Isolation, identification and virulence of indigenous entomopathogenic fungal strains against the peach-potato aphid, Myzus persicae Sulzer (Hemiptera: Aphididae), and the fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

As different biogeographic strains and isolates of entomopathogenic fungi vary in their genetic, enzymatic and pathogenic characteristics, this study assessed the virulence of 2 indigenous strains of Beauveria bassiana (Balsam) Vuillemin and Metarhizium anisopliae (Metschn.) Sorokin (Ascomycota, Hypocreales: Clavicipitaceae), isolated from naturally infected insect cadavers, against the 3rd instar nymphs of Myzus persicae (Sulzer) (Hemiptera: Aphididae) and 3rd instar larvae of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) using leaf-dip and larval-dip methods, respectively. Both fungal isolates exhibited considerable pathogenicity against M. persicae and S. frugiperda. Mortality in all bioassays was conidial concentration and exposure time dependent and increased significantly along with both factors (R2 = 0.86–0.99 for B. bassiana and 0.82–0.94 for M. anisopliae). Moreover, M. anisopliae isolate appeared more virulent to S. frugiperda larvae than B. bassiana isolate, while the later fungal isolate was more lethal to M. persicae nymphs than the former one. At the highest conidial concentration (1.0 × 109 conidia/ml), M. anisopliae caused maximum mean mortality of S. frugiperda (88%) and M. persicae (65%) and B. bassiana exhibited maximum mean mortality of S. frugiperda (76%) and M. persicae (94%). Moreover, probit regression analyses showed LT50 values for M. persicae of 4.57 and 6.86 days at 1.0 × 109 conidia/ml for the isolates of B. bassiana and M. anisopliae, respectively, while LC50 values were 7.75 × 106 and 8.70 × 107 conidia/ml after 10th day of application, for the isolates of B. bassiana and M. anisopliae, respectively, against M. persicae. Similarly, LT50 values for S. frugiperda were 7.75 and 7.03 days for 1.0 × 109 conidia/ml concentration and LC50 values were 2.84 × 107 and 8.84 × 105 conidia/ml at 10th day data for the isolates of B. bassiana and M. anisopliae, respectively. Overall study results demonstrated the effectiveness of B. bassiana and M. anisopliae against M. persicae and S. frugiperda, respectively. However, field evaluations of these indigenously isolated promising fungal strains against these insect pests.


Background
Almost all field and forage, fruit and vegetable crops and forest and ornamental plantations are attacked by a wide range of sucking and chewing insect pests (Lehmann et al. 2020). Aphids (Hemiptera; Aphididae) and armyworms (Lepidoptera; Noctuidae) are among the destructive and economically important insect pests throughout the world (Singh and Singh 2020).
Among aphids, the peach-potato aphid Myzus persicae (Sulzer) (Hemiptera: Aphididae) is a highly polyphagous pest species that infests and damages about 400 plant species from 40 families around the globe including Pakistan (Hlaoui et al. 2019). Moreover, M. persicae vectors about 100 plant viruses. Similarly, fall armyworm Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) is one of the economically important and notorious lepidopterous pests. It is native to tropical and subtropical Americas but has recently been dispersed to African and Asian countries including Pakistan (Gilal et al. 2020). It infests more than 80 agricultural crops, particularly rice, maize, cotton, millet, sorghum and sugarcane crops (De Groote et al. 2020).
Farmers in Pakistan rely primarily on synthetic pesticides to combat aphid and armyworm infestations on their crops. However, extensive use of broad-spectrum persistent synthetic chemicals has led to many issues such as insecticides resistance (Zhang et al. 2021), secondary pest outbreaks (Gross and Rosenheim 2011), eradication of beneficial fauna (Bueno et al. 2017), environmental contamination and human health hazards (Gomes et al. 2020). In view of these emerging ecological consequences of synthetic insecticides, it is imperative to seek out relatively safer and environment-friendly pest control options such as entomopathogenic fungi (EPF).
A wide range of EPF, particularly belonging to Beauveria, Isaria, Lecanicillium and Metarhizium genera, have been evidenced as potential biological control agents exhibiting significant virulence and pathogenicity against various insect pests. M. persicae and S. frugiperda are naturally infected by many species of EPF, some of which are very effective biocontrol agents (Firake and Behere 2020). The occurrence of such EPF naturally in an environment or agro-ecosystem indicates their potential role as biotic factors regulating insect pest populations in the field (Meyling and Eilenberg 2007).
Moreover, as EPF have a great genetic variation among their different biogeographic strains, the virulence and pathogenicity of different isolates to target insect pests may vary considerably due to their differential enzymatic and molecular characteristics (Maistrou et al. 2020). The present laboratory study aimed to isolate, identify and assess the virulence and entomopathogenicity of indigenous strains of EPF against the aphid M. persicae and the fall armyworm S. frugiperda as model mandibulate and haustellate phytophagous pests of economic importance, respectively.

