Legume Research

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Legume Research, volume 45 issue 12 (december 2022) : 1572-1579

Efficacy of Three Entomopathogenic Fungi Beauveria bassiana, Metarhizium anisopliae and Lecanicillium lecanii Isolates against Black Bean Aphid, Aphis fabae (Scop.) (Hemiptera: Aphididae) on Faba bean (Vicia faba L.)

Tawfiq Qubbaj1,*, Rana Samara2
1Department of Plant Production and Protection, Faculty of Agriculture and Veterinary Medicine, An-Najah National University, Nablus P.O. Box 7, Palestine.
2Department of Horticulture and Agricultural Extension, Faculty of Agricultural Sciences and Technology, Palestine Technical University-Kadoorie (PTUK), Tulkarm, Palestine.
  • Submitted24-06-2022|

  • Accepted01-09-2022|

  • First Online 14-09-2022|

  • doi 10.18805/LRF-706

Cite article:- Qubbaj Tawfiq, Samara Rana (2022). Efficacy of Three Entomopathogenic Fungi Beauveria bassiana, Metarhizium anisopliae and Lecanicillium lecanii Isolates against Black Bean Aphid, Aphis fabae (Scop.) (Hemiptera: Aphididae) on Faba bean (Vicia faba L.) . Legume Research. 45(12): 1572-1579. doi: 10.18805/LRF-706.
Background: A laboratory bioassay study was conducted to evaluate the in vitro pathogenicity of different isolates of B. bassiana, M. anisopliae and L. lecanii, against the adults of black bean aphid. 

Methods: The PCR-based method was used to identify the different isolates molecularly using sequence information from the ITS region. The total genomic DNA of the 19 fungal isolates was recovered from aphid cadavers using CTAB. The amplified DNA using QRT-PCR showed no significant differences in the ANOVA that tested mean cycle threshold (CT) values from the control. Post-molecular identification of the isolated entomopathogen was approved. The single discriminative concentration bioassay was carried out to determine LT50 values for each of twelve isolates to determine the most virulent for further studies. 

Result: LT50 values for B. bassiana, M. anisopliae and V. lecanii isolates varied from 110-113, 71-75 and 64-77 h, respectively. B. bassiana isolate BBK2, M. anisopliae isolate MAA2 and V. lecanii isolates VLJ2 were selected for further experiments based on their discriminating concentration values. LC50 of BBA post-exposure to isolates of V. lecanii, M. anisopliae and B. bassiana was 46, 269 and 251 ppm, respectively. A significant difference in cumulative mortality was recorded between the three EPF. M. anisopliae showed a higher significant cumulative mortality during the first and second days post-application. Then V. lecanii recorded higher significant cumulative mortality from the third until the seventh-day post-application. V. lecanii showed higher virulence among the other entomopathogenic isolates.
Faba bean (Vicia faba L.) is an important winter legume crop that originated in West Asia and is considered as a cheap and primary protein source for the population in the Mediterranean region (Rahate et al., 2021). They play an essential role in fixing atmospheric nitrogen through the symbiotic relationship with Rhizobium bacteria and improving the soil’s nitrogen status (Köpke et al., 2010). Aphids are a large group of phloem-feeding insects that cause severe damage to major crops worldwide (Maketon et al., 2013). Black bean aphid (BBA), Aphis fabae (Scop.) (Hemiptera: Aphididae) is a significant insect pest of V. faba and Beta vulgaris L. crops (Almogdad and Semaškienë, 2021). BBA adults and nymphs feed directly on the plant sap and transmit many plant viruses such as bean yellow mosaic (BYMV) and pea leaf roll (PLRV) virus and other mosaic viruses (Almogdad and Semaškienë, 2021). Entomopathogenic fungi (EPF) gained a significant role in insect pest management programs (Reddy et al., 2021). More than 700 species belonging to more than 90 genera are classified as entomopathogens. EPF strains, such as Lecanicillium lecanii (Zimm.) Viegas, Beauveria bassiana (Bals.) Vuill., Isaria fumosorosea (Wize) and Metarhizium anisopliae (Metsch.) Sorokin. were used worldwide as a natural enemy for controlling a wide range of insects species, including Helicoverpa armigera, Alphitobius diaperinus, Plutella xylostella, Laniifera cyclades, Prostephanus truncatus, Nilaparvata lugens, Polyphagotarsonemus latus and Bemisia tabaci (Atta et al., 2020; Ojha et al., 2018; Singh and Joshi, 2020).
 
