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. 2019 Feb 13;9(3):80. doi: 10.1007/s13205-019-1602-2

Screening of high-virulent entomopathogenic fungal strains to infect Xylotrechus rusticus larvae

Yan-chen Wang 1, De-fu Chi 1,✉
PMCID: PMC6374243  PMID: 30800591

Abstract

The gray tiger longicorn beetle, Xylotrechus rusticus Linnaeus (Coleoptera: Cerambycidae) is a stem-boring pest that can inhibit not only the transportation of nutrients in the trunk but also the tree growth, increasing the risk of tree breakage and causing economic losses. It is distributed in China, Iran, Turkey, Russia, Korea, Japan, and Southern Europe. This study aimed to investigate selected Beauveria strains that could be used as entomopathogenic fungi for the biological control of this pest. The high-virulence strains were screened among the selected strains by cumulative mortality, correct mortality, and lethal time to 50% mortality (LT50). These screened high-virulence strains were Bb01, CFCC83486, and CFCC81428. Bb01 exhibited 96.96% cumulative mortality, with an estimated LT50 of 3.28 days. CFCC83486 and CFCC81428 caused 89.29% and 75.74% cumulative mortality, with an estimated LT50 of 3.45 and 4.28 days, respectively. Pathogenicity at different concentrations and lethal concentration of 50% (LC50) of these high-virulence strains were investigated. The pathogenicity was found to be positively correlated with suspension concentration, and LC50 was negatively correlated with infection time. These suspensions of high-virulence strains at different concentrations were also investigated in the forest by brushing the suspensions on the poplar tree trunk infested with X. rusticus L. larvae. The most effective strain was found to be Bb01, whose cumulative mortality reached 76.33% at 1.32 × 108 conidia mL−1, followed by the strain CFCC83486, whose cumulative mortality reached 65.17% at 1.32 × 108 conidia mL−1. This study provides an important basis for using B. bassiana in the biological control of X. rusticus L.

Keywords: Beauveria bassiana, Beauveria brongniartii, Pathogenicity, Fungal, Gray tiger longicorn beetle, Biocontrol

Introduction

The gray tiger longicorn beetle, Xylotrechus rusticus Linnaeus (Coleoptera: Cerambycidae), is one of the most destructive pests. It is a stem-boring pest feeding mainly on poplar trees. It is found in northeast Asia where poplar trees are present, including China (Heilongjiang, Jilin, Liaoning, Inner Mongolia, and Jiangsu provinces), Iran, Turkey, Russia, Korea, Japan, and Southern Europe. X. rusticus L. inhibits the transportation of nutrients and growth by boring the trunk of poplar trees, increasing the risk of tree breakage. This pest was listed as a forestry quarantine pest by the State Forestry Administration of China after 2004 (Wang et al. 2003; Hu et al. 2004; Wang and Liu 2005; Li et al. 2014). Poplar species have strong economic value; the bark and leaves are valuable ingredients in traditional Chinese medicine and the trunk in paper, wood composite board, chopsticks, and so on. X. rusticus L. causes serious environmental and economic losses (Zhao 2011).

At present, X. rusticus L. can be managed using biological control, which includes inundative releases of the natural enemies [Dastarcus helophoroides (Fairmaire) and Scleroderma guani Xiao et Wu] (Ding et al. 2016). Chemical methods include spraying or brushing insecticides (omethoate, chlorbenzuron, and sumithion) on the poplar trunk to kill larvae (Wang et al. 2002; Wen and Wang 2010). Long-term use of chemical pesticides increases pest resistance and resurgence to these chemical pesticides, and also causes severe environmental and ecological damage. Therefore, discovering a strategy that can minimize negative environmental effects and effectively control X. rusticus L. is becoming extremely urgent.

Entomopathogenic fungi have a great potential to be an environment friendly and sustainable alternative to control pests by parasitizing insects. These entomopathogenic fungi include Beauveria bassiana (Balsamo) Vuillemin (Hypocreales: Cordycipitaceae), B. brongniartii (Saccardo) Petch (Hypocreales: Cordycipitaceae), Metarhizium brunneum Petch (Hypocreales: Clavicipitaceae), M. flavoviride var. minus, Isaria farinosa (Holmskjold) Fries (Hypocreales: Clavicipitaceae), and I.poprawskii sp. nov. (Hypocreales: Cordycipitaceae) (Cabanillas and Jones 2009; Popa et al. 2012; Cabanillas et al. 2013; Woruba et al. 2014; Brabbs et al. 2015).

