Pathogenic Response of Entomopathogenic Fungal Strains on Larvae of Fall Armyworm (Spodoptera frugiperda)

INTRODUCTION

Fall armyworm, technically known as Spodoptera rugiperda (Lepidoptera: Noctuidae) a serious migratory lepidopteron insect pest [1]. It is polyphagous and feeds on about 80 recorded plant species in 26 plant families, including the most economically important crops sorghum, maize, rice, millet, sugarcane, and sugarcane [2-4]. Previous studies indicate that in 2018, the first fall armyworm was reported in China in Yunnan province [5]. Because of the wide range of host plants and high reproduction rate, the pest spread out within a few years and became a serious pest of the maize crop in the northeast and northwest of China [6]. This region is famous for corn production, with an area of 13 million hectares. It is considered that the FAW is more serious than Helicoverpa armigera (Hübner) [7] and causes more losses to the maize crop. It was observed that, due to rapid control, most of the farmer community depends on chemical pesticides to control FAW [8]. It has been observed that the time of the chemical application greatly affects its efficiency, and FAW caterpillars are typically located in corn whorls where they are mainly shielded from pesticide treatments [9]. Due to the extensive application of chemical pesticides, FAW has developed resistance to commonly used chemical pesticides [10, 11]. It is estimated that FAW has developed resistance to more than 30 active ingredients of chemical pesticides from well-known classes. (https://www.pesticideresistance.org/). So new green control approaches are needed to minimize pest infestation and the adverse effects of pesticides on the environment [12]. Among chemical and biocontrol agents for pest control, entomopathogenic fungi are the most efficient and easy to use; about 1000 insect-pathogenic fungal strains are known globally [13]. Whereas, over 100 microbial insecticides are available on the market to control insect pests [14, 15]. The entomopathogenic fungus Isaria fumosorosea, Metarhizium anisopliae, and Beauveria bassiana are widely used against different insects [16]. Previous literature also confirmed the potential of entomopathogenic fungi against such insect pests [17, 18]. These fungi have special characteristics to infect their host insect through their cuticle [19]. After penetration into the cuticle, the host dies within 3-6 days In most cases, the fungal efficiency depends on environmental factors; among other environmental factors, humidity is known as a key factor for successful infection [20]. The host insect and its life stages are also important for successful infection [16, 20]. Therefore, in this study, we investigated different entomopathogenic fungi to evaluate the potential entomopathogenic fungal strains against (FAW) under laboratory conditions. We hope the results will be helpful for researchers who are working on microbial control of insect pests in their IPM program.

MATERIALS AND METHODS

Entomopathogenic fungal strain selection

The multiple strains of entomopathogenic fungi, e.g., Beauveria bassiana strain (Bb885 & 86), Cordyceps cicadae, Metarhizium anisopliae strain (73 & 42) and Paecilomyces fumosoroseus, were obtained from the fungal bank of the Research Center on Entomogenous Fungi (RCEF) at Anhui Agricultural University, Hefei, China (Latitude 31° N and Longitude 117° E) for the bioassay test. These strains were preserved at -70 °C before use.

Preparation of fungal spore suspension

Two hundred microliters of conidial suspension tube were obtained from the fungal bank and inoculated with Sabouraud dextrose agar (SDA) in 9 cm-diameter Petri dishes, containing 20 g of agar, 10 g of peptone, and 40 g of dextrose. The mixture was then maintained at 24 ± 1 °C in an incubation chamber for 12 days. Penicillin/streptomycin (2.5 ml/l), potassium (0.5 mg), and cycloheximide (40 g) were added to the medium to support bacterial growth before plating. Completely developed conidia were obtained by scraping the upper layer of the culture and then diluted using 100 milliliters of 0.05% Tween®80 in a 200-milliliter conical flask. For five minutes, the flask containing the conidia was homogenized in a vortex. The diluted conidia were transferred into a sterile beaker using cotton and a sterile 30 ml syringe. Using a hemocytometer and a microscope, suspensions were brought to predetermined concentrations. The standardization of conidia suspensions was done at 1 x 10⁵, 1 x 10⁶, 1 x 107, and 1 × 10⁸ conidia /ml [21].

Insect collection and bioassay procedures

Newly hatched larvae of the fall armyworm (early 2^nd and 3^rd instar) were collected from an already established insect culture in the laboratory of the Institute of Plant Protection, Agro-Products Safety, Anhui Academy of Agricultural Sciences, and Hefei China. The collected FAW larvae were brought to the Anhui Provincial Key Laboratory of Microbial Control, Anhui Agricultural University, Hefei, China, for the fungal bioassay test. Two different bioassay methods, i.e., direct larvae dipping and the plant leaf inoculation method, were used for the bioassay test. Each treatment was repeated four times, along with the control treatment. We used four different concentrations with four replications, with 6 larvae/ replicate; a total of 24 larvae for each concentration and 120 for each treatment, along with a control treatment. We haven’t found significant results against the 3^rd instar at the initial test, so data is not mentioned in this manuscript.

