摘要
林果枝干病害是林果上一类重要的生物灾害,危害严重,我国对于林果枝干病害的防治研究,主要采用栽培措施和化学防治措施,虽然取得了一定的防治效果,但枝干病害的化学防治带来的环境污染等缺陷使化学防治受到了极大限制,寻求新的病害控制方法已经成为林果枝干病害控制的关键。
生物防治方法作为一种有效的病害防治措施,新的生物源药剂的挖掘、开发和制剂的创制成为目前生物防治方法研究的热点和焦点,目前,我国对于林果枝干病害的生物源制剂的研究和开发报道很少,本研究以来自石榴果实(生防菌株A)和国槐树皮中(生防菌株B)的两株生防细菌为研究对象,研究了细菌的形态、培养特性、生理生化及16S rDNA的分子特征,明确了生防菌株的分类地位;同时以林果常见的六种枝干和果实病害的病原菌为参试菌株,研究了两株生防细菌的抗菌作用,最后对两种生防细菌的作用机制进行了初步研究,主要结果和结论如下:
1.两株生防细菌在PDA培养基上的单菌落为圆形、表面光滑、中央隆起、易乳化、在折射和透射光下观察,具有乳光、菌落不透明并呈奶油状、菌落边缘完整;两株生防细菌均为杆状,菌株A大小平均为1.03×0.44μm,菌株B大小平均为1.0×0.37μm,两者没有显著差异,革兰氏染色均为阳性;11项生理生化指标测定表明:两株生防细菌都能够利用葡萄糖和丙二酸盐,能够使硝酸盐还原和进行接触酶反应,均为好氧性细菌,同时能够耐盐和液化明胶,并能够产H_2S,但两菌株不能利用柠檬酸,V-P和M.R试验也表现为阴性反应,上述的研究结果表明了两株生防细菌的生理生化特性完全相同;16s rDNA序列表明,两菌株序列长度为1530bp和1531bp,仅有三个碱基的区别,其同源性达到了99.8%。系统树构建及上述所有结果表明:两菌株为同种枯草芽孢杆菌(Bacillus subtilus)。
2.两株生防细菌抗菌作用的测定和作用机制初步研究表明:两株生防细菌能够不同程度地对林果枝干和果实上的病原菌具有抑制作用,其中对杨树溃疡病菌和石榴疮痂病菌的抑制作用最为明显,而对其它几种病菌也有不同程度的抑制作用;抗菌机制初步研究表明,两株生防细菌对于由Botryosphaeria dothidea引起的林果枝干和果实病害的病菌主要表现为菌丝顶端大量细胞膨大、细胞器增厚、停止生长等特征,但没有引起菌丝细胞的死亡,而对于由Valsa sordida引起的杨树腐烂病菌而言,两株生防细菌均可以引起病菌菌丝顶端细胞的分解和死亡,但不出现顶端细胞膨大和细胞壁增厚现象。
Forest fruits and trunk's disease is a kind of important biological disaster and damage is very serious. Research on the control of forest fruit and branches disease in china mainly uses cultivation measures and chemical control measures at present, despite it had got a certain effect, but the chemical control of branch diseases had polluted the environment and had some other deficiencies, the chemical control was extremely limited. so seeking new disease control methods have become the key point on the forest fruit and branches disease.
The biological control method is an effective disease control measures. The new biological source medicament discovery, exploitation and the creation had become hot spot and focus for biological control method. At present, biological medicament discovery, exploitation and the creation in tree and forest fruit branch diseases were rarely reported in china. This paper mainly research on the two antagonistic bacteria from pomegranate fruit (antagonistic bacteria A) and the bark of Sophora japonica L(antagonistic bacteria B), research on the culture characteristics, the morphological features, physiological and biochemical characteristics and 16S rDNA molecular characteristics and clarify the taxonomic status,at the same time, the author use the six pathogen of important branches and fruit diseases for the tests, studied on the two antagonism of the antibacterial activity, finally,the author also preliminy study on the resisting mechanism for two antagonistics bacteria. The major results and the conclusions are as follows:
1.The cultivated, morphological and physiological and biochemical characteristics of the two antagonistic bacteria results indicated that the two antagonistic bacteria single colony are round,smooth in surface,ridgy in center,easy in emulsification on the PDA culture medium,when obserbed in the refraction and transmission,they have breastical lights, opacity bacteria groups like butter and full brim; the measure of morphological characteristics indicated that these two antagonistic bacteria are rod shape, the antagonistic strain A is 1.03×0.44μm on average, but the antagonistics strain B is 1.0×0.37μm on average, litter smaller than the antagonistics strain A, the result of physiological and biochemical characteristics are showed that the Gram's of two antagonistic bacteria are all masculine,they can use dextrose and diacid salt,can make nitrate deoxidized and process contactical enzyme reactivity.these two antagonistic bacterias all like oxygen,they are able to endure salt and liquid glutin in the same time, and also can create H_2S, but the two bacteria plants can't use citric acid, the V-P and M.R tests also indicated lunar reactivity. 16S rDNA 's molecule size of the two bacteria are 1530bp and 1531bp and only three bases are different, these DNA sequences had 99.8% identity; the results of construction phylogenetic tree and others indicated that two antagonistic bacterias belong to different strains which one species of Bacillus sublitis.
2. The measures of antibacterial action and antibacterial mechanism indicated that two bacteria could inhibit the pathogen of the branches and fruit pathogen to some extents, the effect on the pathogen to canker disease of forest fruit's trunk and branches disease of pomegranate scab are obvious, meanwhile the inhibitory effect had some to other pathogens. Two antagonistic bacteria can lead the mostly top cells of mycelium of the bacteria which caused by Botryosphaeria dothidea on forest branches and fruits to enlarge and thickene cell wall and stop grow as well, but they can not cause the cells of mycelium die, as far as the pahtogen caused by poplar rotten disease,two antagonistic bacteria can cause the top cells of mycelium die, meanwhile there is no phenomenon that enlargement of top cells of mycelium and cell wall thickening.
引文
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