论文部分内容阅读
Abstract [Objectives]This study was conducted to investigate the safety of surfactants AOS and Tween 80 on Bt and their effects on Bt’s adhesion amount on crop leaves and its field efficacy.[Methods]Certain concentrations of AOS and Tween 80 were added to Bt liquid to check the effects of the surfactants on Bt spore germination， the adhesion amount of Bt on vegetable leaves and the field efficacy on beet armyworm.[Results]After treatment with surfactants AOS and Tween 80， Bt spores germinated normally. After adding AOS， the adhesion amounts of Bt spores on scallion， kohlrabi， radish and Chinese cabbage leaves were 209.75， 249.00， 274.00 and 281.75， respectively， which was 3.8， 2.2， 2.0 and 1.6 times higher than the CK. After adding Tween 80， the adhesion amounts of Bt spores on scallion， kohlrabi， radish and Chinese cabbage leaves were 198.25， 203.25， 233.00 and 236.75， respectively， which were 3.6， 1.8， 1.7 and 1.4 times higher than the CK， respectively. The average field insecticidal effect of the Bt liquid reached 81.3% and 79.5% after the addition of surfactants AOS and Tween 80， respectively， while the average insecticidal effect of the CK without the addition of a surfactant was only 53.3%， which was remarkably lower than above two values.[Conclusions]Surfactants AOS and Tween 80 are safe for Bt; and the addition of surfactants AOS and Tween 80 significantly increased the amount of Bt attached to vegetable leaves， and significantly improved the control efficacy of pests on plants rich in cuticle.
Key words Bacillus thuringiensis; Surfactant; Alpha olefine sulfonate; Adhesion amount; Control efficacy
Bacillus thuringiensis （Bt） is harmless to humans and animals， and has many advantages such as ecological environment safety， making it the most widely used biological pesticide[1]. Because Bt only has the effect of stomach toxicity， but no combined effects of chemical pesticides， the amount of Bt attached to plant surface will directly affect the intake of pests and the control efficacy in field[2]. The addition of surfactant to Bt can increase its adhesion amount on plant surface and increase the amount of Bt fed by pests， thereby improving the insecticidal effect[3]. Since the surface of plant has a variety of membranelike cuticles， which will reduce the adhesion of Bt on the surface of crops， the same Bt has different control efficacy on different plants[4]. Studies have shown that the use of surfactants to improve the field efficacy of Bt is a convenient， simple， effective and environmentally friendly method[5]. In this paper， through the preliminary study， AOS （alpha olefine sulfonate）， which is an anionic surfactant， and Tween 80， which is a nonionic surfactant， were selected as representatives of green surfactants for the present study. Materials and Methods
Materials
Bt wettable powder， produced by Beijing Zhongnong Yitai Technology Development Co.， Ltd.; alpha olefine sulfonate （AOS）， produced by Shanghai Chuxing Chemical Co.， Ltd.; Tween 80， produced by Jiangsu Haian Petrochemical Plant; Nutrient agar， produced by Hangzhou Tianhe Microbial Reagent Co.， Ltd.
Methods
Safety testing
The purchased vegetable seeds were planted in the greenhouse of the Agricultural College of Yanbian University， routinely managed， and used for testing after growing up.
Preparation of nutrient agar medium： A certain amount of agar （20 g） was weighed into a 1 000 ml beaker with a balance， and added with 500 ml of distilled water， followed by mixing well， sealing with tin foil and sterilizing in a high temperature sterilizer. The medium was taken out after routine sterilization， and poured into plates on a clean bench， and the ultraviolet lamp was turned on after the medium was solidified.
On a clean bench， Bt was diluted to 200 cfu/ml with sterile water. Then， 10 ml was added into three 20 ml tubes， respectively， 10 l of AOS and Tween 80 were taken and added into the tubes， respectively， to dilute AOS and Tween 80 by 1 000 times， and the last tube served as the control （CK）， which was formed by adding AOS and Tween 80 but no surfactant and thoroughly mixing. Then， 200 l of corresponding test tube solution was applied to the nutrient agar medium， followed by culturing in an incubator at 27 for 2 d to observe the development of the colony， and the number of colonies was recorded. Each treatment had 6 replicates.
