Trait Variation between Two Wild Specimens of Pleurotus ostreatus and Their Progeny in the Context of Usefulness in Nematode Control

Abstract

Pleurotus ostreatus is one of the most widespread mushrooms in the world. It is a valuable and widely cultivated edible mushroom with nematicidal properties. The mycelium of this mushroom produces a toxin that paralyzes nematodes, after which the nematode becomes infected with the filament, resulting in its death. This feature can be put to practical use as a natural nematicidal agent. In this paper, we have described studies on two wild strains of P. ostreatus (PO1, PO2) and the monokaryotic progeny obtained from their fruiting bodies. Monokaryons were crossed with each other within the progeny of one strain (PO1xPO1; PO2xPO2) and of two different strains (PO2xPO1). The growth conditions, morphological and molecular characteristics, toxicity and ability to form hyphal knobs of the maternal strains, monokaryons and those obtained by crossing were studied. The toxicity of mycelium was tested against the model organism Caenorhabditis elegans. The results of the study allowed the selection of five progeny heterokaryotic mycelia with the best parameters useful for in-vitro control of nematodes.

1. Introduction

Mushrooms that inhabit the environment are exposed not only to the adverse effects of the environment, but also to other organisms that inhabit it. In the course of evolution, fungi have adapted to the prevailing environmental conditions and have developed many defensive traits against organisms that threaten them. These organisms are mycophagous organisms, which include nematodes.

The main defenses against nematodes are the ability to synthesize toxic substances and create traps, the ability to parasitize their tormentor, and the ability to act with enzymes on nematode tissues [1,2,3,4]. Defensive properties have been described for more than 200 different species of fungi, including the genera Arthrobotrys, Coprinus, Harposporium, Climacodon, Lentinus, Pleurotus, and others [1,2,5,6]. The defensive methods used by fungi can be used to reduce the abundance of nematodes in soils for plant cultivation.

Mushrooms of the genus Pleurotus are the most common edible mushrooms in the world [7,8]. Of the entire Pleurotus genus, Pleurotus ostreatus (the oyster mushroom) is the most widely cultivated. It is a heterothallic fungus with mating controlled by a bifactorial tetrapolar genetic system [9,10,11,12]. It produces fruiting bodies at low temperatures, but in cultivation there are different varieties that produce fruiting bodies at different temperature ranges. Therefore, the production of its fruiting bodies can take place all year round. In terms of commercial aspects, special attention is paid mainly to the possibility of non-sporulation, which significantly extends their durability and safety of use [3,6,13]. Despite its widespread commercial use and cultivation on a global scale, as well as fairly good knowledge of its nematicidal effect, no practical use for the oyster mushroom has yet been found [14]. Its main defense mechanism against nematodes is, as reported by Kwok et al. [15], the synthesis of the toxin trans-2-decenoic acid, which is secreted as droplets at the ends of knobs located on the hyphae [13,16,17]. The secreted toxin was named ‘ostreatin’ [15]. Once a nematode comes into contact with a droplet of the toxin, it becomes paralyzed and dies, providing an ideal source of nutrients for the oyster mushroom [18,19,20]. The hyphal knobs producing toxin may be considered vestigial blastoconidia [2]. More recently, other compounds were described as toxins produced by P. ostreatus and identified as a ribotoxin-like protein [21] and protein ‘ostreolysin’ [22]. These features can be used to protect against parasitic nematodes. Pleurotus ostreatus is highly effective against a wide range of nematode species, thanks to the paralysis-inducing substances produced by its mycelium [23]. Similar properties are exhibited by other species of oyster mushrooms, but few studies have been devoted to comparing them, and progeny obtained by sexual reproduction has not been studied to date. In nature, there are many versions of the various features which characterize organisms, and one of them is the possibility to produce toxic compounds as a defense mechanism. We decided to check if the differences are present in various mycelia of P. ostreatus, with a special emphasis on their progenies. This seems important because P. ostreatus is a widely produced edible mushroom with great amounts of mushroom spawn, which has to be utilized after the cultivation process. Its potential utilization in agriculture as a nutritional and protective agent will improve the protective measures against nematodes, however, the betterment of the protective mycelial parameters should be developed.

We hypothesize that suitability for protective use will be demonstrated by mushrooms with higher mycelial growth rates and greater capacity for toxin production/paralyzing effect on nematodes. The aim of this study was to determine the lethal capabilities of two strains of P. ostreatus and their progeny against the model nematode Caenorhabditis elegans. Our research concentrated on a model nematode and is a foundation from which to start further experiments with other nematode species.

