绿僵菌防治甘蔗螟虫的初步研究
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摘要
绿僵菌(Metarhizium ssp.)是一种广谱性杀虫真菌,可广泛寄生8个目、30个科中的200余种昆虫,也能寄生螨类和线虫,经过100多年的研究,目前在许多国家绿僵菌已作为重要的生物防治作用物,用于害虫的防治。与传统的化学农药相比,绿僵茵寄主范围广,致病力强,具有对人、畜、农作物无毒,无残留,菌剂易生产,持效期长,害虫不产生抗药性等优点,具有广阔的应用前景,而应用绿僵菌防治甘蔗螟虫是对螟虫进行生物防治的基本方法之一。本研究在分离筛选出高产孢量、强耐热性以及高毒力的绿僵菌菌株基础上,经大规模发酵后将其应用于田间试验,探索利用绿僵菌防治甘蔗螟虫的有效途径,主要研究结果如下：
1.通过采集样品进行分离筛选,得到两株新的绿僵菌菌株HS1和LY2。
2.经形态学分析和ITS分子鉴定,确认这两株菌株属于金龟子绿僵菌小孢变种,且有可能分属不同亚种。
3.分析了绿僵菌菌株在3种不同培养基上培养时其产孢量的2个影响因素(初始接种浓度和相对湿度),得出菌株在3种不同培养基上培养时须分别配以适宜的接种浓度和相对湿度,以获取最大产孢量(燕麦培养基：1×107cfu/ml,65％; PPDA培养基：1×106cfu/ml,85%；玉米粉酵母浸膏培养基：1×108cfu/ml,75％)。另外,随着继代培养代数增加,菌株在这3种培养基上前五代的产孢量比较稳定,第七代及之后的产孢量明显下降。根据3种培养基功能方面的差别,在PPDA培养基上培养菌株时要着重注意不同继代培养代数对产孢量的影响,其它2种培养基培养菌株一般不超过五代,产孢量相对比较稳定。
4.通过耐高温菌株的诱导筛选,使绿僵菌孢子的耐热性得到很大提高,同温度下培养时,经过诱导的菌株产孢量明显高于未经诱导的菌株产孢量,而且经过诱导的菌株的产孢量在34℃前随温度升高产孢量有所增加,35℃时产孢量开始显著下降,所以该方法也可有效提高菌株在35℃前的产孢量。
5.分析了菌株液固双相发酵中液体发酵罐的转速与通气量对绿僵菌产孢量的影响,得出随发酵时间增加,通气量与转速应设定为：0-24h保持通气流量为1.4m3/h；24-48h保持通气流量为1.6m3/h；48h后保持通气流量为1.8m3／h,转速始终维持在160r/min,此条件下菌株发酵的产孢量较高。
6.对实验室经常用来进行菌种保存的PPDA斜面保存法和20%甘油保存法所保存的菌株进行萌发率测定,认为PPDA斜面保存的菌株应半年左右转接一次,20%甘油保存的菌株也应在一年左右转接一次,避免菌株失活。同时对发酵后孢子粉的两种保存方式进行萌发率测定,认为发酵后应将孢子粉冷冻干燥处理后再放入冰箱冷藏。另外,冷冻干燥法也可用于保存实验室菌株。
7.在室内用喷雾法测定了4株绿僵菌对甘蔗螟虫3-4龄若虫的致病力。结果表明,绿僵菌致病力强弱与菌株种类和接种剂量有密切关系。先接虫后喷施菌剂时,菌株对甘蔗螟虫若虫致病力的强弱顺序为JF813>HS1>MA4>JF883;先喷施菌剂后接虫时,菌株对甘蔗螟虫若虫致病力的强弱顺序为MA4>HS1> JF813>JF883。在接种各菌株孢子浓度为1×108cfu·ml-1时,菌剂对甘蔗螟虫若虫的致死率高。其中JF813菌株对甘蔗螟虫若虫具有很强的二次侵染能力,HS1菌株在两种处理方式中致病力稳定。
8.在联合毒力测定中三种化学杀虫剂都对绿僵菌孢子萌发有一定抑制作用,浓度越大,毒性越强,抑制作用明显。三种化学杀虫剂致死剂量的混配剂虽然杀虫效果较好,但对绿僵菌孢子的抑制率很高,不利于绿僵菌发挥作用。而亚致死剂量呋喃丹的抑制率明显低于三种致死剂量的抑制率,另外从杀虫效果看,亚致死剂量呋喃丹混配剂仅次于三种致死剂量混配剂,优于其他处理,因此,综合考虑后,选择亚致死剂量呋喃丹与绿僵菌的混配剂为最佳处理。
9.综合分析小区试验的枯心率和产量情况,参考土壤宿存能力的监测结果,选择水剂JF813和水剂+甘蔗渣MA4为最佳处理进行大田试验。分析大田试验枯心率和产量情况,结合不同处理的菌株土壤宿存情况和甘蔗螟虫动态的初步监测,验证了所使用的三种绿僵菌菌剂在大田试验中对甘蔗螟虫的防治效果与三种化学杀虫剂的防治效果基本相当,可以替代三种化学杀虫剂进行甘蔗螟虫的防治使用。为防止螟虫对菌剂产生抗性,可将三种菌剂轮换使用。另外,在施用菌剂时,带甘蔗渣的处理施用后用树枝划一遍效果较好,种植后两个半月时可增加一次菌剂的喷施,可更有效抑制螟虫的发生。
Metarhizium anisopliae (Metschn.) Sorokin is one of the species of broad-spectrum insecticidal fungi. It can parasitize more than 200 kinds of insects belonging to 8 Orders,30 Families, as well as mites and nematodes. Research on Metarhizium anisopliae has been more than 100 years. M. anisopliae has a lot of advantages over the conventional chemical pesticides such as broad host range, high pathogenicity, and no toxicity to human being, livestock, and crops. Using Metarhizium spp. as a biocontrol agent is an basic measure to against Sugarcane Stemborer in biocontrol. Based on the Metarhizium spp. strains which isolated and have high sporification, strong heat resistance, high virulence, after large-scale fermentation the strains of Metarhizium spp.,we did the field tests. Through these efforts, we preliminary explored the effective ways of using Metarhizium spp. against Sugarcane Stemborer. The main results were showed as followings:
1. Two strains of Metarhizium (HS1 and LY2)were isolated from soils samples and cadavers.
2. Morphological and ITS sequences identification shows these two strains originally belonged to Metarhizium anisopliae var.anisopliae and were from different subspecies.
3. Metarhizium spp. is a broad spectrum entomopathogenic fungus, and it is thus of great significance to increase sporification of the fungus. Two factors [inoculation concentrations and relative humidity (RH)] affecting the sporification of Metarhizium spp. strauns grown in 3 different mediums were determined. The results showed that these two factors were very important for maximum sporification of this fungus on these three mediums. Maximum sporification occurred when the fungus were cultured on the oats medium at the inoculation concentration of 1×107 cfu·ml-1 and RH 65%; on PPDA medium at the inoculation concentration of 1×106 cfu·ml-1 and RH 85%; or on the corn powder and yeast soaking flour medium at the 1×108 cfu·ml-1 and RH 75%. Moreover, this fungus when subcultured under these mediums and conditions had higher and stable volume of spores in the first 5 generations of the subculture but produced less spores in and after the 7th generation of subculture. According to the different functions of the 3 mediums, the subculture generation greatly affected the sporification in the PPDA medium, and lightly affected other two mediums unless the subculture overtakes the fifth generation.
4. Screen the strains by high temperature cultur to increase the heat resistance of strains. When cultured in the same temperature, the quantity of sporification of the induced strains was more than those of none induced. And the quantity increased as the temperature raised when under 34℃, but significantly decreased at 35℃. So this method can be used to increase the quantity of sporification of strains below 35℃.
5. We went through the influence of two factors(rotation speed and ventilation of the fermentation tank in diphasic fermentation technology)on the quantity of sporification of the Metarhizium spp.strains in liquid fermentor. The results showed that the best conditionswas that:0-24h, the ventilation is 1.4 m3/h; 24-48h, the ventilation is 1.6 m3/h; after 48h, the ventilation is 1.8 m3/h, the rotation speed is 160 r/min all the time.
6. Analysis the germination of the Metarhizium spp.strains saved by PPDA slant and 20% glycerol in the lab. The results showed that the strains saved by PPDA slant should transfer to a new PPDA slant per half a year, and the strains saved by 20% glycerol should transfer to a new PPDA slant per year, otherwise the strains will lost its activity. Meanwhile, we analysis the germination of the Metarhizium spp.strains spores which were saved by two ways in fridge at 4℃. The results showed that spores should be freeze drying firstly before saved in fridge at 4℃. Moreover, freeze drying was applicable to save separated strains.
