Isolation, molecular characterization of indigenous Metarhizium anisopliae (Metchnikoff) isolate, using ITS-5.8s rDNA region, and its efficacy against the Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae)

Abstract

Background

As a biological control agent, entomopathogenic fungus (EPF) can give an alternative to high-risk pesticides. Metarhizium anisopliae is one of the most promising pest controls EPF in the worldwide.

Result

On fungi-specific selective media, the EPF isolate SVPUAT was isolated from Western Uttar Pradesh, India. The isolate was identified as M. anisopliae (GenBank accession no. OP962431) after molecular screening utilizing PCR amplification with the internal transcribed spacer (ITS) region primer. In a contact toxicity experiment, the isolate SVPUAT was tested against the 4th instar larvae of Helicoverpa armigera (Hubner) at concentrations ranging from 10³ to 10¹⁰ spores ml⁻¹. The lethal time LT₅₀ and LT₉₀ values for the 4th instar larvae of H. armigera injected with 1 × 10¹⁰ spores ml⁻¹ were 3.46 and 5.54 days, respectively.

Conclusions

The present M. anisopliae isolate SVPUAT was identified using the ITS-5.8s rDNA region from GenBank and has showed significant pathogenicity to H. armigera. More research is needed to prove the efficacy against various pests of economic important as a legitimate choice for an integrated pest management program.

Background

One of the most destructive insect pests in the world is Helicoverpa armigera (Lepidoptera: Noctuidae), which is estimated to cause annual global economic losses of over 3 billion US dollars. Cotton, tomato, soybean, grain crops including corn and sorghum, chickpea and other pulse crops are among the most affected crops. In most cropping regions, this species' adults complete 4–6 generations per year with large migratory capacities (> 2000 km), high fertility and quick reproductive rates. The larvae are also polyphagous, with a wide variety of hosts, and have the capacity to enter diapause in order to survive unfavorable environmental conditions (Riaz et al. 2021).

Chemical pesticides have been used extensively to prevent harmful crop-attacking pathogens and insects, but this has had a detrimental impact on soil microorganisms and human health (Damalas et al. 2011). It can be replaced by efficient microbial control agents. The majority of toxic materials produced by microbial pathogens have been recognized as peptides, yet their structure, toxicity and specificity are significantly varied. In order to control insects, biological control uses biopesticides made from fungi. The demand for creating microbial bio-pesticides as a significant part of integrated pest management (IPM) programs and spurred necessity for chemical-free pesticides is rising around the world (Ravensberg 2015). The entomopathogenic fungus (EPF) met the requirements for a potential natural insect mortality factor (Boomsma et al. 2014). An established model for studying the biological control of pests by fungi is the entomopathogen M. anisopliae (Metchnikoff), which is well known to cause pathogenesis for many insects and has been used as mycobiocontrol agents for the biological control of agricultural insects worldwide (Sandhu et al. 2012). Quite recently, the internal transcribed spacer (ITS), a short gene sequence, was extracted from a standardized region of the genome and can be recovered and characterized as a unique identification marker for all biological identifications and diagnostic species, including fungi (Fergani and Yehia 2020). By locating the sequence in the international database that is most similar to the sample sequence, nearest neighbor algorithms are frequently used to match an unknown sample to a recognized species (Saitou and Nei 1987). The National Center of Biotechnology Information (NCBI) offers the Basic Local Alignment Search Tool (BLAST), a popular matching tool that looks for similarities between a query sequence and a sequence library (Altschul et al. 1990). The major goal of this study was to identify a new EPF isolate from Western Uttar Pradesh, India, and assess its effectiveness against the American bollworm or tomato fruit borer, Helicoverpa armigera (Hubner), in the laboratory conditions.

Methods

Sample collection

Several soil samples collected in late winter 2021 from various field in Western Uttar Pradesh, India, by gathering topsoil down to 40 cm depth with a metal shovel. Each site's samples were packed in sterile plastic bags, transported to the laboratory and stored at 4–8 °C until used.

