Characterization and Evaluation of Metarhizium spp. (Metsch.) Sorokin Isolates for Their Temperature Tolerance

A field survey was done in teak (Tectona grandis F.) forests in South India to explore the entomopathogenic effect of Metarhizium anisopliae (Ascomycota: Sordariomycetes) against teak defoliator, Hyblaea puera (Lepidoptera: Hyblaeidae). About 300 soils and infected insect samples were collected during the survey and thirty-six fungal isolates were isolated from soil and insect samples and characterized. The fungi were cultured on PDAY with dodine and antibiotics. Generally, the EPF culture was incubated at 27 °C in darkness for 15 days. Virulence of the Entomopathogenic Fungi (EPF) ability to germinate under cold and heat temperatures was assessed in a culture impregnated with conidia. In the experiment, it was found that for the first time Metarhizium quizhouense, Metarhizium robertsii, and Metarhizium majus species caused significantly higher mortality to hosts. These isolates of M. anisopliae, M. robertsii, M. majus, and M. quizhouense were all considered to be effective virulent and environmentally adaptive. The Metarhizium isolates were recommended as effective bio-control agents through the field investigation of teak defoliator Hyblaea puera from South India forest. This study paves the way to utilize the indigenous isolates of EPF for the control of teak defoliator and to combat the pests thatare resistant to insecticide.


Introduction
Entomopathogenic fungi (EPF) are widely used as bio-agents against various insect pests in agriculture, forest, other crop pests, and aquatic invertebrates. For about 100 years, myco-insecticides have been established [1] as one of the most promising pest biocontrol options in important insects or other arthropods [2,3]. However, it increases the low growth rate of opportunistic fungi [4][5][6].
The use of EPF as a pest management tool is affected by many biotic and abiotic factors. During conidia germination, they are affected by enhanced activity of the secondary metabolism [7][8][9][10][11]. Thermotolerance is an important factor that influences the survival, growth, development, and pathogenicity of EPF [12]. The Metarhizium sp., culture showed delayed conidial germination and mycelia growth when it was exposed to lower light [13,14]. Conversely, the EPF tolerated heat temperature during mycelia growth, which induces the high-protein metabolism [15,16].
During the survey, 300 soil samples were collected from 20 different climatic locations of South India, and 16 infected cadavers were collected [28] (Figure 1). The fungus was isolated from the soil using the Galleria bait method. The fungal infected was subsequently purified on Veen's medium containing [29] Dodine, subjected to selective media PDAY supplemented with antibiotics with the following composition (1% yeast extract (0.6 g), Chloramphenicol (100 µg/mL), Streptomycin (50 µg/mL), crystal violet (2 mg) in Potato Dextrose Agar with Yeast extract (PDAY), and then incubated at 27 ± 2 • C for up to 5 to 14 days with RH 80% to evaluate the fungal viability. EPF isolates were continuously sub-cultured on Sabouraud Dextrose Agar with Yeast extract (SDAY) slants at 28 • C and maintained at 4 • C. Further, microscopic permanent slide cultures were prepared for the identification of the Metarhizium sp. [30].

Insect Culture
The insect cultures of teak defoliator (Hyblaea puera) were maintained at ICFRE-Institute of Wood Science and Technology, Bengaluru, Karnataka, India. The culture was maintained on the natural host plant (teak) for two generations. For establishing the insect culture, larvae were grown on fresh tender teak leaf. The leaves were collected and stored in the plastic box that contained moist filter paper disc. The leaves were changed every 10 days. After establishment of the culture under laboratory conditions, the larvae were maintained on artificial media. The larvae were reared on ingredients of artificial diet (Kabuligram flour, Multivitamin and mineral mixture, Vitamin E, Streptomycin sulphate, Tetracycline hydrochloride, Sorbic acid, Ascorbic acid, and Formaldehyde) under laboratory conditions [31]. Individual larvae were then reared separately in sterilized plastic tubes (7 cm × 2.5 cm) [25]. The cups were covered with aluminum foil to prevent the larvae from escaping. The culture was maintained at the temperature at 28 ± 2 • C and 75 ± 5% RH.