Adhesion amount testing
A certain amount of Bt （1 g） was mixed with 1 000 ml of sterile water thoroughly. Then， 200 ml was pipetted into two 500 ml handheld sprayers， respectively， and 50 l of AOS and Tween 80 were added into one of the two sprayers， respectively， followed by mixing well. Clean leaves of four kinds of vegetables （scallion， kohlrabi， radish and Chinese cabbage） were selected. Each leaf was held on hand obliquely and sprayed evenly for 6 times with corresponding handheld sprayer 20 cm from the leaf. The leaves were then dried on a table in the shade. The dry leaves were punched with a 9 mm hole puncher. Five pieces were added into a test tube and added with 10 ml of sterile water， followed by mixing well. Next， 200 l was pipetted and applied on nutrient agar， and cultured in an incubator at 27 for 2 d. Finally， the number of Bt spores was recorded. Each treatment included four replicates， and Bt without the addition of a surfactant was also set as a control （CK）. Field efficacy testing
In the greenhouse， the scallion field was divided into several 5 m2 （5 m≠1 m） plots. After diluting the concentration of Bt to 2≠106 cfu/ml， 500 ml of the solution was added into two 500 ml handheld sprayers， respectively， and 250 l of AOS and Tween 80 were added into one of the two sprayers， respectively， and mixed well with the Bt solution， to dilute AOS and Tween 80 by 2 000 times. Scallion leaves were sprayed for 3 times at intervals of 1 week. Before each time of spraying and 1 week after spraying， the number of beet armyworm larvae on 30 scallion leaves was randomly investigated in each plot， to calculate field efficacy. The test was repeated for 3 times， with Bt without the addition of a surfactant as a control （CK）.
Data analysis method
Data were analyzed with software SPSS17.0.
Results and Analysis
Safety to Bt spores
Bt is a biopesticide， and it is important to screen surfactants that are safe for Bt. The test results of the two surfactants AOS and Tween 80 on Bt spore germination are shown in Table 1 and Fig. 1.
It could be seen from Table 1 that whether for the surfactant AOS or Tween 80 treatment， there was no significant difference between the number of colonies after germination of Bt spores and the number of colonies in the CK， indicating that the two surfactants have no adverse effects on Bt spore germination.
As shown in Fig. 1， after treatment with AOS and Tween 80， the periphery of Bt colonies was irregular， with radial folds， and the colonies were slightly moist and yellowish， indicating that the development of Bt colonies was completely normal. Therefore， the two kinds of surfactants are safe for Bt.
Adhesion of Bt
The test results of the effects of surfactants AOS and Tween 80 on the adhesion of Bt on the four kinds of vegetable leaves are shown in Table 2.
It could be seen from Table 2 that the addition of AOS or Tween 80 to the Bt solution significantly increased the amount of Bt attached to the four kinds of vegetable leaves. After adding AOS， the adhesion amounts of Bt spores on scallion， kohlrabi， radish and Chinese cabbage leaves were 209.75， 249.00， 274.00 and 281.75， respectively， which was 3.8， 2.2， 2.0 and 1.6 times higher than the CK. After adding Tween 80， the adhesion amounts of Bt spores on scallion， kohlrabi， radish and Chinese cabbage leaves were 198.25， 203.25， 233.00 and 236.75， respectively， which were 3.6， 1.8， 1.7 and 1.4 times higher than the CK， respectively. The results show that the adhesion effect of surfactant AOS is better than that of Tween 80; and among the four vegetables， onion leaves are most rich in cuticle， followed by kohlrabi leaves and radish leaves sequentially， and Chinese cabbage has the least cuticle. The adhesion effects of the surfactantadded Bt liquids on scallion leaves are shown in Fig. 2.
As shown in Fig. 2， the adhesion effects of Bt added with surfactants AOS and Tween 80 were considerably more remarkable than that of the CK. Furthermore， compared with Tween 80， AOS had a slightly better adhesion effect than Tween 80.
