Pathogenicity of Metarrhizium anisopliae (Metschn.) and Beauveria bassiana (Bals.) against Spodoptera litura (Fabricius)

Introduction

The extensive use of conventional insecticides for decades has disrupted biological control systems by natural enemies, led to the development of resistance and fostered concern over possible environmental and health hazards. Therefore, in recent years, various researchers have been concentrating their efforts on the search of natural products derived from plants and microorganisms as an alternative to conventional chemical insecticides for insect control. A common feature of some microorganisms, especially fungi, is their natural ability to produce metabolic compounds that may be toxic against insect pests. These bio-control agents kill wide range of insect pests, spread quickly, safe to non-target organisms including man, compatible with other means of pest control, amenable for mass multiplication on readily available cheap substrate. Because most of the fungi invade the host insect through the exoskeleton, they are the only significant naturally occurring entomopathogens of phytophagous insects. Among several fungal pathogens, Metarrhizium anisopliae (Metschn.) Sorokin and Beauveria bassiana (Bals.) Vuill. have been found to be promising in the control of several agricultural pests (Lingappa et al., 2005) [1] . M. anisopliae is a potential entmopathogenic fungus belonging to the class Hyphomycetes, which reproduce by spores. The diseases caused by this fungus is called green muscardine disease because of green colour of its spores (conidia). It is known to attack a wide range of arthropods, greater than 200 species in over 50 families. It is used to control a number of pests viz., grasshoppers, termites, thrips, caterpillars, beetles, aphids, mealy bugs, scale insects, hoppers and mosquitoes (Chaudhari, 2012; Gurav, 2012) [2,3] . B. bassiana is a hypomycete insect-pathogenic fungus in the subdivision Deuteromycotina which occurs worldwide. Over 200 species of insects in nine orders, mainly Lepidoptera and Coleoptera have since been recorded as hosts (Li et al., 1988) [4] . It is found naturally on some plants and in soils as is regarded as a safe biopesticide (Uma Devi et al., 2008) [5] than 120 host plants (Ramana et.al., 1988) [10] . The major one includes tobacco, cotton, groundnut, jute, lucerne, maize, rice, soybean, tea, cauliflower, cabbage, capsicum, potato and castor etc. (Sharma et al., 2008) [11] . The extent of damage caused by it varies from 60 to 100 per cent (Chari, 1987) [12] and 25.8 to 100 per cent (Dhir et al., 1992) [13] , respectively. Pest is sporadic in occurrence but when left unattended causes complete failure of the crop. It is resistant against almost all the insecticide groups (Kranthi et al., 2002) [14] . Hence, microbial pesticides are alternative to tackle insecticide resistance populations of S. litura (Pawar et al., 2005) [15] .

Materials and methods

Sources of fungal inoculum

Preparation of solution of entomopathogenic fungi

The spores of the laboratory cultured fungus viz., M. anisopliae and B. bassiana were taken in 10 ml water. One milliliter solution of the desired fungus were taken on haemocytometer, cover slip were placed over it for measuring the spores. Five squares were randomly selected for the spore count and then average numbers of spores were calculated for each square. The values obtained were put in following formula given by Knuckles et al. (1990) [16] to know the concentration of solution.

No. of spores / ml = 𝐷 × 𝑋 𝑁 × 𝐾

Where, D = Dilution factor X = Total number of spore counted N = Number of small squares counted K = Volume above one small square in cm 3

Culture of S. litura

The initial culture of S. litura was obtained by collecting a few larvae from field which were reared in the laboratory in a 25 cm high and 17 cm diameter glass jar containing 5 cm thick layer of soil at the bottom to facilitate pupation (Plate II). Fresh castor leaves were supplied daily as food to the larvae after removing the excreta and uneaten parts of leaves till pupation. The newly formed pupae were collected and placed in a glass jar (25 cm x 17 cm). The open end of the jar was closed with muslin cloth, which was secured firmly in position with a rubber band. Freshly emerged males and females were released in another glass jar (25 cm x 17 cm). The open end of the jar was closed with muslin cloth secured firmly with rubber band. A cotton swab soaked in 5 per cent honey solution was kept hanging in the jar as food for the moths. Food was changed daily. Crumpled pieces of blotting paper were kept inside the jar for egg laying. The blotting paper was removed and cut into pieces each containing egg mass and such cut pieces carrying eggs were placed for hatching in a 10 cm diameter petridish containing a round piece of blotting paper at the bottom.

