Obtaining positive controls for the detection of two polymorphic regions in Cuban strains of ‘Candidatus Liberibacter asiaticus’

Introduction

“Huanglongbing” (HLB), also known as citrus greening, is the most serious disease threating the citrus industry worldwide (11. Bové J-M. “Huanglongbing” or yellow shoot, a disease of Gondwanan origin: will it destroy citrus worldwide? Phytoparasitica. 2014; 42: 579-583. DOI: 20.2007/s12600-014-0415-4 ). Its occurrence has been associated with the presence of three bacteria within the ‘Candidatus Liberibacter’ genus: ‘Ca. L. asiaticus’, ‘Ca. L. africanus’ and ‘Ca. L. americanus’ (22. Da Graça JV, Douhan GW, Halbert SE, Keremane ML, Lee RF, Vidalakis G. et al. “Huanglongbing”: an overview of a complex pathosystem ravaging the world’s citrus. Journal of Integrative Plant Biology. 2016; 58: 373-387. DOI: 10.1111/jjpb.12437 ). In Cuba, ‘Ca. L. asiaticus’ has been the only liberibacter detected since 2007 (33. Luis M, Collazo C, Llauger R, Blanco E, Peña I, López D, et al. Occurrence of citrus “huanglongbing” in Cuba and association of the disease with ‘Candidatus Liberibacter asiaticus’. Journal of Plant Pathology. 2009; 91(3): 709-712. Disponible en: http://www.jstor.org/stable/41998692 , 44. Luis-Pantoja M, Paredes-Tomás C, Uneau Y, Myrie W, Morillon R, Satta E, et al. Identification of ‘Candidatus Phytoplasma’ species in “huanglongbing” infected citrus orchards in the Caribbean. European Journal of Plant Pathology. 2021. DOI: 10.1007/s10658-021-02234-7 ). The lack of cultivation of this bacterium in artificial medium encouraged the active contribution of molecular tools in most of the related research (55. Deng X, Chen J, Feng Z, Shan Z, Guo H, Zhu J, et al. Identification and characterization of the “huanglongbing” bacterium in pummelo from multiple locations in Guangdong. P. R. China. Plant Disease. 2008; 92: 513-518. DOI: 10.1094/PDIS-92-4-0513 ). Its detection, identification and genetic diversity studies have mainly been focused on the use of housekeeping genes, as well as of the 16S rRNA, the intergenic region 16S-23S (66. Jagoueix S, Bové J-M, Garnier M. The phloem-limited bacterium of greening disease of citrus is a member of the α-subdivision of the Proteobacteria. International Journal of Systematic Bacteriology. 1994; 44(3): 379-386. DOI: 10.1099/00207713-44-3-379 , 77. Jagoueix S, Bové J-M, Garnier M. 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. International Journal of Systematic Bacteriology. 1997; 47: 224-227. DOI: 10.1099/00207713-47-1-224 , 88. Li W, Hartung JS, Levy L. Quantitative real-time PCR for detection and identification of ‘Candidatus Liberobacter’ species associated with citrus “huanglongbing”. Journal of Microbiological Methods. 