Renjie Fu · Luxiang Zhou · Kai Feng · Xiaoyu Lu · Jian Luo · Fang Tang

Abstract Odontotermes formosanus (Shiraki), a blackwinged subterranean termite, is a common forest pest. A red pigment-producing bacterial strain isolated from the termite was identif ied as Serratia marcescens and named SM1. A bioassay of SM1 on O. formosanus show that the LD 50 ranged from 1.77 × 10 4 to 10.82 × 10 4 cells/termite over 21-39 h. Three biological control agents, Beauveria bassiana (2 × 10 10 cells/mL), Metarhizium anisopliae (1 × 10 10 cells/mL) and Bacillus thuringiensis (1.6 × 10 8 IU/mL), were used for an O. formosanus bioassay. The results show that the insecticidal Effect of B. bassiana was stronger than that of M. anisopliae. In addition, two mixtures were obtained by combining B. bassiana (2 × 10 10 cells/mL) with SM1(1.5 × 10 10 cells/mL), and M. anisopliae (1 × 10 10 cells/mL)with S. marcescens (SM1) (1.5 × 10 10 cells/mL) in equal volumes. The results show that B. bassiana and SM1 was less Effective than SM1 alone. However, the insecticidal Effect of M. anisopliae and SM1 was better than that of M. anisopliae or SM1 individually. These studies provide an important contribution for termite biocontrol.

Keywords Odontotermes formosanus (Shiraki) · Serratia marcescens strain SM1 · LD 50 · Biological control agents

Introduction

Current control methods forOdontotermes formosanus(Shiraki) rely mainly on chemical treatments, including spraying liquid chemicals, feeding bait, dusting and grouting. The active ingredients in these chemicals are synthetic organic pesticides that present diff erent levels of threat to soil, water and the human body. Therefore, alternative, environmentally friendly termite control agents such as biological control agents need to be developed (Liang et al. 2012). An active bacteria strain isolated fromO. formosanus, was identif ied asSerratia marcescens(SM1) (Fu et al. 2019).

S. marcescens, is a Gram-negative bacterium and an important member of theEnterobacteriaceae.It can Effectively control a number of pests and pathogenic bacteria in the f ield of biological control (Babashpour et al. 2012;Wang et al. 2013) and can also enhance plant resistance to pathogenic bacteria (Nobutaka et al. 2005; Lavania et al.2006; Chakraborty et al. 2010; Ting et al. 2010). In recent years, it has been found thatS. marcescenshas insecticidal properties. Liu et al. ( 1988) found it in diseased planthoppers. Wang et al. ( 2010) isolated a strain ofS.marcescens, KI3, which showed an obvious toxic Effect to aphids. In addition, a strain ofS. marcescensisolated from the cotton bollworm,Helicoverpa armigeraHübner, had a strong pathogenicity and control onH. armigera, on the cabbage white worm,Pieris rapaeL. and beet armyworm,Spodoptera exigua(Hübner) (Chen et al. 2001). The development and utilization ofS. marcescensbacterial strains and their mechanism of action on plant pests has attracted increasing attention from researchers worldwide. However,the application ofS. marcescenstoO. formosanushas not yet been studied.

Mixing pesticides may increase their toxicity, and the combined use of diff erent bacteria may also increase the preventive Effects of pesticide treatment. Yin et al. ( 2004) found that chitinase enzymes produced byS. marcescenshydrolyze chitin in the midgut peritrophic membranes of locusts and perforate intestinal membranes, which makes insecticidal proteins ofS. marcescensmore likely to penetrate into the insect body and destroy the digestive tract, thereby greatly improving the insecticidal Effect on the locusts. At the same time, chitinases produced byS. marcescensalso cause signif icant damage to allow other pathogens to invade locusts and increase the infection and fatality rates. Chitinases that is benef icial for Bt infection have a synergistic Effect onBacillus thuringiensis(Bt) (Sampson and Gooday 1998). In addition, Liu et al. ( 1988) reported a synergizing Effect from the mixed use ofS. marcescensandB. thuringiensisstrain HD-1. However, the toxicity ofS. marcescenstoO. formosanushas not been reported. Therefore, the SM1 strain ofS.marcescens, which we isolated and identif ied, was used to(1) determine the degree of toxicity toO. formosanus; and,(2) determine whether the toxicity of the mixture increased afterS. marcescenswas mixed with other biological control agents.

