Efficacy of Entomopathogenic Fungi for Sustainable Biocontrol of Fungus Gnat (Bradysia difformis) in Peat-Free Substrates: A Laboratory Study

Abstract

Bradysia difformis Frey (Diptera: Sciaridae) is a fungus gnat that poses a significant threat to greenhouse cultures, and is attracted to soils devoid of peat. Fungal strains from the German Collection of Microorganisms and Cell Culture (DSMZ), such as Beauveria bassiana, Metarhizium flavoviride, Mucor hiemalis, and Niesslia tinuis, as well as Serendipita indica, were screened for entomopathogenic activity against B. difformis and their capacity to colonize Petunia hybrida cv. “Mitchell” and Ocimum basilicum plants. The survival rates of Bradysia difformis (three instar larvae) treated with Metarhizium flavoviride were 45.33% at 14 days following inoculation with 1 × 10⁶ spores/mL of each fungal strain, when compared to others. We concluded that the fungal strain M. flavoviride could serve as an entomopathogenic fungus with the highest virulence against B. difformis larvae. Although M. flavoviride did not show a beneficial effect as an endophyte, interestingly, the strain Niesslia tinuis exhibited plant growth benefits in Petunia hybrida cv. “Mitchell” by enhancing its shoot length up to 13.18 ± 0.72 cm, whereas the control treatment had a shoot length up to 10.68 ± 0.39. Enzymatic assays confirmed the ability of M. flavoviride to produce cuticle-degrading enzymes such as chitinase and protease. Together, these findings highlight the potential of EMPF—particularly M. flavoviride—as a sustainable biocontrol tool well-suited for peat-free horticultural systems, offering an eco-friendly alternative to chemical insecticides where fungus gnat pressure is typically high.

1. Introduction

Black fungus gnats, also known as sciarids (Diptera, Sciaridae), are small insects with adult lengths of up to 8 mm. They are predominantly dark in color, and their larvae typically develop among rotting plant remains that have been infested with fungal hyphae [1]. They are among the most prevalent insect pests of greenhouse ornamentals and production nurseries, especially Bradysia sp. (Diptera: Sciaridae) [2,3]. Two fungus gnat species, Bradysia coprophila Lintner and Bradysia difformis Frey (=B. impatiens Johansen), are regarded as significant greenhouse pests in Europe and the United States [4]. Although their primary food sources are fungi and organic materials, fungus gnat maggots can harm a variety of ornamental plants [5]. Plant roots and tunnel stems are frequently chewed or stripped by these maggots [6]. Severe damage to plants affects their ability to absorb water and nutrients, which causes them to lose vitality, turn off-color, and eventually die. Furthermore, adult flies are a nuisance to humans and spread fungal spores from plants to plants as they move around the greenhouse [7], whereas maggots can spread fungal pathogens (Fusarium, Phoma, Pythium, and Verticillium) during feeding [8]. During their lifetime, adult females frequently produce up to 1000 eggs, which they then deposit on the growth media surface [9]. Within four to six days, eggs hatch; within 12 to 14 days, maggots move through four instars, pupate in the soil for three to four days, and then emerge as adults. Accordingly, at 20–25 °C, the egg-to-egg life cycle can be finished in 20–25 d [9,10].

Bradysia species continue to be major greenhouse pests due to their capacity to transmit various plant pathogens and cause direct root damage, leading to significant crop losses. Their preference for moist, microbially rich growing media makes peat-free substrates, increasingly used for sustainability reasons, especially susceptible to infestations by B. difformis [11,12]. These organic-rich media provide ideal conditions for fungus gnat oviposition and larval development, highlighting the urgent need for effective biological control methods compatible with environmentally friendly horticultural practices. The horticultural industry’s transition to peat-free substrates, driven by environmental sustainability objectives, has inadvertently resulted in a marked increase in the population of fungus gnats (Diptera: Sciaridae) in greenhouse cultures. The high organic content and microbial activity of peat-free media, such as composted green waste and other organic-rich materials, attract these pests, especially species like Bradysia difformis. It has been demonstrated by means of empirical observation that the substrates in question provide a favorable environment for fungus gnat oviposition and larval development, thus resulting in infestations in greenhouse environments. Notwithstanding the fact that fungus gnat larvae primarily consume decomposing organic matter, they have been observed to cause harm to plant roots and root hairs. This has the potential to compromise the health and vitality of the plants cultivated in such systems. This phenomenon assumes particular significance in systems devoid of peat, where the abundance of organic waste serves to stimulate the proliferation of gnats. Consequently, the management of fungus gnat populations has become a significant challenge in the context of environmentally sustainable horticultural practices [12].

Administering organophosphates, carbamates, and neonicotinoid pesticides is currently the most widely used method for controlling fungus gnat larvae [13]. However, owing to the development of pesticide resistance in larvae following years of treatment, the level of control has been inadequate [14]. Concerns about human health and environmental contamination have led to growing restrictions on the use of conventional chemical pesticides [15]. Thus, investigating biological control options is crucial for reducing the use of pesticides, lowering environmental pollution levels, and enhancing the control of pest populations.

