绿僵菌与宿主蛴螬田间互作及种群续存研究
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摘要
绿僵菌施用后与蛴螬共存于土壤环境中，复杂的生态关系影响绿僵菌生存、定植、增殖及致病作用。本文研究了绿僵菌与蛴螬田间互作关系及种群续存动态，可为研制高效产品、建立稳定防治技术提供参考。研究结果如下：
1.绿僵菌对蛴螬致病性初选得到9个菌株，再对金龟子卵和不同龄期幼虫检测，表明菌株致病力存在显著差异，并与菌株剂量显著正相关；确定3个高毒力菌株5-8、Br818和2-2；证明铜绿丽金龟幼虫低龄比高龄敏感，对卵致死率和僵卵率达68.3%和51.5%；大黑鳃金龟无明显虫龄敏感性规律。田间防治蛴螬必备高效菌株，应把握蛴螬发育阶段施菌。
2.蛴螬对绿僵菌和植株的行为趋向性证明蛴螬在土壤中为随机无定向移动；试验的蛴螬分别有83~86%和55~69%移动到花生植株和草坪草附近，表现明显的寄主趋向性；蛴螬选择施菌区和对照区的数量大致相同、分布相似，说明对绿僵菌无趋避性。因此，田间应当将菌剂施用到植物根部附近土壤中，以提高蛴螬接触菌剂几率，增加感染机会。
3.绿僵菌田间施用技术1）防治花生蛴螬盆栽试验表明，播种期施用绿僵菌到开花期仍保持对蛴螬的控制作用，可使花生虫果率比对照减少22.4%，好果数增加31.2%，果重增加33.7%；施用辛硫磷虫果率减少58.9%，但好果数和果重也减少30.0%和26.7%，说明辛硫磷能控制蛴螬，但可能对植株有药害；减半剂量菌药混用，不能有效控制蛴螬，而对植株仍有药害。田间施用绿僵菌的花生好果数和果重都提高约40%，虫果率降低10.9%；菌加肥处理，虫果率和好果数比对照无显著差异，果重增加，说明肥对荚果发育有促进作用。2）防治草坪蛴螬笼罩试验，绿僵菌可减少红脚绿丽金龟新生幼虫27.3~87.3％；对1龄幼虫作用最强，在66.7×108孢子/kg土壤条件下6d致死率超过90％，对2、3龄幼虫LT50需11d和18d。小区试验表明，喷洒和投撒施菌方式适用于草坪。大区防治效果达到78.8~90.7%。
4.绿僵菌田间续存种群动态1）确定优良选择性培养基CA，分离效率72.6%，直接稀释法的分离效率高且数据稳定。2）185周监测表明，绿僵菌施用土壤后种群数量呈现先快下降到相对平衡的过程，经38周衰减后逐渐稳定，2~3年内可保持初始量10~20%，半衰期于26.7周。3）花生播种到收获期间，在不同深度和与植株不同距离位置上，绿僵菌种群表现为30~60d内快速下降，随后减缓，90d左右下降到初始量的10~20%，之后维持相对平衡或缓慢恢复；种群在3cm深度比15cm深度降幅小，距植株近处恢复较快；根际120d时可恢复到56.7%；30cm深度未施菌土层，没有向下渗透的绿僵菌。4）3个施菌量经150d衰减后种群趋于相近水平；增施有机肥和农药的，种群下降快但恢复较早，44d CFU比单施菌的高1倍；开花期接入蛴螬，80d内对绿僵菌种群无明显影响。5）距植株11.5cm分界，界外施菌的，30d时界内侧绿僵菌数量提高6.5~7.4倍，反之界内施菌的外侧菌量无明显变化，说明有向内生长趋势。种群数量下降与平衡机理需进一步研究，以正确指导田间应用。
5.绿僵菌颗粒剂研制及种群恢复确定2%海藻酸钠、0.2~0.3M Ca~(++)为适宜胶粒浓度；填充4~10%凹凸棒土支撑胶网内部有利于孢子存活和回复生长;添加0.5%玉米粉或糊精使孢子增殖率提高60%；海藻胶与聚乙烯醇联合外膜对抵御外界微生物侵袭有一定作用。
Metarhizium anisopliae, an entomopathogenic fungus, was applied in soil to control white grubs. Weinvestigated interactions of the fungus with white grubs and population persistence in field. Thepurpose was to understand impact factors of control effect and possibility of technical manipulation.