Rearing of S. frugiperda
Late instar larvae of fall armyworm (S. frugiperda) were collected from the maize crop (Zea mays L.; hybrid cultivar Pioneer-32B33) grown in the farm area of Agriculture College, University of Sargodha (32°07′54" N; 72°41′35" E). The collected larvae were reared individually in a solitary manner in glass Petri plates (90 mm diameter) under controlled conditions (25 ± 2 °C, 65 ± 5% RH and 14-h: 10-h photoperiod). Larvae were fed on chickpea-based artificial diet prepared according to protocol of Jin et al. (2020) with slight modifications. Diet was changed daily until pupation and the newly formed pupae were placed on moistened filter paper discs lined in glass Petri plates (90 mm diameter). Newly emerged adults were shifted to rearing cages (30 × 30 × 30 cm; Bugdorm-I, Taiwan) for mating and egg lying and were provided with 10% honey solution soaked in cotton swabs as food. Egg clusters were shifted to glass Petri plates (90 mm diameter) on artificial diet. The culture of S. frugiperda was reared in the laboratory up to F 4 generation prior to their utilization in experimentation.

Rearing of M. persicae
Colonies of the peach-potato aphid (M. persicae) were randomly collected from the potato crop (Solanum tuberosum L.; white-skin cultivar Diamant) cultivated in the vicinity of Agriculture College, University of Sargodha (32°07′58" N; 72°41′32" E). This aphid population was reared on potted cabbage (Brassica oleracea L. var. botrytis) plants grown under controlled conditions (60 ± 10% RH and 27 ± 2 °C and 14-h: 10-h photoperiod). Old plants were replaced with new ones every week. The culture of M. persicae was reared in the laboratory for several generations before its utilization in the bioassays. Healthy and active insect individuals were used in all bioassays.

Isolation of EPF from naturally dead insects
Cadavers of naturally infected insects were collected from the leaf litter around agricultural field banks and from the sideway areas of an irrigation canal junction (32°08′0.5" N; 72°40′46" E). These sites were selected because of known insect pest activities with no application of insecticides and fungicides for the last few months. Insect cadavers were collected and placed in zip-lock polythene bags and were brought to the laboratory for isolation of EPF. Field-collected insect cadavers, potentially having a fungal infection, were surface-sterilized with 0.5% aqueous solution of sodium hypochlorite, followed by 3 rinsing with sterile distilled water, and then were dried by placing and rolling them on sterile filter paper sheet. Subsequently, these larvae were placed in glass Petri plates (90 mm diameter) lined with moistened sterile filter paper discs and were incubated at 25 ± 2 °C to stimulate the conidial germination (Herlinda et al. 2008). Fungal growth on insect cadavers was monitored on daily basis. When a fungal growth was observed, the conidia and hyphae were transferred to glass Petri plates (90 mm diameter) lined with potato dextrose agar (PDA) medium with the help of a sterile inoculation needle for purification and identification. Petri plates were incubated at 25 ± 2 °C and were inspected daily for fungal growth. At the point when more than one type of growth was developed on the same plate, they were isolated by sub-culturing. For further purification, small inoculum of fungal mycelia was cut out with a sterile inoculation needle and was moved to new Petri plates lined with Sabouraud dextrose agar (SDA) medium.

Identification of entomopathogenic fungi
Identification of isolated fungal strains was based on cultural and morphological features and was done by observing the growth pattern and colony formation of the fungal cultures. Slides were prepared from each isolate to identify its morpho-taxonomic characters using an inverted trinocular microscope (XDS-3, Optika SRL, Italy) by inspecting conidial structure and morphology according to the available literature and identification keys (Mongkolsamrit et al. 2020). Two cultures were identified as EPF: Beauveria bassiana (Balsam) Vuillemin and Metarhizium anisopliae (Metschn.) Sorokin (Ascomycota, Hypocreales: Clavicipitaceae) and were selected for further determination of their entomo-virulence against the model insect pests i.e., M. persicae and S. frugiperda.

