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

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Abstract

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 β-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.

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Data availability

The data that support the findings of this study are available from the corresponding author upon reasonable request.

References

  • Adkar-Purushothama CR, Quaglino F, Casati P, Ramanayaka JG, Bianco PA (2009) Genetic diversity among ‘Candidatus Liberibacter asiaticus’ isolates based on single nucleotide polymorphisms in 16S rRNA and ribosomal protein genes. Ann Microbial 59:681–688

    Article  CAS  Google Scholar 

  • Azzaro A, Leocata S, Polizzi G (1993) Greening spread in two locations of Uttar Pradesh (India). Proceeding of 12th Conference IOCV. IOCV, Riverside, pp 443–444

    Google Scholar 

  • Bove JM (2006) Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. J Plant Pathol 88:7–37

    Google Scholar 

  • Chen J, Deng X, Sun X, Jones D, Irey M, Civerolo E (2010) Guangdong and Florida populations of ‘Candidatus Liberibacter asiaticus’ distinguished by a genomic locus with short tandem repeats. Phytopathol 100:567–572

    Article  CAS  Google Scholar 

  • Coletta-Filho HD, Takita MA, Targon MLPN, Machado MA (2005) Analysis of 16S rDNA sequences from citrus huanglongbing bacteria reveal a different “Ca. Liberibacter” strain associated with citrus disease in Sao Paulo. Plant Dis 89:848–852

    Article  CAS  Google Scholar 

  • da Graça JV, Douhan GW, Halbert SE, Keremane ML, Lee RF, Vidalakis G, Zhao H (2016) Huanglongbing: an overview of a complex pathosystem ravaging the world’s citrus. J Integr Plant Biol 58:373–387

    Article  Google Scholar 

  • Dala-Paula BM, Plotto A, Bai J, Manthey JA, Baldwin EA, Ferrarezi RS, Gloria MB (2019) Effect of huanglongbing or greening disease on orange juice quality, a review. Front Plant Sci 9:1976

    Article  Google Scholar 

  • Duan Y, Zhou L, Hall DG, Li W, Doddapaneni H, Lin H, Liu L, Vahling CM, Gabriel DW, Williams KP, Dickerman A (2009) Complete genome sequence of citrus Huanglongbing bacterium, ‘Candidatus Liberibacter asiaticus’ obtained through metagenomics. Mol Plant-Microbe Interact 22:1011–1020

    Article  CAS  Google Scholar 

  • Fraser LR, Singh D (1968) Citrus dieback in India-the contribution of greening virus. Proceeding of 4th Conference IOCV. IOCV, Riverside, pp 141–144

    Google Scholar 

  • Garnier M, Jagoueix-Eveillard S, Cronje PR, Le Roux HF, Bove JM (2000) Genomic characterization of a liberibacter present in an ornamental rutaceous tree, Calodendrum capense, in the Western Cape province of South Africa. Proposal of Candidatus Liberibacter africanus subsp. capensis’. Int J Syst Evol Microbiol 50:2119–2125

    Article  CAS  Google Scholar 

  • Ghosh D, Bhose S, Mukherjee K, Baranwal VK (2013) Sequence and evolutionary analysis of ribosomal DNA from Huanglongbing (HLB) isolates of Western India. Phytoparasitica 41:295–305

    Article  Google Scholar 

  • Ghosh DK, Bhose S, Motghare M, Warghane A, Mukherjee K, Ghosh DK Sr, Sharma AK, Ladaniya MS, Gowda S (2015) Genetic diversity of the Indian populations of ‘Candidatus liberibacter asiaticus’ based on the tandem repeat variability in a genomic locus. Phytopathol 105:1043–1049

    Article  CAS  Google Scholar 

  • Ghosh DK, Kokane S, Kumar P, Ozcan A, Warghane A, Motghare M, Santra S, Sharma AK (2018a) Antimicrobial nano-zinc oxide-2S albumin protein formulation significantly inhibits growth of “Candidatus Liberibacter asiaticus” in planta. PLoS ONE 13(10):e0204702

    Article  Google Scholar 

  • Ghosh DK, Kokane SB, Kokane AD, Warghane AJ, Motghare MR, Bhose S, Sharma AK, Reddy MK (2018b) Development of a recombinase polymerase based isothermal amplification combined with lateral flow assay (HLB-RPA-LFA) for rapid detection of “Candidatus Liberibacter asiaticus”. PLoS ONE 13(12):e0208530