1. Pathogenicity of Metarhizium anisopliae against white grubs There were significantly differencebetween pathogenicity of M. anisopliae isolates based on bioassays with eggs and various larvalinstars. Grub mortality was positive correlation with fungal dosage. Three isolates (5-8, Br818and2-2) were high virulent to Holotrichia parallela, H. oblita and Anomala corpulenta.1st-instar larvaeof A. corpulenta were much more sensitive than2nd-and3rd-instar ones. But there were no diversityin larvae of H. oblita. Mortality and viewable infection of A. corpulenta eggs were to68.3%and51.5%. So high virulent isolate was need and grub development should be inspected for pest control.
2. Behavior taxis of white grubs to M. anisopliae and plant White grubs moved random in soil.When with peanut plant,83~86%of grubs assembled around the plant. When with golf turf,55~69%of grubs did under and around them. It indicated that grubs strongly trend to host plants but had nopositive or negative tendency to the fungus. So the fungal agent should be delivered round the rootsto increase opportunity of grub infection.
3. Application technique of M. anisopliae in field a) For peanut grubs: In potted treatments whenseeding, harvest survey showed that bad fruits decreased22.4%，amount and weight of fruitsincreased31.2%and33.7%in the fungal treatment compared to control. For phoxim treatment, badfruits decreased58.9%，but amount and weight of fruits decreased30.0%and26.7%. It indicated thatthe chemical effectively depressed grubs, but may be harmful to peanut. Mixture of half dosage ofthe fungus and phoxim could not suppress grubs effectively but still harmful to peanut. In fieldexperiment, bad fruits decreased10.9%, both amount and weight of fruits increased about40%.When the fungus and manure were used together, fruit amount had no significant difference to thecontrol, but fruit weight evidently higher. It means manure could weaken the fungal action but makefor peanut growth. b) For lawn grubs: In cage treatments, M. anisopliae could decrease27.3-87.3％neonate of lawn scarab A. cupripes under three dosages. The fungus presented strong infection in1st-instar larvae,90%mortality in6days at6.67×109spores/kg soil. LT50for2nd-and3rd-instars were11d and18d respectively. Therefore, fungal release at younger phase of grubs would be a preferablestrategy to enhance efficiency. For fungal release mode，sprinkling or scattering was better thanburying and stiletto-scattering. In large area, the effect of control grubs were78.8~90.7%.
4. Population dynamic of M. anisopliae in field a) A selective medium, CA, was determined forisolating and quantity evaluating for M. anisopliae from soil. Isolating efficiency was72.6%.Directly dilute extraction provide a higher and more stable isolating efficiency than sugarcentrifugation. b) The soil inspection was carried out17times lasting for185weeks after inoculatingfungus. The results showed that M. anisopliae population in soil descend obviously at the trend ofstarting down slowly-went on down quickly-decelerated gradually-fluctuated or revived up. The attenuation process underwent38weeks then gradually stabilized at10~20%lever of originalpopulation for2~3years. Half quantity was at26.7weeks. c) From fungal inoculation when seedingto harvest time, the quantities of M. anisopliae at various places of different depth and distance topeanut plant all declined quickly in30d-60d, then relaxed. The quantities would descend to20%~10%of original inoculation at90d or about, then maintained or return appreciably. Thedecrease extent at3cm depth was smaller than at15cm depth. The fungal population would returnearlier and more quickly at12.5cm distance to peanut plant than those places more far from plant. d)Although three dosage of the fungus were inoculated in soil at seeding time, their quantity remain anear lever at last. Adding manure or phoxim, the fungal population would bring forward comingdown and reviving. Inoculation of grubs at anthesis did not impact obviously on the fungalquantity at lasting80days. e）At the dividing line around plant of11.5cm radius, the fungus couldhave the trend of growing from outside soil inoculated the fungus to inside soil without the fungus.
5. Preparation of M. anisopliae granule and improvement of population revival Alginate gelpellets were formed with reaction of2%alginate sodium and0.2~0.3M Ca~(++).4~10%of attapulgitewas filled in gel to uphold inner space.0.5%corn powder or dextrin added into the gel pelletscould advance60%of spore multiplication. The outer membrane formed by alginate and polyvinylalcohol could resist strike from environmental microbes and improve population reviving.
