Novel Entomopathogenic Fungi of Tamil Nadu Soils and Their Pathogenicity on Waxmoth Larva (Galleria mellonella)

Pest concerns are a fundamental component of present-day agricultural practices and are typically brought about by agroecosystems that are too simplified in addition to the development of less stable natural ecosystems. Natural enemies, which keep pests in check, are annihilated when broad-spectrum pesticides are used. Because of this, scientists are now urging to focus on developing environmentally beneficial alternatives. Biocontrol is the most effective substitute among them. In a sustainable pest management project, biological plant protection with entomopathogenic fungi is vital. Entomopathogenic fungi offer an indispensable part in the microbial control of insect pests. According to Sinha et al., (2016), entomopathogenic fungi play an imperative role in the microbial management of insect pests. Although there are other biological control strategies involving bacteria, viruses and protozoa, EPF is the most significant because of a variety of characteristics, such as simple production processes, the availability of numerous strains that have already been identified and the over-expression of endogenous and exogenous toxins and proteins (St Leger and Wang, 2010). The two basic requirements to successfully employ an entomopathogenic fungus as a myco biocontrol agent are the insect’s sensitivity and the fungus’s virulence.
       
However, it is not a novel concept to use microbes to eradicate pests. The first entomopathogenic fungus that brings about the insect disease white muscardine was discovered and described by Agastino Bassi. This fungus was later given the name Beauveria bassiana. Entomopathogenic fungi still have many unrealized potential benefits despite being commercialized recently (Mantzoukas et al., 2022). Entomopathogens are preferred to kill insects at different phases of their life cycles due to their biopersistence and eco-friendliness.
       
Clonostachys rosea is a filamentous fungus found in diverse environments featuring different kinds of soil types and decaying plant material. It was formerly known as Gliocladium roseum. The morphology, ecology, teleomorph and DNA sequence data of Gliocladium roseum were significantly different from those of other Gliocladium species, which led to the reclassification of Gliocladium roseum as Clonostachys rosea (Sun et al., 2020). It is known as a parasite of nematodes and fungal pathogens and invades living plants as an endophyte and a saprophyte, respectively, as well as consuming soil-based substances. Purpureocillium lilacinum formerly called Paecilomyces lilacinus is an excellent soil fungus for biological control. The effectiveness of this species has been comparable to that of commonly used nematicides and it is also effective at controlling insects.
       
Talaromyces was previously considered as the genus Penicillium. Relying on their traditional classification in the guild of fungal antagonists, Talaromyces sp. (Eurotiales: Trichocomaceae) have merely sometimes been associated with insects. Species such as T. flavus, T. pinophilus and T. purpureogenus are already known to produce bioactive chemicals and play a crucial part in the antagonism against plant diseases (Nicoletti and Becchimanzi, 2022). The genus Penicillium contains fungi that have been identified for their entomopathogenic behavior and certain species have been suggested as efficient biocontrol agents. At least 62 Penicillium species have been discovered in conjunction with insects (Nicoletti et al., 2023).
       
On the entomopathogenic potential of Clonostachys rosea and Purpureocillium lilacinum, there is very little research. Whether Talaromyces pinophilus and Penicillium simplicissimum exhibit an impact on insects, is not yet known. Hence, it is necessary to investigate novel fungal species to use them for pest management. The current investigation’s goal was to assess novel native Tamil Nadu entomopathogenic fungus strains pathogenic potential.

The present study was conducted at Insectary, Tamil Nadu Agricultural University, Coimbatore during 2021-2022. Fungi were isolated from the soils of Tamil Nadu using the soil baiting method described by Zimmermann (Inglis et al., 2012). Soil samples were collected from a depth of 15 cm, processed and preserved for further use. The test insect Galleria mellonella was cultured at Insectary, Tamil Nadu Agricultural University, Coimbatore, at 25°C. The artificial diet recommended by Gitanjali (2021) was modified and used for rearing Galleria mellonella.
       
