The experiment was conducted during the 2021-2022 at Tamil Nadu Agricultural University, Coimbatore. Soil samples were collected from Manipur and were processed for further studies at Insectary, Department of Agricultural Entomology, Nadu Agricultural University, Coimbatore.
Soil sample collection
The soil samples were collected from various sites across 10 districts of Manipur (Fig 1), ranging from cultivated lands to forest areas. The geographic coordinates of each sample collection site were recorded (Table 1). From each district, a total of 10 samples were collected, with each sample being a mixture of five different soil samples. Approximately 200 grams of soil were collected from each site, from a depth of 10-15 cm below the ground, in five randomly selected points. These soil samples were mixed together and stored in double zip lock bags. Overall, 10 samples were collected from each district, resulting in 100 samples in total, which were subsequently transported to Tamil Nadu Agricultural University, Coimbatore for further analysis. The transportation process took several days, necessitating the preparation of the samples for transport without compromising the viability of entomopathogenic fungi, as per the methods outlined by
Clasen et al., 2020. All the soil samples were stored in a refrigerator at 4-5
oC until further processing. In the laboratory, each bag containing soil was thoroughly mixed and homogenized manually.
Isolation of entomopathogenic fungi from soil samples
Entomopathogenic fungi were isolated from the collected soil samples using ‘
Galleria bait method’ developed by
Zimmermann (1986).
Rearing wax moth
Greater Wax moth (
Galleria mellonella) larvae were obtained from Insectary at Tamil Nadu Agricultural University, Coimbatore. These larvae were reared in plastic boxes. An artificial diet was prepared for feeding the larvae of the wax moth while the adult moths were fed on 30% honey solution. The diet formulation was prepared following the method provided by
Singh et al., (2019), with slight modification. The ingredients used for making artificial diet included wheat flour, corn flour, milk powder, yeast, honey, glycerine, wheat bran, bees-wax, vitamin E capsule and Streptomycin sulphate. The diet was stored at 4
oC in a refrigerator for further use.
Soil baiting
Prior to baiting, the wax moth larvae were subjected to heat treatment to prevent webbing in soil. Water was heated to 56
oC in water bath and then larvae were treated for 10 seconds in the hot water. Afterward, the larvae were placed on a dry tissue paper and kept in dark condition for 4-5 hours, following the method described by
Woodring and Kaya (1988). The soil was then transferred from the bag to transparent plastic cups, leaving some air space at the top. If the soil was excessively dried, then it was moistened with water to provide required moisture for the growth of entomopathogenic fungi. Subsequently, five larvae were placed in each box. The boxes were then incubated at a temperature of 25±5
oC for a period of 21 days. Initially for seven days, the boxes were inverted once a day for increasing the exposure of more surfaces of larvae and left undisturbed for the remaining days. After 21 days, the larvae that showed signs of fungal infection were treated with 1% sodium hypochlorite solution for 2 minutes, followed by washing in distilled water three times. The larvae were then placed on potato dextrose agar media in petri plates to facilitate further growth of entomopathogenic fungi.
Preliminary pathogenicity test (Koch’s postulates)
The isolated fungi were first grown on Potato dextrose agar media for 10 to 15 days until sporulation. The Fourth and fifth instar larvae of
Galleria mellonella were rolled across a sporulating culture with the help of forceps. The infected larvae were then transferred into 5 cm diameter petridishes containing moistened filter paper to encourage spore germination on the insect cuticle. Lids of the petridishes were kept with moistened filter paper to encourage spore germination on the insect cuticle. The lids of the petridishes were sealed with parafilm® to maintain a suitable relative humidity and incubated at a temperature of 25
oC in darkness. These larvae were inspected daily until larval death or pupation occurred. Fungus recovery was attempted using the afore-mentioned procedures and the experiment was replicated four times to confirm the pathogenicity of the fungi.
Identification and characterization of common entomopathogenic fungal isolates
The isolated and confirmed entomopathogenic fungi were identified morphologically by preparing slides for light microscopy at a magnification of 400x. The molecular characterization was performed by comparing rDNA ITS sequences for similarity.
Morphological identification
Macroscopic characteristics of fungi, including colony colour (front and reverse side), colony texture and colony appearance were recorded. Observations on the microscopic characters were made to examine shape of the spores.
Fungal DNA extraction
Genomic DNA extraction from fungal mycelium was carried out using CTAB method
(Zhang et al., 2010). Fungal mycelium (10 mg) was scraped from 15-day old fungal plates and crushed in mortar and pestle using 1 ml of CTAB buffer. Crushed mycelium was then transferred to an Eppendorf® tube and incubated in water bath at 65
oC for 1 hour. Afterwards, the tube was centrifuged at 12000 rpm for 10 minutes at 4
oC and the supernatant was collected. An equal volume of Phenol: Chloroform: Isoamyl alcohol (25:24:1) was added to the supernatant and vortexed. The mixture was centrifuged at 12000 rpm for 10 minutes at 4
oC, resulting in phase separation. The supernatant was collected and an equal volume of ice-cold isopropanol was added. The mixture was incubated overnight at -20
oC. The next day, the mixture was centrifuged at 13000 rpm for 15 minutes at 4!. The DNA pellet was washed in 70% ethanol, air dried and dissolved in 50 µl of nuclease free water.
Amplification and sequencing
The ITS1-5.8S-ITS2 region of extracted rDNA was PCR amplified and sequenced using the primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3'). The PCR mixture (30 µl) consisted of 15 µl of universal master mix, 12 µl of molecular water, 1 µl of primers ITS1 and ITS4 each and 1 µl of extracted DNA. PCR amplification was carried out with specific temperatures, described by
Gandarilla-Pacheco et al., (2021). After amplification, the PCR products were run on 1% Agarose gel with Ethidium bromide and then bands were visualized under UV light using a Gel documentation unit. The amplified PCR products were then processed at Syngenome (OPC) Private Limited, Coimbatore and sequences were obtained. Sequences were edited using BIOEDIT software 7.2 and compared with sequences in NCBI database using Basic Local Alignment Search Tool (BLAST). MEGA 11.0 software was used for the construction of phylogenetic tree, using Neighbour joining tree statistical method and Kimura-2 parameter model. For this, ITS sequences of this study were compared with already published sequences present in NCBI database.
Evaluation of diversity indices
The diversity of isolated entomopathogenic fungi was determined by using Simpson’s index, Shannon-Wiener index and species richness
(Yakubu et al., 2022). These indices provide measures of species diversity, abundance and evenness within the fungal community.
Where:
P= No of individual of specific species / Total no of individuals.
∑= Sum of calculations.
ln= Natural log.
S = No of species.
Where:
S= No of species.
N= Total no of isolates.