Biodiversity and virulence characterization of entomopathogenic fungi isolated from soils in different regions of Nigeria

Studying the diversity of the indigenous entomopathogenic fungi (EPF) is a prerequisite to effective insect pest control. The distribution and occurrence of EPF from the soil across Nigeria were evaluated. Three subsoil samples from three locations in each of the 11 states were used to isolate the fungi by the insect bait method. Three fungal rates, 1.0 × 102, 5.0 × 101 and 7.5 × 101 spores/ml, were applied to the Galleria larvae. EPF occurred in 41.1% of the soil sampled. These fungi belong to five and an unidentified genus. High species diversity was observed in Abuja with 6 species, and a uniform diversity of 5 species in Gombe, Ibadan, Jos, Kano, Lagos, Nasarawa and Port Harcourt. The Shannon–Wiener index ranges from 2.48 to 1.84. Similarly, species evenness showed a distinct similarity across the 11 sampling states and ranges from 0.99 to 0.92. Simpson Diversity Index was found to be highest in Bauchi with 0.31 and lowest in Abuja with 0.19. The mortality of the Galleria mellonella (L.) larvae differed significantly (P < 0.01) with EPF species and time of exposure. There was also a strong (P < 0.001) relationship between larval mortality and conidial concentration within and among the species. The virulence of the isolated fungi on Galleria mellonella larvae based on LC50 differs with states (sampling site) even within the same species. The present study uncovered the diversity and occurrence of EPF in soil across Nigeria. In addition, the results can be useful in selecting the suitable soils and best adapted EPF in a particular soil across the study area, or it could be the beginning of a molecular study.


Background
Entomopathogenic fungi (EPF) are natural enemies of insect. They play a vital role in regulating the insect pest population. This group of fungi can be found in five out of the eight fungal phyla (Araújo and Hughes 2016). Important genera of EPF are Metarhizium, Beauveria and Lecanicillium. EPF lower insect density, thereby reducing their disease-producing activities and consequently damage to crops (Sinha et al. 2016). The mode of actions deployed by EPF is unique from that of bacteria and viruses, it does not require ingestion, and rather they need contact action to invade their host directly through the cuticle.
Agricultural losses due to insect pests are a major threat to food security worldwide (FAO 2021). Crop losses in Nigeria and other African countries as a result of insect pests are estimated to be 49% of the expected total crop yield (Sharma et al. 2017), causing a loss of approximately U$$ 200 billion each year (Akinfenwa 2022).
Modern agricultural practices promote pests' development by the creation of a less stable natural ecosystem.
Page 2 of 8 Yakubu et al. Egyptian Journal of Biological Pest Control (2022) 32:93 Monocropping encouraged ideal food reserves which support pests' populations to develop higher densities than they would under a natural ecosystem (Sinha et al. 2016). Also, wide-spectrum synthetic chemical pesticides kill the natural enemies that regulate pests' populations. Despite the expensive nature of these chemical pesticides, their effect is temporal as the natural ability of insects allows them to develop resistance. Presently, over 500 insect species are resistant to one or more of these chemicals. These among other issues stimulate the renowned interest is the use of EPF in the control of insect pests. Nearly, all insects order is prone to fungal disease. EPF have diversity in mode of nutrition from biotrophy to necrotrophy and a general tendency to overcome insect defence (Sharma et al. 2020). Most of the research in this field focuses on the pathogenicity of EPF against specific insect pests. However, pathogenicity may vary with genera and strains. Therefore, there is a need to explore the diversity and virulence in the native EPF as an alternative method of integrated pest management.

Description of soil sample collection sites and sample collection techniques
Ninety-nine soil samples were collected from five geopolitical zones in Nigeria, namely Northcentral, Northwest, Northeast, South-south and Southwest. This includes Kano and Sokoto states; Bauchi, Gombe and Borno states; Rivers states; and Oyo and Lagos states, respectively (Table 1). Three subsoil samples of approximately 100 g were collected from three locations in each of the states. Table 1 shows the soil sample collection sites and the geographical locations. At each site, rhizospheric soil was collected using a hand auger at a depth of 10-15 cm. The three subsoils from each sampling site were mixed thoroughly to make a pooled sample. These samples were brought to the Ecology laboratory, Abubakar Tafawa Balewa University, and stored at 4 °C until further use.