1.绿僵菌对蛴螬致病性初选得到9个菌株，再对金龟子卵和不同龄期幼虫检测，表明菌株致病力存在显著差异，并与菌株剂量显著正相关；确定3个高毒力菌株5-8、Br818和2-2；证明铜绿丽金龟幼虫低龄比高龄敏感，对卵致死率和僵卵率达68.3%和51.5%；大黑鳃金龟无明显虫龄敏感性规律。田间防治蛴螬必备高效菌株，应把握蛴螬发育阶段施菌。
2.蛴螬对绿僵菌和植株的行为趋向性证明蛴螬在土壤中为随机无定向移动；试验的蛴螬分别有83~86%和55~69%移动到花生植株和草坪草附近，表现明显的寄主趋向性；蛴螬选择施菌区和对照区的数量大致相同、分布相似，说明对绿僵菌无趋避性。因此，田间应当将菌剂施用到植物根部附近土壤中，以提高蛴螬接触菌剂几率，增加感染机会。
3.绿僵菌田间施用技术1）防治花生蛴螬盆栽试验表明，播种期施用绿僵菌到开花期仍保持对蛴螬的控制作用，可使花生虫果率比对照减少22.4%，好果数增加31.2%，果重增加33.7%；施用辛硫磷虫果率减少58.9%，但好果数和果重也减少30.0%和26.7%，说明辛硫磷能控制蛴螬，但可能对植株有药害；减半剂量菌药混用，不能有效控制蛴螬，而对植株仍有药害。田间施用绿僵菌的花生好果数和果重都提高约40%，虫果率降低10.9%；菌加肥处理，虫果率和好果数比对照无显著差异，果重增加，说明肥对荚果发育有促进作用。2）防治草坪蛴螬笼罩试验，绿僵菌可减少红脚绿丽金龟新生幼虫27.3~87.3％；对1龄幼虫作用最强，在66.7×108孢子/kg土壤条件下6d致死率超过90％，对2、3龄幼虫LT50需11d和18d。小区试验表明，喷洒和投撒施菌方式适用于草坪。大区防治效果达到78.8~90.7%。
4.绿僵菌田间续存种群动态1）确定优良选择性培养基CA，分离效率72.6%，直接稀释法的分离效率高且数据稳定。2）185周监测表明，绿僵菌施用土壤后种群数量呈现先快下降到相对平衡的过程，经38周衰减后逐渐稳定，2~3年内可保持初始量10~20%，半衰期于26.7周。3）花生播种到收获期间，在不同深度和与植株不同距离位置上，绿僵菌种群表现为30~60d内快速下降，随后减缓，90d左右下降到初始量的10~20%，之后维持相对平衡或缓慢恢复；种群在3cm深度比15cm深度降幅小，距植株近处恢复较快；根际120d时可恢复到56.7%；30cm深度未施菌土层，没有向下渗透的绿僵菌。4）3个施菌量经150d衰减后种群趋于相近水平；增施有机肥和农药的，种群下降快但恢复较早，44d CFU比单施菌的高1倍；开花期接入蛴螬，80d内对绿僵菌种群无明显影响。5）距植株11.5cm分界，界外施菌的，30d时界内侧绿僵菌数量提高6.5~7.4倍，反之界内施菌的外侧菌量无明显变化，说明有向内生长趋势。种群数量下降与平衡机理需进一步研究，以正确指导田间应用。
5.绿僵菌颗粒剂研制及种群恢复确定2%海藻酸钠、0.2~0.3M Ca~(++)为适宜胶粒浓度；填充4~10%凹凸棒土支撑胶网内部有利于孢子存活和回复生长;添加0.5%玉米粉或糊精使孢子增殖率提高60%；海藻胶与聚乙烯醇联合外膜对抵御外界微生物侵袭有一定作用。
Metarhizium anisopliae, an entomopathogenic fungus, was applied in soil to control white grubs. Weinvestigated interactions of the fungus with white grubs and population persistence in field. Thepurpose was to understand impact factors of control effect and possibility of technical manipulation.
