Canadian Journal of Forest Research
Phenology and cultivation of Suillus bovinus, an edible mycorrhizal fungus, in a Pinus massoniana plantation
Journal: Canadian Journal of Forest Research
Manuscript ID cjfr-2018-0405.R2
Manuscript Type: Article
Date Submitted by the 10-Mar-2019 Author:
Complete List of Authors: Sun, Xueguang; Guizhou University, Institute for Forest Resources & Environment of Guizhou Feng, Wanyan; Guizhou University Li, Min; Guizhou University, College of Forestry Shi, Jing; GuizhouDraft Minzu University Ding, Guijie; Guizhou University, College of Forestry
Suillus bovinus, sporocarp phenology, Pinus massoniana, Keyword: ectomycorrhiza, morphological features
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1 Phenology and cultivation of Suillus bovinus, an edible mycorrhizal fungus, in a
2 Pinus massoniana plantation
3 Xueguang Sun1, 2, *, Wanyan Feng1, 2, Min Li1, 2, Jing Shi3, Guijie Ding1, 2
4 1 Institute for Forest Resources & Environment of Guizhou, Guizhou University,
5 Guiyang, China; [email protected] (WF), [email protected] (ML),
6 [email protected] (GD)
7 2 College of Forestry, Guizhou University, Guiyang, China
8 3 Guizhou Minzu University, Guiyang, China; [email protected] (JS)
9 * Corresponding author:
10 E-mail: [email protected] (XS)Draft
11
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12 Abstract:
13 Edible mycorrhizal fungi (EMF) are a valuable forest resource, both in terms of their
14 ecological functions as a symbiont of host trees and the economic value of their edible
15 sporocarps. In this study, we characterized the morphological features of Suillus
16 bovinus, a dominant EMF associated with Pinus massoniana, and conducted a
17 three-year field survey to clarify the phenology of S. bovinus sporocarps. The
18 morphological features of S. bovinus were generally in accordance with those reported
19 by previous studies, and S. bovinus sporocarp production was largely influenced by
20 meteorological conditions and the intra-annual growth rhythm of P. massoniana. The
21 monthly fruiting pattern of sporocarpsDraft correlated with monthly variations in
22 temperature (P<0.01) and precipitation (P<0.05). Sporocarp productivity increased as
23 the temperature or precipitation increased and decreased as the temperature or
24 precipitation decreased. The correlation between P. massoniana growth stages and the
25 production of mature sporocarps was also significant (P<0.01): sporocarps were
26 mainly formed during the shoot elongation stage, shoot growth ceased stage, and
27 vegetative growth period. The sporocarp productivity data plus the economic
28 evaluation indicate that a considerable amount of S. bovinus sporocarps could be
29 produced in P. massoniana plantations which merit further commercial exploitation.
30 To facilitate commercial use, both the morphological features of the association
31 formed by S. bovinus and P. massoniana and the influences that determine S. bovinus
32 fruiting should be taken into consideration.
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33 Key words:
34 Suillus bovinus; sporocarp phenology; Pinus massoniana; ectomycorrhiza
35
Draft
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36 Introduction
37 Ectomycorrhizal fungi form mutually beneficial relationships with a series of tree
38 species, including pines, that efficiently improve the performance of the host trees and
39 contribute to forest health (Smith and Read 2008). Edible mycorrhizal fungi (EMF)
40 are ectomycorrhizal fungi that produce edible sporocarps. Many of these fungi are
41 considered ecologically and economically important for forestry development (Murat
42 et al. 2008): for example, Tuber spp., Boletus spp., Cantharellus spp., Tricholoma
43 matsutake, and Lactarius deliciosus are important non-wood forest products (NWFP)
44 worldwide (Kranabetter and Kroeger 2001; De Roman and Boa 2006; Martínez-Peña
45 et al. 2012; Tomao et al. 2017). In Draftsome regions, including Southwest China, the
46 income derived from selling these mushrooms can account for more than half of their
47 income for the local people. Indeed, in some cases, the economic value of
48 mushroom-based ecosystem services can be much higher than the economic profit
49 traditionally obtained from timber-oriented forestry (Palahí et al. 2009; Martínez de
50 Aragón et al. 2011).
51 The market for wild edible fungi is rapidly growing worldwide owing to
52 international free trade policies (Bonet et al. 2012). However, in addition to those
53 species that have already been successfully marketed, there are still a large number of
54 species (especially EMF) with high nutritional and gastronomic values and
55 considerable yields that could be commercially exploited. Although factors that
56 influence the production and distribution of several EMF, such as Tuber spp., Boletus
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57 edulis, and L. deliciosus, have been thoroughly investigated worldwide, other species
58 have received little attention. Understanding the mechanisms of sporocarp production
59 is vitally important for mushroom pickers and forest managers (Egli et al. 2010). As
60 with saprotrophic fungi, the duration of the EMF fruiting season and the frequency of
61 sporocarp emergence are affected by various environmental factors (Boddy et al.
62 2014; Alday et al. 2017; Karavani et al. 2018). Climatic factors, especially
63 temperature and precipitation, greatly influence fungal growth and ultimately
64 mushroom production (Bonet et al. 2012; Alday et al. 2017; Karavani et al. 2018).