Insect Culture
The insect cultures of teak defoliator (Hyblaea puera) were maintained at ICFRE-Institute of Wood Science and Technology, Bengaluru, Karnataka, India. The culture was maintained on the natural host plant (teak) for two generations. For establishing the insect culture, larvae were grown on fresh tender teak leaf. The leaves were collected and stored in the plastic box that contained moist filter paper disc. The leaves were changed every 10 days. After establishment of the culture under laboratory conditions, the larvae were maintained on artificial media. The larvae were reared on ingredients of artificial diet (Kabuligram flour, Multivitamin and mineral mixture, Vitamin E, Streptomycin sulphate, Tetracycline hydrochloride, Sorbic acid, Ascorbic acid, and Formaldehyde) under laboratory conditions [31]. Individual larvae were then reared separately in sterilized plastic tubes (7 cm × 2.5 cm) [25]. The cups were covered with aluminum foil to prevent the larvae from escaping. The culture was maintained at the temperature at 28 ± 2 °C and 75 ± 5% RH.

Bioassay of Metarhizium spp. against Hyblaea puera
Out of the 36 isolates that were screened, nine EPF isolates were evaluated through the YPD medium (0.2% yeast extract, 1% peptone, 2% dextrose with 1% casein/10 g chitin purified powder (25 ± 2 °C, 75 ± 5% RH) incubated at 15 days post inoculation and some modification in Pontecorvo [32]. Based on the preliminary pathogenicity studies, the virulent isolates of nine Metarhizium sp. were used for the bioassay against 3rd instar larvae of H. puera, using for the concentration of 1 × 10 3 to 1 × 10 6 conidial/mL to determine their pathogenicity. Each experiment was replicated three times. The conidial suspension was prepared using the 15 days old culture by scrapping the mycelial growth in sterile water

Bioassay of Metarhizium spp. against Hyblaea puera
Out of the 36 isolates that were screened, nine EPF isolates were evaluated through the YPD medium (0.2% yeast extract, 1% peptone, 2% dextrose with 1% casein/10 g chitin purified powder (25 ± 2 • C, 75 ± 5% RH) incubated at 15 days post inoculation and some modification in Pontecorvo [32]. Based on the preliminary pathogenicity studies, the virulent isolates of nine Metarhizium sp. were used for the bioassay against 3rd instar larvae of H. puera, using for the concentration of 1 × 10 3 to 1 × 10 6 conidial/mL to determine their pathogenicity. Each experiment was replicated three times. The conidial suspension was prepared using the 15 days old culture by scrapping the mycelial growth in sterile water and then filtering in double layer muslin cloth. The stock conidial suspension was prepared by counting the conidia using the improved Neubauer Heamocytometer. Uniform dispersion of conidial suspension was achieved by adding the wetting agents (0.2% Tween-80) to the stock solution. The teak leaf was dipped in the conidial suspension and provided for feeding to the 3rd instars larvae. In each replication, 15 larvae were used. The mortality counts were taken daily for 10 days. The dead larvae were placed on moist filter paper in Petri plates.

Production of Cuticle-Degradation Enzymes
To obtain the insect cuticle, infected 3rd insect larvae of the H. puera cuticles were used. The internal materials of the dead insect larvae were removed and dried to a constant weight in an oven at 80 • C. Then, the exoskeleton was macerated. The resulting powder was sieved and stored at −20 • C. At the time of experiment, the powder was suspended in an aqueous solution of potassium tetraborate (1%). The extract was subjected to a flowing steam for 20 min [33], dried in a sterile-air-flow cabinet to avoid contamination, and inoculated with 50 µL distilled water, which contained 5000 conidia. Following incubation (60 h) at 27 ± 5 • C, the cuticles were checked microscopically to ensure there was no bacterial contamination and enzymes were extracted as described [33]. The cuticle extract was placed on performed enzyme specific medium and incubated at 27 • C for 15 days after growth of the EPF species.

