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Biocontrol Potential of Some Entomopathogenic Fungal Strains Against Bean Aphid Megoura japonica (Matsumura)

Biocontrol Potential of Some Entomopathogenic Fungal Strains Against Bean Aphid Megoura japonica... agriculture Article Biocontrol Potential of Some Entomopathogenic Fungal Strains Against Bean Aphid Megoura japonica (Matsumura) 1 , 2 , 2 1 , Duy Nam Trinh *, Thi Kim Lien Ha and Dewen Qiu * State Key Laboratory of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China Plant Protection Research Institute, Hanoi, Vietnam; kimlientta@gmail.com * Correspondence: trinhduynam09@yahoo.com.vn (D.N.T.); qiudewen@caas.cn (D.Q.); Tel.: +86-10-8210-5929 (D.Q.) Received: 4 February 2020; Accepted: 31 March 2020; Published: 4 April 2020 Abstract: This research reported the in vitro pathogenicity of Verticillium lecanii strains, L2 and L5, and Beauveria bassiana strains, B76 and B252, against Megoura japonica using leaf-dip method. The virulence potential of these four entomopathogenic fungal strains of V. lecanii and of B. bassiana were 6 7 8 1 compared between fungi conidia (concentrations 1  10 , 1  10 , and 1  10 conidia mL ) and culture filtrate. Moreover, binary combination of four di erent fungal strains (L2 + B76, B76 + L5, L2 + B252, and L2 + B76 + B252 + L5) were evaluated against M. japonica under control condition. Aphid mortality was recorded after two, four, six, and eight days of post-treatment. In the conidial concentration bioassay, strain B76 showed maximal mortality (85.3%) against bean aphid, and strain 8 1 L5 showed the lowest e ect (60.0%) at the highest concentration (1  10 conidia mL ) at eight days post-treatment. Whereas, binary combinations of B76 and L2 strains showed the highest e ect against M. japonica (90.5%) than other combinations. Moreover, in comparison with the e ect of filtrate and conidia bioassay, 91.4% and 84.1% were achieved in strain B76, and the lowest e ect (63.8% and 55.1%) was recorded in strain L5. Keywords: Verticillium lecanii; Beauveria bassiana; Megoura japonica; binary combination; filtrates; conidial 1. Introduction The bean aphid Megoura japonica (Matsumura) is one of the most dangerous agricultural insect pests on legume plants, such as common bean, soybean, and mung bean [1]. Bean aphid is distributed widely all over the world [2]. They suck cell sap from plants and also transmit various viruses in di erent crops [3]. The management of M. japonica is through synthetic pesticides. However, the unselective application of pesticides has resulted in many visible problems, such as resistance to pesticides, killing natural enemies, environmental pollution, and human health issues [4]. To overcome problems related to widespread use of chemical pesticides, alternative methods such as biocontrol substances have extensively been researched in the world. Many microbial insecticides based on pathogenic organisms, such as virus, bacteria, fungus, and nematode, have played a significant role in the field of crop protection and are being used to control an extensive range of insects [5–9]. Entomopathogenic fungal strains, including Verticillium lecanii, Beauveria bassiana, Isaria fumosorosea, and Metarhizium anisopliae, were used as the specific biological pesticides, which are environmentally friendly and can be used against many sucking insect pests [5,10–12]. Spores germinated after attaching to the epidermis of the host insects, and the hyphae penetrate the body of the insects, which causes the death of the host within a few days [13,14]. In addition, these entomopathogenic fungi have no or Agriculture 2020, 10, 114; doi:10.3390/agriculture10040114 www.mdpi.com/journal/agriculture Agriculture 2020, 10, 114 2 of 10 little harmfulness on mammals. Their residuals are target specific and less vulnerable to resistance evolution [15,16]. These virulent fungi were focused on by researchers in the past decades for their potential as biological control agents around the world, and these could exist at the epizootic or enzootic levels in their host population. V. lecanii and B. bassiana are among the most well researched virulent entomopathogenic fungi belonging to order of Hypocreals. They have a wide range of insect pest colonization [17,18]. These two kinds of fungal strains are easily collected from the phylloplane of vegetation, as well as from infected insects and soil [19,20]. As a bio-insecticide, V. lecanii has been used to control black bean aphid Aphis fabae (Hemiptera: Aphididae) under control conditions [21]. The entomopathogenic fungi have been developed as one of the major new bioactive agents for plant pathogen and insect pest control [22–24]. Fungal conidia are produced asexually and become the basis of infection in insect pests of crops. Infection through conidia starts when they are attached to the host cuticle, then germinate following the activation of the enzymatic reaction and invaded the body of the insects by germ tube, appressoria, and penetration pegs [25]. Refined culture filtrates of entomopathogenic fungi, V. lecanii and B. bassiana, decreased the reproductive rate of aphids [26,27] and prevented feeding of the larva of Spodoptera littoralis and Bemisia tabaci [28,29]. Increased fungus concentration decreased the number of adult parasitoids and also negatively a ected its developmental stages [30]. Filtrate culture contains many enzymes like chitinases, lipases, and protease, and these enzymes help in the infection process by degrading the cuticle of insects. The concentration of an enzyme can be enhanced by the use of di erent additives in the culture media, like colloidal chitin [27]. This paper focused on the evaluation of the ecacy of our collected fungal strains (V. lecanii and B. bassiana) with di erent application materials (conidia, filtrate) on bean aphids. Furthermore, we also aimed to determine the combined e ect of di erent entomopathogenic fungal strains against bean aphids. This result could be helpful for establishing an e ective integrated pest management method which could reduce bean aphid population below economic thresholds, while minimizing the use of synthetic chemical pesticides. 2. Results 2.1. Inhibition E ect of Di erent Concentrations of the Fungal Spore on Bean Aphid The conidial bioassay results showed the significant e ect of the di erent fungal trains (p < 0.01; Table 1). Moreover, the factorial analysis of variance revealed a significant e ect of di erent concentrations (F = 296.72, p < 0.01), di erent time intervals (F = ******, p < 0.01), and their interaction (p < 0.01, Table 1). All strains of B. bassiana and V. lecanii caused significant bean aphid mortality at all di erent concentrations. The maximum e ect of the fungal strain (85.3%) was collected on the 8 1 eighth day after treatment with the concentration of 1  10 conidia mL from strain B76, whereas the minimum e ect (71.4%) was collected at the lowest concentration of B76 (i.e., 1  10 conidia mL , Figure 1). The virulence from fungal strain B252 against M. japonica showed a similar tendency to the condition of strain B76. The highest mean e ect (66.0%) of strain B252 was collected with a 8 1 concentration of 1  10 conidia mL at 8 dpi. The minimum mean e ect of strain B252 (49.9%) was 6 1 collected at the lowest concentration (1  10 conidia mL ). Similarly, the e ect of V. lecanii strains, L2 and L5, on M. japonica presented that corrected mortality of bean aphid improved with the exposure time and concentration of fungal strains (Figure 1). Agriculture 2020, 10, 114 3 of 10 Table 1. Analysis of variance (ANOVA) for the mortality of M. japonica by three di erent concentrations from four entomopathogenic fungi strains at di erent times against bean aphids. Source of Variation DF Sums of Squares Mean Squares F Ratio Prob Treatment 4 39,204.6 9801.15 ****** 0.000 Concentration 2 3151.77 1575.88 296.72 0.000 Time 3 57,007.1 19002.4 ****** 0.000 Treatment  Concentration 8 849.592 106.199 20.00 0.000 Treatment  Time 12 12,258.7 1021.56 192.34 0.000 Concentration  Time 6 625.468 104.245 19.63 0.000 Treatment  Concentration  Time 24 454.597 18.9415 3.57 0.000 RESIDUAL 120 637.332 5.31110 Total 179 114,189. 