Helicoverpa armigera (Hub.) rearing

The H. armigera larvae were brought to the lab from the tomato, chickpea and pigeon pea crop that is showed in the experimental field of Sardar Vallabhbhai Patel University of Agriculture and Technology, Meerut, Uttar Pradesh, India. According to the procedure provided by Wakil et al. 2011, it was raised on an artificial diet containing chickpea flour, red kidney beans, canned tomato paste, yeast, agar, vitamin mixture mixed in distilled water. The rearing condition maintained at a temperature of 27 ± 2 °C, 75 ± 5% RH.

Insect bait method

Insect bait technique recommended by (Zimmermann 1986) was adopted to screen and isolate the native species of EPF, using larvae of the wax moth, Galleria mellonella. Larvae were treated by warm water to avoid extensive webbing in the soil (Meyling and Eilenberg 2006). Soil samples were moistened and kept in petri dishes. Twenty medium-sized larvae were used for each soil sample. Samples were incubated at 25 ± 2 °C in the dark and inverted every day. Soil samples were examined after 5 days; departed bait larvae were collected and surface sterilized with 1% Na-hypochlorite to prevent external saprophytic fungi from growing on the dead cadaver. Dead larvae were placed in petri dish lined with a single layer of wet filter paper until signs of green muscardine were perceived. The fungal spore was grown on Sabouraud dextrose yeast agar (SDAY) medium. The petri dishes (5 cm × 1 cm) were incubated at 28 °C for 3–7 days. For extra refinement, single spore cultures were plated out from multispore cultures. Fungal strain showing good growth and spore production traits was selected, purified and identified according to microscopic observations following the taxonomic keys, using color atlas of pathogenic fungi for Metarhizium genus (Frey et al. 1979; Webster and Weber 2007).

Molecular identification of fungal isolate

Mycelia and conidia from the isolated fungal strain were inoculated, and a single spore colony was grown on Sabouraud dextrose yeast agar (SDAY) and incubated on a shaker (150 rpm) at 20 °C for 5–7 days. DNA extraction was done, following the manufacturer’s protocol, using GeneJET Genomic DNA Purification Kit (Thermo Scientific, Waltham, MA, USA). Qubit (Life Technologies, Carlsbad, CA, USA) was used to quantify the quantity of extracted DNA, and a Bioanalyzer was used to evaluate its quality (Agilent Technologies, Santa Clara, CA, USA). For the ITS1-5.8S-ITS2, PCR amplification and sequencing were performed. ITS1F (5′-TCCGTA GGTGAACCTGCGG-3′) and ITS4R (5′-TCCTCCGCTTATTGATATGC-3′) primers were used to amplify the partial gene region (White et al. 1990). For the PCR amplification, the final 25 µL reaction volume contained 1 × reaction buffer, 10 µM of each forward and reverse primers and 12.5 µL of DreamTaq PCR MasterMix (Thermo Scientific, Waltham, MA, USA). The reaction conditions were as follows: initial denaturation at 94 °C for 10 min, 35 cycles of 94 °C for 30 s, 55 °C for 45 s, 72 °C for 1 min followed by final extension at 72 °C for 10 min. PCR amplicons were purified and subjected to bidirectional Sanger sequencing at Centyle Biotech Pvt., Ltd., New Delhi, India.

A BLASTN search was conducted on the NCBI database to classify associated sequences. The BioEdit 4.8.9 software (Hall 1999) was used, and the alignment was corrected manually. CLUSTAL-X was used to perform multiple sequences alignment. A phylogenetic tree was reconstructed using MEGA ver. X (Kumar et al. 2018) by maximum likelihood method based on the Jukes–Cantor model. Branch support was estimated with 1000 bootstrap replicates under appropriate substitution models. Thereafter, tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances were used to infer phylogenetic tree.

