烟草抗根结线虫内生微生物的筛选
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摘要
本研究旨在探明烟草植株中内生微生物的分布规律,从而筛选活性内生菌防治烟草根结线虫病害。通过对4个优质常规烟草品种(K326、G-80、红花大金元、净叶黄)的种子、根、茎和叶进行内生菌的分离计数、初步分类、抗菌与抗根结线虫筛选研究。得以下主要结论:
(1)4个烟草品种内生微生物分布规律探究表明:烟草中有大量的内生菌分布,以细菌最多,放线菌次之,真菌最少,与土壤中的分布规律一致。烟草各器官中内生菌的分布表现为根和种子中数量相当且较多,茎次之,叶最少的规律。各烟草品种间的分布为:种子中是K326和红花大金元内生菌分布较多,净叶黄次之,G-80最少;苗期植株中4个品种的内生菌数基本相同;大田期是净叶黄的内生菌数量最少。烟草内生细菌以革兰氏阴性细菌为主,而种子中内生细菌的芽孢杆菌数量明显多于植株中的。
(2)内生菌对大肠杆菌和金黄色葡萄球菌的抑菌试验结果表明:烟草内生细菌发酵液抑菌效果不明显;烟草内生放线菌的菌体和发酵液抑菌效果都较强,其中,内生放线菌Y12对金黄色葡萄球菌的抑菌圈直径达15mm,A7对大肠杆菌的抑菌圈达21mm。从红花大金元品种中筛选到有抑菌活性的菌株5株,从净叶黄和K326中分别筛选到1株。
(3)对筛选出来的拮抗性较好的菌株进行了抑杀根结线虫2龄幼虫实验表明:内生菌菌体抑杀根结线虫2龄幼虫没有效果,内生细菌发酵液抑杀根结线虫效果突出,抑杀率达到100%的菌株有3株,抑杀效果最好H3号菌株,在30%浓度下对根结线虫2龄幼虫的抑杀率达到了100%。内生防线菌除Y12外,其余菌株效果均不佳。
(4)盆栽试验结果:选取H1、H3、K6和Y12进行防治烟草根结线虫的盆栽试验,结果表明:内生细菌发酵液的防治效果优于内生细菌菌体,内生放线菌则相反。内生菌均对植物具有促生作用,但内生菌菌体的促生效果优于内生菌的发酵液。H3处理内生细菌对根结线虫防治效果最好,防治效果为91.68%,明显优于空白对照,且略优于阿维菌素处理,H3内生细菌处理的烟草形态指标明显优于阿维菌素处理。
(5)鉴定结果:对H3菌株进行了16S rDNA以及常规鉴定。16S rDNA测序比对以及形态和生理生化指标测定表明H3菌株可能为蜡样芽孢杆菌。
This paper is designed to discover the distributing pattern of the endophytes in tobacco plants, and select bioactive endophytes which have the capability to be anti-tobacco rot-knot nematodes. And four species of high-quality tobaccos, namely K326, G-80, Hong Hua Da Jing Yuan and Jing Ye Huang are used to conduct the experiment, in which the endophytes in their seeds, roots, stems and leaves are isolated, counted and selected. The results are as follows:
First, it is found that there are a large amount of endophytes in tobacco, of which endophytic bacterias takes the largest percentage, endophytic actinomycetes the next and endophytic fungus the least. The distribution of them in the soil is the same. Concerning the numbers of the endophytes in different parts of the plant, it is almost the same in the roots and the seeds. Caudexes have the medium amount of endophytes, and leaves have the least. In different species of tobaccos, the distribution of endophytes also varies. When they are still seeds, the number of endophytes in K326 and Hong Hua Da Jing Yuan is considerably high. In Jing Ye Huang, the number is also not small. And G-80 has the least. During the seedling period, there is no obvious distinction among the mentioned four species. However, when they are planted in the field, the number of endophytes in Jing Ye Huang is the smallest. In the plants of tobacco, the kind of endophytes that occupies a large quantity is Gram-negative bacterium, while the number of spore bacteria is higher in seeds.