Estimation of Extracellular Enzyme Activity on H. puera
Among the nine isolates selected, Metarhizium spp. was grown in a selective medium for 12 days as described above. Protease activity was measured using casein as a substrate 0.6% (w/v). Casein solution (prepared by mixing 6 mg/mL casein with the reaction mixture) contained 100 µL of Tris-HCl buffer (20 mM, pH 8) and was inoculated with 1 mL of the enzyme at 30 • C for 10 min [22,23]. A measure of 0.5 mL of the supernatant was added (1.25 mL of 0.4 M Na 2 CO 3 and 0.25 mL of 2N Folin). It was then added to 0.4 M trichloroacetic acid (TCA) solution and the absorbance was measured at 650 nm in a spectrophotometer, which was incubated at 30 • C for 30 min. The amount of amino acids released was calculated from a standard curve plotted against a range of known concentrations of tyrosine. Afterwards, the product was centrifuged for 5 min at 10,000 rpm. One unit of enzyme was defined as the amount of enzyme that released 1 µg of tyrosine mL −1 of crude enzyme.
Chitinase enzyme activity of the culture supernatant was estimated using the substrate acid-swollen chitin [20,34]. To prepare the acid-swollen chitin, 10 g of chitin (purified powder from crab shells (HiMedia, Mumbai, Maharashtra) was suspended in 100 mL of HCl (35%, w/v) and the mixture was stirred at 5 min intervals for 1 h at room temperature in a fume hood. One unit of chitins was defined as the amount of enzyme that released Nacetyl D-glucosamine, or 100 µL dinitrosalicylic acid was added with incubation for 10 min inboiling water. Absorbance of the reaction mixture at 582 nm (A582) was measured after cooling the room temperature [35]. The procedure is described above, and the bioassay was conducted according to [32]. The relative germination of EPF at high temperature (45 • C & 48 • C) was checked on the 9 virulent isolates of Metarhizium spp., Out of 36 isolates, nine isolates has been found to be virulent isolates. The EPF was cultured on PDAY medium and kept in the dark at 27 ± 1 • C for 15 days. Mycelium of the fungus was harvested with a microbiological loop in sterile water and immediately suspended in Tween 80 solution. The suspension was filtered out through double layer muslin cloth to get conidia. The conidial concentration was prepared by counting the conidia under the Neubauer hemocytometer and the concentration of 10 6 conidia/mL was made. The 2 mL filtered suspension was transferred to a Borosil glass screw cap tube and kept in water bath at 45 • C and 48 • C for 5 ± 1 min. After 2, 4, 6, 8, 10, 12, 24, 36, 48, and 60 h of exposure with 45 • C and 48 • C, a 20 µL aliquot was placed on 4 mL of medium (PDAY) and one drop of lactophenol methyl blue was added, after which a coverslip was placed on it [36]. The germination was observed at 400× magnification. For the control, the conidial suspension was not exposed at 45 • C and 48 • C temperatures. All of the culture was incubated at 27 ± 2 • C. The observation was taken on the 300 conidia and the germination percentage was calculated by comparing the germination of the heated conidia with the control [37]. Only germinated isolates were taken for assessment.

Effect of Cold Temperature on Relative Germination (RG)
To know the effect of cold temperature (5 • C, 10 • C, and 20 • C) on relative germination, the 2 mL of filtered suspension was transferred to a (Borosil glass) screw cap tube and exposed to cold temperature at 5 • C, 10 • C, and 20 • C. After 5, 10, and 15 days of exposure, 20 µL of conidial suspension of each isolate was poured on 4 mL of medium by following the procedure that was used for the effect of heat temperature. For the control, the conidia were grown in ambient temperature (28 ± 2 • C) without exposure to cold temperature [38].

Analysis of Results
Significant mortality of the larvae (percent) was recorded from the bioassay and corrected according to Abbott's formula. Two-way ANOVA was used to compare the effects of the experimental and control treatments on the larval mortality. Prior to statistical analysis, data expressed as proportions were subjected to a regular arc transformation (one way analysis p < 0.001). The data were then subjected to analysis of variance (ANOVA), and the means were compared by Tukey test at the (p < 0.001) probability level using (Graph was generated using Origin Lab Professional Version 2021b software; URL link: www.OriginLab.com/2021b, accessed on 29 September 2021).

Isolation and Identification of Fungi
A total 36 isolates were obtained from forest soils and infected cadavers, which were collected from various locations [39]. There were four species of entomopathogenic fungi, viz., M. robertsii, M. quizhouense, M. majus, and M. anisopliae, from the soil and infected cadaver sample (Table 1). At the same time, other entomopathogenic fungi were grown in culture medium but failed to infect the host insects and were, therefore, not taken for further study. Moreover, during the isolation on PDAY culture medium, opportunistic fungi, viz., Aspergillus, Penicillium, Colletotrichum, and Scopulariopsis, were dominant and these fungi were not considered for further study in this identification. All these isolates were molecularly characterized using ribosomal internal transcribed spacer region (ITS) and DNA-directed RNA polymerase II subunit (RPB1) [39], and the Elongation factor 1-alpha (EP1α) genes sequence was analyzed as well, although it could not yet be submitted (NCBI).

Bioassay Tested Metarhiziumsp. Isolates against H. puera
Overall, these results revealed that the three isolates of M. anisopliae (DhMz4R), M. quizhouense (ArMz1W), and M. majus (VjMz1W) showed higher mortality, viz., 94.4 ± 2.03, 92 ± 4.19, and 90.8 ± 3.2 per cent, respectively, against the larvae of H. puera. However, there was no significant difference in the pathogenicity of isolates during 10 days after treatment (Figure 2). At 8 days after treatment, there was a significant difference in the pathogenicity among the isolates. The isolate of M. quizhouense (ArMz1W) showed significant percent mortality against the 3rd instar larvae of H. puera (Figure 2).

