Draft - University of Toronto T-Space · 27 Morchella spp. (morel) is a prized edible and nutritional fungus and its fruit-body (ascocarp) ... 33 outdoor cultivation of morels has

Draft

Analyses of artificial morel soil bacterial community structure and mineral element contents in ascocarp and the

cultivated soil

Journal: Canadian Journal of Microbiology

Manuscript ID cjm-2018-0600.R2

Manuscript Type: Article

Date Submitted by the Author: 27-Apr-2019

Complete List of Authors: Zhang, Fusheng; College of Life Sciences, Sichuan University, Chengdu 610064, P.R. ChinaLong, Li; College of Life Sciences, Sichuan University, Chengdu 610064, P.R. ChinaZongyue, Hu; College of Life Sciences, Sichuan University, Chengdu 610064, P.R. ChinaXiaorui, Yu; College of Life Sciences, Sichuan University, Chengdu 610064, P.R. ChinaLiu, Qingya; College of Life Sciences, Sichuan University, Chengdu 610064, P.R. ChinaBao, Jinku; College of Life Sciences, Sichuan University, Chengdu 610064, P.R. ChinaLong, Zhangfu; College of Life Sciences, Sichuan University, Chengdu 610064, P.R. China,

Keyword: morel, high-throughput sequencing, soil bacterial community structure, bioconcentration factor

Is the invited manuscript for consideration in a Special

Issue? :Not applicable (regular submission)

https://mc06.manuscriptcentral.com/cjm-pubs

Canadian Journal of Microbiology

Draft

Manuscript

1

1 Analyses of artificial morel soil bacterial community structure and

2 mineral element contents in ascocarp and the cultivated soil

3 Fusheng Zhang1, Li Long1①, Zongyue Hu1, Xiaorui Yu1, Qingya Liu1, Jinku Bao1, Zhangfu Long1①

4 1. Key Laboratory of Bio-resources and Eco-environment (Ministry of Education), College of Life

5 Sciences, Sichuan University, Chengdu 610064, P.R. China

① The author contributed equally to the first author in this work

① Corresponding author E-mail address: [email protected]

Page 1 of 33



mailto:[email protected]

Draft

Manuscript

2

7 Abstract: This study explored the differences among different artificial morel cultivations and the

8 influential factors, including soil bacterial community structure, yield, and mineral element contents of

9 ascocarp and the cultivated soil. High-throughput sequencing results revealed that the dominant

10 bacterial phyla in all the samples, including Proteobacteria, Acidobacteria, Chloroflexi, Bacteroides and

11 Gemmatimonadetes, were found not only in morel soils (experimental group) but also in wheat soil

12 (control group), while the highest richness and diversity in the soil bacteria were observed during the

13 primordial differentiation stage. M6 group exhibited the highest yield (271.8g/m2) and had an

14 unexpectedly high proportion of Pseudomonas (25.30%) during the primordial differentiation stage,

15 which was 1.77 ～ 194.62 times more than the proportion of Pseudomonas in other samples.

16 Pseudomonas may influence the growth of morel. Mineral element contents of the varied soil groups

17 and ascocarp were determined using electro thermal digestion and inductively coupled plasma mass

18 spectrometry. The results revealed that morel had high enrichment effects on Phosphorus (P,

19 Bioconcentration factor = 16.83), Potassium (K, 2.18), Boron (B, 1.47), Zinc (Zn, 1.36), Copper (Cu, 1.15)

20 and Selenium (Se, 2.27). P levels were the highest followed by Se and K, and the mineral element

21 contents in ascocarp were positively correlated with the soil element contents.

22 Keywords: morel, high-throughput sequencing, soil bacterial community structure, bioconcentration

23 factor

24

Page 2 of 33



Draft

Manuscript

3

26 1. Introduction

27 Morchella spp. (morel) is a prized edible and nutritional fungus and its fruit-body (ascocarp) is rich in

28 polysaccharides, amino acids, vitamins, and other nutrients (Vieira et al. 2016; Su et al. 2013). Numerous

29 studies have demonstrated that the polysaccharides from morel extracts have great potential in medical

30 applications, such as remarkable anti-tumor, antioxidant, and immunity enhancement properties (Nitha et

31 al. 2007; Mau. 2004; Greve et al. 2010; Li et al. 2017).

32 The first indoor cultivation of morels was reported by Ower (Ower. 1982; Ower et al. 1986), and

33 outdoor cultivation of morels has been developed and practiced on a large scale in China in recent years.

34 Many studies have reported that morel mycelial growth and fruit-body formation are influenced by

35 numerous factors, including temperature, humidity, illumination, air, pH, and nutrition (Kalyoncu et al.

36 2009; Richard. 2006; Du et al. 2012). Considering that morel can only fruit in soil and the sowing process

37 needs to be covered by the soil, we speculate that certain substances in the soil may play a key role in

38 morel ascocarp formation. Microorganisms are essential components of soils which participate in the

39 transport and cycling of materials in the soils, in addition to engineering and maintaining soil structure and

40 fertility (Gans et al. 2005; Torsvik et al. 2002). Bacteria are an important component of soil

41 microorganisms and are closely related to other soil biota, which is essential for maintaining the stability

42 and balance of soil microbial community structure (Rousk et al. 2015; Liu et al. 2017; Lin et al. 2014).

43 Numerous studies have observed that there are close symbiotic relationships between plants and

44 rhizosphere soil microbes (Johansson et al. 2004; Nazir et al. 2017; Rudnick et al. 2015), with these

45 interactions also observed between fungi and bacteria (Warmink et al. 2011; Boersma et al. 2010). Pion et

46 al. (2013) in their study observed that when the mycelia of Morchella esculenta were co-cultured with

Page 3 of 33



Draft

Manuscript

4

47 Pseudomonas putida, the bacteria could spread around the mycelia and the bacterial secretions facilitated

48 mycelial growth (Pion et al. 2013). Studies have shown that Pseudomonas can stimulate the growth of

49 Agaricus bisporus mycelia, reduce the production of ethylene, and induce the formation of Agaricus

50 bispores primordia (Chen et al. 2013; Noble et al. 2009). However, little is known regarding whether the

51 formation of morel fruiting bodies is similar to Agaricus bisporus, and whether some soil bacteria or their

52 secondary metabolites could stimulate morel fruiting.

53 In a single farmland soil ecosystem, the content of elements in soil does not great fluctuate in a

54 short period of time under natural conditions. At present, there are few reports about the

55 bioconcentration of different mineral elements in soil by morel, and whether the mineral elements in soil

56 have an effect on the yields difference of different varieties of morel. Nevertheless, previous studies

57 conducted in our laboratory have observed that different elements significantly influence soil bacterial

58 community structure and morel yield (Liu et al. 2017; Li et al. 2013). Inorganic fertilizers are commonly

59 used in farmland management and every trace element has critical physiological functions that could

60 influence fungal growth rates and soil bacterial community structures (Li et al. 2013; Zhao et al. 2014;Liu

61 et al. 2017). Relevant studies also show that zinc and iron in soil trace elements are valuable nutrients to

62 promote the growth of morel(Liu et al. 2017; Liu et al. 2015). However, whether the levels of elements in

63 the soil influence the element content in fruiting bodies or growth rates of morels remains unclear.

64 Little is known to date about the key mechanism of morel ascocarp formation, which severely limits the

65 further development of the industry of Morchella. However, some studies have shown a close relationship

66 between soil habitats and morel ascocarp formation. Therefore, in the present study, we design an

67 experiment to investigate the role of soil in the growth of Morchella and its influence on yield.

