Nematocidal Properties of Wild Strains of Pleurotus ostreatus Progeny Derived from Buller Phenomenon Crosses

Abstract

Plant pathogenic nematodes play an important role in crop production and are difficult to control. One of them is Heterodera schachtii—a cyst nematode, pathogenic to sugar beet. Nature suggests a natural way to resolve this problem by using nematode-trapping fungi, one of which is Pleurotus ostreatus. It is one of the most widespread mushrooms in the world. It is a valuable and widely cultivated edible mushroom with nematocidal properties. The mycelium of this mushroom produces toxin droplets that paralyze nematodes, after which the nematodes become infected with the hyphae filament, resulting in their death. This feature can be put to practical use as a natural nematocidal agent. In this paper, we have described studies on the variability of the nematocidal properties in the progeny of three wild strains of P. ostreatus (Po1, Po2, Po4), obtained by crossing dikaryons with monokaryons (Buller phenomenon). The toxicity of mycelium was tested against the model organism Caenorhabditis elegans and against sugar beet pest—H. schachtii. The results of the study allowed the selection of the progeny heterokaryotic mycelia with the best parameters useful for in vitro control of both nematodes. They differed in the activity against C. elegans and H. schachtii, and in the toxic knobs production. The results indicated that the progeny of the Po1 strain presented a good ability to produce hyphal knobs in the presence of C. elegans, and progenies of the Po4 strain presented good quality of growth in preferred temperatures (10–25 °C). Three strains Po1 5dix27, Po2 20dix21, and Po4 2dix1, as well as a maternal strain Po4 controlled H. schachtii by entwining cysts better than other strains. These strains were moderately effective against C. elegans. Strains Po2 15dix17, Po4 1dix18, and Po4 1dix30 may be considered good controlling isolates against both tested organisms. The results of the research also clearly suggest that the killing properties of P. ostreatus mycelia should not be assessed only by their ability to produce toxic hyphal knobs. Their nematocidal properties also depend on other mechanisms developed by mycelia, which is observed as the lethality of nematodes. The results of this research will lead to a natural way to protect plants against nematodes. The research also proved the nematocidal properties of the wild strains to reduce the H. schachtii population in the soil.

1. Introduction

Nematodes play an important role in the environment, and they are also significant for agriculture. Among them, many species are severe plant pathogens, which reduce crop yield. One of them is Heterodera schachtii, a cyst nematode pathogenic to sugar beets. This nematode significantly reduces root and sugar yields and the sugar content in roots. H. schachtii has a very wide host range (over 200 plant species) and a very long lifespan in the soil (up to twenty years) [1]. Despite the threat this pathogen presents in sugar beet crops, it is still difficult to control. This pest attacks beet plants throughout the entire growing season, producing two and sometimes three generations per year in a moderate climate. The dynamics of its development are significantly influenced by soil temperature and humidity. If conditions are favorable for early hatching of larvae, the roots of young beet plants are damaged. This reduces field stands, increasing the sensitivity of plants to other soil pathogens and drought, and also promotes subsequent root damage, which results in crop losses. The population of the beetroot cyst nematode H. schachtii increases very quickly in fields where beets and cruciferous plants are cultivated in crop rotation. The basic rule in the fight against this pest is to prevent the introduction of the cyst nematode into the field, to grow plants tolerant to the cyst nematode (particularly in the case of sugar beets), and to introduce a minimum three-year break in the cultivation of sugar beets and other host plants. Another supportive method is the cultivation of pre-crops, called “anti-nematode catch crops”, such as white mustard and oil radish. Nematode populations can also be reduced by growing other plant species (e.g., rye or corn) that activate cyst nematodes and prevent them from developing a full life cycle. Taking into account the limited ways of controlling the sugar beet cyst nematode H. schachtii and other nematodes that are harmful to plants, which are limited only to biological methods, it is worth testing other antagonistic factors for pathogenic nematodes. The scientific literature provides numerous methods of combating various nematodes using fungi and bacteria [2,3]. One such example is the nematocidal activity of the oyster mushroom (Pleurotus ostreatus), which, in the presence of nematodes, produces small structures, hyphal knobs, containing toxins directed against nematodes. This ability protects hyphae against mycophagy and provides an additional source of nitrogen for the developing vegetative mycelium [3,4,5]. Other species of fungi produce traps in the form of loops of hyphae and sticky projections [6].

Toxins produced by P. ostreatus vegetative mycelium in tiny protrusions containing droplets of toxin (hyphal knobs, toxocysts) immobilize nematodes in as little as 30 s [3,4,7]. The toxin was reported as a compound with a strong effect because within 1 h, at a concentration of 300 ppm, it can immobilize 95% of nematodes. This toxin, called ostreatin, was identified as trans-2-decenedioic acid [8]. Ostreatin is secreted by vegetative hyphae and does not occur in the fruiting bodies of Pleurotus sp. Its main function is to protect the mycelium against mites, tardigrades, and springtails [9]. The toxin’s mode of action is based on affecting the permeability of the cell membrane, which disrupts the function of nerve and muscle cells. Trans-2-decenedioic acid is a simple natural compound, which is important for its subsequent biodegradation [8]. Also, other substances were considered as active molecules produced by mycelium, including the ribotoxin-like protein that is also named ostreatin [9,10]. Despite the discovery of this phenomenon quite a long time ago, so far not many studies have been carried out on the variability of the toxic properties within P. ostreatus populations and its offspring. However, data from the research of Lee and coworkers [10], published last time, showed another compound responsible for the toxic activity of P. ostreatus. This substance was identified as 3-octanone, a volatile compound of toxic droplets produced by hyphal knobs [10]. Various publications describing toxic compounds elicited by P. ostreatus prove that the activity of the mycelium is not dependent on one substance and one mode of action; however, the final result is the same—nematodes’ death and decomposition. Toxic abilities are also known for other Pleurotus species [4].