Bioassay Tested Metarhiziumsp. Isolates against H. puera
Overall, these results revealed that the three isolates of M. anisopliae (DhMz4R quizhouense (ArMz1W), and M. majus (VjMz1W) showed higher mortality, viz., 94.4 ± 92 ± 4.19, and 90.8 ± 3.2 per cent, respectively, against the larvae of H. puera. How there was no significant difference in the pathogenicity of isolates during 10 days treatment (Figure 2). At 8 days after treatment, there was a significant difference in pathogenicity among the isolates. The isolate of M. quizhouense (ArMz1W) showed si icant percent mortality against the 3rd instar larvae of H. puera (Figure 2).

Evaluation of Metarhizium sp. Isolates Effected Non-Extracellur Enzymes
The four species of Metarhizium sp., viz., M. robertsii, M. quizhouense, M. majus and M. anisopliae, were previously screened for pathogenicity [28]. The larvae were evaluated in four concentrations spanning from 1 × 10 3 to 1 × 10 6 conidia/mL (Table 2). It was observed that the mortality was increased as the concentration increased. Protease response during the observation (1 ×    Table 2).
The results indicate that M. quizhouense (ArMz1W) was most effective in causing the mortality against the 3rd instar larvae of H. puera through the production of chitinase and protease.

Extracellur Protease Enzymes Activity
During the experiment, the effect of protease enzymes on virulence levels was evaluated. Protease activities reached a maximum after 5 days of incubation and decreased thereafter for the control. The protease activity of nine isolates of Metarhizium sp. was significantly different from each other (Table 3; Figure 3). Through comparative analysis, the present study demonstrated that the isolates, viz., M. quizhouense (ArMz1W) and M. robertsii (ArMz6W), showed the highest significant on par protease activity compared with other isolates (22.4 ± 1.0 and 22 ± 0.7 U/mL), respectively, whereas the isolate of M. anisopliae (BgMz2S) showed the lowest protease activity.

Extracellur-Chitinases Enzymes Activity
Chitinase activity of M. anisopliae, M. majus, M. robertsii, and M. quizhouensewas varied significantly between the nine pathogenic isolates on 5 days post inoculation in chitin medium (CD(0.01) = 2.684). The comparative analysis demonstrated that the isolates, viz., M. robertsii (ArMz3S, ArMz3R, and ArMz1W) and M. anisopliae (WnMz1S), showed statistically on par chitinase activity. The chitinase activity was found to be at its maximum during the 5th day of inoculation (Table 4; Figure 4).

Extracellur-Chitinases Enzymes Activity
Chitinase activity of M. anisopliae, M. majus, M. robertsii, and M. quizhouense was varied significantly between the nine pathogenic isolates on 5 days post inoculation in chitin medium (CD(0.01) = 2.684). The comparative analysis demonstrated that the isolates, viz., M. robertsii (ArMz3S, ArMz3R, and ArMz1W) and M. anisopliae (WnMz1S), showed statistically on par chitinase activity. The chitinase activity was found to be at its maximum during the 5th day of inoculation (Table 4; Figure 4). Within the same row, mean ± S.E.M. followed by different letters are significantly different f each other followed by adjusted Tukey test.

Heat Tolerance and Cold Activity
High variability in conidia thermo-tolerance was observed among the Metar isolates. However, Metarhizium sp. isolates had a maximum germination after bei posed to cold germination temperatures at 20 °C for 15 days in-between (26.92 to 28 for M. anisopliae (Dhz14R) and M. majus (VjMz1W), respectively. In contrast, the s high variability in cold activity was observed when conidia wasexplored at 10 °C days, with results ranging from (20.77 to 24.05%) in M. anisopliae (DhMz4R) and M. (VjMz1W).Similarly conidial germination was greatly reduced at 5 °C for 15 days w exceptions of M. anisopliae (DhMz4R) and M. majus (VjMz1W), which had approxim lower germination ( Figure 5A-C).
Among the 9 Metarhizium spp., which were exposed at 45 °C and 48 °C for 2 h h, 8 h,and 10 h, the isolates of Metarhizium sp. varied in their conidial germination (F

Heat Tolerance and Cold Activity
High variability in conidia thermo-tolerance was observed among the Metarhizium isolates. However, Metarhizium sp. isolates had a maximum germination after being exposed to cold germination temperatures at 20 • C for 15 days in-between (26.92 to 28.22%) for M. anisopliae (Dhz14R) and M. majus (VjMz1W), respectively. In contrast, the second high variability in cold activity was observed when conidia wasexplored at 10 • C for 15 days, with results ranging from (20.77 to 24.05%) in M. anisopliae (DhMz4R) and M. majus (VjMz1W). Similarly conidial germination was greatly reduced at 5 • C for 15 days with the exceptions of M. anisopliae (DhMz4R) and M. majus (VjMz1W), which had approximately lower germination ( Figure 5A-C).