Direct dipping method

The newly hatched 2^nd instar larvae of FAW were dipped for 3 seconds into different fungal spore suspensions, such as 1 x 10⁵, 1 x 10⁶, 1 x 10⁷, and 1 × 10⁸. After inoculation, the larvae were placed on tissue paper to remove excessive liquid spores and placed into plastic boxes (with six holes to avoid the action of cannibalism). A small piece of green Chinese cabbage leaf was cut about 1 square inch and placed in each whole as food with a controlled relative humidity of 70 ± 5% in a rearing chamber at 24 ± 1 °C. Old leaves were replaced after 24 hours with fresh leaves. Boxes were covered with lids to avoid the larvae escaping. Two cotton swabs were placed in each box to maintain humidity and promote fungal infection. For 10 days, daily observations were noted. The dead insects were taken out of the boxes and put onto Petri dishes with moist filter paper so that the fungal infection could be confirmed by mycelial growth.

Plant leaf inoculation method

In the plant leaf inoculation method, the Chinese cabbage leaves were cut into small pieces of about 1 inch and dipped into different fungal spore suspensions, such as 1 x 10⁵, 1 x 10⁶, 1 x 10⁷, and 1 × 10⁸. After dipping leaves, pieces were placed on tissue paper to remove excessive water and offered to newly hatched 2^nd  instar larvae of FAW. The larvae were placed into plastic boxes with six holes to avoid cannibalism. The inoculated small piece of Chinese cabbage leaves (1 inch) was offered to the larvae as food, and the larvae were kept at a relative humidity of 70 ± 5% in a rearing chamber at 24 ± 1 °C. Boxes were covered with lids to avoid the larvae escaping. Two cotton swabs were placed in each box to maintain humidity and promote fungal infection. Old leaves inoculated with fungal spores were replaced after 24 hours with fresh leaves. For 10 days, daily observations were noted. The dead insects were taken out of the boxes and put onto Petri dishes with moist filter paper so that the fungal infection could be confirmed by mycelial growth.

Data analysis

The data was statistically analyzed using one-way analysis of variance (ANOVA) using Minitab statistical software, while the effectiveness of the fungi was compared by Fisher's least significant difference (LSD) test to determine differences among fungal concentrations at a significance level of P ≤ 0.05.

RESULTS AND DISCUSSION

We noticed that the leaf dipping method was more effective than the larval dipping method after conducting an initial bioassay screening of fungal strains and testing on FAW larvae. Therefore, we carried out additional experiments using the leaf dipping method with three virulent strains, namely B. bassiana (Bb885), C. cicadae, and M. anisopliae strains (73), which were found to be more lethal to FAW larvae (Figure 1).

According to our findings, the larval population was significantly infected when Chinese cabbage leaves were dipped in B. bassiana (Bb885) fungal spores.

Our results showed that the highest mortality rate, 66.67%, was observed in the conidial rate of 1 × 108 spores/ml. Subsequently, mortality rates of 20.83%, 29.17%, and 45.83% were noticed at conidial concentrations of 1 x 105, 1 x 106, and 1 x 107 spores/ml, respectively (Figure 2). Whereas, 9.38% was observed in the control treatment which was significantly less than other treatments observed by the LSD test (Df = 4, F = 3.45, P = 0.009) (Figure 3).

Whereas, maximum cumulative mortality of larvae 54.17 %, 41.67%, 37.50%, and 16.67 was observed on 1 x 10⁵/ ml, 1 x 10⁶/ ml, 1 x 10⁷ / ml, and 1 × 10⁸ spores/ml respectively on cordyceps cicadae treatment (Figure 4). We found a significant difference among all concentrations. However, all concentrations were also significantly different from the control treatment when compared with the LSD test (Df = 4, F = 2.85, P = 0.009) (Figure 5).

The M. anisopliae strains also performed good pathogenicity and observed maximum mortally rates of 45.83%, 37.50%, 20.83%, and 12.50% was observed on conidial concentrations of 1 x 10⁵, 1 x 10⁶, 1 x 10⁷, and 1 × 10⁸/ ml, respectively (Figure 6).  We observed a great difference in pathogenicity between the lowest and highest concentrations. However, all the concentrations were statistically different from the control treatment (Df = 4, F = 2.23, P = 0.067) (Figure 7).