1. Pathogenicity of Metarhizium anisopliae against white grubs There were significantly differencebetween pathogenicity of M. anisopliae isolates based on bioassays with eggs and various larvalinstars. Grub mortality was positive correlation with fungal dosage. Three isolates (5-8, Br818and2-2) were high virulent to Holotrichia parallela, H. oblita and Anomala corpulenta.1st-instar larvaeof A. corpulenta were much more sensitive than2nd-and3rd-instar ones. But there were no diversityin larvae of H. oblita. Mortality and viewable infection of A. corpulenta eggs were to68.3%and51.5%. So high virulent isolate was need and grub development should be inspected for pest control.
2. Behavior taxis of white grubs to M. anisopliae and plant White grubs moved random in soil.When with peanut plant,83~86%of grubs assembled around the plant. When with golf turf,55~69%of grubs did under and around them. It indicated that grubs strongly trend to host plants but had nopositive or negative tendency to the fungus. So the fungal agent should be delivered round the rootsto increase opportunity of grub infection.
3. Application technique of M. anisopliae in field a) For peanut grubs: In potted treatments whenseeding, harvest survey showed that bad fruits decreased22.4%，amount and weight of fruitsincreased31.2%and33.7%in the fungal treatment compared to control. For phoxim treatment, badfruits decreased58.9%，but amount and weight of fruits decreased30.0%and26.7%. It indicated thatthe chemical effectively depressed grubs, but may be harmful to peanut. Mixture of half dosage ofthe fungus and phoxim could not suppress grubs effectively but still harmful to peanut. In fieldexperiment, bad fruits decreased10.9%, both amount and weight of fruits increased about40%.When the fungus and manure were used together, fruit amount had no significant difference to thecontrol, but fruit weight evidently higher. It means manure could weaken the fungal action but makefor peanut growth. b) For lawn grubs: In cage treatments, M. anisopliae could decrease27.3-87.3％neonate of lawn scarab A. cupripes under three dosages. The fungus presented strong infection in1st-instar larvae,90%mortality in6days at6.67×109spores/kg soil. LT50for2nd-and3rd-instars were11d and18d respectively. Therefore, fungal release at younger phase of grubs would be a preferablestrategy to enhance efficiency. For fungal release mode，sprinkling or scattering was better thanburying and stiletto-scattering. In large area, the effect of control grubs were78.8~90.7%.
4. Population dynamic of M. anisopliae in field a) A selective medium, CA, was determined forisolating and quantity evaluating for M. anisopliae from soil. Isolating efficiency was72.6%.Directly dilute extraction provide a higher and more stable isolating efficiency than sugarcentrifugation. b) The soil inspection was carried out17times lasting for185weeks after inoculatingfungus. The results showed that M. anisopliae population in soil descend obviously at the trend ofstarting down slowly-went on down quickly-decelerated gradually-fluctuated or revived up. The attenuation process underwent38weeks then gradually stabilized at10~20%lever of originalpopulation for2~3years. Half quantity was at26.7weeks. c) From fungal inoculation when seedingto harvest time, the quantities of M. anisopliae at various places of different depth and distance topeanut plant all declined quickly in30d-60d, then relaxed. The quantities would descend to20%~10%of original inoculation at90d or about, then maintained or return appreciably. Thedecrease extent at3cm depth was smaller than at15cm depth. The fungal population would returnearlier and more quickly at12.5cm distance to peanut plant than those places more far from plant. d)Although three dosage of the fungus were inoculated in soil at seeding time, their quantity remain anear lever at last. Adding manure or phoxim, the fungal population would bring forward comingdown and reviving. Inoculation of grubs at anthesis did not impact obviously on the fungalquantity at lasting80days. e）At the dividing line around plant of11.5cm radius, the fungus couldhave the trend of growing from outside soil inoculated the fungus to inside soil without the fungus.
5. Preparation of M. anisopliae granule and improvement of population revival Alginate gelpellets were formed with reaction of2%alginate sodium and0.2~0.3M Ca~(++).4~10%of attapulgitewas filled in gel to uphold inner space.0.5%corn powder or dextrin added into the gel pelletscould advance60%of spore multiplication. The outer membrane formed by alginate and polyvinylalcohol could resist strike from environmental microbes and improve population reviving.
引文
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