A fundamental component of Integrated Pest Management (IPM) is biological control, which includes the use of natural enemies such as predators, parasitoids, entomopathogenic fungi, nematodes, bacteria, and insect viruses, due to their effectiveness and low environmental impact [16,17,18]. Entomopathogenic fungi (EMPF) are parasitic microorganisms that can infect and significantly increase the mortality of arthropod populations. They are widely used in the biological control of insects because of their biology, which allows them to infect them through touch, pierce their cuticles, and multiply in the hemocoel [19]. A diverse group of EMPF includes potential biocontrol agents targeting agriculturally important pest species [20,21]. Entomopathogenic fungi used for pest control include Beauveria bassiana (Balsamo), Paecilomyces fumosoroseus (Wize), Nomuraea rileyi (Farlow), Verticillium lecanii (Zimmerman), and Metarhizium anisopliae (Metschnikoff) [22]. Only a few entomopathogenic fungal species have been successfully developed into commercial biopesticides, where they have shown promising results against fungus gnats (Diptera: Sciaridae), which are significant pests in greenhouse and nursery production. For example, Andreadis et al. [23] reported that Beauveria bassiana formulations caused 100% mortality in adult Lycoriella ingenua, demonstrating strong pathogenicity in mushroom cultivation environments. In greenhouse trials, Metarhizium brunneum was found to significantly reduce larval survival and adult emergence of Bradysia impatiens when applied to growing substrates. Furthermore, the combined application of M. brunneum with botanical compounds enhanced control efficacy, indicating potential for integrated biopesticide strategies [24]. These studies highlight the direct applicability of entomopathogenic fungi for controlling fungus gnats, supporting their integration into sustainable pest management programs. These examples illustrate the diverse potential of entomopathogenic fungi to manage different arthropod pests through various mechanisms, making them valuable tools in integrated pest management strategies. As highlighted by Tóthné Bogdányi et al. (2019) [25], M. flavoviride exhibits broad host specificity, effective cuticle degradation, and a notable potential for sustainable pest control. However, its performance in peat-free horticultural systems remains underexplored.

In addition to their role as biological insecticides, increasing evidence suggests that numerous EMPF species have the ability to colonize the tissues of specific plants. A plethora of endeavors have been undertaken to artificially induce the presence of electromagnetic pulses (EMPs) in plants, employing a range of methodologies. However, only a limited number of EMPF species have been identified as naturally occurring endophytes [26,27]. As it has been shown to increase plant growth and decrease pest infestation in a variety of economically important crops, this natural or artificial colonization may be advantageous for the plant [28,29,30,31]. EMPF endophytes have been found in hundreds of plants, such as a number of significant crops, including wheat, soybeans, bananas, and tomatoes [30,32,33], and as beneficial rhizosphere colonizers [34,35] and plant growth promoters [36,37,38,39]. Nevertheless, the diverse functions of fungal entomopathogens can also be economically and subsequently utilized in sustainable agriculture, such as microbial control agents against plant diseases and arthropod pests [39,40,41,42] as vertically transmitted fungal endophytes that can enhance host plant fitness, induce systemic resistance, and provide long-term pest and disease suppression [38,43,44,45].

The objective of this study was to evaluate the potential of selected fungal strains obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ), namely Beauveria bassiana (strain 1344), Metarhizium flavoviride (strain 21704), Mucor hiemalis (strain 2655), and Niesslia tinuis (strain 3481). In this group, B. bassiana, M. flavoviride, and M. hiemalis are well-established entomopathogenic fungi, whereas N. tinuis represents a novel species that has not previously been investigated for its potential as either an entomopathogen or an endophyte. The objective of the present study was to evaluate the potential of the fungal strains under investigation, in conjunction with Serendipita indica, a well-documented plant endophyte, to regulate the population dynamics of Bradysia difformis and to establish a colonizing presence within the root systems of Petunia hybrida cv. “Mitchell” and Ocimum basilicum plants.

2. Materials and Methods

2.1. Efficacy of Entomopathogenic Fungi to Manage Fungus Gnat Larvae from Becoming Adults

Five fungal strains such as Beauveria bassiana (1344), Metarhizium flavoviride (21704), Mucor hiemalis (2655), and Niesslia tinuis (3481), obtained from DSMZ and Serendipita indica, were assessed for their ability to affect the population of fungus gnat larvae to become adults. The sterile regular coconut block with potting soil substrate used for the fungus gnat culture was placed in 750 mL plastic cups and treated with fungal spore solution at a concentration of 1 × 10⁶ spores/mL. A negative control, consisting of sterile water, and a positive control using 0.1% Neemazal were included in the experimental design. The boxes were incubated for 24 h at 16:8 light: dark and 25 ± 1 °C. After 24 h of incubation, a fine paintbrush was used to place 25 freshly molted third-instar fungus gnat larvae into each treated control cup. The cups were kept in a growth room with a 16:8 light-dark photoperiod at 25 ± 1 °C and 65 ± 5% relative humidity (RH). A lid was placed on each cup to prevent contamination and larval escape. A yellow sticky trap was attached to the top of each cup to monitor adult emergence, and the moisture content of the treatments was maintained by spraying sterilized distilled water every other day. The number of adults caught in the traps was recorded throughout the observation period. Observations were made over a two-week period to evaluate adult emergence, fungal effectiveness, and larval development in adults, where six replicates of each treatment were used. The trial was terminated when no new adults were counted within five days. The mortality rate and control efficiency were calculated according to Equations (1) and (2) [46,47]. Prior to conducting ANOVA, data were tested for normality using the Shapiro–Wilk test, and for homogeneity of variances using the Brown–Forsythe test. Statistical analyses were performed using GraphPad Prism version 10.5, and treatment means were compared using Tukey’s HSD test at a significance level of p ≤ 0.05.

$$Mortality rate={\displaystyle \frac{\mathrm{Number}\mathrm{of}\mathrm{dead}\mathrm{larave}}{\mathrm{Total}\mathrm{Number}\mathrm{of}\mathrm{treated}\mathrm{larvae}}}\times 100$$

(1)

$$Control efficiency={\displaystyle \frac{\mathrm{Mortality}\mathrm{rate}\mathrm{of}\mathrm{control}\mathrm{group}-\mathrm{Mortlity}\mathrm{rate}\mathrm{of}\mathrm{treatment}\mathrm{group}}{100-\mathrm{Mortality}\mathrm{rate}\mathrm{of}\mathrm{control}\mathrm{group}}}\times 100$$

(2)

2.2. Larval Survival on Pure Fungal Cultures

Initially, 25 third-instar larvae of Bradysia difformis, were placed on water agar plates (Carl Roth GmbH and Co. KG, Karlsruhe, Germany) for 6 h to cleanse their guts. The larvae were then moved with a small brush to fungal cultures, where they stayed to finish growing and become adults. Each control treatment consisted of PDA plates without fungal growth. Every day, larval death and adult emergence were carefully observed with a binocular microscope, while all plates were kept in a climate chamber at 25 ± 1 °C in darkness. Eight replicates, each containing 25 larvae, were created for each treatment, with 200 larvae per treatment. Fungal plates of each strain without B. difformis larvae and sterile PDA plates with larvae inoculated without fungal inoculation were also used as controls.

