In vitro compatibility of entomopathogenic fungi, Beauveria bassiana (Balsam) with insecticides and fungicides

Introduction

In many parts of the world, agricultural development has a lengthy history. Around 10,000 years ago, agricultural activities started in the Fertile Crescent of Mesopotamia, which roughly corresponds to the majority of modern-day Iraq, Turkey, Syria, and Jordan. Individuals who lived in these regions and gathered edible seeds using techniques like forest gardening and firestick farming. Large quantities of wheat, barley, peas, lentils, chickpeas, bitter vetch, and flax were grown as the people grew more sedentary and lived on farms. Around 7500 years ago, sorghum and rice were first cultivated in the Sahel region of Africa (Davies, 1968) [1] . Furthermore, according to Davies (1968) [1] , several indigenous crops were independently domesticated approximately 7500 years ago in Ethiopia, New Guinea, and West Africa. China was the country that domesticated millet and rice. Sunflowers, potatoes, corn, and squash were all domesticated by Americans on their own. Pests, weeds, and illnesses frequently plague farmed crops, which can significantly reduce crop productivity. Without the use of pesticides, diseases and pests would cause up to 78%, 54%, and 32% of the loss of fruits, vegetables, and cereals, respectively (Zhang et al., 2011) [2] . Thus, it is imperative that researchers and the general public find solutions to the issues brought on by illnesses and pests. In many areas, high-yielding cultivars, better soil and water management, fertilization, and other farming practices can boost crop productivity. However, larger crop output potential is frequently linked to increased susceptibility to pest attack, which raises absolute losses and loss rates. Pre-harvest pests cost farmers around the world an average of 35% of their potential crop output. Conventional pesticides are thousands of years behind the evolutionary relationships between farmers and pests. Different levels of loss can be distinguished, such as direct and indirect losses or primary and secondary losses, demonstrating that pests can have an impact on the supply of food and feed, the economies of rural areas, and even entire nations, in addition to threatening crop productivity and lowering farmers' net income. To meet the rising food demand, herbicides, insecti cides, fungicides, and nematicides are frequently used to lower the pest pressure (Sarkar et al., 2021) [3] . Pesticides are thought to be crucial for both crop protection and the treatment of human diseases. Pesticides are categorized as either organism targets, repellents, or destroyers. Many insects and pests cannot be controlled by store-bought insecticides. Large quantities of commercial insecticides are also utilized to manage pests. Depending on the type of pest and the methods used for control, pests are treated both preventively and remedially. Insecticides, herbicides, fungicides, molluscicides, nematicides, plant growth regulators, and rodenticides are among the pesticides categorized by the species they target. The environment is negatively impacted by the widespread usage of pesticides. Research indicates that pesticides have a negative impact on biodiversity and human health. Usually stable and soluble in water, pesticides have an impact on aquatic life, wildlife, and native plants. Pesticides are harmful in agricultural applications and expose workers to occupational hazards during the manufacturing process. Because chemical pesticides are persistent in the environment and can infiltrate the food chain, they have a negative impact on human health. Chemicals from pesticides can enter the human food chain through a variety of sources, including contaminated water, polluted air, and vegetables and fruits. Fish, birds, mammals, and beneficial insects are all harmed by a number of pesticides. The toxicity of pesticides, their quantity, timing, frequency, application technique, weather, and soil type all affect wildlife poisoning. The use of carbamate and organophosphate has significantly expanded over the last forty years. Endosulfan and other organochlorines are quite persistent in the environment in the south. The bulk of the population has decreased as a result of pesticide poisoning as a result of habitat alteration. Entomopathogenic fungi show great promise in controlling a range of arthropod pests. However, their field application may result in inconsistent control because biotic factors, such as interactions with antagonistic bacteria, and abiotic factors, such as temperature and humidity, can easily affect infections of hosts with insect pathogenic fungus. Entomopathogenic fungi have been used in conjunction with botanicals and sublethal chemical dosages to boost efficacy and accelerate insect mortality. Pesticides. In many instances, the effectiveness of entomopathogenic fungi in combating insect pests has been enhanced by the application of certain insecticides. The effects of insecticides and fungicides on mycelial development and sporulation of fungi have been the focus of most fungus-insecticide compatibility studies. While the effects of insecticides on conidial germination, which are commonly overlooked, are the most significant factor to consider when evaluating insecticide compatibility since it is the first stage of the infection. Beauveria bassiana (Balsam) Vuillemin is a necrotrophic fungus that lives in soil and is commonly referred to as white muscardine. This fungus is used for its entomopathogenic qualities as well as its other advantageous qualities as plant endophytes, plant disease antagonists, helpful rhizosphere colonizers, and plant growth boosters (Vega et al., 2009) [4] . Based on above mentioned problem, research is required to examine which insecticides and fungicides compatible with entomopathogenic fungus B. bassiana. Thus, the purpose of this study was to determine the compatibility of various insecticides and fungicides at particular doses to entomopathogenic fungus B. bassiana.

