Short Communication
Effect of sodium chloride on basidiospore germination and vegetative mycelial growth of the ectomycorrhizal fungus Rhizopogon roseolus
2022 Volume 63 Issue 3 Pages 96-101
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2022 Volume 63 Issue 3 Pages 96-101
Rhizopogon roseolus is a basidiomycetous ectomycorrhizal fungus that inhabits mainly coastal areas. Understanding the response of this fungus to salinity at each stage of its life cycle will lead to elucidation of the strategies for its propagation. This study examined the effect of sodium chloride (NaCl) on basidiospore germination and mycelial growth of both homokaryotic and heterokaryotic strains of R. roseolus, on nutrient agar media with varying concentrations of NaCl (0, 50, 150, and 300 mM). Regardless of the presence of NaCl, R. roseolus basidiospores germinated and the germlings grew, forming compatible fusions. In addition, all multispore strains, including homokaryons and heterokaryons, grew under these NaCl conditions. Most of these strains had an effective concentration inhibiting mycelial growth by 50% value greater than 300 mM of NaCl. These results indicate that R. roseolus can germinate, grow, and mate in the presence of NaCl, allowing it to propagate in saline habitats.
The basidiomycetous ectomycorrhizal (EM) fungus Rhizopogon roseolus (Corda) Th. M. Fr. (=R. rubescens Tul. & C. Tul.), distributed in Japan, mainly inhabits coastal sand dunes and forms a symbiotic association with Pinus thunbergii (Nagasawa, 2000). This fungus produces hypogeous basidiocarps (edible mushrooms) in spring and autumn. It has been artificially cultivated by using pine seedlings colonized by EM roots using basidiospores (hereafter referred to as spores) and pure-cultured mycelia (Yamada, Ogura, & Ohmasa, 2001; Shimomura, Matsuda, Ariyoshi, & Aimi, 2012b; Wang, Cummings, & Guerin-Laguette, 2012).
The putative life cycle of this fungus is considered as follows (Sawada et al., 2014). After spores are dispersed, they germinate in soil; subsequently, the germlings as homokaryons grow and mate with compatible homokaryons. Then, the generated heterokaryons form EM roots on host plants and produce basidiocarps. Coastal sandy areas represent harsh growing conditions, such as high salinity resulting from the inflow of seawater (Ishikawa, Furukawa, & Oikawa, 1995), potentially limiting R. roseolus from germinating, mating, and growing in this habitat. Therefore, understanding the response to salinity at each stage of the life cycle will lead to elucidation of the strategies for its propagation. Several researchers have studied the response to salinity of this fungus in vitro; for example, Matsuda, Sugiyama, Nakanishi, and Ito (2006) reported that the wild-type heterokaryotic strain isolated from the fruiting body tissue showed salinity tolerance on the basis of biomass yield. Nakano, Sawada, Gao, Aimi, and Shimomura (2015) reported the intraspecific variation in growth response to salinity of homokaryotic strains obtained by single-spore isolation and of heterokaryotic strains produced by crossing these homokaryotic strains. However, there is still limited knowledge on the effect of salinity on the vegetative growth stage. Furthermore, the response to salinity in the spore stage remains unknown.
To the best of our knowledge, there are no reports on the effect of salinity on spore germination of EM fungi, including R. roseolus. This is probably because spores of most EM fungal species do not easily germinate on nutrient agar media (Fries, 1987). However, R. roseolus is an EM fungus whose axenic protocol for spore germination has been established (Martín & Gràcia, 2000; Nakano, Gao, Aimi, & Shimomura, 2016, 2017). In this study, we examined the effect of sodium chloride (NaCl) on spore germination and subsequent colony development of R. roseolus on nutrient agar media containing different concentrations of NaCl. We also examined the effect of NaCl on the growth of multispore strains, including homokaryons and heterokaryons, isolated from nutrient agar media with or without NaCl to confirm the hypothesis that the strains from agar media with NaCl would have a higher adapted tolerance to NaCl. Based on these results, the salinity tolerance and propagation strategy of R. roseolus are discussed. In this study, salinity tolerance of R. roseolus strains was determined when they maintained more than 50% of their mycelial growth on NaCl stress medium compared with NaCl stress-free medium, referred to Chen, Ellul, Herdman, and Cairney (2001) and Matsuda et al. (2006).
