Atta, Sagheer

‘Candidatus Liberibacter asiaticus’ (CLas) is a devastating pathogen of citrus associated with Huanglongbing (HLB, citrus greening disease). HLB is economically significant in Asia and has destroyed millions of citrus trees worldwide during the last century. Since 2007, when the first molecular evidence for the presence of CLas in North-West Frontier Province of Pakistan (Khyber Pakhtunkhwa) were presented, there have been a limited number of studies reporting the pathogen in different citrus growing districts of the Punjab Province, one of the major citrus producing areas of the country. In this study, a small-scale survey was conducted in citrus groves exhibiting HLB-like symptoms in different districts of Punjab Province. The aim was to obtain current information on the incidence of CLas in the area and complement the previous studies. Conventional and real time quantitative polymerase chain reaction (PCR and qPCR) assays were used for the detection of CLas in the 94 samples tested, followed by sequencing of the PCR amplicons. Overall CLas was detected in 43% (40/94) of the tested samples from Kinnow mandarin (n= 22), sweet orange (n= 12), grapefruit (n= 3) and Feutrell’s early mandarin (n= 3). In agreement with previous reports, this survey confirmed the presence of HLB in the Punjab Province of Pakistan and provided updated molecular evidence for the presence of CLas and disease incidence for four citrus cultivars from five citrus growing districts of the province.

‘Candidatus Liberibacter asiaticus’ (CLas) is a pathogen causing Huanglongbing (HLB, yellow shoot disease), which is highly destructive to citrus production. The CLas strains harbor prophages. We identified two unique prophages, designated as P-PA19-1 and P-PA19-2, in CLas strain PA19 from Pakistan using next-generation sequencing analysis. P-PA19-1 prophage has high sequence similarity (identity: 78.23%) at the early-gene region of prophage SC1 (Type 1), but it is significantly divergent in the late-gene region (identity: 62.03%). P-PA19-2 was highly similar to SC2 (Type 2) in the late gene region (identity: 97.96%), and also in the early gene region except for a deletion of a 7,179-bp nucleotide sequence that contains a CRISPR/cas system in SC2. Both P-PA19-1 and P-PA19-2 had circular plasmid forms, and only P-PA19-2 was found integrated in the PA19 chromosome. The two new prophages were only found in Pakistani samples. Identification of prophages enhances our understanding of CLas genomic diversity and also the biology and evolution of CLas prophages.

‘Candidatus Liberibacter asiaticus’ (CLas) is an unculturable, phloem-restricted αProteobacteria, associated with citrus Huanglongbing (HLB), which is one of the most destructive diseases in citrus production worldwide. Here, we present the genome sequences of CLas strains PA19 and PA20 from HLB-affected kinnow trees in Multan, Punjab Province, Pakistan. The CLas genomes of PA19 and PA20 comprise 1,224,156 bp and 1,226,225 bp, respectively, with an average GC content of 36.4%. Both harbored the Type 2 prophage. In this study, we report two CLas genomes from Pakistan, which extends the sequence database of CLas and will contribute to CLas biology and HLB management.