637.928 CV% 7.0 CV%: Coecient of variation. Agriculture 2020, 10, x FOR PEER REVIEW 3 of 10 Figure 1. The mortality of Megoura japonica by di erent concentrations of Verticillium lecanii (L2 and L5) Figure 1. The mortality of Megoura japonica by different concentrations of Verticillium lecanii (L2 and and Beauveria bassiana (B76 and B252) spores at di erent time intervals. Columns show the mortality of L5) and Beauveria bassiana (B76 and B252) spores at different time intervals. Columns show the aphids with di erent fungal strains  SE (n = 50). The di erent letters express significant di erences mortality of aphids with different fungal strains ± SE (n = 50). The different letters express significant between di erent treatments (three-way factorial analysis of variance (ANOVA); least significant differences between different treatments (three‐way factorial analysis of variance (ANOVA); least di erence (LSD) test at = 0.05). DAT: Days after treatment. significant difference (LSD) test at α = 0.05). DAT: Days after treatment. 2.2. Fungal Strain Combinations Against Bean Aphid Table 1. Analysis of variance (ANOVA) for the mortality of M. japonica by three different In the combination bioassays test, the results showed a high significant e ect of all treatments (F = concentrations from four entomopathogenic fungi strains at different times against bean aphids. 618.56; p < 0.01), the di erent time intervals (F = 775.30, p < 0.01), and the interaction between di erent Source of Variation DF Sums of Squares Mean Squares F Ratio Prob times and di erent treatments on fungal strains (F = 40.60, p < 0.01, Table 2). The highest inhibition Treatment 4 39204.6 9801.15 ****** 0.000 e ect of the fungal strain (90.5%) was observed for L2 + B76, while the e ect of the combination of Concentration 2 3151.77 1575.88 296.72 0.000 B76 + L5 was 81.1%. Binary combination of L2 + B252 caused 71.6% corrected bean aphid mortality Time 3 57007.1 19002.4 ****** 0.000 (Figure 2). Nevertheless, for the combination of four di erent fungal strains (L2 + L5 + B76 + B252), Treatment × Concentration 8 849.592 106.199 20.00 0.000 the bean aphid mortality rate was recorded as 69.4%. This phenomenon was most probably due to Treatment × Time 12 12258.7 1021.56 192.34 0.000 the reduced number of spores of the high virulence strain (B76 and L2) in the combination (B76 + Concentration × Time 6 625.468 104.245 19.63 0.000 L2 + B252 + L4). So, its e ect was reduced to be significantly lower against M. japonica than other Treatment × Concentration × Time 24 454.597 18.9415 3.57 0.000 combinations (Figure 2). RESIDUAL 120 637.332 5.31110 Total 179 114189. 637.928 CV% 7.0 CV%: Coefficient of variation. 2.2. Fungal Strain Combinations Against Bean Aphid In the combination bioassays test, the results showed a high significant effect of all treatments (F = 618.56; p < 0.01), the different time intervals (F = 775.30, p < 0.01), and the interaction between different times and different treatments on fungal strains (F = 40.60, p < 0.01, Table 3). The highest inhibition effect of the fungal strain (90.5%) was observed for L2 + B76, while the effect of the combination of B76 + L5 was 81.1%. Binary combination of L2 + B252 caused 71.6% corrected bean aphid mortality (Figure 2). Nevertheless, for the combination of four different fungal strains (L2 + L5 + B76 + B252), the bean aphid mortality rate was recorded as 69.4%. This phenomenon was most probably due to the reduced number of spores of the high virulence strain (B76 and L2) in the combination (B76 + L2 + B252 + L4). So, its effect was reduced to be significantly lower against M. japonica than other combinations (Figure 2). Agriculture 2020, 10, 114 4 of 10 Table 2. Analysis of variance (ANOVA) for the mortality of M. japonica by the binary combinations from four fungal strains of V. lecanii and B. bassiana at di erent time intervals. Source of Variation DF Sums of Squares Mean Squares F Ratio Prob Treatment 4 20,964.9 5241.22 618.56 0.000 Time 3 19,708.0 6569.34 775.30 0.000 Treatment  Time 12 4127.90 343.992 40.60 0.000 RESIDUAL 40 338.932 8.47329 Total 59 45,139.7 765.080 CV% 7.2 CV%: Coecient of variation. Agriculture 2020, 10, x FOR PEER REVIEW 4 of 10 Figure 2. The mortality of Megoura japonica by the binary combinations from strains of Verticillium Figure 2. The mortality of Megoura japonica by the binary combinations from strains of Verticillium lecanii and Beauveria bassiana (L2 + B76, B76 + L5, L2 + B252, L2 + B76 + B252 + L5, and control) recorded lecanii and Beauveria bassiana (L2 + B76, B76 + L5, L2 + B252, L2 + B76 + B252 + L5, and control) recorded at di erent time intervals. Columns show the bean aphid mortality with di erent fungal strains  SE at different time intervals. Columns show the bean aphid mortality with different fungal strains ± SE (n = 50). The di erent letters express the significant di erence between di erent treatments (two-way (n = 50). The different letters express the significant difference between different treatments (two‐way factorial analysis of variance (ANOVA); least significant di erence (LSD) test at = 0.05). DAT: Days factorial analysis of variance (ANOVA); least significant difference (LSD) test at α = 0.05). DAT: Days after treatment. after treatment. 2.3. Comparison of the Mortality of Bean Aphid by Fungi Filtrate and Conidia Table 2. Analysis of variance (ANOVA) for the mortality of M. japonica by the binary combinations The filtrate bioassay results revealed that the overall mean e ect of di erent fungi filtrate against from four fungal strains of V. lecanii and B. bassiana at different time intervals. bean aphid was higher in comparison to their conidial treatments. Statistical processing results showed Source of Variation DF Sums of Squares Mean Squares F Ratio Prob that there was a significant e ect of fungal conidia and filtrates on corrected bean aphid mortality (F = Treatment 4 20964.9 5241.22 618.56 0.000 117.71, p < 0.01, Table 3). The highest e ect of the filtrate solution was strain B76, with 91% recorded on Time 3 19708.0 6569.34 775.30 0.000 the eighth day of treatment, while the e ect of strain B76 conidia was recorded as 84% on the eighth 12 4127.90 343.992 40.60 0.000 Treatment × Time day of treatment (p < 0.01). Other fungal strains had similar results. However, the lowest e ect of RESIDUAL 40 338.932 8.47329 filtrates and conidia was in fungal strain L5, recorded as 63.8% and 55.1%, respectively (Figure 3). Total 59 45139.7 765.080 Table 3. Analysis of variance (ANOVA) for the comparison of the mortality of M. japonica by filtrate CV% 7.2 and the conidia from di erent fungal strains (V. lecanii and B. bassiana). CV%: Coefficient of variation. Source of Variation DF Sums of Squares Mean Squares F Ratio Prob 2.3. Comparison of the Mortality of Bean Aphid by Fungi Filtrate and Conidia Treatment 8 15,365.3 1920.66 117.71 0.000 The filtrate RESIDUAL bioassay results reve 18 aled tha 293.692 t the overall mean ef 16.3162 fect of different fungi filtrate against Total 26 15,659.0 602.268 bean aphid was higher in comparison to their conidial treatments. Statistical processing results CV% 6.0 showed that there was a significant effect of fungal conidia and filtrates on corrected bean aphid CV%: Coecient of variation. mortality (F = 117.71, p < 0.01, Table 4). The highest effect of the filtrate solution was strain B76, with 91% recorded on the eighth day of treatment, while the effect of strain B76 conidia was recorded as 84% on the eighth day of treatment (p < 0.01). Other fungal strains had similar results. However, the lowest effect of filtrates and conidia was in fungal strain L5, recorded as 63.8% and 55.1%, respectively (Figure 3). Agriculture 2020, 10, x FOR PEER REVIEW 5 of 10 Agriculture 2020, 10, 114 5 of 10 Figure 3. Comparison of the mortality of M. japonica by filtrate and the conidia from di erent fungal strains (V. lecanii and B. bassiana). Columns show the mortality of aphids by di erent fungal strains  SE Figure 3. Comparison of the mortality of M. japonica by filtrate and the conidia from different fungal (n = 50). The di erent letters express the significant di erence between di erent treatments (one-way strains (V. lecanii and B. bassiana). Columns show the mortality of aphids by different fungal strains ± factorial analysis of variance (ANOVA); least significant di erence (LSD) test at = 0.05). SE (n = 50). The different letters express the significant difference between different treatments (one‐ way factorial analysis of variance (ANOVA); least significant difference (LSD) test at α = 0.05). 3. Discussion Developing biological control methods, based on entomopathogenic fungi with resistance to Table 3. Analysis of variance (ANOVA) for the comparison of the mortality of M. japonica by filtrate pathogens and the co and niinsect dia fropests, m diffe is reone nt fung of the al stra cor ins e ar (V. eas lecani of curr i and ent B. biological bassiana). control research. Many virulent fungal strains were found to be e ective against di erent insect pests [31,32]. This study determined Source of Variation DF Sums of Squares Mean Squares F Ratio Prob the e ect of di erent entomopathogenic fungi against bean aphid. The results of this study presented Treatment 8 15365.3 1920.66 117.71 0.000 that four strains, V. lecanii (L2 and L5) and B. bassiana (B76 and B252), could be e ective biocontrol RESIDUAL 18 293.692 16.3162 agents against bean aphids. This study result is also similar to previous research showing the e ect of Total 26 15659.0 602.268 di erent strains of V. lecanii and B. bassiana against aphids [26,33]. CV% 6.0 In all experiments, the e ect of fungal strains appeared to be dose and time-dependent and CV%: Coefficient of variation. increased by increasing time after application and the conidia concentrations of fungus, [34] which was also demonstrated in our conidial treatment study. They use di erent concentrations of the 3. Discussion entomopathogenic fungus, B. bassiana, against Metopeurum fuscoviride and A. fabae, and reported that 8 1 Developing biological control methods, based on entomopathogenic fungi with resistance to the highest concentration (1  10 spores mL ) exhibited the highest mortality percent on seventh pathogens and insect pests, is one of the core areas of current biological control research. Many day post-treatment. It was also demonstrated that the mortality of aphids increased with time and virulent fungal strains were found to be effective against different insect pests [31,32]. This study conidial concentration exposure [35,36]. Similarly, it was described that the mortality percentage determined the effect of different entomopathogenic fungi against bean aphid. The results of this was a ected by the concentration of conidia, temperature, and exposure time [37]. The mortality study presented that four strains, V. lecanii (L2 and L5) and B. bassiana (B76 and B252), could be percentage was directly proportional to the concentration of conidia using di erent concentrations 6 7 8 1 effective biocontrol agents against bean aphids. This study result is also similar to previous research of B. bassiana (1  10 , 1  10 and 1  10 conidia mL ) Rhopalosiphum padi, Schizaphis graminum, showing the effect of different strains of V. lecanii and B. bassiana against aphids [26,33]. Lipaphis erysimi, and Brevicoryne brassicae. All concentrations were e ective for the control of aphids, 8 1 In all experiments, the effect of fungal strains appeared to be dose and time‐dependent and but the highest concentration (1  10 conidia mL ) caused the highest percentage mortality [38]. increased by increasing time after application and the conidia concentrations of fungus, [34] which The research was conducted for the determination of the virulence of V. lecanii and its result showed 8 1 was also demonstrated in our conidial treatment study. They use different concentrations of the that concentration of 1  10 conidia mL had the best e ect, with an 86% mortality rate of nymphal 7 8 entomopathogenic fungus, B. bassiana, against Metopeurum fuscoviride and A. fabae, and reported that observed after five days of application [39]. It was also reported that at concentrations of 10 and 10 8 −1 the highest concentration (1 × 10 spores mL ) exhibited the highest mortality percent on seventh day conidia mL of V. lecanii, the maximum mortality rate of M. persicae was recorded as 100% after 12 post‐treatment. It was also demonstrated that the mortality of aphids increased with time and days of application [40]. Similarly, the e ect of B. bassiana on M. persicae was also determined under the 7 1 conidial concentration exposure [35,36]. Similarly, it was described that the mortality percentage was controlled condition with the concentration of 1  10 conidia mL and showed that three strains of affected by the concentration of conidia, temperature, and exposure time [37]. The mortality B. bassiana (BAU019, BAU004, and BAU018) showed high e ect on aphids, with a mortality of over percentage was directly proportional to the concentration of conidia using different concentrations 75% [34,41–44]. According to filtrate treatment, our results are similar to results which revealed that 6 7 8 −1 of B. bassiana (1 x 10 , 1 x 10 and 1 x 10 conidia mL ) Rhopalosiphum padi, Schizaphis graminum, Lipaphis degradation of the insect body was observed at higher infiltrate treated aphids, and the reduction of erysimi, and Brevicoryne brassicae. All concentrations were effective for the control of aphids, but the aphid population was higher in higher dose filtrate treatment [27]. The maximum ecacy was due 8 −1 highest concentration (1 x 10 conidia mL ) caused the highest percentage mortality [38]. The research to the production of metabolites in the