Effect of Fungal Extracts on Bacillus thuringiensis Serotypes Causing Flacherie Disease in Mulberry Silkworm, Bombyx mori L

Diseases are the most important limiting factor affecting silkworm, Bombyx mori L., known for the production of silk cocoons are generally affected by viral, fungal and protozoan pathogens among which bacterial pathogens alone cause cocoon loss about 7.5 to 20.0 percent (Selvakumar, 2013). The major fact responsible for bacterial flacherie was the rearing conditions, rise in temperature and humidity in rearing place, insufficient bed spacing leads to dysfunction of alimentary canal which encourages flacherie (Nataraju et al., 2005). Bacillus thuringiensis (Bt) is one of the causative agents for flacherie disease and the impact was high in silkworm larvae (Nishiit sutsuj et al., 1979). Bt is a Gram positive spore forming bacterium characterized by the formation of parasporal inclusions during sporulation (Ohba, 1996). After being ingested by the larvae, the parasporal inclusions are dissolved in larval midgut juice and release protoxins. The International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 2 (2020) Journal homepage: http://www.ijcmas.com


Introduction
Diseases are the most important limiting factor affecting silkworm, Bombyx mori L., known for the production of silk cocoons are generally affected by viral, fungal and protozoan pathogens among which bacterial pathogens alone cause cocoon loss about 7.5 to 20.0 percent (Selvakumar, 2013). The major fact responsible for bacterial flacherie was the rearing conditions, rise in temperature and humidity in rearing place, insufficient bed spacing leads to dysfunction of alimentary canal which encourages flacherie (Nataraju et al., 2005). Bacillus thuringiensis (Bt) is one of the causative agents for flacherie disease and the impact was high in silkworm larvae (Nishiit sutsuj et al., 1979). Bt is a Gram positive spore forming bacterium characterized by the formation of parasporal inclusions during sporulation (Ohba, 1996). After being ingested by the larvae, the parasporal inclusions are dissolved in larval midgut juice and release protoxins. The

ISSN: 2319-7706 Volume 9 Number 2 (2020)
Journal homepage: http://www.ijcmas.com Antibacterial activity of six different species of fungal extracts viz., Aspergillus ochraceous, Paecilomyces variotii, Schizophyllum sp., Epicoccum sp., Metarhizium sp., Aspergillus niger were evaluated against ten serotypes of Bacillus thuringiensis to develop new bioactive secondary metabolites from the microbial sources. Fungal crude extracts were isolated by ethyl acetate solvent extraction method and product yields varied from 0.150g to 0.538g. Thin Layer Chromatography was carried out for detecting the small molecule compounds present in the crude extracts. Antibacterial activities of fungal extracts were analyzed by disc diffusion method. All ten B.t serotypes were inhibited by six fungal extracts with zone of inhibition ranging from 12 ± 1.73 to 36 ± 1.53. Among the six extracts Paecilomyces variotii showed highest inhibition of 36 mm against B.t sotto. In vitro and in vivo studies conducted for the effect of extracts with different concentration and exposure duration reveals that effective against all serotypes. The results suggested that the Microbial derived extracts could be a new source of alternative in developing an effective bioactive molecule for the management of silkworm bacterial diseases. activated toxins interact with the larval epithelial membrane and induce pore formation in the membrane, which ultimately leads to insect death (Gill et al., 1992). Larvae affected by Bt lose their appetite, undergo convulsions and their bodies become stretched and cracked. The cadavers gradually become brown to black-brown and finally when rotting they turn black with fowling smell (Aruga, 1994). Researchers have come out with different sources of bioactive compounds/crude extracts from plants to prevent the silkworm bacterial pathogens (Selvakumar et al., 2001;Manimegalai and Chandramohan, 2005;Priyadharshini, 2008) but limited attempts were made for the use of microbial metabolites. In the present study, the efficacy of fungal extracts controlling the ten serotypes of Bt causing flacherie disease in mulberry silkworm was evaluated to develop a effective bioactive molecules for the control of disease.

Preparation of fungal extract
Fungal strains were grown in PDA culture medium at 28 °C for 5 days after that cultures were cut into solid discs (1cm 2 ) and inoculated into 1000 ml Potato dextrose broth in Erlenmeyer flasks. These cultures were grown with continuous shaking of 200 rpm at 25 ± 2 °C in shaking incubator for fifteen days. At the end of incubation, the fungal mycelium and the culture medium were separated by filtration using Whatmann filter No.3. The filtered supernatant was extracted thrice with equal volume of ethyl acetate. The combined organic phase was dried with anhydrous sodium sulphate and concentrated in reduced vacuum evaporator at 45°C. The crude extract dissolved in 10% dimethylsulfoxide (DMSO) and kept in 4 °C for further use.

Identification of small molecules
Thin Layer Chromatography technique was used for detecting the small molecule compounds present in the fungal crude extracts. The extract samples were spotted on silica plates and eluted in a TLC chamber with ethyl acetate and hexane as the mobile phase in the ratio 8:2. The plates were then air dried and visualized by exposing to iodine vapor.

In vitro antibacterial activity by disc diffusion
Antimicrobial susceptibility test was carried out by the disc diffusion method. Overnight incubated Bt cultures (1x10 8 spores/ml) spread on a Muller Hinton Agar plate. Whatman No.1 filter paper discs (6 mm diameter) dipped in the extract concentration of 50mg/ml solution and kept on inoculated plates. Ampicillin (10 mcg) was used as positive control. The plates inoculated with bacteria were made in triplicate and incubated at 32±1°C for 48 hours. The development of zone around the disc diameter was measured in millimeters. All data were analyzed through Microsoft Excel for Windows.

