages are important components of the genome of ‘Candidatus Liberibacter asiaticus’ (CLas), an unculturable alphaproteobacterium associated with citrus huanglongbing (HLB) disease. Phage variations have significant contributions to CLas strain diversity research, which provide critical information for HLB management. In this study, prophage variations among selected CLas strains from southern Texas were studied. The CLas strains were collected from three different CLas inhabitant environments: citrus leaf, citrus root, and Asian citrus psyllid (ACP), the vector of CLas. Regardless of the different habitats and time span, more than 80% of CLas strains consistently had both Type 1 and Type 2 prophages, the same prophage type profile as in CLas strains from Florida but different to those reported in California and China. Further studies were performed on prophage type diversity. Analyses on Type 1-specific PCR amplicon sequences (encoding an endolysin protein) revealed the presence of two groups: Type 1-A, clustered around prophage SC1 originating from Florida, and Type 1-B, clustered with prophage P-SGCA5-1 originating in California. Type 1-B strains were mostly from ACP of nearby citrus orchards. On the other hand, analyses on Type 2-specific PCR amplicon sequences (encoding a putative hypothetical protein) showed a single group clustering around prophage SC2 originated from Florida, although a different Type 2 prophage has been reported in California. The presence of two distinct Type 1 prophage groups suggested the possibility of two different CLas introductions in southern Texas. The results from this study provide an initial baseline of information on genomic and population diversity of CLas in Texas.

AbstractCandidatus Liberibacter asiaticus (CLas) has been associated with Huanglongbing, a lethal vector-borne disease affecting citrus crops worldwide. While comparative genomics has provided preliminary insights into the metabolic capabilities of this uncultured microorganism, a comprehensive functional characterization is currently lacking. Here, we reconstructed and manually curated genome-scale metabolic models for the six CLas strains A4, FL17, gxpsy, Ishi-1, psy62, and YCPsy, in addition to a model of the closest related culturable microorganism, L. crescens BT-1. Predictions about nutrient requirements and changes in growth phenotypes of CLas were confirmed using in vitro hairy root-based assays, while the L. crescens BT-1 model was validated using cultivation assays. Host-dependent metabolic phenotypes were revealed using expression data obtained from CLas-infected citrus trees and from the CLas-harboring psyllid Diaphorina citri Kuwayama. These results identified conserved and unique metabolic traits, as well as strain-specific interactions between CLas and its hosts, laying the foundation for the development of model-driven Huanglongbing management strategies.

Globally, citrus is threatened by huanglongbing (HLB), and the lack of effective control measures is a major concern of farmers, markets, and consumers. There is compelling evidence that plant health is a function of the activities of the plant's associated microbiome. Using Liberibacter crescens , a culturable surrogate for the unculturable HLB-associated bacterium “ Candidatus Liberibacter asiaticus,” we tested the hypothesis that members of the citrus microbiome produce potential anti-“ Ca . Liberibacter asiaticus” natural products with potential anti-“ Ca . Liberibacter asiaticus” activity. A subset of isolates obtained from the microbiome inhibited L. crescens growth in an agar diffusion inhibition assay. Further fractionation experiments linked the inhibitory activity of the fungus Cladosporium cladosporioides to the fungus-produced natural products cladosporols A, C, and D, demonstrating dose-dependent antagonism to L. crescens .

The 22–amino acid (flg22) pathogen-associated molecular pattern from the flagellin of Xanthomonas citri subsp. citri has been shown to induce defense responses correlated with citrus canker resistance. Here, flg22 of ‘Candidatus Liberibacter asiaticus’, the putative causal agent of Huanglongbing (HLB), elicited differential defense responses that were weaker than those from Xcc-flg22, between those of the HLB-tolerant mandarin cultivar Sun Chu Sha and susceptible grapefruit cultivar Duncan. Transcriptomics was used to compare the effect of CLas-flg22 and Xcc-flg22 between the citrus genotypes and identified 86 genes induced only by CLas-flg22 in the tolerant mandarin. Expression of 16 selected genes was validated, by reverse transcription-quantitative polymerase chain reaction, and was evaluated in citrus during ‘Ca. L. asiaticus’ infection. Differential expression of a number of genes occurred between tolerant and susceptible citrus infected with ‘Ca. L. asiaticus’, suggesting their involvement in HLB tolerance. In addition, several genes were similarly regulated by CLas-flg22 and ‘Ca. L. asiaticus’ treatments, while others were oppositely regulated in the tolerant mandarin, suggesting similarity and interplay between CLas-flg22 and ‘Ca. L. asiaticus’–triggered defenses. Genes identified are valuable in furthering the study of HLB tolerance mechanisms and, potentially, for screening for HLB-tolerant citrus using CLas-flg22 as a pathogen proxy.

Citrus production throughout the world is being severely threatened by Huanglongbing (HLB), which is a disease associated with the bacteria ‘Candidatus Liberibacter asiaticus’ (CLas), africanus, and americanus. This Resource Announcement provides amplicon-based next generation sequencing (NGS) datasets of the bacterial and fungal rRNA internal transcribed spacer (ITS) region from CLas-infected citrus budwood, leaves, and roots from five orchards located in different geographical regions in Florida (USA). To our knowledge, this is the first amplicon-based NGS study (i) that describes the fungal taxa associated with citrus and (ii) that provides comparative analyses of the bacterial and fungal taxa associated with budwood, leaves, and roots from the same citrus trees. This report also provides the sample metadata linked to these sequence datasets including HLB severity rating, tissue type, citrus rootstock, citrus scion, geographical region, and year trees were planted. When analyzed with other similar datasets, we anticipate that researchers will be able to obtain a greater understanding of the factors that shape the citrus microbiome as well as identify individual microorganisms or consortia of microorganisms that play a role in HLB suppression or exacerbation.

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.

Incidence and severity of Huanglongbing (HLB) disease were assessed in Apr. 2010 among eight citrus cultivars representing diverse scion types growing in commercial groves in Florida's Indian River region, an area with a high incidence of HLB. In each grove, 20 trees of each cultivar were rated for visual HLB symptoms and leaves were collected for quantitative polymerase chain reaction quantification of Candidatus Liberibacter asiaticus (CLas), the presumptive causal agent of HLB. There was a strong correlation between HLB rating and CLas titer (titer represented by Ct, r2 = 0.37 and 0.40, for whole tree and leaf sample, respectively, both with P < 0.0001) across all cultivars and groves. Although incidence and severity of HLB varied considerably among the groves, scion-specific differences were apparent, even when analyses excluded potentially confounding grove effects. ‘Temple’ tangor showed the most consistently low incidence of HLB symptoms and CLas titer; in contrast, ‘Murcott’ tangor and ‘Minneola’ tangelo had the highest incidence of HLB symptoms and highest CLas titer. These results suggest useful resistance to HLB with reduced symptoms and reduced CLas titer may be found in conventional scion cultivars and further work is needed to assess this potential and its commercial value.