Page 4 of 33



Draft

Manuscript

5

68

69 2. Materials and methods

70 2.1 Experimental strains

71 The experimental strains used in the present study were collected from Jinchuan County, Longzhou

72 mountain, located in Huili county, and the artificial morel base of Chongzhou. These locations are located

73 in Sichuan Province, P. R. China. The strains were identified by their ITS sequences, including Morchella

74 sp. (M2, GenBank accession No: MH100896), Morchella galilaea (M3, GenBank accession No:

75 MH100895), and Morchella sextelata (M6, GenBank accession No: MH137040). All strains were

76 preserved at 4 ˚C in the laboratory using PDA medium (200 g of potato, 20 g of glucose, 20 g of agar, and

77 1 L of ddH2O; natural pH value).

78

79 2.2 Analysis of high-throughput sequencing of soil bacterial community structure

80 2.2.1 Field experiment and soil samples collection

81 The field experiment site was in Chongzhou, Sichuan, P.R. China. Before cultivation, the field was

82 levelled and ploughed, 9 split plot was set up, the shape of each split plot was1.5m × 15m, and a drainage

83 ditch of 10cm between the two-compartments. Each experiment group selected the adjacent

84 3-compartment to cultivate the same kind of morel. The mycelia were reactivated using PDA media

85 (potato 200g/L, glucose 20g/L, agar 18g/L, nature pH value) and then were inoculated into the newly

86 sterile spawn media (70% cooked wheat, 30% vermiculite and soil sealing, sterilization 4 h under 121 oC).

87 When numerous sclerotia grew around the culture bottle, and the fungus turned yellow and brown, the

88 seeds were seeded into a ploughed field with 1.5 of these bottles per square meter. The seeds were sown in

Page 5 of 33



Draft

Manuscript

6

89 a line instead of being spread out and were then covered with soil (3 ～ 5 cm depth) to facilitate the

90 subsequent production of morels. After 10 days, nutrition bags (70% cooked wheat, 30% vermiculite) were

91 added into the soil to restore the mycelium. The soil was regularly irrigated with mist to maintain soil

92 moisture between 30%-40% under the sun-shade net.

93 Soil samples were collected on November 25, 2016 (the mycelium growth stage), January 8, 2017

94 (the primordial differentiation stage), and February 13, 2017 (the ascocarp growth stage) for the three

95 varieties at the artificial morel cultivation base in Chongzhou. Each experimental group selected

96 three sampling sites on each split, the surface soil and shallow soil with depth of 5 cm were

97 respectively collected by tweezers and about 1 g soil put into sterile plastic bags，a total of

98 about 18 g of soil was sufficiently mixed uniform as soil samples(Sig.1).Soil samples of M2

99 strain from the three growth phases were respectively marked with M2-1, M2-2 and M2-3. M3 and M6

100 strain samples were labelled similarly. The wheat field soil (non-morel cultivation，W) was selected as the

101 control and labelled W1, W2, and W3 at the corresponding stages. The collected soil samples were

102 encapsulated with sterile polyethylene plastic bags, preserved at 4°C, and DNA was extracted within 24 h.

103

104 2.2.2 Soil genomic DNA extraction

105 DNA was extracted from the fresh soil samples using the Ezup column soil DNA extraction kit (Sangon

106 Biotech, Shanghai, China), according to the manufacturer’s instructions. The universal primers 515F

107 (5’-gtgccagcmgccgcggtaa-3’) and 909R (5’-ccccgycaattcmtttragt-3’) with a 12-nt unique barcode were

108 used to amplify the V4 hyper variable regions of the 16S rRNA genes for pyrosequencing using a MiSeq

109 sequencer (Caporaso et al. 2011; Caporaso et al. 2012). Each sample was amplified in a 25 μL reaction

Page 6 of 33



Draft

Manuscript

7

110 according the protocol reported in the study by Tamaki et al. (2011). Two PCR reactions were conducted

111 for each sample and their products combined and subjected to electrophoresis using a 1.5% agarose gel.

112 The bands with the correct sizes were separately excised and were purified using AXYGEN gel recovery

113 kit (Sangon Biotech, Shanghai, China). The products were quantified using Nanodrop 2000, and the

114 samples were pooled together in equal molar amounts.

115

116 2.2.3 High-throughput sequencing and data analysis

117 The Sequencing libraries were prepared using Illumina's TruSeq Nano DNA LT Library Prep Kit

118 (Illumina, Shanghai, China) and sequenced on the Illumina MiSeq sequencing platform. Before

119 on-machine sequencing, the library was checked on an Agilent Bioanalyzer using the Agilent High

120 Sensitivity DNA Kit (Illumina, Shanghai, China). The sequencing libraries were qualified on the machine

121 after gradient dilution based on the 30% of final DNA amount, , and denatured into single chains by 0.1N

122 NaOH on-machine sequencing. The 2 × 300 bp double-stranded sequencing was performed using a MiSeq

123 sequencer with MiSeq Reagent Kit V3 (600 cycles) (Illumina, Shanghai, China), according to

124 manufacturer’s instructions.

125 Paired-end sequencing of the community DNA fragments was performed on the Illumina MiSeq platform.

126 To integrate the original two-terminal sequencing data, the two-terminal sequence in FASTQ format was

127 screened using the sliding window method one by one (the average mass of the window base ≥ Q20; the

128 length of the sequence after truncation ≥ 150bp and without Ambiguous base N). Subsequently, the

129 double-stranded sequence that passed the quality screening was paired with overlapping bases using

130 FLASH software (v1.2.7, http://ccb.jhu.edu/software/FLASH/) (Magoc et al. 2011) (Read 1 and Read 2.

Page 7 of 33



Draft

Manuscript

8

131 The overlapping length of two sequences is ≥ 10bp, and base mismatch is not allowed). Finally, all the

132 sequence reads were trimmed and assigned to each sample based on their barcodes. QIIME software

133 (Quantitative Insights into Microbial Ecology, v1.8.0, http://qiime.org/) was used to identify the query

134 sequences (Caporaso et al. 2010). USEARCH (v5.2.236, http://www.drive5.com/usearch/) was invoked

135 using QIIME software (v1.8.0, http://qiime.org/) to check and delete the chimera sequences. High quality

136 sequences (length > 150bp, without ambiguous base 'N' and an average base quality score > 30) were used

137 for downstream analysis.

138 Most 16S rRNA gene-based studies on the diversity of bacterial structures usually use 97% sequence

139 similarity as Operational Taxonomic Units (OUT) to divide the threshold (Edgar et al. 2011). We

140 conducted alpha-diversity (rarefaction curves, Alpha-diversity index calculation, and Taxonomy

141 composition analysis) and beta-diversity (PCA) analyses for the observed species. Sequences obtained

142 from the present study were submitted in the NCBI Sequence Read Archive (SRA) with accession number

143 SRP132138.

144

145 2.3 Mineral elements determination of the morel ascocarp and soil

146 When the ascocarp began to mature (in early March), the M2, M3, and M6 morels were collected

147 until there was no more fruiting on the soil (in the end of March), and the yields were calculated separately.