65 This explains why previous studies have reported that sporocarp morphogenesis is
66 always correlated with climatic factors,Draft usually temperature and precipitation (Egli et
67 al. 2010). Mycorrhizal fungi mostly depend on photosynthetically fixed carbon
68 produced by the associated host trees to extend their vegetative mycelium in the soil,
69 and for the formation of mycorrhizas as well as sporocarps for sexual reproduction
70 (Högberg et al. 2008; Egli et al. 2010). Thus, the tree’s growth status affects the
71 growth of the EMF and, ultimately, sporocarp formation. Many studies have reported
72 that sporocarps of mycorrhizal fungi are more abundant in young stands with higher
73 growth rates than those of old stands (Bonet et al. 2008; Tahvanainen et al. 2016). A
74 thinning experiment conducted by Egli et al. (2010) indicated that annual tree growth
75 affects sporocarp production. However, the effects of intra-annual variations in host
76 tree growth are largely unknown. Although numerous variables influencing
77 mushroom production have been identified, there are few general conclusions that
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78 apply to all species. As the ecology of fungal species can vary considerably even
79 within the same genus, environmental factors can have different effects on different
80 fungal species (Tomao et al. 2017). Therefore, when considering the
81 commercialization of a specific EMF species, the factors influencing sporocarp
82 production need to be thoroughly investigated.
83 Studies of mycorrhizal effects and sporocarp productivity have generally been
84 considered separately. Unlike the characteristics of EMF sporocarps, the anatomical
85 features of the mycelium and its various specialized structures, especially
86 ectomycorrhizas, have mostly been – and often still are – ignored when sporocarps are
87 collected in the field (Buyck et al. Draft2018). The inner tissues of ectomycorrhizas, i.e.,
88 the Hartig net structure, are important for the intercellular symbiosis of mycorrhiza
89 (Yamada et al. 2001). It is believed that the inner tissue and outer tissue structures are
90 largely determined by the causal fungi and are stable within the combination of a
91 fungal species and a specific host species (Godbout and Fortin 1985; Agerer 1991).
92 Based on a morphological investigation of ectomycorrhizas, we may be able to
93 identify the EMF and, hence, predict the production of sporocarps in the wild.
94 Furthermore, when EMF have been applied to host seedlings for EMF cultivation
95 purposes, ectomycorrhizal structures are routinely used to validate mycorrhization
96 with a specific EMF. To do this, natural mycorrhizas also need to be compared with
97 those synthesized in vitro (Yamada et al. 2001).
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98 Pinus massoniana Lamb. is one of the most widely distributed native species in the
99 subtropical forests of China. Over the past decades an increasing amount of P.
100 massoniana has been planted in China owing to its high economic and ecological
101 values (Zhou 2001). Several EMF are known to form mutual symbiotic associations
102 with P. massoniana, including L. deliciosus, Cantharellus cibarius, and Suillus luteus
103 (Chen 1989). A survey of P. massoniana forest in the Guizhou district (2015–2017)
104 revealed that Suillus bovinus (L.: Fr.) Roussel is the dominant EMF species, and that
105 it has a fruiting season that can last for ten to eleven months annually (Sun et al.,
106 unpublished data). This EMF species was widely introduced worldwide and forms
107 ectomycorrhizal associations with DraftPinus trees (Fries and Sun 1992; Dahlberg and
108 Stenlid 1994). There is a long history of S. bovinus sporocarps being eaten by local
109 people, and in some districts, they are collected for sale (Tong 2006). However, there
110 is scarcely any information about the S. bovinus and P. massoniana symbiotic
111 relationship or the S. bovinus fruiting patterns. Many poisonous and edible
112 mushrooms have similar morphological features and, hence, poisoning incidents
113 frequently occur all around the world during mushroom fruiting season, especially in
114 Southwest China (Watling et al. 2005; Chen et al. 2014). Suillus granulatus (L.) Snell
115 has very similar morphological features to those of S. bovinus. Although S. granulatus
116 is considered to be an edible species, sporocarp consumption can cause
117 gastrointestinal toxic reactions (Volk 2004; Mao 2009; Greenwood 2011). To avoid
118 intoxication incidents, and for better exploitation and utilization of S. bovinus in P.
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119 massoniana forests, the morphological and anatomical features of both the sporocarps
120 and ectomycorrhizas need to be characterized. Furthermore, to market S. bovinus
121 sporocarps, the main factors that influence fruiting need to be identified to determine
122 sporocarp production regularity.
123 The aim of this study was to provide a foundation of knowledge for future studies
124 of P. massoniana mycorrhization with S. bovinus and for the mycosilviculture of its
125 edible sporocarps in the P. massoniana forests of China. A field survey and a
126 laboratory experiment were conducted to investigate: firstly, the characteristics of S.
127 bovinus associated with P. massoniana by studying the morphological features of
128 pure culture, sporocarps, and ectomycorrhiza;Draft secondly, and more importantly, the
129 phenology of S. bovinus fruiting in P. massoniana plantations.
130
131 Materials and methods
132 Sampling area description
133 Sporocarp collection, the investigation of fruiting regularity and ectomycorrhizal root
134 sampling were conducted in a pure stand of P. massoniana located in Longli County,
135 Guizhou Province, China (location: 107°00′37.0″ E, 26°28′00" N; altitude: 1180 m
136 asl). The forest covers an area of ca. 324 ha (800 acres), and the stand was 18 years
137 old at the start of this investigation. Furthermore, previous studies have reported that
138 sporocarps of mycorrhizal fungi are more abundant in young stands than in old stands
139 (Egli et al. 2010; Tahvanainen et al. 2016; Tomao et al. 2017).