Conidial suspensions preparation
Isolated cultures of B. bassiana and M. anisopliae were further mass cultured on Sabouraud dextrose agar yeast extract (SDAY) medium lined in glass Petri plates (90 mm diameter) incubated at 25 ± 2 °C. Conidial suspensions of both fungal isolates were prepared by harvesting their 15-day-old cultures with the help of a sterile inoculation loop and suspending them in 10 ml sterile doubledistilled water in sterile vials containing 0.1% Tween-80 as surfactant. The solution was gently mixed and filtered through a three-layer sterile muslin cloth to eliminate other mycelial mass. Conidial concentrations of both fungal filtrates were determined by improved Neubauer's hemocytometer as described by Ibrahim et al. (2016). From the stock solution, serial dilutions were made by the concentrations of 1.0 × 10 9 , 1.0 × 10 8 , 1.0 × 10 7 and 1.0 × 10 6 conidia/ml and were stored in refrigerator at 4 ºC until their downstream utilization in pathogenicity bioassays.

Bioassay of isolated EPF strains against M. persicae
Leaf-dip bioassay method as described by Nazir et al. (2019) was followed for determining the virulence of promising isolates of B. bassiana and M. anisopliae against M. persicae. In brief, leaf discs (50 mm diameter) were prepared from freshly clipped cabbage (B. oleracea var. botrytis) leaves and were dipped in each conidial concentration for 15 s. These treated leaf discs were placed on sterile filter paper sheet to remove excessive solution and were then shifted in sterile glass Petri plates (60 mm diameter) lined with 1.0% agar solution. In the control treatment, leaf discs were dipped in sterile doubledistilled water containing 0.1% Tween-80 solution. Ten late 3rd instar nymphs of M. persicae were released on the treated leaf discs using sterile camel hair brush, and Petri plates were incubated under controlled conditions (65 ± 5% RH and 25 ± 2 °C). Mortality of exposed aphid individuals was recorded on 3rd, 5th, 7th and 10th day post-exposure. Dead aphid nymphs were removed and placed immediately on moistened filter paper discs lined in glass Petri plates (60 mm diameter) and were inspected daily for the development of fungal mycelia in order to confirm their fungus infection-induced death (Additional file 1: Fig. S1).

Bioassay of EPF isolates against S. frugiperda
The virulence of B. bassiana and M. anisopliae isolates was determined against S. frugiperda by larval-dip bioassay method according to a previously described protocol (Ramanujam et al. 2020). In brief, freshly molted (0-6 h old) 3rd instar larvae of S. frugiperda were immersed in conidial concentrations for approximately 15 s. In control treatments, larvae were dipped in sterile double-distilled water having 0.1% Tween-80. Treated larvae were transferred in glass Petri plates (90 mm diameter) containing freshly cut leaves of cauliflower (B. oleracea var. botrytis) lined on 1.0% agar solution. Ten 3rd instar larvae of S. frugiperda were exposed in each Petri plate, and 5 replications were maintained for each treatment. Petri plates were incubated for 10 days under controlled conditions (27 ± 2 °C and 60 ± 5% RH). Leaves inside plates were changed every 2nd-or 3rd-day intervals. Larval mortality was recorded on 3rd, 5th, 7th and 10th day postexposure. Dead larvae were removed and inspected for the fungal infection as described above (Additional file 1: Fig. S1).

Statistical analysis
All bioassays were conducted according to the completely randomized design (CRD) with 5 replications for each treatment. Mean percent mortality of M. persicae nymphs was conidial concentration and time dependent as it increased along with the increase of conidial concentrations and exposure time. In case of B. bassiana isolate, aphid nymphal mortality increased significantly from day 3 to day 10 at all concentrations (R 2 = 0.97-0.99). Maximum nymphal mortality (94%) was exhibited by the highest concentration (1.0 × 10 9 conidia/ml) of B. bassiana recorded on 10th day of bioassay, while minimum aphid mortality (8-31%) was observed for the lowest concentrations (1.0 × 10 6 and 1.0 × 10 7 conidia/ml) on 3rd and 5th day of bioassay (Fig. 1). Similar trend of gradual and significant increase in the mortality of M. persicae nymphs was recorded for M. anisopliae isolate. Nymphal mortality increased significantly from day 3 to day 10 for all conidial concentrations (R 2 = 0.86-0.96). Maximum percent mortality (65%) was found at the highest conidial concentration (1.0 × 10 9 conidia/ml) on 10th day of bioassay, while the minimum aphid mortality values (6 -24%) were recorded for low concentrations (1.0 × 10 6 and 1.0 × 10 7 conidia/ml) at 3rd and 5th day of bioassay (Fig. 2).