Although many EPF strains are commercially mass-produced and available as bioagents, the native isolates may still be adapted to the dominant environmental conditions. Therefore, the present study aims to evaluate the bio-efficacy and effectiveness of three Palestinian EPF isolates, B. bassiana, M. anisopliae  and V. lecanii, as potential biological control agents against A. fabae in faba bean (V. faba ). Furthermore, we also aimed to investigate fast and reliable molecular identification tools for the three Palestinian EPF isolates. These results could help to establish an effective integrated pest management method that is eco-friendly and cost-effective to mass-produce locally, reducing BBA population below economic thresholds while minimizing the use of synthetic chemical insecticides .
Sources and preparation of fungal isolates
 
The eleven virulent isolates of the EPF B. bassiana, M. anisopliae and V. lecanii were collected and maintained in the PTUK laboratories during 2021-22. EPF isolates were sub-cultured and incubated by a single spore method (Sufyan et al., 2019) for 7-10 days at 28±2°C on PDA media (Fig 1 A-C). A pathogenicity test was carried out after the 6-7 subcultures to maintain the virulance of entomopathogen isolates. The spore concentration was prepared following the method of Samara (2016). Under the microscope, the spore concentration was then determined using a hemocytometer. All the cultures were adjusted to 1 × 1010 spores ml-1, from which the lower concentrations were prepared by serial dilution technique for bioassay studies.
 
Insect culture
 
BBA adults were collected from a broad bean field at Najah National University Farm, An-Nassarya field station. Then reared and maintained on the young broad bean plants in the PTUK glasshouses at 25±5°C, 65±5% RH and 16:8h L:D. The mature aphids were kept on plants for 24 h, resulting in neonate nymphs with an age of 0-24 h that were used throughout the bioassay experiments.
 
Molecular identification of the EPF
 
Genomic DNA of the fungal isolates was extracted from the growing fungal mycelium from insect cadavers using modified cetyl-trimethyl-ammonium bromide (CTAB) protocol (Reineke et al., 1998). A positive source of the EPF used in this experiment was obtained from the culture maintained at PTUK lab. The PCR-based method was amplified using sequence information from the ITS region from all fungal isolates. Preparation of the qPCR reaction mixture of the final volume of 20 μl; each PCR reaction contains 1 μl of DNA isolate, SsoAdvancedTM SYBR® Green Supermix (Bio-Rad Lab. Inc.) and 0.3 μM of the forward specific primers and 0.3 μM of the reverse specific primers. For B. bassiana (Hegedus and Khachatourians, 1996), for M. anisopliae (Destéfano et al., 2004) and V. lecanii were used (Nam et al., 2020). The 96 well plates were loaded using two technical replicates for each isolate: positive, negative and water control (Husien, 2019). Plates were then placed in CFX Connect Real-Time PCR Detection System (CFX Connect®; Bio-Rad, Hercules, CA, USA). Data analysis was carried out using CFX ManagerTM Software (Bio-Rad) with auto-calculated baseline and fixed threshold fluorescence units (RFU) settings. Once the identity was confirmed, isolated samples were used in the next bio-assays.
 
