On the virulence of the entomopathogenic fungi, Beauveria bassiana and Metarhizium anisopliae (Ascomycota: Hypocreales), against the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae)

Background: The diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is a noxious pest of cruciferous crops all over the world causing serious economic damage. Management of insect pest generally depends on chemical control; however, due to development of resistance against all types of insecticides, alternative approaches especially utilization of a microbial agent is inevitable. Results: Potential of 2 entomopathogenic fungi (EPF), viz., Beauveria bassiana and Metarhizium anisopliae, was evaluated against 2 and 3 larval instars of P. xylostella by adopting leaf dip and direct spraying methods under laboratory conditions. Significant mortality rate was achieved by each fungus under adopted methodologies. However, B. bassiana was found to be more effective in both conditions than M. anisopliae. Highest mean corrected mortality (77.80%) was recorded, when spores of B. bassiana were sprayed on the 2 instar larvae (LC50= 1.78×10/ml) after the 6th day of treatment. Similarly, incase of M. anisopliae LC50 for the 2 nd instar at the same methodology was 2.78×10/ml with a mortality percentage of 70.0%. Offspring sex ratio was non-significantly related to treatment concentration and methodology, except for the control. Conclusion: Beauveria bassiana and M. anisopliae had potential to suppress P. xylostella infestations when applied appropriately. Present findings suggested that B. bassiana and M. anisopliae when sprayed on immatures of host insect had more effect as compared to leaf dip procedure. Furthermore, no significant effect of concentrations was observed on sex ratio.

Application of highly toxic, broad-spectrum synthetic insecticides is the primary management strategy against insect pests; however, P. xylostella is on the frontline in developing resistance to every class of insecticides (Ridland and Endersby 2011). Another drawback of overuse of insecticides is the environmental hazards and adverse effects on natural enemies (Peng et al. 2010). Therefore, exploration of eco-friendly management strategies to cope with resistant DBM population is inevitable. Alternatively, entomopathogenic fungi (EPF), like Beauveria bassiana (Balsamo-Crivelli) Vuillemin (Ascomycota: Hypocreales), Metarhizium anisopliae (Metschn.) Sorokin (Hypocreales: Clavicipitaceae), Isaria fumosorosea Wize (Hypocreales: Cordyciptaceae), Verticillium lecanii (Zimm.) (Deuteromycotina: Hyphomycetes), and Nomuraea rileyi (Farl.), have been found to be promising tools for controlling several agricultural insect pests (Trdan et al. 2020). The most efficient and well-studied EPF are the soil-borne fungi mainly B. bassiana and M. anisopliae that have a wide range of insect hosts (Javed et al. 2019). These 2 fungi are compatible with each other and showed significant results against lepidopteran insect pests (El Husseini 2019). Several studies have been conducted to evaluate efficiency of EPF against DBM immatures either alone or in combination with other EPF (Correa-Cuadros et al. 2016). However, to obtain sufficient results in insect pest management by EPF, proper methods of application are mandatory. Therefore, this study was conducted to evaluate the bioefficacy of B. bassiana and M. anisopliae against P. xylostella under the best feasible method of their application.

Insect culture
Plutella xylostella culture was reared on natural diet (cabbage leaves) in cages (60×60×60 cm) at 25°C, 65±5% RH, and 16L:8D photoperiod. Emerging adults were fed on 20% honey solution soaked in cotton balls. To obtain homogenous population, moths were collected and placed in separate cages for mating and oviposition on cabbage plants. After 6 h, plants were removed and shifted to new cages for egg hatching. Neonate 2 nd and 3 rd larval instars were collected for pathogenicity trials.