2. Materials and Methods

2.1. Mycelia and Growing Tests

Pleurotus ostreatus mycelia were extracted from two different fruiting bodies from the wild and were identified based on morphological characteristics and identified molecularly based on the sequence of the ITS1-ITS2 region. In the study, they were treated as maternal mycelia and were named P. ostreatus 1 (PO1) and P. ostreatus 2 (PO2). In order to obtain fruiting bodies from these mycelia, they were grown on a medium consisting of cut straw mixed with oat flakes (oats constituting 10% of the volume of the straw). Previously, the straw, after being cut into finer fragments, was rinsed several times in sterile demineralized water. The finished medium was sterilized in an autoclave. The inoculum for infection of this medium consisted of wheat grains overgrown with PO1 or PO2 strains. After three weeks and following typical cultivation rules, fruiting bodies were obtained. Basidiospores were harvested from PO1 and PO2 fruiting bodies using the following methods: printing—this involves lining a section of the mature fruiting body with water agar (WA); suspending the fruiting body over a container containing sterile physiological fluid; suspending the fruiting body over a container with sterile distilled water; washing basidiospores directly from between the lamellae of the fruiting body with physiological fluid applied with an automatic pipette; shaking basidiospores from the fruiting body directly onto the WA [11,24].

Mycelia obtained from basidiospores were transferred to a PDA medium. The monokaryoticity of the obtained mycelia was confirmed by microscopic observations to exclude the presence of clump connections. After excluding dikaryotic mycelia, monokaryotic mycelia were designated for storage on PDA slants.

The monokaryotic progeny mycelia obtained in this way from PO1 and PO2 were crossed in two-organism cultures. The crossing was carried out within PO1, within PO2 and between PO1 and PO2 (Supplementary Figure S1). Dikaryotic mycelia were initially isolated macroscopically from the interface between the two crossed components and then microscopically evaluated for the presence of clump connections. Heterokaryons (dikaryons) were detached onto a pure PDA medium and stored similarly to monokaryons [25].

The obtained homokaryotic and heterokaryotic strains were described morphologically in a culture on PDA medium, and their growth rates at different temperatures were determined: 10, 15, 20, 25 ± 2 °C. Measurements of mycelial growth were taken every three days. The final morphological description was made after two weeks of growing the mycelium. The growing tests were realized in four replications for each temperature. The morphological evaluation took into account the structure of the mycelium (fluffiness/compactness), color, the presence of specific structures, and zoning of the mycelium. Morphological features of each mycelium were grouped and compared using NTSYS software by introducing a dendrogram using the UPGMA method. Twenty features were distinguished and noted as present or absent.

The ability to produce hyphal knobs (Supplementary Figure S2) was determined for mycelia growing at 20 °C. Observations were made in microscope slides prepared from the middle part of the mycelium, and knobs were counted in the field of view, in five slides which were treated as experimental repetitions. The following scale was adopted: 0—no hyphal knobs, 1—from 1 to 3 hyphal knobs in the field of view, 2—more than three hyphal knobs in the field of view. The results were presented as an index of effectiveness (IW) calculated according to the formula:

$$IW=\frac{{{\displaystyle \sum }}_{n=0}^i \left(a \times n\right)}{i \times N}$$

(1)

where: a—number of measurements in a given degree of the scale; i—highest step of the scale; n—degree of scale; N—the sum of measurements in the sample.

2.2. Toxicity Tests

In order to determine the nematicidal capacity of the tested mycelia and the frequency of their production of hyphal knobs, they were grafted onto WA [20,26]. After the entire mycelial medium was overgrown, it was covered with a suspension containing model nematodes Caenorhabditis elegans N2 (wild isolate). After 1 h, 2 h, 3 h, 24 h, and 48 h, the motility of the nematodes and the intensity of production of hyphal knobs within the applied drop of the suspension were observed. To determine nematode motility, the following scale was used: (0)—the majority of nematodes (75–100%) immotile and overgrown with mycelium or in a state of decomposition, the rest (0–25%) immotile and not overgrown with mycelium, single nematodes motile; (1)—the majority of nematodes (75–100%) immotile, the rest (0–25%) with slowed motility, individual nematodes dead, individual nematodes with normal motility; (2)—most individuals (75–100%) with slowed motility, the rest (0–25%) immotile or with normal motility, individual nematodes dead; (3)—most individuals (75–100%) viable with normal motility, the rest (0–25%) with slowed motility. Supplementary Figure S3 shows nematodes partly overgrown and paralyzed by mycelium.