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140 The basic physical and chemical properties of the top soil (0–20cm) were as
141 follows: pH 4.4, 21.46 g kg−1 of soil organic matter, 0.68 g kg−1 of total nitrogen, 62.2
142 mg kg−1 of available nitrogen, 0.11 g kg−1 of total phosphorus, 0.86 mg kg−1 of
143 available phosphorus, and 62.7 mg kg−1 of available potassium (Li et al. 2016). The
144 climate in this region is classified as a humid subtropical climate, i.e., humid with a
145 mean annual rainfall of 1100 mm and an average annual temperature of 14.8C with a
146 range from 4.8C to 23.5C.
147
148 Sampling and morphological analysis of S. bovinus sporocarps and
149 ectomycorrhizas Draft
150 Fresh sporocarp specimens for identification and in vitro cultivation were collected
151 in June. Macroscopic features (color of cap, stipe, pores, and flesh; shape, size, and
152 texture of both cap and stipe) were recorded based on fresh materials and field
153 photographs before transporting the fresh materials to the laboratory for further
154 observation and analysis. Sporocarps were either observed directly under a
155 stereomicroscope (M205FA, Leica Microsystems, Wetzlar, Germany) or sectioned by
156 hand, mounted in filtered water (sterilized using a 0.22 μm filter membrane) and
157 observed under a light microscope (DM3000, Leica Microsystems, Wetzlar,
158 Germany).
159 Fine roots connected to sporocarps via rhizomorphs or hyphae were carefully
160 traced and collected. After the ectomycorrhizas had been carefully washed with tap
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161 water, they were observed under a stereomicroscope (M205FA, Leica Microsystems,
162 Wetzlar, Germany). The shape, color, attachment, and surface texture were recorded.
163 After that, longitudinal and transversal cross-sections of at least 20 independent
164 ectomycorrhizas were obtained using a razor blade. The roots were cleared to make
165 them transparent (5% KOH solution, 90C for 2 h), acidified (1% HCl solution w/v,
166 10 min at room temperature), and then stained with 0.05% Trypan Blue dye (90C for
167 20 min). The stained sections were then mounted in glycerol and the microscopic
168 features were observed under a light microscope (DM3000, Leica Microsystems,
169 Wetzlar, Germany).
170 Draft
171 Isolation and identification of S. bovinus
172 Suillus bovinus sporocarps collected at the study site were used for the isolation of
173 dikaryotic mycelium in pure culture following the protocol described by Brundrett et
174 al. (1996). Immature sporocarps were first brushed free of adhering soil particles and
175 then fractured carefully to ensure that their internal surfaces were not contaminated by
176 dirt or handling. Insect-infected tissue, as indicated by the presence of bruising or the
177 formation of burrows, was discarded. A small amount of tissue from either the apex of
178 the stem or the cap just above the gills (approximately 2 mm3) was removed with fine
179 forceps that had been flame sterilized after immersion in 70% ethanol using an
180 alcohol lamp. The tissue was placed on Pachlewski medium (Pachlewski and
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181 Pachlewska 1974) in Petri dishes. Cultures were incubated at 25°C in the dark. In
182 total, five isolates were isolated from the five sporocarps.
183 Total DNA was isolated from fresh sporocarp tissue, hyphae of 2-week-old pure
184 culture and five to ten ectomycorrhizal tips using the CTAB method. Two primer
185 pairs, ITS1 (5-TCCGTAGGTGAACCTGCGC-3) and ITS4
186 (5-TCCTCCGCTTATTGATATGC-3) (White et al. 1990), and NL1
187 (5-GCATATCAATAAGCGGAGGAAAAG-3) and NL4
188 (5-GGTCCGTGTTTCAAGACGG-3) (Kurtzman and Robnett 1998), were used to
189 amplify the fungal rDNA internal transcribed spacer (ITS) and partial 28S region,
190 respectively. The PCR reactions wereDraft performed in a T100 Thermal Cycler (Bio-Rad,
191 Hercules, CA, USA). PCR was performed in 50-µl reaction volumes with an initial
192 denaturation at 95°C for 3 min, followed by 35 cycles of denaturation for 1 min at
193 95°C, annealing for 45 s at 55°C, and extension for 2 min at 72°C, and then a final
194 extension phase for 10 min at 72°C. PCR products were separated on 1.0% agarose
195 gel and then purified with an E.Z.N.A.® Gel extraction kit (Omega Bio-Tek, Inc.,
196 Norcross, GA, USA). Sequencing was carried out by Nanjing GenScript Corporation
197 (China). DNA sequences were edited and compared with the available sequences
198 from the National Center for Biotechnology Information (NCBI) using the basic local
199 alignment search tool (online BLAST), and were submitted to GenBank under
200 accession numbers MH681579, MH681581, and MK239979–MK239984.
201
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202 Synthesis of ectomycorrhizas between S. bovinus and P. massoniana roots
203 Pinus massoniana seeds (line: Huang No. 1) were collected from a first-generation
204 orchard of the Duyun Forestry Station, Duyun, Guizhou Province. The seeds were
205 surface sterilized with 0.5% KMnO4 solution and then rinsed three times in sterilized
206 water. After that, the seeds were left to germinate in damp vermiculite (sterilized by
207 autoclaving at 121°C for 1 h before use) at 25C. Approximately one-month-old P.