Control efficacy on beet armyworm
Since scallion leaves are most rich in cuticle among the four kinds of vegetables， scallion was selected for the control of beet armyworm. The control efficacy of surfactantadded Bt on beet armyworm in scallion field is shown in Table 3.
As shown in Table 3， the average field insecticidal effect of the Bt liquid added with surfactant AOS was 81.3%; the average insecticidal effect of the Bt liquid added with surfactant Tween 80 was 79.5% in field; and the average insecticidal effect of the CK without the addition of a surfactant was only 53.3%， indicating that the addition of surfactant AOS or Tween 80 to vegetables rich in cuticle can significantly improve the control efficacy in field.
Conclusions
The experimental results showed that compared with the CK， surfactants AOS and Tween 80 did not adversely affect Bt spore germination， so AOS and Tween 80 are safe for Bt.
After adding surfactants AOS and Tween 80， the adhesion of Bt on vegetable leaves ranked as scallion>kohlrabi>radish>Chinese cabbage. This result shows that scallion is most rich in cuticle， followed by B. kohlrabi and radishm， and Chinese cabbage has the least cuticle.
At the same dilution ratio， the adhesion amounts of surfactant AOS on the four kinds of vegetables were always higher than Tween 80， and the field efficacy was slightly higher than Tween 80， indicating that AOS has a broad application prospect.
Selecting suitable surfactants when spraying Bt pesticide can not only improve the control efficacy of Bt， but also can improve the use efficiency of the pesticide， thereby reducing the application amount of the pesticide and significantly reducing the impact on ecological environment.
References
[1]BRAR SK， VERMA M， TYAGI RD， et al. Recent advances in downstream processing and formulations of Bacillus thuringiensis based biopesticides[J]. Process Biochemistry， 2006， 41（2）： 323-342.
[2]JIN DY， QUAN XL， LI XG， et al. Effects of Tween 80 on spreading of Bacillus thuringiensis on crop leaves and its control efficacy against Spodoptera exigua in scallion fields[J]. Plant Protection， 2012， 38（5）： 143-146. （in Chinese）
[3]YOU ZG， LI BL， QU LL， et al. Interaction of surfactants， spraying volume and weed leaf angle in droplet retention of imazethapyr[J]. Journal of Plant Protection， 1994， 21（3）： 283-287. （in Chinese）
[4]ZHANG HY， YU ZN. Factors affecting the insecticidal effect of Bacillus thuringiensis preparations[J]. Plant Protection， 1997， 23（4）： 41-44. （in Chinese）
[5]LI Z， JIN DY. Effects of 4 surfactants on adhesion amount of Bacillus thuringiensis on surface of vegetable leaves[J]. Agrochemicals， 2014， 53（10）： 625-627. （in Chinese）
Key words Bacillus thuringiensis; Surfactant; Alpha olefine sulfonate; Adhesion amount; Control efficacy
Bacillus thuringiensis （Bt） is harmless to humans and animals， and has many advantages such as ecological environment safety， making it the most widely used biological pesticide[1]. Because Bt only has the effect of stomach toxicity， but no combined effects of chemical pesticides， the amount of Bt attached to plant surface will directly affect the intake of pests and the control efficacy in field[2]. The addition of surfactant to Bt can increase its adhesion amount on plant surface and increase the amount of Bt fed by pests， thereby improving the insecticidal effect[3]. Since the surface of plant has a variety of membranelike cuticles， which will reduce the adhesion of Bt on the surface of crops， the same Bt has different control efficacy on different plants[4]. Studies have shown that the use of surfactants to improve the field efficacy of Bt is a convenient， simple， effective and environmentally friendly method[5]. In this paper， through the preliminary study， AOS （alpha olefine sulfonate）， which is an anionic surfactant， and Tween 80， which is a nonionic surfactant， were selected as representatives of green surfactants for the present study. Materials and Methods
Materials
Bt wettable powder， produced by Beijing Zhongnong Yitai Technology Development Co.， Ltd.; alpha olefine sulfonate （AOS）， produced by Shanghai Chuxing Chemical Co.， Ltd.; Tween 80， produced by Jiangsu Haian Petrochemical Plant; Nutrient agar， produced by Hangzhou Tianhe Microbial Reagent Co.， Ltd.