culture filtrate that helped to degrade the cuticle and deform was conducted for the determination of the virulence of V. lecanii and its result showed that the hemocoel. As compared with the filtrate, conidia need more time to germinate and release the 8 −1 concentration of 1 x 10 conidia mL had the best effect, with an 86% mortality rate of nymphal required enzymes for the degradation of the insect body. It was demonstrated that the use of filtrate 7 8 observed after five days of application [39]. It was also reported that at concentrations of 10 and 10 Agriculture 2020, 10, 114 6 of 10 application for the control of insects was the best method, and also e ective for insects which had a short life cycle because these insects have more chances to shed the conidia from their body by molting, since germination of conidia needs a specific time [45,46]. According to their findings, our results were similar. Filtrate had more ability to control the insect population, because they contained toxic enzymes for the degradation of the insect body. Through filtrate optimization, the production of enzymes could be enhanced easily with fungal genetic manipulation. Filtrate production, storage, and transportation are more suitable than conidia, and it meets the commercialization requirements. In this study, the e ect of di erent fungal strains was found to be time dependent in cases of conidia, filtrate suspension, and combinations. The maximal e ect was recorded after eight days, while the minimal e ect was recorded after two days. These results are similar to research that showed an increase in the e ect with the increase in time and concentration [39]. Regarding the pathogenicity of di erent fungal combinations, our results showed that the highest synergistic e ects were a combination of fungal strains (B76 + L5, and L2 + B76). However, there was no synergistic e ect for the combination of fungal strains L2 + B252 and L2 + B76 + B252 + L5. These fungal strains may show synergies if applied in a sequential combination, as demonstrated in the case of nematodes and pathogenic fungi [47]. Therefore, the use of the entomopathogenic fungi is considered safe and environmentally friendly compared to chemical pesticides [48], so they are recommended against harmful pests, such as aphids. 4. Materials and Methods 4.1. Insect Culture Bean aphids were collected from the Institute of Plant Protection, Beijing, China. Then, they were reared on Chinese cabbage plants (Brassica rapa) placed in cages in the growth chamber at 50–60% relative humidity and 25  2 C, with a 16:8 h light:dark photoperiod. The first instar aphid was used for all bioassays in this study. 4.2. Fungal Isolates All fungal isolates (Table 4) were collected from fields in China and cultured on potato dextrose agar (PDA) medium (20.0 g agar, 200.0 g potatoes, 20.0 g dextrose, and 1 L distilled water) on Petri-dishes for 20 days in the dark at 25  2 C. Table 4. Name of fungal strains, hosts and geographical of the entomopathogenic fungi. Name Symbols Geographical Origin Verticilliun lecanii 2 L2 Institute of Plant Protection, Beijing, China Verticilliun lecanii 5 L5 Institute of Plant Protection, Beijing, China Beauveria bassiana 76 B76 Institute of Plant Protection, Beijing, China Beauveria bassiana 252 B252 Institute of Plant Protection, Beijing, China 4.3. Conidial Suspension The conidia were collected from PDA dishes in 0.02% Tween solution after 20 days culture and were filtered using sterile cheesecloth. The spore concentrations of all fungal strains were counted under the microscope using a hemocytometer. The conidia viability was checked before using for the design of the bioassays experiment [49]. 4.4. Fungal Filtrate The primary culture of the four strains (B76, B252, L2, and L5) was prepared by mixing 100 mL of Adamek liquid medium (ALM) with 5 mL of conidial suspension and shaken for 3 days at 150 rpm. The secondary culture (1.0%) was prepared by mixing 250 mL of ALM with 2.5 mL of the primary culture medium and shaken for 6 days at 150 rpm, 25 C. The mycelium was removed by centrifugation Agriculture 2020, 10, 114 7 of 10 for 15 min at 12,000 rpm, 4 C and then the supernatant was filtered through the 0.45 m pore-size filter (Millipore Corp) to get the filtrate. 4.5. Pathogenicity Bioassays The e ect of the entomopathogenic fungal strains (B76, B252, L2, and L5) against bean aphids was measured by conducting their conidial and filtrate bioassays, and evaluating the binary combinations of di erent fungal strains. All bioassays were measured using the leaf-dip method [50] with slight modifications. All treatments were performed on 90 mm Petri dishes containing a thin layer of 1.0% agar, and a 60 mm detached leaf disk of Chinese cabbage was placed on the Petri dish. The control leaves were dipped only in 0.02% Tween. Fifty newly molted bean aphids up to 12 h old (first instar) were released on the treated and control leaf on a Petri dish, and then they were stored at 50–60% relative humidity and 25  2 C with a 16:8 h light:dark photoperiod. All the treatments were replicated 3 times (each treatment has 5 Petri dishes for each time). For the bioassay treatment 8 1 of binary combination, the uppermost concentration 1  10 conidia mL of each fungal strain was mixed with the 1 mL conidia of each other fungal strain for all combinations, and then a 2 mL mixed combination sample for each treatment was collected. The combinations, L2 + B76, B76 + L5, L2 + B525, and L2 + B76 + B252 + L5 were designed. For all treatments, the e ect was recorded on 2, 4, 6 and 8 days. All dead bean aphids from each experiment were maintained at 25  2 C and 90% relative humidity in dark to confirm mortality by the pathogens. The e ect of all fungal strains was determined using Abbott’s formula: E ect (%) = (X Y)/X)  100 (X: the percent living in the control; Y: the percent living in the treatment) [51]. 4.6. Data Analysis The data was analyzed using Statistix 8.1 (Analytical Software, Tallahassee, FL, USA). Comparisons of the treatment means were performed, using variances (ANOVA) to determine the significance of individual di erences of the least significant di erence (LSD) test at = 0.05 level. 5. Conclusions In brief, this study shows the e ect of four di erent fungal strains of V. lecanii (L2 and L5) and B. bassiana (B76 and B252) against M. japonica. Through a series of bioassays, the results demonstrated that di erent fungal strains of V. lecanii (L2 and L5) and B. bassiana (B76 and B252) have di erent virulence potential. The di erent application materials and their dosages also a ect pathogenicity on bean aphid. Filtrate application is the most suitable material for the control of M. japonica. In the combination of bioassays, the binary combination of strains of B76 and L2 exhibited high mortality of bean aphid (90.5%). Thus, the results of this study suggest that these fungal strains may be used as novel biological control agents against bean aphids. Author Contributions: Conceptualization, D.Q. and D.N.T.; Investigation, D.N.T.; Data Curation, D.N.T. and T.K.L.H.; Writing Original Draft Preparation, D.N.T.; Writing—Review & Editing, D.Q. and D.N.T.; Supervision, D.Q.; Funding Acquisition, D.Q. All authors have read and agreed to the published version of the manuscript. Acknowledgments: We are especially thankful to the China Scholarship Council (CSC) for providing a Ph.D. scholarship. Conflicts of Interest: The authors declare no conflict of interest. References 1. Zhang, S.Z.; Cao, Z.; Wang, Q.L.; Zhang, F.; Liu, T.X. Exposing eggs to high temperatures a ects the development, survival and reproduction of Harmonia axyridis. J. Therm. Biol. 2014, 39, 40–44. [CrossRef] 2. 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Biocontrol Potential of Some Entomopathogenic Fungal Strains Against Bean Aphid Megoura japonica (Matsumura)