Additionally, the dead larvae and adults obtained from the petri dish underwent two rinses in sterile distilled water and a 30 s immersion in 0.5% sodium hypochlorite for surface sterilization. Following surface sterilization, the larvae were placed on PDA medium to observe the fungal outgrowth.

2.3. Chitinase Production Assay

2.3.1. Preparation of Culture Filtrates

A basal medium composed of (per liter): 0.3 g MgSO₄ × 7H₂O, 3.0 g (NH₄)₂SO₄, 2.0 g KH₂PO₄, 0.91 g citric acid, 200 μL Tween-80, and 4.5 g colloidal chitin was prepared, the pH was adjusted to 5.0, and the mixture was autoclaved at 121 °C for 20 min. After the sterilization process, the medium was inoculated with a culture disk (6 mm diameter) taken from the growing margins of fungi on a PDA plate. The inoculated flasks were then placed on a rotary shaker at 200 rpm and 28 °C. Each treatment was replicated five times. The culture filtrate (250 mL) was used as the crude enzyme extract after incubation for 5, 10, and 20 d. The culture broths from both media were filtered through a 0.22 μM bacterial-proof filter. Culture filtrates were used as sources of crude chitinase and kept throughout the experiment at −20 °C for further experiments.

2.3.2. Preparation of Colloidal Chitin

A modified protocol from Roberts and Selitrennikoff [48] was used to make colloidal chitin. Briefly, 5 g of chitin powder derived from crab shells (Carl Roth GmbH and Co. KG, Karlsruhe, Germany) was slowly added to 60 mL of concentrated HCl (Carl Roth GmbH and Co. KG, Karlsruhe, Germany) under vigorous stirring and allowed to stand at room temperature for at least three hours. The resulting mixture was then poured into 200 mL of ice-cold 95% ethanol and stirred vigorously overnight at room temperature to facilitate precipitation. The precipitated colloidal chitin was collected by centrifugation at 4000× g for 20 min at 4 °C. The pellet was transferred to a glass funnel fitted with filter paper and washed repeatedly with distilled water using a vacuum filtration system until the pH of the filtrate reached 5.0. The washed and pH adjusted colloidal chitin retained on the filter paper was scraped off, weighed, and stored at 4 °C in the dark until further use.

2.3.3. Qualitative Detection of Chitinase Enzyme

Qualitative detection of chitinase enzyme was performed using the protocol described by [49] with minor modifications. A chitinase detection medium was prepared using a basal medium composed of (per liter): 0.3 g MgSO₄·7H₂O, 3.0 g (NH₄)₂SO₄, 2.0 g KH₂PO₄, 0.91 g citric acid, 15 g agar, 200 μL Tween-80, 4.5 g colloidal chitin, and 0.15 g bromocresol purple. The pH was adjusted to 5.0, and the mixture was autoclaved at 121 °C for 15 min. The sterilized lukewarm medium was poured into Petri dishes and allowed to solidify. To assess chitinase activity, fresh culture plugs of the isolates were introduced into the solidified medium and incubated at 25 ± 2 °C. Plates were observed for the formation of purple-colored zones around the colonies. In parallel, isolates were inoculated into colloidal chitin broth without bromocresol purple and incubated for 20 days. The resulting supernatant was filtered through a 0.22 μm bacterial-proof filter on the 5th, 10th, and 20th days to verify the reproducibility of the results. Wells were prepared in solid media Petri plates supplemented with colloidal chitin, and each well was filled with the filtered supernatant. A commercially available chitinase enzyme derived from Trichoderma harzianum (ASA Spezial enzyme GmbH, Wolfenbüttel, Germany) was used as the positive control, while colloidal chitin broth without fungal inoculation served as the negative control. Both controls were incubated at room temperature for 15 min. The appearance of a purple zone surrounding the wells was considered indicative of chitinase activity.

2.3.4. Assay for Chitinase Activity

Quantification of endochitinase activity was determined by mixing colloidal chitin with the enzyme solution in a 1:1 ratio and incubated at 37 °C for 1, 2.5, 4, 5.5, 7, and 8.5 h. The reduction in turbidity of the colloidal chitin suspension, measured as absorbance at 510 nm was used as an indicator of enzyme activity, following the method of Tronsmo and Harman [50]. The colloidal chitin suspensions, sterilized by autoclaving, consisted of 1% (w/v) colloidal chitin in 100 mM acetate buffer at pH 5.0. Chitinase activity was expressed as the percentage decrease in turbidity relative to a control suspension containing water in place of the enzyme solution. One enzyme unit was defined as the amount of enzyme required to reduce the turbidity of a chitin suspension by 5%, as described by [50].

2.4. Protease Production Assay

The method described by Zghair [51] was performed to detect protease production in fungi with slight modification. Skim milk agar was prepared by dissolving 10 g of skim milk powder (Carl Roth GmbH and Co. KG, Karlsruhe, Germany) in 100 mL distilled water with gentle stirring at approximately 37–40 °C to aid solubility without denaturing milk proteins. The basal media consisting of 10 g of agar and 2 g of yeast extract were dissolved separately in 900 mL of distilled water. Subsequently, the pH of yeast extract-agar solution was adjusted to 7 and autoclaved at 121 °C for 20 min. The skim milk solution was sterilized separately by filter sterilization using a 0.22 μm bacterial-proof filter. Once the autoclaved agar media was cooled to approximately 45 °C, sterile skim milk solution was added aseptically and mixed thoroughly and poured into the Petri plates. To assess protease activity, fresh culture plugs of the isolates were introduced into the solidified medium and incubated at 26 °C for 3 to 7 days. Protein breakdown (casein in milk) by the production of protease enzyme was detected when a transparent halo around the fungal colonies appeared.

