Nuclear conditions of basidiospores and hyphal cells in the edible mushroom Oudemansiella aparlosarca

Abstract Oudemansiella aparlosarca is an edible mushroom possessing medicinal and health benefits. Although there are studies on the cultivation of O. aparlosarca, only a few studies have focused on its genetics and life cycle. Therefore, the main objective of this study was to identify the nuclear conditions of basidiospores and homokaryotic and heterokaryotic hyphal cells and to determine the influence of different nuclear conditions on basidiospore diameter in O. aparlosarca. Two parental strains: strain‐55 and strain‐81 were used. Staining of basidiospores and hyphal cells in the apical region was performed. We observed the following nuclear conditions: non‐nucleate, mononucleate, binucleate, and multinucleate. In both parental strains, binucleate spores were predominant, while the number of non‐nucleate spores was the lowest. The diameter of non‐nucleate spores was the smallest, being 11.52 µm and 12.15 µm in parental strain‐81 and strain‐55, respectively, while multinucleate spores had the largest diameter, being 14.78 µm in both parental strains. Both homokaryotic and heterokaryotic strains were identified in isolated single spores from parental strains. Binucleate cells were majorly present in heterokaryotic hyphal cells, and multinucleate cells were predominant in homokaryotic hyphal cells. We conclude that O. aparlosarca contains homokaryotic and heterokaryotic basidiospores, which indicates an amphithallic life cycle. The observed binucleate spores might be the result of post‐meiotic mitosis.

To enhance the production and quality of edible mushrooms, several genetic studies have been conducted on mushrooms to optimize strains and breeding strategies (Kothe, 2001). Although efforts to cultivate O. aparlosarca have been successful, a limited number of morphological and nuclear genetic studies have been conducted on basidiospores and homokaryotic and heterokaryotic hyphae from this species (Corner, 1948;Webster & Weber, 2007). Additionally, few studies have been conducted on their nuclear condition; hence, adequate information on the genetics and life cycle of Oudemansiella is not available.
Nucleus-based studies are necessary to elucidate the life cycle of mushrooms. Therefore, we conducted this study to describe the different nuclear conditions in basidiospores and homokaryotic and heterokaryotic hyphal cells, and to determine the influence of different nuclear conditions on the diameter of basidiospores of two parental strains of O. aparlosarca. We expect that the findings of this study will support future breeding and genetic studies as well as the production of quality mushrooms from Oudemansiella species.
These two parental strains were identified by Dr. Hao Yanjia of the Kunming Institute of Botany, Chinese Academy of Sciences, following the method reported by Hao et al. (2016). Strains 55 and 81 were collected from Dadugang, Yunnan Province, China, in 2011, andBaiyun Mountain, Guangdong Province, China, in 2013, respectively, and  One kilogram of the substrate was put in a polyethylene bag (16 cm × 32 cm × 0.04 cm) and autoclaved at 121°C for 120 min.
The sterilized substrate was inoculated by spreading the spawn on its surface at a concentration of 2% (w/w) fresh weight of the substrate. The bags were kept in a spawn running room at 25°C and 70% relative humidity (RH) under dark conditions. After completion of mycelium running, the bags were unfolded to induce fruiting body development and then maintained at 22°C and 85% RH. Fruiting bodies were harvested at the same mature stage when mushroom caps were open.

| Spore suspension and single spore isolation
Six plastic bottles, each containing 100 ml of distilled water, were autoclaved at 121°C for 30 min. Mature fruiting bodies of both parental strains were harvested using a sterile knife, and the mushroom surface was sterilized using cotton soaked in 75% alcohol under sterile conditions in a laminar flow cabinet. The stipe of the mushroom was shortened using a sterilized blade, and it was hung on a hook, which was then fixed to an autoclaved bottle lid, with the mushroom gills facing downwards. These procedures were performed in a laminar flow cabinet. Spore suspensions were collected after two days. One part of the suspension was used for staining, and the other part was subjected to sequential dilution with sterilized water to obtain a concentration of approximately 1 × 10 3 spores/ml. One hundred microliters of the spore suspen-  The sequences of the renamed parental strains (this study) and the isolated single spore strains (hereafter, referred to as progeny, originating from a single spore) were checked with the original sequence of O. canarii, which is available in the NCBI GenBank database under the accession number MK336783.1. Sigma-Aldrich, St. Louis, MO, USA) and 2 PPM calcofluor white (Sigma-Aldrich). The glass slide was covered with an alcoholcleaned coverslip, and the edges were sealed with transparent nail polish. To enhance stain penetration, the prepared slides were heated at 50°C for 10 s and immediately cooled inside an icebox for 1 min. Again, the slides were heated at 50°C for 30 s and cooled for 1 min; this procedure was repeated three times. Finally, the stained slides were observed under a UV fluorescence microscope (Olympus IX70 Multi-parameter Fluorescence Microscope;

