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.
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.