2.5. Root Colonizing Ability of EMPFs

2.5.1. Preparation of ½ Murashige and Skoog (MS) Medium

To prepare Murashige and Skoog (½ MS) agar medium, 1.1 g of MS vitamins, 5 g of sucrose, and 3.5 g of plant agar purchased from Duchefa Biochemie, Haarlem, The Netherlands were dissolved in 500 mL of distilled water. The pH was set to 5.8 before autoclaving at 121 °C for 20 min. The sterilized material was then poured into sterile Petri plates and 24 well plates (5 mL per well) and stored aseptically until needed.

2.5.2. Seed Sterilization and Germination

Seeds of Petunia hybrida cv “Mitchell” and Ocimum basilicum underwent surface sterilization with immersion in 70% ethanol for 3 minutes followed by a 10 min treatment with 10% of sodium hypochlorite (NaOCl). After treating with NaOCl, seeds were rinsed three to four times with sterile distilled water. Sterilized seeds (20–25 per plate) were germinated on ½ MS agar plates under controlled conditions in a phyto-chamber at 24 °C, 16:8 light/dark (L:D) photoperiod for ten days. After ten days of incubation, uniform healthy seedlings were selected for further study.

2.5.3. Inoculation of Test Fungi

In this study, direct mycelial inoculation method was used for the analysis of fungal root colonization and plant growth benefits. Fungal discs of test fungi measuring 6 mm were excised from the actively growing margin of the freshly cultivated fungal plate with PDA as culture media. Each seedling was inoculated with a single fungal disc in the root zone ensuring the fungal mycelia and the root tissues are in intimate contact. The plant saplings were planted in 24-well plates containing 5 mL of ½ MS agar medium in each well. The fungal strains acquired from DSMZ were used as test treatments, and the control groups included a negative control that was not inoculated with any fungi, and seedlings treated with Serendipita indica were used as a positive control. Each treatment was replicated six times. The plants were incubated at 24 °C with a 16:8 L:D photoperiod in a climate-controlled growth chamber for 20 d.

2.5.4. Assessment of Root Colonization and Plant Growth Effects

The plants were harvested and subjected to growth examination for confirming the fungal colonization in the root, which included measurement of shoot length, root length, and the number of leaves produced. After being sequentially washed with sterile distilled water, the root samples were treated with 70% ethanol for 3 min, 2.5% NaOCl for 6 min, and three additional rinses with sterile distilled water. Surface sterilized roots were chopped into 1 cm long pieces and placed on PDA plates. The plates were incubated at 26 °C in the dark, and fungal development from the roots was observed. To confirm colonization, DNA was extracted from the outgrown fungi according to Cenis methodology [52] and sequenced for identity confirmation (Eurofins, Genomics, GmbH, Konstanz, Germany and NCBI; Blast search).

3. Results

3.1. Effect of Entomopathogenic Fungi to Control the Bradysia Difformis Larvae

The four DSMZ strains that were selected for this experiment, Beauveria bassiana (1344), Metarhizium flavoviride (21704), Mucor hiemalis (2655), and Niesslia tinuis (3481) along with Serendipita indica were examined for their ability to lower the survival rate of B. difformis larvae, with fewer adults emerging from the treated substrate containing pure fungal spores than from the sterile water control and 0.1% Neemazal. When compared to the spore solutions (1 × 10⁶ spores/mL) of other fungal strains, B. difformis larvae were significantly susceptible to the treatment fungal culture M. flavoviride (

Table 1. Means of survival and mortality rates and control efficiency by 1 × 10⁶ spores/mL of selected fungal isolates of DSMZ along with S. indica with estimated control efficiency third-instar larval stages of B. difformis. (SE = standard error).

Treatments	Mortality Rate (%) ± SE	Control Efficiency (%) ± SE
A. dest (sterile)	13.33 ± 1.8	0
0.1% Neemazal	58.00 ± 3.5	51.54 ± 4.08
B. bassiana	21.33 ± 5.3	9.23 ± 6.7
N. tinuis	19.33 ± 4.05	6.92 ± 4.7
M. flavoviride	54.66 ± 3.2	44.61 ± 3.7
M. hiemalis	24.20 ± 4.9	12.27 ± 5.7
S. indica	24.66 ± 4.05	13.08 ± 4.6

). The emergence of adults in substrate was significantly inhibited by M. flavoviride up to 44.61% in comparison to the control treatments (0.1% Neemazal = 51.54%, p < 0.001). In terms of control efficiency, the number of fungus gnat adults that emerged from B. bassiana (9.23%), M. hiemalis (12.27%), N. tinuis (6.92%), and S. indica (13.08%) was substantially higher than the number of adults that emerged from the control treatment (0.1% Neemazal = 51.54, p < 0.001) and did not significantly lower the survival rate of larvae, resulting in a higher emergence rate of adults with a survival rate of 78.66% (p < 0.4057), 72% (p < 0.102), 80.66% (p < 0.6640), and 13.08% (p < 0.132). In contrast, the number of adults emerging from M. flavoviride treatment was lower with a survival rate of 45.33% (p < 0.001), while 0.1% Neemazal, the positive treatment of this study, showed a survival rate of 42% (p < 0.001) (Figure 1A,B). The outcomes were confirmed by examining the impact of varying M. flavoviride concentrations (1 × 10³ spores/mL, 1 × 10⁵ spores/mL, and 1 × 10⁷ spores/mL) on the emergence of B. difformis larvae into adults. As shown in

Table 2. Means of survival and mortality rates by different concentrations of M. flavoviride (1 × 10³ spores/mL, 1 × 10⁵ spores/mL and 1 × 10⁷ spores/mL), with estimated control efficiency in third-instar larval stages of B. difformis. (SE = standard error).