Methods
Safety testing
The purchased vegetable seeds were planted in the greenhouse of the Agricultural College of Yanbian University， routinely managed， and used for testing after growing up.
Preparation of nutrient agar medium： A certain amount of agar （20 g） was weighed into a 1 000 ml beaker with a balance， and added with 500 ml of distilled water， followed by mixing well， sealing with tin foil and sterilizing in a high temperature sterilizer. The medium was taken out after routine sterilization， and poured into plates on a clean bench， and the ultraviolet lamp was turned on after the medium was solidified.
On a clean bench， Bt was diluted to 200 cfu/ml with sterile water. Then， 10 ml was added into three 20 ml tubes， respectively， 10 l of AOS and Tween 80 were taken and added into the tubes， respectively， to dilute AOS and Tween 80 by 1 000 times， and the last tube served as the control （CK）， which was formed by adding AOS and Tween 80 but no surfactant and thoroughly mixing. Then， 200 l of corresponding test tube solution was applied to the nutrient agar medium， followed by culturing in an incubator at 27 for 2 d to observe the development of the colony， and the number of colonies was recorded. Each treatment had 6 replicates.
Adhesion amount testing
A certain amount of Bt （1 g） was mixed with 1 000 ml of sterile water thoroughly. Then， 200 ml was pipetted into two 500 ml handheld sprayers， respectively， and 50 l of AOS and Tween 80 were added into one of the two sprayers， respectively， followed by mixing well. Clean leaves of four kinds of vegetables （scallion， kohlrabi， radish and Chinese cabbage） were selected. Each leaf was held on hand obliquely and sprayed evenly for 6 times with corresponding handheld sprayer 20 cm from the leaf. The leaves were then dried on a table in the shade. The dry leaves were punched with a 9 mm hole puncher. Five pieces were added into a test tube and added with 10 ml of sterile water， followed by mixing well. Next， 200 l was pipetted and applied on nutrient agar， and cultured in an incubator at 27 for 2 d. Finally， the number of Bt spores was recorded. Each treatment included four replicates， and Bt without the addition of a surfactant was also set as a control （CK）. Field efficacy testing
In the greenhouse， the scallion field was divided into several 5 m2 （5 m≠1 m） plots. After diluting the concentration of Bt to 2≠106 cfu/ml， 500 ml of the solution was added into two 500 ml handheld sprayers， respectively， and 250 l of AOS and Tween 80 were added into one of the two sprayers， respectively， and mixed well with the Bt solution， to dilute AOS and Tween 80 by 2 000 times. Scallion leaves were sprayed for 3 times at intervals of 1 week. Before each time of spraying and 1 week after spraying， the number of beet armyworm larvae on 30 scallion leaves was randomly investigated in each plot， to calculate field efficacy. The test was repeated for 3 times， with Bt without the addition of a surfactant as a control （CK）.
Data analysis method
Data were analyzed with software SPSS17.0.
Results and Analysis
Safety to Bt spores
Bt is a biopesticide， and it is important to screen surfactants that are safe for Bt. The test results of the two surfactants AOS and Tween 80 on Bt spore germination are shown in Table 1 and Fig. 1.
It could be seen from Table 1 that whether for the surfactant AOS or Tween 80 treatment， there was no significant difference between the number of colonies after germination of Bt spores and the number of colonies in the CK， indicating that the two surfactants have no adverse effects on Bt spore germination.
As shown in Fig. 1， after treatment with AOS and Tween 80， the periphery of Bt colonies was irregular， with radial folds， and the colonies were slightly moist and yellowish， indicating that the development of Bt colonies was completely normal. Therefore， the two kinds of surfactants are safe for Bt.
Adhesion of Bt
The test results of the effects of surfactants AOS and Tween 80 on the adhesion of Bt on the four kinds of vegetable leaves are shown in Table 2.
