Côté Marie-José, Tardif Marie-Claude, Meldrum Allison J
Canadian Food Inspection Agency, Ottawa Laboratory (Fallowfield), Centre for Plant Quarantine Pests, 3851 Fallowfield Road, Ottawa, Ontario, Canada, K2H 8P9.
Health Canada, Food Directorate, Building #7, Tunney's Pasture, P.L. 0700E1, Ottawa, Ontario, Canada K1A 0L2.
Plant Dis. 2004 Nov;88(11):1219-1225. doi: 10.1094/PDIS.2004.88.11.1219.
Monilinia fructigena, M. fructicola, M. laxa, and Monilia polystroma each have a different regulatory status. To monitor imported and exported fruit for the presence of quarantined Monilinia or Monilia species, a timely identification method is required. Random amplified polymorphic DNA analysis was used to generate an M. fructigena-specific band that was characterized by sequencing. Using the sequence obtained, primers were designed to amplify bands in the same genomic region of M. fructicola and M. laxa. These bands were also characterized by sequencing. From all three sequences, a multiplex polymerase chain reaction (PCR) method based on a common reverse primer (MO368-5) and three species-specific forward primers (MO368-8R, MO368-10R, and Laxa-R2) was established for the differentiation of the three Monilinia species. The multiplex PCR was tested with additional isolates and consistently produced a 402-bp PCR product for M. fructigena, a 535-bp product for M. fructicola, and a 351-bp product for M. laxa. The method was also used with isolates of the recently characterized Monilia polystroma, and all isolates amplified a 425-bp PCR product. The identification method was shown to amplify a PCR product directly from inoculated apples, and the PCR band produced was specific to the inoculated Monilinia or Monilia species. Furthermore, the multiplex PCR was used to identify Monilinia species on naturally infected stone fruits. The method correctly identified infections by both M. laxa and M. fructicola by successful amplification of corresponding PCR products for each species.
果生链核盘菌、果核链核盘菌、核果链核盘菌和多室链核盘菌各自具有不同的监管状态。为监测进出口水果中是否存在检疫性链核盘菌或链核盘菌属物种，需要一种及时的鉴定方法。利用随机扩增多态性DNA分析产生了一条果生链核盘菌特异性条带，并对其进行了测序表征。利用获得的序列设计引物，以扩增果核链核盘菌和核果链核盘菌相同基因组区域的条带。这些条带也通过测序进行了表征。基于一条通用反向引物（MO368 - 5）和三条物种特异性正向引物（MO368 - 8R、MO368 - 10R和Laxa - R2），建立了一种多重聚合酶链反应（PCR）方法，用于区分这三种链核盘菌物种。用其他分离株对多重PCR进行了测试，结果始终为果生链核盘菌产生一条402 bp的PCR产物，为果核链核盘菌产生一条535 bp的产物，为核果链核盘菌产生一条351 bp的产物。该方法还用于最近鉴定的多室链核盘菌的分离株，所有分离株均扩增出一条425 bp的PCR产物。该鉴定方法被证明可直接从接种的苹果中扩增出PCR产物，产生的PCR条带对接种的链核盘菌或链核盘菌属物种具有特异性。此外，多重PCR被用于鉴定自然感染核果上的链核盘菌物种。该方法通过成功扩增每个物种相应的PCR产物，正确鉴定了核果链核盘菌和果核链核盘菌的感染情况。