    Article  CAS  Google Scholar 

  • Ghosh DK, Motghare M, Gowda S (2018c) Citrus greening: overview of the most severe disease of citrus. Adv Agric Res Technol J 2:83–100

    Google Scholar 

  • Halbert S (2005) The discovery of huanglongbing in Florida. In: Proceedings of 2nd international citrus canker and huanglongbing research workshop, Orlando, H-3. https://swfrec.ifas.ufl.edu/hlb/database/pdf/22_CankerHuang_05.pdf

  • Halbert SE, Manjunath KL (2004) Asian citrus psyllids (Sternorrhyncha: Psyllidae) and greening disease of citrus: a literature review and assessment of risk in Florida. Fla Entomol 87:330–353

    Article  Google Scholar 

  • Jagoueix S, Bove JM, Garnier M (1997) Comparison of the 16S/23S ribosomal intergenic regions of “Candidatus Liberobacter asiaticum” and “Candidatus Liberobacter africanum”, the two species associated with citrus huanglongbing (greening) disease. Int J Syst Bacteriol 47:224–227

    Article  CAS  Google Scholar 

  • Lopes SA, Bertolini E, Frare GF, Martins EC, Wulff NA, Teixeira DC, FernandesCambra NGM (2009) Graft transmission efficiencies and multiplication of ‘Candidatus Liberibacter americanus’ and ‘Candidatus Liberibacter asiaticus’ in citrus plants. Phytopathol 99:301–306

    Article  CAS  Google Scholar 

  • National Horticulture Mission (Annual Report) (2014) Ministry of Agriculture & Farmers welfare. Government of India, New Delhi, 2013–2014. https://hortnet.gov.in/

  • Razi MF, Keremane ML, Ramadugu C, Roose M, Khan IA, Lee RF (2014) Detection of citrus huanglongbing-associated ‘Candidatus Liberibacter asiaticus’ in citrus and Diaphorina citri in Pakistan, seasonal variability, and implications for disease management. Phytopathol 104:257–268

    Article  CAS  Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739

    Article  CAS  Google Scholar 

  • Teixeira CD, Saillard C, Eveillard S, Danet JL, Ayres AJ, Bove J (2005) ‘Candidatus Liberibacter americanus’, associated with citrus huanglongbing (greening disease) in Sao Paulo State, Brazil. Int J Syst Evol Microbiol 55:1857–1862

    Article  CAS  Google Scholar 

  • Tokajian S, Issa N, Salloum T, Ibrahim J, Farah M (2016) 16S–23S rRNA gene intergenic spacer region variability helps resolve closely related sphingomonads. Front Microbiol 7:149

    Article  Google Scholar 

  • Tomimura K, Miyata SI, Furuya N, Kubota K, Okuda M, Subandiyah S, Hung TH, Su HJ, Iwanami T (2009) Evaluation of genetic diversity among ‘Candidatus Liberibacter asiaticus’ isolates collected in Southeast Asia. Phytopathol 99:1062–1069

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This research was funded by Indian Council of Agricultural Research- Consortia Research Platform (CRP) on Vaccines and Diagnostics, ICAR, Government of India, New Delhi. Authors thank Dr. Arun Kumar Dhar, University of Arizona, Tucson, Arizona 8572, USA for reviewing this manuscript. The authors declare there are no competing interests.

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  1. Sumit Bhose

    Present address: Sea6Energy Private Limited, C-CAMP, TIFR-NCBS Campus, Belary Road, Bengaluru, Karnataka, 560065, India

Authors and Affiliations

  1. Plant Virology Laboratory, ICAR-Central Citrus Research Institute, Nagpur, Maharashtra, India

    Sunil B. Kokane, Sumit Bhose, Amol Kokane, Mrugendra Gubyad & Dilip Kumar Ghosh

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  1. Sunil B. Kokane
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  2. Sumit Bhose
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  4. Mrugendra Gubyad
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Contributions

DK, SB, and SK conceived and designed the experiments. SB, SK, AK and MG performed the experiments and analyzed the data. DK, SB and SK wrote and edited the manuscript. All authors read and approved the final manuscript.

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Correspondence to Sumit Bhose or Dilip Kumar Ghosh.

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Kokane, S.B., Bhose, S., Kokane, A. et al. Molecular detection, identification, and sequence analysis of ‘Candidatus Liberibacter asiaticus’ associated with Huanglongbing disease of citrus in North India. 3 Biotech 10, 341 (2020). https://doi.org/10.1007/s13205-020-02334-x

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