苏云金芽孢杆菌毒杀松材线虫活性研究及线虫冷冻保存
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摘要
松材线虫病是由松材线虫(Bursaphelenchus xylophilus)引起的松树上的一种毁灭性病害,又称松萎蔫病、松树的癌症。松材线虫是目前中国林业生产上最具威胁性的有害生物,也是世界林业检疫有害生物之一。筛选松材线虫拮抗细菌是防治松材线虫病的一条途径,为此,对松材线虫的拮抗细菌进行了筛选,并探索了松材线虫低温冷冻保存的方法。
2008-2010年间,广泛采集浙江省各松材线虫病疫区的土壤、健康和患病马尾松、黑松的枝干和松墨天牛,共采集样品120份,分离到细菌210株,其中产生芽孢的细菌有70株。以松材线虫为靶标,通过96孔细胞培养板法,进行生物测定,得到8株具杀松材线虫活性的芽孢杆菌,其中四株细菌对松材线虫的毒杀活性较高,提取4株细菌的伴胞晶体蛋白,测试其对松材线虫的杀线活性,结果发现其中4株对松材线虫具较高杀线活性,其中菌株zjfc85杀线活性最高,作用48h,校正死亡率达95.3%,其次为菌株zjfc14、zjfc65、zjfc67,其校正死亡率分别为92.6%、82.3%、77.8%。
结合细菌形态特征、生理生化特性、16S rDNA鉴定结果,确定该菌为苏云金芽孢杆菌。通过SDS - PAGE (十二烷基硫酸钠-聚丙烯酰胺凝胶电泳)对4株细菌的伴胞晶体蛋白的分子量进行测定,结果表明4株细菌的伴胞晶体蛋白各不相同。
对实验室条件下松材线虫的保存方法进行了研究,摸索了-80℃下松材线虫的冷冻保存方法,研究了不同冷冻保护剂对松材线虫低温保存的保护效果,以及解冻后松材线虫的繁殖情况。结果表明:15%甘油对松材线虫保护效果最好,其次为10%甘油、20%甘油、25%甘油,保护效果最差的是1%二甲基亚砜(DMSO),解冻后存活率达36.5%。解冻后线虫能够正常生长繁殖,冻存3d和冻存30d的线虫存活率和繁殖无显著性差异。
本研究筛选到了4株具较高杀松材线虫活性的苏云金芽孢杆菌,为松材线虫病的生物防治提供了材料,探索了松材线虫在实验室条件下的低温冷冻保存方法,解决了在松材线虫病的研究中虫源不稳定的问题,可避免多次继代培养松材线虫活性和致病性降低的发生,为实验室条件下保存松材线虫提供了方法参考。
Pine wood nematode disease (also called pine wilt disease) caused by Bursaphelenchus xylophilus(Steiner et Buhrer), is a destructive disease of pine trees. In present, Bursaphelenchus xylophilus is a threat to China forestry, and it is one of the quarantine pests in the world. Bacteria were shown nematicidal activity to other nematodes, like Caenorhabditis elegans. Screening nematicidal bacteria against B. xylophilus were performed. For the purpose of convenient culture of B. xylophilus, cryoperservation of this nematode at -80℃was established.
In the first place, 210 samples were isolated from the soil, dead pine tissue and healthy pine plant tissue from Zhejiang forest. After assay for nematicidal activities in 96 wells, 8 strains among them were shown the significant nematicidal activities against B. xylophilus. All of the 8 strains produce the endospores. For further, Among these 8 strians, 4 Bt strains’crystal proteins tested on Bursaphelenchus xylophilus, were found to had higher nematicidal activity. The bioassay result showed that the mortality after 48h of zjfc85, zjfc14, zjfc65 and zjfc67 were 90.91%,85.58%, 75.80% and 60.00%.
During 2008 to 2010, 120 exzamples including soil, diseased and healthy trunks of P. massoniana, Black pinus and Monochamus alternatus, were collected from the areas that pine wood nematode disease occurrenced in Zhejiang province. 210 isolates were obtained, 70 isolates that had spores were included. 96 well plate were used to test the nematicidal activity of all isolates. The results showed that 8 strains had nematicidal activity, 4 of the 8 strains showed higher activity, named zjfc14, zjfc65, zjfc67, zjfc85. Crystal proteins were extracted and their nematicidal activity against Bursaphelenchus xylophilus were tested. The bioassay result showed that the mortality of zjfc14, zjfc65, zjfc67, zjfc85 after 48h, were92.6%, 82.3%, 77.8% and 95.3%. Strain zjfc85 had the highest nematicidal activity.
The colony morphology, physiologic and biochemical analysis results and the result of 16srDNA proved that the 4 strains were Bacillus thuringiensis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) result showed that the crystal proteins of the four Bt strains were different from each other.
In this paper, cryoperservation of Bursaphelenchus xylophilus at -80℃was estabilished. Followed by investigation of different cryoprotectants affecting the survival and propagation, cryoperservation with 15% (V/V) glycerol as the protectant was found as the best for cryoperservation, followed by 10% (V/V), 20% (V/V) and 25%(V/V). The last was 1% DMSO. Under the circumstance of 15% (V/V) glycerol, the survival rate maintained as high as 36.5% after defrosting, and nematodes remained normal at propagation. There was no significant difference between 3 days and 30 days later after defrosting, which indicated that cryoperservation with 15% (V/V) glycerol was suitable for B. xylophilus at -80℃, substantially.