Rearing of Galleria mellonella
Larvae of G. mellonella were reared at the Biological Science laboratory, Abubakar Tafawa Balewa University, according to the method described by Jorjão et al. (2018) with slight modification, using baby food cereal instead of wheat bran. Distilled water was heated to 80 °C, and honey, glycerol and bee wax were separately added while swirling vigorously. While still warm 50 ml of the baby cereal was added. 100 g of the medium above was placed in each 500 ml bottle, and 300 adult moths were added. The lid of the bottle was replaced by a wire mesh for ventilation, and the bottles were then wrapped with aluminium foil and incubated at 30 °C. After four weeks, 4th instar larvae became available and in additional 7-8 days, and the 5th instar were present.

Isolation of entomopathogenic fungi from soil
Thirty grams of soil sample was added to 300 ml of distilled water. The content was thoroughly mixed for half an hour using an electric stirrer. The supernatant was serially diluted up to 10 −6 dilutions. 100 µl of the sample from 10 −2 and 10 −4 dilutions was inoculated and smeared on a Potatoes (PDA) dextrose agar containing chloramphenicol (600 mg/l) and dodine (100 µl/500 ml). The Petri dishes were incubated for 5-7 days at 25 °C. The fungal colonies identical to the targeted fungi were sub-cultured onto fresh PDA and identified microscopically a using standard identification key. Pure isolates were stored in an agar slant and refrigerated at 4 °C.

Culture characterization
EPF isolates were culturally examined based on characteristics such as the pattern of growth, colony colour, shape and texture on PDA.

Morphological characterization
The isolated fungal cultures were identified microscopically using the slide culture technique (Fazeli-Dinan et al. 2016). A block of 1 cm 2 of potato dextrose agar was placed into a sterile petri dish containing a glass slide. A seven-day-old fungal culture was inoculated on the agar block and covered with a cover slide. The culture was incubated for five days at 28 °C. Once the fungal structure was observed, the cover slide was carefully removed and a drop of lactophenol cotton blue with lactic acid was added. The slide was viewed under a microscope, and images were captured. Morphological identification keys of Zare and Walter (2007) were used to identify the fungi. Each fungal species was identified based on conidia shape and size, conidiogenous cell and colony growth rate.

Fungal spores' suspension preparation
The spore suspensions of all isolates were prepared by growing the isolates on potato dextrose agar (PDA) for 14 days at 25 °C. The fungal spores were scraped using a sterile spatula and transferred into 10 ml of sterile distilled water containing 0.1% Tween 80. Mycelium was removed by filtering the suspensions with layers of cheesecloth. Spore concentrations of the filtrates were determined by enumerating the number of spores in 10 µl of each fungal suspension using Hemocytometer at 400 × magnification.

Estimation of entomopathogenic fungi diversity
The EPF diversity in the study area was determined using the ComEcoPac software version 1.0 (Drozd 2010). The diversity parameters evaluated include the Shannon-Wiener index (H'), Simpson's index (D), species abundance, species richness (R) and evenness of fungal communities (E H ). These diversity indices were measured according to the formula: where p is the number (n/N) of individuals of specific species found (n) divided by the total number of , Σ is the sum of the calculations, ln is the natural log, and S is the number of species.
where H' is the calculated Shannon-Wiener diversity; H max = In(s) species diversity under maximum equitability conditions.

Pathogenicity assay of selected isolates against Galleria mellonella
The pathogenicity of the major and consistent isolates was tested against G. mellonella larvae as described by Yakubu et al. (2018). Twelve larvae of 3rd to 4th instar were selected and placed in a Petri dish lined with moistened filter paper. Ten larvae were immersed in 2 ml of conidial suspension of different concentrations of 1.0 × 10 2 , 5.0 × 10 1 and 7.5 × 10 1 spores/ml. Each isolate was replicated four times. Control was maintained by dipping the larvae in 2 ml sterile distilled water. The treated larvae alone with the container were kept at 25 °C. The number of dead larvae was recorded at 24 h intervals for the following 10 days.