Freshly hatched larvae were reared on castor leaves. Thus the mass culture of pest was prepared and maintained in the laboratory (Surve, 2003). A precaution was taken to disinfect the rearing jars and petridishes with 40 per cent formalin to avoid infection. The 2 nd and 4 th instar larvae of same age were selected randomly from the culture and were used for testing the pathogenicity and efficacy of M. anisopliae and B. bassiana along with botanicals.

Methodology

A laboratory experiments were conducted in the Department of Agricultural Entomology, College of Agriculture, Dapoli with a view to study the pathogenicity of M. anisopliae and B. bassiana at different doses against two different instars (2 nd and 4 th ) of S. litura larvae during the year 2025-26. The details of the experiments were as follows.

The culture of M. anisopliae and B. bassiana developed on PDA media were used for testing the pathogenicity of both the fungi at different doses viz., 1.5 × 10 8 , 1.5 × 10 7 , 1.5 × 10 6 , 1.5 × 10 5 and 1.5 × 10 4 conidia/ml prepared after serial dilution from stock suspension of 1.5 × 10 9 conidia/ml. The larvae along with fresh castor leaves as their food were released in petridish of 20 cm diameter and were directly sprayed with spore suspension with a hand atomizer (Kulkarni et al., 2002) [17] . Then, they were air dried and transferred into petridishes. The larvae in control treatment received a spray of sterilized distilled water. The larvae were provided with untreated fresh castor leaves daily after 48 hours of treatment (Vimala Devi, 1994) [18] .

Experimental details

Design: Completely Randomized Design (CRD) Replication: Three Treatments: Seven 1) 1.5 × 10 9 conidia/ml 2) 1.5 × 10 8 conidia/ml 3) 1.5 × 10 7 conidia/ml 4) 1.5 × 10 6 conidia/ml 5) 1.5 × 10 5 conidia/ml 6) 1.5 × 10 4 conidia/ml 7) Control (Sterilized water spray) Instars to be tested: 1) Second instar 2) Fourth instar Number of larvae/ treatment/replication: 10

Observations recorded

The larvae were observed daily for the symptoms of fungal infection and mortality. In case of second instar, four observations on larval mortality i.e. on 5 th , 7 th , 9 th and 11 th DAT were recorded. While in case of fourth instar, three observations were recorded i.e. on 6 th , 8 th and 10 th DAT, respectively, for both the fungal pathogens. Based on healthy and dead larvae, per cent mortality were worked out and subjected to statistical analysis after following arc sine transformation. The dead larvae were removed on the day of observation and maintained in a humid chamber prepared with a petriplate covered with moist blotting paper from all the sides to allow the outgrowth of fungus for confirming its infection.

Results and discussion

Pathogenicity of M. anisopliae and B. bassiana against second instar larvae of S. litura

Observations on pathogenicity of M. anisopliae at various doses against second instar larvae of S. litura recorded on 5 th , 7 th , 9 th and 11 th Day after treatment (DAT) are presented in Table 3.1. The observations recorded on 5 th DAT revealed that the lower doses of 1.5 × 10⁴, 1.5 × 10⁵ and 1.5 × 10⁶ conidia/ml did not appear to be pathogenic as no larval mortality was observed in these treatments against second instar larvae of S. litura. However, the higher doses of 1.5 × 10⁷, 1.5 × 10⁸ and 1.5 × 10⁹ conidia/ml recorded 6.66, 13.33 and 16.66 per cent larval mortality respectively, thereby indicating pathogenic effect of the fungus. Among these treatments, the highest dose of 1.5 × 10⁹ conidia/ml recorded comparatively higher mortality though the differences among higher doses were not pronounced during the initial stage of observation. The data recorded on 7 th DAT showed that M. anisopliae exhibited pathogenicity at all the doses by causing significantly higher larval mortality in second instar larvae than untreated control. The observations clearly indicated that the pathogenicity increased with increase in conidial concentration. The highest dose of 1.5 × 10⁹ conidia/ml was found to be the most pathogenic as it recorded maximum larval mortality of 56.66 per cent. However, it was at par with the mortality caused by 1.5 × 10 8

