Phytoparasitic Nematodes Affecting Kyrgyzstan’s Vegetable Crops and Examining the Predatory Fungus Arthrobotrys oligospora as a Biological Control Agent

1.1 The knowledge level and prevalence of plant-parasitic nematodes in the agroecological systems of Kyrgyzstan

According to a research by Dzhunusov [1], a group of soil-dwelling nematodes called devisaprobionts has been identified in wheat and barley. This group consists of 14 genera and 27 species. The most common genera in this group include Acrobeles, Acrobeloides, Eucephalobus, Cephalobus, Chiloplacus, and Panagrolaimus. Additionally, there are phytohelminths with non-specific pathogenic effects, encompassing seven genera and 16 species, with the most prevalent genera being Tylenchus and Aphelenchus. Groups of ectoparasitic mycohelminths, perforators, and phytohelminths with specific pathogenic effects are represented by 12 genera and 19 species. Notable genera in this category include Aphelenchoides, Ditylenchus, Hrlicotylenchus, Tylenchorhynchus, and Pratylenchus. In the sugar beet fields of the Chui Valley, researchers identified 14 species of parasitic nematodes belonging to eight different genera. The most commonly found species include Pratylenchus crenatus, Pratylenchus pratensis, Paratylenchus nanus, Rotylenchus robustus, and Heterodera schachtii. Notably, the sugar beet cyst nematode (H. schachtii) and the worm-like parasitic nematodes of Pratylenchus were present in all sugar beet cultivation areas throughout the Chui Valley [2].

In terms of ecological groups and the qualitative composition of nematodes associated with sugar beets grown in crop rotation, the distribution of individuals in both the root-soil and the root system of the sugar beet is as follows: pararhizobionts constitute 15.4% in the root-soil and 0% in the root system. Deisaprobes comprise 20.9% in the root-soil and 56.3% in the root system. Eusaprobes account for 21.3% of the root soil and 0.9% of the root system [3].

The dominant nematode species in potato plantings include Aphelenchoides parietinus, Panagrolaimus rigidus, and others. Among the true parasites, the root-knot nematode (Meloidogine incognita) and the potato stem nematode (Ditylenchus dipsaci) have been recorded in significant quantities in the soil and underground organs. The latter is responsible for plant helminthiasis in several surveyed farms in Kyrgyzstan’s Issyk-Kul and Chui regions. The composition of nematodes found in vegetable crops in Kyrgyzstan was analyzed, focusing specifically on phytoparasitic species [1, 4, 5]. The most nematode species were identified in tomato, onion, and garlic plants, while the lowest was observed in pepper and cabbage. The dominant families across all plant organs and their rhizospheres were Cephalobidae, Panagrolaimidae, and Aphelenchidae. The most abundant species included Panagrolaimus rigidus, Acrobeloides nanus, Cephalobus persegnia, and Ditylenchus intermedia. Notably, the phytoparasite Pratylenchus pratensis was found to affect the roots of cabbage, onion, and garlic plants. Additionally, species from the Ditylenchus and Helicotylenchus genera were detected in the roots and root-soil of cabbage, pepper, and tomato plants [5].

In the parasitic nematode fauna associated with alfalfa, over 50 species from 15 genera have been identified. However, only a few of these are causative agents of nematode diseases. The main nematodes responsible for such diseases include stem nematodes (Ditylenchus spp.), root-knot nematodes (Meloidogyne spp.), and short-bodied nematodes (Pratylenchus spp.) [6].

The nematode fauna complex associated with alfalfa phytohelminths comprises several genera, each with specific percentages identified in the root-soil and root systems. The genera and their respective percentages are as follows: - Pratylenchus: 6.4% in root-soil and 22.7% in root systems; Aphelenchus: 6.8% in root-soil and 4.8% in root systems; Aphelenchoides: 2.9% in root-soil and 6.1% in root systems; Ditylenchus: 11.7% in root-soil and 3.5% in root systems; Tylenchus: 10.8% in root-soil and 11.4% in root systems; Helicotylenchus: 8.3% in root-soil and 4.5% in root systems; Tylenchorhynchus: 11.9% in root-soil and 2.9% in root systems [7].