2006; 66(1): 104-115. DOI: 10.16//j.mimet.2005.10.018 ), the omp gene (99. Bastianel C, Garnier-Semancik M, Renaudin J, Bové J-M, Eveillard S. Diversity of ‘Candidatus Liberibacter asiaticus’, based on the omp gene sequence. Applied and Environmental Microbiology. 2005; 71(11): 6473-6478. DOI: 10.1128/AEM.71.11.6473-6478.2005 ), or the operon β genes (1010. Teixeira DC, Eveillard S, Sirand-Pugnet P, Wulff A, Saillard C, Ayres AJ, et al. The tufB-secE-nusG-rplKALJ-rpoB gene cluster of the liberibacters: sequence comparisons, phylogeny and speciation. International Journal of Systematic and Evolutionary Microbiology. 2008; 58: 1414-1421. DOI: 10.1099/ijs.0.65641-0 ). These approaches allowed the differentiation of the three known species (66. Jagoueix S, Bové J-M, Garnier M. The phloem-limited bacterium of greening disease of citrus is a member of the α-subdivision of the Proteobacteria. International Journal of Systematic Bacteriology. 1994; 44(3): 379-386. DOI: 10.1099/00207713-44-3-379 , 77. Jagoueix S, Bové J-M, Garnier M. 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. International Journal of Systematic Bacteriology. 1997; 47: 224-227. DOI: 10.1099/00207713-47-1-224 , 99. Bastianel C, Garnier-Semancik M, Renaudin J, Bové J-M, Eveillard S. Diversity of ‘Candidatus Liberibacter asiaticus’, based on the omp gene sequence. Applied and Environmental Microbiology. 2005; 71(11): 6473-6478. DOI: 10.1128/AEM.71.11.6473-6478.2005 , 1111. Teixeira DC, Danet J-L, Eveillard S, Martins Cintra de Jesús VJ, Yamamoto P, Lopes SA, et al. Citrus “huanglongbing” in Sao Paulo State, Brazil: PCR detection of the ‘Candidatus Liberibacter’ species associated with the disease. Molecular and Cellular Probes. 2005; 19, 173-179. DOI: 10.1016/j.mcp.2004.11.002 ). However, the high conserved nature of these genes limits the discriminatory power for closely related bacteria (i.e., strains of the same species) (33. Luis M, Collazo C, Llauger R, Blanco E, Peña I, López D, et al. Occurrence of citrus “huanglongbing” in Cuba and association of the disease with ‘Candidatus Liberibacter asiaticus’. Journal of Plant Pathology. 2009; 91(3): 709-712. Disponible en: http://www.jstor.org/stable/41998692 , 1212. Adkar-Purushothama CR, Quaglino F, Casati P, Ramanayaka JG, Bianco PA. Genetic diversity among ‘Candidatus Liberibacter asiaticus’ isolated based on single nucleotide polymorphisms in 16S rRNA and ribosomal protein genes. Annals of Microbiology. 2002; 59(4): 681-688. DOI: 10.1007/BF03179208 ).