Data analysis
The spore production data were analysed using a oneway analysis of variance. The percentage mortality data were normalized using arcsine transformation (Khan et al. 2015) and subjected to analysis of variance using Minitab version 17 (Minitab Inc., USA). The significant difference between the means was determined by Tukey's test at P < 0.05. The LC 50 and LC 90 values were determined by probit analysis (Finney 1971).

Biodiversity and morphological characterization of fungi
A total of 111 (41.1%) EPF species belonging to five and an unidentified genus were identified from 127 soil samples. Based on morphological features, Metarhizium, Beauveria, Verticillium, Aspergillus, Fusarium and others were identified (Fig. 1A). The cultural characteristics of the Metarhizium isolates revealed greenish colour colonies on the top side, whereas the backs of the colonies were either brownish, white or yellowish-white. Metarhizium colonies had a round, thin to thick adpressed texture and flat to slightly raise elevation (Fig. 1A).
Beauveria colonies are white to yellowish with a smooth powdery to cottony texture, round in shape and showed a dispersed pattern of growth (Fig. 1B). Lecanicillium colonies were whitish to pale yellow, pinkishbrown, red, green or yellow. The reverses of the colonies Page 4 of 8 Yakubu et al. Egyptian Journal of Biological Pest Control (2022) 32:93 were either colourless, yellow or reddish-brown. Conidiospores are well-differentiated and erect, vertically branch-bearing whorls of slender awl-shaped divergent phialides. Aspergillus colonies were initially white or bluish-green powdery becoming black or yellow-green powdery later with darkly pigmented conidia. The reverses of the colonies are pale yellowish. The surface of the colonies is suede-like consisting of a dense felt of conidiospores (Fig. 1C).

Distribution and occurrence of the entomopathogenic fungi
The distribution and occurrence of the EPF isolated from the 11 states across Nigeria are summarized in Table 2.

Diversity indices
The variation pattern of the EPF isolated from the different sampling sites was evaluated, and species richness, species abundance, species diversity indices and evenness were determined as shown in Table 3. Species richness is the measure of different kinds of species in an area. The data obtained showed a relatively low species diversity in the 11 states: 6 species in Abuja, a uniform diversity of 5 species in Gombe, Ibadan, Jos, Kano, Lagos, Nasarawa  and Port Harcourt, while Bauchi and Sokoto recorded the lowest species richness with 4 species each.
The results of species abundance revealed a higher number of species in Gombe and Kano with 14 species each, followed by Lagos, Nasarawa and Port Harcourt with 12 species, Abuja with 11 species, Ibadan and Sokoto with 10 species each, and the lowest abundance was observed in Bauchi and Jos with 9 and 7 species, respectively (Table 3).
Shannon-Wiener index is the most preferred diversity index, is a measure of randomness and was relatively the same across all the states. The Shannon index ranged from 2.48 to 1.84. Similarly, species evenness showed a distinct similarity across the 11 sampling states and ranged from 0.99 to 0.92. Simpson's Diversity Index measures the probability of two individuals randomly selected belonging to a species. Simpson index was found to be highest in Bauchi with 0.31 and lowest in Abuja with 0.19 but was relatively the same in the rest of the states ( Table 3).

