Application of newly designed primer for Huanglongbing disease detection in citrus plants

¹ Graduate Program of Plant Breeding and Biotechnology, Faculty of Agriculture, IPB University, Jalan Meranti, Dramaga Campus, Bogor 16680, Indonesia
² Department of Agronomy and Horticulture, Faculty of Agriculture, IPB University, Jalan Meranti, Dramaga Campus, Bogor 16680, Indonesia
³ Research Center for Horticulture, Research Organization for Agriculture and Food, National Research and Innovation Agency, Jalan Raya Jakarta-Bogor Km. 46, Cibinong, Bogor 16911, Indonesia
⁴ PMB Lab, Department of Agronomy and Horticulture, Faculty of Agriculture, IPB University, Jalan Meranti, Dramaga Campus, Bogor 16680, Indonesia
⁵ Research Center for Applied Botany, Research Organization for Life Sciences and Environment, National Research and Innovation Agency, Jalan Raya Jakarta-Bogor Km. 46, Cibinong, Bogor 16911, Indonesia

^* Corresponding author: apurwito@apps.ipb.ac.id

Abstract

The symptoms of Huanglongbing (HLB) disease are occasionally misunderstood with nutrient deficiency symptoms. The study aimed to develop HLB specific primer targeting the CLIBASIA_RS01220 gene sequence for HLB detection in citrus plants collected around Cimanggu, Bogor. A total of 21 citrus leaves showing HLB symptoms were subjected to the bacterial DNA isolation and subsequently were amplified with the newly designed primer, namely YihY primers, and the reference A2/J5 primers. Of these, 15 out of 21 collected citrus leaves exhibited positive detection of HLB yielded a band size of 930 bp as targeted amplicon of the YihY primer. On the other side, the primer A2/J5 succesfully detected positive amplicon band with a size of 703 bp from 17 out of 21 citrus leaves. Based on the homology sequences analysis, all of the HLB isolate sequences both amplified by YihY and A2/J5 primers had similarity with Candidatus Liberibacter asiaticus. The phylogenetic analysis, both based on YihY and A2/J5 primers showed the mixed between bacterial isolates collected from Japansche citroen maintained in the Cikeumeuh greenhouse and lime trees in Cimanggu greenhouse, assuming the HLB isolate has been transferred between the two greenhouses by the ability of Diaphorina citri to fly in far distance.