Until now, no one tested various mycelia within the same species of the Pleurotus genus against their toxic and predatory activities. We hypothesize that, among the offspring of P. ostreatus, it is possible to find strains that present a greater capacity for toxin production and/or paralyzing effect on nematodes. Thus, it is possible to select suitable strains for use in plant protection. The goal of this research was to obtain a new generation of offspring of three wild strains of P. ostreatus and to check their nematocidal activity against a model nematode Caenorhabditis elegans N2 and a phytopathogenic cyst nematode Heterodera schachtii. The P. ostreatus offspring were obtained by crossing heterokaryons and monokaryons according to the Buller phenomenon [11]. We believe that the implementation of these results will be helpful in plant protection against parasitic nematodes.

2. Materials and Methods

2.1. Fungal Strains

The research was realized using three wild strains of Pleurotus ostreatus. All of these were collected in southwest Poland, in the Opole Voivodeship. Strains were assigned as Po1, Po2, and Po4, and they were called “mother mycelia”. The Po1 and Po2 strains were previously characterized by Kudrys et al. [12], and the Po4 strain was a newly collected one.

2.2. Molecular Identification of Po4 Strain

The Po4 strain was identified according to the morphological features and by sequencing the ITS1-ITS region and comparing the sequence to the GenBank database [12]. For this purpose, the mycelium was cultured in a liquid peptone medium (10 g peptone dissolved in 1 dm³), and the DNA was isolated from it using an isolation kit (Bead-Beat Micro AX Gravity, A&A Biotechnology, Gdynia, Poland). The genomic DNA was used for amplifying the ITS1-ITS2 region in a PCR reaction using primers ITS1 and ITS4. The reaction conditions were as follows: initial denaturation at 94 °C for 2 min; 30 cycles of denaturation at 94 °C for 60 s, annealing at 50 °C for 60 s, and extension at 72 °C for 3 min; followed by a final extension for 10 min of 72 °C. Amplification of the DNA fragments was performed in a Bio-RadT100™ Thermal Cycler (Tokyo, Japan). Sequencing of obtained DNA fragments was performed in Genomed (Gdańsk, Poland), and in the next step, sequences were compared to the NCBI database. Six sequences were used in the analysis: EU424300.2, MG819161.1, MG819736.1, MT644908.1, ON869376.1, and KF309193.1 and they were applied to build a phylogenetic tree. Phylogenetic analysis was realized in MEGA X software using the Likelihood method and the Tamura–Nei model with 1000 bootstrap repetitions [13].

2.3. Basidiospore Collecting, Assessment, and Development

In the laboratory, the mother mycelia were grown to obtain fruiting bodies and then both monokaryotic basidiospores and dikaryotic basidiospores were collected. Basidiospores were initially incubated on water agar (WA) and then the initial mycelia were transferred on the PDA (Potato Dextrose Agar) medium.

Some of the basidiospores naturally presented the two nuclei appearance, and the frequency of their occurrence was assessed. The result was given in terms of the percentage of binucleate basidiospores in a total sample of basidiospores. For this assessment, samples of basidiospores were stained with safranin O using the Bandoni method [14] and observed under a light microscope. The assessment was performed in eight replications for each mother strain.

New mycelia were grown from both types of basidiospores—mononucleate and binucleate ones—and used in crossing.

2.4. Crossing Mycelia

Mononucleate mycelia were crossed with binucleate mycelia developed from the binucleate basidiospores, according to the Buller phenomenon, which is named as such because the phenomenon was first described by Buller [11] as an effect of nuclei exchange. Crossing led to new types of heterokaryons. All cultures were incubated on the PDA medium. Cultures were secured on agar slants and wheat grain and stored in the refrigerator.

2.5. Mycelia Characteristics

All mycelia were characterized morphologically, taking into account their color, structure, and growth rate in the temperature range of 10–25 °C. For this purpose, they were grown on the PDA medium. The differences and similarities were analyzed and presented in the form of a dendrogram made in the NTSYS program by the UPGMA method.

The native DNA samples of tested strains were used in the molecular characterization of them. The isolation procedure was the same as it was described above in Section 2.2. The molecular characteristics of the tested strains were determined using a comparison of the gel profiles obtained in the ISSR method described by Kudrys et al. [12]. ISSR primers used in the test were as follows: (5′-3′): GTG with the sequence (GTG)5, GAT with the sequence (GA)8T, CTC with the sequence (CTC)6, and GAC with the sequence (GA)8C [15]. Visualization of the amplification effects was performed on an agarose gel (1.0–1.5%) and recorded in the documentation system (BioRad, Tokyo, Japan). Product profiles were compared using the NTSYS software program and the UPGMA method (version No. NTSYSpc 2.21w).

2.6. Nematodes Used in the Research

In the laboratory research, the model nematode Caenorhabditis elegans N2 and a phytopathogenic cyst nematode Heterodera schachtii were used.

The Caenorhabditis elegans N2 model culture was purchased from the Caenorhabditis Genetics Center (CGC) and propagated according to the methodology provided at https://www.ncbi.nlm.nih.gov/books/NBK19649/#A7971 (accessed on 3 September 2024).

In order to obtain test individuals of H. schachtii, soil samples (black earth of Kujawy region, Poland) heavily inhabited with nematodes were collected from a depth of 0–25 cm, cleaned of stones and larger organic parts, rinsed with water in the Seinhorst apparatus, and directed to a funnel with a filter paper [16]. Under a binocular, the blotting material was examined, and with a dissecting needle, young light brown beet nematode cysts were selected and placed in a separate vessel for further use in the tests.

2.7. Nematocidal Abilities

For the collected mycelia the nematocidal properties were checked against the model nematode C. elegans N2 strain and beet pathogen—H. schachtii. Tests against C. elegans were prepared on water agar. When mycelia were sufficiently developed, usually after two weeks, they were treated with drops of a suspension containing C. elegans nematodes. After periods of 1, 3, 5, 24, and 48 h, the viability of the nematodes was determined, taking into account their mobility. The mycelium reaction was assessed by checking the toxin-producing processes. Both studies were assessed using two scales (0–3) developed in the previous study [12]. The experiment was carried out in three replications. Data of toxin production were presented as means in the whole experiment vs. controls, and the mobility results for the 5th and 24th hours of the test were shown on the graphs together with means of the mobility assessments in the whole experiment (1–48 h).