Fungi culture
Already established cultures of B. bassiana and M. anisopliae were used in the bioassays. To check the hyphal viability, the obtained cultures were grown on potato dextrose agar (PDA) media in Petri plates (9-cm diameter) and incubated at a temperature of 28°C, 65±5% relative humidity (RH), and a photoperiod of 12:12 (L:D) h for 15 days. Conidia were extracted from each culture plate by adding 20 ml sterile distilled water with 0.05% Tween-80 suspension. Conidia in each Petri dish were harvested from the suspension with the help of 90-μm mesh sieve, the number of conidia/ml were determined, using Neubauer chamber, and concentrations of 10 8 , 10 7 , 10 6 , 10 5 , 10 4 conidia/ml were prepared.

Pathogenicity of entomopathogenic fungal isolates
The virulence of EPF against 2 nd and 3 rd larval instars of P. xylostella was determined using direct spraying of fungal inoculum (methodology I) and leaf immersion method (methodology II) on host insect immatures. Each applied treatment was replicated 3 times. Thirty larvae of P. xylostella were used for each experimental unit. In the 1st methodology, 30 individuals of 2 nd or 3 rd larval instars were placed in Petri plates, and 2 ml of fungal suspension from each concentration was sprayed directly onto the larvae, using a sprayer with droplet size about 200-300 μm. After that, larvae were air dried, shifted into the Petri plates having moistened filter paper provided with cabbage leaf disc. Afterwards, treated larvae were placed in growth chamber under the abovementioned conditions.
In leaf immersion method, leaf discs of cabbage (Brassica oleracea L.) cv. Asha (approx. 8-cm diameter) were prepared. Leaf discs were dipped in 2-ml fungal suspension from each concentration (10 8 , 10 7 , 10 6 , 10 5 , 10 4 spores/ml of water) for 1min. After air drying, individual leaves were placed in Petri dishes having moistened filter papers at their bottoms (8-cm diameter). In control treatment, leaves were dipped in distilled water only. A soft brush was used to transfer 30 individuals of 2nd or 3rd larval instars of P. xylostella to each leaf disc, and the dishes were placed in a growth chamber at 25°C, 65±5% RH, and a photoperiod of 16L: 8D h. Mortality rates were recorded after every 24 h, starting 48 h of post-application up to one week. As significant mortality occured after 120 h and 144 h of post treatment application, therefore data was analyzed according to these time intervals. The larvae, unable to move on poking, were considered dead and were placed in a humid chamber to confirm whether the larvae were killed by the entomopathogen or not. The humid chamber consisted of sterilized and moistened filter paper disc (8-cm diameter) and foam, placed on Petri dishes (9-cm diameter). Confirmation of mortality was performed after 10 days of incubation in the humid chamber by observing the conidial structures of the fungus with the help of a microscope.

Sex ratio of emerged adults
The larvae that remained viable and developed to pupal stage were recorded, and afterward number of emerged moths was counted. On the basis of emerged adults, sex ratio of the progeny was assessed as percentage of females as described by Maia & Alfredo (2000).

Statistical analysis
Total mortality rates were corrected, using Abbot's formula (Abbott 1925). The experiment was conducted in a completely randomized design, and the data were subjected to analysis of variance. Means were compared by Tukey's test (P < 0.05; PROC GLM, SAS Institute 2002). The lethal concentrations (LC 50 ) were determined using Probit analysis (P > 0.05) by the R statistic software.