148 Three randomly selected ascocarp varieties and 12 soil samples were processed using electrothermal

149 digestion, Samples were dried at 55 °C for 24 h and homogenized using an agate pestle. Then, a 0.1 g

150 sample was placed into a graphite crucible and digested as described in literature (Liu et al. 2012). The

151 residue was dissolved with 0.2% (w/w) HNO3 and diluted up to 10 mLThe digestion solutions were

Page 8 of 33



Draft

Manuscript

9

152 assayed to determine the concentrations of 11 mineral elements (calcium, ferric, boron, copper, selenium,

153 potassium, magnesium, manganese, phosphorus, zinc and sodium) using inductively coupled plasma mass

154 spectrometry (ICP-MS, Shimadzu, Japan), according to in the manufacturer’s instructions.

155 The bioconcentration factor (BCF) was calculated as the ratio of the content of mineral element in the

156 samples to that in the soil. When a BCF ≤ 1, it indicates that the morel can only absorb but not accumulate

157 mineral element ; when a BCF > 1, it shows that morel can accumulate mineral element(Liu et al. 2009)

158 The mineral element concentrations and yields of each morel were analyzed using SPSS.21.

159

160 3. Results

161 3.1 Experimental morel species

162 Following the observation of the morphology of the three kinds of morel (Fig. 1a), it was observed that

163 there were major differences in their appearance, particularly with regard to ascocarp color and shape.

164 When pileus color was compared, M2 was flesh colored, M3 was yellow, and M6 was gray brown. With

165 regard to stem color, M3 stems had a light yellow color, while those of M2 and M6 were beige. In addition,

166 M3 pileus had cylindrical shape, while pileus in M2 and M6 were conical.

167 Sclerotia formation in the three morel strains after seven days inoculation in PDA medium is shown in

168 Fig. 1b. M2 did not form a sclerotium structure, and the sclerotium of M3 was concentrated near the

169 inoculation block, while the sclerotium of M6 was more and evenly dispersed on the surface of the entire

170 medium.

171

172 3.2 OTU classification and Alpha-diversity index analysis

Page 9 of 33



Draft

Manuscript

10

173 According to the mi-seq platform sequencing results, 351,303 valid sequences of the 16S rRNA genes

174 were generated from 12 samples and 27,684 OTUs were obtained. The sequence information of the

175 samples and the calculated microbial diversity indices are listed in Table 1. The rarefaction analysis was

176 used to standardize and compare the observed taxa on richness between samples, and to identify whether

177 the samples were unequally sampled (Zhang et al. 2016). The rarefaction curves (Sig. 2) of the 12 samples

178 gradually exhibited a gentle trend, which implied that the sequenced depths in the current study were

179 adequate to reflect the diversity of the samples.

180 The numbers OTUs, Ace, highest species richness (Chao 1), the Shannon-Weaver Index, and Simpson

181 Index at 3% cut-off were similar did not exhibit changes among the three stages of M2, but exhibited

182 changes in the M3-2, M6-2, and W2, among their other two stages. M3, M6, and W were significantly

183 more enriched in the primordial differentiation stage than in the mycelial growth stage and the ascocarp

184 growth stage. Among the valid sequences, M6-2 was the largest among all groups, and its valid sequences

185 were 3.55 and 4.05-fold greater than those in M6-1 and M6-3, respectively, and were 1.44 to 3.78-fold

186 greater than those in the other samples However, the OTUs of M6-2 were only 1.26 and 1.34-fold more

187 than those of M6-1 and M6-2, respectively, while they were only 0.93 and 1.34-fold more than in the other

188 samples. This could imply that the quantities of some microorganisms rapidly increase in the primordial

189 stage of M6, which results in the highest sequence and OTU numbers.

190

191 3.3 Bacterial community structure at phylum level

192 Bacterial community structure was influenced by the different stage and strains (Fig. 2). Eight dominant

193 bacterial phyla, including Proteobacteria, Acidobacteria, Chloroflexi, Bacteroides, Gemmatimonadetes,

Page 10 of 33



https://www.sciencedirect.com/science/article/pii/S0304389415300297#tbl0005

https://www.sciencedirect.com/science/article/pii/S0304389415300297#fig0010

Draft

Manuscript

11

194 Planctomycetes, Actinomyces and Nitrospira, were observed in all groups, and their percentages ranged

195 from 71.20% to 94.92% (Table 2). The bacterial percentage was similar across all samples, ranging from

196 90.22% to 94.92%, except for in the M6-3 samples (which were only 71.20%). Proteobacteria and

197 Acidobacteria were the most dominant and the second most dominant bacterial phyla in most samples,

198 respectively. The proportions of the two bacterial phyla ranged from 43.21% (M6-3) to 61.24% (M6-1).

199 When the bacterial community structures in the four groups were compared at similar stages, the

200 proportion of Proteobacteria in M6-1 was the highest (49.84%), and was 1.15 ～ 1.79 times those

201 proportions in M2-1, M3-1 and W1. The proportion of Bacteroidetes in M6-2 (25.27%) was the highest

202 and was 3.11 ～ 3.42 times greater than the proportions of Bacteroidetes in M2-2, M3-2, and W2. In

203 addition, the proportion of Acidobacteria in M2-3 (20.62%) was the highest and was 1.10～1.69 times

204 more than those proportions of Acidobacteria in M3-3, M6-3, and W3. At different growth stages,

205 dominant bacterial phyla were varied and were distributed across different experimental samples.

206 When the three stages in each group were compared, it was found that the bacteria percentages increased

207 from the mycelia growth stage to the primordial differentiation stage, and decreased from the primordial

208 differentiation stage to the fruiting body growth stage (except in the M2 group). From the first stage to the

209 second stage, Proteobacteria exhibited an increasing trend in M2 (6.19%), M3 (52.76%) and W (12.18%),

210 but decreased in M6 (14.73%). Acidobacteria (16.69%～24.78%), Chloroflexi (10.79%～40.33%), and

211 Gemmatimonadetes (1.43%～38.29%) all decreased while Actinobacteria (37.84%～93.13%) increased in

212 all groups, except in M6 (decreased 24.21%). Bacteroidetes (28.50%～162.96%) exhibited an increasing

213 trend in all groups except in M3 (decreased 2.52%); Nitrospirae (44.89%～53.90%) showed an upward

214 trend in all groups except in M2 (decreased 59.84%). From the primordial differentiation stage to the

Page 11 of 33



Draft

Manuscript

12

215 ascocarp growth stage, the Proteobanteria (12.12% ～ 27.05%) and Actinobacteria (37.84% ～ 93.13%)

216 decreased while the Chloroflexi (2.67%～49.53%) and Gemmatimonadetes (1.60%～78.70%) increased in

217 all groups. Acidobacteria (2.03% ～66.45%) exhibited an increasing trend in all groups except in the M3

218 (decreased 26.27%). In addition, Bacteroides (1.65%～78.79%) exhibited an increasing trend in all groups

219 except in M6 (decreased 73.15%). There were minimal differences in the percentages of dominant

220 bacterial phyla in each of the groups, and the changes in each dominant bacterium were in the dynamic

221 states.

222

223 3.4 Bacterial community structure at genus level

224 The dominant genera were Pseudomonas, Pedobacter, Candidatus Solibacter, Rhodopseudomonas,

225 Geobacter, and Planctomyces (Table 3). Pseudomonas, Rhodoplanes, and Geobacter belong to

226 Proteobacteria. Candidatus Solibacter and Planctomyces belong to Acidobacteria and the Planctomycetes,

227 respectively. The proportion of Pseudomonas and Pedobacter varied considerably in different groups.

