In Vitro Pathogenicity of Some Entomopathogenic Fungal Strains against Green Peach Aphid Myzus persicae ( Homoptera : Aphididae )

Green peach aphid, Myzus persicae (Hemiptera: Aphididae), is an economically important pest of crops within more than 40 plant families all over the world. This study encompasses in-vitro pathogenicity of two strains of Beauveria bassiana (BB-72 and BB-252) and one strain of Lecanicillium lecanii (V-4) against green peach aphid (M. persicae). Using a leaf-dip method, three different bioassays were conducted comprised of filtrates and conidial concentrations of BB-72, BB-252 and V-4 fungal strains and their binary combinations. Infiltrate bioassays, 2 mL fungal filtrate of each strain was used. In conidial bioassays, three different concentrations (i.e., 1 × 106, 1 × 107 and 1 × 108 conidia mL−1) of each fungal strain were used, while in binary combination bioassays, LC50 and LC33 of these fungal strains were evaluated. According to the results, maximum pathogenicity against test aphids was observed at 10th day of all treatments. Both strains of B. bassiana (BB-72 and BB-252) exhibited higher mortality of aphids (up to 95 and 91%, respectively) than L. lecanii (V-4) strain (87%) in all three types of bioassays. Moreover, binary combinations of BB-72 and BB-252 strains showed significantly higher aphid mortality (94%) than other combinations. Reduced mortality in case of fungal combinations of V-4 strain might be due to the antagonistic effect of L. lecanii strain along with both strains of B. bassiana. Nevertheless, evaluation of combined pathogenicity of the fungal strains constitutes the novelty of this in-vitro study which revealed that both strains of B. bassiana (i.e., BB-72 and BB-252) are mutually compatible and can be utilized together as new biocontrol tools against destructive insect pests such as M. persicae.


Introduction
In temperate regions, aphids are the most damaging and economically important insect pests of many crops.They suck cell sap and nutrients from plants and also spread many viral diseases in various field and horticultural crops [1].Myzus persicae Sulzer, commonly known as the green peach aphid, is considered as one of the most important pest species.This aphid has been found attacking more than 800 plant species worldwide.Moreover, its incidence is gradually increasing on different fruit and vegetable crops [2].Primarily, the control of M. persicae relies on its management through hazardous synthetic insecticides.However, indiscriminate use of pesticides has resulted in many conspicuous problems including pesticide resistance, the resurgence of secondary pests, disruption of beneficial fauna and other environmental and human health issues [3].For instance, M. persicae populations have been reported to acquire resistance against various carbamate, organophosphate and pyrethroid pesticides [2].
To overcome these problems associated with the extensive use of chemical insecticides, alternative approaches such as the use of biological control agents have been widely studied in many countries.Many microbial bio-pesticides based on entomopathogenic organisms such as viruses, nematodes, fungi and bacteria have been playing important roles in the field of plant protection and are being used against a wide range of insect pests [4][5][6][7][8].Entomopathogenic fungi such as Beauveria bassiana, Lecanicillium lecanii, Metarhizium anisopliae, Isaria fumosorosea are appearing to be effective, environment-friendly and target-specific biocontrol tools against many sucking insect pest species [5,[9][10][11].Upon germination of spores attached to the cuticle of a target insect, fungal hyphae penetrate the insect body and cause host death within a few days [12][13][14].Moreover, entomopathogenic fungi have no or low mammalian toxicity or residual activity and are target specific and less vulnerable to resistance development [15,16].These fungi have been focused for rigorous research in past decades for being important bio-control agents all over the world and can exist at enzootic or epizootic levels in the populations of their hosts.
B. bassiana Balsamo and L. lecanii Zimmerman are among the most studied and virulent entomopathogenic fungi belonging to order Hypocreals (Fungi: Ascomycota).Both these fungal species are ubiquitous and are distributed in a wide range of habitats including aquatic, urban, forest, agricultural, pasture and desert habitats.They have a wide range of target host species of insect pests [17,18].Strains of both these fungi can be easily isolated even from phylloplanes of vegetation as well as from soil and infected insect bodies [19,20].Keeping in view the ecological and environmental issues of synthetic chemical insecticides and current need of developing new alternate biological control tools, the present study aimed to assess the virulence and pathogenicity of isolates of B. bassiana and L. lecanii against M. persicae under laboratory conditions.Moreover, as there is a lack of information about any possible combined effect of different entomopathogenic fungal strains; it was also aimed to determine if these fungal strains can act synergistically against M. persicae.We hypothesized that the combination of fungal strains would have greater efficiency than a single strain.

