Molecular detection, identification, and sequence analysis of 'Candidatus Liberibacter asiaticus' associated with Huanglongbing disease of citrus in North India.

Huanglongbing (HLB, Citrus greening), caused by a phloem-limited fastidious gram-negative bacterium, "Candidatus Liberibacter spp.", is one of the devastating diseases of citrus worldwide. The pathogen belongs to the alpha-proteobacteria group and is classified on the basis of its geographical origin and 16S rRNA sequence diversity. Although the disease has been reported from all citrus growing states of India, the status and the molecular variability among the isolates from the Northern part of the country is unknown. A total of five different HLB isolates originating from Northern India showing variable symptoms were studied. The genomic regions of four different genes, i.e., 16S rRNA, intergenic 16S/23S rRNA spacer region, rplA-rplJ, and CLIBASIA_01645 were amplified by PCR, sequenced, and variations in these sequences were assessed. Analysis of 16S rRNA clearly indicated that all five isolates fit in to 'Candidatus Liberibacter asiaticus' (CLas) group. However, 16S/23S rRNA intergenic spacer region-based analysis failed to segregate these isolates beyond species level. Sequence analysis of rplA-rplJ gene and CLIBASIA_01645 loci also confirmed the existence of diversity among the 'CLas' in the surveyed areas. Further, 16S rRNA and rplA-rplJ-based SNP analysis revealed that some isolates segregated into three new lineages, two on the basis of 16Sr (16Sr-XV and 16Sr-XVI), and one based on ß-rp (rp-IV), respectively. A tandem repeat number (TRN) at CLIBASIA_01645 region were TRN = 5, 6 and 13; with TRN = 6 being common in three 'CLas' isolates. Overall, the study demonstrated that all examined five HLB isolates belonged to 'CLas' group. However, these isolates showed distinct sequence variability in three out of four genomic regions. The results provide a robust framework for understanding differences in pathogenicity among different HLB isolates as it is plausibly related to their genomic variation, and evolutionary history.

Development of a recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) for rapid detection of "Candidatus Liberibacter asiaticus".

Huanglongbing (HLB) or citrus greening is highly destructive disease that is affecting the citrus industry worldwide and it has killed millions of citrus plants globally. HLB is caused by the phloem limited, Gram negative, non-culturable, alpha-proteobacterium, 'Candidatus Liberibacter asiaticus'. Currently, polymerase chain reaction (PCR) and real time PCR have been the gold standard techniques used for detection of 'Ca. L. asiaticus'. These diagnostic methods are expensive, require well equipped laboratories, not user-friendly and not suitable for on-site detection of the pathogen. In this study, a sensitive, reliable, quick and low cost recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) technique has been developed as a diagnostic tool for detection of 'Ca. L. asiaticus'. The assay was standardized by designing the specific primer pair and probe based on the conserved 16S rRNA gene of 'Ca. L. asiaticus'. The assay was optimized for temperature and reaction time by using purified DNA and crude plant extracts and the best HLB-RPA-LFA was achieved at the isothermal temperature of 38°C for 20 to 30 min. The efficacy and sensitivity of the assay was carried out by using field grown, HLB-infected, HLB-doubtful and healthy citrus cultivars including mandarin, sweet orange cv. mosambi, and acid lime. The HLB-RPA-LFA did not show cross-reactivity with other citrus pathogens and is simple, cost-effective, rapid, user-friendly and sensitive. Thus, the HLB-RPA-LFA method has great potential to provide an improved diagnostic tool for detection of 'Ca. L. asiaticus' for the farmers, nurserymen, disease surveyors, mobile plant pathology laboratories, bud-wood certification and quarantine programs.

Development of a simple and rapid reverse transcription-loop mediated isothermal amplification (RT-LAMP) assay for sensitive detection of Citrus tristeza virus.

Tristeza is a devastating disease of citrus and reported to be present in almost all countries where it is cultivated as a commercial crop. The etiological agent of this disease is Citrus tristeza virus (CTV), a member of the genus Closterovirus with in the family Closteroviridae. The pathogen is restricted to the phloem tissue of the infected citrus plant and has a monopartite ss (+) RNA genome of ∼20kb size. Till date, there is no effective control measure available for this virus. Management of tristeza depends on destruction of CTV infected field plants, production of virus-free planting material for new orchard establishment and controlling viruliferous aphid vectors responsible for field spread of the pathogen. Availability of rapid diagnostic assay is essential for rapid and efficient detection of the pathogen. In the present investigation, RT-LAMP (reverse transcription-loop mediated isothermal amplification), a highly sensitive, robust and low cost assay has been developed for rapid detection of CTV in infected citrus plant samples. Based on conserved nucleotide sequences available in GenBank and specific to p25 gene (major coat protein gene) of predominant CTV isolates of India, four primer sets (CTV-F3, CTV-B3, CTV-FIP and CTV-BIP) ware designed and custom synthesized. The amplified LAMP products obtained after maintaining isothermal condition of 65°C for 60min duration could be visible easily with necked eyes in presence of SYBR Green I (100X). Subsequently, LAMP products were verified by electrophoresis run in 1.5% agarose gel. The RT-LAMP results obtained with known CTV isolates maintained in screen house of CCRI, Nagpur were validated using field samples and thereafter it was further confirmed by conventional RT-PCR (reveres transcription-polymerase chain reaction) assay. The sensitivity of CTV-RT-LAMP protocol standardized in the present study was 100 times more than conventional one step RT-PCR assay. It also has maximum detection limit up to 0.0001ng RNA in individual reaction mixture. CTV-RT-LAMP assay is a simple, sensitive, rapid and less costly detection technique. This assay could be used for CTV diagnosis in pathology laboratories having limited facility and resources and even by citrus nurseries situated in remote locations. As per our knowledge and available literature, the present study reports first time about the usefulness of RT-LAMP assay for detection of CTV from India.