Tests against H. schachtii were carried out according to the modified methodology used for C. elegans. Whole cysts of the beet nematode H. schachtii were briefly placed on the mycelium. Only young cysts filled with eggs and/or larvae were used in the study. In this way, the dynamics of the process of entwining of the H. schachtii cysts by the mycelium of P. ostreatus was investigated.

Tests were carried out on Petri dishes overgrown with two-week-old cultures of P. ostreatus mycelia developed on WA. Three nematode cysts were placed on the mycelium using a dissecting needle. The tests were performed in 3 replications. Plates were incubated at room temperature (20 °C) with natural sunlight. The nematocidal ability of tested P. ostreatus strains was determined by assessing the degree of entwinement of H. schachtii cysts by P. ostreatus mycelium using a scale of 0–3 (microscopic observations), where (0) means no reaction of the hyphae to the presence of the cyst; (1) hyphae growing directed towards the cyst; (2) fine hyphae entwining the cyst; and (3) cyst completely entwined by growing hyphae [Supplementary Figure S1].

Mycelia were assessed according to the development of toxin-producing knobs in the presence of cysts using a scale of 0–3 (microscopic observations), where (0) means no hyphal knobs produced; (1) denotes few hyphal knobs appearing, (2) denotes an average number of toxin-knobs, and (3) denotes the high intensity of their occurrence. Microscopic observations were made 1, 2, 5, 24, and 48 h after the test started [Supplementary Figure S2].

2.8. Mycelium Killing Capacity against Nematodes—Pot/Greenhouse Test (on Soil Substrate)

The experiment was prepared in the laboratory growing chamber at 22 °C and relative air humidity of about 65%, using plastic pots (11 × 11 cm) of 1 dm³ capacity. In this test, three heterokaryotic strains of oyster mushroom (Po1 5dix27; Po2 15dix17; Po4 2dix1) were used. Mycelia were predominantly grown on sterilized barley seeds. Pots were filled on with the soil naturally infested with the cysts of nematode H. schachtii. The soil used in the experiment was characterized by a neutral pH, medium nitrogen, potassium content, and a high phosphorus content. In each pot, approximately, a layer of 7 cm of H. schachtii infested soil was added. Next, pots were filled with barley straw and inoculated by 30 seeds overgrown by particular mycelium. When the straw was well colonized by mycelia, pots were filled with the remained soil layer also infested with H. schachtii cysts. Control pots were not treated by any mycelium. The experiment consisted of two variants: (I) pots without growing any plants and (II) pots in which sugar beets (cv. Fantazja) were sown (6 plants per pot). The experiment was prepared in triplicate. The experiment lasted 90 days. As a result, the H. schachtii population was examined in each variant of the experiment.

The soil used in the experiment was initially and in the end of the experiment evaluated to assess the population of beet cyst nematodes (eggs and larvae). For this purpose, the soil was removed from pots, dried at room temperature, sieved through a 2 mm sieve to remove any straw residues, and beet cyst nematodes were obtained from it according to the Kaczorowski method (1992) [16]. The cysts were then transferred to a microscope slide and crushed with a metal spatula in a drop of distilled water to obtain a suspension of nematode eggs and larvae. Then, microscopic observations were carried out (magnification 40×) to determine the population size of H. schachtii at the beginning (Pi) and after the end of the experiment (Pf). Based on the obtained results, the increase/decrease in the number of eggs and larvae was calculated and the Pf/Pi coefficient was determined (the ratio of the number of eggs and larvae of beet cyst nematodes after the end of the experiment to their number in the initial population).

2.9. Field Experiment

The field experiment was carried out in cooperation with the sugar beet breeding company KHBC (Kutnowska Hodowla Buraka Cukrowego, Straszków, Poland). The experiment was carried out on microplots, measuring 8 × 4 m, within the cultivation tents. Growing tents in which a high level of soil infestation was detected were used for this study. Soil infestation by H. schachtii, for this purpose, was assessed in March 2023, before the experiment started. Soil in tents was artificially watered by a system of drip irrigation and weeded by hand. In this experiment, the P. ostreatus Po4 mother mycelium was used as the model mycelium. The dosage of the substrate consisting of straw overgrown with mycelium was set at a dose corresponding to approx. 18 t/ha. Cubes of straw substrate overgrown with mycelium were pre-crushed by hand and then mixed with the soil using a tiller. The mycelium was produced under conditions typical for the production of substrate for oyster mushroom cultivation. Plots were sown with sugar beet cv. Janetka in the beginning of April 2023. The experiment consisted of four variants: P. ostreatus + sugar beet; P. ostreatus; sugar beet (control); black fallow (control). The experiment was made in four replicates. As a result, the H. schachtii population was examined in each variant of the experiment.

2.10. Calculations and Statistics

Statistical analysis calculations were performed using the Statistica v.13.3 package, the results were assessed using analysis of variance (p = 0.05), and the significance of differences was assessed using the Duncan test. Cluster analysis of K-means was applied. This method involves grouping elements into relatively homogeneous classes. In figures, standard deviations were included.

3. Results

3.1. Fungal Strains and Their Characteristics

The species affiliation of the Po4 strain to the Pleurotus ostreatus species was confirmed based on morphological characteristics (winter natural fruiting) (Supplementary Figure S3). Molecular identification by sequencing the ITS1-ITS2 region confirmed our morphological observations (Figure 1).

In the laboratory, mushrooms of all three strains were produced, and they then served as the sources of basidiospores. Basidiospores were collected directly from the caps of all maternal strains according to the method described by Kudrys et al. [12]. The frequency of binucleate basidiospores (Figure 2) created by maternal strains was determined to be at the level of 3–3.35% for all strains. Mononucleate and binucleate spores were grown to obtain mycelia, which were crossed according to the Buller phenomenon (n + n × n) using one dikaryotic and one monokaryotic mycelium in each trial. Crossing within these progenies resulted in a collection of a new generation of heterokaryons (dikaryons).

The morphological comparison of the tested mycelia, heterokaryons, and maternal strains using the UPGMA method resulted in a dendrogram, on which two clusters, A and B, could be distinguished. Cluster B contains the majority of the studied strains, while cluster A contains mother strains and three dikaryons of the Po2 progeny group and six dikaryons of the Po1 progeny group (Figure 3).