208 massoniana plantlets were transplanted into sterilized soil (autoclaved at 121°C for 1
209 h) collected from the surface of the forest floor of the P. massoniana forest field site.
210 One-month-old S. bovinus agar cultures were mashed up and then mixed thoroughly
211 with sterilized soil. The control plantsDraft were inoculated with sterilized cultures. The
212 progress of ectomycorrhizal synthesis was checked every month for six months.
213 When ectomycorrhizas started to form (from three months onward), the
214 morphological features of the ectomycorrhizas were recorded as described above.
215
216 Outbreak dynamics of S. bovinus sporocarps
217 Monthly surveys of sporocarp production for phenology analysis were carried out at
218 the study site from January 2015 until December 2017. At least one survey was
219 performed per month, and two to four surveys were performed per month from May
220 to September. All surveys were conducted during the day. For each survey, we
221 walked along a linear census route (ca. 1 km), recording all S. bovinus sporocarps
222 present within 2 m of the line on both sides of the line. Four independent walks were
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223 conducted each time. The sporocarps were collected by carefully picking the bottom
224 of the stalk while subjecting the forest floor to the least amount of trampling to
225 minimize damage to the belowground mycelium networks.
226 All the sporocarps observed at the time of each survey were collected and counted
227 regardless of their stage of development. However, to maximize commercial
228 productivity, immature sporocarps should be left to keep on growing. Thus, to better
229 estimate sporocarp productivity in terms of potential commercial productivity, we
230 used the number of sporocarps instead of weight to represent the production of
231 sporocarps. We also recorded the time required for S. bovinus to develop from the
232 button phase (approximately 2 mmDraft in height) to the fully opened phase. Based on
233 these data, we calculated monthly sporocarp production per acre.
234 The growth status of P. massoniana was also recorded according to the description
235 of Yu (1959). According to his study, the intra-annual growth of P. massoniana can
236 be divided into six stages, i.e., the quiescent stage (Qs), terminal bud germination
237 stage (Tbg), shoot elongation stage (Se), shoot growth ceased stage (Sgc), vegetative
238 growth period (Vgp), and terminal bud development stage (Tbd). The durations of
239 these stages were observed while collecting the sporocarps. Monthly climatic
240 variables (minimum, maximum, and mean temperatures and total precipitation) were
241 obtained for the 2015–2017 period from the Weather Bureau of Longli County.
242
243 Data analysis
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244 The results were analyzed using the statistical software SPSS 17.0.0 (Statistical
245 Product and Service Solutions, SPSS Inc., Chicago, IL, USA). Prior to the analyses,
246 all data were checked for normality and homogeneity of variance. Correlation analysis
247 was preformed using Spearman’s correlation analysis. Data are presented as means of
248 four replications.
249
250 Results
251 Identification and characteristics of S. bovinus in pure culture
252 We obtained five isolates of S. bovinus from fresh sporocarps. The isolates produced
253 white colonies that were elliptical inDraft shape (the reverse side of the colony changed to
254 yellow-brown or dark brown as the hyphae senesced) (Fig. 1a and 1b). The hyphae
255 were almost straight and light brown, seldom septate, and about 2–6 µm wide (Fig.
256 1c). No spores were produced in pure culture.
257 The molecular identification of the pure culture was carried out by amplifying and
258 sequencing the ITS1-5.8S rDNA-ITS2 region and the partial 28S region. The
259 sequences were deposited in GenBank under accession numbers MH681579 (partial
260 28S region) and MH681581 (ITS1-5.8S rDNA-ITS2 region). A BLAST search
261 revealed that both sequences shared 99% identity with S. bovinus sequences already
262 deposited in the database. We also obtained the same sequences using DNA extracted
263 from sporocarps and ectomycorrhizas. These sequences were also deposited in
264 GenBank under accession numbers MK239979–MK239984: MK239979 and
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265 MK239981 are the ITS and 28S sequences, respectively, of ectomycorrhizas
266 synthesized in the laboratory; MK239980 and MK239982 are the ITS and 28S
267 sequences, respectively, of sporocarp tissue; MK239984 and MK239983 are the ITS
268 and 28S sequences, respectively, of ectomycorrhizas collected in the wild.
269
270 Morphological characteristics of S. bovinus sporocarps
271 In general, the morphological features of the sporocarps corresponded to the type
272 description of S. bovinus (L. : Fr.). Roussel. We characterized Suillus bovinus as a
273 gregarious bolete with caps of 3 to 10 cm in diameter (the statistical results of 50
274 sporocarps, Fig. 1d). The colors ofDraft the cap vary from pale yellow to deep orange and,
275 in general, its flesh color varies from white to clay pink and does not change. The
276 tubes terminate in large compound pores (which are usually divided into two
277 compartments) (Fig. S1a) and have rod-like cheilocystidia (Fig. 1e). The sporocarp
278 stipe is club-shaped, 6 to 10 cm in diameter and 5 to 8 cm tall (the statistical results of
279 50 sporocarps), clay-colored with whitish flesh and a pink tinge near the base of the
280 stem; the stipe lacks a stem ring (Figs. 1d and S1b). The spore print is brown (Fig.