To assess the ability of the mycelia to form hyphal knobs, a scale of 1 to 3 was used, where (1) denotes few hyphal knobs appearing, and (3) denotes the high intensity of their occurrence. The controls were WA plates overgrown with the tested mycelia, without the addition of nematodes, and nematode droplets applied to pure WA. The tests were performed in triplicate.

C. elegans was purchased from CGC (Caenorhabditis Genetics Center) of the Minnesota University, USA. The methodology contained in WormBook was followed for its multiplication and storage [27].

2.3. Molecular Tests

Molecular identification of both strains was performed based on sequences of rDNA. This was completed by amplifying the ITS1-ITS2 region in a PCR reaction using primers ITS1 and ITS4. The reaction conditions were: initial denaturation at 94 °C for 2 min; 30 cycles of denaturation 94 °C for 60 s, annealing 50 °C for 60 s, and extension 72 °C for 3 min; followed by a final extension for 10 min at 72 °C. Sequencing was performed in Genomed (Poland), and in the next step they were compared to the NCBI database. The phylogenetic tree was built in MEGA 11 software using the Likelihood method and the Tamura-Nei model with 2000 bootstrap repetitions. DNA obtained from the commercial fruiting body of P. ostreatus identified in this study as PO3 was used for comparison (Supplementary material).

The differentiation and similarity of the tested mycelia was determined molecularly using inter-simple sequence repeat (ISSR) markers. Four ISSR primers with sequences GTG-(GTG)5; GAT-(GA)8T; CTC-(CTC)6; GAC-(GA)8C were selected for the study [28]. For this purpose, the mycelia were cultured in a liquid peptone medium (10 g peptone dissolved in 1 dm³), and the DNA was isolated from them using an isolation kit (Bead-Beat Micro AX Gravity, A&A Biotechnology). Amplification of the DNA fragments was performed in a Bio-RadT100™ ThermalCycler. A commercial ColorTaq PCR Master Mix (2x) (EURx) kit was used for the PCR reaction. Amplification was performed in a 50 µL reaction mixture consisting of: 25.0 µL Color Taq PCR Master Mix (containing 1.5 mM MgCl2, 0.2 mM dNTPs, 1.25 U TaqDNA polymerase, optimized buffer and two dyes to track the PCR product on the gel); 1.25 µL of one of the primers (0.5 µM); 5.0 µL native DNA; 18.75 µL water. Reactions were carried out under the following conditions: initial denaturation at 94 °C (2 min); denaturation 94 °C (60 s); annealing 50 °C (60 s); elongation 72 °C (3 min); final elongation 72 °C (10 min); number of cycles—30. The products obtained in the PCR reaction were separated by electrophoresis and visualized with the GelDoc EZ System (Bio-Rad). The size of the bands was evaluated using ImageJ software. The obtained profiles were compared using NTSYS software by entering a dendrogram using the UPGMA method.

3. Statistics

The research used cluster analysis, which is a tool for exploratory data analysis. This method involves grouping elements into relatively homogeneous classes. The basis of grouping in most algorithms is the similarity between the elements, expressed using a similarity function. It aims for cluster heterogeneity by linking objects together based on the calculated distances between them. Of the many possible definitions for determining distances, the commonly used Euclidean distance was adopted. The significance of the differences was assessed by analysis of variance. The study was conducted using the Statistica v.13.3 package.