Efficacy of minimum antibacterial concentration
The effect of different concentrations of fungal product for minimum germicidal activity was tested in vitro against B. thuringiensis serotypes (1x10 8 cells/ml). These pathogens were suspended separately in different concentrations of fungal product (0.25 to 4%) for different durations 30 min, 1 hour and 2 hour at room temperature. After this incubation, samples were inoculated in Louria Bertani agar plate and incubated at 32±1°C for 48 hours. The experiment conducted in triplicate. The minimum bactericidal activity was determined by the lowest concentration of fungal product incubated with pathogens were completely killed.

In vivo activity of fungal extract
The antibacterial activity of the product with different concentrations tested in vivo against Bacillus thuringiensis serotypes (1x10 8 cells/ml). These pathogens suspended to the various concentrations of product and incubated at 25±2°C for different duration (30 min, 1 hour and 2 hour). After the treatment, samples centrifuged, washed and used for inoculation to determine the infectivity of the pathogens. 0.5 ml of this sample orally inoculated to third instar larvae.
In positive control, viable pathogens inoculated without treatment to the larvae and in negative control, without inoculation larvae reared for comparative study in three replications. All the batches were reared till spinning and allowed to spin the cocoons. Observations recorded for disease development and mortality. Treatment effects were analyzed to determine the differences between the treatment means of extract concentration.

Screening of small molecules
Antibacterial activities of the fungal extracts isolated from six different fungi were analyzed to derive high active biomolecules for treatment of Bt serotypes. The crude extract of fungal product yields varied from 0.150g to 0.538g. A range of yields among extracts was observed depending on the fungal material used.
The maximum extraction yield was obtained from Schizophyllum sp. with 0.538g and P. variotii exhibited the lowest extraction yield of 0.150g. Thin Layer Chromatography was carried out for detecting the small molecule compounds present in the crude extracts and 2 to 5 product spots were appeared in all fungal extract in the range of 0.2 to 0.84 retention factor (Rf) with ethyl acetate as mobile phase for the extract.

In vitro antibacterial activity
In vitro studies results showed that all six fungal extracts exhibited antibacterial activity in the range of 12 ± 1.73 to 36 ± 1.53 against Bt serotypes (Table 1), (Fig.2 & 3). The maximum activity of 36± 1.53 mm observed in P. variotii and 29 ± 1.00 in A. ochraceus against sotto and followed by 28 ± 0.58 mm in diameter against japonensis by Epicoccum sp. In particular, P. variotii and Epicoccum sp. showed above 20 mm inhibition against all serotypes of Bt except kenyae and tolworthi respectively. Among the six extracts, Metarhizium sp. exhibited lowest activities observed in all Bt bacteria except against sotto which was 25 ± 1.00 mm in diameter of growth inhibition. Ampicillin (10 mg) was used as positive control and results indicated that the extracts showed almost equivalent activity at low concentrations.      Table 2). In that 30 min exposure duration with the extract, 2 and 4% concentrations are effective and observed devoid of colonies. Up to 1% concentration was not effective and 10 to 50 bacterial colonies observed in 0.5, 1% and more than 300 colonies was observed in 0.025 % extract on the LB medium incubated for 48 hours. In 1 hour treatment similar trend was observed but number of colonies was less than the 30 min duration. In 2 hour duration, 1% extract onwards effective in all serotypes.

In vivo activity of extracts
An in vivo study reveals that 1% concentration of P. variotii extract effective for only 2 hour incubation, 2% and 4% extract was effective for all three exposed durations against Bt serotypes (Table 3). In positive control, without treatment larvae were infected by the pathogens but mortality rate was varied for all Bt serotypes. Bt morrsoni infected larvae died within 24 hours after inoculation of pathogen followed by japonensis, tolworthi, aizawai, sotto, alesti and less mortality rate was observed in kenyae, israelensis, kurstaki, thuringiensis with the influence of toxicity, temperature and/or food quality. Similar results reported by Selvakumar, (2013) stated that environmental factors have little impact on the mortality through toxicity and not infection by the bacteria.
Bacillus thuringiensis is a widely distributed facultative entomogenous bacterium well known for bio pesticide and employed extensively against insect pests in agriculture but they are pathogenic to the silkworm (Selvakumar et al., 1999). Mahmoud et al., (2019) reported that black seed and basil leaves extracts treated with Bacillus thuriengiensis infected larvae significantly decreased larval mortality in B. mori.
Similarly, aqueous extract of Thuja orientalis (Manimegalai and Chandramohan, 2005), leaf extract of Aegle marmelos and bark extracts of T. orientalis (Manimegalai et al., 2010) were effective in managing Bt infected B. mori. Similar observation was indicated by Mohanta et al., (2013) extracts of C. zeylenicum, C. longa, Z. officinale showed a strong antibacterial activity against Flacherie pathogens. The secondary metabolites have no apparent metabolic, physiologic and structural role in the producer, but have effects on other organisms function as biochemical defence (Jain et al., 2004). Zhang et al., (2011) isolated four active secondary metabolite (Cerebroside C, Cerebroside D, 2-Hydroxybenzyl alcohol, and 2-(4-Hydrroxypheny) ethanol) from the Paecilomyces sp. In the present study P. Variotii showed highest activity against all Bt pathogens and this might be presence of these compounds in the extract. Similar results reported by Oliveira et al., (2009) P. variotii extracts inhibited eight clinical isolates of entericococcs bacterial in the ranging from 25 to 35.25mm.
These results indicated that the possibility of the presence of highly active bioactive compounds/ secondary metabolites in the fungal crude extract. It is concluded that further studies are required to find out the bioactive compounds/ secondary metabolite responsible for the activity. Microbial derived products may be a source of alternative active compounds for control of silkworm diseases and it is a new approach in silkworm management.