281 S1c). The basidiospores are subfusiform, smooth, and 8.24 ± 1.15 μm × 3.20 ± 0.33
282 μm in size (means from more than 60 randomly selected spores, ranging in length
283 from 5.63~10.70 μm, and in width from 2.55~3.90 μm) (Fig. 1f). Sporocarps of
284 Gomphidius roseus were regularly found near the sporocarps of S. bovinus (Fig. S2).
285
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286 Morphological characteristics of ectomycorrhizas formed between S. bovinus
287 and P. massoniana roots
288 The formation of an ectomycorrhizal association between the roots of P. massoniana
289 and S. bovinus was confirmed using the molecular method mentioned above. We
290 characterized the ectomycorrhizas formed by S. bovinus and roots of P. massoniana
291 as usually coralloid in shape and yellowish to yellowish brown in color (Fig. 1g and
292 1h). The surface of the ectomycorrhiza is smooth without any attachments, and
293 sometimes there are constrictions present on the branches. The mantle is composed of
294 5–8 layers of hyphae and is 38.64 ± 3.87 μm thick (means from 50 intersections of at
295 least 20 independent ectomycorrhizas;Draft range, 26.21~74.07 μm) (Fig. 1i and 1j).
296 Ectomycorrhizas synthesized in the laboratory had the same characteristics as those
297 collected in the forests (Fig. 1h). Ectomycorrhizas formed under controlled conditions
298 initially developed an expanded dichotomous branching shape (enlarged image in the
299 top-right of Fig. 1h) and then ramified into a coralloid shape in six months.
300
301 Suillus bovinus fruiting dynamics associated with temperature and precipitation
302 variations and annual growth periods of P. massoniana
303 Suillus bovinus developed from the button phase to the fully opened phase in about
304 2 weeks.
305 For the past 3 years, the annual variations of monthly mean temperature, maximum
306 temperature, and minimum temperature were similar, and the monthly production of
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307 the sporocarps showed similar trends (Fig. 2). Generally, the monthly fruiting pattern
308 coincided with monthly temperature variations. Sporocarp production increased as the
309 temperature rose and decreased as the temperature fell. Monthly variations in the
310 mean temperature, maximum temperature, and minimum temperature showed similar
311 trends over the three years. Fruiting appeared to require a minimum temperature of
312 5C given that no button phase or mature sporocarps were found when the
313 temperature was less than 5C. By contrast, the highest monthly production of
314 sporocarps always occurred in the month with the highest mean temperature or
315 maximum temperature (usually June or July).
316 Guizhou is located in the subtropicalDraft humid monsoon climate zone, with the main
317 precipitation falling between April and July (Fig. 3). Unlike temperature, there were
318 large variations in monthly precipitation over the three years. Generally, the monthly
319 fruiting pattern of S. bovinus sporocarps coincided with monthly variations in
320 precipitation. Furthermore, the highest monthly production of sporocarps usually
321 occurred in the month with the highest precipitation level (except in 2017). However,
322 the minimum level of precipitation required for fruiting could not be determined on
323 the basis of the 2015–2017 precipitation data given that sporocarp production was still
324 observed in the month with the least precipitation.
325 For the period 2015–2017, the durations of the plant phenology stages of P.
326 massoniana were as follows: Qs, December, January, and February; Tbg, March; Se,
327 April and May; Sgc, June; Vgp, July, August, and September; Tbd, October and
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328 November. Pinus massoniana growth affected sporocarp production (Fig. 4).
329 Sporocarps were mainly generated during the Se, Sgc and Vgp stages. The lowest
330 monthly sporocarp production levels occurred during the Qs period.
331 The correlation analysis showed that the mean temperature, the maximum
332 temperature, and the minimum temperature were all positively correlated with the
333 production of sporocarps (P<0.01) (Fig. 5). Precipitation was positively correlated
334 with the production of sporocarps at the 0.05 level. The correlation between P.
335 massoniana growth stages and the production of sporocarps was also significant
336 (P<0.01) (Fig. 5).
337 Given that the average weight ofDraft a mature sporocarp is about 28 g (means of 50
338 mature sporocarps), the annual production of sporocarps was approximately 14.6 kg
339 ha-1 for 2015, 17.8 kg ha-1 for 2016 and 21.0 kg ha-1 for 2017.
340
341 Discussion
342 EMF are important for forestry development because they can promote host tree
343 growth and produce edible sporocarps (Murat et al. 2008). Although S. bovinus is
344 commonly associated with P. massoniana in China, the exploitation and utilization of
345 this EMF has been hampered by gaps in our knowledge regarding the characteristics
346 of S. bovinus in pure culture, and those of the ectomycorrhiza and sporocarps, and of
347 the factors influencing sporocarp production.
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348 To compare the morphological features of ectomycorrhizas formed in the wild with
349 those synthesized in the laboratory, we isolated S. bovinus from fresh sporocarps to
350 obtain a pure culture, and the isolates were then identified by analyzing the sequences
351 of the ITS1-5.8S-ITS2 and 28S regions of the nuclear rDNA. The morphological
352 features of the pure culture described in this study are generally consistent with those
353 described by Tong (2006) and Zhang (2012), although they did not give detailed
354 information about the colony shape and color or the hyphal morphotypes. The
355 formation of an ectomycorrhizal association between the isolate and seedlings of P.
356 massoniana further confirmed that S. bovinus is a typical ectomycorrhizal fungus.
357 Mushroom poisoning is the mainDraft cause of mortality in food poisoning incidents in
358 China (Chen et al. 2014). Edible and poisonous mushrooms are frequently confused
359 owing to the limited information on distinguishing morphological features.
