Advertisement

Loop-mediated isothermal amplification (LAMP) based method for rapid and sensitive detection of ‘Candidatus Liberibacter asiaticus’ in citrus and the psyllid vector, Diaphorina citri Kuwayama

  • Dilip Kumar GhoshEmail author
  • Sumit Bhose
  • Ashish Warghane
  • Manali Motghare
  • Ashwani Kumar Sharma
  • Arun Kumar Dhar
  • Siddarame Gowda
Short Communication

Abstract

Citrus greening is a destructive disease of citrus in India and many citrus-growing regions around the world. The disease is associated with three Gram negative, fastidious and phloem-limited bacteria in the genus ‘Candidatus Liberibacter’. ‘Ca. L. asiaticus’ is the most wide spread and destructive species. Currently, there is no effective control method available to manage this disease, thus rapid detection, control of its psyllid vector population and removal of affected trees are commonly recommended to manage citrus greening . The present study was conducted to standardize a rapid and sensitive loop-mediated isothermal amplification (LAMP) protocol to detect ‘Ca. L. asiaticus’ in citrus and the psyllid vector Diaphorina citri Kuwayama. A set of six primers were identified from 16S rDNA region of Indian ‘Ca. L. asiaticus’ and the amplification reaction was optimized to 65 °C for 60 min. The amplified DNA produced a ladder-like band pattern on agarose gels, and visually produced an intense green color upon staining with SYBR green. The results were subsequently validated by PCR (polymerase chain reaction) and sequencing of the amplicon. The optimized LAMP protocol is rapid, highly sensitive and cost-effective method for the early detection of citrus greening in citrus groves and nurseries, and could be performed even in small laboratories located in remote places with limited resources.

Keywords

Huanglongbing LAMP Candidatus Liberibacter asiaticus Diaphorina citri Citrus greening disease 