Median lethal time (LT50) assessment
 
V. faba seedlings were grown in plastic pots in the greenhouse at Najah Farm, An-Nassarya. Two-week-old seedlings were used in this bioassay study. A single discriminative concentration bioassay was carried out to determine LT50 values for each of the 12 isolates to select the most virulent for further studies (Samara 2016). For each, an aqueous suspension containing 1 × 1010 spores conidia ml-1  was prepared in Tween 80 (0.05% v/v). Sub-samples were plated onto Sabouraud dextrose agar (SDA) and the germination percentage was counted after 24 h. LT50 was assessed at 24 h intervals for a 7-day mentoring period (Fig 1 D-F). Each date was replicated three times. Ten aphids were transferred to a broad bean seedling using a camel hairbrush. A Total of 210 aphids were used per EPF isolates per bioassay. Every plant infected with aphids was sprayed with an aqueous suspension containing 1 × 1010 spores conidia ml-1 per EPF isolates by a hand atomize. Dead aphids were collected daily and transferred to a moist filter paper in a self-sealed Petri dish; once the dead aphid produced mycelial growth, they were considered for the mortality count (Pegu et al., 2017). The mortality data were then corrected using Abbott’s formula (Abbott 1925).
 
Median lethal concentration (LC50) assessment
 
B. bassiana isolate BBK2, M. anisopliae isolate MAA2 and V. lecanii isolates VLJ2 were selected for the further experiments based on their discriminating dose bioassay carried out in the previous experiment. Seven serial dilutions (50, 100, 200, 350, 500, 750, 1000 ppm) were prepared as described above and each dilution was replicated three times. Ten aphids were transferred to a broad bean seedling using a camel hairbrush. A total of 30 aphids were used per concentration and a total of 210 aphids were used per bioassay. Every three plants infected with aphids were sprayed with one of the seven dilutions of the fungal spore suspensions by a hand atomize. The mortality of aphids was counted every 24 h up to seven days (Fig 1 D-F). Dead aphids were collected daily and transferred to a moist filter paper in a self-sealed Petri dish; once the dead aphid produced mycelial growth, they were considered for the mortality count (Parveen et al., 2021). The mortality data were then corrected using Abbott’s formula (Abbott 1925). Data were analysed by Probit analysis and LC50 and LT50 values and their 95% confidence limits (CL 95%) were calculated from Probit regressions using SAS software.
 
Cumulative mortality
 
Same EPF isolates were used to assess the cumulative mortality of B. bassiana isolate BBK2, M. anisopliae isolate MAA2 and V. lecanii isolates VLJ2 on BBA. Ten aphids were transferred to a self-sealed petri dish with wetted filter paper using a camel hairbrush after spraying an aqueous suspension containing 1 × 1010 spores conidia ml-1 per EPF isolates by a hand atomize. A total of 100 aphids were used per EPF isolates per bioassay (Samara 2016). Dead aphids were assessed as described above. The mortality data were then corrected using Abbott’s formula (Abbott 1925).
 
Statistical analysis
 
All the data were analysed by Probit analysis and LC50 and the LT50 values and their 95% confidence limits (CL 95%) were calculated from Probit regressions using SAS software. Each fungus’s per cent corrected cumulative mortality was analyzed using ANOVA as a general linear model (PROC GLM) procedure. The significance level was determined by applying the Student-Newman-Keuls test at P = 0.05.
Molecular identification of the EPF
 
Amplification of the isolated DNA sample using QRT-PCR results is presented in Fig 4. The statistical analysis of seven EPF isolates with qPCR amplification experiments showed no significant differences in the ANOVA that tested mean cycle threshold (CT) values from the positive (Fig 4). The Real-time amplification of the PCR product generated by specific primers confirmed that the three EPF isolates of B. bassiana, M. anisopliae and V. lecanii isolates under 20 cycles. Using this protocol, amplifying the control samples began around 20 cycles in a real-time experiment, whereas the target DNA (i.e., B. bassiana, M. anisopliae and V. lecanii isolates) was detected later to 21 cycles. Three isolated samples, B. bassiana (isolate BBK2), M. anisopliae (isolate MAA2) and V. lecanii (isolate VLJ2), were used in the next bio-assays.
 