Trinh, Duy Nam; Ha, Thi Kim Lien; Qiu, Dewen
Agriculture , Volume 10 (4) – Apr 4, 2020
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agriculture Article Biocontrol Potential of Some Entomopathogenic Fungal Strains Against Bean Aphid Megoura japonica (Matsumura) 1 , 2 , 2 1 , Duy Nam Trinh *, Thi Kim Lien Ha and Dewen Qiu * State Key Laboratory of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China Plant Protection Research Institute, Hanoi, Vietnam; kimlientta@gmail.com * Correspondence: trinhduynam09@yahoo.com.vn (D.N.T.); qiudewen@caas.cn (D.Q.); Tel.: +86-10-8210-5929 (D.Q.) Received: 4 February 2020; Accepted: 31 March 2020; Published: 4 April 2020 Abstract: This research reported the in vitro pathogenicity of Verticillium lecanii strains, L2 and L5, and Beauveria bassiana strains, B76 and B252, against Megoura japonica using leaf-dip method. The virulence potential of these four entomopathogenic fungal strains of V. lecanii and of B. bassiana were 6 7 8 1 compared between fungi conidia (concentrations 1  10 , 1  10 , and 1  10 conidia mL ) and culture filtrate. Moreover, binary combination of four di erent fungal strains (L2 + B76, B76 + L5, L2 + B252, and L2 + B76 + B252 + L5) were evaluated against M. japonica under control condition. Aphid mortality was recorded after two, four, six, and eight days of post-treatment. In the conidial concentration bioassay, strain B76 showed maximal mortality (85.3%) against bean aphid, and strain 8 1 L5 showed the lowest e ect (60.0%) at the highest concentration (1  10 conidia mL ) at eight days post-treatment. Whereas, binary combinations of B76 and L2 strains showed the highest e ect against M. japonica (90.5%) than other combinations. Moreover, in comparison with the e ect of filtrate and conidia bioassay, 91.4% and 84.1% were achieved in strain B76, and the lowest e ect (63.8% and 55.1%) was recorded in strain L5. Keywords: Verticillium lecanii; Beauveria bassiana; Megoura japonica; binary combination; filtrates; conidial 1. Introduction The bean aphid Megoura japonica (Matsumura) is one of the most dangerous agricultural insect pests on legume plants, such as common bean, soybean, and mung bean [1]. Bean aphid is distributed widely all over the world [2]. They suck cell sap from plants and also transmit various viruses in di erent crops [3]. The management of M. japonica is through synthetic pesticides. However, the unselective application of pesticides has resulted in many visible problems, such as resistance to pesticides, killing natural enemies, environmental pollution, and human health issues [4]. To overcome problems related to widespread use of chemical pesticides, alternative methods such as biocontrol substances have extensively been researched in the world. Many microbial insecticides based on pathogenic organisms, such as virus, bacteria, fungus, and nematode, have played a significant role in the field of crop protection and are being used to control an extensive range of insects [5–9]. Entomopathogenic fungal strains, including Verticillium lecanii, Beauveria bassiana, Isaria fumosorosea, and Metarhizium anisopliae, were used as the specific biological pesticides, which are environmentally friendly and can be used against many sucking insect pests [5,10–12]. Spores germinated after attaching to the epidermis of the host insects, and the hyphae penetrate the body of the insects, which causes the death of the host within a few days [13,14]. In addition, these entomopathogenic fungi have no or Agriculture 2020, 10, 114; doi:10.3390/agriculture10040114 www.mdpi.com/journal/agriculture Agriculture 2020, 10, 114 2 of 10 little harmfulness on mammals. Their residuals are target specific and less vulnerable to resistance evolution [15,16]. These virulent fungi were focused on by researchers in the past decades for their potential as biological control agents around the world, and these could exist at the epizootic or enzootic levels in their host population. V. lecanii and B. bassiana are among the most well researched virulent entomopathogenic fungi belonging to order of Hypocreals. They have a wide range of insect pest colonization [17,18]. These two kinds of fungal strains are easily collected from the phylloplane of vegetation, as well as from infected insects and soil [19,20]. As a bio-insecticide, V. lecanii has been used to control black bean aphid Aphis fabae (Hemiptera: Aphididae) under control conditions [21]. The entomopathogenic fungi have been developed as one of the major new bioactive agents for plant pathogen and insect pest control [22–24]. Fungal conidia are produced asexually and become the basis of infection in insect pests of crops. Infection through conidia starts when they are attached to the host cuticle, then germinate following the activation of the enzymatic reaction and invaded the body of the insects by germ tube, appressoria, and penetration pegs [25]. Refined culture filtrates of entomopathogenic fungi, V. lecanii and B. bassiana, decreased the reproductive rate of aphids [26,27] and prevented feeding of the larva of Spodoptera littoralis and Bemisia tabaci [28,29]. Increased fungus concentration decreased the number of adult parasitoids and also negatively a ected its developmental stages [30]. Filtrate culture contains many enzymes like chitinases, lipases, and protease, and these enzymes help in the infection process by degrading the cuticle of insects. The concentration of an enzyme can be enhanced by the use of di erent additives in the culture media, like colloidal chitin [27]. This paper focused on the evaluation of the ecacy of our collected fungal strains (V. lecanii and B. bassiana) with di erent application materials (conidia, filtrate) on bean aphids. Furthermore, we also aimed to determine the combined e ect of di erent entomopathogenic fungal strains against bean aphids. This result could be helpful for establishing an e ective integrated pest management method which could reduce bean aphid population below economic thresholds, while minimizing the use of synthetic chemical pesticides. 