. The dose of 1.5 × 10⁸ was found to be significantly more pathogenic than 1.5 × 10⁷ and 1.5 × 10⁶ with 40.00 and 26.66 per cent mortality, respectively. The lower doses of 1.5 × 10⁵ and 1.5 × 10⁴ conidia/ml were observed to be least pathogenic as they recorded significantly the least per cent larval mortality (16.66 and 10.00). Thus, the observations indicated that increase in the dose of conidia per ml of water increased the fungal pathogenicity. On 9 th DAT, M. anisopliae at all the doses was found to be more pathogenic than observed on 7 th DAT as the larval mortality was ranged from 16.66 to 80.00 per cent as against 10.00 to 56.66 per cent mortality on 7 th DAT. The highest dose of 1.5 × 10⁹ was found to be most pathogenic as it caused significantly highest larval mortality (80.00 %), however, it was at par with 1.5 × 10 8 (73.33 %) followed by 1.5 × 10 7

(60.00 %). Each higher dose appeared to be significantly more pathogenic than its lower dose with an exception of 1.5 × 10 6 and 1.5 × 10 5 which remain at par with each other. The lowest dose of 1.5 × 10⁴ conidia/ml was observed to be the least pathogenic as it caused significantly the least mortality of 16.66 per cent. Further observations revealed that M. anisopliae remained pathogenic even on 11 th DAT as significantly higher larval mortality was observed at all the doses than untreated control. Again, the highest dose of 1.5 × 10⁹ conidia/ml was observed to be the most pathogenic as it produced significantly the highest mortality of 93.33 per cent, however, it was at par with 1.5 × 10 8 (86.67 %). The doses 1.5 × 10 8 and 1.5 × 10 7 , 1.5 × 10 7 and 1.5 × 10 6 , 1.5 × 10 6 and 1.5 × 10 5 remained equally pathogenic, as there were no significant differences in the mortality. However, all these doses were appeared to be significantly more pathogenic to 1.5 × 10 4 (20.00 %). The observations also indicated that pathogenicity of the fungus increased as the period after treatment extended and appeared to be directly proportional to the dose applied.

Pathogenicity of B. bassiana against second instar larvae of S. litura

Observations on pathogenicity of B. bassiana at various doses against second instar larvae of S. litura recorded on 5 th , 7 th , 9 th and 11 th Day after treatment (DAT) are presented in Table 3.1. The data on 5 th DAT indicated that the fungus B. bassiana showed its pathogenic activity only at higher doses viz., 1.5 × 10 7 , 1.5 × 10 8 and 1.5 × 10⁹ showing 3.33, 6.66 and 13.33 per cent mortality in second instar larvae of S. litura, respectively, without any significant differences. However, the mortality was not very high. The highest dose of 1.5 × 10 9 caused significantly higher mortality than control. The fungus at lower doses viz., 1.5 × 10 4 , 1.5 × 10 5 and 1.5 × 10 6 showed no pathogenic activity, as it caused zero per cent mortality. On 7 th DAT, it was appeared that the fungus at all the doses, except 1.5 × 10 4 was pathogenic, however, it caused significantly higher mortality only at 1.5 × 10 9 and 1.5 × 10 8 over rest of the doses. The highest dose of 1.5 × 10 9 produced significantly highest mortality of 46.66 per cent. It was followed by 1.5 × 10 8 (30.00 %) and 1.5 × 10 7 (26.66 %) which were at par with each other. There were no significant differences observed between doses of 1.5 × 10 8 and 1.5 × 10 7 , 1.5 × 10 7 and 1.5 × 10 6 were at par with each other. The lowest dose of 1.5 × 10 4 appeared to be non-pathogenic as it recorded 0.00 per cent larval mortality along with untreated control. Further, on 7 th DAT, the fungal pathogenicity found to be increased than that on 5 th DAT as the cumulative mortality caused by the fungus at various doses ranged from 6.66 to 46.66 on 7 th DAT as against 3.33 to 13.33 per cent on 5 th DAT. Besides this, increase in dose increased the pathogenicity of the fungus. Observations of 9 th DAT revealed that the fungus at all the doses was found to be more pathogenic as the larval mortality in second instar ranged from 10.00 to 76.66 per cent as compared to 0.00 to 46.66 per cent observed on 7 th DAT. The highest dose of 1.5 × 10⁹ appeared to be the most pathogenic as it caused significantly highest cumulative mortality of 76.66 per cent followed by 1.5 × 10 8 (60.00 %), 1.5 × 10 7 (56.66 %) and 1.5 × 10 6 (36.66 %). There were no significant differences between the fungal doses of 1.5 × 10 8 and 1.5 × 10 7 , 1.5 × 10 5 and 1.5 × 10 4 . The fungus at 1.5 × 10 6 was also found to be significantly superior to 1.5 × 10 5 and 1.5 × 10 4 . Whereas, the control showed zero per cent larval mortality. Further observations indicated that the fungus, B. bassiana continued to show its pathogenicity even on 11 th DAT as all the treatments recorded significantly higher larval mortality than untreated control. The highest dose of 1.5 × 10⁹ was found to be most pathogenic and recorded maximum larval mortality of 86.66 per cent followed by 1.5 × 10⁸ (80.00 %) and 1.5 × 10 7 (73.33 %) and all these three treatments were at par and equally effective in showing their high pathogenicity against second instar larvae of S. litura. Among remaining treatments, the treatment of 1.5 × 10 6 (50.00) was found significantly superior to 1.5 × 10⁵ (26.66 %) and 1.5 × 10⁴ (16.66 %) in causing larval mortality. It is evident from the observations recorded on 5 th DAT, 7 th DAT, 9 th DAT and 11 th DAT that the pathogenicity of the fungus increased as the period after treatment advanced and the pathogenicity was directly proportional to the dose.