7. The third to fourth instar nymphs of Sugarcane Stemborers were sprayed with solutions of 4 strains of fungus Metarhiziums spp. in laboratory to observe the pathogenicity of each strain against the nymphs. The bioassay showed that the pathogenicity of the fungus was closely related with the strains and their inoculation rate. The pathogenicity of the fungus was in the order of the strains JF813> HS1> MA4> JF883 when sugarcane was inoculated with the larvae of Sugarcane Stemborers first and then with the fungus, and MA4> HS1> JF813> JF883 when vice versa. The mortality of Sugarcane Stemborers were the highest when sugarcane was inoculated with fungus at a concentration of 1×108spores·ml-1.The strain JF813 had a very high ability in infecting the second time, and the HS1 had a stable pathogenicity.
8. Joint Toxicollogical Testing of 3 chemical pesticides and Metarhizium spp. against the third to fourth instar nymphs of Sugarcane Stemborer by spraying. The results showed that the chemical pesticides inhibited the strain germination, when the concentration of the chemical pesticides raised, the inhibition significantlyincreased. In spite of good performance of the joint pathogenicity of the mixtures in desinsection, they shows strong inhibition to the germinationof the strains. So these mixtures are not suitable for this experiment. The sublethal dose mixture of carbofuran haveweaker inhibition to the germinationof the strains, and the joint pathogenicity of this mixture is a little lower than the lethal dose mixtures, but better than any other mixture. So, the sublethal dose mixture of carbofuran is the best choice.
9. Take the accommodation of the Metarhizium spp. strain soil into account the cane NPI and yield of plot experiments were comprehensive analysised.The results showed that agent JF813 and agent+bagasse MA4 were the best treatment and could be used into field experiments. Then analysis the cane NPI and yield, refer to the accommodationof the Metarhizium spp. strain soil and the trends of Sugarcane Stemborer in the field. The results showed that the control effect of the three Metarhizium spp. formulations was substantially the same as the control effect of the 3 chemical pesticides. So, the three Metarhizium spp. formulations could be applied to against Sugarcane Stemborer to replace the 3 chemical pesticides. And the three Metarhizium spp. formulations would be used alternatives in a rotation strategy to manage the development of the resistance in Sugarcane Stemborer. Furthermore, it is better to scratch the bagasse with a branch afterapplication on the agent+bagasse MA4. And after planting, to spray the Metarhizium spp. agent two month later for inhibite the growth of Sugarcane Stemborer.
1.通过采集样品进行分离筛选,得到两株新的绿僵菌菌株HS1和LY2。
2.经形态学分析和ITS分子鉴定,确认这两株菌株属于金龟子绿僵菌小孢变种,且有可能分属不同亚种。
3.分析了绿僵菌菌株在3种不同培养基上培养时其产孢量的2个影响因素(初始接种浓度和相对湿度),得出菌株在3种不同培养基上培养时须分别配以适宜的接种浓度和相对湿度,以获取最大产孢量(燕麦培养基：1×107cfu/ml,65％; PPDA培养基：1×106cfu/ml,85%；玉米粉酵母浸膏培养基：1×108cfu/ml,75％)。另外,随着继代培养代数增加,菌株在这3种培养基上前五代的产孢量比较稳定,第七代及之后的产孢量明显下降。根据3种培养基功能方面的差别,在PPDA培养基上培养菌株时要着重注意不同继代培养代数对产孢量的影响,其它2种培养基培养菌株一般不超过五代,产孢量相对比较稳定。
4.通过耐高温菌株的诱导筛选,使绿僵菌孢子的耐热性得到很大提高,同温度下培养时,经过诱导的菌株产孢量明显高于未经诱导的菌株产孢量,而且经过诱导的菌株的产孢量在34℃前随温度升高产孢量有所增加,35℃时产孢量开始显著下降,所以该方法也可有效提高菌株在35℃前的产孢量。
5.分析了菌株液固双相发酵中液体发酵罐的转速与通气量对绿僵菌产孢量的影响,得出随发酵时间增加,通气量与转速应设定为：0-24h保持通气流量为1.4m3/h；24-48h保持通气流量为1.6m3/h；48h后保持通气流量为1.8m3／h,转速始终维持在160r/min,此条件下菌株发酵的产孢量较高。