Materials and methods

Isolation and culture of the S. marcescens strain, SM1

S. marcescensstrain SM1 was isolated from infectedO. formosanusand was deposited at Nanjing Forestry University in Nanjing, China. Solid bacterial medium (1 L) consists of peptone 10 g, beef extract 20 g, NaCl 2 g, K2HPO42 g, agar 18 g and H2O1 L, pH 7.2-7.4.; Seed medium (1 L) consists of peptone 10 g, yeast extract 20 g, NaCl 2 g, K2HPO42 g and H2O1 L; Zymotic medium (1 L) consists of peptone 10 g, soybean oil 30 g, NaCl 2 g, K2HPO42 g and H2O1 L.The isolated SM1 strain was cultured on solid media without light at 27 °C. A single colony was isolated, placed in a 250 mL Erlenmeyer f lask containing 50 mL sterilized seed medium and incubated for 12 h at 30 °C and 200 r/min. The 70 mL seed solution was added to 250 mL zymotic medium and cultured in a shaking incubator at a rotational speed of 200 r/min at 30 °C for 36 h (Zhang et al. 2015).

Insects

Eight colonies ofO. formosanuswere collected from Jurong County in Zhenjiang, Jiangsu Province and kept in sealed plastic containers in total darkness at 27 ± 1 °C and 75 ± 1% relative humidity. All colonies were maintained under laboratory conditions without soil and with moist f ilter paper for 1 day before treatments.

Determination and dilution of the concentration of SM1

The zymotic medium was used as stock liquid and diluted and quantif ied. A drop of the bacterial liquid was used to f ill the counting area made up of 25 squares, and the number of bacteria in f ive squares (upper left, lower left,upper right, lower right and centre) was counted according to their diagonal position.

Calculation formula:

where, (The number of bacteria in the f ive squares)/5 is the average number of bacteria in the f ive middle squares (pink),and N/5 × 25 the total number of bacteria in the center (i.e.,the total number of bacteria in 0.1 mm 3 ). N/5 × 25 × 10 is the total number of bacteria in 1 mm 3 , and N/5 × 25 × 10 × 10 6 the total number of bacteria in 1 L.

The concentration of the stock liquor was 1.5 × 10 10 cells/mL, diluted to 10, 10 2 , 10 3 and 10 4 to obtain 1.5 × 10 9 , 1.5 × 10 8 , 1.5 × 10 7 and 1.5 × 10 6 cells/mL.

Source and concentration of three biological agents

Beauveria bassianaandMetarhizium anisopliaewere obtained from Yancheng Shenwei Microbiological Strain Technology Co. Ltd., andB. thuringiensisfrom Hubei Kangxin Agricultural Pharmaceutical Co., Ltd. The three were diluted as suspensions so thatB. bassianawas 2 × 10 10 cells/mL,M. anisopliae1 × 10 10 cells/mL andB.thuringiensis1.6 × 10 8 IU/mL.

Bioassay of O. formosanus with SM1 and three biological agents

TenO. formosanusworkers of similar size were placed in a 7-cm petri dish on a wet f ilter paper. A 0.12 μL quantity of the diff erent concentrations of biocontrol agents was titrated on the pronotum of each worker; 0.12 μL water was titrated as the control. Each experiment was repeated three times and mortality was observed and recorded every hour in the dark at 27 °C. When the mortality rate of the control group was approximately 20%, the experiment was concluded.

To study the combination of the two biological agents and SM1, another tenO. formosanusworkers of similar size were placed in a 7-cm petri dish, on wet f ilter paper, andB. bassiana(2 × 10 10 cells/mL) orM. anisopliae(1 × 10 10 cells/mL) mixed with SM1 (1.5 × 10 10 cells/mL) (volume ratio = 1:1). The same method was used for the bioassay experiments. Each experiment was repeated three times and mortality recorded hourly in the dark at 27 °C. When the mortality rate of the control group was approximately 20%,the experiment ended.

Statistical analysis

Data were processed by DPS 4.5 software and LD50with a 95% conf idence interval was obtained. The data were subjected to analysis of variance using InStat software (Graph-Pad, San Diego, CA) with signif icance def ined asP< 0.05.

Results

Determination of SM1 toxicity to O. formosanus

The bioassay results show that SM1 clearly aff ectedO. formosanus, and the higher the concentration, the greater the toxicity. In addition, the longer the processing time of SM1,the stronger the toxic Effect. The LD50of SM1 at 24 h was 6.66 × 10 4 cells/termite, and at 39 h 1.77 × 10 4 cells/termite(Table 1).

Toxicity of B. bassiana, M. anisopliae and B.thuringiensis to O. formosanus

Comparing the titration results for these three biological agents, toxicity levels of theB. bassiana(2 × 10 10 cells/mL)andM. anisopliae(1 × 10 10 cells/mL) were signif icantly different from the control.B. bassianaandM. anisopliaewere more toxic toO. formosanus;B. thuringiensis(1.6 × 10 8 IU/mL) was not diff erent from the controls, i.e., there was no toxicity (Table 2).