Pathogenicity of M. anisopliae against fourth instar larvae of S. litura

The observations on pathogenicity of M. anisopliae at various doses against fourth instar larvae of S. litura recorded on 6 th , 8 th and 10 th Day after treatment (DAT) are presented in Table 3.2. The results indicated that M. anisopliae started to exhibit its pathogenic activity against fourth instar larvae of S. litura at 6 th Day after treatment (DAT). However, the pathogenicity was very low as the fungus at the highest dose of 1.5 × 10⁹ caused 13.33 per cent mortality and it was significantly superior to control and rest of the doses except 1.5 × 10 8 (10.00 %) which was at par with it. More or less similar trend in the pathogenicity of the fungus was also observed on 8 th DAT and 10 th DAT. Again it was appeared that the fungal pathogenicity increased as the period after treatment extended, since the cumulative mortality caused by the fungus at highest dose of 1.5 × 10⁹ on 8 th DAT was 36.66 per cent was more than that observed on 6 th DAT (13.33 %). Moreover, the doses of 1.5 × 10⁹, 1.5 × 10 8 and 1.5 × 10 7 remained at par with each other. Lower doses of 1.5 × 10 6 and 1.5 × 10 5 recorded per cent larval mortality of 13.33 and 6.66, respectively, whereas, dose of 1.5 × 10 4 and control showed 0.00 per cent mortality. Further, it was also noticed that as the dose of fungus increased, its pathogenicity also increased. At 10 th DAT the per cent larval mortality ranged from 3.33 to 53.33 per cent showing an increase in the pathogenicity of the fungus as compared to observation on 8 th DAT where the mortality ranged from 0.00 to 36.66 per cent. Among the treatments, the highest concentration of 1.5 × 10⁹ was found to be the most effective treatment causing 53.33 per cent larval mortality followed by 1.5 × 10 8 (46.67 %), 1.5 × 10 7 (33.33 %). However, doses of 1.5 × 10⁹ and 1.5 × 10 8 , 1.5 × 10 8 and 1.5 × 10 7 were found at par with each and equally effective it their pathogenicity against the test insect. The doses of 1.5 × 10 6 and 1.5 × 10 5 recorded per cent larval mortality of 20.00 and 13.33 per cent, respectively and were at par and superior to lowest dose 1.5 × 10 4 (3.33 %). The untreated control showed zero per cent larval mortality.

