Controlling Strawberry Mites: Comparison of New Bio-Pesticides and Conventional Chemical Pesticides

BACKGROUND Tetranychus cinnabarinus is one of the pest insects most severely influencing strawberry production. It has a high attack rate and causes severe economic losses. Laboratory toxicity tests and greenhouse experiments were carried out using 13 acaricides to determine their efficacy and potential mechanisms of action. RESULTS Abamectin showed the highest efficacy against T. cinnabarinus female mites; its LC50 value was 0.18mg L-1. Pyridaben, cyhalothrin, veratrine, and carbosulfan showed reasonably high efficacy; their LC50 values were 2.69 mg L-1, 3.94 mg L-1, 5.98 mg L-1, and 6.75 mg L-1, respectively. Less effective were hexythiazox and bifenthrin, their LC50 values were 9.82 mg L-1 and 19.09 mg L-1, respectively. Other acaricides such as spirodiclofen, chlorantraniliprole, chlorfenapyr, spinosad, and bifenazate did not show good efficacy. The status of female mites treated with avermectin, pyridaben, kanghebio and cyhalothrin changed significantly under a fluorescence microscope. There were no significant differences among female mites treated with spirotetramat, chlorantraniliprole, spinosad and bifenazate. Enzyme activity tests showed that Kanghebio and cyhalothrin obviously inhibited Ca2+-adenosine triphosphatase (Ca2+-ATPase), while veratrine and kanghebi obviously inhibited acetylcholinesterase (TChE) and monoamine oxidase (MAO). Cyhalothrinexerted an auxo-action on MAO. Greenhouse experiment indicated that abamectin showed the best efficacy, as well as the longest duration of efficacy, pyridaben, cyhalothrin, veratrine, and kanghebio followed, while carbosulfan, hexythiazox, and bifenthrin performed the worst. CONCLUSIONS Our study provided a scientific basis for chemical pesticides to be replaced by these and potentially other new bio-pesticides.


INTRODUCTION 17
Sweet Charlie. Eight acaricides (abamectin, pyridaben, cyhalothrin, veratrine, 1 carbosulfan, hexythiazox, bifenazate, and kanghebio) were tested, and all treatments 2 were repeated three times. Twenty-eight replicated plots were set up. A back-mounted 3 hand sprayer was used to spraying the acaricides. Ten strawberry plants were used in 4 each experiment, and the number of strawberry mites on 5 leaves of each plant was 5 counted before spraying. The number of remaining live strawberry mites was counted 6 after 3d, 7d, 14d, and 21d using a microscope. 7

Preparation of enzyme solution 9
Twenty-four hours after acaricide treatment, 100 female adult mites were 10 homogenized with 0.25mL normal saline solution (NS) for 15min in a 4°C ice-water 11 bath at 2500rpm,and the resulting supernatant was collected. 12

Enzyme source protein concentration 13
G-250 Coomassie Brilliant Blue (5 mL) was mixed with enzyme liquid (0.2 mL) at 14 25°C . Optical density (OD) was measured three times at 595nm, and the protein 15 content was calculated according to a standard curve. 16

Acetylcholinesterase (TChE) test 17
The TChE checker board method was followed the instructions of enzyme activity 18 test kit produced by Nanjing Jiancheng Bioengineering Institute. OD was measured 19 three times at 412nm and zero set with double distilled water (DDW). 20

Ca 2+ -ATPase test 21 7
The ultra-micro ATPase checkerboard method was followed the instructions of 1 enzyme activity test kit produced by Nanjing Jiancheng Bioengineering Institute. OD 2 was measured three times at 412nm and zero set with distilled water. 3

Monoamine oxidase (MAO) test 4
The monoamine oxidase checkerboard method was followed the instructions of 5 enzyme activity test kit produced by Nanjing Jiancheng Bioengineering Institute. OD 6 was measured three times at 242nm and the enzyme activity calculated. 7