The mycelia of tested strains were usually white, quite fluffy, and of medium height, with zoning or lumps visible on their surface.

The molecular similarity of tested strains was assessed on the basis of ISSR band patterns. The comparison of band patterns was performed in the NTSYS program using the UPGMA method to build a dendrogram showing the similarities and differences in the examined mycelia (Figure 4).

The dendrogram shows that the tested mycelia present quite little diversity according to the ISSR starters. Most of them clustered close, except Po1 and Po2, with Po4 and Po1-5dix30 and Po4-2dix6 and Po2-20dix23 clustering separately (Figure 4). The result of the study indicates a low genetic diversity among the tested mycelia, which can be investigated with the ISSR primer set used.

3.2. Effect of Various Temperatures on the Growth of Tested Strains

Temperatures significantly affected the growth of the tested mycelia, and although different types of mycelia responded differently to different temperatures, this was not true for growth at 10 °C (Figure 5 and Figure 6,

Table 1. The average growth of mycelia in groups of Buller progenies (signed as dixMon) of Po1, Po2, and Po4 (p = 0.05).

Strains’ Group	10 °C	15 °C	20 °C	25 °C
Po1 dixMon	17.15 a *	28.95 a	65.45 c	67.20 b
Po2 dixMon	17.00 a	34.70 b	44.35 a	82.70 a
Po4 dixMon	18.60 a	41.50 c	55.70 b	85.40 a

* data labeled with the same letter do not differ significantly in columns.

). Increasing the temperature accelerated the growth of the mycelia, and although, in the case of the Po1 progenies group, the difference between the growth rate at 20 and 25 °C was small, these mycelia reached high growth values at higher temperatures (Figure 5, Table 1).

A different growth pattern was observed in the Po2 progenies group, in which case a small difference in growth was observed at temperatures of 15 and 20 °C (Figure 5 and Figure 6, Table 1). The mycelia from the Po4 progenies group behaved in a somewhat similar manner. The mycelia from this group were characterized by the best growth parameters at temperatures of 15 and 25 °C (Figure 7 and Figure 8, Table 1). Compared to the mother mycelia, better growth parameters were obtained in the progeny, which was particularly visible in the Po4 progeny group, almost all of which showed good adaptation to growth at 15 °C (Figure 5 and Figure 6, Table 1).

At 10 °C, the mycelia of the Po2 progeny and Po4 progeny groups were white, without characteristic clumps, medium in height, fluffy, and with characteristic zones. At a temperature of 25 °C, the mycelia of the Po4 progeny group were white, of medium height, fluffy, and had characteristic clumps. The mycelia of the Po1 progeny group and the other two groups at 15 and 20 °C had both types of morphology.

Differences in growth at individual temperatures for individual mycelia allow the identification of those mycelia characterized by an individual faster growth rate, such as Po4 3dix7 and Po4 2dix5, which are characterized by good growth at temperatures of 15, 20, and 25 °C, better than the other tested mycelia (Figure 5 and Figure 6).

3.3. Toxic Properties of P. ostreatus Strains against C. elegans—Hyphal Knobs Production

The tested strains differed in their toxic properties against the model organism C. elegans. Each tested strain showed the natural presence of toxic knobs on the mycelium without any induction. In the whole test period, the mean level of toxic knobs presence for most of the strains was assessed in our scale as 1 (Figure 7). However, some of them presented natural predisposition to create higher number of hyphal knobs without the presence of nematodes, which were assessed at the level of 2.00 (Po1 26dix27, Po1 26dix32, Po1 42dix32, Po2 14dix17). Almost all tested strains showed increased production of hyphal knobs after contact with C. elegans nematodes. The best activity was observed for the strain Po1 26dix32, for which the mean level of toxic hyphal knob production was 2.4 (Figure 7). The results show that Po1 progenies produced more toxin droplets than other progenies groups, particularly when they were treated by nematodes (Figure 9). In the case of the control, without nematodes’ induction, the three tested groups did not differ significantly (Figure 9). Cluster analysis by K-mean indicated that cluster 1 consists of 13 of the most active strains (Figure 8,

Table 2. Elements of individual clusters—toxin-producing hyphal knobs.

Elements of Cluster 1	Elements of Cluster 2
Po1 36dix32	Po2 14dix21
Po1 5dix30	Po2 15dix22
Po1 26dix27	Po2 14dix17
Po1 42dix27	Po2 20dix21
Po1 5dix27	Po2 20dix17
Po1 26dix32	Po2 20dix23
Po1 26dix30	Po2 15dix23
Po1 42dix32	Po2 15dix17
Po1 36dix30	Po2 14dix23
Po1 5dix32	Po2 26dix27
Po4 3dix17	Po4 3dix5
Po4 1dix30	Po4 2dix17
Po4 2dix5	Po4 3dix7
	Po4 1dix18
	Po4 2dix1
	Po4 1dix5
	Po4 2dix6

).

3.4. Toxic Properties of P. ostreatus Strains against C. elegans—Influence on the Nematode Mobility and Killing Properties

The toxic and killing ability of tested mycelia resulted in slowing nematode movement. Disruptions in the nematode mobility started usually shortly after the first contact with a toxin, and it was quite uniform. After 5 h from the beginning of the tests, the movement of C. elegans was rated at 1.33 to 1.00, except for the strain Po4 1dix18, which resulted in the movement of C. elegans being assessed at 2.00 (Figure 10). Usually, after 24 h, nematodes showed no movement (score = 0), although this was worse for the Po2 and Po4 offspring, and the Po4 progeny was performing worse than the other two groups of mycelia (Figure 10). In some cases, alive and weakly mobile individuals were observed, but usually they were larvae born from killed adults. These larvae died within next few hours. In the case of all tested mycelia, after 48 h from contact with P. ostreatus, nematodes were digested and their bodies were filled with hyphae. Around the residues of the nematode bodies, numerous hyphal knobs were visible (Supplementary Figures S4 and S5).