Pathogenicity of B. bassiana against fourth instar larvae of S. litura

The observations on pathogenicity of B. bassiana at various doses against fourth instar larvae of S. litura recorded on 6 th , 8 th and 10 th Day after treatment (DAT) are presented in Table 3.2. Observations recorded on the 6 th DAT revealed that the lower doses of 1.5 × 10 6 , 1.5 × 10 5 and 1.5 × 10 4 did not show any pathogenic effect, as no larval mortality was observed in these treatments. However, the higher doses of fungus viz., 1.5 × 10 9 , 1.5 × 10 8 and 1.5 × 10 7 caused 10.00, 3.33 and 3.33 per cent larval mortality respectively, indicating the starting of initial pathogenic effect of the entomopathogenic fungus, B. bassiana. Among these treatments, the DAT indicated that the fungus exhibited progressive pathogenicity across all the tested doses by causing higher larval mortality than the untreated control where the per cent larval mortality ranged from 3.33 to 30.00. The highest dose of 1.5 × 10 9 proved to be the most lethal, recording a maximum larval mortality of 30.00 per cent, however, it was at par and equally effective with 1.5 × 10 8 inciting 20.00 per cent mortality. However, there were no significant differences between 1.5 × 10 8 , 1.5 × 10 7 and 1.5 × 10 6 . The lower doses of 1.5 × 10 6 , 1.5 × 10 5 and 1.5 × 10 4 recorded comparatively low mortality of 10.00, 6.66 and 3.33 per cent, respectively. These results clearly indicated that the pest mortality peaks directly with an increase in conidial concentration. Further observations on 10 th DAT confirmed a substantial increase in fungal pathogenicity at all doses as larval mortality among these different doses ranged from 3.33 to 46.67 as compared to the lower mortality ranges seen during previous intervals of 6 th DAT and 8 th DAT. The highest dose of 1.5 × 10 9 proved to be the most pathogenic, resulting in a highest larval mortality of 46.67 per cent but was at par and equally effective in its pathogenicity with 1.5 × 10 8 (36.67 %) followed by 1.5 × 10 7 (26.66 %). However, 1.5 × 10 8 and 1.5 × 10 7 were at par and equally effective. The lower doses of 1.5 × 10 5 and 1.5 × 10 4 resulted in comparatively lower mortality of 10.00 and 3.33 per cent, respectively as compared to 0.00 in control. The present results on pathogenicity of M. anisopliae and B. bassiana against S. litura are in close agreement with Bekheit et al. (2002) [19] who reported that the number of days taken for mummification to occur in treated 2 nd instar larvae was shorter than that obtained with the 4 th instar larvae and the mortality of S. littoralis larvae increased with increasing dosage of M. anisopliae. Dayakar et al. (2003) [20] showed that the LC 50 values of B. bassiana and M. anisopliae against second instar larvae of S. litura were 6.4 and 13.97 conidia/ml, respectively. Wraight et al. (2010) [21] found that elevated concentrations of B. bassiana resulted in higher mortality of lepidopteran larvae and also accelerated the infection process. Moorthi et al. (2011) [22] observed that the mortality of 66.67, 73.33 and 80.0 per cent with respect to the B. bassiana isolates Bb 02 , Bb 09 and Bb 10 , respectively, in larvae of S. litura was obtained four days after treatment. The LC 50 and LT 50 values for Bb 02 , Bb 09 and Bb 10 isolates were 2.1 × 10 6 , 3.6 × 10 7 , 1.2 × 10 7 conidia ml -1 and 4.8, 4.8 and 4.0 days @ 10 8 spore ml -1 , respectively. Bb 10 strain was the most virulent with potential for the management of S. litura. Petlamul et al. (2012) [23] revealed that all five strains each of M. anisopliae and B. bassiana were pathogenic to S. litura larvae. The mortality of larvae started after 2 days incubation and reached over 50 per cent at 4 th day and total death (100.0 %) was occurred at 5 th day from M. anisopliae (M33, M36, M6 strains) and B. bassiana (BNBCRC, BPMC) and 6 th days for M. anisopliae (M8, MNBCRC) and B. bassiana (B14841, B16041, B14532). Asi et al. (2013) [24] reported that the virulence potential of the entomopathogenic fungi varied with different biological stages of S. litura. The early larval instars were found to be more susceptible than older larvae and the susceptibility of the pest to entomopathogenic fungi decreased with the advancement in age of larvae. The highest mortality was caused by B. bassiana 25 followed by M. anisopliae L6 whereas, the median lethal concentration (LC 50 ) for 3 rd instar larvae was 1.11 × 10 7 conidia ml -1 in