产生杀线虫活性物质的放线菌菌株筛选
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摘要
从南京、淮阴和上海等地的30个土样中分离得到344株放线菌，离体条件下测定了各菌株振荡培养滤液对根结线虫二龄幼虫的击倒活性，初步筛选出LH004、LH010、LH018、LH053、LH054、LH082、LH117、LH120、LH121、LH132、LH139、SH037、NJ001、NJ024、NJ026、NJ051、NJ095等17个菌株，其培养滤液稀释8倍后对根结线虫二龄幼虫的击倒率仍达85％以上。将上述17个菌株培养滤液用于防治番茄根结线虫，得到能显著抑制根结和卵块形成数量的3个菌株：NJ026、LH117和LH121，2ml培养滤液稀释5倍后对根结形成的抑制率分别达到62.3％、85.1％和67.0％，对卵块形成的抑制率分别为63.6％、82.7％和68.2％。将3菌株继代培养6代，研究了它们产生杀线活性物质的遗传稳定性，结果显示NJ026很快丧失了杀线活性，LH117产生活性物质的能力也有所下降，LH121能保持稳定的杀线活性。根据以上结果，笔者认为LH117和LH121是有潜力应用于生产上防治蔬菜根结线虫的菌株。两菌株的发酵滤液除了具有杀线活性外，研究还发现两菌株对辣椒黑色炭疽(Colletotrichum gloeosporioides)、辣椒红色炭疽(Gloeosporium piperatum)、黄瓜早疫病菌(Alternaria solani)、黄瓜叶霉病菌(Cladosporium cucumerinum)、黄瓜炭疽病菌(Colletotrichum orbiculare)等5种重要的蔬菜病原真菌的生长有较强的抑制作用。
初步探索了LH117和LH121防治根结线虫的作用方式。LH121在4h内即对二龄幼虫有100％的击倒率，但清水处理后能恢复活性；12h后活性不能恢复，24h后虫体发生电解质渗漏，虫体透明。LH117在8h内对二龄幼虫达到90％击倒率，清水处理能恢复活性，24h时仍有26.5％的恢复活动性。二菌株对卵块孵化也有影响，且抑制率随时间变化逐渐增加，72h后LH121处理的卵块完全停止孵化，并且变为红褐色；LH117处理的卵块72h后对卵块孵化的抑制率达到82.3％。温室盆钵试验表明，两菌株主要是通过杀死未侵入的二龄线虫或降低其侵入活性而控制根结形成，对于已经侵入植物根系建立了寄生关系的线虫的发育影响不大。
优化了二菌株的发酵条件。LH117和LH121二菌株的最佳发酵条件很接近，采用1号培养液、PH7-8、转速170rpm、菌龄6d左右、振荡培养4-5d发酵滤液对松材线虫的击倒活性最强；可溶性淀粉、燕麦片等复杂碳源对产生活性物质优于葡萄糖、蔗糖等简单碳源；牛肉膏、蛋白胨等有机复杂氮源优于无机氮源。
根据国际链霉菌计划(ISP)推荐的方法对LH117和LH121进行了菌株鉴定。
产生不残活件物质oj jJg线由植优9＄选
对其形态特征、培养特征和生理生化传性进行了研冗，与已知的种比较，鉴定二
菌株分别为尖抱链霉菌（）tr。ptoll］，c，s c。lspjdospo。。，s Hlgashlde，t。j，1966）
和玫瑰暗黄链霉菌（价。，尸N／！y。，。，，。，o八jj。，fi。P。ebrazllell。kara，19，7）。
Culture filtrates of 344 actinomyces strains isolated from 30 soil samples were tested of knock-down efficiency in vitro against J2 of root-knot nematodes. Seventeen strains among them, coded as LH004, LH010, LH018, LH053, LH054, LH082, LH117, LH120, LH121, LH132, LH139, SH037, NJ001, NJ024, NJ026, NJ051 and NJ095 respectively, showed strong nematicidal activity .All of their 8-fold diluted culture filtrates had more than 85% knock-down efficiency.
Then the 17strains' culture filtrates were further assayed control effect on root-knot nematodes.3 strains, which were NJ026, LH117 and LH121 respectively, could inhibit gall and egg mass formation most significantly, 2ml culture filtrates reduced 62.3%, 85.1%, 67.0% gall formation and 63.6%, 82.7%, 68.2 % egg mass formation. Then successive 6 generations of the 3 strains were examined genetic stability producing active nematicidal substance, results showed that NJ026 lost its active substance producing ability within 6 generations, LH117 remained nematicidal activity after 6 generations but the decreasing trend may be discerned, only LH121 always demonstrated stable strong nematicidal activity. Based on the above experiment, LH117 and LH121 were considered as potential to be used in practice for root-knot nematodes control. Besides nematicidal activity, culture filtrates of LH117 and LH121 both could inhibit growth of five important vegetable pathogenic fungi, namely Colletotrichum gloeosporioide,Gloeospori
um piperatwn, Alternaria solani, Cladosporium cucumerinum and Colletotrichum orbiculare.
Action mode of the two strains against root-knot nematodes was probed. Culture filtrates of LH121 could completely knock-down all the J2 tested within 4h, but most recovered their movements in fresh water; 12h later, no recovery was observed; 24h later, all of the individuals turned transparent observed under dissect microscope, which revealed that electrolyte leakage occurred. LH117 knocked down 90% of J2 within 8h but most recovered movements in fresh water, 24h later 26.3% of J2 could still recover. Both strains affected egg hatching. 72h treatment with LH121 no egg hatched any longer and the egg mass turned to red-brown; 72h treatment with LH117 the inhibition ratio was 82.3% compared with CK. Experiments in vivo revealed that both strains control root-knot
nematode mainly through killing J2 or affecting their activity, they cannot influence nematode development after the nematode-plant endoparasite relationship has been established.