4. Results

4.1. Mycelia and Molecular Tests

All techniques for obtaining basidiospores from P. ostreatus fruiting bodies were successful, but the best results were obtained by direct shaking of basidiospores on WA. Morphological species identification of maternal strains (PO1 and PO2) as Pleurotus ostreatus was confirmed molecularly by comparing ribosomal DNA sequences to those deposited in the NCBI database (Supplementary Figure S4). The obtained progeny mycelia (monokaryons/homokaryons) were characterized by a diversity of morphological features (Figure 1). Large differences were noted for the growth intensity of the tested mycelia. They also differed in terms of morphological features such as mycelial structure (fluffiness, presence of tufts) and the presence of concentric zonation. The mycelia were generally white, but some of them produced orange or yellow areas. It was observed that monokaryotic strains formed taller and fluffier mycelia, while heterokaryons formed compact and dense mycelia. Grouping of the studied strains based on morphological characteristics (Figure 1) allowed for the creation of a dendrogram in which two clusters were distinguished. Cluster 1 consists of three subclusters (1-A, 1-B, 1-C). Subcluster 1-A groups both maternal isolates PO1 and PO2 and monokaryotic mycelia derived from strain PO1- (2,3,9,13,16,17,22,29,30,32) and strain PO2- (3,4,12,16,21,25,31), as well as a few heterokaryons derived from crossing PO2 and PO1 progeny: 2-6x1-9, 2-1x1-7, 2-1x1-9, and one heterokaryon from crossing PO2 progeny: PO2-19x2-24. Subcluster 1-B also contains representatives of PO1 monokaryons, four PO2 monokaryons, and heterokaryons from crossing within PO1 progeny, and between PO2 and PO1 progeny. Subcluster 1-C includes three heterokaryons and one monokaryon PO1-39. In cluster 1, four examples of strains with identical morphological characteristics can be distinguished, with a grouping of two isolates in each, which represent all of the groups studied. The fifth group of identical strains consisted of three isolates, two monokaryotic PO1 (16 and 22) and heterokaryon 2-6x1-9. In cluster 2, two subclusters 2-A and 2-B were distinguished, which also include representatives of monokaryons as well as heterokaryons. Identical features were shown by strains PO1-23 and PO1-24, as well as PO2-24 and PO1-15x1-21. The outgroup for both clusters consists of two strains PO2-19 and PO1-28 which are located separately (Figure 1). It can be noted that the morphological characteristics observed among the tested mycelia were not correlated with any of the mycelia’s nuclear status and origin.

The genetic diversity of the tested mycelia was compared using four ISSR markers, which generally yielded a maximum of eight bands for a single primer and up to a maximum of twenty-two bands in total for all primers used per isolate. The most effective primer was the GTG primer, and the least effective was GAT. The GTG primer yielded up to a maximum of eight bands per isolate, as did CAC, with the GTG primer on average yielding four to six bands per isolate, and CTC a rather variable number of bands per isolate, with some isolates showing no markers of this type. Dendrogram analysis (Figure 2) of the studied mycelia allowed for the identification of three main clusters and one smaller cluster located between clusters I and II. Cluster III groups monokaryons of both parental mycelia along with heterokaryons derived from all types of crossing. In this cluster, two groups of isolates with identical ISSR profiles can be distinguished—one of them includes PO1-31, PO1-20x21, and the other, larger one includes PO1-39, PO2- monokaryons (1,3,4,21,24,25,28), two heterokaryons PO1-12x1-18 and PO1-14x1-18 and PO2-23x2-9, as well as a heterokaryon resulting from crossing the progeny of both maternal mycelia PO2-5xPO1-7. Cluster II groups only PO1 progeny monokaryons, and cluster I groups heterokaryons derived from crossing PO2 and PO1 progeny. The small cluster located between clusters I and II includes two PO2 monokaryons (PO2-13, PO2-18) (Figure 2). Examining molecular traits allowed us to conclude that, in some of the progeny (especially PO1 monokaryons), genetic traits were not highly differentiated, similarly as for heterokaryons formed from crossing the progeny of both maternal mycelia (cluster I). At the same time, it is possible to identify a group of mycelia that, despite their different origins, shows some similarities at the molecular level; sometimes, the similarity is very high (cluster III).

4.2. Growing Tests

Examining the physiological parameters of the mycelia allowed it to be concluded that temperatures affected the growth of the tested strains differently, and the rate at which the tested strains grew varied and depended mainly on the incubation temperature and on the individual characteristics of the mycelia themselves.

The tested mycelia grew by far the slowest at 10 °C, from 1.2 to 1.7 mm/day (Figure 3). Mycelia that tolerate higher temperatures well are grouped in clusters 1, 4, and 5. They include heterokaryons obtained from crossing between progeny of both maternal strains and heterokaryons obtained from crossing within their own PO1 or PO2 progeny, as well as a small group of monokaryons. Most monokaryons are gathered in clusters 2 and 3, which grow well at 20 °C. Strains characterized by good growth at 15 °C are found in cluster 1, and at the same time, they grow well at 25 °C and perform best at 10 °C, which predisposes them to better adaptation to changing environmental conditions. Maternal PO1 and PO2 strains are found in cluster 2 (Figure 3, Figure 4 and Figure 5;

Table 1. Elements of individual clusters—mycelial growth.