360 Furthermore, some edible species (especially species belonging to the Boletus or
361 Suillus genera) are easily confused owing to similarities in their appearance, which
362 could impede their commercialization (Watling et al. 2005). In this study, we
363 characterized the morphological features of the edible sporocarp of S. bovinus, which
364 should help collectors to distinguish this species from others. The morphological
365 features described in this study are generally consistent with those described by
366 Watling et al. (2005). Sporocarps of G. roseus were also regularly found near to
367 sporocarps of S. bovinus, which raises the possibility that P. massoniana may
368 establish a three-way relationship with these two fungal species. It would be
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369 intriguing to investigate the relationship between these two fungal species and P.
370 massoniana in the future, especially given that previous studies suggest that G. roseus
371 may play a parasitic role in this relationship (Olsson et al. 2000; Watling et al. 2005).
372 Ectomycorrhizal structures, such as the Hartig net, fungal sheath, rhizomorph, and
373 extraradical mycelium, are important for the maintenance and functions of symbiosis
374 (Yamada et al. 2001). The ectomycorrhizas formed by S. bovinus and P. massoniana
375 roots are usually coralloid in shape and have well-developed fungal sheaths and a
376 Hartig net. The ectomycorrhizas synthesized in the laboratory had the same
377 characteristics as those collected in the forest. The inner tissue and outer tissue
378 structures of a fungal species and aDraft specific host species are thought to be stable
379 (Godbout and Fortin 1985; Agerer 1991). However, a specific ectomycorrhizal fungus
380 forms ectomycorrhizas of different phenotypes with different host plants (Kinoshita et
381 al. 2018). The morphological features of the ectomycorrhizas that we observed in this
382 study were not entirely consistent with those reported by Yamada et al. (2001) and
383 Mleczko and Ronikier (2007). For example, they observed hyphae emanating from
384 the surface of ectomycorrhizas, whereas in our study we hardly found any emanating
385 hyphae. A possible reason for this discrepancy may be that they did not use P.
386 massoniana as the host plant. The morphological features associated with S. bovinus
387 could be used to identify the presence of an ectomycorrhizal association between S.
388 bovinus and P. massoniana and would help to predict the presence of S. bovinus
389 sporocarps in a P. massoniana forest during the non-fruiting period.
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390 Previous studies have reported that climatic variables (especially temperature and
391 precipitation) are key drivers of sporocarp production (Hernández-Rodríguez et al.
392 2015). The monthly fruiting pattern of S. bovinus sporocarps coincided with monthly
393 variations of temperature, with sporocarp production increasing as temperature rose
394 and decreasing as temperature declined. Similar results have been reported for B.
395 edulis and L. deliciosus growing in Pinus sylvestris forests (Martínez-Peña et al.
396 2012). In this study, almost no sporocarps were found when the temperature was less
397 than 5C, suggesting that 5C is the minimum temperature for S. bovinus sporocarp
398 production. Hernández-Rodríguez et al. (2015) also reported that B. edulis sporocarp
399 production had a minimum temperatureDraft limit. Sporocarp production is also strongly
400 related to mean monthly precipitation (Krebs et al. 2008; Taye et al. 2016). Generally,
401 the monthly fruiting pattern of S. bovinus sporocarps coincided with monthly
402 variations of precipitation, and the maximum monthly sporocarp production usually
403 occurred in the month with the highest precipitation level. However, we did not
404 identify a minimum limit that determines the production of sporocarps. Similarly,
405 Hernández-Rodríguez et al. (2015) concluded that temperature rather than
406 precipitation was the limiting factor for the production of B. edulis in Cistus ladanifer
407 scrublands. Overall, temperature seems to be more crucial than rainfall for explaining
408 sporocarp production (Martínez-Peña et al. 2012). The sampling area in this study is
409 located in the humid subtropical region of China (with precipitation of over 800 mm
410 annually and bell-like distribution without prolonged extreme drought periods), and,
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411 hence, precipitation could not be a limiting factor for fruiting even in the month with
412 the minimum precipitation. In contrast, due to the relatively high altitude (1180 m
413 asl), the minimum temperature in a year could be lower than 5C, at which
414 temperature most microbial activities are constrained.
415 However, some researchers have suggested that climatic conditions alone do not
416 completely explain sporocarp occurrence because the interactions between individual
417 meteorological variables and other ecosystem variables are quite complex (Krebs et
418 al. 2008; Egli et al. 2010; Egli 2011). The production of EMF sporocarps requires a
419 large input of organic nutrition. As symbiotic fungi, their growth mainly depends on
420 carbon supplements derived from theDraft photosynthetic activity of the host plant (Leake
421 et al. 2001; Högberg et al. 2008; Egli et al. 2010). Furthermore, high EMF sporocarp
422 yields are usually observed during the period when the host tree shows the greatest
423 growth (Bonet et al. 2012), which suggests that the amount of photosynthates
424 allocated to EMF mycelia determines the productivity of EMF sporocarps. In this
425 study, we found that the tree phenology of P. massoniana affects sporocarp
426 production, and that the button phase or mature sporocarps were mainly produced
427 during the Se, Sgc, and Vgp stages. During these periods, P. massoniana have high
428 levels of photosynthetic efficiency (Zeng et al. 1999), and it is possible that there are
429 considerable surplus photosynthates after self-consumption. As host species grow fast
430 and require more nutrients, mycorrhizal fungal species associated with their roots
431 receive more carbohydrates, which may enhance mushroom fruiting (Ortega-Martínez
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432 et al. 2011; Sato et al. 2012). The lowest monthly production of sporocarps occurred
433 during the Qs, which is likely to be at least partially because host growth has ceased
434 and the photosynthetic activity of the host plant is low. Factors that influence tree
435 growth and physiological processes such as photosynthetic activity and carbohydrate
436 balance are also likely to affect the associated mycorrhizal colonization and sporocarp
437 production (Egli et al. 2010). Thus, the effects of temperature and rainfall on
438 sporocarp production may be indirect effects of their direct influence on P.
