Turunçgil Antraknoz Etmeni Colletotrichum gloeosporioides’in LAMP (Loop-Mediated İsothermal Amplification) Tekniği Kullanılarak Hızlı Tanısı

Abstract

Anthracnose disease in citrus occurs as dieback in shoots, spot on leaves, early leaf and fruit drop and anthracnose symptoms in fruits. Colletotrichum gloeosporioides, a causal agent of anthracnose, causes severe infections in many citrus species and varieties, including lemon. Fungal pathogen C. gloeosporioides can be diagnosed by morphological, molecular methods and protein-based spectrum analyzes. This study was conducted to perform a rapid identification of C. gloeosporioides, using the LAMP technique. Specific primers for the LAMP method were designed using the computer program (Primary Primer Explorer V3) (http://primerexplorer.jp/e), with primer sets specific to each gene region (F3 / B3 and FIP / BIP) of the gene sequences of the C. gloeosporioides strain. The specificity of the primers intended for use in the LAMP reaction was evaluated by using different three isolates of C. gloeosporioides and Fs4 isolates of Fusarium solani using a total of 3 DNA samples. Genomic DNA was used in the amount of 1 ng µl-1 in the PCR study and 10 fg µl-1 in the LAMP study. As a result, C. gloeosporioides isolates glowed in tubes when examined with fluorescent dye. However, no glare was observed in the tube with F. solani. LAMP analysis showed that it successfully detected genomic DNA from C. gloeosporioides, but F. solani was unable to detect the genomic DNA obtained. After 2 hours of analysis with LAMP technique, the pathogen was detected in a short time with the successful results.