A fresh fruiting body of R. roseolus was collected from the P. thunbergii forests in Tottori Prefecture, Japan. The fruiting body was divided into two parts by hand. The gleba color of the fruiting body was white to beige. One of the parts was used to prepare the spore suspension as follows. Tissue fragments were cut from the gleba and immersed in sterile distilled water. The spore suspension was then filtered through a 100-μm cell strainer to remove tissue debris. The spore concentration of the resulting suspension was adjusted to 106-107 spores/mL using a hemocytometer. The other half of the fruiting body was used to obtain tissue culture isolates as the wild-type heterokaryotic strain Tottori University Fungal Collection (TUFC) 101244. Vegetative mycelia were obtained by placing the inner tissue fragments of the fruiting body on malt extract agar medium (Becton Dickinson Co., Sparks, MD, USA). The dried specimens were deposited in the Fungus/Mushroom Resource and Research Center of the Faculty of Agriculture at Tottori University and assigned a specimen number Tottori University Mycological Herbarium (TUMH) 63209.
The basal medium for spore germination and mycelial growth was 1/5 diluted modified Melin-Norkrans (5D-MMN) agar medium, in which the MMN medium (Marx, 1969) was diluted with distilled water to 1/5 before the addition of 2.0% (w/v) agar. The pH of the medium was adjusted to 5.5 with 1 N HCl. The medium was sterilized by autoclaving at 120 °C for 20 min, and 20 mL of molten agar was poured into a plastic dish (9 cm diameter). The spore suspension (100 μL) from the fruiting body was spread onto 5D-MMN agar medium with different concentrations of NaCl (0, 50, 150, and 300 mM), and the plates were incubated at 25 °C in the dark. Five plates were prepared for each NaCl treatment. After 20 d of incubation, the number of colony-forming units was counted visually. Then, the numbers of germinated and ungerminated spores for a total of 1000 spores per plate were measured under a light microscope. The germination process of spores in R. roseolus has been investigated in detail (Martín & Gràcia, 2000; Nakano et al., 2016). In this study, spores showing morphological events such as protrusion stage, swelling stage, and germ tube extension stage were considered as germinated spores, and the germination rate was calculated as follows: Spore germination rate (%) = (germinated spores/observed spores) × 100.
Individual mycelial colonies on 5D-MMN agar medium containing different concentrations of NaCl were randomly isolated and cultured on fresh 5D-MMN agar medium at 25 °C in the dark. To verify if the strains were homokaryotic or heterokaryotic, we cultured the isolates on the TM7 agar medium developed by Shimomura, Egi, Sawada, and Aimi (2012a) containing 0.2% (w/v) Tween 80 (Wako Pure Chemicals, Tokyo, Japan), 0.1% (w/v) malt extract, and 2.0% (w/v) agar, with pH adjusted to 7.0, with 1 N NaOH to help visualize the formation of the clamp connection. Isolates without clamps were used as homokaryons, and those with clamps were used as heterokaryons. Inocula of the two homokaryotic strains were co-cultured on 5D-MMN agar medium. After 10-14 d of incubation at 25 °C in the dark, the mycelia were taken from the interface of two mycelial colonies that crossed each other. These hyphal samples were then transferred to TM7 agar medium. After 3-7 d of incubation, the fungal hyphae were examined under a light microscope. Mycelia with clamps were used as the heterokaryons.
Fungal colonies of homokaryotic and heterokaryotic strains pre-cultured on 5D-MMN agar medium for 15-20 d were removed with a 4-mm cork borer and transferred to 5D-MMN agar media with different concentrations of NaCl (0, 50, 150, and 300 mM). The diameter of the mycelial colonies of each strain was measured after 16 d of incubation at 25 °C in the dark. Relative mycelial growth was calculated by comparing the colony diameter on the NaCl stress medium with that on the NaCl stress-free medium. The effective concentration inhibiting mycelial growth by 50% (EC50) value of homokaryotic and heterokaryotic strains was defined as NaCl concentration needed to reduce the mycelial growth by 50% in comparison with the NaCl stress-free condition.