The analysis of cluster averages by K-means, considering nematode C. elegans mobility and viability as a result of the toxic properties of the tested mycelia developed in 5–24 h of the tests, resulted in the separation of two clusters. Among the tested strains, the mycelia included in cluster 2 (lower mean nematode mobility) developed better killing properties against C. elegans than those included in cluster 1. Good toxic and killing features were presented by strains Po1 5dix27, Po1 5dix32, Po2 14dix21, Po2 20dix21, and Po2 15dix17. Among Po4 progenies, the best properties were displayed by Po4 1dix30 and Po4 3dix7, but they were grouped in cluster 1, which means that they performed worse than those from cluster 2 (Figure 10 and Figure 11,

Table 3. Elements of individual clusters—nematode C. elegans mobility as a result of toxic properties of mycelia.

Elements of Cluster 1	Elements of Cluster 2
Po2 15dix22	Po1 36dix32
Po2 14dix17	Po1 5dix30
Po2 20dix17	Po1 26dix27
Po2 20dix23	Po1 42dix27
Po2 15dix23	Po1 5dix27
Po2 14dix23	Po1 26dix32
Po2 26dix27	Po1 26dix30
Po4 3dix5	Po1 42dix32
Po4 2dix17	Po1 36dix30
Po4 3dix7	Po1 5dix32
Po4 1dix18	Po2 14dix21
Po4 3dix17	Po2 20dix21
Po4 1dix30	Po2 15dix17
Po4 2dix1
Po4 2dix5
Po4 1dix5
Po4 2dix6

).

The results of the research clearly suggest that the killing properties of P. ostreatus mycelia should not be assessed only by their ability to produce toxic hyphal knobs. Their nematocidal properties also depend on other mechanisms developed by mycelia, which is observed as the lethality of nematodes.

3.5. Toxic Properties of P. ostreatus Strains against H. schachtii—Cysts’ Entwining by Hyphae

The toxic ability of the tested strains against H. schachtii could not be assessed based on the nematode mobility. H. schachtii is a cyst nematode, and its life cycle differs from that of C. elegans. The larvae of H. schachtii are typically grouped in cysts, and, usually, they were not mobile in our tests. It was also possible to observe a small number of larvae, which potentially left the interior of the cysts (Supplementary Figures S6 and S7). However, their natural mobility was also slower compared to C. elegans, and they seemed to be less sensitive to the toxin produced by the mycelium than C. elegans. P. ostreatus worked by entwining cysts of H. schachtii by hypha and then colonizing them. Thus, the experiments with H. schachtii concerned the ability of the P. ostreatus mycelia to entwine cysts of that nematode. Observations were conducted for 48 h, starting 1 h after the placement of the cysts on the mycelium surface. The hyphae’s reaction to the presence of cysts was observed shortly after the beginning of the test and it developed during the experiment, reaching a high level of entwinement between 24 and 48 h (Figure 12). After the first hour of the test, the strains did not differ significantly; the next hour of the test showed a minute differentiation among the tested strains, but the significance of the difference was low (Supplementary Table S1). In the end of the experiment, 19 strains, including mother strains Po2 and Po4, reached entwining activity at the level of 3, and according to the variance analysis and comparison of significance by Duncan’s test, Po1 36dix30 showed the highest activity, significantly different from other strains (Supplementary Table S1). However, the cluster analysis by K-means with the designation of three clusters showed that the best activity in first two hours of the test was characteristic for the majority of strains grouped in cluster 1 (Figure 13). The cluster analysis within 5 to 48 h showed that four strains from cluster 3, Po4, Po1 5dix27, Po2 20dix21, and Po4 2dix1, may be considered the best strains in this test (Figure 14). All of them were grouped previously in cluster 1, which confirms the result (Figure 13 and Figure 14,

Table 4. Elements of individual clusters—entwinement of cysts by tested mycelia developed in 1–2 h.

Elements of Cluster 1		Elements of Cluster 2	Elements of Cluster 3
Po1	Po2 14dix23	Po1 5dix30	Po2 14dix21
Po2	Po2 15dix22	Po1 5dix32
Po4	Po2 20dix21	Po1 36dix30
Po1 5dix27	Po2 20dix23	Po2 15dix17
Po1 26dix27	Po4 1dix5	Po2 15dix23
Po1 26dix30	Po4 1dix18	Po2 20dix17
Po1 26dix32	Po4 2dix1	Po2 26dix27
Po1 36dx32	Po4 2dix5	Po4 1dix30
Po1 42dixx27	Po4 2dix6	Po4 3dix5
Po1 42dix30	Po4 2dix17
Po2 14dix17	Po4 3dix7
	Po4 3dix17

and

Table 5. Elements of individual clusters—entwinement of cysts by tested mycelia developed in time 5 h, 24 h, and 48 h.

Elements of Cluster 1	Elements of Cluster 2	Elements of Cluster 3
Po1	Po1 5dix30	Po4
Po2	Po1 5dix32	Po1 5dix27
Po1 26dix27	Po1 36dix30	Po2 20dix21
Po1 26dix30	Po2 15dix17	Po4 2dix1
Po1 26dix32	Po2 15dix22
Po1 36dix32	Po2 15dix23
Po1 42dix27	Po2 20dix23
Po1 42dix30	Po4 1dix5
Po2 14dix17	Po4 1dix18
Po2 14dix21
Po2 14dix23
Po2 20dix17
Po2 26dix27
Po4 1dix30

).

The properties to entwine nematodes’ cysts by hyphae seem to be one of the most important abilities of the tested mycelia. Thus, we decided to consider the strains in cluster 3 (Table 5) as the most promising strains. The other properties of these strains, tested in the current research, in general, were also good. However, the other strains also may be useful in controlling cyst nematodes.