The process of B. bassiana infection can be described in five steps as follows: first, B. bassiana attaches to the host body by the conidia. Second, an infection structure is formed. Third, B. bassiana penetrates into the host body cavity and invades hemolymph. Fourth, mycelium grows within the host body and produces toxins against the immune system of the host, killing the host. Fifth, the conidia are formed on the surface of the host body after a period of parasitic growth, and a new infection cycle is formed (Latge et al. 1987; Clarkson and Charnley 1996; Ding et al. 2015).

The pest control efficiency of B. bassiana and B. brongniartii has been reported in many studies. B. bassiana (Hyphomycetes) has been studied to control turpentine beetle, Dendroctonus valens LeConte. It killed all the infected insects at a concentration of 1 × 108 conidia mL−1, and the median lethal time [lethal time to 50% mortality (LT50)] was 4.06 days (Zhang et al. 2011). Sabbahi et al. (2008) found that B. bassiana (Bals.) Vuill can be used to control a tarnished plant bug, Lygus lineolaris L. Dubois et al. (2004) reported that fiber bands impregnated with B. brongniartii can be used to control Asian long-horned beetle, Anoplophora glabripennis. Being inspired by these studies, the researchers aimed to explore an ecological and environment-friendly strategy to control X. rusticus L. using B. bassiana and B. brongniartii in the present study.

This study included five strains of B. bassiana and one strain of B. brongniartii to screen high-virulent entomopathogenic fungal strains infecting larvae of X. rusticus L. The most effective entomopathogenic fungal strains (determined by the cumulative mortality rate, correct mortality rate, and LT50 at the same concentration of each strain) were formulated into various concentrations to evaluate their lethal concentration of 50% (LC50) and their effect (cumulative mortality rate and correct mortality rate) in the laboratory and forest. In this way, the most lethal and the most optimal concentration was determined to serve as a potential biological and environment-friendly strategy to control X. rusticus L.

Materials and methods

Preparation of insects

Xylotrechus rusticus L. larvae were collected from the poplar tree infested with X. rusticus L. larva, from Bin county, Harbin, China, and transferred to the Key Laboratory of Forest Pest Biology (KLFPB), Northeast Forestry University (Harbin, Heilongjiang Province, China). X. rusticus L. larvae were incubated at 25 °C ± 0.5 °C, 75% ± 10% relative humidity (RH), darkness, and fed with artificial diet for continuous rearing of X. rusticus L. (Ding et al. 2015). The dead or sick larvae were removed. Healthy fifth instar larvae of uniform size were used for the experiments.

Preparation of Beauveria strains

The B. bassiana strains CFCC83486, CFCC87298, CFCC83116, and CFCC81428 and B. brongniartii strain CFCC83487 were obtained from BeNa Culture Collection, Beijing. B. bassiana strain Bb01 was obtained from KLFPB (Table 1). All the strains were cultured using an improved Martin culture medium (200 g L−1 potato extraction, 20 g L−1 glucose, 3 g L−1 KH2PO4, 1.5 g L−1 MgSO4·7H2O, 8 mg L−1 vitamin B1, and 20 g L−1 agar, pH 6).

Table 1.