In this study 4 different Bt strains that had high nematicidal activity were obtained. They can be basic materials in theresearch of biocontrol of pine wood nematode disease. And the cryoperservation method of Bursaphelenchus xylophilus were primarily constructed, which can solve the problems in the research of Bursaphelenchus xylophilus.
2008-2010年间,广泛采集浙江省各松材线虫病疫区的土壤、健康和患病马尾松、黑松的枝干和松墨天牛,共采集样品120份,分离到细菌210株,其中产生芽孢的细菌有70株。以松材线虫为靶标,通过96孔细胞培养板法,进行生物测定,得到8株具杀松材线虫活性的芽孢杆菌,其中四株细菌对松材线虫的毒杀活性较高,提取4株细菌的伴胞晶体蛋白,测试其对松材线虫的杀线活性,结果发现其中4株对松材线虫具较高杀线活性,其中菌株zjfc85杀线活性最高,作用48h,校正死亡率达95.3%,其次为菌株zjfc14、zjfc65、zjfc67,其校正死亡率分别为92.6%、82.3%、77.8%。
结合细菌形态特征、生理生化特性、16S rDNA鉴定结果,确定该菌为苏云金芽孢杆菌。通过SDS - PAGE (十二烷基硫酸钠-聚丙烯酰胺凝胶电泳)对4株细菌的伴胞晶体蛋白的分子量进行测定,结果表明4株细菌的伴胞晶体蛋白各不相同。
对实验室条件下松材线虫的保存方法进行了研究,摸索了-80℃下松材线虫的冷冻保存方法,研究了不同冷冻保护剂对松材线虫低温保存的保护效果,以及解冻后松材线虫的繁殖情况。结果表明:15%甘油对松材线虫保护效果最好,其次为10%甘油、20%甘油、25%甘油,保护效果最差的是1%二甲基亚砜(DMSO),解冻后存活率达36.5%。解冻后线虫能够正常生长繁殖,冻存3d和冻存30d的线虫存活率和繁殖无显著性差异。
本研究筛选到了4株具较高杀松材线虫活性的苏云金芽孢杆菌,为松材线虫病的生物防治提供了材料,探索了松材线虫在实验室条件下的低温冷冻保存方法,解决了在松材线虫病的研究中虫源不稳定的问题,可避免多次继代培养松材线虫活性和致病性降低的发生,为实验室条件下保存松材线虫提供了方法参考。
Pine wood nematode disease (also called pine wilt disease) caused by Bursaphelenchus xylophilus(Steiner et Buhrer), is a destructive disease of pine trees. In present, Bursaphelenchus xylophilus is a threat to China forestry, and it is one of the quarantine pests in the world. Bacteria were shown nematicidal activity to other nematodes, like Caenorhabditis elegans. Screening nematicidal bacteria against B. xylophilus were performed. For the purpose of convenient culture of B. xylophilus, cryoperservation of this nematode at -80℃was established.
In the first place, 210 samples were isolated from the soil, dead pine tissue and healthy pine plant tissue from Zhejiang forest. After assay for nematicidal activities in 96 wells, 8 strains among them were shown the significant nematicidal activities against B. xylophilus. All of the 8 strains produce the endospores. For further, Among these 8 strians, 4 Bt strains’crystal proteins tested on Bursaphelenchus xylophilus, were found to had higher nematicidal activity. The bioassay result showed that the mortality after 48h of zjfc85, zjfc14, zjfc65 and zjfc67 were 90.91%,85.58%, 75.80% and 60.00%.
During 2008 to 2010, 120 exzamples including soil, diseased and healthy trunks of P. massoniana, Black pinus and Monochamus alternatus, were collected from the areas that pine wood nematode disease occurrenced in Zhejiang province. 210 isolates were obtained, 70 isolates that had spores were included. 96 well plate were used to test the nematicidal activity of all isolates. The results showed that 8 strains had nematicidal activity, 4 of the 8 strains showed higher activity, named zjfc14, zjfc65, zjfc67, zjfc85. Crystal proteins were extracted and their nematicidal activity against Bursaphelenchus xylophilus were tested. The bioassay result showed that the mortality of zjfc14, zjfc65, zjfc67, zjfc85 after 48h, were92.6%, 82.3%, 77.8% and 95.3%. Strain zjfc85 had the highest nematicidal activity.
The colony morphology, physiologic and biochemical analysis results and the result of 16srDNA proved that the 4 strains were Bacillus thuringiensis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) result showed that the crystal proteins of the four Bt strains were different from each other.
In this paper, cryoperservation of Bursaphelenchus xylophilus at -80℃was estabilished. Followed by investigation of different cryoprotectants affecting the survival and propagation, cryoperservation with 15% (V/V) glycerol as the protectant was found as the best for cryoperservation, followed by 10% (V/V), 20% (V/V) and 25%(V/V). The last was 1% DMSO. Under the circumstance of 15% (V/V) glycerol, the survival rate maintained as high as 36.5% after defrosting, and nematodes remained normal at propagation. There was no significant difference between 3 days and 30 days later after defrosting, which indicated that cryoperservation with 15% (V/V) glycerol was suitable for B. xylophilus at -80℃, substantially.
In this study 4 different Bt strains that had high nematicidal activity were obtained. They can be basic materials in theresearch of biocontrol of pine wood nematode disease. And the cryoperservation method of Bursaphelenchus xylophilus were primarily constructed, which can solve the problems in the research of Bursaphelenchus xylophilus.
引文
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