Studies on the efficacy of Germitol - A general room disinfectant in sericulture industry

Introduction

The mulberry silkworm, Bombyx mori L. (Lepidoptera: Bombycidae), is vulnerable to several diseases that pose significant challenges in achieving optimal cocoon yield and productivity. Some of the most common diseases affecting silkworms include Nuclear polyhedrosis, Bacterial and Viral Flacherie, Muscardine and Pebrine. Nuclear polyhedrosis is caused by Bombyx mori Nucleopolyhedrovirus (BmNPV). Viral flacherie is triggered by multiple viruses, including Bombyx mori infectious flacherie virus (BmIFV), Bombyx mori densonucliovirus (BmDNV) and Bombyx mori cytoplasmic polyhedrosis virus (BmCPV). Bacterial flacherie caused by bacteria such as Bacillus thuringiensis, Serratia marcescens, Streptococcus and Staphylococcus species. Muscardine, a fungal disease caused by Beauveria bassiana and Pebrine caused by Nosema bombycis, a microsporidian pathogen. These silkworm diseases occurs in all major silk-producing countries, with a reported loss of 15-20 kg of cocoons per 100 dfls, accounting for up to 25% of total production losses (Selvakumar et al., 2002) [17] . Sometimes, it has been noticed that most of the farmers have lost their complete crop due to viral diseases. Pathogens can spread through various means into the rearing environment, including contaminated air, mulberry leaves, rearing equipment and direct contact with infected silkworms or through the rearing personnel leading to disease outbreaks and substantial economic losses (Baig et al., 1990) [2] . Effective disease control is essential for increasing silk production by up to 25% without expanding the mulberry cultivation area (Nagarajan and Radha, 1999) [12] . Despite strict rearing techniques, controlling diseases remains crucial for sustainable sericulture practices. At present, no effective curative methods are available to manage silkworm diseases, making preventive and prophylactic measures are the most reliable approach for disease control. Hence, disinfection and hygiene are integral parts of successful silkworm rearing. Proper disinfection of the silkworm rearing room and its appliances plays a crucial role in minimizing pathogen load and preventing disease outbreaks thereby ensuring healthy cocoon production. Various disinfectants viz., Formalin (Kagawa, 1980), Bleaching powder (Balavenkatasubbaiah et al., 1994 a,b) [4] , Iodine compounds (Kawakami, 1970; Venkata Reddy et al., 1990) [10] , Calcium hydroxide (Ishiwata & Zhou, 1968) [8] , Chlorine dioxide (Sanitech) in combination with lime (Balavenkatasubbaiah et al., 1999; 2006) [3] , Astra (Venugopal et al., 2012) [21] , Decol (Santha et al., 2012) [16] have been used in sericulture industry for disinfection of rearing houses and appliances. However, many of these disinfectants have certain limitations, such as being corrosive, carcinogenic or causing irritation to the eyes and throat of the users, particularly for sericulture farmers. Germitol, a new spray disinfectant developed by RV Seri Agro Vet in Malur, Kolar district, Karnataka has been introduced for disinfecting rearing houses, surrounding areas and equipments. This phenol-based disinfectant is reported to be effective against a broad spectrum of silkworm pathogens. This broad-spectrum disinfectant offers promising potential for silkworm rearing due to its non-corrosiveness and cost-effective properties. Germitol is formulated as a synergistic blend of multiple active compounds, based on selected phenolic derivatives. In addition to its antimicrobial efficacy, it is designed to be less toxic than traditional phenol-based disinfectants. When used at the recommended concentration, Germitol remains effective across a range of temperatures while minimizing environmental impact. This study aims to evaluate the efficacy of Germitol in reducing microbial contamination in the silkworm rearing environment and to assess its impact on silkworm health and cocoon yield.

Material and Methods

This study was conducted at the Andhra Pradesh State Sericulture Research and Development Institute (APSSRDI) in Hindupur, Andhra Pradesh. Germitol was evaluated under laboratory and field conditions to assess its efficacy against silkworm pathogens and its impact on silkworm productivity through a series of in vitro, in vivo and field trials. The germicidal activity of Germitol was tested in vitro against Bacillus thuringiensis (1 × 10 8 spores/ml) and Beauveria bassiana (1×10 8 conidia/ml). In each case, pathogen suspensions were centrifuged at 5000 rpm for 10 minutes and the resulting pellets were exposed to a 1% Germitol solution for 30 minutes at room temperature. After treatment, the pathogens were centrifuged again and the pellets were resuspended in 1 ml of sterilized distilled water. Viability was assessed by inoculating the treated pathogens onto Nutrient Agar (NA) for bacteria and Potato Dextrose Agar (PDA) for fungi. The plates were incubated at 28-30 °C for 24-48 hours for bacteria and at 25-28 °C for 48-72 hours for fungi (Anonymous, 2019). A control group, consisting of untreated pathogens, was processed in parallel for comparison. The number of Colony Forming Units per millilitre (CFU/ml) was calculated using the formula:

Number of colonies counted x Dilution factor CFU/ml=

The percentage reduction in microbial load was calculated using the following formula:

The in-vivo germicidal efficacy of Germitol was evaluated under simulated silkworm rearing conditions. Four major silkworm pathogens were selected for the study viz., BmNPV, BmCPV, Bacillus thuringiensis and Beauveria bassiana. Pathogen suspensions were prepared using standardized concentrations of 1×10 7 POBs/ml for BmNPV, 1×10 7 polyhedra/ml for BmCPV, 1×10 8 spores/ml for B. thuringiensis and 1×10 7 conidia/ml for B. bassiana. The rearing room was artificially contaminated by uniformly spraying the pathogen suspensions uniformly over the rearing bed, walls, racks and surrounding equipment. After allowing the pathogens to settle for 30 minutes, the contaminated surfaces were sprayed thoroughly with a 1% Germitol solution and left to dry completely before initiating rearing. Following disinfection, healthy third instar larvae of the FC1 × FC2 hybrid were introduced into the treated environment and reared under standard environmental conditions Each treatment was replicated three times with 100 larvae per replication. A separate control group was maintained in an identically contaminated rearing room without the application of Germitol. Larvae were observed daily for ten days to monitor the disease incidence and larval mortality.

To further assess the effectiveness of Germitol, FC1 × FC2 reared under three conditions viz., disinfection of rearing room and appliances with 1% Germitol (1 liter of Germitol in 99 liters of water, applied at 150 ml per square foot or 1.5 liters per square meter) as per the recommendation of the product developer. Disinfection using bleaching powder (2.5% concentration) and an untreated control group for comparison (table 1). Each treatment had three replications with 5 dfls per replicate. Silkworm rearing was conducted following the standard rearing practices (Dandin and Giridhar, 2014) and V1 mulberry leaves were fed to silkworm till spinning. Rearing and cocoon parameters viz., larval mortality (%), fifth instar duration (h), fifth instar larval weight (g), cocoon yield by number (per 10,000 larvae), cocoon yield by weight (kg/per 10,000 larvae), pupation rate(%), single cocoon weight, shell weight (g) and shell ratio (%) were recorded.

Table 1: Treatment details

T1

Rearing room was disinfected with 1% Germitol solution.

T2

Rearing room treated with standard disinfectant Bleaching powder (control)

T3

No disinfection treatment (untreated control for baseline pathogen load comparison.

The efficacy of Germitol was also evaluated at farmers rearing houses in Sri Sathya district of Andhra Pradesh and Chikkaballapur, Kolar districts of Karnataka. Rearing houses were disinfected with a 1% Germitol solution and observations were recorded on cocoon yield and other parameters. A total of 20 silkworm cocoon crops, involving 4108 dfls with different silkworm breeds/hybrids (CSR2, CB, FC2, FC1 and FC1 × FC2) were assessed. Further, an in-house evaluation was conducted over five silkworm rearing crops involving 125 DFLs and the rearing performance viz., single cocoon weight (g), single shell weight (g), shell ratio (%), yield/100 dfls (kg) was recorded. All laboratory and field experiments were conducted under standardized environmental conditions. Data was analysed using a completely randomized design (CRD) with OPSTAT statistical software.