Treatments	Mortality Rate (%) ± SE	Control Efficiency (%) ± SE
A. dest (sterile)	17.71 ± 7.2	0
0.1% Neemazal	59.42 ± 4.4	50.69 ± 5.3
M. flavoviride 1 × 103	37.14 ± 6.5	23.61 ± 7.9
M. flavoviride 1 × 105	57.71 ± 6.3	48.61 ± 7.6
M. flavoviride 1 × 107	74.28 ± 5.3	68.75 ± 6.5

, the highest activity in controlling the larvae to emerge as adults was significantly exhibited by 1 × 10⁷ spores/mL with control efficiency 68.75%, which showed 25.71% of survival rate, followed by 1 × 10⁵ with control efficiency 48.61%, which showed 48.28% of survival rate, while 1 × 10³ spores/mL with control efficiency 23.61%, which showed 62.85% of survival rate, did not show any significant reduction in adult emergence (Figure 2A,B).

3.2. Larval Survival on Pure Fungal Cultures

Survival rates of larvae progressively declined from the first day after treatment when exposed to the different fungal cultures (Chisq = 25, df = 4, p < 0.001; Figure 1). The control group of larvae, reared on pure PDA, survived only for four days, which was significantly shorter than in all other fungal treatments. In comparison, larvae exposed to fungal cultures of M. flavoviride (21704), B. bassiana (1344), M. hiemalis (2655), and N. tinuis (3481) survived for 8, 14, 19, and 16 days post-exposure, respectively. Larvae treated with M. flavoviride were the most susceptible, with only 7.85 ± 0.95% adult emergence (total N = 200), significantly lower than all other treatments. Exposure to B. bassiana resulted in intermediate survivorship, with 18.62 ± 1.71% (N = 200) of larvae successfully completing their life cycle to adult flies. A significant difference in adult emergence rates was observed between these two treatments (p = 0.0013). Larvae exposed to N. tinuis and M. hiemalis exhibited prolonged survival for up to 16 days post-infection, with adult emergence rates of 20.37% and 20.87% (total N = 200), respectively. Although differences in emergence rates between these two treatments were not significant, both demonstrated significantly higher survival rates compared to the M. flavoviride treatment (p = 0.0002) (Figure 3).

3.2.1. Qualitative Detection of Chitinase Activity

Chitinase activity was assessed by inoculating fresh fungal plugs and culture filtrates into basal chitinase detection medium, as described by Agrawal and Kotasthane [49], supplemented with colloidal chitin (4.5 g/L) and bromocresol purple (0.15 g/L). As illustrated in Figure 4 and Figure 5, the plate assay revealed that Beauveria bassiana and Metarhizium flavoviride, both culture plugs and supernatants, exhibited a pronounced purple coloration, indicating high chitinase activity. In contrast, Mucor hiemalis culture plugs displayed minimal activity. Additionally, culture supernatants collected at different incubation intervals (5th, 10th, and 20th days) were analyzed to confirm chitinase production. The results confirmed preliminary screening with fresh fungal plugs, revealing that B. bassiana produced a more intense purple zone around the wells compared to M. flavoviride, while no zone formation was observed for the supernatant from M. hiemalis. Notably, the intensity of the purple zones surrounding the wells of M. flavoviride increased progressively with incubation time, with supernatants collected on day 20 producing the highest intensity, followed by those obtained on days 10 and 5 (Figure 4 and Figure 5).

3.2.2. Assay for Chitinase Activity

Chitinase activity in culture filtrates was assessed using colloidal chitin as the substrate and incubating the reaction mixture at 37 °C for time intervals ranging from 1 to 8.5 h. A consistent increase in chitinase activity was observed across all incubation durations (5th, 10th, and 20th day filtrates). Among the tested strains, Beauveria bassiana exhibited the highest chitinase activity, increasing from 19.8 ± 0.05 to 31.6 ± 2.9 U/mL, with the 10-day culture filtrate reaching a peak activity of 38.4 ± 4.3 U/mL, although a notable decline was recorded at the 7 h time point, followed by M. flavoviride showing activity ranging from 19.8 ± 0.16 to 28.64 ± 2.1 U/mL, as well as a similar drop at the 7 h mark. In contrast, Mucor hiemalis demonstrated the lowest chitinase activity among the three fungi, with values increasing from 8.7 ± 1.3 to 24.5 ± 4.5 U/mL across the tested incubation periods. Overall, B. bassiana filtrates (10 and 20 days) exhibited higher chitinase activity, followed by M. flavoviride, while M. hiemalis consistently showed the lowest activity levels (Figure 6,

Table 3. Chitinase activity (mean ± SE, five independent replicates) exhibited by the selected DSMZ strains for 5 d, 10 d and 20 d of incubation (SE = Standard Error).

Culture Filtrates	Chitinase Activity (Units/mL)
	5 d	10 d	20 d
B. bassiana	19.8 ± 0.05 a	38.4 ± 4.3 a	31.6 ± 2.9 a
M. flavoviride	19.8 ± 0.16 a	27.70 ± 3.4 a	28.64 ± 2.1 a
M. hiemalis	8.7 ± 1.3 a	11.78 ± 0.25 b	14.5 ± 4.5 b

Means in the same columns followed by different lowercase superscripts are significantly different from each other (Tukey’s HSD, p ≤ 0.05).

).