The comparison of the frequency of individual genera of phytonematodes in fields with crop rotation versus those with permanent alfalfa cultivation revealed a notable contrast. In the crop rotation fields, 13 out of 29 detected genera were present in more than one-third of the samples. In contrast, in fields with permanent alfalfa cultivation, a significantly higher number of genera—18 out of 29—were found with the exact prevalence [7]. The presence of dangerous root parasites of the genus Pratylenchus in the examined crops is noteworthy. Additionally, many nematodes from the Aphelenchoides and Aphelenchus genera across all fields suggest that phytoparasitic fungi may be present in soil. The high proportion of the Ditylenchus genus is likely due to an outbreak of this disease in the studied crops and fields. It is well documented that Ditylenchus dipsaci has over 40 races and can affect a wide range of cultivated and weed plant species [7].

1.2 Biological traits and harmful effects of the stem nematode (Ditylenchus dipsaci) and tuber nematode (Ditylenchus destructor) in crops

The stem nematode, Ditylenchus dipsaci, is a type of roundworm (phytohelminth) that affects the underground parts of various plants. Approximately 450 races of this nematode infect around 450 plant species. D. dipsaci can infest important crops such as sugar beets, table beets, tomatoes, eggplants, carrots, onions, peppers, garlic, and various pumpkins, including cucumbers. It also impacts sunflowers, cereals, legumes, and hops and is found in various weeds. This nematode can infect ornamental crops and particularly damages potatoes [8]. D. dipsaci is a microscopic worm that measures approximately 1.5 mm in length. It can penetrate plants from the soil, through infected planting material, or sometimes from seeds. These worms inhabit the intercellular spaces of plants, particularly between the scales of onion and garlic bulbs. They feed on cell sap and can lay up to 250 eggs per season. Under optimal temperature conditions, ranging from 15 to 20°C, up to six generations can develop within a single season [9]. Research has identified the stem nematode Ditylenchus dipsaci as one of the many parasites affecting vegetable crops in Kyrgyzstan, particularly onions and garlic. This polyphagous plant-parasitic nematode is found worldwide and comprises over 20 biological races that primarily differ in their host plant preferences. In Kyrgyzstan, surveys indicate that Ditylenchus dipsaci is consistently present in nearly all areas where onions are cultivated intensively, especially in the Chui region. In areas with continuous onion farming, such as the Sokuluk and Moscow regions, damage from this nematode has been observed to range from 60 to 90%. Consequently, crop losses often amount to 15–25%, with total losses ranging from 58 to 77 quintals per hectare [5].

The tube nematode (Ditylenchus destructor) is a type of roundworm, a phytohelminth. It primarily affects the underground parts of plants. This nematode thrives in temperatures ranging from +5 to +34°C and can develop within 18 to 68 days. It typically overwinters at either the egg or fourth larval stage. In recent years, damage to potato tubers caused by stem nematodes has been recorded annually. The harm caused by these nematodes lies in their ability to damage tubers, leading to secondary infections. As a result, the tubers may rot or decline in their commercial and seed quality [10]. The potato stem nematode (Ditylenchus destructor) significantly reduces seed tuber quality and increases potato waste during storage. This situation underscores the need for collective action, as only 2 of the 12 farms surveyed in the Jety-Oguz and Ak-Suu districts of Issyk-Kul, as well as in the Alamedin, Sokuluk, and Moskovsky districts of the Chui region, were free of this nematode [1, 4].