Materials and Methods

The present investigation was conducted at plant pathology laboratory, R. A. K. College of Agriculture, RVSKVV, Sehore, Madhya Pradesh. The impact of these chemical insecticides and fungicides on the vegetative growth and spore germination of B. bassiana was assessed using poisoned food technique (Grover and Moore, 1962) [5] . The list of various insecticides and fungicides are used which showed in Table -1.

Maintenance of pure culture of B. bassiana

For the current investigations, a pure fungal culture of B. bassiana was obtained from ICAR-NBAIR. A loopful of the inoculum from the mother culture slants of B. bassiana was moved to PDA slants and Petri plates in order to preserve the pure culture. The PDA medium was autoclaved for 30 minutes at 121 ℃ and 15 psi of pressure. The autoclaved PDA medium was then transferred to sanitized Petri plates, chilled, and aseptically injected with a pure culture of B. bassiana. After that, the plates were cultured for fourteen days at room temperature (25±2 ℃). After 12-15 days of incubation, fungal mat was blended and filtered through muslin cloth and diluted with sterile distilled water. The required concentration of conidia for compatibility studies were made by using hemocytometer.

Assessment of vegetative growth (VG)

Required concentrations of each test insecticides and fungicides were incorporated into 200 ml of melted sterile PDA media under aseptic condition, mixed thoroughly, poured into 9 cm diameter sterile petri plates and allowed to solidify under laminar air flow cabinet. Using a 5 mm diameter cork borer, the mycelium mat and agar disc of B. bassiana were cored from the edges of the fourteen-day-old colony and positioned in the middle of the PDA plate. PDA media treated as an untreated control that was infected with mycelial disc but did not include pesticides. In order to allow for radical growth, Parafin sealed Petri dishes were incubated for two days at 25±1 ℃, 60±5% relative humidity, and 12:12 hours of photo:dark phase. Each treatment was replicated four times. The radial growth (mm) of entomopathogenic fungus B. bassiana was measured using caliper rule when when control plates attained full growth. Per cent inhibition of B. bassiana growth in the poisoned media was calculated using the formula suggested by Vincent (1927).

Percent inhibition = C-T/C × 100

Where, C = Diameter of fungal growth in control T = Diameter of fungal growth in treatment Spore (conidia) production After vegetative growth measurement, the central 5 colony discs from each test were drawn for conidia production analysis. Each disc was placed in test tube containing 10ml of sterile distilled water with Tween 80 (0.02 per cent). The tubes were vortexed for 30 seconds to extract the conidia from the disc. Number of conidia per ml were counted using haemocytometer.