228 Through a heatmap (Fig. 3) analysis, we observed that the dominant genus varied greatly among different

229 samples, and the differences in the same stage in different groups were substantial.

230 The varying trends in dominant bacteria genera were analyzed from the mycelium growth stage to the

231 primordial differentiation stage. Pedobacter increased (80.43%～2420.00%) while Candidatus Solibacter

232 decreased (28.96%～38.52%) in all groups. The levels of Pseudomonas increased in M2 (884.61%) and

233 M3 (703.70%) but decreased in M6 (84.11%) and W (25.43%). In addition, Rhodoplanes decreased in M2

234 (18.03%) but exhibited a decrease in the other groups (8.60% ～ 29.76%). Both Planctomyces and

235 Geobacter exhibited an upward trend in M2 (21.88% and 6.48%) and M6 (188.46% and 33.33%), while

Page 12 of 33



Draft

Manuscript

13

236 they showed a downward trend in M3 (44.88% and 53.13%) and W (31.81% and 68.23%).

237 A comparison of the varying trends in the dominant bacteria genera from the primordial differentiation

238 stage to the fruiting body stage, Candidatus solibacter (18.25%～88.64%) revealed an increasing trend,

239 while Pseudomonas (64.98%～98.78%) and Pedobacter (28.90%～91.72%) showed decreasing trends in

240 all groups. Rhodoplanes and Geobacter showed a decreasing trend in M2 (61.00% and 33.91%) and M3

241 (1.87% and 38.33%), and increasing trends in M6 (2.75% and 36.11%) and W (52.48% and 176.92%).

242 Planctomyces exhibited an increase in W (73.33%), decreases in M2 (59.83%) and M6 (24.00%), and

243 remained stable in M3. The percentages of the dominant genuses in different experimental groups were in

244 dynamic states.

245

246 3.5 Principal component analysis

247 According to the map (Fig. 4), the first axis explained 53.11% of the cumulative percentage variance

248 of the species and 22.16% of the cumulative percentage variance was explained by the second axis.

249 Therefore, 75.27% variance in the species could be explained by the two axes. The results of the PCA

250 analysis suggested that there were differences in the bacterial communities in M6-1, M6-3, W1, and W2,

251 while the bacterial communities were similar among the respective three periods in M2 and M3. In

252 addition, the bacterial communities among the periods in M6 were fairly different. There were minimal

253 differences among M6-2, M2, and M3. However, the difference between M6-1 and M6-3 was considerable

254 compared with the other experimental groups, and the differences between M6-1 and M6-3 were very

255 significant. The bacterial communities in W1, W2, and W3 were distinctly different, but the difference was

256 not much greater when compared with the bacterial communities in M6. The differences in bacterial

Page 13 of 33



Draft

Manuscript

14

257 communities among W3, M2, and M3 were minimal, and there was a distinct difference between bacterial

258 communities of W1 and W2.

259

260 3.6 Mineral elements concentrations in morel ascocarp and soil

261 According to the data (Table 4), the concentrations of mineral elements in the ascocarp were proportional

262 to the concentrations in soil, with the higher contents in soil corresponding with higher concentrations in

263 the ascocarp, and lower concentrations in the soil corresponding with generally lower concentrations in the

264 ascocarp. The order of the mineral element contents in the fruiting body were as follows: Potassium (K) >

265 Phosphorus (P) > Calcium (Ca) > Magnesium (Mg) > Ferric (Fe) > Sodium (Na) > Manganese (Mn) >

266 Zinc (Zn) > Boron (B) > Copper (Cu) > Selenium (Se). The value of BCF was greater than one and the

267 fruiting body had a high enrichment effect on this kind of metal. Therefore, among the 11 mineral elements,

268 P, K, B, Zn, Cu, and Se had a high enrichment effect on the fruiting bodies. The BCF of P was the highest

269 and averaged up to 16.83, which was 6.08–14.6 times that of the other highly adsorptive metals. In

270 comparing the contents among the three groups, the nine mineral element contents in M3 ascacorps were

271 the highest (except for Fe and B) than were M2 and M6, the mineral elements BCF also were the highest

272 (except P, Fe, Na, and Mn).

273 The relative yields of artificial morels demonstrated the following trend: M6 > M3 > M2 (Fig 5). The

274 yield of M6 was the highest, up to 271.8g/m2, 6.7 times that of M2 and 3.9 times that of M3.

275

276 4. Discussion

277 Large-scale cultivation of morels in China is usually field based. The field cultivation successfully

Page 14 of 33



Draft

Manuscript

15

278 owes to simulating the habitat conditions of wild morel. Consequently, morel yields were greatly

279 influenced by numerous factors, including cultivars, natural habitats, soil properties, and soil microbes.

280 Numerous studies have reported the most suitable morel cultivation conditions (Kalyoncu, et al. 2009;

281 Richard et al. 2006; Obst et al. 2000). However, we still know little about the mechanism of morel

282 development from primordial differentiation to the fruiting body. Soil may play a critical role in the

283 process. The relationships between nutrient elements and morel yield have been explored by numerous

284 reports (Liu et al. 2017; Li et al. 2013; Liu et al. 2015), but only a few reports have specifically examined

285 what kinds of microbes and nutrient elements influence morel fruiting.

286 High-throughput sequencing results showed that the bacteria in artificial morel cultivation soil

287 underwent dynamic changes. According to the changes in bacterial community structure in each group, the

288 richness and diversity of bacterial communities during the primordial differentiation stage were almost the

289 highest in all groups, which could be due to the addition of secondary nutrition bags to the soil, which

290 supplies more energy sources for the growth of microbes. The comparison of bacteria in morel soil and

291 wheat soil revealed that there was no genus that was observed exclusively in morel soil and not in the

292 wheat field, which indicated that the bacteria that stimulated morel development were not specific. It may

293 also explain why morel species may grow in soils that have never been planted with morel, and may fruit

294 after sowing. Analysis of the dominant bacteria, Rhodoplanes and Geobacter as the dominant genus, which

295 belong to Proteobacteria, reveals that they could exploit organic compounds as carbon sources for electron

296 transfer, which is critical for the transformation of material (Bond et al. 2003; Childers et al. 2002).

297 Acidobacteria play a critical role in the maintenance of soil pH. Therefore, changes in their proportions

298 could also influence the growth of other bacteria (Long et al. 2010; Jones et al. 2009). Nitrobacter and

Page 15 of 33



Draft

Manuscript

16

299 Carbendazim have been reported to be the dominant bacterial groups in wastewater treatment systems,

300 which play a critical role in the degradation of organic chlorides and of ammonia nitrogen (An et al. 2013;

301 Nelson et al. 2011). The bacteria played critical roles in the maintenance of soil physical and chemical

302 properties, and provided a suitable environment for the growth of morels.

303 Comparing the bacterial community structure of M2, M3, and M6 reveals that the bacterial community

304 structure of the three stages in M6 were highly different from the others, especially in the proportions of

305 Pseudomonas. Pseudomonas was the predominant genus in M6-1 and accounted for the largest proportion

306 of bacteria (25.30%). It was 97.3 times more than was that in M2-1 (0.26%) and 93.7 times more than was

307 that in M3-1 (0.27%). In addition, the yield in M6 (271.6g/m2) was 6.7 times that in M2 (40.5g/m2) and 3.9

308 times that in M3 (69.6g/m2). According to the analysis of the correlation between yield and the proportion

309 of Pseudomonas, the high Pseudomonas bacteria contents could have contributed to the improvement in

310 ascocarp yield. Experiments by Pion et al. (2013) also demonstrated that morel and Pseudomonas putida

311 co-culture were beneficial for both. This is also consistent with the findings of our previous study (Liu et al.