1.3 Predatory fungi Arthrobotrys oligospora as a biological agent against soil parasitic nematodes

Annual cycles of plant nematode infestations result in a significant buildup of soil parasites, making effective, sustainable control crucial. While chemical methods using nematicides can be effective, they often harm the environment and non-parasitic soil organisms [11, 12]. Additionally, frequent use of anthelmintics can lead to parasite resistance, which may require higher doses or alternative products, further raising environmental concerns. This underscores the need to develop environmentally friendly control alternatives [13, 14]. Nematophagous fungi play a vital role in naturally controlling parasitic nematodes, offering an ecological solution to this issue. Approximately 200 fungal species can attack active nematodes. These fungi use unique mechanisms, such as specialized traps and toxic compounds, to infect and eliminate their nematode hosts [15, 16]. One notable example of a fungus that targets living nematodes is Arthrobotrys oligospora Fres., which shows significant promise as a biological control agent against parasitic plant nematodes [17, 18]. Its effectiveness stems from its antagonistic, parasitic, and predatory behaviors in the soil, relying on the complexity of the traps it forms to capture nematodes. Arthrobotrys oligospora can develop various mycelial structures for predation, including conidial traps, hyphal spirals, appressoria, and three-dimensional sticky networks [16, 19]. The initial step in capturing nematodes by A. oligospora involves the chemotaxis of fungal hyphae, which release volatile compounds to attract their prey [20, 21]. Additionally, the fungus produces antibiotics and toxic substances to digest the nematode’ s organs and eliminate competing microorganisms. It also releases toxic terpene compounds that form the sticky substances used in traps [22]. The glue produced by nematophagous fungi is a sticky material composed of proteins and carbohydrates. These carbohydrates coat nematode surfaces and facilitate communication via chemotaxis and pectin. Specifically, A. oligospora recognizes nematodes through the pectin component Gal-Nac [23, 24]. Fungi compete for limited food resources and can often switch from a saprophytic lifestyle to a parasitic one. Laboratory experiments have demonstrated that a low carbon-to-nitrogen (C:N) ratio can trigger this transition from saprophytism to predation. Specifically, the addition of ammonia (NH3) within a specific concentration range, along with steroids such as lanosterol, ergosterol, phytosterol, β-sitosterol, or cortisone acetate, has been shown to stimulate trap formation in these fungi [23, 25, 26]. Conversely, high concentrations of carbon dioxide (51.0%), exposure to light, and phosphate levels above 30 μM have been found to inhibit trap formation. There may also be regional differences in the activity of various isolates of nematophagous fungi [27].

In Kyrgyzstan, little attention has been paid to studying these unique predatory fungi and their potential use against soil-dwelling parasitic nematodes, which cause significant crop production losses. Vegetable and field crops in many regions and climates are at risk from harmful stem and root nematodes. In recent years, large amounts of chemical nematicides, crop rotation practices, and resistant plant varieties have been employed to combat these parasitic nematodes. However, these methods have drawbacks, including environmental pollution, limited crop rotation options, and a shortage of resistant varieties. As a result, there is an urgent need for alternative control methods.

Isolating local predatory fungal strains with high activity to control parasitic nematodes in crops is essential as an alternative to chemical nematicides. Local species of predatory fungi are considered well-adapted to the climatic conditions of various regions’ soil habitats.

The main aim of this study was to isolate predatory fungi from natural soil environments using classical and molecular biological methods and to select highly active strains against harmful parasitic nematodes.

2.1 Isolation of nematodes for screening tests

To isolate the garlic nematode Ditylenchus dipsaci, the Baermann funnel technique was employed to extract it from garlic and potato samples. The isolated nematodes were maintained on cornmeal agar plates at 25°C. They were subcultured every 2 weeks and regularly monitored under a light microscope for morphological and viability characteristics. The garlic nematode, Ditylenchus dipsaci, was identified using the identification keys described in the sources by Mai and Mullin [28].