Since 2009, the availability of the complete genome of different ‘Ca. L. asiaticus’ strains from different countries has increased exponentially (1313. Duan Y, Zhou L, Hall DG, Li W, Doddapaneni H, Lin H, et al. Complete genome sequence of citrus huanglongbing bacterium ‘Candidatus Liberibacter asiaticus’ obtained through metagenomics. Molecular Plant Microbe Interactions. 2009; 22: 1011-1020. DOI: 10.1094/MPMI-22-8-1011 , 1414. Lin H, Han CS, Liu B, Lou B, Bai X, Deng C, et al. Complete genome sequence of a Chinese strain of ‘Candidatus Liberibacter asiaticus’. Genome Announcements. 2013; 1: e00184-13. DOI: 10.1128/genomea.00184-13 , 1515. Zheng Z, Sun X, Deng X, Chen J. Whole-genome sequence of ‘Candidatus Liberibacter asiaticus’ from a “huanglongbing”-affected citrus tree in Central Florida. Genome Announcements. 2015; 3: e00169-15. DOI: 10.1128/genomea.00169-15 ). Subsequently, genomic regions with greater variability have been successfully implemented, including microsatellites (1212. Adkar-Purushothama CR, Quaglino F, Casati P, Ramanayaka JG, Bianco PA. Genetic diversity among ‘Candidatus Liberibacter asiaticus’ isolated based on single nucleotide polymorphisms in 16S rRNA and ribosomal protein genes. Annals of Microbiology. 2002; 59(4): 681-688. DOI: 10.1007/BF03179208 , 1616. Chen J, Deng X, Sun X, Jones D, Irey M, Civerolo E. Guangdong and Florida populations of ‘Candidatus Liberibacter asiaticus’ distinguished by a genomic locus with short tandem repeats. Phytopathology. 2010; 100: 567-572. DOI: 10.1094/PHYTO-100-6-0567 , 1717. Katoh H, Subandiyah S, Tomimura K, Okuda M, Su HJ, Iwanami T. Differentiation of ‘Candidatus Liberibacter asiaticus’ isolates by variable-number tandem-repeat analysis. Applied and Environmental Microbiology. 2011; 77(5): 1910-1917. DOI: 10.1128/AEM.01571-10 ), prophage genes (1818. Liu R, Zhang P, Pu X, Xing X, Chen J, Deng X. Analysis of a prophage gene frequency revealed population variation of ‘Candidatus Liberibacter asiaticus’ from two citrus-growing provinces in China. Plant Disease. 2011; 95: 431-435. DOI: 10.1094/PDIS-04-10-0300 , 1919. Wang X, Zhou C, Deng X, Su H, Chen J. Molecular characterization of a mosaic locus in the genome of ‘Candidatus Liberibacter asiaticus’. BMC Microbiology. 2012; 12: 18. DOI: 10/1186/1471-2180-12-18 , 2020. Zheng Z, Bao M, Wu F, Van Horn C, Chen J, Deng X. A type 3 prophage of ‘Candidatus Liberibacter asiaticus’ carrying a restriction-modification system. Phytopathology. 2018; 108: 454-461. DOI: 10.1094/PHYTO-08-17-0282-R ), and miniature inverted-repeat transposable elements (MITEs) (2121. Wang X, Tan J, Bai Z, Su H, Deng X, Li Z, et al. Detection and characterization of Miniature Inverted-repeat Transposable Elements in ‘Candidatus Liberibacter asiaticus’. Journal of Bacteriology. 2013; 195(17): 3979-3986. DOI: 10.1128/jb.00413-13 ) identified inside the genome of these bacteria. In Cuba, previous strain differentiation works were based on the study of these conserved regions (33. Luis M, Collazo C, Llauger R, Blanco E, Peña I, López D, et al. Occurrence of citrus “huanglongbing” in Cuba and association of the disease with ‘Candidatus Liberibacter asiaticus’. Journal of Plant Pathology. 2009; 91(3): 709-712. Disponible en: http://www.jstor.org/stable/41998692 , 44. Luis-Pantoja M, Paredes-Tomás C, Uneau Y, Myrie W, Morillon R, Satta E, et al. Identification of ‘Candidatus Phytoplasma’ species in “huanglongbing” infected citrus orchards in the Caribbean. European Journal of Plant Pathology. 2021. DOI: 10.1007/s10658-021-02234-7 ). Due to their greater variability, polymorphic regions are more suitable tools to evaluate genetic variability within populations of the same species. However, to guarantee the correct amplification and interpretation of the results, it is necessary to improve these techniques by including positive controls.

