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1、三种红树林植物健康与非健康叶片中真菌的比较 基金项目：国家自然科学基金项目（31560727）；广西自然科学基金项目（2014GXNSFBA118135）；钦州市科技开发项目（201322034）；广西海洋生物技术重点实验室开放基金资助(GLMBT-201505)。supported by National Natural Science Foundation of China (31560727); Guangxi Natural Science Foundation (2014GXNSFBA118135); Science and technology development projec
2、t of Qinzhou (201322034); Open Research Fund Program of Guangxi Key Laboratory of Marine Biotechnology(GLMBT-201505)作者简介：龚斌(1979-)，男，湖北宜昌人，博士，副教授，从事微生物学和生物技术制药研究，Tel：15278742065，（E-mail）。缺出生年，籍贯龚斌1*，方怀义1, 2，刘小雪1，廖日权1，宋静静1，林啟福1（1. 广西钦州学院海洋学院，广西 钦州 535000；2. 广西海洋生物技术重点实验室，广西 北海 536000）摘要： 红树林叶片感染真菌后会向
3、非健康状态变化，为了比较三种红树林植物海漆、秋茄和桐花树健康与非健康叶片真菌类群，明确其中真菌类群的差异，该研究从广西茅尾海国家红树林自然保护区采集海漆、秋茄和桐花树健康与非健康叶片。对叶片中真菌分离纯化并进行形态学鉴定，提取真菌的DNA，采用RAPD多样性、ITS序列对真菌进行分子鉴定。经过初步分析，从海漆、秋茄和桐花树中共分离到157株真菌，经过形态学和RAPD分析，可能为19种不同的真菌。采用真菌ITS序列对19种真菌代表性菌株进行分析，结果表明19种真菌都属于子囊菌门，有15株与已有的真菌ITS序列相似性在97%以上；有4株相似性低于95%，可能为新种。海漆健康和非健康叶片分别分离到真
4、菌7种和5种，健康叶片含了非健康叶片中所有真菌；秋茄健康和非健康叶片分离到真菌1种和9种，且健康叶片中分离到的内生真菌Dothiorella aegiceri也包含于非健康叶片分离的真菌中；桐花树健康和非健康叶片分离到真菌1种和3种，且桐花树分离的内生真菌也是Dothiorella aegiceri，其包含于非健康叶片中分离到的真菌。本研究证明，海漆内生真菌同时也可能是植物病原菌，内生真菌与植物病原菌没有明显界限；另一方面，本研究发现秋茄和桐花树叶片的病变是由外部的病原真菌感染引起，感染的原因可能是昆虫叮咬红树林植物后留下了创口，这些创口有利于真菌的感染。关键词关键词之间用逗号隔开 ： 内生真
5、菌；，RAPD多样性；，ITS；，病原真菌The comparative study of fungi in healthy and unhealthy leaves of three kinds of mangrove plants GONGong Bin1*, FANGang Huai-yi1,2, LIUiu Xiao-xue1, LIAOiao R-quan1, SONGong Jing-jing1, LINin Qi-fu请按照本刊的格式修改1(1.Qinzhou University, Ocean department, Qinzhou 535000, China; 2. Guan
6、gxi Key Laboratory of Marine Biotechnology, Beihai 536000, China)Abstract: Mangrove become unhealthy after infected by fungi. In order to compare the fungi in healthy and unhealthy leaves of mangrove plants Excoecaria agallocha, Kandelia candel and Aegiceras corniculata, the healthy and unhealthy le
7、aves of Excoecaria agallocha, Kandelia candel and Aegiceras corniculata were collected from Maowei Sea National Mangrove Reserve, including 82, 82 and 81 of them from healthy leaves, and 90, 86 and 90 from healthy leaves respectively. The fungi were isolated and purified from leaves and identified b
8、y morphological characters, then the DNA of fungi were extracted and analysed by RAPD and ITS sequence. After preliminary analysis, 157 strains of endophytic fungi were isolated, and they are classified into 19 kinds through morphology and RAPD analysis. The 19 strains of fungi were analysed by ITS
9、sequence. The results show that 19 strains belong to Ascomycetes, and the similarity between 15 isolates and the existed ITS sequences in GenBank is more than 97%. Among them, the ITS sequences of 4 endophytic fungi share less than 95% similarity with the ITS sequences available in GenBank, so they
10、may belong to new species. Seven species of fungi were isolated from healthy leaves of Excoecaria agallocha, and five from unhealthy leaves, and the fungi of unhealthy leaves was included in the fungi from healthy leaves. One fungus was isolated from healthy leaves of Kandelia candel, and nine from
11、unhealthy leaves, and the fungus of unhealthy leaves was included in the fungi from healthy leaves, and Dothiorella aegiceri was both isolated in healthy and unhealthy leaves; One fungus was isolated from healthy leaves of Aegiceras corniculata, and three from unhealthy leaves, and Dothiorella aegic
12、eri was both isolated in healthy and unhealthy leaves too. Our result is interesting. Firstly, our research demonstrated that the discriminate between fungi pathogen and endophytic fungi was not very clear in some plants, because the endophytic fungi of Excoecaria agallocha developed into plants pat
13、hogens; secondly, we found that the pathological changes in Kandelia candel and Aegiceras corniculata was caused by external fungi pathogen, and this was followed by biting of insect on leaves, and the wound were in favor of infections of fungi pathogen. Key words: endophytic fungi, RAPD, ITS, fungi
14、 pahogen基金项目 国家自然科学基金项目（31560727）；广西自然科学基金项目（2014GXNSFBA118135）；钦州市科技开发项目（201322034）；广西海洋生物技术重点实验室开放基金资助(GLMBT-201505)。supported by National Natural Science Foundation of China (31560727); Guangxi Natural Science Foundation (2014GXNSFBA118135); Science and technology development project of Qinzhou (
15、201322034); Open Research Fund Program of Guangxi Key Laboratory of Marine Biotechnology(GLMBT-201505)通讯作者*龚斌，博士，副教授，从事微生物学和生物技术制药研究，Tel：15278742065，E-mail:红树林是生长于热带、亚热带海湾、河口泥滩上特有的胎生植物，具有防风、固堤、净化海岸带环境、养殖和旅游等重要的经济价值（Turner, 2015；Ahmed et al, 2016）。我国东南沿海的广东、福建、广西和海南等省是红树林的主要分布区。近几年，红树林虫害频发（Kathiresan
16、 et al, 2003），进一步引起真菌病害的继发感染(Devarajan et al, 2006)，使大片红树林植物受灾，例如李云对危害海桑苗的灰霉病进行研究，发现病原菌为灰葡萄孢（李云等，1996）。周志权报道了广西沿海的山口、钦州、北仑河口 3个主要红树林分布区的病原真菌，发现其主要是炭疽菌、拟盘多毛孢菌、交链孢菌和叶点霉菌（周志权等，2001）。方镇福对福建漳江口红树林自然保护区红树林叶部病害进行调查，发现真菌病害包括交链孢属、镰刀菌属、盘多毛孢属、曲霉属、青霉属等2 纲3目4科5属（方镇福等，2008）。已有的研究对于我们认识红树林病原真菌具有重要意义，但是笔者认为以上研究存在以下两个缺陷：首先，病原真菌分离纯化时有采用PDA培养法，该法分离真菌时对植物有益的内生真菌也可以生长，因此不能通过结果判断分离出的到底是病原菌还是内生真菌；其次，病原真菌鉴定时用的是形态学