3.3. Qualitative Identification of Protease Production

Protease activity was assessed by inoculating fresh fungal plugs onto skim milk agar plates. Protease producing fungi among the DSMZ strains Beauveria bassiana, Metarhizium flavoviride and Mucor hiemalis were identified by the formation of a clear zone around the fungal colonies, indicating casein hydrolysis. As shown in Figure 7, the skim milk agar plate assay revealed that M. flavoviride exhibited the largest clear zone around its colony (17 ± 0.57 mm), followed by B. bassiana (8 ± 0.57 mm), both indicating significant protease activity. In contrast, M. hiemalis produced only a narrow zone of clearance, indicating minimal protease secretion. The pronounced difference in the diameter of the clear zones suggests that M. flavoviride and B. bassiana possess higher extracellular protease activity under the tested conditions compared to the other isolates.

3.4. Effect of Fungal Colonization in Petunia and Basil Plants

The impact of different fungal inoculations on Petunia hybrida cv. “Mitchell” growth was evaluated by measuring shoot length, root length, and number of leaves after 20 days of incubation (Figure 8, Supplementary Table S2). A significant increase in shoot length was observed in seedlings treated with N. tinuis (13.18 ± 0.72 cm) compared to the control (10.68 ± 0.39 cm; p ≤ 0.05). In contrast, M. flavoviride and M. hiemalis treatments significantly reduced shoot length to 3.18 ± 0.57 cm and 0.88 ± 0.08 cm, respectively (p ≤ 0.05). No statistically significant differences in root length were detected between the control and any of the fungal treatments (p > 0.05). In terms of leaf production, M. hiemalis inoculation resulted in a significant reduction in the number of leaves (7.00 ± 0.36) compared to the control (11.33 ± 0.42; p ≤ 0.05). Other treatments, including N. tinuis and M. flavoviride, did not show a significant impact on leaf number. Overall, N. tinuis promoted shoot elongation in Petunia hybrida, while M. flavoviride and M. hiemalis exerted inhibitory effects on growth parameters. Similarly, in Ocimum basilicum, a significant reduction in shoot length was recorded in seedlings treated with M. flavoviride (3.28 ± 0.47) and M. hiemalis (2.93 ± 0.38 cm) compared to the control (5.70 ± 0.47; p ≤ 0.05). Both fungal strains also significantly reduced root length, with values of 5.51 ± 0.48 cm for M. flavoviride and 5.95 ± 0.69 cm for M. hiemalis, relative to the control (12.48 ± 0.47; p ≤ 0.05). Moreover, the number of leaves was significantly decreased in plants inoculated with M. flavoviride (8.33 ± 1.05) and M. hiemalis (5.16 ± 0.54) compared to control seedlings (12.33 ± 1.33; p ≤ 0.05). These results suggest that while M. flavoviride and M. hiemalis negatively impacted Ocimum basilicum growth, other tested fungi did not significantly alter growth parameters (Figure 9, Supplementary Table S3). Surface sterilization and subsequent incubation of plant roots confirmed that M. flavoviride and N. tinuis were capable of colonizing the plant roots. However, among these, only N. tinuis demonstrated a significant influence on the growth of Petunia hybrida cv. “Mitchell”, specifically by promoting an increase in shoot length. Molecular identification through DNA extraction and sequencing further validated the successful colonization of these fungal strains within the plant root tissues (Supplementary Table S1).

4. Discussion

In this study, we checked selected fungal strains from German Collection of Microorganisms and Cell Culture (DSMZ) namely Beauveria bassiana, Metarhizium flavoviride, Mucor hiemalis, and Niesslia tinuis for their ability to act as entomopathogens against the fungus gnat, Bradysia difformis along with its ability to act as endophytes in Petunia hybrida cv. “Mitchell” and Ocimum basilicum. The results of the bioassay revealed that the significant entomopathogenic potential of M. flavoviride in controlling the population of B. difformis, a species of fungus gnat. Among the four selected DSMZ fungal strains and Serendipita indica tested, only M. flavoviride exhibited a significant inhibitory effect on adult emergence, leading to a significant reduction in the survival rate of larvae. This finding confirms previous findings that M. anisophile, a well-documented entomopathogen belonging to the same genus as M. flavoviride, is effective against the insect pests including those within the order Diptera (Fungus gnats) [53]. The effectiveness of B. bassiana and M. anisopliae against the spiny bollworm (Earias insulana) on cotton plants was assessed in a study published in the Egyptian Journal of Biological Pest Control [54]. The results indicate that in some situations, Metarhizium sp. may provide better control than B. bassiana. In this case, M. flavoviride’s considerable pathogenicity was noticeably greater than that of the other evaluated fungal strains. In line with previous research indicating that these fungi might not always be as efficient against specific pest species under controlled settings, B. bassiana and M. hiemalis did not exhibit a substantial decrease in larval survival or adult emergence [47]. Remarkably, in the current investigation, the DSMZ-obtained strain of Mucor hiemalis showed no pathogenicity towards fungus gnat populations which stands in contrast to research conducted [55] where they who found that the isolated strain of M. hiemalis BO-1 was significantly harmful to the fungus gnat Bradysia odoriphaga (Diptera: Sciaridae). Notably, higher spore concentrations (1 × 10⁷ spores/mL) of M. flavoviride significantly reduced adult emergence of B. difformis, which was accompanied by a lower survival rate of 25.71%. This concentration-dependent effect of M. flavoviride was also noted in line with recent research that found that larger concentrations of M. flavoviride result in better control of insect populations, where it shows that the concentration of the entomopathogenic fungus is a critical factor in increasing its efficacy [56]. Finding the ideal spore concentration for efficient pest control is crucial, as lower concentrations (1 × 10³ spores/mL) did not exhibit any discernible effects.