Insect Culture
Green peach aphids (M.persicae) were collected from the seedlings of Chinese cabbage (Brassica rapa subsp.pekinensis) grown in the greenhouse at Chinese Academy of Agricultural Sciences (CAAS) Beijing, China.The aphid population was reared on the seedlings of same cabbage subspecies grown in pots placed in cages at 55-60% relative humidity and 27 ± 2 • C temperature with 16:8 h light:dark (L:D) photoperiod.Plants were replaced every week with new ones during the whole study period.

Fungal Isolates and Conidial Suspensions Preparation
Three fungal isolates (Table 1), two of B. bassiana (BB-72 and BB-252) and one of L. lecanii (V-4), were maintained on refined potato dextrose agar (PDA) medium (200 gL −1 potatoes, 20 gL −1 dextrose and 20 gL −1 agar) in polystyrene petri-plates (90 mm diameter) for 3 weeks in the dark at 25 ± 2 • C. Sporulating mycelia of these fungal strains were carefully selected and identified according to their standard morphological descriptions [21].For each fungal strain, stock suspension of conidia was prepared in 250 mL reagent bottle with the addition of mass sporulating culture in 100 mL of distilled sterile water having 0.01% (w/v) Tween 80 ® (Sigma-Aldrich, Saint Louis, MO, USA).The mixture was shaken vigorously by hand for two min and the filtrate was blended to isolate the hyphal debris through sterilized Miracloth (EMD Milipore Corp., Billerica, MA, USA).Improved Neubauer haemocytometer (Brand GmbH, Wertheim, Germany) was used for the determination of the conidial concentration.
Fungal filtrates were prepared by adding 2 mL of conidial suspension of each fungal strain in 50 mL of Adamek liquid medium (40 g yeast extract (Difco, Detroit, MI, USA), 40 g dextrose and 30 g corn steep liquor (Sigma-Aldrich, Saint Louis, MO, USA) for primary cultures of all three fungal strains.Culture incubation was carried out for three days at 140 rpm and secondary cultures were prepared by the addition of 2 mL from primary culture of each strain into 200 mL of the Adamek liquid medium via incubation at 25 • C and 140 rpm for six days.Centrifugation of the samples was then carried out at 4 • C and 12,000 rpm for 20 min.Later, filtration of the subsequent supernatant was taken through 0.45 µm pore size filter (Millipore Corp., Billerica, MA, USA) in order to get the filtrate.The pH of the filtrate was maintained at 6.

Pathogenicity Bioassays
The susceptibility of test aphids to these entomopathogenic fungal strains (BB-72, BB-252 and V-4) was determined by conducting their filtrate and conidial bioassays and by evaluating the binary combinations of their LC 50 and LC 33 (lethal concentrations causing 50 and 33% mortality of the exposed aphid individuals, respectively).The leaf-dip method was used for all bioassays as described previously [23] after slight modifications.For each treatment, leaf discs of 50 mm diameter were obtained from healthy cabbage plants and were dipped for 10 s in 5 mL of conidial and fungal filtrate suspensions.Then, leaf discs were placed on sterilized filter paper for 15-20 min in order to remove the excessive suspension.Leaf discs in control treatments were dipped in 0.05% Tween 80 only.Ten newly molted (up to 12 h old) non-alate adults of laboratory-maintained M. persicae were released on treated and untreated leaf discs with the sterilized camel hair brush and these petri-plates were then incubated at 25 ± 2 • C and at a photoperiod of 16:8 h of Light: Dark.Each treatment was replicated 10 times.In binary combination bioassays, LC 50 values of each fungal strain, as obtained from their respective conidial bioassays, were used for the combinations of BB-72 × BB-252, BB-72 × V-4 and BB-252 × V-4 and LC 33 for BB-72 × BB-252 × V-4 combination.Aphid mortality was recorded on 3rd, 5th, 7th and 10th day post-treatment.Dead aphids were collected from petri-plates and were kept in dark at 90% relative humidity to promote fungal development and sporulation in order to confirm that aphids died due to infection by each particular fungal strain.