The information of fungal strain used in the experiments

Strain no. Strain Host Geographic origin
Bb01 Beauveria bassiana Xylotrechus rusticus L Heilongjiang, China
CFCC83486 Beauveria bassiana Cerambycidae pupa Liaoning, China
CFCC83487 Beauveria brongniartii Holotrichia diomphalia Heilongjiang, China
CFCC87298 Beauveria bassiana Trunk borer pupa Jilin, China
CFCC83116 Beauveria bassiana Anoplophora glabripennis Inner Mongolia, China
CFCC81428 Beauveria bassiana Cerambycidae larvae Anhui, China
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Pathogenicity and LT50 of the six Beauveria strains to X. rusticus L. larvae

Conidia from the six strains were isolated and scraped into 0.1% Tween-80 sterile solutions to make bacteria suspensions at 1.32 × 108 conidia mL−1. The X. rusticus L. larvae were immersed in 1.32 × 108 conidia mL−1 suspension for 30 s and then incubated at 26 °C, 75% ± 10% RH, and darkness for 10 days. The control group was immersed in 0.1% Tween-80 sterile solution for 30 s and incubated at the same condition mentioned earlier. Thirty larvae were tested in each group, and the procedure was repeated 3 times. The post-infection larval mortality was observed on the second, fourth, sixth, eighth, and tenth days. The mortality rates were recorded, and the calculated LT50 was taken as a basis to select the highest virulence for further study. The mortality rates were adjusted as follows. Corrected mortality (%) = (C1 − C)/(100 − C) × 100%, where C1 is the cumulative mortality rate of the treated larvae and C is the cumulative mortality rate of the control (Ding et al. 2015).

Pathogenicity at different concentrations and LC50 of the high-virulence strains to X. rusticus L. larvae

The suspensions of high-virulence strains were diluted to1.32 × 108, 1.32 × 107, 1.32 × 106, 1.32 × 105, and 1.32 × 104 conidia mL−1 using a hemacytometer (XB. K. 25, Qiujing, Shanghai, China) and 0.1% Tween-80 sterile solution. The 0.1% Tween-80 sterile solution served as the control. The larvae were treated using the same methods described in the “Pathogenicity and LT50 of the six Beauveria strains to X. rusticus L. larvae” section. The post-infection larval mortality was observed on the second, fourth, sixth, eighth, and tenth days. Ten larvae were tested in each group, and the procedure was repeated three times.

Forest trial of the pathogenicity of high-virulence strains at different concentrations

Forest trial was conducted from June 15 to June 30, 2016, in Lindian county, Daqing, China, where trees infested with X. rusticus L. were presented in the forest (124.86°E, 47.17°N). The annual average temperature was 4 °C, annual precipitation was 550–600 mm, and altitude was 142.7–172.4 m.

The suspensions of high-virulence strains were diluted into 1.32 × 108, 1.32 × 107,1.32 × 106, 1.32 × 105, and 1.32 × 104 conidia mL−1 by the same method described in the “Pathogenicity and LT50 of the six Beauveria strains to X. rusticus L. larvae” section. The Tween-80 sterile solution served as the control. The diluted suspensions were brushed on the poplar tree trunk infested with X. rusticus L. larvae (2 m high from the ground) and covered with perforated plastic film. Five larvae-infested poplar trees were tested in each group, and the procedure was repeated three times. Different treated groups were separated by more than 4 m from untreated tree belts. The average height of the tree was 40 m with an average 35 cm diameter at breast height. The post-infection larval mortality was observed by dissecting these treated trees, accounting the living and dead larvae on the 15th day after treatment. The average climate temperature was 28 °C during the forest trial.

Statistical analysis

Cumulative mortality data was conducted using the one-way analysis of variance followed by Duncan’s multiple range test. LT50 and LC50 were calculated by SPSS v.19.0 (IBM-SPSS, NY, USA) (Cao and Chi 2017). The cumulative mortality rate was calculated by Microsoft Office 2017 Excel (Microsoft, WA, USA).

Results

Pathogenicity of the six Beauveria strains to X. rusticus L. larvae

The dynamic changes in the cumulative mortality of X. rusticus L. larvae treated by six fungal strains are illustrated in Fig. 1. All the six strains exhibited pathogenicity after the larvae were treated with 1.32 × 108 conidia mL−1 suspension. The strain Bb01 appeared to have the highest cumulative mortality among other strains (P < 0.01). On the tenth day, the cumulative mortality reached up to 96.96% ± 1.19%. The cumulative mortality of CFCC83486 increased faster from the second day to the eighth day (P < 0.05, P < 0.01). On the tenth day, the cumulative mortality reached 89.29% ± 0.17%. The cumulative mortality of CFCC81428 increased fast from the second day to the sixth day and then increased slowly from the sixth day to the tenth day (P < 0.05, P < 0.01), respectively. On the tenth day, the cumulative mortality reached up to 75.74% ± 0.13%. The cumulative mortality of the other three strains (CFCC83116, CFCC87298, and CFCC83487) increased slowly and was lower than 70% on the tenth day. Therefore, the most effective strains were Bb01, CFCC83486, and CFCC81428.