All statistical analyses were performed using BellCurve for Excel (Social Survey Research Information, Tokyo, Japan). To compare the spore germination rate and colony diameter of homokaryotic and heterokaryotic strains at different concentrations of NaCl, we conducted one-way analyses of variance followed by Tukey's honestly significant difference test. For the test, the significance threshold was set to p < 0.05.
Spore germination and subsequent colony development were affected by the addition of NaCl (Table 1, Fig. 1). In the 0 mM treatment, 35.5% of spores germinated to various stages, including 1.9% of spores with protrusion, 31.8% of spores with swelling, and 1.7% of spores with germ tube (Table 1). In the presence of additional NaCl, 32.6% of spores germinated with 50 mM NaCl (4.8% of spores had protrusion, 25.8% of spores had swelling and 2.0% of spores had germ tube), 7.4% with 150 mM NaCl (2.2%, 5.2%, and 0.0%, respectively), and 0.5% with 300 mM NaCl (0.3%, 0.1%, and 0.0%, respectively). The total germination rate significantly decreased as the NaCl concentration increased. When compared with the germination rate in each germination stage, the rate in the swelling stage also significantly decreased with increasing concentration of NaCl, while the highest rate in the protrusion stage was recorded in 50 mM NaCl, and the rate in the germ tube stage was not significantly different between 0 and 50 mM NaCl (Table 1). When the number of colony-forming units was counted, more than 500 colonies per 5 plates were recorded in 0 mM and 50 mM NaCl, and 135 colonies per 5 plates with 150 mM NaCl, while only 3 colonies per 5 plates were observed in 300 mM NaCl. Multispore strains from 0, 50, and 150 mM NaCl included both homokaryons and heterokaryons (Supplementary Table S1 and Supplementary Table S2), but three strains from 300 mM NaCl (TUFC101244-Ho43, TUFC101244-Ho44, and TUFC101244-Ho45) were homokaryons. Thus, two heterokaryotic strains (TUFC100244-He46 and TUFC100244-He47) were generated by self-mating between these homokaryotic strains (Supplementary Table S2).
| NaCl concentration (mM) | Basidiospore germination rate (%) | Number of colony-forming units | |||
| Protrusion | Swelling | Germ tube | Total | ||
| 0 | 1.9 ± 0.3b | 31.8 ± 0.3a | 1.7 ± 0.2a | 35.5 ± 0.4a | 580 |
| 50 | 4.8 ± 0.3a | 25.8 ± 0.1b | 2.0 ± 0.1a | 32.6 ± 0.5b | 510 |
| 150 | 2.2 ± 0.6b | 5.2 ± 0.4c | 0.0 ± 0.0b | 7.4 ± 1.0c | 135 |
| 300 | 0.3 ± 0.1c | 0.1 ± 0.0d | 0.0 ± 0.0b | 0.5 ± 0.1d | 3 |
Each value of the spore germination rate represents the mean ± standard error (n = 5). Different letters in spore germination rate indicate significant differences among NaCl concentrations (Tukey's test, p < 0.05). The number of colony-forming units represents the total number from 5 plates.
Basidiospores showing morphological events such as protrusion and swelling (A), germ tube extension (B), and germling growth (C) in 50 mM NaCl treatment. Mycelial colony developments on (D) 0 mM NaCl treatment, (E) 50 mM NaCl treatment, (F) 150 mM NaCl treatment, and (G) 300 mM NaCl treatment. Bars: (A)-(C), 10 µm; (D)-(G), 1 cm.