2.2 Isolation of predatory fungi from natural sources

A total of 50 samples, including crop residues, compost, and soil, were collected to isolate predatory fungi from various ecosystems in Kyrgyzstan (Table 1). The samples were gathered between September and November and April and June, spanning the years 2016 to 2019. Individual samples were placed in plastic bags and stored at 4°C until analysis. Modified versions of established methods were utilized to isolate A. oligospora fungi [29, 30]. Each sample was coarsely ground and thoroughly mixed using a blender.

To isolate the primary colonies of these fungi, a cornmeal agar medium (CMA) containing 1% streptomycin to inhibit bacterial growth was utilized. Three grams of a slurry composite sample were placed on the surface of the CMA. As bait, 100 healthy garlic nematodes (Ditylenchus dipsaci) were added to each plate, with a variation of ±32 (n = 3).

The plates were incubated at room temperature (26°C) for several weeks. The plates were examined under a dissecting microscope during the incubation, and fungal colonies were observed and recorded. Subsequently, fungal colonies were purified, and single-spore isolations were performed on CMA medium to obtain pure fungal cultures, and at least three subcultures were prepared. A specific microculture slide technique was employed to determine the shape and size of conidia, conidiophores, and other capturing structures. Taxa were identified morphologically using established identification keys [31, 32].

2.3 The solid and liquid media used to cultivate predatory fungi

Several nutrient media were used to determine the morphological and cultural properties of predatory fungi.

Corn agar: Corn flour 20.0 g; agar-agar 15 g; distilled water 1 L; pH 6.2; Czapek medium: Sucrose 20.0 g; NaNO3 2 g; KH2PO4 1 g; MgSO4 × 7H2O 0.5 g; KCl 0.5 g; FeSO4 0.01 g; agar-agar 20 g; distilled water 1 L; pH 6.5;

Rice and sorghum medium: Rice 15 g; sorghum 15 g; distilled water 200 mL; pH 7.2.

Richard’s medium: Sucrose 50 g; KNO3 10 g; K2HPO4 5 g; MgSO4 × 7H2O 0.5 g; FeCl3 0.02 g; pH 6.0;

Yeast extract, peptone, and soluble starch medium: Soluble starch 20 g; yeast extract 4 g; K2HPO4 0.5 g; MgSO4 × 7H2O 0.2 g; pH 7.0.

2.4 In vitro test for evaluation of fungal activity against living nematodes

A piece (5 mm in diameter) was cut from the margin of A. oligospora fungal colonies and transferred to the centre of a Petri dish containing 1.5% water agar medium. The Petri dishes were incubated in the dark at 25°C for 1 week.

Following this incubation, the Petri dishes were inoculated with a 1 ml suspension of Ditylenchus dipsaci nematodes containing 100 to 150 individuals. Four to five drops of the nematode suspension were distributed around the fungal colonies.

Control plates that did not contain fungi or nematodes were also prepared. The formation of traps was observed and recorded under a stereomicroscope (EMZ-5 with Swf10x, MEIJI, Zoom Stereo Microscope, Japan). The average number of trap rings formed was calculated for 10 randomly selected microscopic fields. Four replicates were conducted to evaluate trap formation over 96 hours. Microscopic images were captured using an ML5000 MEIJI TECHNO microscope fitted with a Euromex CMEX-32000 digital camera system.

2.5 Evaluation of fungal activity on live nematodes in the rhizosphere soil of potato plants

For the assessment of fungal activity against nematodes, three pots per treatment containing mixed soil and humus in a 3:1 ratio were utilized.

Potato buds were planted at a depth of 5 to 6 cm in pots. The colonies of A. oligospora, in the form of 5 mm diameter disks, were mixed with soil—four fungal colony disks per 500 g of soil—and incubated at 25°C in the dark for 1 week under sterile conditions.

Three experimental options were introduced to assess the effectiveness of predatory fungi against nematodes in the potato root system:

Control 1: Plants without any treatment, nematodes, or nematophagous fungi. Control 2: Plants with introduced nematodes but without nematophagous fungi. In these control pots, 1 ml of a suspension containing mixed life stages of the nematode Ditylenchus dipsaci (approximately 1000 worms) was added to the soil, which was then incubated under natural conditions.