The direct fungal feed assay results support the pathogenicity assay results because M. flavoviride (21704) exhibits high virulence, which results in a significantly reduced adult emergence rate of 7.85%. This emphasizes the potential of the strain as an efficient biocontrol agent against fungus gnats. This is in line with earlier studies showing M. flavoviride’s effectiveness in managing a range of insect pests. Liu et al. (2022) [57], for example, demonstrated the broad-spectrum insecticidal abilities of M. flavoviride by reporting significant pathogenicity against Spodoptera litura. On the contrary, B. bassiana (1344), which had an adult emergence rate of 18.62%, exhibited moderate efficacy. This is compatible with the results of Cloyd et al. [58], who observed that although B. bassiana can be useful against specific fungus gnat stages, its overall efficacy varies based on the developmental stage that is targeted. Additionally, as mentioned earlier, Mucor hiemalis (2655) exhibited an adult emergence rate of 20.87% and demonstrated little pathogenicity in our research. However, Ref. [55] discovered a strain of M. hiemalis (BO-1) that was significantly virulent against Bradysia odoriphaga. At concentrations more than 1 × 10⁷ spores/mL, the strain achieved over 80% control efficiency in just five days. This disparity emphasizes how crucial strain-specific analyses are to biocontrol studies.

A qualitative assessment of the chitinase synthesis in the entomopathogenic fungi B. bassiana and M. flavoviride was conducted using bromocresol purple indicator agar medium. After 5, 10, and 20 days of incubation, a distinct purple ring appeared around the wells containing fungal extracts, confirming the isolates’ capacity to manufacture the extracellular chitinase enzyme. Commercially available chitinase from Trichoderma sp. worked well as a positive control, but M. hiemalis notably had no such activity during any of the incubation periods. Chitinase activity in M. flavoviride and B. bassiana is in line with a number of other discoveries. There is a strong correlation between chitinase synthesis and insecticidal capacity, as Juliya [59] showed in a study that isolates of Beauveria and Metarhizium with increased extracellular chitinase activity exhibited enhanced pathogenicity against the cowpea aphid (Aphis craccivora). Further supporting the critical role of chitinase in fungal virulence, a study by Gebremariam et al. [60] found that higher chitinase activity in M. anisopliae and B. bassiana corresponded with increased mortality in whiteflies (Bemisia tabaci and Trialeurodes vaporariorum). Furthermore, the purple ring’s increasing intensity over longer incubation times in this study is consistent with the chitinase genes’ temporal expression pattern, which was studied by Fang et al. [61]. They found that the late exponential and stationary phases of growth were when B. bassiana produced the most chitinase. Fungal biomass accumulation and the adaptive need for chitin degradation during host invasion and nutrition acquisition are probably correlated with the steady rise in enzyme synthesis. The chitinase activity of Beauveria bassiana, which reached a peak of 38.4 ± 4.3 Units/mL in 10-day culture filtrates, is consistent with previous findings demonstrating the role of chitinolytic enzymes in enhancing fungal virulence. Notably, the Bbchit1 gene encoding an endochitinase was purified and characterized from B. bassiana, and its overexpression significantly increased virulence against aphids by reducing both the lethal concentration and the time to mortality [61]. In contrast, M. flavoviride exhibited moderate chitinase activity, ranging from 19.8 ± 0.16 to 28.64 ± 2.1 U/mL, while Mucor hiemalis showed the lowest activity levels (8.7 ± 1.3 to 24.5 ± 4.5 U/mL). These findings indicate that while B. bassiana’s strong chitinolytic activity likely contributes to its entomopathogenicity, M. flavoviride and M. hiemalis may utilize alternative or complementary mechanisms, as their lower chitinase activity does not directly correspond to reduced pathogenic effects. The decline in chitinase activity observed at the 7 h incubation point across all three fungi may indicate a regulatory mechanism or substrate depletion, warranting further investigation. The higher chitinase activity observed in B. bassiana and M. flavoviride may contribute significantly to their larvicidal effects. Chitinases are hydrolytic enzymes that degrade the chitin polymer found in the insect cuticle and peritrophic matrix, facilitating fungal penetration and ultimately causing host death. Previous research has shown a direct correlation between chitinase activity and fungal virulence [61]. The increasing enzyme activity over incubation time also aligns with the progressively enhanced mortality seen in bioassays, suggesting that enzymatic degradation plays a critical role in larval susceptibility [62]. The exogenous production of cuticle-degrading enzymes such as chitinase by Metarhizium flavoviride, as demonstrated in this study, offers a practical advantage over gene-driven pest control strategies that require endogenous genetic manipulation. While genetic approaches aimed at inducing chitinase expression in insects face challenges related to population-level gene regulation, entomopathogenic fungi achieve similar outcomes externally, simplifying implementation and avoiding potential ecological complications [63]. Overall, the comparative analysis underscores the significance of chitinase activity in fungal virulence and highlights potential avenues for enhancing biocontrol efficacy through genetic or environmental modulation of enzyme production.

Protease activity assays demonstrated significant variability among the tested fungal strains. M. flavoviride exhibited the highest proteolytic activity, followed by B. bassiana, while Mucor hiemalis showed no detectable casein hydrolysis. These findings are consistent with previous studies highlighting the importance of extracellular proteases in entomopathogenic fungi for degrading host cuticles and facilitating infection [64]. In particular, B. bassiana is known to secrete subtilisin-like proteases, which are critical for both pathogenicity and nutrient acquisition [64]. Conversely, the minimal activity observed in M. hiemalis aligns with earlier reports indicating that protease production in Mucor species tends to be cell-associated or exhibits limited activity under similar experimental conditions [65]. These findings align with Tóthné Bogdányi et al. (2019) [25], who reported that M. flavoviride relies heavily on extracellular enzymes such as chitinases and proteases for cuticle degradation, which facilitates host infection and contributes to its broad insecticidal efficacy. These functional disparities suggest that the fungi under study employ distinct ecological and physiological strategies, with potential implications for their application in plant–microbe interaction studies and biological control initiatives.