Homokaryotic strains from the progeny of TUFC101244 showed a wide variation in their mycelial growth under NaCl stress-free or NaCl stress conditions (Supplementary Table S1). Some strains showed decreasing colony diameter and relative growth along with the concentration of NaCl, while other strains did not show such a monotonous decrease. Ten strains, TUFC101244-Ho8, Ho15, Ho25, Ho28, Ho30, Ho31, Ho34, Ho35, Ho36, and Ho44, grew better under NaCl stress conditions than under NaCl stress-free conditions. The EC50 value of most homokaryotic strains was more than 300 mM (Supplementary Table S1). When all replications from each 5D-MMN agar medium with or without NaCl were pooled, similar levels of colony diameter were observed in each NaCl treatment among the groups (Fig. 2A).
Boxes indicate median for each group (horizontal line in the box), and the 25th and 75th percentiles (lower and upper edges of the box). Ho-0, Ho-50, Ho-150 and Ho-300 for homokaryotic strain and He-0, He-50, He-150 and He-300 for heterokaryotic strain show the population of strains isolated from 1/5 diluted MMN agar medium containing 0 mM, 50 mM, 150 mM and 300 mM NaCl, respectively. All replications of homokaryotic and heterokaryotic strains in each treatment were pooled within each NaCl concentration.
The growth responses to NaCl in the heterokaryotic strains from the progeny of TUFC101244 also varied among the strains (Supplementary Table S2). Some heterokaryotic strains showed greater colony diameter than their parental wild-type heterokaryotic strain TUFC101244 under both NaCl stress-free and NaCl stress conditions. Similar to the homokaryotic strains, growth responses that decreased colony diameter and relative growth along with the concentration of NaCl were observed in some heterokaryotic strains, but not in others. Five strains, TUFC101244-He5, He15, He18, He19, and He23, grew better under NaCl stress conditions than under NaCl stress-free conditions. The EC50 values of most heterokaryotic strains were more than 300 mM, whereas those of a few strains were between 150 and 300 mM (Supplementary Table S2). When all replications from each 5D-MMN agar medium with or without NaCl were pooled, the group of heterokaryotic strains from 300 mM NaCl showed greater colony diameters than those of the heterokaryotic strains isolated from other treatments (Fig. 2B).
Germination of spores of arbuscular mycorrhizal (AM) fungi can be described by the following four steps: hydration, activation, germ tube emergence, and growth of mycelia (Tommerup, 1984). Several studies reported that spores of AM fungi normally hydrate and the germlings can grow at approximately 50 mM NaCl, whereas the germination process is eventually inhibited above a concentration of 100 mM NaCl (Hirrel 1981; Juniper & Abbott, 1993; Campagnac & Khasa, 2014). This is also the case for the spores of the EM fungus R. roseolus. As the concentration of NaCl increases, the osmotic potential decreases, and the ionic toxicity increases (Tester & Davenport, 2003). This study is the first to demonstrate the effect of NaCl on spore germination in EM fungi.
The genus Rhizopogon propagates primarily through spore dispersal and vegetative mycelial expansion (Kretzer, Dunham, Molina, & Spatafora, 2005; Grubisha, Bergemann, & Bruns, 2007; Abe, Tabuchi, Okuda, Matsumoto, & Nara, 2017). Therefore, successful germling growth followed by spore germination and subsequent vegetative growth can be of great significance for the development and maintenance of R. roseolus populations. In this study, spores of R. roseolus germinated, and the germlings grew and formed compatible fusions in the presence of additional NaCl. Furthermore, all multispore strains, including homokaryons and heterokaryons, grew under these NaCl conditions. These can be important traits for this fungus to propagate via both sexual reproduction and clonal vegetative growth in its habitats exposed to salinity. At higher concentrations of NaCl, however, almost all spores of this fungus did not germinate. Fungal spores function for survival of individuals, and therefore remain ungerminated when environmental conditions are unsuitable, but subsequently germinate when the conditions become amenable to growth (Cooke & Whipps, 1993). Several studies reported that ungerminated spores of AM fungi began to germinate after transferring from NaCl stress media to NaCl stress-free media (Hirrel, 1981). Further study is required whether ungerminated spores of R. roseolus subjected to higher concentrations of NaCl germinate after transferring to NaCl stress-free media.
