Entomopathogenic fungi as a promising biological control agent against banana fruit scarring beetle, Basilepta subcostata (Jac.) (Chrysomelidae: Coleoptera)

Banana fruit scarring beetle (BFSB), Basilepta subcostata (Jac.) (Chrysomelidae:Coleoptera), is an important insect pest feeds on leaf and fingers, which affects the cosmetic value of the fruit. The pest is distributed in Assam, Bihar, West Bengal, Chhattisgarh, and North-eastern Hill regions of India. The pest is currently managed by foliar spray with insecticides. In order to identify eco-friendly control of the pest, attempts were made to isolate microbial agent and evaluate their potential to control the pest. A total of 27 entomopathogenic fugal isolates were obtained from Odoiporus longicollis (Oliver), Cosmopolites sordidus (Germar), Basilepta subcostata (Jac), and other insect Galleria mellonella (Fabr). Based on colony morphology, the collected fungal isolates were identified as Metarhizium spp. (17) and Beauveria spp. (10). Through ITS sequencing, the fungal isolates were further characterized at species level as B. bassiana (8), B. brongniartii (2), M. anisopliae (8), M. robertsii (6), M. guizhouense (2), and M. pinghaense (1). Their sequences were submitted in GenBank and obtained accession numbers. Among 27 isolates tested against B. subcostata under laboratory conditions, 3 isolates (M. anisopliae NRCBEPF-36, M. pinghaense NRCBEPF-7 and B. brongniartii NRCBEPF-27) recorded 100% beetle mortality, followed by 11 isolates with 95–99% and 13 isolates with 88–93% within 8 days of treatment. This study highlights the two native North East India isolates B. brongniartii NRCBEPF-27 (MT151781) and M. anisopliae NRCBEPF-36 (MT140308) showed the significance to use as potential biocontrol agents against banana fruit scarring beetle B. subcostata. Further experiments under field conditions are required to evaluate their biological control efficacy against the pest.


Background
Banana is the most important fruit crop and cultivated across 130 countries in the tropical and subtropical region. More than 200 species of insect and non-insect pests have been reported (Simmonds, 1966). The banana leaf and fruit scarring beetle Basilepta subcostata (Jacoby) (Chrysomelidae: Coleoptera) is considered as one of the most economically important pests in North-Eastern India (Mishra et al., 2015 andBhagabati andDeka, 2016). Recently, taxonomic descriptions with illustrations of the genitalia of banana fruit and leaf scarring beetle led to correct identification of B. subcostata (Prathapan et al., 2019).
Entomopathogenic fungal (EPF) strains have been commercialized as a biocontrol agent, and the majority of them have been developed from Ascomycota, Beauveria spp. (Bals-Criv.) Vuill. (Hypocreales: Clavicipitaceae) and Metarhizium spp. (Metschn.) Sorok. (Hypocreales: Clavicipitaceae) (Rehner et al., 2011 andVelavan et al., 2017). The efficacy of Beauveria spp. has been well established as a potential biocontrol agent of Cosmopolites sordidus (Akello et al., 2008 andLopes et al., 2013). EPF have been isolated and evaluated against banana stem weevil, Odoiporus longicollis and showed promising results Alagesan et al., 2019). Additionally, the combined use of myco-insecticides and the full or reduced concentration of botanical/chemical insecticides is a promising pest-control option for minimizing adverse chemical effects (Samui et al., 2004 andChoudhary et al., 2010). In this study, an attempt was made to collect, isolate and identify promising EPF as a biocontrol agent for managing scarring beetle of banana.