6.对实验室经常用来进行菌种保存的PPDA斜面保存法和20%甘油保存法所保存的菌株进行萌发率测定,认为PPDA斜面保存的菌株应半年左右转接一次,20%甘油保存的菌株也应在一年左右转接一次,避免菌株失活。同时对发酵后孢子粉的两种保存方式进行萌发率测定,认为发酵后应将孢子粉冷冻干燥处理后再放入冰箱冷藏。另外,冷冻干燥法也可用于保存实验室菌株。
7.在室内用喷雾法测定了4株绿僵菌对甘蔗螟虫3-4龄若虫的致病力。结果表明,绿僵菌致病力强弱与菌株种类和接种剂量有密切关系。先接虫后喷施菌剂时,菌株对甘蔗螟虫若虫致病力的强弱顺序为JF813>HS1>MA4>JF883;先喷施菌剂后接虫时,菌株对甘蔗螟虫若虫致病力的强弱顺序为MA4>HS1> JF813>JF883。在接种各菌株孢子浓度为1×108cfu·ml-1时,菌剂对甘蔗螟虫若虫的致死率高。其中JF813菌株对甘蔗螟虫若虫具有很强的二次侵染能力,HS1菌株在两种处理方式中致病力稳定。
8.在联合毒力测定中三种化学杀虫剂都对绿僵菌孢子萌发有一定抑制作用,浓度越大,毒性越强,抑制作用明显。三种化学杀虫剂致死剂量的混配剂虽然杀虫效果较好,但对绿僵菌孢子的抑制率很高,不利于绿僵菌发挥作用。而亚致死剂量呋喃丹的抑制率明显低于三种致死剂量的抑制率,另外从杀虫效果看,亚致死剂量呋喃丹混配剂仅次于三种致死剂量混配剂,优于其他处理,因此,综合考虑后,选择亚致死剂量呋喃丹与绿僵菌的混配剂为最佳处理。
9.综合分析小区试验的枯心率和产量情况,参考土壤宿存能力的监测结果,选择水剂JF813和水剂+甘蔗渣MA4为最佳处理进行大田试验。分析大田试验枯心率和产量情况,结合不同处理的菌株土壤宿存情况和甘蔗螟虫动态的初步监测,验证了所使用的三种绿僵菌菌剂在大田试验中对甘蔗螟虫的防治效果与三种化学杀虫剂的防治效果基本相当,可以替代三种化学杀虫剂进行甘蔗螟虫的防治使用。为防止螟虫对菌剂产生抗性,可将三种菌剂轮换使用。另外,在施用菌剂时,带甘蔗渣的处理施用后用树枝划一遍效果较好,种植后两个半月时可增加一次菌剂的喷施,可更有效抑制螟虫的发生。
Metarhizium anisopliae (Metschn.) Sorokin is one of the species of broad-spectrum insecticidal fungi. It can parasitize more than 200 kinds of insects belonging to 8 Orders,30 Families, as well as mites and nematodes. Research on Metarhizium anisopliae has been more than 100 years. M. anisopliae has a lot of advantages over the conventional chemical pesticides such as broad host range, high pathogenicity, and no toxicity to human being, livestock, and crops. Using Metarhizium spp. as a biocontrol agent is an basic measure to against Sugarcane Stemborer in biocontrol. Based on the Metarhizium spp. strains which isolated and have high sporification, strong heat resistance, high virulence, after large-scale fermentation the strains of Metarhizium spp.,we did the field tests. Through these efforts, we preliminary explored the effective ways of using Metarhizium spp. against Sugarcane Stemborer. The main results were showed as followings:
1. Two strains of Metarhizium (HS1 and LY2)were isolated from soils samples and cadavers.
2. Morphological and ITS sequences identification shows these two strains originally belonged to Metarhizium anisopliae var.anisopliae and were from different subspecies.
3. Metarhizium spp. is a broad spectrum entomopathogenic fungus, and it is thus of great significance to increase sporification of the fungus. Two factors [inoculation concentrations and relative humidity (RH)] affecting the sporification of Metarhizium spp. strauns grown in 3 different mediums were determined. The results showed that these two factors were very important for maximum sporification of this fungus on these three mediums. Maximum sporification occurred when the fungus were cultured on the oats medium at the inoculation concentration of 1×107 cfu·ml-1 and RH 65%; on PPDA medium at the inoculation concentration of 1×106 cfu·ml-1 and RH 85%; or on the corn powder and yeast soaking flour medium at the 1×108 cfu·ml-1 and RH 75%. Moreover, this fungus when subcultured under these mediums and conditions had higher and stable volume of spores in the first 5 generations of the subculture but produced less spores in and after the 7th generation of subculture. According to the different functions of the 3 mediums, the subculture generation greatly affected the sporification in the PPDA medium, and lightly affected other two mediums unless the subculture overtakes the fifth generation.