Second, in antagonistic experiment and antiblastic emperiments of Escherichia coli and Staphyloccus aureus, the results show that Endophyte bacterias fermentation broth's effect on controlling bacterias is not clear. At least, it can not be detected from the experiment.But endophytic actinomycetes and its bacterias fermentation broth are effective.The inhibition diameters of the endophytic actinomycetes Y12 is as wide as 15 mm. in the anti-Staphyloccus aureus experiment, and that of the plant A7 has reached 21 mm in the anti-Escherichia coli experiment. Besides, there are 5 Hong Hua Da Jing Yuan, 1 Jing Ye Huang and 1 K326, which are found to be antiblastically active. All these indicate that the anti-botanic disease and antagonistic characteristics of the four species are to some extent connected with each other.
Third, the selected plants which are more antagonistically effective are then used to conduct bacterias fermentation broth antagonistic and the secondary females rot-knot nematodes controlling experiment. It turns out that the Endophyte has no controlling and killing ability. However, its bacterias fermentation broth is obviously effective. In the preliminary experiments, there are 3 plants that can kill all the bactierias, among which the plant H3 is the most effective. When put into 30% bacterias fermentation broth, the plant H3's ability of killing rot-knot nematodes can reach as high as 100 percent. But the results of endophytic actinomycetes Y12 is not desirable.
Forth, H1, H3, K6 and Y12 are selected to conduct root-knot nematodes controlling experiment. It turns out that Endophyte bacterias fermentation broth is more effective than its thallus. But the result of endophytic actinomycetes is the opposite. Ordinarily speaking, Endophyte can promote the growth of plants. But the promoting effect of the Endophyte thallus is larger than that of its bacterias fermentation broth. With disease index of 4.16, controling effect is 91.68%, the H3 Endophyte is best in controlling rot-knot nematodes , which is much better than blank CK and is also better than CK Avermectins. Moreover, the H3 Endophyte's root-knot nematodes indicator of tobacco is obviously higher than CK Avermectins'.
Finally, as the plant H3's effect is so obvious in the above experiments, it is then used to carry out 16S rDNA identification and ordinary identification. According to 16S rDNA order comparison and the result of morphological, physiological and biochemical characteristics, it is concluded that the plant H3 is probably bacillus cereus.
(1)4个烟草品种内生微生物分布规律探究表明:烟草中有大量的内生菌分布,以细菌最多,放线菌次之,真菌最少,与土壤中的分布规律一致。烟草各器官中内生菌的分布表现为根和种子中数量相当且较多,茎次之,叶最少的规律。各烟草品种间的分布为:种子中是K326和红花大金元内生菌分布较多,净叶黄次之,G-80最少;苗期植株中4个品种的内生菌数基本相同;大田期是净叶黄的内生菌数量最少。烟草内生细菌以革兰氏阴性细菌为主,而种子中内生细菌的芽孢杆菌数量明显多于植株中的。
(2)内生菌对大肠杆菌和金黄色葡萄球菌的抑菌试验结果表明:烟草内生细菌发酵液抑菌效果不明显;烟草内生放线菌的菌体和发酵液抑菌效果都较强,其中,内生放线菌Y12对金黄色葡萄球菌的抑菌圈直径达15mm,A7对大肠杆菌的抑菌圈达21mm。从红花大金元品种中筛选到有抑菌活性的菌株5株,从净叶黄和K326中分别筛选到1株。
(3)对筛选出来的拮抗性较好的菌株进行了抑杀根结线虫2龄幼虫实验表明:内生菌菌体抑杀根结线虫2龄幼虫没有效果,内生细菌发酵液抑杀根结线虫效果突出,抑杀率达到100%的菌株有3株,抑杀效果最好H3号菌株,在30%浓度下对根结线虫2龄幼虫的抑杀率达到了100%。内生防线菌除Y12外,其余菌株效果均不佳。
(4)盆栽试验结果:选取H1、H3、K6和Y12进行防治烟草根结线虫的盆栽试验,结果表明:内生细菌发酵液的防治效果优于内生细菌菌体,内生放线菌则相反。内生菌均对植物具有促生作用,但内生菌菌体的促生效果优于内生菌的发酵液。H3处理内生细菌对根结线虫防治效果最好,防治效果为91.68%,明显优于空白对照,且略优于阿维菌素处理,H3内生细菌处理的烟草形态指标明显优于阿维菌素处理。
(5)鉴定结果:对H3菌株进行了16S rDNA以及常规鉴定。16S rDNA测序比对以及形态和生理生化指标测定表明H3菌株可能为蜡样芽孢杆菌。
This paper is designed to discover the distributing pattern of the endophytes in tobacco plants, and select bioactive endophytes which have the capability to be anti-tobacco rot-knot nematodes. And four species of high-quality tobaccos, namely K326, G-80, Hong Hua Da Jing Yuan and Jing Ye Huang are used to conduct the experiment, in which the endophytes in their seeds, roots, stems and leaves are isolated, counted and selected. The results are as follows:
First, it is found that there are a large amount of endophytes in tobacco, of which endophytic bacterias takes the largest percentage, endophytic actinomycetes the next and endophytic fungus the least. The distribution of them in the soil is the same. Concerning the numbers of the endophytes in different parts of the plant, it is almost the same in the roots and the seeds. Caudexes have the medium amount of endophytes, and leaves have the least. In different species of tobaccos, the distribution of endophytes also varies. When they are still seeds, the number of endophytes in K326 and Hong Hua Da Jing Yuan is considerably high. In Jing Ye Huang, the number is also not small. And G-80 has the least. During the seedling period, there is no obvious distinction among the mentioned four species. However, when they are planted in the field, the number of endophytes in Jing Ye Huang is the smallest. In the plants of tobacco, the kind of endophytes that occupies a large quantity is Gram-negative bacterium, while the number of spore bacteria is higher in seeds.
Second, in antagonistic experiment and antiblastic emperiments of Escherichia coli and Staphyloccus aureus, the results show that Endophyte bacterias fermentation broth's effect on controlling bacterias is not clear. At least, it can not be detected from the experiment.But endophytic actinomycetes and its bacterias fermentation broth are effective.The inhibition diameters of the endophytic actinomycetes Y12 is as wide as 15 mm. in the anti-Staphyloccus aureus experiment, and that of the plant A7 has reached 21 mm in the anti-Escherichia coli experiment. Besides, there are 5 Hong Hua Da Jing Yuan, 1 Jing Ye Huang and 1 K326, which are found to be antiblastically active. All these indicate that the anti-botanic disease and antagonistic characteristics of the four species are to some extent connected with each other.
Third, the selected plants which are more antagonistically effective are then used to conduct bacterias fermentation broth antagonistic and the secondary females rot-knot nematodes controlling experiment. It turns out that the Endophyte has no controlling and killing ability. However, its bacterias fermentation broth is obviously effective. In the preliminary experiments, there are 3 plants that can kill all the bactierias, among which the plant H3 is the most effective. When put into 30% bacterias fermentation broth, the plant H3's ability of killing rot-knot nematodes can reach as high as 100 percent. But the results of endophytic actinomycetes Y12 is not desirable.
Forth, H1, H3, K6 and Y12 are selected to conduct root-knot nematodes controlling experiment. It turns out that Endophyte bacterias fermentation broth is more effective than its thallus. But the result of endophytic actinomycetes is the opposite. Ordinarily speaking, Endophyte can promote the growth of plants. But the promoting effect of the Endophyte thallus is larger than that of its bacterias fermentation broth. With disease index of 4.16, controling effect is 91.68%, the H3 Endophyte is best in controlling rot-knot nematodes , which is much better than blank CK and is also better than CK Avermectins. Moreover, the H3 Endophyte's root-knot nematodes indicator of tobacco is obviously higher than CK Avermectins'.
Finally, as the plant H3's effect is so obvious in the above experiments, it is then used to carry out 16S rDNA identification and ordinary identification. According to 16S rDNA order comparison and the result of morphological, physiological and biochemical characteristics, it is concluded that the plant H3 is probably bacillus cereus.
引文
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