2. Results 2.1. Inhibition E ect of Di erent Concentrations of the Fungal Spore on Bean Aphid The conidial bioassay results showed the significant e ect of the di erent fungal trains (p < 0.01; Table 1). Moreover, the factorial analysis of variance revealed a significant e ect of di erent concentrations (F = 296.72, p < 0.01), di erent time intervals (F = ******, p < 0.01), and their interaction (p < 0.01, Table 1). All strains of B. bassiana and V. lecanii caused significant bean aphid mortality at all di erent concentrations. The maximum e ect of the fungal strain (85.3%) was collected on the 8 1 eighth day after treatment with the concentration of 1  10 conidia mL from strain B76, whereas the minimum e ect (71.4%) was collected at the lowest concentration of B76 (i.e., 1  10 conidia mL , Figure 1). The virulence from fungal strain B252 against M. japonica showed a similar tendency to the condition of strain B76. The highest mean e ect (66.0%) of strain B252 was collected with a 8 1 concentration of 1  10 conidia mL at 8 dpi. The minimum mean e ect of strain B252 (49.9%) was 6 1 collected at the lowest concentration (1  10 conidia mL ). Similarly, the e ect of V. lecanii strains, L2 and L5, on M. japonica presented that corrected mortality of bean aphid improved with the exposure time and concentration of fungal strains (Figure 1). Agriculture 2020, 10, 114 3 of 10 Table 1. Analysis of variance (ANOVA) for the mortality of M. japonica by three di erent concentrations from four entomopathogenic fungi strains at di erent times against bean aphids. Source of Variation DF Sums of Squares Mean Squares F Ratio Prob Treatment 4 39,204.6 9801.15 ****** 0.000 Concentration 2 3151.77 1575.88 296.72 0.000 Time 3 57,007.1 19002.4 ****** 0.000 Treatment  Concentration 8 849.592 106.199 20.00 0.000 Treatment  Time 12 12,258.7 1021.56 192.34 0.000 Concentration  Time 6 625.468 104.245 19.63 0.000 Treatment  Concentration  Time 24 454.597 18.9415 3.57 0.000 RESIDUAL 120 637.332 5.31110 Total 179 114,189. 637.928 CV% 7.0 CV%: Coecient of variation. Agriculture 2020, 10, x FOR PEER REVIEW 3 of 10 Figure 1. The mortality of Megoura japonica by di erent concentrations of Verticillium lecanii (L2 and L5) Figure 1. The mortality of Megoura japonica by different concentrations of Verticillium lecanii (L2 and and Beauveria bassiana (B76 and B252) spores at di erent time intervals. Columns show the mortality of L5) and Beauveria bassiana (B76 and B252) spores at different time intervals. Columns show the aphids with di erent fungal strains  SE (n = 50). The di erent letters express significant di erences mortality of aphids with different fungal strains ± SE (n = 50). The different letters express significant between di erent treatments (three-way factorial analysis of variance (ANOVA); least significant differences between different treatments (three‐way factorial analysis of variance (ANOVA); least di erence (LSD) test at = 0.05). DAT: Days after treatment. significant difference (LSD) test at α = 0.05). DAT: Days after treatment. 2.2. Fungal Strain Combinations Against Bean Aphid Table 1. Analysis of variance (ANOVA) for the mortality of M. japonica by three different In the combination bioassays test, the results showed a high significant e ect of all treatments (F = concentrations from four entomopathogenic fungi strains at different times against bean aphids. 618.56; p < 0.01), the di erent time intervals (F = 775.30, p < 0.01), and the interaction between di erent Source of Variation DF Sums of Squares Mean Squares F Ratio Prob times and di erent treatments on fungal strains (F = 40.60, p < 0.01, Table 2). The highest inhibition Treatment 4 39204.6 9801.15 ****** 0.000 e ect of the fungal strain (90.5%) was observed for L2 + B76, while the e ect of the combination of Concentration 2 3151.77 1575.88 296.72 0.000 B76 + L5 was 81.1%. Binary combination of L2 + B252 caused 71.6% corrected bean aphid mortality Time 3 57007.1 19002.4 ****** 0.000 (Figure 2). Nevertheless, for the combination of four di erent fungal strains (L2 + L5 + B76 + B252), Treatment × Concentration 8 849.592 106.199 20.00 0.000 the bean aphid mortality rate was recorded as 69.4%. This phenomenon was most probably due to Treatment × Time 12 12258.7 1021.56 192.34 0.000 the reduced number of spores of the high virulence strain (B76 and L2) in the combination (B76 + Concentration × Time 6 625.468 104.245 19.63 0.000 L2 + B252 + L4). So, its e ect was reduced to be significantly lower against M. japonica than other Treatment × Concentration × Time 24 454.597 18.9415 3.57 0.000 combinations (Figure 2). RESIDUAL 120 637.332 5.31110 Total 179 114189. 637.928 CV% 7.0 CV%: Coefficient of variation. 2.2. Fungal Strain Combinations Against Bean Aphid In the combination bioassays test, the results showed a high significant effect of all treatments (F = 618.56; p < 0.01), the different time intervals (F = 775.30, p < 0.01), and the interaction between different times and different treatments on fungal strains (F = 40.60, p < 0.01, Table 3). The highest inhibition effect of the fungal strain (90.5%) was observed for L2 + B76, while the effect of the combination of B76 + L5 was 81.1%. Binary combination of L2 + B252 caused 71.6% corrected bean aphid mortality (Figure 2). Nevertheless, for the combination of four different fungal strains (L2 + L5 + B76 + B252), the bean aphid mortality rate was recorded as 69.4%. This phenomenon was most probably due to the reduced number of spores of the high virulence strain (B76 and L2) in the combination (B76 + L2 + B252 + L4). So, its effect was reduced to be significantly lower against M. japonica than other combinations (Figure 2). Agriculture 2020, 10, 114 4 of 10 Table 2. Analysis of variance (ANOVA) for the mortality of M. japonica by the binary combinations from four fungal strains of V. lecanii and B. bassiana at di erent time intervals. Source of Variation DF Sums of Squares Mean Squares F Ratio Prob Treatment 4 20,964.9 5241.22 618.56 0.000 Time 3 19,708.0 6569.34 775.30 0.000 Treatment  Time 12 4127.90 343.992 40.60 0.000 RESIDUAL 40 338.932 8.47329 Total 59 45,139.7 765.080 CV% 7.2 CV%: Coecient of variation. Agriculture 2020, 10, x FOR PEER REVIEW 4 of 10 Figure 2. The mortality of Megoura japonica by the binary combinations from strains of Verticillium Figure 2. The mortality of Megoura japonica by the binary combinations from strains of Verticillium lecanii and Beauveria bassiana (L2 + B76, B76 + L5, L2 + B252, L2 + B76 + B252 + L5, and control) recorded lecanii and Beauveria bassiana (L2 + B76, B76 + L5, L2 + B252, L2 + B76 + B252 + L5, and control) recorded at di erent time intervals. Columns show the bean aphid mortality with di erent fungal strains  SE at different time intervals. Columns show the bean aphid mortality with different fungal strains ± SE (n = 50). The di erent letters express the significant di erence between di erent treatments (two-way (n = 50). The different letters express the significant difference between different treatments (two‐way factorial analysis of variance (ANOVA); least significant di erence (LSD) test at = 0.05). DAT: Days factorial analysis of variance (ANOVA); least significant difference (LSD) test at α = 0.05). DAT: Days after treatment. after treatment. 