Efficacy of the M. anisopliae strain against 4th instar larvae of H. armigera

The 4th instar larvae of H. armigera were used in the present study. A total of 45 larvae (15 larvae per replication) were dipped for 20 s in prepared concentrations (10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹ and 10¹⁰ spores ml⁻¹), water as control and released in fresh chickpea food including tender leaves, shoots and pods. The extra moisture of treated larvae was soaked on sterilized tissue paper. The experiment was conducted in the plastic transparent cage with complete randomized design (CRD) with four replications at room temperature. Moreover, the method was repeated twice to further confirm the results. The mortality rate of different concentration of M. anisopliae was recorded on daily basis till five days, and percent mortalities were calculated by Abbot’s formula as follows:

$$\frac{{{\text{Survival}}\;{\text{percent}}\;{\text{in}}\;{\text{control}} - {\text{Survival}}\;{\text{percent}}\;{\text{in}}\;{\text{treatments}}}}{{{\text{Survival}}\;{\text{percent}}\;{\text{in}}\;{\text{control}}}} \times 100$$

Statistical analysis

According to Feng et al. 1992, the percentage of mortality was computed. SPSS (ver. 21) program was used to determine the lethal time LT₅₀ and LT₉₀ values, which were necessary to kill 50 and 90% of the population, respectively (Finney 1971).

Results

Isolation and morphological characterization

The dead bait larvae of G. mellonella were separated, sterilized and stored in a petri dish until infection signs appeared after 5 days of soil sample inspection. The infection test revealed that the dead bait larvae had signs of infection, were less energetic feeders and moved slowly and sluggishly. After three days or more, green mycelium developed on the outer surface and intersegmental surface of deceased larvae. For morphological characterization on selected Sabouraud dextrose yeast broth, fungal samples were obtained, and these samples revealed morphological and cultural characteristics resembling those of Metarhizium species. Circular ring colonies were formed with green powdery appearance; conidiospores were short, one celled, smooth, hyaline and densely grouped in whorls. Instead of plating on medium, the isolation of EPF using the Galleria bait approach is proved to be an efficient strategy for screening of native species.

Molecular characterization

The ITS1-5.8S-ITS2 gene region from the present M. anisopliae isolate SVPUAT was deposited in GenBank as accession no. OP962431 with a total length of 557 bp and a 49.90% GC content. Homology comparison of the submitted sequence of the present isolate with a various of different genotypes belonging to the Hypocreomycetidae subclass revealed a unique genetic sequence. Significant degree of similarity (> 90%) between this isolate and others from GenBank was determined by calculating the percentage of sequence identity. The maximum sequence identity (99.20%) with lowest divergent value was recorded between the present isolate and M. anisopliae (MW599203), followed by 95.40–92.60% with other M. anisopliae isolates (Table 1).

Table 1 Pairwise percent sequence identity of Metarhizium anisopliae isolate SVPUAT (OP962431) with other the closely related species

Phylogenetic analyses were performed on the basis of partial and complete ITS1-5.8S-ITS2 sequence alignments using the maximum likelihood technique based on the Jukes–Cantor model representing two orders, Hypocreales and Glomerellales (Fig. 1). The ML tree revealed a cluster including all of the sordariomycetes taxa, clearly separated into two different clades (Fig. 1). The first clade was separated into two subclades, one of which had all of the Clavicipitaceae species, while the other comprised other Cordycipitaceae, Nectriaceae, Incertae sedis and Bionectriaceae taxa. Colletotrichum graminicola belonged to the Glomerellaceae, which was part of the Glomerellales. The present M. anisopliae isolate SVPUAT revealed a well-resolved unique clade with members belonging to the family Clavicipitaceae and deeply embedded inside the genus Metarhizium with close relationship to the previously identified M. anisopliae (MW599203, KU364600, MN727141, OP268209, OM373005, MK046658, KX809518, AF218207, LT220715, KU983799, HM055446, KU593545, LR792769 and LR792750) (Fig. 1).