Statistical analysis

The compatibility data were analyzed using Completely Randomised Design (CRD) with Analysis of Variance (ANOVA). Toxicity of each insecticide was calculated using formula proposed by Alves et al., (1998). In this formula calculation of vegetative growth (VG) and sporulation (SP) is made in relation to control (100%): T = [20 (VG) + 80 (SP)]/100. Where T = 0 to 30 (very toxic); 31 to 45 (toxic); 46 to 60 (moderately toxic); >60 (compatible). bassiana that would be a limiting factor in their future applications in the IPM programs (Anderson & Roberts, 1983) [6] . The degree to which the insecticides inhibited B. bassiana germination, vegetative growth, and sporulation varied and was mostly influenced by the chemical characteristics and concentrations of each compound.

Because the ingredients in the insecticide formulations can be employed directly as nutrients, the greatest vegetative growth and spore yield seen with flonicamid 50 WG treatment boosting the pathogen's conidia generation and vegetative development.

The current finding that buprofezin, a chitin inhibitor, was relatively safe at higher concentrations is supported by Anderson et al. (1989) [8] , who also reported that insecticides of microbial origin and chitin inhibitors like abamectin, thuringiensin, and triflumuron were compatible with B. bassiana even at higher doses. Additionally, Andrew et al. (2005) verified that buprofezin, the active ingredient, offered a satisfactory degree of L. muscarium spore germination. According to Deb et al. (2017) [9] , the isolate of Beauveria BP1.1 exhibited the highest growth in copper oxychloride (80.50 mm), followed by neem leaf extract (74.75 mm) and neem seed kernel extract (70.75 mm). In contrast, the fungicide carbendazim showed the highest inhibition of fungal growth. Due to its multisite activity, copper oxychloride prevents fungal growth by disrupting the spore and mycelium's enzyme systems. As the copper binding protein (CuBp) of B. bassiana is a member of metallothioneins, a cysteine-rich protein devoid of aromatic residues and composed of copper (I)-thiolate cluster, Challa and Savinda (2014) reported that B. bassiana was found to be highly compatible with copper oxycloride. B. bassiana has a higher affinity for copper because of this. Similarly, in the present study, copper oxychloride 50WP was found as the most compatible fungicide with 45.20 mm radial growth of B. bassiana.

In our study carbendazim showed significant inhibition of entomopathogenic fungi and found very toxic to B. bassiana according to toxicity classification, which might be due to carbendazim inhibits the development of fungus by interfering with spindle formation at mitosis during cell division, thus, inhibiting germtube development, formation of appresorium and growth of mycelia. Butters et al. (2003) [11] has also reported the abnormal bent appearance of the germination peg of B. bassiana with bursting at the tip in the presence of bavistin. Similarly, in the present study, the total inhibition of mycelial growth was observed in carbendazim 50WP at all three concentrations.

Spore yield

The data from Table-2 indicated that the total quantity of conidial spore produced by various pesticides varied significantly. With 5.63×10 4 conidial spores/ml, the control treatment had the highest spore yield. This was followed by flonicamid 50 WG and copper oxychloride 50 WP, which had 5.15×10 4 [13] . However, on days 14 and 30 following inoculation, imidacloprid, thiamethoxam, acetamiprid, clothianidin, acephate, fipronil, buprofezin, flonicamid, and azadirachtin were shown to be compatible. According to Oliveira et al. (2003) [14] , the fungus was compatible with formulations of thiamethoxam at all three concentrations and cyclfluthrin at the two lowest values. The Alpha-Cypermethrin formulation, the Fenpropathrin and Deltamethrin formulations at the lowest concentrations, and the Thiamethoxam and Cyfluthrin formulations can all be regarded as compatible when germination/viability is taken into account as a compatibility factor.

Conclusion

Since the onset of epizootics depends on these structures' ability to germinate on the host, conidial germination is a crucial stage in fungal pest management. However, conidial vigor is necessary for the entomopathogenic fungus to succeed. With the exception of carbendazim and propiconazole, our investigation revealed that the majority of pesticides were compatible with B. bassiana. However, the highest spore production and vegetative growth have been observed with flonicamid and copper oxychloride treatment. However, field investigation of the interactions between B. bassiana and insecticides/fungicides should be under taken to examine their impact on pests and beneficial insects.

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