No.	Elements of Cluster 1	Elements of Cluster 2	Elements of Cluster 3	Elements of Cluster 4	Elements of Cluster 5
1	PO1-11x13	PO1	PO1-30	PO2-4	PO1-23
2	PO1-12x18	PO1-1	PO1-32	PO2-16	PO1-28
3	PO1-7x11	PO1-2	PO1-34	PO2-25	PO1-39
4	PO2-13x9	PO1-3	PO2-1	PO1-14x18	PO2-3
5	PO2-17x13	PO1-7	PO2-5	PO1-15x21	PO2-24
6	PO2-23x25	PO1-9	PO2-6	PO1-20x21	PO2-27
7	PO2-23x9	PO1-11	PO2-7	PO1-9x16	PO2-16x21
8	PO2-1xPO1-1	PO1-12	PO2-8	PO2-13x12	PO2-19x24
9	PO2-1xPO1-2	PO1-13	PO2-9	PO2-22x13	PO2-1xPO1-12
10	PO2-4xPO1-11	PO1-16	PO2-12	PO2-1xPO1-7	PO2-1xPO1-9
11	PO2-5xPO1-9	PO1-17	PO2-13	PO2-4xPO1-2	PO2-4xPO1-12
12	PO2-6xPO1-2	PO1-18	PO2-17	PO2-5xPO1-7	PO2-4xPO1-3
13		PO1-19	PO2-18	PO2-6xPO1-11	PO2-9xPO1-1
14		PO1-20	PO2-19	PO2-6xPO1-12	PO2-9xPO1-3
15		PO1-21	PO2-21	PO2-6xPO1-9	PO2-9xPO1-7
16		PO1-22	PO2-22	PO2-9xPO1-2
17		PO1-24	PO2-23
18		PO1-25	PO2-28
19		PO1-27	PO2-31
20		PO1-29	PO2-32
21		PO1-31
22		PO2

). The analysis of variance indicates the significance of the results obtained (Supplementary Table S1).

4.3. Toxicity Tests

Hyphal knobs observed on mycelia cultured on PDA medium were very few, ranging from 1 to a few knobs in the microscope’s field of view. During the observations, a large variation was found due to the presence of hyphal knobs between monokaryons and heterokaryons. The highest value of the IW coefficient of hyphal knob formation of 0.9 was achieved by two isolates, PO1-23 and PO2-24. Most of the isolates reached an IW value in the range of 0.6–0.8, and for the rest, this coefficient was lower or equal to 0 (Figure 6). Isolates that reached the value of IW = 0 were not included in Figure 6.

An analysis of the ability of the mycelia to form hyphal knobs on water agar in the presence of C. elegans (Figure 7 and Figure 8) indicates a certain level of variation as to this parameter, and, thus, strains with weaker and stronger abilities to produce hyphal knobs can be distinguished (Figure 9;

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

No.	Elements of Cluster 1	Elements of Cluster 2	Elements of Cluster 3	Elements of Cluster 4
1	PO1-11	PO1-2	PO1-16	PO1
2	PO1-12	PO1-39	PO1-17	PO1-1
3	PO1-21	PO2-12	PO1-19	PO1-3
4	PO1-24	PO2-17	PO1-20	PO1-7
5	PO1-31	PO2-18	PO1-22	PO1-9
6	PO1-32	PO2-21	PO1-23	PO1-13
7	PO1-34	PO2-22	PO1-25	PO1-18
8	PO2	PO2-31	PO2-1	PO1-27
9	PO2-8	PO2-32	PO2-3	PO1-28
10	PO2-13	PO1-20x21	PO2-4	PO1-29
11	PO2-19	PO1-9x16	PO2-5	PO1-30
12	PO2-25	PO2-1xPO1-12	PO2-7	PO2-6
13	PO2-28	PO2-6xPO1-12	PO2-9	PO2-16
14	PO2-13x12	PO2-9xPO1-2	PO2-23	PO2-24
15	PO2-13x9		PO1-12x18	PO1-11x13
16	PO2-17x13		PO2-22x13	PO1-14x18
17	PO2-23x25		PO2-1xPO1-7	PO1-15x21
18	PO2-23x9		PO2-1xPO1-9	PO1-7x11
19	PO2-1xPO1-1			PO2-19x24
20	PO2-1xPO1-2			PO2-4xPO1-11
21	PO2-4xPO1-12			PO2-4xPO1-2
22	PO2-9xPO1-1			PO2-4xPO1-3
23	PO2-9xPO1-7			PO2-5xPO1-7
24				PO2-5xPO1-9
25				PO2-6xPO1-11
26				PO2-6xPO1-2
27				PO2-6xPO1-9
28				PO2-9xPO1-3