Fig. 1

Molecular phylogenetic analysis by maximum likelihood method based on the Jukes–Cantor model. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying the Maximum Parsimony method. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. Evolutionary analyses were conducted in MEGA X

Efficacy of the M. anisopliae strain against 4th instar larvae of H. armigera

H. armigera larvae showed extensive straight mortality percentage when inoculated with increasing concentrations (10³, 10⁴, 10⁵, 10⁶, 10⁷, 10⁸, 10⁹ and 10¹⁰ spores ml⁻¹) of the native M. anisopliae strain SVPUAT (Table 2). The highest concentration (10¹⁰ spores ml⁻¹) showed highest mortality percentages against 4th instar larvae of H. armigera ranged from 0.00 to 87.77% after 24 and 120 h, while the least concentration 10³ spores ml⁻¹ showed mortality percentages ranged from 0.00 to 41.11% after 24 and 120 h. Typical symptoms developed on the infected larvae when kept under high humidity in petri dishes within 5 to 8 days with complete sporulation. The lethal time LT₅₀ and LT₉₀ value for the 4th instar larvae of H. armigera inoculated with the concentration of 10¹⁰ spores ml⁻¹ was 3.46 and 5.54 days, respectively. The results indicated that the mortality rate was directly proportional to increasing spore concentration and exposure time.

Table 2 Efficacy of Metarhizium anisopliae strain against 4th instar larvae of Helicoverpa armigera

Discussion

Expansion and extreme use of chemical pesticides to control serious crop-attacking pathogens and insects have led to many negative side effects on human health and environmental biodiversity, including soil microorganisms (Damalas et al. 2011). Therefore, using beneficial microorganisms as a biocontrol agent or as an effective and ecofriendly alternative to chemical applications has emerged as a powerful tool with auspicious success under several experimental and field conditions (Shah et al. 2003; Li et al. 2010; Ramanujam et al. 2020).

Keller et al. (2003) and Meyling and Eilenberg (2006) suggested that isolation and identification of indigenous isolates found in soil could be obtained by baiting soil samples with G. mellonella larvae. Since species-level identification based only on physical features was ineffective, the genetic diversity of Metarhizium species is detected utilizing the ITS and additional genomic loci (Pérez-González et al. 2014; Korosi et al. 2019; Mongkolsamrit et al. 2020).

The internal transcribed spacer region (ITS1-5.8S-ITS2) was used for this study in accordance with White et al. (1990), Curran et al. (1994). According to Zare and Gams (2008), the nuclear ITS1-5.8S-ITS2 region has been discovered as a helpful tool for providing molecular sequence data appropriate for building fungal phylogenies. The targeted gene region was amplified by PCR using the ITS1F/4R primers, which were recommended by White et al. (1990) as universal primers for fungal DNA barcode.

Nguyen et al. (2007) reported 87.00% mortality against 4th instar larvae of H. armigera after two weeks of exposure, and Fite et al. (2020) reported 71.00% mortality at 1 × 10⁹ spores ml⁻¹ after eleven days of post-treatment. Similarly, Rijal et al. (2008) and Sabry et al. (2011) reported that conidial concentration 1 × 10⁷ spores ml⁻¹, 65.33%, 86.67%, 60.00% and 80.00% mortality of second and third instar larval after six, seven and ten days post-treatment, respectively. The present findings are also supported with Taliyan et al. 2020 reported that 78.15% larval motility at 1.8 × 10⁹ conidia ml⁻¹. The LT₅₀ value increased with decrease in conidial concentration and 6.20 days & 3.71 days at 1 × 10⁹ spores ml⁻¹ conidial concentration reported by Tahir et al. (2019) and Fite et al. (2020), respectively. Nahar et al. (2008) and Nguyen et al. (2007) found 3.4 days and 3.00 days for 1.0 × 10⁷ ml⁻¹ conidial concentration, respectively. In present study, the lethal action of M. anisopliae against the 4th instar larvae of H. armigera is proportionally dependent on the concentration. The highest concentration is more efficacious to the insect (Yasin, et al. 2019).

Conclusion

The present M. anisopliae isolate SVPUAT was identified using the ITS-5.8s rDNA region deposited in GenBank and shown considerable pathogenicity toward H. armigera. Further, more research is needed to assess its efficiency as a biological pest control agent in India.