PREVALENCE AND DIVERSITY OF CANDIDATUS LIBERIBACTER SPECIES IN MAIN CITRUS GROWING AREAS OF MALAKAND IN NORTHWEST PAKISTAN

Abstract

Malakand Division in northwest Pakistan is known for delicious sweet orange, locally called Malta (Citrus sinensis). However, both quality and quantity of the citrus produce has seen a decline over the past few decades. Among production constraints, Huanglongbing (HLB) caused by phloem-limited alpha proteobacterium Candidatus Liberibacter asiaticus (CLas) -an endosymbiont of Asian Citrus Psyllid (Diaphorina citri), is of great economic significance. Therefore, we assessed HLB incidence through symptom-based surveys in major citrus growing areas of Khyber Pakhtunkhwa (KP), Northwest Pakistan. The presence or absence of CLas in the collected citrus samples was confirmed by PCR (Polymerase Chain Reaction) using three set of primers (LSS/LAS606, OI1/OI2C “16S rDNA” and MH353/MH354 “operon “nusG-rplK”). An average disease incidence of 92% and severity of 34% were recorded from main production zones in district Swat, KP. Likewise, in district Lower Dir, HLB had disease incidence and severity of 64% and 24%, respectively. Molecular diagnostics using 16S rDNA-specific primer LSS/LAS606 and OI1/OI2C, yielded characteristic bands of 500 bp and 1160 bp, from all five pooled samples from Swat district, while only two samples of Dir tested positive for both primers. Using an alternative primer MH353/MH354 (nusG-rplK) showed positive amplification for four samples from Swat and two samples of Dir with an amplicon of 631 bp. To assess the molecular diversity of the CLas, Sanger sequencing was carried out based on16S rDNA gene. Sequence analysis of 16S rDNA gene from all samples revealed maximum similarity with C. Liberibacter asiaticus. Among studied isolates, SwKjuPk-5 (MH374503) and SwMtaPk-2 (MH374500) isolates appeared to be more divergent and grouped in separate clad as compared to remaining isolates of Swat and Dir.