439 massoniana growth.
440 To further investigate the economic importance of selling this edible sporocarp, we
441 estimated the annual production ofDraft S. bovinus sporocarps in this sampling area. Take
442 that of 2016 production levels (17.8 kg ha-1) for example, we estimated that the total
443 sporocarp production of the studied 324 ha area was about 5767 kg. The general
444 market price for S. bovinus sporocarps in this region is about 10 US dollars per kg,
445 therefore selling these edible mushrooms would bring in a total income of about
446 57,600 US dollars. This would be a considerable income for rural people in this area
447 and, hence, marketing of this low-labor-consumption non-wood forest product should
448 be encouraged.
449 In conclusion, we characterized the morphological features of S. bovinus associated
450 with P. massoniana roots and some variables that influence the production of S.
451 bovinus sporocarps. For the first time, we examined the features of pure culture,
452 ectomycorrhizas, and the sporocarp of a specific EMF species associated with P.
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453 massoniana. These results should provide a framework of knowledge for future P.
454 massoniana mycorrhization with S. bovinus as well as the mycosilviculture of its
455 edible sporocarps (Fig. 6). Although factors affecting sporocarp production have been
456 studied for many years, many questions related to fungal ecosystem functioning
457 remain opened with regard to forest management practices to increase mushroom
458 production. The maximal economic profit of sites with potentially high mushroom
459 yields could be achieved by regulating host plant growth using silviculture and forest
460 management techniques (Palahí et al. 2009; Bonet et al. 2012; Tomao et al. 2017).
461 However, the observed pattern of S. bovinus sporocarp occurrence may not
462 necessarily be the same as the patternDraft found for other species because different fungal
463 species have adopted different ecological strategies to survive and face interspecies
464 competition in complex ecosystems.
465
466 Acknowledgments
467 This research was supported by the National Natural Science Foundation of China
468 (31500090) (https://isisn.nsfc.gov.cn/egrantweb/, to XS), the Science and Technology
469 Foundation of Guizhou Province, China [2015]2039
470 (http://xmgl.gzst.gov.cn/lib/login.html;jsessionid=ADA97DD9CBF23832B47A5DB8
471 EB582FBF, to XS), the Guangxi Innovation-driven Development Project, China
472 (AA17204087-4) (http://portal.gxst.gov.cn/egrantweb/, to XS), the Project of
473 First-Class Disciplines Construction in Guizhou Province (NO.GNYL[2017] 007)
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474 (http://www.gzsjyt.gov.cn/index.html, to GD), the Research Foundation of Education
475 Bureau of Guizhou Province, China [2015]442
476 (http://211.149.166.192:811/Admin/Login?ReturnUrl=%2fAdmin, to XS), the
477 Scientific Research Foundation of Human Resource Ministry and Social Security of
478 Guizhou Province for Returned Scholars [2015]09 (http://ptpm.mohrss.gov.cn:8080/,
479 to XS) and the Scientific Research Foundation of Guizhou University (2014065)
480 (http://210.40.3.226/kjweb/, to XS). The funders had no role in study design, data
481 collection and analysis, decision to publish, or preparation of the manuscript. We
482 thank Dan Hu, Jingwei Feng, and Yuqian Chen at the College of Forestry, Guizhou
483 University, for assistance during fieldDraft work.
484
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623 Figure Captions 624
625 Fig. 1. Morphological features of pure culture, sporocarp, and ectomycorrhiza of
626 Suillus bovinus associated with Pinus massoniana. (a) Aerial view of a 2-week-old S.
627 bovinus culture on Pachlewski medium (scale bar = 1 cm) and (b) reverse view (scale
628 bar = 1 cm). (c) Compound microscopy image of transparent hyphae (scale bar = 50
629 μm). (d) Gregarious S. bovinus sporocarps. (e) Tufted rod-like cheilocystidia
630 (indicated by the arrowhead; scale bar = 100 μm). (f) Long elliptical basidiospores
631 (scale bar = 50 μm). (g) Coralloid ectomycorrhizas collected in a P. massoniana
632 forest associated with sporocarps of S. bovinus. The arrowhead indicates a
633 constriction (scale bar = 2 mm). TheDraft enlarged image (top-right) shows a constriction
634 (indicated by the arrowhead) on the ectomycorrhizal branch. (h) Ectomycorrhiza
635 synthesized in the laboratory. Arrowheads indicate constrictions (scale bar = 2 mm).
636 The enlarged image (top-right) shows the dichotomous branching that
637 ectomycorrhizas formed initially under controlled conditions. (i) Transversal
638 cross-section of a stained ectomycorrhiza with a black arrowhead indicating the
639 mantle; the white arrowhead in the enlargement indicates the Hartig net (scale bar =
640 100 μm). (j) Longitudinal cross-section of a stained ectomycorrhiza with a black
641 arrowhead indicating the mantle and a white arrowhead indicating the Hartig net
642 (scale bar = 100 μm).