@figure4
 
Identifying entomopathogenic bio-agents is one of the main challenges in using indigenous microbial pesticides (Samada and Tambunan, 2020). Morphological, developmental and physiological characteristics were the sole methods used for identification, but they required taxonomical experiences and took a long time (Hetjens et al., 2021). Recently DNA and RNA-based molecular techniques have been used for taxonomical hierarchy and phonological classification (Goettel and Glare, 2005). The QRT-PCR protocols with specific primers have proven to be very sensitive in detecting and identifying the EPF isolates (Sabbahi et al., 2009). They are also considered a standard approach for accurately and rapidly identifying microorganisms. The modified specific primers for PCR have been used effectively to detect and differentiate plant pathogenic fungi in the current study. The genomic DNA of the fungal isolates was isolated to obtain pure DNA from cadavers using CTAB.
 
LT50 assessment
 
BBA LT50 and LT90 with the corresponding 95% confidence limits after exposure to twelve isolates of B. bassiana. M. anisopliae and V. lecanii are presented in Table 1, along with the value of Pearson Chi-square, degree of freedom (df) and regression equations. Mortality in the control treatments was consistently below 10%. LT50 values for B. bassiana isolates varied from 110.39-113.74 h, while LT50 values for M. anisopliae varied from 71.88-75.27 h and LT50 values for V. lecanii isolates varied from 64.86-77.17 h.
 

Table 1: LT50 and LT90 values (with corresponding 95% confidence limits) for BBA adults after exposure to broad bean leaf sprayed with different concentrations of the EPF B. bassiana. M. anisopliae and V. lecanii.


 
LT50 of BBA post-exposure to VLJ2 isolates of V. lecanii, was the shortest 64.86 h, while the longest for VLK2 isolates 77.17 h. There were no significant differences between the four isolates of V. lecanii based on the non-overlapping of the fiducial limits 95%. LT50 for MAA2 isolate of M. anisopliae, was the shortest 71.88 h, while LT50 for MAJ2 isolate of M. anisopliae, was the longest 75.27 h. No significant differences between the four isolates of M. anisopliae. As for B. bassiana BBK2 isolates LT50 was the shortest 110.39 h, while BBA1 isolates LT50 was the longest 113.74 h. No significant differences between the four isolates of B. bassiana. Similar results were found with LT90 of BBA post-exposure to isolates of V. lecanii, M. anisopliae and B. bassiana. Based on the discriminating dose bioassay, B. bassiana isolate BBK2, M. anisopliae isolate MAA2 and V. lecanii isolates VLJ2 were selected for further experiments.
       
LC50 assessment
 
The probit and logit analysis graph for BBA (Fig 3) and LC50 and LC90 with the corresponding 95% confidence limits after exposure to isolates of B. bassiana. M. anisopliae and V. lecanii are presented in Table 2, along with the value of Pearson Chi-square, degree of freedom (df) and regression equations. Mortality in the control treatments was consistently below 10%. LC50 of BBA post-exposure to isolates of V. lecanii was 46.47 ppm, M. anisopliae 269.53 ppm and B. bassiana was 251.48 ppm. LC90 of BBA post-exposure to isolates of V. lecanii was 215 ppm, M. anisopliae 1311 ppm and B. bassiana was 1274 ppm.
 

Fig 3: The output of the probit graph for the probability of BBA mortality at different doses of the EPF B. bassiana isolate BBK2 (A), M. anisopliae isolate MAA2 (B) and V. lecanii isolate VLJ2 (C).


 

Table 2: LC50 and LC90 values (with corresponding 95% confidence limits) for BBA adults after exposure to broad bean leaf sprayed with different concentrations of the EPF B. bassiana (BBK2), M. anisopliae (MAA2) and V. lecanii (VLJ2).


 
Cumulative mortality
 
BBA cumulative post-treatments mortality with the isolates of the EPF B. bassiana, M. anisopliae and V. lecanii is presented in Fig 2. A significant difference in cumulative mortality was recorded between the three EPF. M. anisopliae showed a higher significant cumulative mortality during the first and second days post-application. Then V. lecanii recorded higher significant cumulative mortality starting from the third unit the seventh-day post-application. The 24 h monitoring intervals showed that V. lecanii, M. anisopliae and B. bassiana caused 50% cumulative death by the 3rd, 4th and 5th day, respectively. V. lecanii, M. anisopliae and B. bassiana cumulative mortality of BBA on the 7th day were 92, 84.8 and 73.8%, respectively.
 