Results
Pathogenicity of B. bassiana on the 2 nd and 3 rd instars of P. xylostella A direct relationship was found between concentration and mortality in case of both methods adopted (P=≤ 0.05) ( Table 1). Mortality induced showed positive relationship to time after treatment application. In the case of the 2 nd instar larvae, significant pathogenicity (63.3%) was recorded, when B. bassiana spores 10 8 were sprayed directly on larvae (methodology 1) after 120 h (5 days). However, the mortality rate reached the maximum value of 77.78% after the 6th day at the highest concentration (10 8 ). In case of low concentration, 10 7 spores /ml, fungal-induced pathogenicity was corresponded to the mortality rate of 61.32% after 144 h. Results revealed that in case of low spore concentrations, i.e., 10 6 , 10 5 , and 10 4 spores/ml, the lethal rates decrease to 52.3%, 42.3%, and 32.3%, respectively (Table 1). However, in case of the leaf dip method (methodology 2), virulence induced in the 2 nd instar larvae were low compared to spraying method. Results indicated that a significant difference in mean-corrected mortality exists between the methodologies corresponding to time interval and concentration. Percentage mortality (51.3%) was observed at high concentration after the 5th time interval, and 70% was the maximum lethal effect induced after the 6th day at the highest concentration of 10 8 . For other concentrations applied of 10 7 , 10 6 , 10 5 , and 10 4 spores/ml, insect pathogenicity ranged between 53.3, 44.4, 37.7, and 28.9%, respectively (Table 1). In case of the 3rd instar larvae, results proclaimed that conidial pathogenicity is relatively lower as compared to earlier instar. Maximum mortality rate (71.3%) corresponded to the highest concentration after the 6th day of treatment application under method 1. However, at the 2nd method, the peak mortality rate (60%) has been recorded after same time interval. Results elaborated significant differences among the lethal rates induced by the 2 different methods. Direct spraying yielded good results than the leaf-dip bioassay.
Pathogenicity of M. anisopliae on the 2 nd and 3 rd instars of P. xylostella In case of M. anisopliae, the highest concentration yielded a maximum lethal effect for both larval instars. Results of the first method were higher than the second on both exposed larval instars. Significant mortality rates were induced at the 1st method in the 2 nd and 3 rd larval instars (70% and 64.7%), respectively. However, for other concentrations, the corrected mortality rates were low and varied from 58.9 to 31.1% at the 2 nd and 3 rd instars, respectively (Table 2). However, in case of the leaf dip bioassay 62.3% was the maximum mortality that was occured in 2 nd instar larvae, 55.7% was the peak mortality corresponding to later instar (Table 2).
In the case of the 2 nd instar larvae, M. anisopliae spores were sprayed directly on the larvae. The maximum mortality rate of the 3 rd instar larvae at same method was 55.7%, which was lower than at the 2nd nd instar. Overall results have showed that B. bassiana performs better in both the methodologies adopted against both larval instars as compared to M. anisopliae.

Sex ratio of emerged adults
Sex ratio, in term of females, was estimated from the emerged adults. Results revealed that progeny sex ratio is independent of treatment application even at high concentration. Therefore, it can be inferred from results that there exists a non-significant relationship among all the treatments including control in terms of sex ratio. Survived adults under control treatment relatively showed higher percentage of females as compared to other treatments in both the methodologies and under both fungal applications. In the case of the 1st method, a significant low percentage was recorded in adults that emerged from 10 4 spores/ml dilution, followed by the 10 8 spores/ml concentration. However, at other treatments, a relatively less number of females were observed.
At the 2nd method, non-significant difference in percent female ratio of emerged adults in both the larval instars has been observed (α=0.05) and was found (Fig. 1).
In case of M. anisopliae, the percent of sex ratio of emerged adults showed that a significantly high number of females emerged from the control treatment of both methods and on both larval instars. Results of the 1st method, however, depicted that relatively less percent of females was recorded from 10 8 spores/ml concentration, survived adults of 2 nd as well as 3 rd instars. At In the 2nd method, female percentage was significantly low in the adults that emerged from treated 3 rd instar larvae at high concentration. However, survived females from other treatments were non-significantaly different. Generally, less female number was observed at the highest concentration (Fig. 2).
Median lethal concentrations LC 50 of the two EPF showed that B. bassiana caused higher pathogenicity comparative to M. anisopliae as its estimated value of LC 50 is lower while M. Anisopliae had more LC 50 value for both instars (Tables 3 and 4).