Discussion
The identification was done on the basis of morphotaxonomic characters revealed by microscopically inspecting the conidia forming mycelia for conidigenious structure. Each isolate was put into regular host passages using the natural hosts on three species ofM. anisopliae, M. robertsii, and M. majus isolate [2,3]. The effect of visible light during mycelia growth factor on the stress tolerance may exhibit an enhanced protein metabolism as suggested by the conidia [7,11,38].
In the present study, nine isolates of Metarhizium sp. were characterized for thermal tolerance and cold activity.For cold activity, the isolates were exposed to low temperature, viz., 5, 10, and 20 °C. M. majus (VjMz1W) showed higher conidial germination in all the cold temperatures. An earlier study reported that the Metarhizium sp., viz., M. flavoviridae and M. frigidium, were able to tolerate the cold temperature and produced conidia at the lowest temperature of their experiments (5 °C).
In the present investigation, chitinase and protease were found to show high correlation with mortality. Earlier literature has been reported that chitinases and proteases were the virulence factors for entomopathogenicity [19,42]. The hydrophobicity of conidia is one of the important parameters influencing fungus-insect interaction as it helps in ad-

Discussion
The identification was done on the basis of morphotaxonomic characters revealed by microscopically inspecting the conidia forming mycelia for conidigenious structure. Each isolate was put into regular host passages using the natural hosts on three species of M. anisopliae, M. robertsii, and M. majus isolate [2,3]. The effect of visible light during mycelia growth factor on the stress tolerance may exhibit an enhanced protein metabolism as suggested by the conidia [7,11,38].
In the present study, nine isolates of Metarhizium sp. were characterized for thermal tolerance and cold activity.For cold activity, the isolates were exposed to low temperature, viz., 5, 10, and 20 • C. M. majus (VjMz1W) showed higher conidial germination in all the cold temperatures. An earlier study reported that the Metarhizium sp., viz., M. flavoviridae and M. frigidium, were able to tolerate the cold temperature and produced conidia at the lowest temperature of their experiments (5 • C).
In the present investigation, chitinase and protease were found to show high correlation with mortality. Earlier literature has been reported that chitinases and proteases were the virulence factors for entomopathogenicity [19,42]. The hydrophobicity of conidia is one of the important parameters influencing fungus-insect interaction as it helps in adhesion of the fungus to the insect cuticle. This plays an important role in providing nutrients before and after the cuticle is penetrated [20,21]. However, chitinase is required only for a brief period during penetration of the host cuticle and is tightly regulated by chitin degradation products [22,23,33]. The fungus produces spores, which, upon germination, produce young germ tubes that penetrate through the cuticle, physically as well as chemically through the enzymes produced, such as proteases, chitinases, and lipases [43][44][45][46][47].
The insecticidal mechanism underlying in the pathogenicity of isolated fungi lies in presence of cuticle degrading enzymes which make paves way for the entry of EPF in the insect body [48][49][50][51][52]. To invade the insect cuticle, which is mainly composed of chitin and several other proteins, entomopathogens must secrete chitinase and protease enzymes [53][54][55][56][57][58]. Mechanism of enzymatic degradation of larval cuticle for hyphal penetrance by this fungus inside larval body is confirmed by the detection of chitinase and protease enzymes thatare secreted by the fungi.
The four species of Metarhizium spp., viz., M. anisopliae, M. robertsii, M. quizhouense, and M. majus, were obtained from insects infected and soil baiting methods. Therefore, EPF isolates revealed that for different parts of South India, indigenous isolates showed thebest mortality against 3rd instar larvae H. puera in the range of 84-92% within 5 days. Our results have indicated that three species, viz., M. robertsii, M. quizhouense, and M. majus, isolates were recorded for the first time and they had comparatively significantly higher mortality rates.

Conclusions
This study has revealed that M. anisopliae, M. robertsii, M. quizhouense, and M. majus are exceptionally effective and can be considered some of the best EPF isolates. Further investigation to control the infestation of teak defoliator H. puera should be performed on plantations in South India. The development of a possible new bio-control agent forthe invasive teak defoliator H. puera pest is now possible with a better understanding ofthe potential use of indigenous entomopathogenic fungi.