3.6. Toxic Properties of P. ostreatus Strains against H. schachtii—Toxic Hyphal Knobs Production

The ability to produce toxic hyphal knobs in the presence of H. schachtii cysts was differentiated for tested strains. Only three strains reached the highest toxin productivity (level 3.0)—Po1 42dix30, Po4 1dix30, and Po4 3dix17 (Figure 15). However, these do not seem to be the most interesting strains, because their toxin-production activity was significantly different from most of other strains, but not from those with the activity at the level, e.g., of 2.33 and 2.67. The dynamics of this activity was different for each of those strains, and in this case only Po4 1dix30 seems to be the most interesting strain. The strains Po2 15dix17 and Po4 behaved very similarly and did not reach the highest level of toxic knob production, but remained very stable during the whole test (Figure 15, Supplementary Table S2). Those last two strains show the ability to produce a high level of hyphal knobs on the poor medium; however, this feature was not confirmed in tests with C. elegans. Cluster analysis by K-means indicated strains with the ability to produce more toxic knobs. The analysis made for the period of 1–2 h indicated two clusters showing good properties: clusters 2 and 3. Cluster 3 consisted of the Po 4 strain and cluster 2 consisted of five strains (Po1 36dix32, Po2 15dix17, Po4 1dix18, Po4 1dix30, and Po4 2dix17) (Figure 16,

Table 6. Elements of individual clusters—toxic hyphal knob production, in the presence of H. schachtii cysts, developed in 1 h and 2 h.

Elements of Cluster 1		Elements of Cluster 2	Elements of Cluster 3
Po1	Po2 15dix22	Po1 36dix32	Po4
Po2	Po2 15dix23	Po2 15dix17
Po1 5dix27	Po2 20dix17	Po4 1dix18
Po1 5dix30	Po2 20dix21	Po4 1dix30
Po1 5dix32	Po2 20dix23	Po4 2dix17
Po1 26dix27	Po2 26dix27
Po1 26dix30	Po4 1dix5
Po1 26dix32	Po4 2dix1
Po1 36dix30	Po4 2dix5
Po1 42dix27	Po4 2dix6
Po1 42dix30	Po4 3dix5
Po2 14dix17	Po4 3dix7
Po2 14dix21	Po4 3dix17
Po2 14dix23

). The cluster analysis for the period of 5–48 h indicated only one cluster consisting of strains with good toxic knob productivity. It contained seven strains: Po 4, Po1 42dix30, Po2 15dix17, Po4 1dix5, Po4 1dix18, Po4 1dix30, and Po4 2dix17 (Figure 17,

Table 7. Elements of individual clusters—toxic hyphal knob production, in the presence of H. schachtii cysts, developed in time 5 h, 24 h, and 48 h.

Elements of Cluster 1		Elements of Cluster 2	Elements of Cluster 3
Po1	Po2 14dix17	Po1 36dix30	Po4
Po2	Po2 14dix23	Po2 14dix21	Po1 42dix30
Po1 5dix27	Po2 15dix23	Po2 15dix22	Po2 15dix17
Po1 5dix30	Po2 20dix21	Po2 20dix17	Po4 1dix5
Po1 5dix32	Po2 20dix23	Po4 2dix5	Po4 1dix18
Po1 26dix27	Po2 26dix27	Po4 3dix5	Po4 1dix30
Po1 26dix30	Po4 2dix1		Po4 2dix17
Po1 26dix32	Po4 2dix6
Po1 36dix32	Po4 3dix7
Po1 42dix27	Po4 3dix17

). Four of these (Po2 15dix17, Po4 1dix18, Po4 1dix30, and Po4 2dix17), and the maternal strain Po4, were clustered in groups containing strains with good wrapping properties in both periods of the analysis. Finally, after 48 h of the test, the best ability to wrap cysts was displayed mainly by the progenies of the Po4 maternal strain (Figure 15, Table 7, Supplementary Table S2).

The ability of the mycelium to entwine H. schachtii cysts in direct contact on the poor substrate depended, in a statistically significantly manner (p = 0.05), on the time of the mutual contact between the mycelium and cysts, and it was also dependent on the mycelium strain. Similarly significant (p = 0.05) was the production of toxin-producing knobs by the tested strains in the presence of H. schachtii; the time of exposure and the individual characteristics of the mycelium were important for this process (Supplementary Table S2).

3.7. The Pot and Field Experiments

In the pot experiment, all tested oyster mushroom strains inoculated to the soil without plant cultivation contributed to a significant reduction in the population of the beet cyst nematode H. shachtii. In the soil without mycelium (black fallow), a 28.1% reduction in the number of nematodes was observed. The mycelia (Po1 5dix27, Po2 15dix17, and Po4 2dix1) introduced into the pots caused a reduction in the population of the beet cyst nematode by 40.9%, 40.2% and 46.9%, respectively (Figure 18). The joint effect of sugar beet plants and Po2 15dix17 mycelium resulted in a slight increase in the number of H. schachtii eggs and larvae (3.7%). However, significantly, the highest increase in the number of nematodes was observed in the control pots (sugar beet plants without any mycelium), which demonstrated 242.6% of the initial infestation; whereas the soil inoculated with Po1 5dix27 mycelium and sown with sugar beets demonstrated 238.1% (Figure 19). The Pf/Pi coefficients confirmed the protective role of the mycelia introduced into the soil, showing the best result for the Po4 Buller progeny (Po4 2dix1), which, even with the combined growth with sugar beet, could decrease the H. schachtii population in the soil (Figure 20).

The microplot experiment realized in the field, in breeding tents, resulted in a significant increase in the population of the beet cyst nematode (by 96.5%) in the variant of cultivation of sugar beet on a site not inoculated with oyster mushroom (Po4) mycelium. As a result of sugar beet cultivation on a site inhabited by P. ostreatus Po4, a 16.9% reduction in the population of H. schachtii was noted, thus achieving a similar phytosanitary effect as in the case of the control variant—black fallow without mycelium, in which a 16.9% decrease in the number of nematode eggs and larvae was achieved. Fallowing the site inhabited with oyster mushroom mycelium resulted in a significant 45.4% decrease in the number of the tested pest, which was confirmed by the lowest Pf/Pi coefficient (Figure 21 and Figure 22).