4. Screen the strains by high temperature cultur to increase the heat resistance of strains. When cultured in the same temperature, the quantity of sporification of the induced strains was more than those of none induced. And the quantity increased as the temperature raised when under 34℃, but significantly decreased at 35℃. So this method can be used to increase the quantity of sporification of strains below 35℃.
5. We went through the influence of two factors(rotation speed and ventilation of the fermentation tank in diphasic fermentation technology)on the quantity of sporification of the Metarhizium spp.strains in liquid fermentor. The results showed that the best conditionswas that:0-24h, the ventilation is 1.4 m3/h; 24-48h, the ventilation is 1.6 m3/h; after 48h, the ventilation is 1.8 m3/h, the rotation speed is 160 r/min all the time.
6. Analysis the germination of the Metarhizium spp.strains saved by PPDA slant and 20% glycerol in the lab. The results showed that the strains saved by PPDA slant should transfer to a new PPDA slant per half a year, and the strains saved by 20% glycerol should transfer to a new PPDA slant per year, otherwise the strains will lost its activity. Meanwhile, we analysis the germination of the Metarhizium spp.strains spores which were saved by two ways in fridge at 4℃. The results showed that spores should be freeze drying firstly before saved in fridge at 4℃. Moreover, freeze drying was applicable to save separated strains.
7. The third to fourth instar nymphs of Sugarcane Stemborers were sprayed with solutions of 4 strains of fungus Metarhiziums spp. in laboratory to observe the pathogenicity of each strain against the nymphs. The bioassay showed that the pathogenicity of the fungus was closely related with the strains and their inoculation rate. The pathogenicity of the fungus was in the order of the strains JF813> HS1> MA4> JF883 when sugarcane was inoculated with the larvae of Sugarcane Stemborers first and then with the fungus, and MA4> HS1> JF813> JF883 when vice versa. The mortality of Sugarcane Stemborers were the highest when sugarcane was inoculated with fungus at a concentration of 1×108spores·ml-1.The strain JF813 had a very high ability in infecting the second time, and the HS1 had a stable pathogenicity.
8. Joint Toxicollogical Testing of 3 chemical pesticides and Metarhizium spp. against the third to fourth instar nymphs of Sugarcane Stemborer by spraying. The results showed that the chemical pesticides inhibited the strain germination, when the concentration of the chemical pesticides raised, the inhibition significantlyincreased. In spite of good performance of the joint pathogenicity of the mixtures in desinsection, they shows strong inhibition to the germinationof the strains. So these mixtures are not suitable for this experiment. The sublethal dose mixture of carbofuran haveweaker inhibition to the germinationof the strains, and the joint pathogenicity of this mixture is a little lower than the lethal dose mixtures, but better than any other mixture. So, the sublethal dose mixture of carbofuran is the best choice.
9. Take the accommodation of the Metarhizium spp. strain soil into account the cane NPI and yield of plot experiments were comprehensive analysised.The results showed that agent JF813 and agent+bagasse MA4 were the best treatment and could be used into field experiments. Then analysis the cane NPI and yield, refer to the accommodationof the Metarhizium spp. strain soil and the trends of Sugarcane Stemborer in the field. The results showed that the control effect of the three Metarhizium spp. formulations was substantially the same as the control effect of the 3 chemical pesticides. So, the three Metarhizium spp. formulations could be applied to against Sugarcane Stemborer to replace the 3 chemical pesticides. And the three Metarhizium spp. formulations would be used alternatives in a rotation strategy to manage the development of the resistance in Sugarcane Stemborer. Furthermore, it is better to scratch the bagasse with a branch afterapplication on the agent+bagasse MA4. And after planting, to spray the Metarhizium spp. agent two month later for inhibite the growth of Sugarcane Stemborer.
引文
[1]蒲蛰龙,李增智.昆虫真菌学[M].安徽：安徽科学技术出版社,1996：95-97.360-368.
[2]高松.绿僵菌研究的新进展[J].中国生物防治,1996,12(4)：182-187.
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