Fig. 1.

Fig. 1

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Dynamic changes of cumulative mortality of larvae Xylotrechus rusticus L. treated by different fungal strains. Capital letters indicate a very significant difference (P < 0.01), lowercase letters indicate a significant difference (P < 0.05)

LT50 of the six Beauveria strains to X. rusticus L. larvae

LT50 of different fungal strains to X. rusticus L. is presented in Table 2. Only the LT50 of strains Bb01, CFCC83486, and CFCC81428 was less than 5 days. The LT50 of strains Bb01, CFCC83486, and CFCC81428 was 3.28, 3.45, and 4.28 days, respectively, indicating that these three strains had faster pathogenic speed compared with other strains. The corrected mortality of strains Bb01, CFCC83486, and CFCC81428 was 96.64%, 88.15%, and 73.14%, respectively. The corrected mortality of strains CFCC83116, CFF87298, and CFCC83487 was lower than 60%.

Table 2.

LT50 of different fungal strains to Xylotrechus rusticus L. larvae

Strains Cumulative mortality rate (%) Corrected mortality (%) Regression equation LT50/days Correlation coefficient
Bb01 96.96 ± 1.19aA 96.64 y = − 1.198 + 1.008x 3.28 0.995
CFCC83486 89.29 ± 0.17bB 88.15 y = − 1.199 + 0.114 x 3.45 0.974
CFCC81428 75.74 ± 0.13cC 73.14 y = − 1.206 + 0.827x 4.28 0.974
CFCC83116 65.73 ± 0.53dD 62.03 y = − 1.199 + 0.743x 5.01 0.925
CFCC87298 54.57 ± 0.22eE 49.71 y = − 1.481 + 0.702x 8.25 0.966
CFCC83487 45.64 ± 0.59fF 39.83 y = − 1.397 + 0.578x 11.23 0.971
CK 9.66 ± 0.11gG
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Capital letters indicate a very significant difference (P < 0.01), lowercase letters indicate a significant difference (P < 0.05). The data format (mean ± SE)

Strains Bb01, CFCC83486, and CFCC81428 were selected for further experiments as high-virulence strains based on the cumulative mortality and LT50.

LC50 and pathogenicity of the high-virulence strains at different concentrations to X. rusticus L. larvae

Cumulative mortality rate of strain Bb01 at different concentrations and its LC50

The dynamic changes of cumulative mortality of X. rusticus L. larvae treated with different concentrations of strain Bb01 are illustrated in Fig. 2. The cumulative mortality rate exhibited a significant difference or the most significant difference at all the concentrations (P < 0.05, P < 0.01) except between 1.32 × 107 and 1.32 × 106 conidia mL−1 on the second day, and between 1.32 × 108 and 1.32 × 107 conidia mL−1 on the tenth day.

Fig. 2.

Fig. 2

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Dynamic changes of cumulative mortality of Xylotrechus rusticus L. larvae treated by different concentration of strains Bb01. Capital letters indicate a very significant difference (P < 0.01), lowercase letters indicate a significant difference (P < 0.05)

The LC50 of strain Bb01 on the second, fourth, sixth, eighth, and tenth days was 2.32 × 1010, 6.07 × 108, 3.05 × 10, 2.63 × 10, and 1.91 conidia mL−1, respectively (Table 3).

Table 3.