; Supplementary Table S2). From the obtained results of the analysis of variance (mainly F values), we conclude that variables 2 h and 3 h constitute the main criterion for belonging to those clusters (Table 2). This means that within 2–3 h of contact with nematodes, increased mycelial activity is observed, and this is more clearly seen in the mycelia gathered in clusters 1 and 2, while in the other clusters this feature is rather constant and the toxigenic capacity is due to the natural characteristics of the mycelia (Figure 9). The mycelia gathered in cluster 4 are characterized by a natural and constant level of high ability to produce hyphal knobs, while those collected in cluster 3 have this parameter at a level that is moderate but higher than for the mycelia gathered in cluster 2 (Figure 9; Table 2). From a practical point of view, in further work on the isolation of mycelia with good killing parameters, it is worth noting the mycelia collected in clusters 1 and 4 show a high level of usefulness (Figure 9; Table 2). The correlation coefficient calculated between hyphal knob production capacity under control conditions (without C. elegans) and the presence of C. elegans was 0.772, confirming the high correlation between the intensity of creating toxin-producing hyphal knobs and the presence of nematodes.

The direct result of the possibility of toxin production by the studied mycelia is their lethal capabilities against the model organism C. elegans. The lethal capability was observed as a paralysis of the movement of C. elegans nematodes applied to mycelia grown on poor water agar (WA) conditions (Figure 10).

Cluster 5 of nematode mobility, grouping the most effective mycelia, consists of four PO1 monokaryotic progeny strains; the rest are heterokaryons, including eight PO2 crosses and three PO1 crosses (

Table 3. Elements of individual clusters—nematode viability/movement under the influence of P. ostreatus mycelia.

No.	Elements of Cluster 1	Elements of Cluster 2	Elements of Cluster 3	Elements of Cluster 4	Elements of Cluster 5
1	PO2-1	PO1-7	PO2-6	PO2-23	PO1-29
2	PO2-3	PO1-13	PO2-9	PO1	PO1-30
3	PO2-4	PO1-20	PO2-16	PO1-28	PO1-31
4	PO2-5	PO1-22	PO2-19	PO1-34	PO1-32
5	PO2-7	PO1-12x18	PO1-1	PO1-39	PO1-11x13
6	PO2-8	PO1-20x21	PO1-2	PO1-15x21	PO1-14x18
7	PO2-12	PO1-9x16	PO1-3	PO2	PO1-7x11
8	PO2-13	PO2-22x13	PO1-9	PO2-13x12	PO2-1xPO1-1
9	PO2-17	PO2-1xPO1-2	PO1-11	PO2-13x9	PO2-1xPO1-12
10	PO2-18	PO2-4xPO1-3	PO1-12	PO2-17x13	PO2-1xPO1-7
11	PO2-21	PO2-5xPO1-9	PO1-16	PO2-19x24	PO2-4xPO1-11
12	PO2-22	PO2-6xPO1-12	PO1-17	PO2-23x25	PO2-4xPO1-2
13	PO2-24	PO2-9xPO1-1	PO1-18	PO2-1xPO1-9	PO2-6xPO1-11
14	PO2-25	PO2-9xPO1-2	PO1-19	PO2-5xPO1-7	PO2-6xPO1-2
15	PO2-28	PO2-9xPO1-7	PO1-21	PO2-9xPO1-3	PO2-6xPO1-9
16	PO2-31		PO1-23
17	PO2-32		PO1-24
18	PO2-4xPO1-12		PO1-25
19			PO1-27
20			PO2-23x9

). The analysis of variance performed for the results (mainly F-values) allows it to be concluded that variable 1h is the main criterion for belonging to those clusters (Supplementary Table S3). From this point on, a slowdown in nematode movements was observed, with the trend increasing up to three hours and being constant after 24 h. After 48 h, there was no difference between the samples—all mycelia had killed the nematodes and decomposed their bodies. During the course of the experiment, moving forms (usually slow-moving) could sometimes be seen, but these were larvae hatched from eggs deposited in the bodies of dead maternal specimens, and dead maternal specimens and moving larvae trapped in their bodies could also be observed. If motile larvae are observed after the second day, it can be assumed that they will be killed no later than the next day. At the same time, in the control cultures, the nematodes remained alive and active throughout the observation period, i.e., for two days. There was no significant correlation between the ability of mycelia to form hyphal knobs on WA (both under control conditions and in the presence of nematodes) and the progression of nematode paralysis (the value of the IW factor), which may prove the action of other factors than just the toxin secreted from hyphal knobs.

A summary of the analyses (

Table 4. Summary of mycelium characteristics—strains that present at least three optimal features to control nematodes.