Experiment 3: In this option, 1 ml of a suspension containing mixed life stages of Ditylenchus dipsaci (1000 worms) was added to each pot, along with the planted material. When the potato leaves developed, the predatory fungi were introduced into the soil. For this, 5 mm-diameter discs of A. oligospora colonies (four pieces) were mixed with the soil and incubated in sterile, dark conditions at room temperature for 1 week. We believe that, during this period and under these conditions, the interaction between nematophagous fungi and nematodes would be intensified in the soil surrounding the potato root system.

In the fourth experiment, a 1 ml suspension containing mixed life stages of the nematode Ditylenchus dipsaci (totaling 1000 worms) was added to the soil. Unlike the plots in the third experiment, in this case, nematophagous fungi were introduced into the soil around the potato root at the time the seed material was planted and again as the leaves began to develop.

2.6 DNA extraction, marker sequence determination and phylogenetic reconstruction

For DNA extraction, fungal isolates were cultivated on Potato Dextrose Agar (PDA) and Yeast Extract Agar at 25°C. Approximately 100 mg of mycelium was placed into a screw-capped 2 ml microcentrifuge tube containing Lysing Matrix C (MP Biomedicals). DNA was extracted using the DNeasy Plant Kit (Qiagen).

Purified DNA was eluted in 100 μL of elution buffer AE and stored at −20°C. DNA sequences were amplified from fungal DNA samples using standard Taq DNA polymerase (New England Biolabs) and the PCR primers listed in Table 2. This process targeted internal partial sequences of several genes: the EF-1α gene, which encodes the translation elongation factor 1-alpha; the RPB2 gene, which encodes the second-largest subunit of RNA polymerase II; and the TUBB gene, which encodes beta-tubulin. Additionally, the complete ribosomal RNA (rrn) operon internal transcribed spacer (ITS) region was amplified, encompassing the ITS1 and ITS2 elements along with the 5.8S rRNA encoding sequence.

To clarify the molecular taxonomy of the KTMU-7 isolate, we compared it with known Arthrobotrys reference sequences from Yang et al. [33]. The sequences generated for the KTMU-7 isolate were used to search GenBank for highly homologous entries. The advanced software tool BLASTn [34] facilitated the search. Among the top 100 hits, sorted by descending maximum identity value, those with at least 80% sequence coverage that were assigned to the genus Arthrobotrys were included in the reference dataset.

2.7 Data analysis

The occurrence frequency (F) of A. oligospora and the total number of fungal isolates from each natural habitat were calculated using the following formula, based on the total number of all species.

If an individual species was isolated from any of the three replicates, it was deemed to have one occurrence. All statistical analyses were carried out using the Statistical Package for the Social Sciences (SPSS). Statistical significance was defined at an alpha level of 0.05.

3.1 Sampling sites

The locations where samples were collected are detailed in Table 1.

3.2 Isolation of garlic endoparasitic nematodes

Identification of nematodes obtained from diseased roots and stems of garlic and potato was conducted using the Determination book [35, 36], and they were morphologically confirmed as Ditylenchus dipsaci. Morphological characteristics of this species include the following: the female body length ranges from 1 to 1.8 mm, while the male body length varies from 1.2 to 1.5 mm. The stylet length is between 10 and 12 μm (Figure 1). The female lays approximately 200–300 eggs. An egg measures 67 to 88 μm in length and 25 to 34 μm in width; the second-stage larvae measure 0.3 mm, and the fourth-stage larvae range from 0.96 to 1.28 mm. The nematode population obtained was maintained at room temperature for further experimental use.

Garlic nematodes are widespread in all garlic-growing regions of Kyrgyzstan, particularly in the Issyk-Kul and Chui areas. These nematodes invade leaves and stalks, acting as parasites and damaging roots. At +15°C, the generation time is 19–23 days.