B. bassiana 25. Mortality of larvae increased with increase in conidial concentrations and time elapsed after treatment. Ummidi et al. (2013) [25] observed that all the strains of M. anisopliae (M19, M20 and M45) and B. bassiana (B51, B52 and B55) at a concentration of 1 × 10 8 conidia/ml were found to be pathogenic to 2 nd instar larvae of S. litura. The mean cumulative mortality due to B. bassiana and M. anisopliae strains ranged between 71.0 to 99.0 per cent. Mycosis was observed in all the strains where the mycosis percentage ranged from 30.0 to 98.0 per cent. B55 strain showing rapid germination also depicted higher mycosis value (98 %) and maximum value of relative virulence index (1.42). Agrawal et al. (2017) [26] revealed that the pathogenicity of B. bassiana varied among different larval stages of tobacco caterpillar, S. litura, with all treatments causing significantly higher mortality than the control. The earlier instars were more susceptible as compared to later instars. The highest dose of 5 per cent (2.3 × 10 6 conidia/ml) resulted in mortality rates of 91.66, 90.00, 88.33, 78.77, 66.11, and 49.99 per cent, respectively in all the six instars, as compared to 11.66, 7.21, 1.10, 0.00, 1.11 and 0.00 mortality, respectively in the untreated control. Overall, the larval mortality in S. litura increased with increasing fungal spore concentration. Indriyanti et al. (2017) [27] showed that the larval mortality in S. litura larvae increased with increasing fungal dose of B. bassiana, with the highest concentration (8g 100 ml¹) causing 100 per cent mortality within 10 to 12 days. . Zulfitri et al. (2018) [28] found that the fungus B. bassiana (B2) caused highest mortality (70 %) against larval stages of S. litura. Dhar et al. (2019) [29] recorded significantly higher mortality rates of tobacco caterpillar, S. litura at all the tested concentrations of conidial suspensions (10 3 to 10 7 spores/ml) of B. bassiana. The mortality rate increased with the increasing number of conidial concentration used. Sonune et al. (2019) [30] reported that the spore suspension of M. anisopliae @ 1 × 10 9 spore ml -1 recorded highest mortality of 91.66 per cent, whereas, the spore suspension of 1 × 10 5 spore ml -1 recorded minimum mortality of 33.33 per cent in larvae of S. litura. Ullah et al. (2019) [31] observed that the entomopathogenic fungi B. bassiana significantly (P < 0.001) reduced relative growth rate (RGR), relative consumption rate (RCR) and efficiency of conversion of ingested food (ECI) in 3 rd and 4 th instar larvae of S. litura. Ramos et al. (2020) [32] found that B. bassiana and M. anisopliae caused 100 per cent mortality in second instar larvae and resulted in 87.0 and 75.0 per cent mortality, respectively in fourth instar larvae of S. frugiperda. Wargane et al. (2020) [33] observed a dose-dependent increase in larval mortality in second instar larvae of S. litura due to B. bassiana. The highest mortality (92.33 %) was recorded at 1 × 10 7 spores/ml. Vivekanandhan et al. (2022) [34] reported that M. flavoviridae conidia caused dose-dependent mortality in third and fourth instar larvae of tobacco caterpillar, S. litura. M. flavoviridae estimated LC 50 and LC 90 values against third instar larvae of S. litura were 3.0 × 10 5 and 1.1 × 10 13 conidia/ml, respectively. Larvae had an 80.0 per cent mortality rate at highest concentration (1 × 10 9 conidia/ml) which was statistically different compared to control. The infection by entomopathogenic fungi significantly reduced total hemocyte count and phenoloxidase activity, indicating suppression of the insect immune system. Antioxidant and detoxifying enzymes such as SOD, CAT, POX, ACP, and ALP were markedly altered following infection, leading to physiological and biochemical imbalance in larvae. Gunjan et al. (2024) [35] showed that among the seven isolates of B. bassiana and M. anisopliae evaluated against S. litura, the isolates UKBSPC Bb 1 and UKMFC Ma showed a highest mortality of 93.33 per cent each at 10 8 conidia/ml. Sharma et al. (2024) [36] observed that the highest concentration of 1.5 per cent of B. bassiana and M. anisopliae against the 3 rd instar larvae of S. litura caused 72 ± 0.21 and 64 ± 0.20 per cent larval mortality after 120 h exposure. Whereas, in 4 th instar larvae of S. litura, the highest concentration of B. bassiana and M. anisopliae (1.5 % each) recorded maximum 70 ± 0.21 and 64 ± 0.20 per cent larval mortality, respectively.

