Evaluation of Prophage Gene Revealed Population Variation of ‘Candidatus Liberibacter Asiaticus’: Bacterial Pathogen of Citrus Huanglongbing (HLB) in Northern Thailand

Abstract

‘Candidatus Liberibacter asiaticus’ is a non-culturable bacterial pathogen, the causal agent of Huanglongbing (HLB, yellow shoot disease, also known as citrus greening disease), a highly destructive disease of citrus (Rutaceae). The pathogen is transmitted by the Asian citrus psyllid: Diaphorina citri Kuwayama. Recent studies, have shown that the HLB pathogen has two prophages, SC1 that has a lytic cycle and SC2 associated with bacterial virulence. This study aimed to search for SC1 and SC2 prophages of HLB in mandarin orange, sweet orange, bitter orange, kumquat, key lime, citron, caviar lime, kaffir lime, pomelo and orange jasmine from five provinces in Northern Thailand. A total of 216 samples collected from Northern Thailand during 2019 and 2020 were studied. The results revealed that 62.04% (134/216) citrus samples were infected with the ‘Ca. L. asiaticus’ the bacterial pathogen associated with citrus HLB. The prophage particles are important genetic elements of bacterial genomes that are involved in lateral gene transfer, pathogenicity, environmental adaptation and interstrain genetic variability. Prophage particles were evaluated in the terminase gene of SC1 and SC2-type prophages. The results of the analysis of the prophage terminase genes of SC1 and SC2 revealed four groups of samples in Northern Thailand.The first group was the population of ‘Ca. L. asiaticus’ which was non-prophage; the frequency was 7.46% (10/134) from the total infected citrus samples. The second and third groups contained one prophage sequence with 1.49% (2/134) containing SC1-type prophage and 64.93% (87/134) containing SC2-type prophage. The fourth group contained both prophage sequence with 26.12% (35/134) containing SC1 and SC2-type prophages.Samples of leaves showing various HLB symptoms were collected from infected citrus trees which correlated with the prophages. In the first group (non-prophage) the symptoms were much less severe than other groups which had prophages. On the other hand, the third group containing the SC2-type prophage and fourth group containing SC1 and SC2-type prophages showed the most severe symptoms. The evaluation of the prophage effect on HLB-induced leaf symptoms showed that isolates with the SC2-type prophage particles produced the most severe symptoms.