In this work, the objective was to obtain positive controls for the prophages and MITEs from Cuban strains of ‘Ca. L. asiaticus’.

Materials and methods

Results and discussion

For detecting prophage type 1 (T1-1F/T1-1R), the amplicon with the expected size (i.e., 1,025 bp) was observed in all the positive samples when amplified with the annealing temperature of 62°C. Similar results were obtained for the prophage type 2, with the primer pair T2-8F/T2-8R with the same temperature for the expected amplicon of 795 bp. These amplifications were obtained with an alternative annealing temperature of 60°C. However, with the additional prophage type 2- specific primers used (T2-1F/T2-1R, with an expected amplicon of 806 bp), no amplification was obtained with any of the three annealing temperatures evaluated (Fig. 1). No amplification was observed with the detection system for prophage type 3 (primers 891-1F/891-1R) (data not shown). With the temperatures tested, none of the primers yielded PCR amplification from DNA extracted from asymptomatic (confirmed non-‘Ca. L. asiaticus’- infected) citrus leaves.

The band with the expected size (1,025 bp) with the primers T1-1F/1R was obtained in all the positive controls with the three annealing temperatures used. Da Silva et al. (2525. Da Silva PA, Fassini CG, Sampaio LS, Dequigiovanni G, Zucchi MI, Wulff NA. Genetic diversity of ‘Candidatus Liberibacter asiaticus’ revealed by short tandem repeats and prophage typing indicates population homogeneity in Brazil. Phytopathology. 2019; 109: 960-971. DOI: 10.1094/PHYTO-08-18-0295-R ) reported the same results using annealing at 55°C with this system. However, in the Cuban samples the best results were obtained with 60°C or 62°C, since a size expected-single band was amplified from all the positive samples. At lower temperature (i.e., 55°C), additional non-specific light bands appeared in some samples (data not shown). For prophage type 2, amplification was only detected with primers T2-8F/T2-8R, while T2-1F/T2-1R did not show bands. This observation was maintained for all the annealing temperatures used. Previous works reported that the set T2-8F/T2-8R, out of eight PCR markers for prophage type 2, amplifyed this prophage from 299 Brazilian strains of ‘Ca. L. asiaticus’ evaluated (2525. Da Silva PA, Fassini CG, Sampaio LS, Dequigiovanni G, Zucchi MI, Wulff NA. Genetic diversity of ‘Candidatus Liberibacter asiaticus’ revealed by short tandem repeats and prophage typing indicates population homogeneity in Brazil. Phytopathology. 2019; 109: 960-971. DOI: 10.1094/PHYTO-08-18-0295-R ). The results with both the Cuban and Brazilian strains highlighted the usefulness of the primers T2-8F/T2-8R. This agrees with their high specificity to prophage type 2 sequence, according to their design (2424. Zheng Z, Bao M, Wu F, Chen J, Deng X. Predominance of a single prophage carrying a CRISPR/cas system in ‘Candidatus Liberibacter asiaticus’ strains in Southern China. PLoS ONE. 2016; 11(1): e0146422. DOI: 10.1371/journal.pone.0146422 ).

Based on the primers of the MITEs in the prophage region, it was possible to identify two types of amplicons, B720 and B720 + B350 (Fig. 2), by using 55°C as annealing temperature. In China, Wang et al. (2121. Wang X, Tan J, Bai Z, Su H, Deng X, Li Z, et al. Detection and characterization of Miniature Inverted-repeat Transposable Elements in ‘Candidatus Liberibacter asiaticus’. Journal of Bacteriology. 2013; 195(17): 3979-3986. DOI: 10.1128/jb.00413-13 ) used this detection system and reported a similar population structure in 10 samples from Guizhou. However, other genotypes were identified when the sample size was increased (2121. Wang X, Tan J, Bai Z, Su H, Deng X, Li Z, et al. Detection and characterization of Miniature Inverted-repeat Transposable Elements in ‘Candidatus Liberibacter asiaticus’. Journal of Bacteriology. 2013; 195(17): 3979-3986. DOI: 10.1128/jb.00413-13 , 2626. Zheng Y, Huang H, Huang Z, Deng X, Zheng Z, Xu M. Prophage region and short tandem repeats of ‘Candidatus Liberibacter asiaticus’ reveal significant population structure in China. Plant Pathology. 2021; 70(4): 959-969. DOI: 10.1111/ppa.13332 ).

These results allowed differentiating several strains from different geographic regions in Cuba through amplification patterns. These samples will therefore be employed as controls in future survey studies. The use of the detection of prophage types and MITEs will allow a deeper and faster characterization of the Cuban population of ‘Ca. L. asiaticus’ strains circulating in the different citrus growing areas of the island. This is also the first detection of polymorphic molecular markers in the Cuban strains of ‘Ca. L. asiaticus’.

Conclusions

Amplification controls were obtained for the first time using diverse types of prophage and MITEs from Cuban strains of ‘Ca. L. asiaticus’. The detection of both markers is a suitable tool to evaluate the population variability of this bacterium from different geographic regions of the country.