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采用室外定点观察,子实体诱导及r DNA ITS、MS204、tef1-α3种分子标记进行系统发育分析等方法,对板栗褐缘叶枯病Phomopsis castaneae-mollissimae的协同致病菌板栗蛇孢日规壳Ophiognomonia castaneae的生活史进行了研究。结果表明,每年7月下旬至8月初叶片发病初期很少分离到O.castaneae,随着病斑扩大该菌的分离频率逐渐增大,至发病后期其分离频率可高达78.5%,甚至可超过致病菌P.castaneae-mollissimae,10月下旬板栗落叶背面的病斑上开始形成O.castaneae的分生孢子盘,11月下旬开始形成O.castaneae的子囊壳原基,次年5、6月越冬落叶背面的病斑上长出子囊壳;带病斑的叶片经室内外诱导,0–25℃范围均可产生成熟子囊壳;湿度是决定子囊壳能否形成的关键因素,强光照不利于子囊壳的产生;分离物的菌丝体在PDA培养基上培养,易产生分生孢子;将分离物分为两种交配型,相互交配后6个月所有处理均未长出该菌的有性型子实体。室外定点观察及r DNA ITS、MS204、tef1-α3种分子标记表明分离物和病斑上的子囊孢子及其萌发菌丝为O.castaneae的不同生长发育阶段。
The phylogenetic analysis was carried out by using outdoor fixed-point observation, fruiting body induction and r DNA ITS, MS204 and tef1-α molecular markers. The symbiotic pathogen Chromosomes of chestnut snake spores on chestnut brown leaf blight Phomopsis castaneae-mollissimae The life history of Ophiognomonia castaneae was studied. The results showed that O. castaneae was seldom isolated from the beginning of July to the beginning of August each year, and the frequency of isolates increased with the spread of the lesion. The frequency of isolation was up to 78.5% P.castaneae-mollissimae, the conidia disk of O.castaneae began to form on the back of chestnut deciduous leaf in late October, and began to form the oocysts shell of O.castaneae in late November. On the back of the deciduous leaves, the ascicle shell grows on the lesion; the leaves with the diseased spot are induced indoors and outdoors, and the mature cysts can be produced in the range of 0-25 ℃. The humidity is the key factor to determine the formation of the orbicle shell. The mycelium of the isolates were cultured on PDA medium, easy to produce conidia; the isolates were divided into two mating types, all the treatments did not grow the bacteria after 6 months of mating Sub-entity. Outdoor fixed point observation and r DNA ITS, MS204 and tef1-α3 molecular markers showed that the ascospores and their mycelial spores on isolates and lesion were different growth stages of O.castaneae.