Data Analysis
Cumulative aphid mortality from dose-response tests was compared with controlled mortality (natural) test.LC 50 and LC 33 values were calculated for each fungal strain by Probit analysis using Polo Plus (Version 2.0, LeOra Software, Berkeley, CA, USA) [24] in association with 95% confidence interval.Furthermore, aphid mortality was analyzed using two-way factorial analysis of the variance (Statistix 8.1v, Tallahassee, FL, USA).Comparisons of the treatment means were performed using Fischer's least significant difference (LSD) test at α = 0.05.

Pathogenicity of Filtrates of Different Strains of B. bassiana and L. lecanii against M. persicae
Results of filtrate bioassay revealed that overall mean mortality of aphids by the isolates of tested fungal strains was higher in comparison to their conidial treatments.There was a significant effect of fungal filtrates, observation time intervals and their interaction on M. persicae mortality (Table 2).Maximum mortality (95%) was recorded on 10th day of treatment of (BB-72).On the other hand, minimum mortality (87%) was recorded on 10th day by filtrate application of strain V-4.Filtrate of fungal strain BB-252 exhibited mean aphid mortality of 91% (Figure 1).Results of the conidial bioassay showed that all three strains tested exhibited significant mortality of aphid individuals (F = 13.21,p < 0.001; Table 3).Furthermore, factorial analysis of variance revealed that there was a significant effect of different concentrations, different time intervals and of their interaction on mean aphid mortality (F = 600.11,p < 0.001, F = 1047.24,p < 0.001 and F = 122.62,p < 0.001, respectively; Table 3).Strain BB-72 of B. bassiana caused considerable aphid mortality at all concentrations.However, maximum mortality (93%) was recorded at 10th day post-treatment at a concentration of 1 × 10 8 conidia mL −1 .In contrast, minimum mortality (74%) was recorded for the lowest concentration of BB-72 (i.e., 1 × 10 6 conidia mL −1 ) (Figure 2).Toxicity of B. bassiana strain BB-252 against M. persicae exhibited a similar trend as in the case of BB-72.Highest mean mortality (86%) of aphids was recorded after the 10th day of the treatment by the concentration of 1 × 10 8 conidia mL −1 (Figure 2).On the other hand, minimum mortality (70%) was recorded at a lowest concentration of 1 × 10 6 conidia mL −1 (Figure 2).Similarly, the entomopathogenicity of strain V-4 of L. lecanii against M. persicae aphids showed that aphid mortality increased along with the fungal concentration and the time of exposure (Figure 2).Minimum mortality (2.5%) was observed at 3rd day post-treatment for all three concentrations, while maximum aphid mortality (79%) was exhibited by the highest conidial concentration at 10th day post-treatment (Figure 2).Mean aphid mortality in control petri-plates remained 6%.Moreover, according to Probit analysis median lethal concentration (LC 50 ) values for strains BB-72, BB-252 and V-4 were recorded as 3.09 × 10 4 , 1.29 × 10 4 and 4.10 × 10 3 conidia mL −1 (Table 4).2. LC50: Lethal concentration (%) of tested entomopathogenic fungal strains that killed 50% of the exposed aphid individuals; FL: 95% Fiducial (confidence) limits, DF: Degree of freedom.X 2 : Chi square   3.  3. LC 50 : Lethal concentration (%) of tested entomopathogenic fungal strains that killed 50% of the exposed aphid individuals; FL: 95% Fiducial (confidence) limits, DF: Degree of freedom.X 2 : Chi square

