Rapid Diagnosis of Citrus Anthracnose Caused by Colletotrichum gloeosporioides Using a LAMP (Loop-Mediated Isothermal Amplification) Assay

Öz

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 1ng/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.

Anahtar Kelimeler

• Anthracnose,C. gloesoporioides,LAMP,İdentification,Citrus

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

Öz

Turunçgillerde antraknoz hastalığı sürgünlerde geriye doğru kuruma, yapraklarda leke, erken dönem yaprak ve meyve dökümleri, meyvelerde antraknoz belirtileri şeklinde ortaya çıkmaktadır. Antraknoz etmeni Colletotrichum gloeosporioides, ülkemizde özellikle limon başta olmak üzere birçok turunçgil tür ve çeşitlerinde şiddetli enfeksiyonlara yol açmaktadır. C. gloeosporioides fungal patojeninin tanısı, morfolojik, moleküler yöntemler ve proteine dayalı spektrum analizleri ile yapılabilmektedir. Bu çalışma, LAMP tekniğini kullanarak turunçgil antraknoz etmeni C.gloeosporioides’in hızlı tanısını gerçekleştirmek için yürütülmüştür. LAMP yöntemi için spesifik primerler, C. gloeosporioides türünün gen dizileri her gen bölgesine özgü primer setleri (F3/B3 ve FIP/BIP) ‘Primer Explorer V3’ adlı bilgisayar programı (http://primerexplorer.jp/e) kullanılarak tasarlanmıştır. LAMP reaksiyonunda kullanılmak üzere tasarlanan primerlerin özgüllükleri C. gloeosporioides’ in 3 farklı izolatları ile Fusarium solani’ nin Fs4 izolatı olmak üzere toplam 3 DNA örneği kullanılarak değerlendirilmiştir. PCR çalışmasında genomik DNA 1ng/µl-1, LAMP çalışmasında 10 fg/µl-1 miktarında kullanılmıştır. Sonuç olarak, Flouresan boya ile incelendiğinde, C. gloeosporioides izolatlarının bulunduğu tüplerde parlama olmuştur. Ancak, F. solani’ nin bulunduğu tüpte parlama gözlenmemiştir. LAMP analizinin C. gloeosporioides’ den elde edilen genomik DNA'yı başarıyla tespit ettiğini göstermiş, fakat F. solani’ elde edilen genomik DNA'yı tespit edememiştir. LAMP tekniği ile 2 saat süren bir analiz sonucunda elde edilen başarılı sonuçlarla kısa süre içerisinde patojenin varlığı tespit edilmiştir.

Anahtar Kelimeler

• Antraknoz,C. gloesoporioides,LAMP,Teşhis

Teşekkür

Bu çalışma, Hatay Mustafa Kemal Üniversitesi Bitki Sağlığı Kliniği Uygulama ve Araştırma Merkezi laboratuvarlarında yürütülmüştür.

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