312 2017) where Pseudomonas could promote the formation and growth of morel ascocarp. Although the

313 content of Pseudomonas increased in M2-2 (884.61%) and M3-2 (703.70%), and decreased in M6-2

314 (84.11%), the contents in M6-2 were twice more than were the contents in M2-2 and M3-2. This could be

315 because of the growth rates, and the capacity to form sclerotia in M6 was higher than was those in M2 and

316 M3. Volk (1989) observed that sclerotia formation was related to fruiting body formation. Ower (Ower,

317 1982; Ower et al. 1986) also established that sclerotia were crucial for fruit-body formation. The number

318 of sclerotia may also affect Pseudomonas content, which is why M. sextelata is widely used in morel field

319 cultivation as a cultivar. The proportions of Pseudomonas in W1 and W2 were also the highest (more than

Page 16 of 33



Draft

Manuscript

17

320 10%), and some studies have found that morel and wheat intercropping result in higher morel yields and

321 wheat secretions could promote the growth of morel (Fan et al. 2013). More growth may also be induced

322 by a similar interdependent relationship between wheat and Pseudomonas, with Pseudomonas influencing

323 morel yield.

324 The present study also revealed the bioconcentration capacity of morel, and the relationship between

325 ascocarp and soil mineral element contents. We found that the contents of trace elements in the ascocarp

326 were positively correlated with the mineral element contents in soil by analyzing data from the three

327 groups. The contents of trace elements in soil influenced the contents of trace elements in the fruiting

328 bodies (Gast et al. 1988; Ma et al. 2013). After fires, which provide abundant trace elements via the ashes

329 of trees and grasses, there are typically high yield morel harvests (Li et al. 2017; Mcfarlane et al. 2005).

330 Previous studies conducted by our laboratory also demonstrated that Zn-Fe elements greatly influenced

331 morel yield (Liu et al. 2017). K contents were the highest in the three varieties, and P contents had the

332 highest value of BCF with an average of up to 16.83. The two elements are both critical for the normal

333 development of morel. Fe is an essential element in morel, with the average value of 696.06 mg/kg that is

334 similar to that in Craterellus odoratus (Nasreddine and Parent, 2002). However, the average BCF of Fe

335 (0.02) was the lowest, which was probably due to its nonionic states in the soil. In summary, when the

336 contents of mineral elements in soil are relatively low, it affects normal morel growth. However, mineral

337 element contents in the soil in the three experimental groups exhibited minimal differences. However, the

338 fruit body yields exhibited significant differences. Therefore, mineral elements in the soil were not factors

339 influencing morel fruiting.

340 Although the role of Pseudomonas species in the differentiation of primordia and the mechanism of

Page 17 of 33



Draft

Manuscript

18

341 symbiosis between Pseudomonas and morel are still not clear, our laboratory isolated nine strains of

342 Pseudomonas that could be cultured from soils of the three M6 stages. Further studies are required to

343 investigate how the Pseudomonas strains influence morel cultivation.

344

345 Acknowledgements

346 The work was financially supported by Sichuan province scientific support program (2016KZ0006),

347 Liangshan state science and agricultural technology innovation program（16NYC0041, 17CXY0015) and

348 Guangyuan Lizhou District research program (13H0923). We also thank Sichuan Wanan Agrobiotech

349 Development Co., LTD for kindly providing morchella spawn and employees joined the field experiment

350 in this study.

351

352 Reference

353 An, L., Chu, Y., Wang, X., Ren, L., Yu, J., Liu, X., et al. 2013. A pyrosequencing-based metagenomic

354 study of methane-producing microbial community in solid-state biogas reactor. Biotechnology for

355 Biofuels. 6(1): 3-3.356 Boersma, FGH., Otten, R., Warmink, JA., Nazir, R., van, Elsas, JD. 2010. Selection of Variovorax 357 paradoxus-like bacteria in the mycosphere and the role of fungal-released compounds. Soil Biol. Bioch358 em. 42: 2137-2145. doi: 10. 1016/j. soilbio. 2010. 08. 009.

359 Bond, DR., Lovley, DR. 2003. Electricity production by Geobacter sulfurreducens attached to electrodes.

360 Applied and Environmental Microbiology. 69(3): 1548-1555.

361 Caporaso, JG., Lauber, CL., Walters, WA., Berg-Lyons, D., Huntley, J., Fierer, N., et al. 2012.

362 Ultra-high-throughput microbial community analysis on the Illumina HiSeq and MiSeq platforms.

363 ISME J. 6(8):1621– 1624. https://doi.org/10.1038/ismej.2012.8 PMID: 22402401 24.

364 Caporaso, JG., Lauber, CL., Walters, WA., Berg-Lyons, D., Lozupone, CA., Turnbaugh, PJ., et al. 2011.

365 Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad

366 Sci USA. 108(25): 4516–4523. 367 Caporaso, JG., Kuczynski, J., Stombaugh, J., Bittinger, K., Bushman, FD., Costello, EK., et al. 2010.

Page 18 of 33



Draft

Manuscript

19

368 QIIME allows analysis of high-throughput community sequencing data. Nat Methods. 7: 335-336.

369 Chen, SC., Qiu, CW., Huang, T., Zhou, WW., Qi, YC., Gao, YQ., et al. 2013. Effect of

370 1-aminocyclopropane-1-carboxylic acid deaminase producing bacteria on the hyphal growth and

371 primordium initiation of Agaricus bisporus. Fungal Ecology. 6(1): 110-118.

372 Childers, S. E., S. Ciulo, and D. R. lovley. 2002. Geobacter metallireducens access Fe(III) oxide by

373 chemotaxis. Nature. 416:767-769.

374 Du, XH., Zhao, Q., O’Donnell, K., Rooney, AP., Yang, ZL. 2012. Multigene molecular phylogenetics

375 reveals true morels (Morel) are especially species rich in China. Fungal Genetics and Biology.

376 49:455-469.

377 Edgar, RC., Haas, BJ., Clemente, JC., Quince, C., Knight, R. 2011. UCHIME improves sensitivity and

378 speed of chimera detection. Bioinformatics. 27(16): 2194-2200.

379 Fan, Zhonghan., Zheng, Lin. 2013. Method of wheat intercropping morels artificial cultivation [P]. CN

380 103141308 A.

381 Gast, C., H, E., Jansen, J., Bierling, L., Haanstra. 1988. Heavy Metals in Mushrooms and Their

382 Relationship with Soil Characteristics. Chromosphere. 17(4): 789-799.

383 Gans, J., Wolinsky, M., Dunbar, J. 2005. Computational improvements reveal great bacterial diversity and

384 high metal toxicity in soil. Science. 309: 1387-1390. doi: 10. 1126/science. 1112665.

385 Greve, H., Mohamed, I. E., Pontius, A., Kehraus, S., Gross, H. and Konig, G. M. 2010. Fungal metabolites:

386 Structural diversity as incentive for anticancer drug development. Phytochem. 9: 537–545.

387 Jeng-Leun, Mau., Chieh-No, Chang., Shih-Jeng, Huang., Chin-Chu, Chen. 2004. Antioxidant properties of

388 methanolic extracts from Grifola frondosa, Morchella esculenta and Termitomyces albuminosus

389 mycelia. In Food Chemistry. 87(1): 111-118.