4. Discussion

The results of our experiments allowed us to select the mycelia of P. ostreatus, which presented the higher speed of growth, especially in the most important temperature ranges, which were 10–15 and 20–25 °C. The lower temperature range corresponds to spring or autumn conditions, and the higher range corresponds to summer temperatures. The isolates that were well adapted to these temperatures were the Po4 dixMon group (Po4 progenies), and they generally presented better growing parameters than the maternal Po4 strain. Our tested strains were the result of crosses performed according to the Buller phenomenon. The phenomenon was documented for the first time by Buller in 1931 [11], and it relies on the fusion of monokaryotic mycelial hyphae with heterokaryotic hyphae, resulting in new heterokaryotic hyphae varieties. This reaction was first called the “Buller phenomenon” by Quintanilha in 1937 [11,17]. The fusion of homokaryotic and heterokaryotic hyphae generates a hybrid heterokaryon that inherits one homokaryon nucleus and one heterokaryon derived nucleus. The nucleus that has been transferred from heterokaryon (donor) to homokaryon (acceptor) must exhibit sexual compatibility with the acceptor hyphae nucleus. This process allows the creation of new types of heterokaryons, and if this is preferred by natural processes, it is possible that a new type of progeny would present better properties than both parental hyphae. The process occurs naturally between heterokaryotic and homokaryotic hyphae. On the other hand, some basidiospores are binucleate, which means that hyphae created from them will give a heterokaryotic mycelium. In our experiments, these binucleate basidiospores were produced by all three maternal strains at the level of ~3.0–3.3%. The mycelia obtained from them easily crossed with monokaryons. The Buller phenomenon seems to be a normal process in nature because, in our tests, its frequency was observed at the level of ~30% between monokaryons and dikaryons of the same origin (developed from basidiospores from the same wild maternal strain). Crosses between monokaryons achieved 22.5% in Po1 progenies, 27.6% in Po2, and 35.5% for crosses of monokaryons Po1 × Po2 (Supplementary Figure S8). Thus, the Buller phenomenon seems to be as desirable as mating between unrelated hyphae [18].

The tested mycelia presented low diversity based on the ISSR markers comparison. Most of them were grouped in two clusters of identical strains and only a few of them, the maternal strains and five progeny strains, were outside of that cluster. This result was surprising because the previously obtained crosses among monokaryons of Po1 and Po2 resulted in a higher ISSR diversity in progenies [12]. The explanation for these results may be that “Buller crosses” led to new genetic arrangements in “Buller offspring”. Some level of diversity among the tested strains was observed based on the morphological and physiological features, but the strains also presented low diversity compared to the typical monokaryotic crosses obtained by Kudrys et al. [12]. Popa et al. [19] demonstrated that, among the European P. ostreatus varieties, the genetic variation is reduced, which is in agreement with our study.

Differences in tested strains were also observed in toxic properties against nematodes. We tested two nematode species—C. elegans, which is a model organism in that group of animals, and the plant parasitic H. schachtii, which infects sugar beets and many other plants, including weeds [1,20]. While our strains were able to capture, kill, and digest C. elegans, the H. schachtii nematode was less susceptible, so we concentrated our study on the cysts, which were trapped and entwined by hyphae. As a result, it was clearly shown that the toxic abilities of hyphae were not uniform in tested strains. Some strains presented higher potential to create toxic hyphal knobs (e.g., Po1 strains) and to kill C. elegans (e.g., all Po1 progenies, Po2 14dix21, Po2 20dix2, and Po2 15dix17) or entwine cysts of H. schachtii (e.g., Po1 5dix30, Po1 5dix32, Po1 36dix30, Po2 15dix17, Po2 15dix22, Po2 15dix23, Po2 20dix23, Po4 1dix5, and Po4 1dix18) than other strains. The mechanism of entwining cysts of H. schachtii seems to be a better method for acquiring nitrogen than capturing nematodes. In the presence of this, pest hyphal knobs were less effectively produced and only a few isolates were able to create a high number of them (Po4, Po1 42dix30, Po2 15dix17, Po4 1dix5, Po4 1dix18, Po4 1dix30, and Po4 2dix17). Entwining and colonization of cysts give a higher dose of nitrogen substrates since cysts are bigger than individual nematodes. Simultaneously, this mode of hyphae action provides better control than killing individual nematodes because cysts are not mobile and contain many larvae. The pot and field experiments showed the possibility of cleaning properties of the Pleurotus mycelia against H. schachtii nematode. However, the mycelium should be carefully chosen, because P. ostreatus strains may differ in their nematocidal properties. This was clearly visible in the case of the pot experiment, in which the strain Po1 5dix27 did not reduce the H. schachtii population, and the variant of the experiment with this mycelium was as ineffective as the cultivation of sugar beets. The suppressive properties of soil against H. schachtii was confirmed by Westphal and Becker [21]. In their experiment, close to one-third of the cysts but no females from suppressive soil were infested with fungi. The most common fungi isolated from infested cysts were Fusarium sp., Fusarium oxysporum, and Dactylella oviparasitica. Some unidentified fungi were also isolated from infested cysts. These results combined with ours may support the importance of soil microbiota’s role in decreasing the pest population. The potential protective role of Pleurotus mycelia were tested with good results by Palizi et al. [22] and Singh et al. [3].

Until now, there have been several studies showing the protective properties of P. ostreatus and other Pleurotus species against Meloidogyne incognita Meloidogyne javanica and Heterodera goldeni and H. schachtii [22,23,24,25,26], and mushrooms or their crude extracts show nematocidal activity against nematodes of genera Pratylenchus, Xiphinema, Tylenchorhynchus, Tylenchus, Helicotylenchus, Ditylenchus, Psilenchus, Aphelenchus, Hoplolaimus, Longidorus, Aphelenchoides, and Paralongidorus [26,27].