Data analysis 8
Comparisons of rates of pest mortality and revised control efficiency were 9 conducted using one-way analysis of variance (one-way ANOVA). Significant 10 differences among means were determined using Fisher's LSD test at P = 0.05 12 , and 11 all analyses were conducted in the statistical program SPSS v 19.0. 12 13 14 3. RESULTS 15

Greenhouse test 19
The same treatments were applied in No.1 and No. 2 greenhouses over the same 20 period. The overall trend was consistent with the laboratory test (Table 3 and 4). Two 21 new bio-pesticides showed better control efficacy than chemical pesticides in 22 greenhouse tests. Of the eight acaricides, abamectin showed the best efficacy, while 1 Pyridaben, kanghebio, cyhalothrin, veratrine, carbosulfan, hexythiazox, and bifenthrin 2 also showed good efficacy. The results of experiments in No.1greenhouse were 3 consistent with those from No.2 greenhouse, and these results were in agreement with 4 those of the laboratory test. However, better control was achieved in No.2 greenhouse 5 than in No.1greenhouse. This was possibly due to moister conditions in No.2 6 greenhouse, which were not suitable for red spider outbreak, while conditions were 7 more suitable for red spider in the No. 1 greenhouse. Eight acaricides were effective 8 in prevention and control, and the efficacy of the biological agents veratrine and 9 kanghebio was good. Combined with laboratory results, this suggests that the 10 biological acaricides veratrine and kanghebio, which are low in toxicity and high 11 effective, could be applied greenhouses to control red spider. 12

Determination of enzyme activity 13
Enzyme activity tests were carried out to explore possible mechanisms of action.   The results showed as follows: of the 13 acaricides tested, those with better efficacy 17 in the control of strawberry mites were Abamectin, Pyridaben, cyhalothrin, veratrine, 18 and kanghebio. Veratrine and kanghebio are bio-pesticides and were as effective as 19 conventional acaricides. They are environment-friendly and effective acaricides with 20 low toxicity. Next, 0.5% of the recommended doses of veratrine and kanghebio are 21 100mlha -1 and 150mlha -1 , respectively. It is recommend that pesticide be applied in 1 early stages of insect attack, spraying once every 3d during severe infestations and 2 spraying once every 7d after insect numbers decrease, then spraying once every 10d 3 for prevention. 4 Spider mites were sensitive to abamectin and have not become resistant to 5 abamectin in the Pinggu area. Some reports 15-19 suggest that strawberry mites have 6 developed pesticide resistance. Therefore, we do not recommend abamectin as a long-7 term option for controlling mites. Of the chemical acaricides, pyridabenand 8 cyhalothrin showed high efficacy and may have unwanted effects on bees during 9 strawberry flowering. They are WHO Class II and not environmentally friendly, so 10 we do not recommend that they be widely applied in the field. We speculate that Ca 2+ -ATPase and TChE inhibition is the mechanism by which these 2 acaricides act. 3 The results obtained in our study show that the bio-pesticides veratrine, kanghebio 4 are effective control agents both in laboratory tests and greenhouse experiments. 5 Furthermore, they are environmentally friendly acaricides, with low toxicity and high 6 efficacy. This research provides a strong basis for using bio-pesticides in place of 7 conventional chemicals to control strawberry spider mites, and provides useful 8 insights into the biological mechanisms by which acaricides act. However, we need to 9 further study their efficacy at different mite developmental stages and clarify the 10 mechanisms of action of these bio-pesticides. 11

CONCLUSION 12
In summary, two new bio-pesticides and several chemical pesticides were 13 compared using laboratory toxicity tests and greenhouse experiments. The bio-14 pesticides showed better control efficacy than chemical acaricides in greenhouse tests. 15 These bio-pesticides have developed a good reputation in international markets in 16 recent years due to their safety, organic nature, environmental friendliness, and lack 17 of propensity to induce pesticide resistance, among other advantages. Our study 18 provides a scientific basis for chemical pesticides to be replaced by these and 19 potentially other new bio-pesticides.           Figure 1. Reductions in numbers of adult female mites following treatment with one of 13 acaricides in slide-dip laboratory tests (6-96h).