LC50 of Bb01 strain to Xylotrechus rusticus L. larvae at different times after treatment

Time/d Regression equation LC50 (conidia mL−1) Correlation coefficient
10 y = − 0.069 + 0.244x 1.91 0.571
8 y = − 0.38 + 0.268x 2.63 × 10 0.556
6 y = − 0.234 + 0.158x 3.05 × 10 0.743
4 y = − 1.184 + 0.135x 6.07 × 108 0.789
2 y = − 1.268 + 0.122x 2.32 × 1010 0.646
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Cumulative mortality rate of strain CFCC83486 at different concentrations and its LC50

The dynamic changes of cumulative mortality of X. rusticus L. larvae treated with different concentrations of strain CFCC83486 are illustrated in Fig. 3. The cumulative mortality rate exhibited a significant difference or the most significant difference at all the concentrations (P < 0.05, P < 0.01) except between 1.32 × 108 and 1.32 × 107 conidia mL−1 on the tenth day.

Fig. 3.

Fig. 3

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Dynamic changes of cumulative mortality of Xylotrechus rusticus L. larvae treated by different concentration of strains CFCC83486. Capital letters indicate a very significant difference (P < 0.01), lowercase letters indicate a significant difference (P < 0.05)

The LC50 of strain CFCC83486 on the second, fourth, sixth, eighth, and tenth days was 1.26 × 1012, 6.97 × 1010, 4.4 × 104, 1.15 × 104, and 1.99 × 103 conidia mL−1, respectively (Table 4).

Table 4.

LC50 of CFCC83486 strain to Xylotrechus rusticus L. larvae at different times after treatment

Time/days Regression equation LC50 (conidia mL−1) Correlation coefficient
10 y = − 0.906 + 0.275x 1.99 × 103 0.602
8 y = − 1.112 + 0.274x 1.15 × 104 0.786
6 y = − 1.07 + 0.23x 4.4 × 104 0.763
4 y = − 1.129 + 0.104x 6.97 × 1010 0.779
2 y = − 1.45 + 0.12x 1.26 × 1012 0.716
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Cumulative mortality rate of strain CFCC81428 at different concentrations and its LC50

The dynamic changes of cumulative mortality of X. rusticus L. larvae treated with different concentrations of strain CFCC81428 are depicted in Fig. 4. The cumulative mortality rates exhibited a significant difference or the most significant difference at all the concentrations (P < 0.05, P < 0.01) except between 1.32 × 107 and 1.32 × 106 conidia mL−1 on the fourth day, and between 1.32 × 108 and 1.32 × 107 conidia mL−1 on the tenth day.

Fig. 4.

Fig. 4

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Dynamic changes of cumulative mortality of Xylotrechus rusticus L. larvae treated by different concentration of strains CFCC81428. Capital letters indicate a very significant difference (P < 0.01), lowercase letters indicate a significant difference (P < 0.05)

The LC50 of strain CFCC81428 on the second, fourth, sixth, eighth, and tenth days was 4.11 × 1012, 5.36 × 107, 1.83 × 105, 3.13 × 104, and 1.5 × 103 conidia mL−1, respectively (Table 5).

Table 5.

LC50 of CFCC81428 strain to Xylotrechus rusticus L. larvae at different times after treatment

Time/days Regression equation LC50 (conidia mL−1) Correlation coefficient
10 y = − 0.448 + 0.141x 1.5 × 103 0.728
8 y = − 0.841 + 0.187x 3.13 × 104 0.754
6 y = − 1.031 + 0.196x 1.83 × 105 0.654
4 y = − 1.405 + 0.182x 5.36 × 107 0.526
2 y = − 1.763 + 0.14x 4.11 × 1012 0.710
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The aforementioned results demonstrated that pathogenicity was positively correlated with suspension concentration and LC50 was negatively correlated with infection time, and dose effect increased.

Pathogenicity of the high-virulence strain at different concentrations in the forest trial

All cumulative mortality rates of different concentrations of Bb01 were significantly higher than other strains and the control (P < 0.01). The correct mortality rate of the strain CFCC83486 was 63.17%, 51.08%, 31.89%, 30.23%, and 17.39% at 1.32 × 108, 1.32 × 107, 1.32 × 106, 1.32 × 105, and 1.32 × 104 conidia mL−1, respectively, which were significantly higher than those of strain CFCC81428 and the control (P < 0.01) but lower than those of strain Bb01 (Table 6). It means that the strain Bb01 was more effective than the strains CFCC83486 and CFCC81428. The pathogenicity of each strain was positively correlated with its conidia concentration.