In order to promote active substance production, fermentation condition of the two actinomyces strains was optimized. Results was that the optimum fermentation condition of the two strains were almost the same. The culture filtrates which utilized No.l nutrition components, with initial PH7-8, shaking 4-5 days at ITOrpm, 6 days strains age around showed the strongest nematicidal activity. Both strains utilized complex carbon sources such as soluble starch and oatments better than simple carbon sources, and complex organic nitric sources such as beef extract and peptone better than inorganic nitric sources.
LH117 and LH121 were identified as Streptomyces cuspidosporus and Streptomyces reseofulvus respectively based on their morphological and physiological properties according to the methods recommended by International Streptomyces Project (ISP).
初步探索了LH117和LH121防治根结线虫的作用方式。LH121在4h内即对二龄幼虫有100％的击倒率，但清水处理后能恢复活性；12h后活性不能恢复，24h后虫体发生电解质渗漏，虫体透明。LH117在8h内对二龄幼虫达到90％击倒率，清水处理能恢复活性，24h时仍有26.5％的恢复活动性。二菌株对卵块孵化也有影响，且抑制率随时间变化逐渐增加，72h后LH121处理的卵块完全停止孵化，并且变为红褐色；LH117处理的卵块72h后对卵块孵化的抑制率达到82.3％。温室盆钵试验表明，两菌株主要是通过杀死未侵入的二龄线虫或降低其侵入活性而控制根结形成，对于已经侵入植物根系建立了寄生关系的线虫的发育影响不大。
优化了二菌株的发酵条件。LH117和LH121二菌株的最佳发酵条件很接近，采用1号培养液、PH7-8、转速170rpm、菌龄6d左右、振荡培养4-5d发酵滤液对松材线虫的击倒活性最强；可溶性淀粉、燕麦片等复杂碳源对产生活性物质优于葡萄糖、蔗糖等简单碳源；牛肉膏、蛋白胨等有机复杂氮源优于无机氮源。
根据国际链霉菌计划(ISP)推荐的方法对LH117和LH121进行了菌株鉴定。
产生不残活件物质oj jJg线由植优9＄选
对其形态特征、培养特征和生理生化传性进行了研冗，与已知的种比较，鉴定二
菌株分别为尖抱链霉菌（）tr。ptoll］，c，s c。lspjdospo。。，s Hlgashlde，t。j，1966）
和玫瑰暗黄链霉菌（价。，尸N／！y。，。，，。，o八jj。，fi。P。ebrazllell。kara，19，7）。
Culture filtrates of 344 actinomyces strains isolated from 30 soil samples were tested of knock-down efficiency in vitro against J2 of root-knot nematodes. Seventeen strains among them, coded as LH004, LH010, LH018, LH053, LH054, LH082, LH117, LH120, LH121, LH132, LH139, SH037, NJ001, NJ024, NJ026, NJ051 and NJ095 respectively, showed strong nematicidal activity .All of their 8-fold diluted culture filtrates had more than 85% knock-down efficiency.
Then the 17strains' culture filtrates were further assayed control effect on root-knot nematodes.3 strains, which were NJ026, LH117 and LH121 respectively, could inhibit gall and egg mass formation most significantly, 2ml culture filtrates reduced 62.3%, 85.1%, 67.0% gall formation and 63.6%, 82.7%, 68.2 % egg mass formation. Then successive 6 generations of the 3 strains were examined genetic stability producing active nematicidal substance, results showed that NJ026 lost its active substance producing ability within 6 generations, LH117 remained nematicidal activity after 6 generations but the decreasing trend may be discerned, only LH121 always demonstrated stable strong nematicidal activity. Based on the above experiment, LH117 and LH121 were considered as potential to be used in practice for root-knot nematodes control. Besides nematicidal activity, culture filtrates of LH117 and LH121 both could inhibit growth of five important vegetable pathogenic fungi, namely Colletotrichum gloeosporioide,Gloeospori
um piperatwn, Alternaria solani, Cladosporium cucumerinum and Colletotrichum orbiculare.
Action mode of the two strains against root-knot nematodes was probed. Culture filtrates of LH121 could completely knock-down all the J2 tested within 4h, but most recovered their movements in fresh water; 12h later, no recovery was observed; 24h later, all of the individuals turned transparent observed under dissect microscope, which revealed that electrolyte leakage occurred. LH117 knocked down 90% of J2 within 8h but most recovered movements in fresh water, 24h later 26.3% of J2 could still recover. Both strains affected egg hatching. 72h treatment with LH121 no egg hatched any longer and the egg mass turned to red-brown; 72h treatment with LH117 the inhibition ratio was 82.3% compared with CK. Experiments in vivo revealed that both strains control root-knot
nematode mainly through killing J2 or affecting their activity, they cannot influence nematode development after the nematode-plant endoparasite relationship has been established.
In order to promote active substance production, fermentation condition of the two actinomyces strains was optimized. Results was that the optimum fermentation condition of the two strains were almost the same. The culture filtrates which utilized No.l nutrition components, with initial PH7-8, shaking 4-5 days at ITOrpm, 6 days strains age around showed the strongest nematicidal activity. Both strains utilized complex carbon sources such as soluble starch and oatments better than simple carbon sources, and complex organic nitric sources such as beef extract and peptone better than inorganic nitric sources.
LH117 and LH121 were identified as Streptomyces cuspidosporus and Streptomyces reseofulvus respectively based on their morphological and physiological properties according to the methods recommended by International Streptomyces Project (ISP).
引文
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