Discussion
Contemporary issues of environmental contamination and health hazards being manifested by the extensive and recurrent use of highly persistent and hazardous synthetic insecticides necessitate looking for relatively safer and environment-friendly pest control options such as EPF which have been effective against a wide number of insect pest species (Litwin et al. 2020). However, these fungi exhibit considerable genetic variations among their biogeographic strains and the virulence and pathogenicity of different isolates to target insect pests may vary according to their differential enzymatic and molecular characteristics (Maistrou et al. 2020).
This laboratory work isolated, identified and assessed the virulence and pathogenicity of 2 promising indigenous strains of B. bassiana and M. anisopliae against M. persicae and S. frugiperda as a model mandibulate and haustellate phytophagous pests of economic importance. B. bassiana and M. anisopliae are ubiquitously found soil-born fungi capable of parasitizing a wide range of insect and mite pests (McGuire and Northfield 2020).
Bioassay results of aphids revealed that the indigenous isolate of B. bassiana was more pathogenic against 3rd instar nymphs exhibiting significantly a high mortality and minimum LT 50 and LC 50 values than those of M. anisopliae. These results are in line with the study of Bugti et al. (2018) in which four hemipteran pests including M. persicae were exposed to different conidial concentrations (1.0 × 10 2 to 6.75 × 10 5 conidia/mm 2 ) of a B. bassiana strain  and demonstrated that B. bassiana showed the highest pathogenicity to M. persicae and caused maximum mortality (100%) with LC 50 and  et al. Egyptian Journal of Biological Pest Control (2022) 32:2 LT 50 values of 6.7 × 10 4 conidia/ml and 5.2 to 8.24 days, respectively. Earlier, Kim et al. (2013) (Nazir et al. 2019), but also have been effective under the field conditions and appeared promising options for sustainable management of S. frugiperda and other lepidopterous insect pests (Mwamburi, 2021) and against sucking insect pests including M. persicae (Dannon et al. 2020). In cage and field experiments, aqueous conidial suspensions of B. bassiana isolates (CG-864 and PL-63) were demonstrated to reduce the M. persicae population and infestation by 60 to 80% than the control (Filho et al. 2011).
However, the present findings are not in line with those of Montecalvo and Navasero (2021) who demonstrated that the virulence of B. bassiana and M. anisopliae varied according to different life stages of S. frugiperda. In this study, B. bassiana appeared more virulent to 1st than 6 th larval instars with LC 50 values of 0.06 × 10 8 to 9.43 × 10 8 conidia/ml, respectively, but LT 50 values were 4.6 to 7.5 days, respectively. However, interestingly M. anisopliae isolate was more pathogenic to 3rd instar S. frugiperda larvae than B. bassiana although their difference was statistically non-significant. Moreover, the virulence of indigenous isolates may vary according to target insect pests. For instance, Gebremariam et al. (2021) showed that the indigenous Ethiopian isolates of B. bassiana were more pathogenic and lethal to G. mellonella than M. anisopliae isolates from the same soil samples.
Moreover, some studies have revealed the potential role of these EPF in phytopathogen antagonism, endophytism, rhizosphere colonization and in triggering the plant growth hormones (Ramos et al. 2020). Similarly, these insect parasitic fungi are also compatible with other pest control tactics including conventional and differential chemistry synthetic insecticides and along with other non-chemical control strategies (Quintela et al. 2013).

Conclusions
It is concluded that the indigenous isolate of M. anisopliae was more virulent to S. frugiperda larvae than B. bassiana isolate, while the later fungal isolate appeared to be more lethal to M. persicae nymphs than the former one, exhibiting significant mortality and minimum LT 50 and LC 50 values. These results corroborate the effectiveness of different strains and isolates of B. bassiana and M. anisopliae against M. persicae and S. frugiperda, respectively, and advocate the significance of considering indigenous isolates of microbial biocontrol agents against native and exotic insect pests. However, field evaluations of these indigenously isolated promising fungal strains against these target insect pests and on their natural enemies constitute the future perspectives of this work.