It could be seen from Table 2 that the addition of AOS or Tween 80 to the Bt solution significantly increased the amount of Bt attached to the four kinds of vegetable leaves. After adding AOS， the adhesion amounts of Bt spores on scallion， kohlrabi， radish and Chinese cabbage leaves were 209.75， 249.00， 274.00 and 281.75， respectively， which was 3.8， 2.2， 2.0 and 1.6 times higher than the CK. After adding Tween 80， the adhesion amounts of Bt spores on scallion， kohlrabi， radish and Chinese cabbage leaves were 198.25， 203.25， 233.00 and 236.75， respectively， which were 3.6， 1.8， 1.7 and 1.4 times higher than the CK， respectively. The results show that the adhesion effect of surfactant AOS is better than that of Tween 80; and among the four vegetables， onion leaves are most rich in cuticle， followed by kohlrabi leaves and radish leaves sequentially， and Chinese cabbage has the least cuticle. The adhesion effects of the surfactantadded Bt liquids on scallion leaves are shown in Fig. 2.
As shown in Fig. 2， the adhesion effects of Bt added with surfactants AOS and Tween 80 were considerably more remarkable than that of the CK. Furthermore， compared with Tween 80， AOS had a slightly better adhesion effect than Tween 80.
Control efficacy on beet armyworm
Since scallion leaves are most rich in cuticle among the four kinds of vegetables， scallion was selected for the control of beet armyworm. The control efficacy of surfactantadded Bt on beet armyworm in scallion field is shown in Table 3.
As shown in Table 3， the average field insecticidal effect of the Bt liquid added with surfactant AOS was 81.3%; the average insecticidal effect of the Bt liquid added with surfactant Tween 80 was 79.5% in field; and the average insecticidal effect of the CK without the addition of a surfactant was only 53.3%， indicating that the addition of surfactant AOS or Tween 80 to vegetables rich in cuticle can significantly improve the control efficacy in field.
Conclusions
The experimental results showed that compared with the CK， surfactants AOS and Tween 80 did not adversely affect Bt spore germination， so AOS and Tween 80 are safe for Bt.
After adding surfactants AOS and Tween 80， the adhesion of Bt on vegetable leaves ranked as scallion>kohlrabi>radish>Chinese cabbage. This result shows that scallion is most rich in cuticle， followed by B. kohlrabi and radishm， and Chinese cabbage has the least cuticle.
At the same dilution ratio， the adhesion amounts of surfactant AOS on the four kinds of vegetables were always higher than Tween 80， and the field efficacy was slightly higher than Tween 80， indicating that AOS has a broad application prospect.
Selecting suitable surfactants when spraying Bt pesticide can not only improve the control efficacy of Bt， but also can improve the use efficiency of the pesticide， thereby reducing the application amount of the pesticide and significantly reducing the impact on ecological environment.
References
[1]BRAR SK， VERMA M， TYAGI RD， et al. Recent advances in downstream processing and formulations of Bacillus thuringiensis based biopesticides[J]. Process Biochemistry， 2006， 41（2）： 323-342.
[2]JIN DY， QUAN XL， LI XG， et al. Effects of Tween 80 on spreading of Bacillus thuringiensis on crop leaves and its control efficacy against Spodoptera exigua in scallion fields[J]. Plant Protection， 2012， 38（5）： 143-146. （in Chinese）
[3]YOU ZG， LI BL， QU LL， et al. Interaction of surfactants， spraying volume and weed leaf angle in droplet retention of imazethapyr[J]. Journal of Plant Protection， 1994， 21（3）： 283-287. （in Chinese）
[4]ZHANG HY， YU ZN. Factors affecting the insecticidal effect of Bacillus thuringiensis preparations[J]. Plant Protection， 1997， 23（4）： 41-44. （in Chinese）
[5]LI Z， JIN DY. Effects of 4 surfactants on adhesion amount of Bacillus thuringiensis on surface of vegetable leaves[J]. Agrochemicals， 2014， 53（10）： 625-627. （in Chinese）