3.3 Isolation of predatory fungi

To ensure long-term viability, the primary colonies of predatory fungi were cultivated using three methods under laboratory conditions: (1) the samples were plated on a solid medium measuring 10 x 10 mm; (2) the samples were combined with live nematodes and placed on solid medium; and (3) the samples were suspended in water containing nematodes. Although all three methods resulted in colony growth, the most effective technique involved mixing the samples with nematodes on solid medium and suspending them in water with nematodes.

From various natural sources studied, approximately 40 primary colonies were obtained. Within a week, these suspected colonies were transferred to a pure medium to facilitate subculturing and obtain a pure culture of the sought fungus. Ultimately, twelve strains of predatory fungi were isolated.

The prevalence of A. oligospora in natural substrates varied significantly. The highest incidence of this fungus was recorded in the residues of damaged potatoes collected from the Jeti-Oguz district, specifically in Kyzyl-Suu village in the Issyk-Kul region, at 36.0 ± 0.091% (P ≤ 0.05). Additionally, garlic crops exhibited a notable population of A. oligospora; in the Sokuluk district of the Chui region, the occurrence was observed at 20.0 ± 0.87%. The garlic-grown soils of the experimental station of the Agriculture Faculty exhibited a fungal frequency of 14.0 ± 0.67% (P ≤ 0.05). In the decomposed plant debris, the frequency of fungi was low, at −10.0 ± 0.51% (P ≤ 0.05). The frequency of A. oligospora was particularly low in wild plant residues, reaching only 6.0 ± 0.37% (P ≤ 0.05).

Figure 2 depicts the isolated primary colonies of A. oligospora from damaged potatoes, along with the conidio-forming mycelium of the fungus.

3.4 Morphological characteristics of A. oligospora isolates

As demonstrated by microscopy (×40, ×100), the obtained isolates exhibit branched vegetative hyphae and a mycelial structure. Conidiophores grow vertically, are pretty long, and their lengths reach 27,270 ± 0.02 μm (n = 50). At the blunt ends of the conidiophores, at the tips of the sterigmata, two-celled, pear- shaped, transparent conidia develop. The average size of the conidia was 27.2 x 16.1 ± 0.031 μm (n = 50) (Figure 3). A single conidiophore branch can produce more than six conidia, which form successive clusters.

Some studies have noted that spores of predatory fungi increase in size when exposed to nematodes [37], whereas others have observed a decrease in spore size under similar conditions [38]. In our research, we found that spores of Arthrobotrys spp. cultures exhibited variations in size and morphology in the presence and absence of nematodes.

In our studies, we found that the size of fungal spores increases when grown on CMA medium; for instance, they reach (31–51 ± 0.019 μm, n = 50). When cultured on a mixed medium (CMA + peptone), both distal and proximal conidia also exhibit larger sizes (30–51 ± 0.23 μm). As noted by other studies [39, 40], fungal conidia attached to nematodes in the wild are larger and more elongated (31–51 ± 0.17 μm). These data are consistent with ours. To obtain larger spores of these fungi, we selected a mixed medium (CMA + peptone), which yields greater fungal spore biomass and may be more effective against nematodes.

It is known that predatory fungi can form chlamydospores with thick cell walls under unfavorable environmental conditions. Other studies note that these structures play a crucial role in the predatory lifestyle of these fungi. Unlike saprophytic fungi, these predatory fungi require additional stimulation to produce chlamydospores. In our studies, we generated intercalary, ellipsoidal chlamydospores on plates containing 2% CMA after 15–18 days of incubation (Figure 4).

3.5 Screening of fungi for predatory activity against live nematodes in vitro

For the application of such fungi in biological control in the field, nematophagous activity must first be screened in vitro. Microscopic observation of slides of co-incubated fungi and nematodes revealed the emergence of a single trapping ring in the fungi, which, over time, were able to form fully developed three-dimensional trapping structures and networks. A. oligospora began to trap nematodes 24 hours after the inoculation of fungal cultures. The number of traps and the percentage of nematodes captured by the fungus increased over time (Figure 5).