Morphological identification of isolated entomopathogenic  fungi was conducted by observing the colour of the fungal colony (front and reverse), texture, appearance and shape of the spore. Fungal isolates were confirmed by following DNA extraction by the Ctab method (Zhang et al., 2010). The extracted DNA was amplified using ITS primers ITS1 (forward) and ITS4 (reverse) and sequencing was conducted at Syngenome (OPC) Private Limited, Coimbatore. The sequences were run through BLAST and compared with data already present in NCBI. Sequences were aligned using BIOEDIT 7.2 software and then submitted to NCBI for accession numbers. A phylogenetic tree was constructed using the MEGA 11.0 software. For this, sequences of isolated fungi and other sequences of fungi from Genbank were aligned using MUSCLE. A neighbor-joining tree was constructed using the Tamura-3 model (Tamura et al., 2007). Bootstrap analysis with 1000 replications was performed.
       
Novel fungal isolates were tested for their virulence against Galleria mellonella. Fifteen larvae were used for each fungal isolate and were replicated four times. The spore suspension was prepared by scraping the fungi into sterile distilled water, homogenized and the number of spores was counted using a haemocytometer. Spore concentration was fixed to 2.5 × 10⁸ spores/ml, 2.5 × 10⁶ spores/ml and 0.1% tween 80 was added as a surfactant. Wax moth larvae were dipped in spore suspension, air-dried and placed in plastic boxes lined with filter paper at the bottom. Control larvae were treated with 0.1% tween 80. Larvae were observed each day for up to 10 days. Isolation of fungi from infected cadavers was performed to obtain a pure culture of fungi. Percent mortality was corrected using Abbott’s formula (Abbott, 1925) and LC₅₀ and LT₅₀ were calculated using the probit analysis method.

Fungal isolates
 
Fungal cultures isolated were identified as Clonostachys rosea, Penicillium simplicissimum, Talaromyces pinophilus from the soils of Ooty (The Nilgiris) and Purpureocillium lilacinum from the soils of Kondayampalayam (Coimbatore) (Table 1) by comparing their sequences with sequences in NCBI database. They were named as TNAU OTD 1 (Cr), TNAU KDP 1 (Pl), TNAU OTY 1 (Tp) and TNAU OTE 1 (Ps). In the phylogenetic tree (Fig 1), Clonostachys rosea split from a common ancestor with Purpureocillium lilacinum, Talaromyces pinophilus and Penicillium simplicissimum at a common node. Subsequently, Purpureocillium lilacinum diverged from a common ancestor with Talaromyces pinophilus and Penicillium simplicissimum at another node. Talaromyces pinophilus and Penicillium simplicissimum on the other hand, share a close evolutionary relationship as they both originated from a common ancestor.
 

 

 
Pathogenicity assay
 
All four isolates caused mortality in Galleria mellonella larvae at both concentrations (2.5 × 10⁸ spores/ml and 2.5 × 10⁶ spores/ ml) and no death was observed in the control (Fig 2). All isolates performed equally well, while TNAU OTE 1 (Ps) caused 95% mortality at higher concentration compared to TNAU OTD 1 (Cr), TNAU KDP 1 (Pl) (91.7%) and TNAU OTY 1 (Tp) (88.3%). The variation in the pathogenic ability of these entomopathogenic fungi against Galleria mellonella may be attributed to variances in the toxins or enzymes released by the fungi, which play a crucial role in the infection process. Additionally, the fungi’s capability to evade the host’s immune response and the impact of environmental factors such as temperature, humidity and pH on their pathogenicity may also contribute to these differences. These fungi also performed well at low concentration by causing nearly 50% mortality in wax moth larvae. These results prove that these fungi were efficient in insect control. Toledo et al., (2006) observed that Oncometopia tucumana experienced a mortality rate of 82.5% within 14 days when exposed to 6 × 10⁵ spores/ml of Clonostachys rosea. Anwar et al., (2018) documented that Clonostachys rosea exhibited virulence against Bemisia tabaci resulting in 50.42% mortality in nymphs and 23.54% mortality in adults after 6 days. Mohammed et al., (2021) reported Clonostachys rosea’s efficacy in controlling coleopteran storage pests, showing mortality rates ranging from 70.7 to 75.7%. Our study’s results complement these findings, demonstrating a mortality rate 45% at a concentration of 2.5 × 10⁶ spores/ ml of Clonostachys rosea, with an LT₅₀ of 4.67 days. Penicillium simplicissimum and Purpureocilium lilacinum exhibited LC₅₀ values of 7.69 × 10⁵ and 8.5 × 10⁵ spores/ml respectively, resulting in 50% population mortality within 3.95 days and 4.37 days (Table 2). A similar finding was reported by Sun et al., (2021) on the pathogenicity of Purpureocillium lilacinum against Bemisia tabaci.