Polymerase Chain Reaction (PCR)- Based Diagnosis of Huanglongbing (Citrus Greening) Disease of Citrus in Nepal

Abstract

Citrus Huanglongbing (HLB) or Citrus Greening disease (CGD) caused by CandidatusLiberibacter asiaticus is the most devastating disease of citrus in Asian countries includingNepal. It affects all citrus cultivars and causes rapid decline of trees. The aim of thisinvestigation was to determine distribution of HLB infestation in various citrusnurseries andorchards of fifteen (Kaski, Syangja, Kathmandu, Doti, Dadeldhura, Baitadi, Salyan,Sindhupalchowk, Lamjung, Kailai, Dhankuta, Okhaldhunga, Rukum, Dailekh and Dhading)different districts of Nepal. Of these 15 districts field visit was carried out (10 August2008) in Syangja, Kaski and Kathmandu districts while from other districts samples were received via National Citrus Development Program, Kirtipur; National Citrus Research Program, Dhankuta, and several District Agriculture Development Offices for HLB detection.Altogether, 183 samples were analyzed for detection of HLB disease using Polymerase ChainReaction (PCR) assay based on amplification of 703bp long fragment of ribosomal proteingene (rpl) using primers f-A2 and r-J5 andother PCR is based on amplification of 1160bplong fragment of 16S rDNA sequences of HLB organism using primers f-OAI, OI1 and r-OI2C. DNA extraction was carried out from the midribs of the suspected leaf samples usingWizard miniprep technique. The PCR reaction was performed in 50µl reaction volume andPCR products were analyzed by using 1% agarose gel in TAE buffer. Among 183 samples used in the study, 18 samples were detected positive for HLB. HLB wasfound to be prevalent in 9 districts out of 15 districts under study. From present investigation,out of 15 districts studied HLB was found to be most prevalent in Kathmandu (33.3%) district followed by Syangja, Salyan and Doti (20% each) districts. HLB was detected positive in both seedling grown trees aswell from grafted trees of suntala (Citrus reticulata) and Junar(C. sinensis) mother plant, grafted grapefruit, and grafted mandarin. Altogether one hundredforty one samples of mandarin (77.04%), thirtynine samples of sweet orange (21.31%), onesample each of pumello (0.55%), grapefruit (0.55%) and muntala species (0.55%) wereanalyzed. Among one hundred eighty three samples of different citrus species studied,thirteen-mandarin, four-sweet orange and one grapefruit show positive HLB infestation. Theresults indicated the rapid spread of HLB in these districts. In addition to HLB several othercitrus diseases such as Phytophthora, Canker, Sooty mould, Felt disease, Leaf minner, Aphidsetc. were also reported in Syangja, Kaski and Kathmandu districts during field visits. Presentstudy has shown that HLB is rapidly spreading to newer areas and is a real threat to citrusindustry of Nepal. If no any control strategies have been taken in time, it will definitelydestroy the citrus orchard and its industry ofNepal. In this context, integrated management ofthis disease has to be followed in which PCR diagnosis plays a pivotal role in order to preventfurther spread of citrus disease.