2.3. Comparison of the Mortality of Bean Aphid by Fungi Filtrate and Conidia Table 2. Analysis of variance (ANOVA) for the mortality of M. japonica by the binary combinations The filtrate bioassay results revealed that the overall mean e ect of di erent fungi filtrate against from four fungal strains of V. lecanii and B. bassiana at different time intervals. bean aphid was higher in comparison to their conidial treatments. Statistical processing results showed Source of Variation DF Sums of Squares Mean Squares F Ratio Prob that there was a significant e ect of fungal conidia and filtrates on corrected bean aphid mortality (F = Treatment 4 20964.9 5241.22 618.56 0.000 117.71, p < 0.01, Table 3). The highest e ect of the filtrate solution was strain B76, with 91% recorded on Time 3 19708.0 6569.34 775.30 0.000 the eighth day of treatment, while the e ect of strain B76 conidia was recorded as 84% on the eighth 12 4127.90 343.992 40.60 0.000 Treatment × Time day of treatment (p < 0.01). Other fungal strains had similar results. However, the lowest e ect of RESIDUAL 40 338.932 8.47329 filtrates and conidia was in fungal strain L5, recorded as 63.8% and 55.1%, respectively (Figure 3). Total 59 45139.7 765.080 Table 3. Analysis of variance (ANOVA) for the comparison of the mortality of M. japonica by filtrate CV% 7.2 and the conidia from di erent fungal strains (V. lecanii and B. bassiana). CV%: Coefficient of variation. Source of Variation DF Sums of Squares Mean Squares F Ratio Prob 2.3. Comparison of the Mortality of Bean Aphid by Fungi Filtrate and Conidia Treatment 8 15,365.3 1920.66 117.71 0.000 The filtrate RESIDUAL bioassay results reve 18 aled tha 293.692 t the overall mean ef 16.3162 fect of different fungi filtrate against Total 26 15,659.0 602.268 bean aphid was higher in comparison to their conidial treatments. Statistical processing results CV% 6.0 showed that there was a significant effect of fungal conidia and filtrates on corrected bean aphid CV%: Coecient of variation. mortality (F = 117.71, p < 0.01, Table 4). The highest effect of the filtrate solution was strain B76, with 91% recorded on the eighth day of treatment, while the effect of strain B76 conidia was recorded as 84% on the eighth day of treatment (p < 0.01). Other fungal strains had similar results. However, the lowest effect of filtrates and conidia was in fungal strain L5, recorded as 63.8% and 55.1%, respectively (Figure 3). Agriculture 2020, 10, x FOR PEER REVIEW 5 of 10 Agriculture 2020, 10, 114 5 of 10 Figure 3. Comparison of the mortality of M. japonica by filtrate and the conidia from di erent fungal strains (V. lecanii and B. bassiana). Columns show the mortality of aphids by di erent fungal strains  SE Figure 3. Comparison of the mortality of M. japonica by filtrate and the conidia from different fungal (n = 50). The di erent letters express the significant di erence between di erent treatments (one-way strains (V. lecanii and B. bassiana). Columns show the mortality of aphids by different fungal strains ± factorial analysis of variance (ANOVA); least significant di erence (LSD) test at = 0.05). SE (n = 50). The different letters express the significant difference between different treatments (one‐ way factorial analysis of variance (ANOVA); least significant difference (LSD) test at α = 0.05). 3. Discussion Developing biological control methods, based on entomopathogenic fungi with resistance to Table 3. Analysis of variance (ANOVA) for the comparison of the mortality of M. japonica by filtrate pathogens and the co and niinsect dia fropests, m diffe is reone nt fung of the al stra cor ins e ar (V. eas lecani of curr i and ent B. biological bassiana). control research. Many virulent fungal strains were found to be e ective against di erent insect pests [31,32]. This study determined Source of Variation DF Sums of Squares Mean Squares F Ratio Prob the e ect of di erent entomopathogenic fungi against bean aphid. The results of this study presented Treatment 8 15365.3 1920.66 117.71 0.000 that four strains, V. lecanii (L2 and L5) and B. bassiana (B76 and B252), could be e ective biocontrol RESIDUAL 18 293.692 16.3162 agents against bean aphids. This study result is also similar to previous research showing the e ect of Total 26 15659.0 602.268 di erent strains of V. lecanii and B. bassiana against aphids [26,33]. CV% 6.0 In all experiments, the e ect of fungal strains appeared to be dose and time-dependent and CV%: Coefficient of variation. increased by increasing time after application and the conidia concentrations of fungus, [34] which was also demonstrated in our conidial treatment study. They use di erent concentrations of the 3. Discussion entomopathogenic fungus, B. bassiana, against Metopeurum fuscoviride and A. fabae, and reported that 8 1 Developing biological control methods, based on entomopathogenic fungi with resistance to the highest concentration (1  10 spores mL ) exhibited the highest mortality percent on seventh pathogens and insect pests, is one of the core areas of current biological control research. Many day post-treatment. It was also demonstrated that the mortality of aphids increased with time and virulent fungal strains were found to be effective against different insect pests [31,32]. This study conidial concentration exposure [35,36]. Similarly, it was described that the mortality percentage determined the effect of different entomopathogenic fungi against bean aphid. The results of this was a ected by the concentration of conidia, temperature, and exposure time [37]. The mortality study presented that four strains, V. lecanii (L2 and L5) and B. bassiana (B76 and B252), could be percentage was directly proportional to the concentration of conidia using di erent concentrations 6 7 8 1 effective biocontrol agents against bean aphids. This study result is also similar to previous research of B. bassiana (1  10 , 1  10 and 1  10 conidia mL ) Rhopalosiphum padi, Schizaphis graminum, showing the effect of different strains of V. lecanii and B. bassiana against aphids [26,33]. Lipaphis erysimi, and Brevicoryne brassicae. All concentrations were e ective for the control of aphids, 8 1 In all experiments, the effect of fungal strains appeared to be dose and time‐dependent and but the highest concentration (1  10 conidia mL ) caused the highest percentage mortality [38]. increased by increasing time after application and the conidia concentrations of fungus, [34] which The research was conducted for the determination of the virulence of V. lecanii and its result showed 8 1 was also demonstrated in our conidial treatment study. They use different concentrations of the that concentration of 1  10 conidia mL had the best e ect, with an 86% mortality rate of nymphal 7 8 entomopathogenic fungus, B. bassiana, against Metopeurum fuscoviride and A. fabae, and reported that observed after five days of application [39]. It was also reported that at concentrations of 10 and 10 8 −1 the highest concentration (1 × 10 spores mL ) exhibited the highest mortality percent on seventh day conidia mL of V. lecanii, the maximum mortality rate of M. persicae was recorded as 100% after 12 post‐treatment. It was also demonstrated that the mortality of aphids increased with time and days of application [40]. Similarly, the e ect of B. bassiana on M. persicae was also determined under the 7 1 conidial concentration exposure [35,36]. Similarly, it was described that the mortality percentage was controlled condition with the concentration of 1  10 conidia mL and showed that three strains of affected by the concentration of conidia, temperature, and exposure time [37]. The mortality B. bassiana (BAU019, BAU004, and BAU018) showed high e ect on aphids, with a mortality of over percentage was directly proportional to the concentration of conidia using different concentrations 75% [34,41–44]. According to filtrate treatment, our results are similar to results which revealed that 6 7 8 −1 of B. bassiana (1 x 10 , 1 x 10 and 1 x 10 conidia mL ) Rhopalosiphum padi, Schizaphis graminum, Lipaphis degradation of the insect body