Table 6.

The results of infecting experiments in the forest trial with three fungal strains

Concentration (conidia mL−1) Strain
Bb01 CFCC83486 CFCC81428 Control
1.32 × 108
 Cumulative mortality (%) 76.33 ± 0.97aA 65.17 ± 1.6bB 57.89 ± 1.98cC 5.43 ± 0.73dD
 Corrected mortality (%) 74.97 63.17 55.47
1.32 × 107
 Cumulative mortality (%) 64.75 ± 0.81aA 53.74 ± 1.32bB 46.81 ± 0.47cC 5.43 ± 0.73dD
 Corrected mortality (%) 62.73 51.08 43.76
1.32 × 106
 Cumulative mortality (%) 43.82 ± 0.74aA 35.59 ± 0.34bB 29.49 ± 0.87cC 5.43 ± 0.73dD
 Corrected mortality (%) 40.49 31.89 25.47
1.32 × 105
 Cumulative mortality (%) 34.12 ± 0.79aA 34.02 ± 0.84aA 23.61 ± 1.49bB 5.43 ± 0.73cC
 Corrected mortality (%) 30.34 30.23 19.22
1.32 × 104
 Cumulative mortality (%) 23.05 ± 1.28aA 21.88 ± 1.59aA 14.55 ± 0.62bB 5.43 ± 0.73cC
 Corrected mortality (%) 18.63 17.39 9.64
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Capital letters indicate a very significant difference (P < 0.01), lowercase letters indicate a significant difference (P < 0.05). The data format (mean ± SE)

Discussion

The Beauveria strains could infect Coleoptera, Diptera, Lepidoptera, Homoptera, Hymenoptera, Orthoptera species, and so on (Su et al. 2009). For instance, B. bassiana was effective for control Monochamus alternatus Hope; the percentage of the pest-infested trees in the treated plot declined to 31.1% (Hu et al. 2006). Another research reported that B. bassiana could also control cowpea weevil, Callosobruchus maculates (Fabricius), in the farm scale (Cherry et al. 2007). In the study by Sabbahi et al. (2008), B. bassiana at the rate of 1 × 1013 conidia ha−1 triggered a significant reduction in the number of tarnished plant bug, Lygus lineolaris L., in strawberry fruit plots. Verde et al. (2015) found that a strain of B. bassiana isolated from infected insects collected on dead palms triggered a significant pathogenicity on Rhynchophorus ferrugineus (Olivier). Therefore, Beauveria strains could be a biological and environment-friendly strategy to control pests.

In the present study, the pathogenicity of five strains of B. bassiana and one strain of B. brongniartii was evaluated. These six fungal strains varied in their cumulative mortality rate, corrected mortality rate, and LT50 at a concentration of 1.32 × 108 conidia mL−1. The strain Bb01, which was isolated from infected X. rusticus L., triggered the highest cumulative mortality rate (96.96% ± 1.19%), corrected mortality rate (96.64%), and the shortest LT50 (3.28 days) compared with the other five strains. Li et al. (2007) also found that the B. bassiana strain of Bi05, which was isolated from infected mulberry longicorn, Apriona germari Hope, caused the highest cumulative mortality rate and the shortest LT50 to A. germari compared with other strains. The reason might be attributed to the fact that both Bb01 and Bi05 were isolated from their original host pest. These two strains had the stronger ability to infect their original host compared with other strains isolated from other host insects. The B. bassiana strains of CFCC83486 and CFCC81428 also have an ideal pathological effect. The reason might be that these two strains were isolated from Cerambycidae, which X. rusticus L. belonged to. The B. brongniartii strain of CFCC83487 triggered the lowest cumulative mortality rate (45.64% ± 0.59%), corrected mortality rate (39.83%), and the longest LT50 (11.23 days) compared with other B. bassiana strains. Gu et al. (2009) found that the pathogenicity of B. bassiana was more significant than that of B. brongniartii, which was consistent with the results of the present study. The reason might be that B. brongniartii could only germinate and expand when it infests its specific host, called host specificity. The reason of host specificity is biological long-term co-evolution, and mutual adaptation limited the choices of parasites for the host (Guillebeau 1994).