Survey and collection of fungal infected insects
Survey was conducted to collect healthy and infected insects in Tamil Nadu, Bihar, and North-Eastern region (Assam) in India (Table1). Naturally infested banana stem weevils (Odoiporus longicollis Oliver), rhizome weevils (Cosmopolites sordidus Germar), several insect cadavers of leaf scarring beetle (Basilepta subcostata Jac), and other insect pests of the greater wax moth (Galleria mellonella L.) were collected (Table 1) and surface-sterilized with 1% sodium hypochlorite (NaOCl) for 30 s, followed by 3 washes with sterile distilled water to prevent external saprophytic contaminations. Dead insects were kept in Petri plate lined with a single layer of wet filter paper until signs of muscardine were observed. After that, they were dissected and placed into Petri plates containing potato dextrose agar yeast extract (PDAY) medium amended with antibiotic (1% yeast extract 0.6 g, 100 μg/ml chloramphenicol, 50 μg/ml streptomycin, 2 mg crystal violet), and the plates were incubated at 28±1°C and 90% RH to facilitate growth and sporulation of the fungus. The cultures were purified using Veen's medium containing Dodine (Veen and Ferron, 1966). Morphologically distinct colonies Beauveria and Metarhizium were picked individually and inoculated on PDAY medium and incubated at 27°C for 15 days. After that, the fungal isolates were transferred to PDAY slants and used for further study. Also, slides were prepared (Riddell, 1950) from 5 days old culture for the identification and the cultures were stained with permanent stain (Cotton blue-Lacto phenol) and mounted using dinbutylphthalata in xylene (DPX). Identification was done on the basis of morphotaxonomic characters through microscopic inspection of conidia and conidiogenous structure (Bischoff et al., 2009 andKepler et al., 2014). Also, for a long-term storage, spores were obtained from 5-dayold culture and mixed in 10% glycerol and stored at −30°C until use.

Molecular characterization through sequencing of ITS region of entomopathogenic fungi (EPF) isolates
DNA was extracted from 50 to 100 g of lyophilized mycelium of the fungus from 10-15-day-old cultures grown in potato dextrose broth (PDB) following cetyltrimethylammonium bromide (CTAB) methods (Rogers and Bendich, 1994). Extracted DNA was suspended in EB buffer (10 mMTris-Hcl, pH 8.5) and stored at -20°C until used. The total DNA concentration was measured in spectrophotometer at 280 nm. PCR amplification of the non-coding region of internal transcribed spacer (ITS) of the nuclear ribosomal DNA was performed using a thermal cycler (Bio-Rad, T100 TM Thermal cycler). Each PCR reaction consisted of 25 μl reaction mixture containing 1X Taq buffer, 0.4-0.6 mM of each primer (ITS1 5′-CTCTCCAAACTCGGTCATTT-3′ and ITS2 5′-ATATGCTTAAGTTCAGCGGGT-3′), 0.2 mM of dNTP mix, 1U of Taq DNA polymerase (GeNei), and 20-50 ng/μl template DNA. The PCR amplification program had one initial cycle at 95°C for 3 min, followed by 35 cycles of denaturation at 95°C for 1 min, annealing 50°C for 45 s extension at 72°C for 2 min, a 2 nd final extension at 72°C for 10 min. The PCR products were separated on 1% agarose gel containing 0.05% of EtBr in 0.5x TBE buffer (45 mM Tris acetate-1 mM EDTA, pH-8). A 100-bp DNA ladder (GeNei) was run on the same gel, and the size marker and bands were visualized under ultraviolet irradiation. The products (650 bp) from ITS region of 27 isolates were eluted from gel and sequenced using forwards and reverse primers, at sequences facility (Chromus Biotech, Bangalore, India). The sequences were analyzed in BLAST https://blast.ncbi.nlm.nih.gov, biosrv.cab.unina.it/webcap3. Based on similarity, the isolates were named and the sequences were submitted in NCBI-GenBank and obtained accession numbers.

Bioassay of EPF on Basilepta subcostata
The banana fruit scarring beetles (B. subcostata) were collected from the infested banana field and reared in entomology laboratory of the institute using perforated plastic containers at room temperature. The leaf midribs of cv. Jahaji (a susceptible host) were utilized for feeding the beetles. The entire test beetles were kept in these containers for at least 1 month before use in the experiments for acclimatization. Whereby beetles were observed using a hand lens and males were differentiated from females on the basis of punctuations on their rostrums spreading beyond the point of antennae insertion. These adult beetles were utilized for the present study.
The 27 indigenous EPF isolates were used for bioassay against B. subcostata. The cultures were grown on PDAY medium for 10 to 15 days at 25 ± 0.5°C. The mycelia mat containing spores were harvested in tube using sterile distilled water 100 ml with continuous stirring the contents in a tube, filtered through a single layer of muslin cloth to remove debris and mycelia. Conidial suspension (CS) was prepared as per the procedure described by Lopes et al. (2013) and Velavan et al. (2017). The conidial concentration was estimated using hemocytometer under light microscope. Subsequently, the conidial suspension was diluted to make a final suspension of 1×10 5 spores/ml with 0.1% Triton X-100, 0.2% Tween 80 and 0.1% glycerol. B. subcostata was transferred aseptically to fresh plastic boxes (10-cm diameter and 30-cm height). The conidial suspension of EPF isolates (27) were swabbed on leaf sheaths (8-10-cm lengths) individually. For comparison, 2 commercial isolates; each M. anisopliae and B. bassiana was also included in the experiment. Each treatment had 5 replications, and each replication consisted of 15 beetles. In control, similar numbers of beetles were introduced on to the leaf sheaths treated with water, 0.1% Triton X-100, 0.2% Tween 80, and 0.1% glycerol alone. Observations were taken on morality of insects at 3, 5, and 8 days after inoculations. Infected insect cadavers were transferred to wet chamber and the fungus was re-isolated and confirmed based on culture spore morphology.