The ability of selected endophytic and entomopathogenic fungi from DSMZ to colonize and their effects on plant growth regulation in Ocimum basilicum and Petunia hybrida cv. “Mitchell” were also examined in this study. When compared to the control, N. tinuis inoculation significantly increased the shoot length in Petunia hybrida. This result is consistent with past studies showing that specific endophytes improved plant growth by influencing hormone levels, enhancing nutrient uptake, and fostering systemic resistance to biotic and abiotic stressors [66,67]. To the best of our knowledge, this is the first publication detailing the influence of N. tinuis on Petunia hybrida cv. “Mitchell”; however, the beneficial effect seen in this study points to its possible function as a plant growth-promoting endophyte (PGPE). While the exact colonization mechanisms of N. tinuis remain unclear, endophytic fungi typically enter plants via root hairs, epidermal cracks, or lateral root emergence sites, forming mutualistic associations by modulating phytohormones and enhancing nutrient uptake. We are planning controlled pot experiments using microscopic imaging and molecular markers to investigate N. tinuis’s colonization patterns, entry routes, and tissue localization. Elucidating its physiological interactions and potential activation of plant defense responses will be essential to understand its endophytic role. These studies will help clarify the mechanisms of N. tinuis endophytism and support its potential use in sustainable horticulture. Similar to their inhibitory effects in Petunia hybrida cv. “Mitchell”, inoculation with M. flavoviride and M. hiemalis in Ocimum basilicum consistently led to considerable reductions in shoot and root length as well as leaf production including the damage to the plants. According to research by Sena et al. [68] and Porras-Alfaro and Baymann [69], some soil fungi and fungal endophytes can have a negative impact on growth, depending on the host species, fungal strain, and environmental conditions, even if they can be advantageous in some plant–fungal systems. Interestingly, other plant development in both species was not significantly impacted by the tested fungal treatments in this study, indicating a strain-and host-specific connection that needs more research. According to Rodriques et al. [70] the increasing understanding that fungal symbioses operate on a mutualism–pathogenicity continuum is reflected in the variation in plant responses to several endophytic and soil fungi that were seen here. These findings highlight the potential of M. flavoviride as effective entomopathogenic agents against fungus gnats, pests that are increasingly problematic in horticultural systems transitioning to peat-free substrates. Peat-free substrates, while environmentally favorable, are known to enhance fungus gnat attraction and infestation due to their higher organic content and moisture retention. Therefore, identifying biological control agents that are not only effective against fungus gnats but also compatible with these substrates is of urgent practical relevance. The substantial chitinase activity observed in M. flavoviride aligns with its insecticidal efficacy in vitro and suggests potential for further development as a biocontrol agent. However, additional studies under greenhouse and field conditions are essential to validate its effectiveness and safety before considering commercial application. Further research is needed to elucidate the plant–fungus interaction mechanisms, especially concerning root colonization and growth promotion. In addition, greenhouse trials should evaluate the performance of these fungal strains in terms of reducing its affinity across various peat-free or peat-reduced substrates under different environmental conditions. These findings lay foundational insights for the possible strategic use of entomopathogenic fungi in sustainable pest management systems. Nonetheless, such applications must be supported by future studies evaluating efficacy across diverse environmental conditions and substrate types.

5. Conclusions

The findings of this study indicate that certain entomopathogenic and endophytic fungal strains, particularly Metarhizium flavoviride, may serve as effective candidates for the environmentally sustainable management of Bradysia difformis (fungus gnats), particularly within peat-free horticultural systems. Among the tested strains, M. flavoviride exhibited superior entomopathogenic activity, significantly reducing larval survival and adult emergence, especially at higher spore concentrations. Concurrently, both M. flavoviride and B. bassiana demonstrated extracellular enzymatic activities, chitinase and protease production, linked to their virulence and insecticidal capabilities. Importantly, the findings also contribute to the growing understanding of plant–fungus interactions. While Niesslia tinuis showed potential as a plant growth-promoting endophyte in Petunia hybrida cv. “Mitchell”, other strains like M. hiemalis demonstrated host-specific pathogenic effects, underlining the complexity of fungal colonization and its outcomes. These observations affirm the ecological and physiological diversity of fungal strains and underscore the need for tailored application strategies. Fungus gnats are a significant greenhouse pest, and their attraction to peat-free substrates that are commonly used for their environmental sustainability poses an increasing challenge for growers. As the horticultural industry transitions away from peat to more eco-friendly alternatives, there is a growing need for effective, substrate-compatible pest control solutions. The entomopathogenic fungi evaluated in this study present a promising biological alternative to conventional insecticides, particularly in systems utilizing peat-free or peat-reduced media.

To maximize their practical application, future research should investigate the complex interactions between fungal strains, host plants, and diverse substrate types under varying environmental conditions. Although the present findings are derived from controlled greenhouse experiments, comprehensive field studies are necessary to substantiate the efficacy and reliability of these fungal strains under practical agricultural conditions. Such investigations will be critical to assess their suitability for incorporation into integrated pest management (IPM) strategies. Advancing such knowledge will support the development of integrated, low-input pest management strategies that align with sustainable horticultural practices. Furthermore, building on the promising results demonstrated in controlled greenhouse conditions, future research should focus on evaluating the efficacy of Metarhizium flavoviride under open-field crop environments to better understand its potential in diverse agricultural settings. An expanding the scope of investigation to include other species of mushroom moth and various soil types will provide valuable insights into the broader applicability of this entomopathogenic fungus. Considering its tropical origin and mode of action, further studies exploring formulation improvements and application strategies could enhance the practical implementation of this biocontrol agent. Additionally, future research should also be focused on testing its efficacy not only against fungus gnats but also against a wider range of insect pests to explore its broader applicability. Finally, integrating molecular and genetic analyses alongside ecological assessments may deepen our understanding of the fungus’s interaction with target pests and contribute to sustainable pest management approaches.