Three high-virulent B. bassiana strains (Bb01, CFCC83486, and CFCC81428) were selected based on the cumulative mortality rate, corrected mortality rate, and LT50 at concentrations of 1.32 × 108 conidia mL−1. These three fungi varied in their cumulative mortality rate at different concentrations. The most significant pathogenicity appeared at the concentration of 1.32 × 108 conidia mL−1 for each strain. For each strain, the cumulative mortality rate increased as the infection time extended. Cao and Chi (2017) found the same pattern in the pathogenicity of B. bassiana to Cryptorhynchus lapathi L. These patterns also confirmed the pathogenicity effect of B. bassiana to Xylosandrus crassiusculus (Motschulsky) and X. germanus (Blandford) (Castrillo et al. 2016). The reason might be that these pests belong to Coleoptera or the host specificity of B. bassiana. However, Bb01, CFCC83486, and CFCC81428 exhibited no significant increase in the cumulative mortality rate from the eighth day to the tenth day at 1.32 × 108 and 1.32 × 107 conidia mL−1. The reason might be that the survived X. rusticus L. had strong resistibility to Bb01 and CFCC81428. However, further studies are required to find out whether other factors are also responsible for this pattern.

In the forest trial, the whole cumulative mortality rate and the corrected mortality rate were lower than those that came from the laboratory, consistent with the results from the forest trial of B. bassiana pathogenicity to C. lapathi (Cao and Chi 2017). The reason might be that temperature affected the pathogenicity of the B. bassiana. In the forest stands, the temperature was fluctuated between day and night time, which was different with the incubation temperature of treating larvae under laboratory condition (26 °C). He et al. (2005) found that temperature could affect the pathogenicity of a strain of beetle-derived B. bassiana to daikon leaf beetle and Phaedon brassicae Baly. Another reason might be that the larvae were immersed in the diluted suspension for 30 s, which means every larva was exposed to a large amount of conidia in the laboratory. However, in the forest trial, the diluted suspensions were brushed on the trunk, making it almost impossible to ensure that every larva was exposed to as many conidia as in the laboratory condition. The sunlight that contains the ultraviolet ray in the forest could also affect the growth and production spores of B. bassiana even though the perforated plastic film was covered on the trunk immediately after brushing the suspension. Qian et al. (2015) found that the growth and production of B. bassiana spores decreased with the increase in exposure time to ultraviolet ray.

The present study demonstrated that the mortality of X. rusticus L. treated by B. bassiana increased with the extension of infection time at the same concentration. The Bb01 strain, which was isolated from X. rusticus L. larvae, exhibited the strongest pathogenicity followed by strains CFCC83486 and CFCC81428. However, the B. brongniartii strain, CFCC83487, exhibited the weakest pathogenicity to X. rusticus L. larvae. The concentration of the suspension of the high-virulent strains Bb01, CFCC83486, and CFCC81428 exhibited a positive effect with the cumulative mortality rate. In the forest trial, Bb01 strain exhibited the strongest pathogenicity among the other high-virulent strains but lower than the bioassay results obtained in the laboratory. The method to increase pathogenicity, as well as the efficiency and stability of high-virulent B. bassiana in the forest, requires further investigation.

Conclusions

This study found that high-virulence strains were Bb01, CFCC83486, and CFCC81428 among the selected strains. The Bb01 strain, which was isolated from X. rusticus L. larvae, exhibited the strongest pathogenicity followed by strains CFCC83486 and CFCC81428. The selected strains was also tested in the forest. Bb01 strain exhibited the strongest pathogenicity among the other high-virulent strains in the forest trial. This study provides an important basis for using B. bassiana in the biological control of X. rusticus L.

Acknowledgements

This article was supported by the National Natural Science Foundation of China (NSFC) [Grant number: 31370649].

Compliance with ethical standards

Conflict of interest

No potential conflict of interest was reported by the authors.

Contributor Information

Yan-chen Wang, Phone: 00+86+045182190362, Email: tony196015249@hotmail.com.

De-fu Chi, Email: chidefu@126.com.

References

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