Mycelial Growth [Cluster 1]	Nematode Movement [Cluster 5]	Hyphal Knobs [Cluster 1 and Cluster 4]	Hyphal Knobs on PDA (IW = 0809)
PO1-11x13	PO1-11x13	PO1-11x13	-
PO1-7x11	PO1-7x11	-	PO1-7x11
PO2-1xPO1-1	PO2-1xPO1-1	PO2-1PO1-1	-
PO2-1xPO1-2	-	PO2-1xPO1-2	PO2-1xPO1-2
PO2-4xPO1-11	PO2-4xPO1-11	PO2-4xPO1-11	-

) contains mycelia extracted from individual clusters and allows potential mycelia with the best parameters to be identified for further research. They were chosen according to their presence in at least three of analyzed clusters. They will be the mycelia growing intensively, including at 15 and 25 °C, capable of producing hyphal knobs well and exhibiting good lethal activity against nematodes. When analyzing the properties of the studied mycelia, it is possible to distinguish several that have these beneficial features. These are three heterokaryotic strains PO1-11x13, PO2-1xPO1-1, and PO2-4xPO1-11, which are characterized by good growth, high lethality against nematodes and the ability to produce hyphal knobs. In contrast, heterokaryon PO1-7x11 shows good growth and lethality against nematodes, as well as the ability to produce hyphal knobs on the PDA medium. Heterokaryon PO2-1xPO1-2 shows similar characteristics to the previous strain, but, in addition to good growth parameters, it produces hyphal knobs well under various conditions (WA and PDA) (Table 4). All those heterokaryons are crosses of only seven individual monokaryons. Three of them belong to the same cluster (cluster 2B) based on the morphology and two others to cluster 1B (Figure 1). According to molecular features they also belong to only two clusters—two of them to cluster III, and three of them to cluster I showing close relationships (Figure 2).

5. Discussion

Nematocidal mushrooms provide an interesting solution for protecting plants against nematodes that are pathogenic for plants. There are many such mushrooms known, but there are not so many practical applications. However, in the era of the need to reduce the use of chemicals, it is worth taking a closer look at the potential applied uses of nematicidal mushrooms. To this end, it is necessary to search for species and strains with the best such properties. We chose to study P. ostreatus and search among its progeny for strains with potentially the best lethal properties. To the best of our knowledge, no such studies have been conducted to date. We considered the best characteristics indicative of mycelial lethality to be rapid vegetative growth at both 15 °C (which will be advantageous under temperate climate conditions) and 25 °C (which may make it easier for the mycelia to survive under warming climate conditions, especially in the summer). Another important feature is the production of a large number of hyphal knobs and high lethal activity (toxic properties) against the model nematode C. elegans. Similar studies have been conducted previously, but the progeny of heterokaryotic mycelia obtained from the environment have not been studied in this respect [16,20,29,30]. The ability to synthesize nematicidal toxins seems to be common in nature; detailed studies have not been conducted to confirm this view, but the properties of representatives of the genera Pleurotus and Coprinus seem to support it [5]. Heydari et al. [29] tested five species of Pleurotus, including P. ostreatus, P. sajor-caju, P. cornucopiae, P. florida, P. eryngii against Meloidogyne javanica, showing similar in vitro results to ours. They achieved complete mycelium colonization of nematode bodies within 24–48 h. Their study also showed that culture filtrates obtained from mushroom cultures conducted on malt extract broth had similar properties. The toxicity varied among species, however, confirming that Basidiomycota can be a source of important substances of practical importance [29,31,32]. The results of such studies encourage research to be undertaken in the search for mycelia with high lethality and the development of procedures for their practical application. Ibrahim et al. [33] and Ibrahim and Handoo [34] obtained satisfactory results using mushroom stems of P. ostreatus as a soil additive against Meloidogyne incognita and Heterodera goldeni on rice plants. M. incognita was also susceptible to aqueous extracts of basidiomycete mushrooms. Among them, Pleurotus ostreatus, P. citrinopileatus, P. pulmonarius, and Boletus sp. showed the highest mortality index in vitro against hatching of second-stage juveniles of root-knot nematode M. incognita. The extracts were also applied into the soil in pots infested with nematodes. Results confirmed that fungal extracts reduced close to 70% of nematode reproduction. Additionally, plants protected by extracts achieved higher fresh mass and their roots were less damaged when extracts of Boletus sp. and P. pulmonarius were used [35]. Research by Comans-Pérez et al. [32] focused (similarly to ours) on evaluating the biological parameters of the mycelia of edible mushrooms, though the subjects of these studies were various mushroom species, including oyster mushrooms. The results of these studies indicate the high suitability of P. ostreatus, as the strain they studied showed rapid mycelial growth and high antihelminthic activity in vitro against Haemonchus contortus, as well as high lethality of the hydroalcoholic extract obtained from the mycelia, applied at a dose of 200 mg/mL. Valdez-Uriostegui et al. [36] obtained similar efficacy for similar extracts obtained from the mycelium of P. ostreatus extracted with ethanol–water (70:30) and basidiomata and the spent substrate extracted with methanol-water (70:30). They observed high efficacy of hydro-alcoholic extracts against larvae and eggs of H. contortus in a wide range of doses, and the lethal result was obtained within 72 h. The efficacy of mycelium extract was reliable, but the efficacy from the spent substrate was related to that of basidiomata extract. Similar anthelmintic results were obtained by Braga [37] for the canine parasite Ancylostoma caninum by using properly prepared P. eryngii culture fluid. The fungus culture was induced by co-incubation with Panagrellus spp. larvae, a free-living nematode. After 21 days of such cultivation, the liquid was harvested along with mycelial fragments and applied to A. caninum, obtaining a 47.5% reduction in the number of live larvae [37]. In our study, we obtained the complete death and colonization of nematodes by the mycelium after 48 h at the latest, but, in many cases, this occurred after just 24 h. Various natural compounds are responsible for the lethal properties of the oyster mushroom [23,38], including ostreatin, about which there have recently been nomenclature inaccuracies. The term was originally used by Barron and Thorn [18] to refer to a substance contained in the toxin drops produced by hyphal knobs but without chemical identification. Subsequently, in this secretion, Kwok et al. [15] identified trans-di-decenedioic acid, which was assigned the name “ostreatin.” More recently, this name has been used for a ribotoxin-like protein, which consists of 131 amino acid residues [21]. Also, another toxic protein, as a pore-forming cytolysin, was identified in oyster mushrooms (P. ostreatus) and called “ostreolysin” (Oly) [22].