Characterization of a bacterioferritin comigratory protein family 1-Cys peroxiredoxin from Candidatus Liberibacter asiaticus.

To defend against the lethality of the reactive oxygen species (ROS), nature has armed microorganisms with a range of antioxidant proteins. These include peroxiredoxin (Prx) super family proteins which are ubiquitous cysteine-based non-heme peroxidases. The phytopathogenic bacterium Candidatus Liberibacter asiaticus (CLA), an etiological agent of citrus plants diseases, posses many genes for defense against oxidative stress. The bacterioferritin comigratory protein (BCP), a member of Prxs, is part of an oxidative stress defense system of CLA. The key residue of these enzymes is peroxidatic Cys (termed CPSH) which is contained within an absolutely conserved PXXX (T/S) XXC motif. In the present study, a 1-Cys Prx enzyme (CLa-BCP), having CPSH/sulfenic acid cysteine (C-46) but lacking the resolving cysteine (CRSH), was characterized from CLA. The peroxidase activity was demonstrated using a non-physiological electron donor DTT against varied substrates. The protein was shown to have the defensive role against peroxide-mediated cell killing and an antioxidant activity. In vitro DNA-binding studies showed that this protein can protect supercoiled DNA from oxidative damage. To the best of our knowledge, this is the first report on a 1-Cys BCPs to have an intracellular reactive oxygen species scavenging activity.

Genetic Diversity of the Indian Populations of 'Candidatus Liberibacter asiaticus' Based on the Tandem Repeat Variability in a Genomic Locus.

Citrus huanglongbing (HLB, citrus greening disease) is an extremely destructive disease affecting citrus and causes severe economic loss to the crop yield worldwide. The disease is caused by a phloem-limited, noncultured, gram-negative bacteria Candidatus Liberibacter spp., the widely present and most destructive species being 'Candidatus Liberibacter asiaticus'. Although the disease has been reported from almost all citrus growing regions of India, knowledge on the molecular variability of the pathogen 'Ca. L. asiaticus' populations from different geographical regions and cultivars is limited. In the present study, variability of the Indian 'Ca. L. asiaticus' based on the tandem repeats at the genomic locus CLIBASIA_01645 was characterized and categorized into four classes based on the tandem repeat number (TRN); Class I (TRN≤5), Class II (TRN>5≤10), Class III (TRN>10≤15), and Class IV (TRN>15). The study revealed that the Indian population of 'Ca. L. asiaticus' is more diverse than reported for Florida and Guangdong populations, which showed less diversity. While Florida and Guangdong populations were dominated by a TRN5 and TRN7 genotype, respectively, the Indian 'Ca. L. asiaticus' populations with TRN copy numbers 9, 10, 11, 12, and 13 were widely distributed throughout the country. Additionally, TRN2 and TRN17 genotypes were also observed among the Indian 'Ca. L. asiaticus' populations. The predominant 'Ca. L. asiaticus' genotypes from the northeastern region of India were TRN6 and TRN7 (53.12%) and surprisingly similar to neighboring South China populations. Preliminary results showed absence of preference of citrus cultivars to any specific 'Ca. L. asiaticus' genotype.

Crystal structure of a periplasmic solute binding protein in metal-free, intermediate and metal-bound states from Candidatus Liberibacter asiaticus.

The Znu system, a member of ABC transporter family, is critical for survival and pathogenesis of Candidatus Liberibacter asiaticus (CLA). Two homologues of this system have been identified in CLA. Here, we report high resolution crystal structure of a periplasmic solute binding protein from second of the two gene clusters of Znu system in CLA (CLas-ZnuA2) in metal-free, intermediate and metal-bound states. CLas-ZnuA2 showed maximum sequence identity to the Mn/Fe-specific solute binding proteins (SBPs) of cluster A-I family. The overall fold of CLas-ZnuA2 is similar to the related cluster A-I family SBPs. The sequence and structure analysis revealed the unique features of CLas-ZnuA2. The comparison of CLas-ZnuA2 structure in three states showed that metal binding and release is facilitated by a large displacement along with a change in orientation of the side chain for one of the metal binding residue (His39) flipped away from metal binding site in metal-free form. The crystal structure captured in intermediate state of metal binding revealed the changes in conformation and interaction of the loop hosting His39 during the metal binding. A rigid body movement of C-domain along with partial unfolding of linker helix at its C-terminal during metal binding, as reported for PsaA, was not observed in CLas-ZnuA2. The present results suggest that despite showing maximum sequence identity to the Mn/Fe-specific SBPs, the mechanistic resemblance of CLas-ZnuA2 seems to be closer to Zn-specific SBPs of cluster A-I family.