| Spore and hyphal cell staining
Olympus, Tokyo, Japan). The experiment was repeated five times, and 2500 spores were counted for each parental strain to observe different nuclear conditions in spores. Additionally, to measure the spore diameter of each nuclear condition, 1000 spores from both parental strains were observed. To categorize the different nuclear conditions in homokaryotic and heterokaryotic hyphal cells in the apical region, the slide culture method was performed as described by Sawada et al. (2014). Briefly, the medium for slide culture was prepared with 1% agar and 0.2% glucose. Petri dishes, forceps, and microscope slides were sterilized. The prepared medium was poured into a small beaker, and sterilized slides were partially dipped into the medium using forceps and kept inside the Petri dishes. Once the medium dried on the slides, a small piece of hypha was placed on each slide and allowed to grow on one half of the slide. Thereafter, the slides were carefully removed from the Petri dishes and gently washed using 0.1 M phosphate- Olympus). A total of 50 hyphal cells in the apical region were manually counted on each culture slide, for both homo-and heterokaryotic hyphae. This procedure was repeated thrice. All hyphal staining experiments were performed on the ninth day of culture.

| Data analysis
A counting instrument was used to count the number of nuclei in spores and hyphal cells. ImageJ software (National Institutes of Health, MD, United States) was used to measure the diameter of spores and for image analysis. Mean values within the nuclear conditions in a parental strain were subjected to one-way ANOVA, and significant differences were analyzed by Duncan's multiple range test at 95% confidence level, using the IBM SPSS Statistics software v25.0. Comparisons and significant differences with respect to each nuclear condition between the parental strains were analyzed using the paired-sample t test at a 95% confidence level.

| RE SULTS
Mature fruiting bodies of both parental strains are shown in Figure 1.
The sequence of parental strains and progeny were highly similar (around 97%-99%) to the original sequence of O. canarii in the NCBI.

| Nuclear conditions of basidiospores
The germination percentage of basidiospores was 88%. We observed a total of 5000 basidiospores of O. aparlosarca, as shown in Values are expressed as percentage means ± SD of five replicates, and 500 basidiospores were observed in each replicate. According to Duncan's multiple range test, means in the same-colored bars followed by the same superscripted letter within the observed nuclear conditions in a parental strain are not significantly different at p < 0.05. Means in the different colored bars followed by the superscripted "*" symbol of the observed non-nucleate condition between the parental strains are significantly different at p < 0.05, according to the paired-sample t test. The error bars denote standard deviation nuclei per spore; Figure 2d), and multinucleate (having three or more nuclei per spore; Figure 2e). Within a parental strain, all the nuclear conditions differed significantly (Figure 3). In addition, the number of binucleate spores was the highest, while the number of non-nucleate spores was lower than that observed under the other nuclear conditions of spores in both parental strains. The percentages of binucleate, mononucleate, multinucleate, and nonnucleate spores in strain-55 were 54.70, 23.50, 16.83, and 4.84%, respectively, and in strain-81, these were 57.38, 20.85, 18.51, and 3.16%, respectively. A significant difference was observed only in non-nucleated spores, while no significant difference was observed in multi-, mono-, and binucleate spores between the parental strains 55 and 81 (Figure 3).