Microscopic observation revealed that, within the first 24 hours, trapping rings and nets began to form on the fungal mycelium originating from the conidia. By 48–72 hours, trap formation intensified, and their numbers increased. Consequently, the number of nematodes captured by the predatory fungi also rose. Nematodes already sedentary in the trap became immobile within 6–10 hours (Figure 6a-d). By 96 hours, active penetration and germination of the fungal mycelium within the body cavity of the nematodes became noticeable. Even after the nematodes were released from the traps, parts of the mycelium still attached to their surfaces could grow and release mycelium into the environment. By 122 hours, the fungal mycelium had multiplied inside the nematodes, completely dissolving their internal contents and leaving behind only traces (Figure 6f).

Microscopic observation revealed that within the first 24 hours, trapping rings and nets began to form on the fungal mycelium g rowing from the conidia. By 48–72 hours, trap formation had intensified and their number had increased, and, accordingly, the number of nematodes caught by the predatory fungi also increased. Nematodes already sedentary in the trap became immobile within 6–10 hours (Figure 6a-d). By 96 hours, active penetration and germination of the fungal mycelium inside the body cavity of the nematodes was noticeable. Microscopic observation revealed that within the first 24 hours, trapping rings and nets began to form on the fungal mycelium growing from the conidia. By 48–72 hours, trap formation had intensified and their number had increased, and, accordingly, the number of nematodes caught by the predatory fungi also increased. Nematodes already sedentary in the trap became immobile within 6–10 h ours (Figure 6a-d). By 96 hours, active penetration and germination of the fungal mycelium inside the body cavity of the nematodes was noticeable. Even when the nematodes were released from the traps, part of the mycelium that remained attached to their surface was able to grow and release mycelium into the environment. By 122 hours, the fungal mycelium had multiplied inside the nematodes, completely dissolving their internal contents and leaving behind only traces (Figure 6f). Even when the nematodes were released from the traps, part of the mycelium that remained attached to their surface was able to grow and release mycelium into the environment. By 122 hours, the fungal mycelium had multiplied inside the nematodes, completely dissolving their internal contents and leaving behind only traces (Figure 6f).

Joint cultivation of in vitro fungi A. oligospora and D. dipsaci was conducted using 1.5% water agar to investigate the formation of trapping rings in predatory fungi in the presence of nematodes; in the control group, tap water was used instead of a nematode suspension. The average number of traps formed in contact with nematodes was counted across 10 fields of view under the microscope. These calculations, performed in ten replicates, showed that the number of traps formed positively correlated with the duration of the previous joint incubation, with values of 39.25 ± 2.14 after 24 h, 157.50 ± 2.9 after 48 h, and 257.50 ± 4.17 after 96 h (P < 0.05). In the control experiments, traps were also formed, but their number was 5–10 times lower than in the experimental group, specifically, 12.25 ± 1.20 after 24 h, 17.50 ± 3.97 after 48 h, and 28.75 ± 9.07 after 96 h (P < 0.05) (Figure 7). Therefore, these results demonstrate that live nematodes enhance the active and dense formation of traps in predatory fungi.

3.6 Determination of the predatory activity of A. oligospora fungi in soil conditions: in the rhizosphere of potato plants

The results of the combined incubation of A. oligospora fungi and the parasitic nematodes D. dipsaci in the rhizosphere soil of potato plants showed significant differences between the variants and the control groups. In the control group, which was not exposed to either nematodes or predatory fungi, the above-ground and below-ground organs of the potato displayed no pathological symptoms: the stems were straight and thick, and the leaf blades were a rich green, free of any spots (Figure 8a). In contrast, plants exposed to nematodes without any suspensions of predatory fungi for protection exhibited clear pathological symptoms: the stems became weak and thin, and the leaf blades were small and yellowing (Figure 8b). Conversely, when plants were exposed to nematodes along with a single treatment of carnivorous fungi, the stems were of medium thickness, the leaves showed no visible signs of disease, and the plants maintained a normal height (Figure 8c). Following the second treatment, the agronomic parameters of the plants improved further: no disease symptoms were observed, the leaves and stems were of medium thickness and standard size, and the plants developed better than in the other experiments (Figure 8d).