The results of the mycelial growth tests showed that the EC50 value for NaCl in heterokaryons of R. roseolus was more than 300 mM, like those of Pisolithus tinctorius and Cenococcum geophilum, which are salinity-tolerant EM fungi (Matsuda et al., 2006; Matsuda, Yamakawa, Inaba, Obase, & Ito, 2017). In addition, the EC50 value in homokaryons was also more than 300 mM NaCl, suggesting that homokaryons of R. roseolus have similar levels of salinity tolerance to heterokaryons in terms of mycelial growth. After spores of this fungus dispersed, the homokaryons from germinating spores in soil must need to spread vegetatively. Given that homokaryons and heterokaryons of R. roseolus are both subject to similar salinity stresses during propagation in coastal sand dunes, it is reasonable to assume that this fungus has salinity tolerance in both stages.
Since the basidiospore formation of basidiomycetous EM fungi is preceded by meiosis in the basidium (Shimomura, Sawada, Aimi, Maekawa, & Matsumoto, 2012c), the germlings exhibit genetic diversity in a given physiological trait, such as indole-3-acetic acid production (Gay & Debaud, 1987), glutamate dehydrogenase activity (Wagner, Gay, & Debaud, 1988), nitrate reductase activity (Wagner, Gay, & Debaud, 1989), mycelial growth (Yamada et al., 2019), and EM colonization (Rosado, Kropp, & Piché, 1994). This is true for salinity tolerance of R. roseolus; the homokaryotic strains derived from the progeny of TUFC101244 exhibited a wide variation in response to salinity. In addition, some heterokaryotic strains generated by self-mating exhibited better salinity tolerance than their parental wild-type strain. This is meaningful for the survival and adaptability to changing salinity levels in coastal areas because genetic variation in a physiological trait in fungi generally reduces the risk of extinction and increases the population fitness in nature (Cooke & Whipps, 1993). The NaCl concentration in coastal soil generally ranges from 10 to 100 mM, but temporarily exceeds 300 mM due to typhoon conditions (Ishikawa et al., 1995). Interestingly, heterokaryotic strains from 300 mM NaCl exhibited vigorous mycelial growth compared to those from 0 mM NaCl. Several studies showed that soil microorganisms isolated from environmentally stressed conditions, such as salinity and high heavy metal concentration, have better potential for stress tolerance than those isolated from non-stressed conditions (Weissenhorn, Leyval, & Berthelin, 1993; Weissenhorn, Glashoff, Leyval, & Berthelin, 1994; Campagnac & Khasa, 2014). Our results imply that certain concentrations of NaCl could induce selection pressure for vigorous mycelial growth of heterokaryons in R. roseolus under NaCl conditions. Branco et al. (2015) reported that EM fungus Suillus brevipes from salinity area was able to tolerate salinity stress by developing differentiated genomic regions with a gene encoding for a membrane Na+/H+ antiporter, a gene associated with salinity stress tolerance.
In conclusion, this study demonstrates that R. roseolus has the potential to germinate, mate, and grow under NaCl stress conditions, and that genetic variation in salinity tolerance arise among strains in both homokaryotic and heterokaryotic stages. These could be important traits for this fungus to propagate and survival in its habitat exposed to salinity. Although R. roseolus can be considered as adapted species to salinity from this study, we recently observed differential responses to salinity in R. roseolus strains originated in inland forests, in comparison to seashore forests; the EC50 value of four heterokaryotic inland-strains was between 50 mM and 150 mM, whereas that of seven heterokaryotic seashore-strains was more than 300 mM NaCl (data not shown). To deeply understand salinity tolerance of this fungus, further research using many strains from different ecological regions is required. Additionally, as this fungus forms EM associations with the host plant roots (e.g., P. thunbergii) in coastal sand dunes, further work will be required to evaluate the salinity response of its EM association stage. This knowledge will lead to elucidation of the propagation strategies of this fungus in coastal sand dunes.
The authors declare no conflicts of interest.
This research was supported in part by the Matching Planner Program from the Japan Science and Technology Agency (JST) and by JSPS KAKENHI grant number 21K05710. We would like to thank Editage (www.editage.com) for English language editing.