Our experiments aimed to select the most effective strains against nematodes. We also wanted to check the diversity of the toxic properties in P. ostreatus wild strains and their progenies, which means that some mechanisms are present in some strains on different levels. This type of studies had not been undertaken before. Previously, the literature reported trans-2-decenedioic acid as a main nematocidal active compound of P. ostreatus [8]. It is deposited in droplets produced by hyphal knobs (toxocysts) on the P. ostreatus mycelium; however, other studies found that this compound may not cause as rapid and effective reaction as was observed before [10,27,28]. Toxic droplets contain many compounds that may vary among species. A typical Pleurotus toxic droplet measures 1.5–3.0 µm and, in the case of P. pulmonarius, may consist of several toxins such as S-coriolic acid, linoleic acid, panisaldehyde, p-anisyl alcohol, 1-(4-methoxyphenyl)-1,2-propanediol, and 2-hydroxy-(40-methoxy)-propiophenone [26,29]. Palizi et al. [22] mentioned that a heat-stable and dialyzable low molecular weight molecule is produced and secreted by the hyphae of Pleurotus spp., which inactivates nematode H. schachtii [22]. They also observed that the larvae of H. schachtii were attacked by hyphae, which had grown towards the nematode and penetrated it through one of the body orifices [22]. This opinion confirms our research, which clearly shows that the number of hyphal knobs (toxocysts) is not directly correlated with the killing ability (paralysis) of P. ostreatus mycelium. This means that strains that do not produce a high number of toxocysts may achieve good nematocidal activity. Also, the main mode of action displayed by our mycelia against H. schachtii cysts agrees with observations of Palizi et al. [22]. The observations by Palizi et al. [22] may be explained, and they remain in agreement with the results of Lee et al. [10], which proved that a volatile ketone, 3-octanone, is produced by P. ostreatus and stored in toxocysts and plays an important role in nematocidal activity. This compound, 3-octanone, disrupts the integrity of the cell membrane in C. elegans N2 tissues (neurons, muscles, hypodermis), which results in a massive calcium influx into the mitochondria and then cell death [10]. Fungivory and mechanical wounding induce defense responses and fungal resistance to mites in P. ostreatus. This involves the expression of a lectin gene, Polec2, which leads to the activation of the reactive oxygen species (ROS)/MAPK signaling pathway, jasmonic acid (JA) regulation, specific gene expression, protein synthesis, and metabolism of toxic substances [29,30,31]. Frangež et al. [32] described the role of other toxins produced by P. ostreatus—Ostreolysin A and Pleurotolysin B (OlyA/PlyB), which are pore-forming cytolysins of a larger group of highly homologous proteins called aegerolysins [32].

The activity of 3-octanone was reported as dose-dependent, which agrees with the observations by Palizi et al. [22]. The comparable activity was also observed for other C8 compounds, which are structurally similar to 3-octanone, namely 2-octanone and 4-octanone [10]. However, the nematocidal activity was correlated with carbon chain length but not with the position of the carbonyl group in the chain. Thus, 3-decanone was more active than eight carbon chain substances, and on the other hand, a six-carbon chain compound, 3-hexanone, showed limited activity [10].

3-octanone is one of the C8 volatile compounds wildly prevalent in many fungi and mushrooms, and it is partially responsible for creating the characteristic mushroom flavor and plays informative roles. 3-octanone acts as a self-inhibitor of spore germination in Penicillium paneum. It is a signal for sporulation in the Trichoderma genus, when it occurs in low concentration; however, it acts as an inhibitor of spores’ sporulation when it is produced in a higher concentration. In P. ostreatus, 3-octanone inhibits various bacteria species at the concentrations naturally found in the fruiting bodies of this mushroom [33,34,35]. All the described mechanisms of self-protection in P. ostreatus are present in the progeny according to the mechanisms of heredity. Thus, the individuals of the progeny may present various levels of self-protection ability, which was shown in our previous research [12] and in the current study. Taking into consideration the strains that were the most effective, it is worth noticing that Po1 5dix27 and the maternal strain Po4 clustered separately, whereas the two other strains, Po2 20dix21 and Po4 2dix1, clustered in the same group based on the genetic features tested as ISSR bands. For these strains, the same relationships are reflected in the morphological and physiological analysis; however, these features showed more differences among the tested mycelia. The three other strains considered effective against nematodes, Po2 15dix17, Po4 1dix18, and Po4 1dix30, clustered in the same big group according to the ISSR patterns, showing molecular similarities; however, they were different according to morphological and physiological features. The ISSR genetic analysis resulted in a rather low number of bands giving some background to consider many similarities in tested mycelia but, on the other hand, the distinct molecular differences were not detected in our tests; this point of the research needs more deep testing. However, the results show two groups of genetic constructions leading to obtaining good predatory properties in P. ostreatus, and these results may be confirmed by more differentiated morphological and physiological features, which also reflect genetic differences.

5. Conclusions

Effective crop control against parasitic nematodes appears to be more difficult than farmers expect. The best results may be achieved by integrated methods, but they should be well developed. Currently, nematode-trapping plants such a white mustard are used to control H. schachtii, but this usually leads to non-uniform and non-significant results achieving no more than 40% nematode reduction [20]. Also, the application of nematocide chemicals frequently fails to affect nematode reproduction. It is possible to obtain better results when a resistant sugar beet variety is grown: this may achieve a reduction in the nematode population of up to 70% [20]. Thus, if the best way to control nematodes is integrated plant protection, then including effective mycelium in this process may result in a more effective program of nematode control [36]. The search for the most effective mycelium, in our laboratory, concentrates on the various types of P. ostreatus progenies. Until now, the study of the differentiation in the progeny according to the self-protecting activity/nematocidal activity has not been conducted, except for the study of Kudrys et al. [12]. Such studies increase the potential for agricultural application of P. ostreatus in the integrated programs of crop protection. However, the choice of the best mycelium seems to be crucial to the success of this idea. Our study clearly shows that the choice of the most effective mycelium is possible but requires careful study, including field trials. According to this research and the results of cluster analyses, we can recommend four strains, Po1 5dix27, Po2 20dix21, Po4 2dix1, and Po4, and also three other strains, Po2 15dix17, Po4 1dix18, and Po4 1dix30 as the most effective ones. The experiments carried out in the soil environment, in pots and breeding tents in the field, showed that heterokaryotic mycelia of P. ostreatus present good potential in soil, cleaning it from the cysts, eggs, and larvae of H. schachtii. Thus, looking for the most effective mycelium makes agronomic sense.

Another future option in plant protection against parasitic nematodes is using the remaining spawn left after the production of P. ostreatus mushrooms; however, the results may not produce the best control of nematodes. The important point is that used compost is also a source of organic substances and mycelium, which is still alive and active. This option should also be carefully studied and some corrections in local law should be arranged because, in Poland, spent oyster mushroom spawn is treated as waste, which must be disposed of instead of used. Composing environmentally safe chemical preparations against nematodes is difficult, and the easiest and most natural way is the application of fungi-based preparations or their cultures. Thus, for this purpose, searching for the best properties of nematocidal activity in mycelia seems to be the best option.