Serological and molecular based detection of graft-transmissible pathogens associated with citrus from non-core areas of Pakistan

Abstract

To assess prevalence of virus and virus-like diseases of citrus, surveys were conducted in citrus groves of Southern Punjab during 2013-15. Based on serological and PCR detection methods, citrus orchards and nurseries were tested for prevalence of Citrus tristeza virus (CTV), Citrus psrosis virus (CPsV), Citrus variegated chlorosis (CVC) caused by Xyellela fastidiosa, huanglongbing (HLB, citrus greening) caused by ‘Candidatus Liberibacter asiaticus’ and citrus stubborn disease (CSD) caused by Spiroplasma citri. Citrus leaves and twigs samples of ‘Kinnow’ and‘Feutrell’s Early’ mandarins (Citrus reticulate Blanco), ‘Mosambi’ sweet oranges (Citrus aurantium L.), ‘Shamber’ grapefruit (Citrus aurantiumL.) were collected from five districts of southern Punjab viz. Multan, Khanewal, Lodhran, Layyah and Bhakkar. Direct antibody sandwich enzyme linked immuno sorbent assay (DAS-ELISA) was employed to detect CTV, CPsV, CVC and CSD, where-as HLB was detected by PCR using specific primers from two regions 16S rDNA intergenic region and ribosomal protein gene of rplKAJL-rpoBCoperon (β-operon) of ‘C. Librebacter asiaticus’. Sequence of 16S rDNA revealed nucleotide identity level upto 98% among different isolates. Disease incidence of CTV, CVC, HLB and CSD was 36.7, 10, 40 and 36.7%, respectively, where-as CPsV was not detected. All ‘citrus species and relatives’ viz., Kinnow, sweet oranges, Feutrell’s early and grapefruit (shamber) were susceptible to these diseases. To our understanding this is first investigation of virus and virus-like diseases in citrus from non-core areas of Punjab, Pakistan.