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.

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.

Indian citrus ringspot virus (ICRSV) is a devastating pathogen that has a particularly deleterious effect on the ‘Kinnow mandarin’, a commercial citrus crop cultivated in the north-west of India. ICRSV belongs to the Mandarivirus genus within the family of Alphaflexiviridae and has a positive sense single-stranded RNA (ssRNA) genome consisting of six open reading frames (ORFs). Severe cases of ICRSV result in a significant reduction in both the yield and quality of crops. Consequently, there is an urgent need to develop methods to detect ICRSV in an accurate and timely manner. Current methods involve a two-step reverse transcriptase-polymerase chain reaction (RT-PCR) that is time-consuming. Here, we describe a novel, one-step, reverse transcription-loop-mediated isothermal amplification (RT-LAMP) method for the sensitive and rapid detection of ICRSV. The RT-LAMP assay was standardized by designing and testing four different primers that targeted the coat protein gene of ICRSV. Amplification results were visualized by a color change after addition of SYBR Green I. The standardized RT-LAMP assay was highly specific and successfully detected all 35 ICRSV isolates tested from the Punjab and Haryana states of India. Furthermore, there was no cross-reaction with 17 isolates of five other citrus pathogens that are common in India. ICRSV-RT-LAMP assay developed in the present study is a simple, rapid, sensitive, and specific, technique. Moreover, the assay consists of only a single step and is more cost-effective than existing methods. This represents the first application of RT-LAMP for the detection of ICRSV. Our RT-LAMP assay is a powerful tool for the detection of ICRSV and will be particularly useful for large scale indexing of field samples in diagnostic laboratories, nurseries, and for quarantine applications.

Huanglongbing (HLB, also known as citrus greening) is considered to be the most devastating disease that has significantly damaged the citrus industry globally. HLB is caused by the Candidatus Liberibacter asiaticus (CLas), the fastidious phloem-restricted gram-negative bacterium, vectored by the asian citrus psyllid. To date, there is no effective control available against CLas. To alleviate the effects of HLB on the industry and protect citrus farmers, there is an urgent need to identify or develop inhibitor molecules to suppress or eradicate CLas from infected citrus plant. In this paper, we demonstrate for the first time an in planta efficacy of two antimicrobial compounds against CLas viz. 2S albumin (a plant based protein; ~12.5 kDa), Nano-Zinc Oxide (Nano-ZnO; ~ 4.0 nm diameter) and their combinations. Aqueous formulations of these compounds were trunk-injected to HLB affected Mosambi plants (Citrus sinensis) grafted on 3-year old rough lemon (C. jambhiri) rootstock with known CLas titer maintained inside an insect-free screen house. The effective concentration of 2S albumin (330 ppm) coupled with the Nano-ZnO (330 ppm) at 1:1 ratio was used. The dynamics of CLas pathogen load of treated Mosambi plants was assessed using TaqMan-qPCR assay every 30 days after treatment (DAT) and monitored till 120 days. We observed that 2S albumin-Nano-ZnO formulation performed the best among all the treatments decreasing CLas population by 96.2%, 97.6%, 95.6%, and 97% of the initial bacterial load (per 12.5 ng of genomic DNA) at 30, 60, 90, and 120 DAT, respectively. Our studies demonstrated the potency of 2S albumin-Nano-ZnO formulation as an antimicrobial treatment for suppressing CLas in planta and could potentially be developed as a novel anti CLas therapeutics to mitigate the HLB severity affecting the citrus industry worldwide.

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