Results and Discussion

Germitol proved significant germicidal efficacy under in vitro and in vivo against major silkworm pathogens. In vitro testing revealed that exposure to 1% Germitol solution for 30 minutes significantly reduced the number of colonyforming units (CFU) viz., 98.83 per cent in B. thuringiensis and 96.91 per cent in B. bassiana (fig. 1). Control plates showed normal colony growth, confirming the viability of untreated pathogens. The near-complete reduction of bacterial and fungal colonies on the treated plates confirms the efficacy of Germitol. The in-vivo evaluation of Germitol under simulated rearing conditions revealed a significant reduction in disease incidence and larval mortality compared to the untreated control. The average cumulative mortality of larvae over the ten day observation period was markedly lower in the Germitol-treated group across all pathogen challenges. In the Germitol-treated batches, the mean larval mortality due to BmNPV, BmCPV, Bacillus thuringiensis and Beauveria bassiana was recorded as 7.95%, 7.72%, 6.13% and 6.45%, respectively (table 2). In contrast, the corresponding mortality in the control group without disinfection was significantly higher, with values of 72.43%, 68.91%, 64.71% and 63.17%, respectively. Among the four pathogens, Bm NPV caused the highest larval mortality in the control group, whereas Bacillus thuringiensis showed relatively lower mortality. Germitol treatment resulted in more than 85% reduction in mortality across all tested pathogens, indicating its broad-spectrum efficacy. No abnormal larval behaviour was observed in the Germitol-treated groups, suggesting the absence of any adverse effects of the disinfectant on silkworm health and development. The results from the room disinfection trials revealed significant improvement in the performance of silkworms reared in Germitol treated (T1) silkworm rearing room across all key parameters (table 3). Larval mortality in the Germitol treated group was significantly lower (4.80%) compared to the bleaching powder (9.71%) and the untreated control group (25.95%). Although the fifth instar duration showed no significant difference among the treatments, it was shortest in the Germitol treated group (149.11 hours) compared to the bleaching powder (155.92 hours) and control groups (153.81 hours). The shorter duration in the Germitol treated group reveals more efficient growth and development, likely due to reduced disease stress and improved hygiene in the rearing environment. Germitol treated group also had the highest fifth instar larval weight (57.21 g), followed by bleaching powder (52.38 g) and control group (49.29 g), which contributed to higher single cocoon weight of 2.23 g, 2.02 g and 1.88 g, respectively. The Germitol-treated group recorded the highest cocoon yield per 10,000 larvae (9,611.39 cocoons with a total weight of 21.94 kg) which was notably higher than the bleaching powder treatment (9,439.27 cocoons weighing 20.02 kg) and the control group, which produced 7,595.23 cocoons with a weight of 14.64 kg. The higher cocoon yield in the Germiol treated group is due to the lower larval mortality and healthier larval growth, both of which can be attributed to better pathogen control in the rearing environment. The pupation rate was 95.20% in the Germitol treated group, compared to 92.28% in the bleaching powder and 73.68% in the control group. Shell weight and shell ratio were also highest in the Germitol treated group (0.45 g and 22.07%) compared to the bleaching powder (0.43 g and 20.09%) and the control group (0.38 g and 18.84%). The promising results in Germitol-treated batches across all parameters highlight its effectiveness in maintaining a pathogen-free rearing environment, reducing larval mortality and enhancing productivity. The bleaching powder treatment (T2) showed moderate efficacy with slightly lower yield and higher disease incidence. The poor performance of the untreated control group (T3) was likely due to a high pathogen load, adversely affecting silkworm health and cocoon production. Field trials conducted at farmer's rearing houses also showed promising results, confirming the laboratory findings with Germitol treated farmers rearing houses showing consistent improvements in cocoon yield and achieved higher market rates per kilogram of cocoons (table 4), resulting in successful and consistent cocoon crop harvests. The data suggest that Germitol is a promising disinfectant for large-scale use, providing enhanced productivity and pathogen control. The significant reduction in disease incidence in large-scale field trials reinforces the laboratory and small-scale findings, establishing Germitol as an effective disinfectant for commercial sericulture operations. [16,15] . The antimicrobial properties of phenolic compounds have been documented by various researchers (Klarmann & Shternov, 1936; Rettger et al., 1929; Read & Miller, 1932; Terleckyj & Axler, 1987), reinforcing the results observed in this study.

Conclusion

The study demonstrated that Germitol is an effective disinfectant for controlling silkworm pathogens under both laboratory and field conditions. With its broad-spectrum biocidal properties, low toxicity and cost-effectiveness when used in recommended concentration, it stands out as a best alternative to traditional disinfectants like bleaching powder. By significantly reducing microbial contamination, it improved larval health and cocoon yield, making it a valuable addition to sericulture practices. Field trials confirmed Germitol practical applicability in commercial sericulture, proving it to be a reliable disinfectant that helps farmers maintain better hygiene standards and reduce disease-related losses. Its successful commercialization for large-scale production further underscores its potential to enhance productivity in the industry.

Acknowledgement

AcknowledgmentThe authors are thankful to RV Seri Agro Vet, Malur, Kolar district for their financial support in facilitating the evaluation of Germitol efficacy and also thank farmers of Andhra Pradesh and Karnataka for their kind cooperation during the study period.

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