The results of our research show the differentiation between the maternal strains of Pleurotus ostreatus, its progeny, as well as strains obtained from crossing between the progeny of the two wild strains. This differentiation is evident in every aspect of the research, as they differ in their preferred optimal growth temperature, mycelial morphology, growth rate, ability to form hyphal knobs and nematicidal capabilities. However, only heterokaryotic strains showed the highest potential usefulness against nematodes. The mechanism of the toxic effect of P. ostreatus on C. elegans was reported by Lee et al. [23], and our results are consistent with their observations. However, they suggest that it is not trans-di-decenedioic acid that is responsible for the paralyzing and lethal properties against C. elegans. At the same time, they point to a strong nematode-killing mechanism that is different from widely used anthelmintic drugs such as ivermectin, levamisole, and aldicarb, which suggests a potentially new way for targeting parasitic nematodes in plants, animals, and humans [23]. The range of optimal temperatures (15–25 °C) favoring the development of some of the mycelia that we studied and indicated as more useful than other mycelia, corresponds to the range of highest accumulation of hatching of Heterodera schachtii, which was observed at temperatures between 15 and 30 °C, and for H. betae between 20 and 30 °C, and should be considered the optimal temperature range for their hatching. Low temperature percentages cause low emergence of juveniles of both beet cyst nematode species [3].

6. Conclusions

The conducted research confirms the nematicidal potential of the oyster mushroom, but at the same time shows a great diversity of traits in the progeny. Taking into account the results of our observations, we isolated the mycelia that occurred in the clusters considered most suitable (Table 4) in terms of the suitability of oyster mushroom traits to combat nematodes. Thus, five heterokaryotic mycelia can be identified that meet expectations. These are the following mycelia: PO1-11x13, PO2-1xPO1-1, PO2-4xPO1-11, PO2-1xPO1-2, and PO1-7x11 with mycelium PO1-7x11 and PO1-7x11 also showing a considerable ability to form toxins on a nutrient-rich PDA medium. These results also indicate that wild strains do not necessarily exhibit the best defense against small soil fauna, as we demonstrated with our wild-type PO1 and PO2 strains. What is surprising, however, is that monokaryotic mycelia do not have high defensive properties, which, in the context of the need to “search” for a suitable mating partner in the environment, seems to be a biologically unfavorable feature. The results obtained can be considered promising and worthy of further verification in subsequent studies, as they create hope for the possibility of using selected strains of oyster mushrooms in agricultural practice to inhibit the population of nematodes harmful to plants. An additional advantage in favor of such a solution is the biodegradability of oyster mushroom toxins and the possibility to use spent mushroom spawn [15,16,38].