Upon examination of the underground organs, root hair nematode infestation was evident. In contrast, the roots of control plants that were not exposed to nematodes were thick, exhibited no symptoms of disease, and had begun to form tubers. However, the roots of nematode-exposed plants without fungal treatment failed to form tubers and instead disintegrated into thin strands. Additionally, the lateral roots of potato plants exposed to nematodes in combination with one or two treatments of A. oligospora formed tubers and displayed no symptoms of disease; after two applications of the carnivorous fungus, the roots of these plants appeared healthier overall and were thicker than those of plants treated only once.

3.7 Field testing of predatory fungi against nematodes

For this experiment, we used three strains known to be effective in the laboratory. Following the scheme outlined below, we applied 1 and 2% liquid preparations of fungi to the nematode-infected garlic field. As illustrated in Figure 9, we introduced the predatory fungi to the garlic field at the beginning, middle, and end of the experimental area. We compared the results with those from the control group.

When introducing a 1–2% suspension of the predatory fungus into the garlic field at the start of treatment, 36–37 ± 0.01 garlic nematodes per soil sample were counted in the initial part of the experimental area. After treatment, 7–8 ± 0.01 nematodes were found 7 days later. In the middle of the field before treatment, there were 45–46 ± 0.01 nematodes, and after treatment, their numbers dropped to between 5 and 10 ± 0.01. At the end of the field, 50–51 ± 0.01 nematodes were counted before treatment, which decreased to 5–10 ± 0.01 afterwards. In contrast, the control variant showed an increase in nematode numbers from 34 to 58 ± 0.01 within 7 days (Table 3). These data demonstrate the effectiveness of three strains of Arthrobotrys oligospora under field conditions against garlic nematodes. Considering various negative abiotic factors in the environment, a 2% suspension of predatory fungi can be recommended; the higher the density of their traps in the soil, the more favorable the results of biological control will be.

Some studies have indicated that environmental conditions may influence nematophagous fungi. Taking this into account, we recommend that a 2% preparation is suitable for pre-treating garlic under field conditions or for spraying during the growing season.

3.8 Molecular taxonomic characterization of the nematophagous fungal isolate KTMU-7

Consensus marker sequences of 602 bp (ITS), 213 bp (ef1a), 275 bp (tubB), and 705 bp (rpb2) were obtained by assembling raw sequence data from genomic DNA isolated from a pure culture of fungal isolate KTMU-7. These sequences have been submitted to Genbank under accession numbers MT559369 and MT568861–MT568863. When queried against the Barcode of Life database, the ITS consensus displayed the highest similarity (96.9–100%) to members of the fungal species Arthrobotrys oligospora. All four consensus sequences identified as top matches in the Genbank records were assigned to the genus Arthrobotrys when used as a query in a BlastN search. In four single-marker phylogenies reconstructed from aligned nucleotide sequences (Figure 10), the sequence from isolate KTMU-7 was most closely related to orthologs representing the taxonomic species Arthrobotrys oligospora, with the corresponding clade receiving relatively high bootstrap support values of 100% (rpb2, tubB), 99% (ITS), and 94% (ef1a). For the ITS marker, pairwise sequence similarity between the KTMU-7 sequence and reference sequences, determined using a p-distance matrix (data not shown), ranged from identical to 96.7% within the putative A. oligospora clade. In contrast, the most closely related reference sequences outside this clade did not demonstrate similarity greater than 94.5%. These genetic data support the classification of KTMU-7 as a taxonomic species of the fungus Arthrobotrys oligospora.