390 Johansson, J F., Paul, L R., Finlay, R D. 2004. Microbial interactions in the mycorrhizosphere and their

391 significance for sustainable agriculture. FEMS Microbiology Ecology. 48 (1): 1-13.

392 Jones, RT., Robeson, MS., Laube,r CL., Hamady, M., Knight, R., Fierer, N. 2009. A comprehensive

393 survey of soil acidobacterial diversity using pyrosequencing and clone library analyses. ISMEJ. 3(4):

394 442-453.

395 Kalyoncu., Fatih, Oskay., Mustafa, Kalyoncu., Meryem. 2009. The Effects of Some Environmental

396 Parameters on Mycelial Growth of Six Morchella species. Journal of Pure and Applied Microbiology.

397 3: 467-472.

398 Li, JJ., Zheng, Y., Yan, JX., Li, HJ., Wang, X., He, JZ., et al. 2013. Effects of different regeneration

399 scenarios and fertilizer treatments on soil microbial ecology in reclaimed opencast mining areas on

400 the Loess Plateau China. PLOS ONE. 8(5): e63275.doi:10.1371/journal.pone.0063275.

Page 19 of 33



Draft

Manuscript

20

401 Li, Q., Xiong, C., Huang, WL., Li, XL. 2017. Controlled surface fire for improving yields of Morchella

402 importuna. Mycol Progress. 16: 1057–1063.

403 Li, S., Gao, A., Dong, S., Chen, Y., Sun, S., Lei, Z., et al. 2017. Purification, antitumor and

404 immunomodulatory activity of polysaccharides from soybean residue fermented with Morchella

405 esculenta. International Journal of Biological Macromolecules. 96:26.

406 Li, X., Chen, ZZ., Peng, CC., Han, Y., Xu, JG., Long, ZF. 2013. Effect of different ferric fertilizers on

407 planting Morchella conica fruit yields and analyses of the microflora and bioactivities of its grown

408 soil. Afr J Microb Res. 7(39): 4707–4716.

409 Lin, YT., Hu, HW., Whitaman, WB., Coleman, DC., Chiu, CY. 2014. Comparision of soil bacterial

410 communities in a natural hardwood forest and coniferous plantations in perhumid subtropical low

411 mountains. Botanical studies. 55: 31-39. 412 Liu, H., Zhang, J., Li, T., Shi, Y., Wang, Y. 2012. Mineral element levels in Wild Edible Mushrooms from 413 Yunnan, China. Biol Trace Elem Res. 147(1): 341–345.

414 Liu, HM., Xu, JJ., Li, X., Zhang, Y., Yin, AQ., Wang, JM., et al. 2015. Effects of microelemental

415 fertilizers on yields, mineral element levels and nutritional compositions of the artificially cultivated

416 Morchella conica. Scientia Horticulturae. 189(1): 86–93.

417 Liu, QY., Liu, HM., Chen, CQ., Wang, J., Han, Y., Long, ZF. 2017. Effects of element complexes

418 containing Fe, Zn and Mn on artificial morel’s biological characteristics and soil bacterial community

419 structures. PLOS ONE. 12(3): e0174618. https://doi.org/10.1371/journal.pone.0174618.

420 Liu, WX., Liu, JW., Wu, MZ., Li, Y., Zhao, Y., Li, SR. 2009. Accumulation and translocation of toxic

421 heavy metals in winter wheat (Triticum aestivum L.) growing in agricultural soil of Zhengzhou, China.

422 Bull Environ Contam Toxicol. 82(3):343–347.423 Long, YH., Xie, L., Liu, N., Ya,n X., Li, MH., Fan, MZ., et al. 2010. Comparison of gut-associated and 424 nest-associated microbial communities of a fungus-growing termite (Odontotermes yunnanensis) [J]. 425 Insect Sci. 17(3): 265-276.

426 M.B, Rudnick., J.A, van Veen., W, de Boer. 2015. Baiting of rhizosphere bacteria with hyphae of common

427 soil fungi reveals a diverse group of potentially mycophagous secondary consumers. Soil Biology and

428 Biochemistry. 88: 73-82.

429 Ma, Garcia & Alonso Díaz, Julián & Melgar, Maria. 2013. Bioconcentration of chromium in edible

430 mushrooms: Influence of environmental and genetic factors. Food and chemical toxicology. 58.

431 10.1016/j.fct.2013.04.049.

432 Magoc, T., Salzberg, SL. 2011. FLASH: fast length adjustment of short reads to improve genome

433 assemblies [J]. Bioinformatics. 27: 2957-2963.

434 Mcfarlane, EM., Pilz, D., Weber, NS. 2005. High-elevation gray morels and other Morchella species

Page 20 of 33



https://doi.org/10.1371/journal.pone.0174618.

Draft

Manuscript

21

435 harvested as non-timber forest products in Idaho and Montana. Mycologist. 19(2): 62–68.

436 Nasreddine, L., Parent-Massin, D. 2002. Food contamination by metals and pesticides in the European

437 Union. Should we worry? Toxicology Letters. 127(3): 29–41.

438 Nazir, Rashid., Mazurier, Sylvie., Yang, Pu., Lemanceau, Philippe., van, Elsas Jan Dirk. 2017. The

439 Ecological Role of Type Three Secretion Systems in the Interaction of Bacteria with Fungi in Soil and

440 Related Habitats Is Diverse and Context-Dependent. Frontiers in Microbiology. 8: 38.

441 Nelson, MC., Morrison, M., Yu, Z. 2011. A meta-analysis of the microbial diversity observed in anaerobic

442 digesters [J]. Bioresource Technology. 102(4): 3730-3739.

443 Nitha, B., Meera, CR., Janardhanan, KK. 2007. Anti-inflammatory and antitumour activities of cultured

444 mycelium of morel mushroom, Morchella esculenta. Current Science. 92(2): 235–239.

445 Noble, Ralph, Dobrovin-Pennington, Andreja, Hobbs, Philip J., et al. 2009. Volatile C8 compounds

446 and pseudomonads influence primordium formation of Agaricus bisporus. Mycologia. 10

447 (5):583-591. doi:10.3852/07-194.

448 Obst, J., Brown, W. 2000. Feasibility of a morel mushroom harvest in the Northwest Territories ［R］.

449 Arctic Ecology & Development Consulting and Deton’cho Corporation, Yellowknife. NWT.

450 402-422.

451 Ower, R., Mills, GL., Malachowski, JA. 1986. Cultivation of Morel. US Patent. No.4594809.

452 Ower, R. 1982. Notes on the development of the morel ascocarp: Morchella esculenta. Mycologia.

453 74(1):142–144.

454 Pion, M., Spangenberg, JE., Simon, A., Bindschedler, S., Flury, C., Chatelain, A., et al. 2013. Bacterial

455 farming by the fungus Morel crassipes. Proc Biol Sci. 280: 20132242.

456 http://dx.doi.org/10.1098/rspb.2013.2242

457 Richard, S., Winder. 2006. Cultural studies of Morchella elata. Mycological Research. 5(110): 612-623.

458 Rousk, J., Frey, SD. 2015. Revisiting the hypothesis that fungal-to-bacterial dominance characterizes

459 turnover of soil organic matter and nutrients. Ecological Monographs. 85(3): 457–472.

460 Su, CA., Xu, XY., Liu, DY., Wu, M., Zeng, FQ., Zeng, MY., et al. 2013. Isolation and characterization of

461 exopolysaccharide with immunomodulatory activity from fermentation broth of Morel conica.