was observed at higher infiltrate treated aphids, and the reduction of erysimi, and Brevicoryne brassicae. All concentrations were effective for the control of aphids, but the aphid population was higher in higher dose filtrate treatment [27]. The maximum ecacy was due 8 −1 highest concentration (1 x 10 conidia mL ) caused the highest percentage mortality [38]. The research to the production of metabolites in the culture filtrate that helped to degrade the cuticle and deform was conducted for the determination of the virulence of V. lecanii and its result showed that the hemocoel. As compared with the filtrate, conidia need more time to germinate and release the 8 −1 concentration of 1 x 10 conidia mL had the best effect, with an 86% mortality rate of nymphal required enzymes for the degradation of the insect body. It was demonstrated that the use of filtrate 7 8 observed after five days of application [39]. It was also reported that at concentrations of 10 and 10 Agriculture 2020, 10, 114 6 of 10 application for the control of insects was the best method, and also e ective for insects which had a short life cycle because these insects have more chances to shed the conidia from their body by molting, since germination of conidia needs a specific time [45,46]. According to their findings, our results were similar. Filtrate had more ability to control the insect population, because they contained toxic enzymes for the degradation of the insect body. Through filtrate optimization, the production of enzymes could be enhanced easily with fungal genetic manipulation. Filtrate production, storage, and transportation are more suitable than conidia, and it meets the commercialization requirements. In this study, the e ect of di erent fungal strains was found to be time dependent in cases of conidia, filtrate suspension, and combinations. The maximal e ect was recorded after eight days, while the minimal e ect was recorded after two days. These results are similar to research that showed an increase in the e ect with the increase in time and concentration [39]. Regarding the pathogenicity of di erent fungal combinations, our results showed that the highest synergistic e ects were a combination of fungal strains (B76 + L5, and L2 + B76). However, there was no synergistic e ect for the combination of fungal strains L2 + B252 and L2 + B76 + B252 + L5. These fungal strains may show synergies if applied in a sequential combination, as demonstrated in the case of nematodes and pathogenic fungi [47]. Therefore, the use of the entomopathogenic fungi is considered safe and environmentally friendly compared to chemical pesticides [48], so they are recommended against harmful pests, such as aphids. 4. Materials and Methods 4.1. Insect Culture Bean aphids were collected from the Institute of Plant Protection, Beijing, China. Then, they were reared on Chinese cabbage plants (Brassica rapa) placed in cages in the growth chamber at 50–60% relative humidity and 25  2 C, with a 16:8 h light:dark photoperiod. The first instar aphid was used for all bioassays in this study. 4.2. Fungal Isolates All fungal isolates (Table 4) were collected from fields in China and cultured on potato dextrose agar (PDA) medium (20.0 g agar, 200.0 g potatoes, 20.0 g dextrose, and 1 L distilled water) on Petri-dishes for 20 days in the dark at 25  2 C. Table 4. Name of fungal strains, hosts and geographical of the entomopathogenic fungi. Name Symbols Geographical Origin Verticilliun lecanii 2 L2 Institute of Plant Protection, Beijing, China Verticilliun lecanii 5 L5 Institute of Plant Protection, Beijing, China Beauveria bassiana 76 B76 Institute of Plant Protection, Beijing, China Beauveria bassiana 252 B252 Institute of Plant Protection, Beijing, China 4.3. Conidial Suspension The conidia were collected from PDA dishes in 0.02% Tween solution after 20 days culture and were filtered using sterile cheesecloth. The spore concentrations of all fungal strains were counted under the microscope using a hemocytometer. The conidia viability was checked before using for the design of the bioassays experiment [49]. 4.4. Fungal Filtrate The primary culture of the four strains (B76, B252, L2, and L5) was prepared by mixing 100 mL of Adamek liquid medium (ALM) with 5 mL of conidial suspension and shaken for 3 days at 150 rpm. The secondary culture (1.0%) was prepared by mixing 250 mL of ALM with 2.5 mL of the primary culture medium and shaken for 6 days at 150 rpm, 25 C. The mycelium was removed by centrifugation Agriculture 2020, 10, 114 7 of 10 for 15 min at 12,000 rpm, 4 C and then the supernatant was filtered through the 0.45 m pore-size filter (Millipore Corp) to get the filtrate. 4.5. Pathogenicity Bioassays The e ect of the entomopathogenic fungal strains (B76, B252, L2, and L5) against bean aphids was measured by conducting their conidial and filtrate bioassays, and evaluating the binary combinations of di erent fungal strains. All bioassays were measured using the leaf-dip method [50] with slight modifications. All treatments were performed on 90 mm Petri dishes containing a thin layer of 1.0% agar, and a 60 mm detached leaf disk of Chinese cabbage was placed on the Petri dish. The control leaves were dipped only in 0.02% Tween. Fifty newly molted bean aphids up to 12 h old (first instar) were released on the treated and control leaf on a Petri dish, and then they were stored at 50–60% relative humidity and 25  2 C with a 16:8 h light:dark photoperiod. All the treatments were replicated 3 times (each treatment has 5 Petri dishes for each time). For the bioassay treatment 8 1 of binary combination, the uppermost concentration 1  10 conidia mL of each fungal strain was mixed with the 1 mL conidia of each other fungal strain for all combinations, and then a 2 mL mixed combination sample for each treatment was collected. The combinations, L2 + B76, B76 + L5, L2 + B525, and L2 + B76 + B252 + L5 were designed. For all treatments, the e ect was recorded on 2, 4, 6 and 8 days. All dead bean aphids from each experiment were maintained at 25  2 C and 90% relative humidity in dark to confirm mortality by the pathogens. The e ect of all fungal strains was determined using Abbott’s formula: E ect (%) = (X Y)/X)  100 (X: the percent living in the control; Y: the percent living in the treatment) [51]. 4.6. Data Analysis The data was analyzed using Statistix 8.1 (Analytical Software, Tallahassee, FL, USA). Comparisons of the treatment means were performed, using variances (ANOVA) to determine the significance of individual di erences of the least significant di erence (LSD) test at = 0.05 level. 5. Conclusions In brief, this study shows the e ect of four di erent fungal strains of V. lecanii (L2 and L5) and B. bassiana (B76 and B252) against M. japonica. Through a series of bioassays, the results demonstrated that di erent fungal strains of V. lecanii (L2 and L5) and B. bassiana (B76 and B252) have di erent virulence potential. The di erent application materials and their dosages also a ect pathogenicity on bean aphid. Filtrate application is the most suitable material for the control of M. japonica. 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Journal

Agriculture – Multidisciplinary Digital Publishing Institute

Published: Apr 4, 2020

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