Pathogenicity of fungal isolates against Galleria mellonella
The analysis of variance of the effects of the EPF on G. mellonella larvae was significantly affected by days (time), EPF and EPF by concentration interaction (Table 4). The mortality of the Galleria larvae showed a progressive increase with an increased number of days, and mortality was significantly (P < 0.001) apparent from the fifth day after inoculation (Fig. 2A). Mortality was significantly higher (P < 0.01) and relatively the same in Metarhizium-, Lecanicillium-and Beauveria-treated larvae than in Aspergillus treatment (Fig. 2B). At the same time, there was a significant (P < 0.001) EPF by concentration interaction, suggesting that larval mortality differed with concentrations between and among the different entomopathogenic fungi. Mortality was significantly higher in larvae treated with 5.0 × 10 1 spores/ml Metarhizium than in 7.5 × 10 1 spores/ml Metarhizium, 5.0 × 10 1 and 7.5 × 10 1 spores/ml Aspergillus and in 5.0 × 10 1 and 1.0 × 10 2 Beauveria treatments (Fig. 2C).
The virulence of the EPF conidia on G. mellonella larvae as expressed in terms of LC 50 showed a significant variation among the isolates (Table 5). However, this variation differed with states (sampling site) even within the same isolates.
Metarhizium strain isolated in Bauchi had the lowest LC 50 (0.551 spores/ml) which signified a highest virulence than those isolated from Kano (5.914 spores/ml) and Sokoto (1202.073 spores/ml). Relatively lower LC 50 was also recorded in Lecanicillium (3.375 spores/ml) and Aspergillus species (7.391 spores/ml). Mean mortality values that did not share the same are significantly different

Discussion
In the present study, Aspergillus niger was found to have the highest rate of occurrence (22.25%), followed by Beauveria and an unidentified species (22.2% each). Ferial et al. (2019) observed Aspergillus species to be associated with a large number of insects. Here, Lecanicillium and Metarhizium recorded a moderate occurrence with 18.5 and 15.3%, respectively. The genus Fusarium recorded the lowest occurrence with 4.5%. This is in contrast with the findings of Bueno-Pallero et al. (2020) who observed that Fusarium had a frequent occurrence rate of 26%. Species in the genus Fusarium exhibited a high diversity of life strategies and types of associations with insects.
Fusarium is a weak virulent EPF, and it acts as s saprophyte on dead insects (Sharma and Marques 2018). The  Page 6 of 8 Yakubu et al. Egyptian Journal of Biological Pest Control (2022) 32:93 diversity indices of the isolated species showed a uniform diversity of five species identified in Gombe, Ibadan, Jos, Kano, Lagos Nasarawa, Kano and Port Harcourt. Bauchi and Sokoto recorded the lowest species richness. However, the highest species abundance was observed in Gombe and Kano, while the lowest was seen in Bauchi and Jos. The distribution of EPF was affected by the geographical location. This may be due to differences in temperature, humidity and soil conditions (e.g. moisture and organic matter). This suggestion is supported by the finding of Abdelghany (2015) who observed that increased organic matter aided infection by EPF. In addition, higher humidity is required by EPF for germination (Sharma et al. 2020). The Shannon-Wiener index, which is a measure of randomness, was found to be generally the same across all the states. While a high Simpson Diversity Index was recorded in Bauchi, it was found to be fairly the same in the rest of the states. All the isolated EPF showed pathogenicity to Galleria mellonella larvae. The larval mortality rate was significantly affected by the time and the EPF genera. The mortality showed a progressive increase with the increased number of days which was apparent from five days after inoculation. This finding is in agreement with Page 7 of 8 Yakubu et al. Egyptian Journal of Biological Pest Control (2022) (Şahin and Yanar 2021). The virulence characteristics of the isolated EPF on the G. mellonella larvae, as indicated in terms of LC 50 , revealed a strong-intraspecies difference among the isolates. Three strains of Metarhizium isolated from different states differed in virulence against G. mellonella larvae. Lecanicillium showed higher virulence than some strains of Metarhizium and Aspergillus species.

Conclusions
Six genera and 111 species of EPF were identified in the present study in soil across Nigeria. The abundance of these strains was directly affected by geographical location physical which may be associated with the spatial variation in soil chemical and physical properties. These indigenous strains of EPF are a promising alternative to the chemical control of insect pests. In addition, the results can be useful in selecting the suitable soils and best adapted EPF in a particular soil across the study area, or it could be the beginning of a molecular study.