Detection of quiescent infection of mango stem end rot pathogen Lasiodiplodia theobromae in shoot and pre-plucked mango fruit by seminested PCR.
Lin YuJu, Wang YenTing, Yang HongRen, Wang PiHan
Author Affiliation: Department of Microbiology, Soochow University, Taipei, Taiwan.
Plant Pathology Bulletin 18 : 225-235
Abstract : A seminested PCR-based method was developed for specific detection of mango (Mangifera indica L.) stem end rot pathogen Lasiodiplodia theobromae Griffon & Maubl. (=Botryodiplodia theobromae Pat.) in plant tissues. A specific primer Lth1 was designed from rDNA internal transcribed spacers (ITS) region of L. theobromae. Under stringent PCR conditions, a 420-bp amplicon formed from L. theobromae DNA but not from other stem end rot pathogens such as Botryosphaeria dothidea, B. ribis, Pestalotiopsis sp., Phomopsis sp., Colletotrichum acutatum, and C. gloeosporioides. Seminested PCR using primer pairs of ITS5/ITS4 and Lth1/ITS4 was sensitive enough to detect 125 fg of genomic DNA. The technique was used to detect L. theobromae and study the colonization of the pathogen in shoots and pre-plucked fruits. The pathogen in asymptomatic old branches was detected by seminested PCR. It survived endophytic ally. During March to May, it grew into new branch and extended to inflorescence. The endophytic pathogen seems to be an important inoculum source of the post-harvest disease. In infected orchards, the pathogen was detected in 45.5% of the base of new shoots, in 63.2% tissues between new shoot and old branch, and in 72.7% old branch top. Pre-plucked mango samples were collected from 7 orchards in Pingtung, Tainan, and Chiayi. The pathogen in fruit stalk and the skin near stalk was detected by seminested PCR. The pre-plucked samples were treated with ethylene and stem end rot symptoms appeared in 9-12 days, significantly after 12 days. In 2004, 0%-82% fruit samples developed stem end rot and the detection rate of L. theobromae was between 6.3%-76%. In 2005, the disease rate of the fruit was 20%-92.9% and the detection rate was about 29.2%-81%. The detection rate of pre-plucked samples was not always consistent with the disease rate. However, when the detection rates in these samples were higher than 10% by seminested PCR, their disease rates were higher than 10%. Based on the standards of mango export orchards, the mango stem end rot disease rates need to be lower than 10%. The seminested PCR assay showed promise as a monitor tool for the mango stem end rot pathogen.