462 DARU J Pharm Sci. 21(1):5. https://doi.org/10.1186/2008-2231-21-5.

463 Tamaki, H., Wright, CL., Li, XZ., Lin, XY., Hwang, CC., Wang, SP., et al. 2011. Analysis of 16S rRNA

464 Amplicon Sequencing Options on the Roche/454 Next-Generation Titanium Sequencing Platform.

465 PLOS ONE. 6(9): e25263. https://doi.org/10.1371/journal. pone.0025263.

466 Torsvik, V., Ovreas, L., Thingstad, TF. 2002. Prokaryotic diversity: magnitude, dynamics, and controlling

467 factors. Science. 296: 1064-1066. doi: 10. 1126/sden«e. 1071698.

Page 21 of 33



http://dx.doi.org/10.3852/07-194

https://doi.org/10.1371/journal.%20pone.0025263

Draft

Manuscript

22

468 Vieira, V., Fernandes, A., Barros, L., Glamoclija, J., Ciric, A., Stojkovic, D. et al. 2016. Wild Morel conica

469 Pers. from different origins: A comparative study of nutritional and bioactive properties. J. Sci. Food

470 Agric. 96:90–98.

471 Volk, TJ., Leonard, TJ. 1989. Experimental studies on the morel Heterokaryon formation between

472 monoascosporous strains of Morchella. Mycologia. 81(4): 523‐531.473 Warmink, JA., Nazir, R., Corten, B., van, Elsas JD. 2011. Hitchhikers on the fungal highway:The helper ef474 fect for bacterial migration via fungal hyphae. Soil Biol. 475 Blodiem. 43: 760-765. doi: 10. 1016/j. soilbio. 2010.12. 009.

476 Zhang, W., Chen, L., Zhang, R., Lin, KL. 2016. High throughput sequencing analysis of the joint effects of

477 BDE209-Pb on soil bacterial community structure [J]. Journal of Hazardous Materials. 301: 1-7.

478 Zhao, J., Ni,T., Li,Y., Xiong,W., Ran,W., Shen,B., et al. 2014. Responses of Bacterial Communities in

479 Arable Soils in a Rice-Wheat Cropping System to Different Fertilizer Regimes and Sampling Times.

480 PLoS ONE 9(1): e85301. https://doi.org/10.1371/journal.pone.0085301.

481

Page 22 of 33



Draft

Manuscript

23

483 Table 1: Microbial community diversity indices in the soil samples.

Sample valid sequence OTUs chao1 ACE shannon Simpson

M2-1 29989±3101ab 4605±611d 1269±101b 1807.14±367.21ab 9.16±0.21a 1±0c

M2-2 32287±3672ab 4299±788b 1225±139ab 1836.88±301.12ab 9.17±0.41a 1±0c

M2-3 30282±3361ab 4332±669b 1209±207ab 1782.93±291.18ab 9.12±0.12a 1±0c

M3-1 37977±4191b 5495±972h 1500±241c 2644.12±477.99c 9.25±0.21a 1±0c

M3-2 37433±3771ab 5819±903i 1537±279c 2576.64±399.21c 9.42±0.71a 1±0c

M3-3 31993±3355ab 5063±793f 1336±244b 1900.04±237.73bc 9.25±0.42a 1±0c

M6-1 31918±3811ab 4316±667b 1204±124ab 1990.13±276.88bc 7.45±0.39a 0.93±0.01a

M6-2 113289±8992e 5420±523g 1369±211b 2331.33±297.81c 8.92±0.14a 0.99±0.005c

M6-3 27987±2651a 4036±677a 1103±133a 1591.28±269.93a 7.94±0.21a 0.96±0.01b

W1 68249±3121c 4491±677c 1202±270ab 1925.18±311.21bc 8.20±0.31a 0.97±0.02bc

W2 78131±5052d 4985±601e 1343±201b 2131.15±334.65c 8.41±0.27a 0.98±0.015bc

W3 3172±3519ab 4436±799b 121±219ab 1712.9±255.27ab 9.12±0.22a 1±0c

484 Note: Values sharing a common superscript letter (a-d) were not significant at P <0.05.

485

Page 23 of 33



Draft

Manuscript

24

487 Table 2: Percentages of predominant bacterial phyla in the soil samples.

488 Note: Pro-Proteobacteria; Aci-Acidobacteria; Chl-Chloroflexi; Bac-Bacteroides; Gem-Gemmatimonadetes;

489 Pla-Planctomycetes; Act-Actinomyces;Nit- Nitrospira; One-way ANOVA analysis of the same bacteria in four soils at the

490 same time; Values sharing a common superscript letter (a-c) were not significant at P <0.05.

Mycelia growth stage（%） Primordial differentiation stage（%） Ascocarp growth stage（%）Phyla

M2-1 M3-1 M6-1 W1 M2-2 M3-2 M6-2 W2 M2-3 M3-3 M6-3 W3

Pro34.27±

5.64a

27.82±

3.31a

49.84±

6.82b

43.27±

5.04b

31.98±

2.47a

34.95±

4.1a31±3.5a

36.65±

5.75a

36.39±3

.79a

42.5±4

.17ab

42.5±3

.34ab

48.54±

5.17b

Aci19.2±1.

4c

18.32±

2.26bc

11.44±

1.74a

14.73±

2.91ab

20.62±

3.21b

13.5±1.

7a

12.21±

2.13a

18.71±

1.73b

16.33±2

.39c

13.78±

1.34bc

9.01±1

.03a

11.24±

2.04ab

Chl14.59±

2.53b

15.67±

1.44b

8.58±0.

64a

9.36±1.

42a

12.58±

2.14b

9.6±1.3

4ab

7.91±0.

73a

10.79±

0.83ab

11.5±1.

65b

9.35±2

.05ab

5.29±2

.13a

8.35±2

.7ab

Bac6.21±1.

37ab

8.32±0.

68bc

9.61±1.

13c

4.1±1.0

5a

9.88±1.

36b

14.5±1.

4c

6.58±1.

46a

7.5±0.4

5ab

7.98±1.

02a

8.11±1

.43a

25.27±

3.14b

7.38±1

.46a

Gem7.24±1.

14b

7.69±1.

23b

5.09±0.

66a7±1.2b

7.32±0.

84a

9.34±1.

06a

6.08±1.

16a

6.96±0.

78a

5.35±1.

24a

7.58±1

.61a

4.09±0

.73a

4.32±1

.06a

Pla5.03±1.

06b

5.03±1.

02b

2.84±0.

74a

3.58±0.

46ab

3.7±0.4

1a

3.54±0.

77a

3.38±0.

74a

3.57±0.

47a

5.71±1.

43b

3.68±0

.76a

4.46±1

.12ab

3.18±0

.26a

Act2.22±0.

62a

3.64±0.

34b

3.18±0.

06ab

5.44±1.

33c

2.9±0.6

5a

4.37±0.

74a

1.9±0.2

5a

4.48±1.

94a

3.06±0.

82a

7.03±1

.01b

2.41±0

.34a

10.36±

1.64c

Nit2.44±0.

71a

3.97±0.

84b

2.25±0.

4a

3.15±0.

85ab

2.67±0.

74a

1.67±0.

3a

2.14±0.

34a

2.94±0.

72a3.9±0.3b

1.83±0

.21a

1.24±0

.24a

1.55±0

.6a

Total91.2±2.