643
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644 Fig. 2. Correlation between monthly fruiting of Suillus bovinus and monthly
645 variations in temperature from 2015 to 2017. Sporocarp production data are the means
646 of four replications. The dashed line indicates the minimum temperature (5C)
647 required for fruiting.
648
649 Fig. 3. Correlation between monthly fruiting of Suillus bovinus and monthly
650 variations in precipitation from 2015 to 2017. Sporocarp production data are the
651 means of four replications.
652
653 Fig. 4. Correlation between monthlyDraft fruiting of Suillus bovinus and Pinus massoniana
654 vegetative growth from 2015 to 2017. Qs, quiescent stage; Tbg, terminal bud
655 germination stage; Se, shoot elongation stage; Sgc, shoot growth ceased stage; Vgp,
656 vegetative growth period; Tbd, terminal bud development stage. The numbers 1–12
657 preceding the different growth stages indicate the months January–December,
658 respectively. Sporocarp production data are the means of four replications.
659
660 Fig. 5. Correlations between fruiting pattern and climatic and biological variables. *
661 indicates correlation is significant at the 0.05 level; ** indicates correlation is
662 significant at the 0.01 level (Spearman’s correlation). Sporocarp production data are
663 the means of four replications.
664
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665 Fig. 6. Diagram showing the framework required to achieve mycosilviculture of an
666 edible mycorrhizal fungus with a specific host. Prior to conducting mycosilvicultural
667 practices with a specific edible mycorrhizal fungus and one of its specific hosts, a
668 series of steps need to be undertaken. First, the identity of the fungus must be clarified
669 through morphological and/or molecular methods. Second, the pure culture of this
670 fungus should be isolated and verified by ectomycorrhizal synthesis with its host. The
671 morphological features of the ectomycorrhiza should also be investigated using roots
672 collected in the field or from the ectomycorrhizal synthesis experiment in the
673 laboratory. Finally, the most important step is to identify variables influencing
674 sporocarp yields. Draft
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Fig. 1. Morphological features of pure culture, sporocarp, and ectomycorrhiza of Suillus bovinus associated with Pinus massoniana. (a) Aerial view of a 2-week-old S. bovinus culture on Pachlewski medium (scale bar = 1 cm) and (b) reverse view (scale bar = 1 cm). (c) Compound microscopy image of transparent hyphae (scale bar = 50 μm). (d) Gregarious S. bovinus sporocarps. (e) Tufted rod-like cheilocystidia (indicated by the arrowhead; scale bar = 100 μm). (f) Long elliptical basidiospores (scale bar = 50 μm). (g) Coralloid ectomycorrhizas collected in a P. massoniana forest associated with sporocarps of S. bovinus. The arrowhead indicates a constriction (scale bar = 2 mm). The enlarged image (top-right) shows a constriction (indicated by the arrowhead) on the ectomycorrhizal branch. (h) Ectomycorrhiza synthesized in the laboratory. Arrowheads indicate constrictions (scale bar = 2 mm). The enlarged image (top-right) shows the dichotomous branching that ectomycorrhizas formed initially under controlled conditions. (i) Transversal cross-section of a stained ectomycorrhiza with a black arrowhead indicating the mantle; the white arrowhead in the enlargement indicates the Hartig net (scale bar = 100 μm). (j) Longitudinal cross-section of a stained ectomycorrhiza with a black arrowhead indicating the mantle and a white arrowhead indicating the Hartig net (scale bar = 100 μm).
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Fig. 2. Correlation between monthly fruiting of Suillus bovinus and monthly variations in temperature from 2015 to 2017. Sporocarp production data are the means of four replications. The dashed line indicates the minimum temperature (5C) required for fruiting.
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Fig. 3. Correlation between monthly fruiting of Suillus bovinus and monthly variations in precipitation from 2015 to 2017. Sporocarp production data are the means of four replications.
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Fig. 4. Correlation between monthly fruiting of Suillus bovinus and Pinus massoniana vegetative growth from 2015 to 2017. Qs, quiescent stage; Tbg, terminal bud germination stage; Se, shoot elongation stage; Sgc, shoot growth ceased stage; Vgp, vegetative growth period; Tbd, terminal bud development stage. The numbers 1–12 preceding the different growth stages indicate the months January–December, respectively. Sporocarp production data are the means of four replications.
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Fig. 5. Correlations between fruiting pattern and climatic and biological variables. * indicates correlation is significant at the 0.05 level; ** indicates correlation is significant at the 0.01 level (Spearman's correlation). Sporocarp production data are the means of four replications.
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Fig. 6. Diagram showing the framework required to achieve mycosilviculture of an edible mycorrhizal fungus with a specific host. Prior to conducting mycosilvicultural practices with a specific edible mycorrhizal fungus and one of its specific hosts, a series of steps need to be undertaken. First, the identity of the fungus must be clarified through morphological and/or molecular methods. Second, the pure culture of this fungus should be isolated and verified by ectomycorrhizal synthesis with its host. The morphological features of the ectomycorrhiza should also be investigated using roots collected in the field or from the ectomycorrhizal synthesis experiment in the laboratory. Finally, the most important step is to identify variables influencing sporocarp yields.
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