| Spore nuclei and spore diameter
In total, we measured 4000 basidiospores. The diameters of different nuclear conditions are presented in Table 1. Non-nucleate spores had the shortest diameter, and the average diameters of parental strains 81 and 55 were 11.52 μm and 12.15 μm, respectively. Multinucleate spores had the largest spore diameter (14.78 μm) in both parental strains. Significant differences were observed within non-nucleate and multinucleate spores, while no significant difference was observed between the diameters of mono-and binucleate spores of parental strain-55. However, in the case of parental strain-81, significant differences were observed between the diameters of spores with all the nuclear conditions (p < 0.05). Although significant differences were detected in the diameters of non-nucleate spores between the parental strains 55 and 81, no significant differences were observed in the diameters of binucleate, mononucleate, and multinucleate spores (Table 1).  Table 2, no significant difference was observed between the nuclear conditions of heterokaryotic parental strain-55 and its progeny. However, the binucleate condition showed significant differences between the heterokaryotic parental strain-81 and its progeny (Table 3). Basidiospores typically contain a single haploid nucleus, and on germination, they produce homokaryotic hyphae with one nucleus per hyphal cell (Nieuwenhuis et al., 2013). Cell fusion between two sexually compatible homokaryotic hyphae leads to the formation of heterokaryotic hyphae, which contain two haploid nuclei with a clamp connection joining two contiguous hyphal cells (Gladfelter & Berman, 2009). Under ideal climatic conditions, the heterokaryotic primordia develop into mushrooms, and the mature fruiting body develops into mononucleate, haploid basidiospores to continue its life cycle (Miles & Chang, 2004 Meiosis is followed by post-meiotic mitosis in secondary homothallism, which occurs in the top portion of the basidium in basidiomycetes.

TA B L E 1 Nuclear conditions and the diameters of the basidiospores of
Occasionally, this occurs inside the sterigmata or pre-mature spore. In pattern D, as described by Duncan and Galbraith (1972), post-meiotic mitosis occurs inside basidiospores and both nuclei persist inside the spores, which are called binucleate spores. In pattern F, post-meiotic mitosis occurs inside the basidium, and two nuclei migrate from the basidium to each spore (Mueller et al., 1993), increasing the number of nuclei in the spores. Additionally, some spores do not develop properly and/or do not receive any post-meiotic nucleus (Ling et al., 2019). In the present study, the occurrence of post-meiotic mitosis might have led to the formation of heterokaryotic or homokaryotic binucleate spores.
In A. bisporus, homokaryotic spores are generally produced in the heterothallic lifecycle, while heterokaryotic spores are vastly produced in the amphithallic life cycle, often referred to as the secondary homothallic life cycle (Callac et al., 1993;Kamzolkina et al., 2006).
The results of the present study demonstrated that approximately 71%-75% of basidiospores were binucleate and multinucleate, and on examination of progeny, we found that only 4%-6% were homokaryons and 94%-96% were heterokaryons. Since both homokaryotic and heterokaryotic progeny were identified, we presumed that O. aparlosarca showed an amphithallic life cycle along with post-meiotic mitosis. We speculated that the predominantly observed binucleate and multinucleate spores might form heterokaryotic progeny.
Asynchronous nuclear migrations, which can lead to unequal distribution of nuclei in spores, have been observed in Pisolithus microcarpus from basidium to basidiospores (Campos & Costa, 2010).
Such occurrence also might have led to the formation of multinucleate basidiospores observed in the present study. A few non-nucleate basidiospores may be produced when they do not receive any postmeiotic nucleus. Here, we observed a spore germination rate of 88%.
Of note, the non-nucleate condition in basidiospores was around 5%, which may explain, in part, the 12% of non-germinated spores. Note: Values are expressed as means ± SD of three replicates, and 50 hyphal cells in the apical region were observed in each replicate. Means in the same column followed by the same superscripted letter within the observed nuclear conditions in parental strain-81/ progeny-81 are not significantly different at p < 0.05, according to Duncan's multiple range tests. Means in the same row followed by the superscripted "*" symbol in the observed binucleate condition among the parental strain-81 and progeny-81 are significantly different at p < 0.05, according to paired-sample t test.
of non-nucleate spores in single spore isolation during the breeding process, and to gain a deep understanding of basidiosporogenesis. and multinucleate cells were more common in the homokaryotic hyphal cells of the progeny than in other nuclear conditions. The nonnucleated condition was the least dominant in both homokaryotic and heterokaryotic hyphal cells of the parent and progeny.

E TH I C S S TATEM ENT
None required.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data generated or analyzed during this study have been pro-