Abbreviations

HLB

Huanglongbing

LAMP

Loop-mediated isothermal amplification

PCR

Polymerase chain reaction

UV

Ultra violet

References

  1. Bové JM (2006) Huanglongbing: a destructive, newly-emerging, century-old disease of citrus. J Plant Pathol 88(1):7–37Google Scholar
  2. Brlansky RH, Rogers ME (2007). Citrus Huanglongbing: Understanding the Vector-Pathogen Interaction for Disease Management. Online. APSnet Features. doi:  10.1094/APSnetFeature-2007-1207
  3. da Graça JV (1991) Citrus greening disease. Annu Rev Phytopathol 29:109–136CrossRefGoogle Scholar
  4. Ghosh DK, Bhose S, Mukherjee K, Baranwal VK (2013) Sequence and evolutionary analysis of ribosomal DNA from Huanglongbing (HLB) isolates of western India. Phytoparasitica 41:295–305CrossRefGoogle Scholar
  5. Ghosh DK, Bhose S, Motghare M, Warghane AJ, 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. Phytopathology 105:1043–1049CrossRefPubMedGoogle Scholar
  6. Gottwald TR (2010) Current epidemiological understanding of citrus Huanglongbing. Annu Rev Phytopathol 48:119–139CrossRefPubMedGoogle Scholar
  7. Grafton-Cardwell EE, Stelinski LL, Stansly PA (2013) Biology and management of Asian citrus psyllid, vector of the huanglongbing pathogens. Annu Rev Entomol 58:413–432CrossRefPubMedGoogle Scholar
  8. Hung TH, Hung SC, Chen CN, Hsu MH, Su HJ (2004) Detection by PCR of Candidatus Liberibacter asiaticus, the bacterium causing citrus huanglongbing in vector psyllids: application to the study of vector-pathogen relationships. Plant Pathol 53:96–102CrossRefGoogle Scholar
  9. Huys G, Vanhoutte T, Joossens M, Mahious AS, De Brandt E, Vermeire S, Swings J (2008) Co-amplification of eukaryotic DNA with 16S rRNA gene-based PCR primers: possible consequences for population fingerprinting of complex microbial communities. Curr Microbiol 56:553–557CrossRefPubMedGoogle Scholar
  10. Jagoueix S, Bové JM, Garnier M (1994) The phloem limited bacterium of greening disease of citrus is a member of the alpha subdivision of the Proteobacteria. Int J of Syst Bact 44:379–386CrossRefGoogle Scholar
  11. Jagoueix S, Bové JM, Garnier M (1996) PCR detection of the two liberobacter species associated with greening disease of citrus. Mol Cell Probes 10:43–50CrossRefPubMedGoogle Scholar
  12. 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 47:224–227Google Scholar
  13. Manjunath KL, Halbert SE, Ramadugu C, Webb S, Lee RF (2008) Detection of ‘Candidatus Liberibacter asiaticus’ in Diaphorina citri and its importance in the management of citrus huanglongbing in Florida. Phytopathology 98(4):387–396CrossRefPubMedGoogle Scholar
  14. Manjunath KL, Ramadugu C, Rodriguez E, Kubota R, Shibata S, Hall DG, Roose ML, Jenkins D, Lee RF (2015) A rapid field detection system for citrus huanglongbing associated ‘Candidatus Liberibacter asiaticus’ from the psyllid vector, Diaphorina citri Kuwayama and its implications in disease management. Crop Prot 68:41–48. doi: 10.1016/j.cropro.2014.10.026 CrossRefGoogle Scholar
  15. Okuda M, Matsumoto M, Tanaka Y, Subandiyah S, Iwanami T (2005) Characterization of the tufB-secE-nusG-rplKAJL-rpoB gene cluster of the citrus greening organism and detection by loop-mediated isothermal amplification. Plant Dis 89:705–711CrossRefGoogle Scholar
  16. Poole CB, Tanner NA, Zhang Y, Evans TC Jr, Carlow CK (2012) Diagnosis of brugian filariasis by loop-mediated isothermal amplification. PLoS Negl Trop Dis 6, e1948. doi: 10.1371/journal.pntd.0001948 PNTD-D-12-00911 CrossRefPubMedPubMedCentralGoogle Scholar
  17. Rigano LA, Malamud F, Orce IG, Filippone MP, Marano MR, do Amaral AM, Castagnaro AP, Vojnov AA (2014) Rapid and sensitive detection of Candidatus Liberibacter asiaticus by loop mediated isothermal amplification combined with a lateral flow dipstick. BMC Microbiol 14:86. doi: 10.1186/1471-2180-14-86 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Tatineni S, Sagaram US, Gowda S, Robertson CJ, Dawson WO, Iwanami T, Wang N (2008) In planta distribution of ‘Candidatus Liberibacter asiaticus’ as revealed by polymerase chain reaction (PCR) and real-time PCR. Phytopathology 98:592–599CrossRefPubMedGoogle Scholar
  19. Teixeira DC, Danet JL, Eveillard S, Martins EC, Jesus Junior WC, Yamamoto PT, Lopes SA, Bassanezi RB, Ayres AJ, Saillard C, Bove JM (2005) Citrus huanglongbing in Sao Paulo State, Brazil: PCR detection of the ‘Candidatus’ Liberibacter species associated with the disease. Mol Cell Probes 19(3):173–179CrossRefGoogle Scholar
  20. Wang N, Trivedi P (2013) Citrus huanglongbing: a newly relevant disease presents unprecedented challenges. Phytopathology 103(7):652–665CrossRefPubMedGoogle Scholar

Copyright information

© Society for Plant Biochemistry and Biotechnology 2015

Authors and Affiliations

  • Dilip Kumar Ghosh
    • 1
    Email author
  • Sumit Bhose
    • 1
  • Ashish Warghane
    • 1
  • Manali Motghare
    • 1
  • Ashwani Kumar Sharma
    • 2
  • Arun Kumar Dhar
    • 3
  • Siddarame Gowda
    • 4
  1. 1.ICAR- Central Citrus Research InstituteShankarnagarIndia
  2. 2.Department of BiotechnologyIndian Institute of TechnologyRoorkeeIndia
  3. 3.BrioBiotechGlenelgUSA
  4. 4.Citrus Research and Education CenterUniversity of FloridaLake AlfredUSA

Personalised recommendations