Fig 2: Mean percent ± S.D. Cumulative mortality of BBA exposed to EPF B. bassiana isolate BBK2, M. anisopliae isolate MAA2 and V. lecanii isolate VLJ2.


 
The current study evaluates the efficacy of EPF in controlling back bean aphids under laboratory environmental conditions. In most soil systems, B. bassiana, M. anisopliae and V. lecanii are naturally occurring EPF. These entomopathogens infect insects when their spores penetrate insect cuticles, produce toxins and cause the death of their host insect (Islam et al., 2021). Identifying the virulence between the different entomopathogen species is one of the critical tools before further genetic, biochemical and environmental risk assessment investigation is carried out (Plantey et al., 2019). On the other hand, these EPF were described to affect their host insects by starving them (Mannino et al., 2019), deteriorating insect tissue (Altinok et al., 2019) and discharging toxic substances (Bamisile et al., 2021). The EPF fungi produce chitinase, protease and lipase enzymes that degrade the insect cuticle (Singh and Joshi, 2020). Once the fungal germ tube penetrates the insect cuticle, they start releasing more mycotoxins in the hemocoel that destroy insect cells and cause their death (Mahankuda and Bhatt, 2019).
 
Screening bioassays of EPF isolates under laboratory conditions is a crucial step toward identifying the most virulence strains prior to field assessments. Due to the constraint status of pesticide registration legislation and regulations in West Bank, the limitations on agrochemical importation and the high market prices of most pesticides. Many local growers showed an increased interest in developing an indigenous EPF over exotic isolates due to political and ecological boundaries.   
 
The current study investigated the toxicity and virulence of three EPF isolates. B. bassiana. M. anisopliae and V. lecanii have been used to control many insects of economic importance, Cylas formicarius (Reddy et al., 2014); Aphis craccivora (Maketon et al., 2013); Leptinotarsa decemlineata (Anderson and Roberts, 1983); Rhynchophorus ferrugineus (Gindin et al., 2006); Agrotis ipsilon (Gabarty et al., 2014); and Spodoptera littoralis (Amer et al., 2008). Similar results were found in several studies; Saranya et al., (2010) reported a 100% mortality of A.craccivora post-application of V. lecanii followed by B.bassiana, M. anisopliae. LC50 value was the highest virulence for V. lecanii compared to B. bassiana, M. anisopliae. Similar to our results, LT50 was the highest for V. lecanii, then M. anisopliae and B. bassiana. These three EPFs have been used wildly against aphids and other insect pests worldwide. They are cheap for mass production, have a broad host range and can tolerate a wide range of temperatures and humid conditions (Milner, 1997). B. bassiana and M. anisopliae are one of the most abundantly and commercially available and used EPF (Peng et al., 2021), but V. lecanii is the only hyphomycete fungi that attack aphids in greenhouses because they need very high humidity (Goettel and Glare, 2005). Javed et al., (2019) reported that V. lecanii have a higher virulence and mortality rate due to their ability to germinate under a broad range of temperatures and humidity, increasing their virulence.
 
LT50 value of V. lecanii against Macrosiphoniella sanborni aphid was three days (Jackson et al., 1985) and they caused higher mortality to Myzus persicae (Sulzer) aphid than B. bassiana (Javed et al., 2019), while M. anisopliae was more efficient than B. bassiana against brown plant hopper (Atta et al., 2020), which is similar to our findings.

EPF for insect pests is one approach to non-chemical crop protection. Studying the bio-efficacy and virulence of the potential EPF is a prerequisite for optimizing the application strategy for controlling insect pests in biological control. V. lecanii, M. anisopliae and B. bassiana were found to be the promising EPF against BBA. They can be used as potential biocontrol agents to manage BBA by further testing their field efficacy as a good alernative insect pest control of aphids for faba bean cultivation.


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