Table 2 Bioassay of three biological agents on O. formosanus

Comparing the Effects ofB. bassianaandM. anisopliaeover 48 to 84 h, the mortality rate withB. bassianareached 50% about 48 h, while it required 84 h for the mortality rate withM. anisopliaeto reach 50%.B. bassiananeeded approximately 72 h andM. anisopliaeapproximately 96 h to achieve an 80% mortality rate. Therefore,B. bassianahad a stronger insecticidal Effect onO. formosanusthanM. anisopliae(Table 2).

Toxicity of combined biological agents to O. formosanus

The results of the bioassay of the combined biological agents showed that the mixture ofB. bassiana(2 × 10 10 cells/mL) and SM1 (1.5 × 10 10 cells/mL) was less Effective than using SM1 alone. The Effect of the mixture ofM. anisopliae(1 × 10 10 cells/mL) and SM1 (1.5 × 10 10 cells/mL) onO. formosanuswas better than the components used separately. It required 30 h to kill 80% of theO. formosanususing only SM1, but only 26 h with the mixture of SM1 andM. anisopliae(Table 3).

Table 1 Toxicity of S. marcescens strain SM1 to O. formosanus

Discussion

Control ofO. formosanusis mainly through chemical means and the Effective components of pesticides are synthetic organic compounds. These compounds present diff erent degrees of toxicity levels to soils, water and to humans.Although development of the practical application of biological controls is progressing slowly, its environmental andpollution-free characteristics suggest that it could be the principal method for controllingO. formosanusin the future.

Table 3 Bioassays of the combination of two biological agents and S. marcescens strain SM1 on O. formosanus

S. marcescensis a naturally occurring ubiquitous bacteria and pathogenic to insects. In recent years, numerous studies have been carried out onS. marcescensfrom various infected insects (e.g.H. armigera,P. rapae,andS. exigua)and signif icant progress has been made (Liu et al. 1988;Chen et al. 2001; Wang et al. 2010). However, the study ofS. marcescensforO. formosanuscontrol has not been reported previously. The strain SM1 was extracted fromO.formosanusinfected byS. marcescens, and toxicity assays with SM1 were carried out. The results show that the SM1 has an obvious toxic Effect.

Of the current biological pesticides forO. formosanuscontrol,B. bassiana,M. anisopliaeandB. thuringiensiswere selected to treatO. formosanuswith 2 × 10 10 cells/mlB.bassiana, 1 × 10 10 cells/mLM. anisopliaeand 1.6 × 10 8 IU/mLB. thuringiensis.B. bassianaandM. anisopliaewere more toxic.B. bassianaachieved 80% mortality after 72 h,andM. anisopliae80% after 96 h. These biological control agents can be used in the prevention and control ofO. formosanusat the early stage of infestation.

The Effects of combined biological agents onO. formosanushave also not been reported before. In this study,B. bassiana(2 × 10 10 cells/mL) andM. anisopliae(1 × 10 10 cells/mL), which had good toxic Effects toO. formosanus,were combined with SM1 (1.5 × 10 10 cells/mL). The toxicity of the combination ofB. bassianaand SM1 was lower than that of SM1 alone, but the toxicity of the combination ofM. anisopliaeand SM1 was higher than that of SM1 alone.These results show that diff erent biological agents, when combined into diff erent formulations, will produce diff erent results. Therefore, laboratory experiments must be undertaken to choose suitable combinations of biological agents to avoid antagonism between agents.

This study showed that theS. marcescensstrain SM1 was signif icantly toxic toO. formosanus, but the specif ic components and mechanisms of this Effect need to be further examined. Research has shown that the pathogenesis ofS.marcescensis mainly due to chitinase enzymes (Regev et al.1996; Zhang et al. 2000; Xu and Peng 2004; Yin et al. 2004;Jin et al. 2005), and Tao ( 2006) has shown that the insecticidal protein ofS. marcescensis a metallic protein that exists in the supernatant of the bacteria. At the same time,both the living bacteria ofS. marcescensand its secretions are toxic to insects (Yang et al. 2012, 2015). Therefore, a titration experiment onO. formosanususing the diff erent constituents ofS. marcescensfermentation will be carried out in a follow-up study.

Based on this study of theS. marcescensstrain SM1,this bacterium has considerable potential for the biological control ofO. formosanusand needs further study. In addition, this experiment includes only an indoor bioassay of theS. marcescensstrain SM1, while the control Effects of SM1 on an entire colony have not been studied and should be the subject of future research.

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