Statistical analyses
Obtained data on beetle mortality rates were Arc Sin transformed and analyzed in Completely Randomized Design using Web Agri Stat Package 2.0 (WASP) https://ccari.res.in/wasp2.0/.

Isolation and characterization of EPF isolates
During 2017-2018 in Tamil Nadu, Bihar, and Assam fungal-infected insects such as corm weevil, pseudostem weevil, and scarring beetle besides Galleria were collected, reared, and identified. In total, 27 fungal isolates were isolated and purified from the dead beetles and Galleria larvae Table 1. Based on colony color and spore morphology, the isolates were broadly classified as Metarhizium (17 isolates) and Beauveria (10 isolates) as they produced green and white colonies, respectively. Among the fungal isolates, 13 isolates consisting of 8 Beauveria and 5 Metarhizium were obtained from dead adult beetles of B. subcostata, while 8 isolates of Metarhizium and one isolate of Beauveria were isolated from Odoiporus longicollis. Also, one isolate of Beauveria and 4 isolates of Metarhizium were obtained from adult of Cosmopolites sordidus and larvae of Galleria, respectively. Observations on morphological characteristic indicated that Metarhizium isolates produced 4 different colonies and conidial morphologies, while Beauveria showed 2 morphological differences (Fig. 1).

Molecular characterization of EPF isolates
Molecular characterization of EPF cultures through sequencing of ITS regions and BLAST analysis with data base sequences revealed that they belonged to B. bassiana (8 isolates), B. brongniartii (2 isolates), M.  (Choudhary et al., 2010;Bhagabati and Deka 2016;Saikia et al., 2018 andDaizy et al., 2019). In India, bananas are consumed majorly as raw fruits and some extent as processed food. Therefore, application of chemical pesticides on banana is hugely discouraged. Alternatively, entomopathogens have been promising in management of pests in different crops. However, success of the biocontrol agent is mainly depending on efficacy of specific strains against target pest and performance of such agents in the given environment. Hence, the present study focused to collect EPF isolates from North, North East, and Southern parts of India and screen against B. subcostata.
In total, 27 EFP isolates were obtained and based on spore and colony morphologies, and they were characterized (Bischoff et al., 2009;Rehner et al., 2011;Ravindran et al., 2015;and Ramanujam et al., 2015) majorly as Metarhizium (17 isolates) and Beauveria (10 isolates). Molecular identification of fungal species is generally carried out through sequencing of ITS regions (Bischoff et al., 2009;Rehner et al., 2011;Lopes et al., 2013;Kepler et al., 2014;Ravindran et al., 2015;and Ramanujam et al., 2015) and BLAST analysis with data base sequences. Similarly, sequencing of ITS region of EPF cultures revealed that they belonged to B. bassiana, B. brongniartii, M. anisopliae, M. robertsii, M. guizhouense, and M. pinghaense. In the present study, majority of isolates were belonging to Metarhizium spp. followed by Beauveria spp. Over all, comparatively Metarhizium isolates out performed than Beauveria isolates against B. subcastata. Though management strategy for lowering infestation of fruit scaring beetle by biopesticide (B. bassiana) was profitable (Samui et al., 2004 andChoudhary et al., 2010), the present study results corroborated with Tuncer et al. (2019) study where Metarhizium isolate showed better results than Beauveria on controlling the coleopteran insect (Ambrosia beetle, Xylosandrus germanus).