Conclusion

The pathogenicity of M. anisopliae against second instar larvae of S. litura indicated that the fungus started to cause second instar mortality from 5 th DAT and that too only at the higher doses of 1.5 × 10 7 , 1.5 × 10 8 and 1.5 × 10 9 only and the pathogenicity was also very low with mortality ranging from 6.66 to 16.66 per cent. The higher dose of 1.5 × 10 9 appeared to be the most pathogenic giving significantly the highest mortality (56.66 %) except 1.5 × 10 8

. The dose of 1.5 × 10 8 appeared to be significantly more pathogenic than 1.5 × 10 4 , 1.5 × 10 5 and 1.5 × 10 6 , while the lower dose of 1.5 × 10 4 was found to be the least pathogenic. Similar pathogenicity trend was also observed on 7 th , 9 th and 11 th DAT. The pathogenicity test against fourth instar larvae indicated that M. anisopliae appeared to be pathogenic to 3 rd instar larvae only from 6 th DAT as against 5 th DAT in second instar. The fungus showed its pathogenicity only at the higher doses of 1.5 × 10 7 , 1.5 × 10 8 and 1.5 × 10 9 causing 3.33, 10.00 and 13.33 per cent mortality. However, at 8 th DAT the fungus at all the doses except 1.5 × 10 4 were found to be pathogenic but it caused significant higher mortality only at 1.5 × 10 8 and 1.5 × 10 9 than control. The fungal pathogenicity was found to be increased on 8 th DAT than on 6 th DAT and on 10 th DAT than on 8 th DAT. The fungus was found to be pathogenic to 4 th instar even on 10 th DAT and that too at all the doses.

Data on pathogenicity of B. bassiana against second instar larvae of S. litura revealed that the fungus started to cause mortality in second instar from 5 th DAT and that too only at the higher doses of 1.5 × 10 7 , 1.5 × 10 8 and 1.5 × 10 9 only and the pathogenicity was also very low with mortality ranging from 3.33 to 13.33 per cent. However, the fungus was found to be pathogenic against 2 nd instar at lower doses, only from 7 th DAT with significantly higher mortality than the control. The higher dose of 1.5 × 10 9 appeared to be the most pathogenic giving significantly the highest mortality (46.66 %). The dose of 1.5 × 10 9 appeared to be significantly more pathogenic than 1.5 × 10 4 , 1.5 × 10 5 and 1.5 × 10 6 , 1.5 × 10 7 and 1.5 × 10 8 while the lower dose of 1.5 × 10 4 was found to be the least pathogenic. Similar pathogenicity trend was also observed on 9 th and 11 th DAT. B. bassiana also showed its pathogenic activity against fourth instar on 6 th DAT and remained pathogenic upto 8 th DAT. However, pathogenicity was very low as the fungus at the highest dose of 1.5 × 10 9 caused only 10.00 and 30.00 per cent mortality on 6 th DAT and 8 th DAT, which was very marginal and 46.67 per cent mortality on 10 DAT which was acceptable.

Limitation

The efficacy of entomopathogenic fungi is highly influenced by environmental factors such as temperature, relative humidity and light intensity, which may affect spore viability, infection rate and persistence. As a result, the performance observed under laboratory or controlled conditions may vary under field conditions. The speed of action of entomopathogenic fungi and botanicals is generally slower compared to chemical insecticides, which may limit their immediate effectiveness against severe pest outbreaks. Mass production, storage and shelf life of entomopathogenic fungi can pose practical challenges, potentially limiting their large-scale adoption by farmers. These limitations highlight the need for further field validation, formulation improvement and long-term studies to fully assess the practical applicability of entomopathogenic fungi and botanicals in integrated pest management programs.

Acknowledgement

AcknowledgementThe authors are thankful to the Head, Department of Agricultural Entomology, College of Agriculture, Dr. Balasaheb Sawant Konkan Krishi Vidyapeeth, Dapoli, Maharashtra, for providing the necessary facilities to conduct the present investigation.

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33. How to cite this article: Chavan A, Mule R, Gurav S, Raut R, Karmarkar M, Zende N. Pathogenicity of Metarrhizium anisopliae (Metschn.) and Beauveria bassiana (Bals.) against Spodoptera litura (Fabricius). International Journal of Advanced Biochemistry Research. 2026;SP-10(9):1965-1971. doi: https://w ww.doi.org/10.33545/26174693.2026.v10.i9Sy.10146