Entomopathogenicity of Binary Combinations of Entomopathogenic Fungal Strains against M. persicae
In case of pathogenicity bioassays performed with the binary combinations of LC 33 and LC 50 values of three fungal strains, there was a significant effect of all treatments and observation time intervals and their interaction on aphid mortality (Table 5).Highest and significant aphid mortality (94%) was observed for BB-72 (LC 50 ) + BB-252 (LC 50 ), whereas 84% mortality was recorded for the combination of BB-72 (LC 50 ) and V-4 (LC 50 ).Binary combination of BB-252 (LC 50 ) and V-4 (LC 50 ) caused 80% aphid mortality (Figure 3).However, in the final tertiary combination of LC 33 values of all three tested fungal strains (BB-72, BB-252 and V-4), mean aphid mortality was recorded as 78% (Figure 3).Table 5. Factorial analysis of variance of mortality of M. persicae bioassayed with binary combinations of three different strains of entomopathogenic fungi.See Figure 3.   5.  5.

Discussion
Developing entomopathogenic fungi based biological control tools with enhanced efficacy against the target pests is one of the core areas of current biocontrol research.Many entomopathogenic fungal strains have been found effective against various insect pests [25][26][27][28].The present study encompassed in-vitro determination of the effectiveness of different strains of entomopathogenic fungi against green peach aphid persicae.The results revealed that all tested strains of B. bassiana and L. lecanii could be effective biocontrol agents against aphids.These results are corroborated by the findings of previous studies reporting the efficiency of different strains of B. bassiana and L. lecanii against M. persicae and other aphid species [29,30].
The virulence of entomopathogenic fungi is usually concentration dependent and target insects' mortality by these fungi also depends on the temperature, exposure time and concentration (conidial suspension) [33,34].The response of similar species of green peach aphid may differ to unlike strains of fungus.Even biotypes of aphid (same species) may have different susceptibility to a fungal suspension [35].Efficacy of B. bassiana against M. persicae aphids was evaluated under controlled conditions with the concentration of 1 × 10 7 conidia mL −1 and found that three isolates of B. bassiana (BAU004, BAU018 and BAU019) showed high virulence against test aphids with a mortality of more than 75% [36].Similarly, the results of our study are in accordance with those found by [29, [37][38][39].However, the findings of some studies are different from ours.This might be because of varied virulence of fungal strains and due to the differential response of target host individuals to that of particular entomopathogenic fungal isolates.
Regarding the pathogenicity of different combinations of these fungal strains, no synergistic effect was observed for any combinations.L. lecanii strain (V-4) caused significant mortality alone but showed an antagonistic action for its combinations with both strains of B. bassiana (BB-72 and BB-252).The later strains showed compatibility and exhibited significantly higher mortality of M. persicae individuals than other combinations.However, these fungal strains might show a synergistic or additive action if applied in a sequential combination as demonstrated in the case of entomopathogenic nematodes and fungi [40].
Nevertheless, aphids have piercing-sucking type mouthparts to suck plant sap directly from the conductive (phloem) tissues.This behavior may prevent the ingestion of microbial agents such as bacteria and viruses because they need to be swallowed for infecting their hosts.On the other side, entomopathogenic fungi as B. bassiana and L. lecanii cause direct infection through the cuticular surface of target insect pests [41].Furthermore, entomopathogenic fungi are considered to be safe and eco-friendly in comparison to the chemical insecticides [42], hence, are recommended against harmful sucking insect pests, such as aphids.

Conclusions
In conclusion, this in-vitro study demonstrates the effectiveness of two different strains of B. bassiana (BB-72 and -252) and a strain of L. lecanii (V-4) against M. persicae, a cosmopolitan pest of winter vegetables and fruits worldwide.Both strains of B. bassiana exhibited higher mortality (91-95%)

Table 1 .
Isolates, strain codes, original hosts and geographical origins of the three entomopathogenic fungal strains used in this study.

Table 2 .
Factorial Analysis of variance of mortality of M. persicae bioassayed with the filtrates of three different strains of entomopathogenic fungi.See Figure1.

Table 3 .
Factorial analysis of variance comparison of mortality of M. persicae bioassayed with three different strains of entomopathogenic fungi.See Figure2.