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).

Fig. 1
figure 1

Front and reverse photograph of some entomopathogenic fungal colonies on PDA with, A Metarhizium anisopliae, B Beauveria bassiana, C Aspergillus niger

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, pinkish-brown, red, green or yellow. The reverses of the colonies 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. Out of the 111 (41.1%) EPF isolated, Aspergillus was the highest in number with 22.5%, followed by Beauveria and an identified genus with (19.8%) each. Lecanicillium (18.5%), Metarhizium and Fusarium had relatively low numbers with (15.3) and (4.5%), respectively. The highest number of isolates was recorded in Gombe and Kano states with (12.6%) each, followed by Lagos, Nasarawa and Port Harcourt with (10.8%) each. Abuja (9.9%), Ibadan and Sokoto recorded (9.0%) each, and Bauchi (8.1%) and Jos recorded the lowest isolates with (6.3%).

Table 2 Distribution of entomopathogenic, Metarhizium, Beauveria, Aspergillus, Fusarium, and unidentified species from different sampling sites

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.

Table 3 Species richness, abundance, Shannon index, evenness and Simpson’s index of six indigenous entomopathogenic fungi isolated from the different geographical zones in Nigeria

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 × 101 spores/ml Metarhizium than in 7.5 × 101 spores/ml Metarhizium, 5.0 × 101 and 7.5 × 101 spores/ml Aspergillus and in 5.0 × 101 and 1.0 × 102 Beauveria treatments (Fig. 2C).

Table 4 F-values for three-way analysis of variance result on the differential effect of entomopathogenic fungi Galleria mellonella larvae
Fig. 2
figure 2

Mean percentage larval mortality with A time after treatment, B entomopathogenic fungi and concentration. The differential effect of the entomopathogenic fungi on Galleria mellonella larvae. Data are mean (n = 4). Bar with different letters is significantly different, Tukey HSDs (P < 0.05)

The virulence of the EPF conidia on G. mellonella larvae as expressed in terms of LC50 showed a significant variation among the isolates (Table 5). However, this variation differed with states (sampling site) even within the same isolates.

Table 5 The percentage mortality and LC50 of the entomopathogenic fungal isolates on Galleria mellonella larvae

Metarhizium strain isolated in Bauchi had the lowest LC50 (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 LC50 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 

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