Discussion
The present study highlighted the virulence of B. bassiana and M. anisopliae against 2 nd and 3 rd larval instars of P. xylostella under 2 different application methods. In the present study, both EPF had lethal effects against P. xylostella as it was reported in the previous studies conducted by Loc and Chi (2007) and Zafar et al. (2020). Results had depicted that in addition to method of treatment application, the virulence was strongly related to concentration exposed to immatures, post-treatment time, and fungal species. In the current study, B. bassiana had a more pathogenic effect on P. xylostella larvae than M. anisopliae using both leaf dip method and foliar spray. This difference might be due to variant sporulation capacity of species as some fungal spores may penetrate more frequently than others (Mannino et al. 2019).
Furthermore, production of bioactive enzymes and other metabolites responsible for the death of the host is also a vital factor toward fungal pathogenicity (Shakeel et al. 2017). It can be inferred from current findings that method 1 showed higher toxic effect than the second one. This suggests that foliar application of spores can significantly suppress P. xylostella. Findings of Beris et al. (2013) and Gabarty et al. (2014) support our findings suggesting that different application methods pose different virulent effects in insect pests including DBM respectively. Another study conducted by Sáenz-Aponte et al. (2020) also testify our results that different methods of fungal spore's application had different virulent activities against host insect. This might be due to the fact that successful sporulation of fungi on P. xylostella larvae depended on spores' adhesion and penetration to the host cuticle, therefore spraying fungal suspension on larvae confirmed a maximum probability that more number of conidia had adhered to insect surface (Qayyum et al. 2019). Overall, an increase in concentrations was found to be more potent in case of both EPF used against P. xylostella. Results obtained by Mehinto et al. (2014) are in compliance with our findings, claimed that 1×10 8 spores/ml showed peak mortality against Maruca vitrata (Lepidoptera: Crambidae) insect pest. Furthermore, Freed et al. (2012) also reported that high EPF concentration against DBM had prompted a significantly higher mortality as compared to low ones. Xia et al. (2013) found the same virulence pattern in P. xylostella. However, non-significant mortality had been observed in case of low concentrations. Actually, the virulence of fungal strain depends on number and growth of spores within the host body (Ali et al. 2010). The sporulation had direct relation to host mortality as suggested by Pauli et al. (2018). Current findings also suggest that after 144 h of posttreatment application, both EPF had instigate the best results oblivious from method of application. Therefore, it could be inferred that fungal spores take time to invade insect cuticle as described by earlier studies of Kim et al. (2002) and Mehinto et al. (2014).
In case of each fungus evaluated for virulence, the mortality prompted in DBM was significantly high against 2 nd instar larvae under both the methods of application. This could be because earlier instars are more susceptible to biocontrol tactics. The study conducted by Hafez et al. (1997) is pursuant to our results who reported that B. bassiana was more pathogenic to earlier instar larvae of potato tuber moth as compared to ensuing instars. Another study conducted by Qayyum et al. (2015) reported that the 2 nd instar larvae were more vulnerable than the later instars. This might be due to hardening of cuticle in later instars as reported by Wraight et al. (2010). Another aspect covered in current study was the sex ratio of survived adults, and results had showed that there was a non-significant difference between both methods. Overall results regarding sex ratio describe that high concentration applied produces relatively low percentage of females. Results of De Souza et al. (2020) are also in line with our current findings predicting that sex ratio of Helicoverpa armigera (Lepidoptera: Noctuidae) is independent to concentration applied. The results of Batcho et al. (2018) also support our obtained results. This fact might be because of that multiple factors in addition to fungal spores plays role in insect sex determination which would be explored after genetic studies and yet to be explored.

Conclusion
Direct spray of the EPF, B. bassiana and/or M. anisopliae, provided higher pathogenicity to the larvae of P. xylostella than the leaf dip method. This method can be recommended to be used under field as well as laboratory conditions for conducting bioassays and successful management of the pest.

Funding
Experimental studies were funded by the Higher Education Commission, Islamabad (Pakistan).