64a

90.46±

2.02a

92.83±

1.79a

90.63±

1.11a

91.65±

2.7b

91.47±

1.04b

71.2±2.

8a

91.6±1.

84b

90.22±0

.74a

93.86±

1.04a

94.27±

2.24a

94.92±

2.66a

Page 24 of 33



Draft

Manuscript

25

492 Table 3: Percentages of predominant bacterial genera in soil samples.

Mycelium growth stage (%)Primordial differentiation stage

(%)Ascocarp growth stage (%)

Genus

M2-1 M3-1 M6-1 W1 M2-2 M3-2 M6-2 W2 M2-3 M3-3 M6-3 W3

Pse0.26±

0.03a

0.27±

0.09a

25.3±2

.72c

14.31±

2.11b

2.56±0

.33a

2.17±

0.41a

4.02±0

.33b

10.67±

2.03c

0.48±

0.14b

0.76±

0.18c

0.3±

0.07b

0.13

±

0.06a

Ped0.1±0

.02a

0.32±

0.08b

3.68±0

.68c

0.1±0.

01a

2.52±0

.25b

2.18±

0.3b

6.64±0

.71c

1.14±0

.11a

0.96±

0.20b

1.55±

0.25c

0.55

±

0.14a

0.72

±

0.18ab

Can1.92±

0.23b

2.44±

0.41d

1.41±0

.22a

2.21±0

.19c

1.26±0

.21ab

1.5±0.

3ab

0.96±0

.13a

1.57±0

.34ab

1.49±

0.19a

1.79±

0.39a

1.64

±

0.32a

2.25

±

0.53b

Rho

1.22±

0.18a

b

0.9±0

.25a

0.84±0

.29a

0.93±0

.22a

1±0.27a

1.07±

0.19a

1.09±0

.27a

1.01±0

.27a

0.39±

0.09a

1.05±

0.31b

1.12

±

0.16b

1.54

±

0.23c

Pla0.96±

0.31b

1.27±

0.21c

0.52±0

.09a

0.44±0

.07a

1.17±0

.21bc

0.7±0.

04b

1.5±0.

32c

0.3±0.

08a

0.47±

0.14a

0.7±0.1

3ab

1.14±

0.22b

0.52±

0.16a

Geo

1.08±

0.19b

c

1.28±

0.33c

0.27±0

.06a

0.82±0

.11b

1.15±0

.14c

0.6±0.

15b

0.36±0

.11a

0.26±0

.09a

0.76±

0.03b

0.37±0.

08a

0.49±

0.13ab

0.72±

0.21b

Total5.54±

1.02a

6.48±

1.26a

32.02±

4.13c

18.81±

2.82b

9.66±1

.57a

8.22±

1.12a

14.57±

1.89b

14.95±

2.81b

4.55±

0.85a

6.22±1.

34a

5.24±

1.17a

5.88±

1.44a

493 Note: Pse-Pseudomonas ； Ped-Pedobacter ； Can-Candidatus Solibacter ； Rho-Rhodopseudomonas; Pla-Planctomyces;

494 Geo-Geobacter; One-way ANOVA analysis of the same bacteria in four soils at the same time; Values sharing a common

495 superscript letter (a-d) were not significant at P <0.05.

Page 25 of 33



Draft

Manuscript

26

497 Table 4: Mineral element contents in morel ascocarp and in cultivated soils (mg/kg dw).

M2 M3 M6

Materials Samples

Contents (mg/kg) BCF Contents (mg/kg) BCF Contents (mg/kg) BCF

Ascocarp 28517.00±363.37e 47055.00±346.41f 33220.00±236.43f

Potassium（K）

Soil 16886.67±207.65c

1.68

17250.00±395.17c

2.727

15540.00±912.03c

2.138

Ascocarp 17566.67±692.63d 21481.67±210.08e 17660.00±271.84e

Phosphorus（P）

Soil 1089.83±124.21a

16.119

1259.83±163.63a

17.051

1020.17±125.81a

17.314

Ascocarp 3775.67±61.81c 5349.00±71.51d 3835.00±39.18d

Calcium（Ca）

Soil 8311.67±423.05b

0.454

8323.33±350.25b

0.620

7691.67±491.74b

0.498

Ascocarp 1368.33±31.92b 1617.33±21.53c 1372.17±29.47cMagnesium

（Mg） Soil 6413±244.81b

0.213

6406.00±189.49b

0.252

5875.00±375.04b

0.234

Ascocarp 876.50±6.51ab 634.17±12.43b 577.50±14.02b

Ferric（Fe）

Soil 35243±2219.6d

0.025

36910.00±2149.84d

0.017

30121.6± 2117.05d

0.019

Ascocarp 149.80±1.08a 240.50±4.57a 167.00±1.85a

Zinc（Zn）

Soil 133.90±3.27a

1.119

138.50±7.63a

1.736

135.43±13.32a

1.233

Ascocarp 743.67±8.57ab 781.67±21.53b 772.00±19.35b

Sodium（Na）

Soil 7300.00±218.30b

0.102

7065.00±253.57b

0.111

6391.67±413.37b

0.121

Ascocarp 27.50±0.63a 37.38±0.40a 30.53±0.39aManganese

（Mn） Soil 282.93±5.96a

0.097

267.28±1.01a

0.14

201.72±28.63a

0.151

Boron（B） Ascocarp 33.48±2.15a 1.262 32.73±1.52a 1.752 30.22±0.31a 1.394

Page 26 of 33



Draft

Manuscript

27

Soil 26.52±2.88a 18.68±5.13a 21.68±0.31a

Ascocarp 26.67±0.52a 40.98±0.82a 28.90±0.71a

Copper（Cu）

Soil 26.78±2.07a

0.996

28.95±5.88a

1.415

27.68±1.76a

1.044

Ascocarp 3.53±0.18a 6.40±0.15a 7.63±0.27a

Selenium（Se）

Soil 3.61±0.42a

0.978

1.51±0.06a

4.240

2.46±0.22a

3.100

498 Note: Values sharing a common superscript letter (a-f) were not significant at P <0.05.

Page 27 of 33



Draft

Manuscript

28

500 Figure captions

501

502 Figure 1: Three morel experimental profiles (a) and growth status of mycelia on PDA medium (b).

503

504 Figure 2: Community composition and abundance distribution at phylum level.

505

506 Figure 3: Cluster analysis of genus community composition.

507

508 Figure 4：Two-dimension distribution figure of PCA analysis results.

509

510 Figure 5: Morel yields among different groups.

Page 28 of 33



Draft

Fig.1: three kind experimental morel profiles (a) and growth status of mycelium on PDA medium (b).

94x28mm (300 x 300 DPI)

Page 29 of 33



Draft

Fig2. Community composition and abundance distribution of phylum level.

47x34mm (300 x 300 DPI)

Page 30 of 33



Draft

Fig3. Cluster analysis of genus community composition.

47x47mm (300 x 300 DPI)

Page 31 of 33



Draft

Fig4. Two dimensions distribution figure of PCA analysis (Each point represents a sample, the closer the distance between two points, indicating that the similarity between the two samples of the microbial

community structure, the smaller the difference).

59x53mm (300 x 300 DPI)

Page 32 of 33



Draft

Fig5. Morel yields of different groups. Values sharing a common capitalized letter (a-c) were not significant at P<0.01.

50x34mm (300 x 300 DPI)

Page 33 of 33



Documents

Draft - University of Toronto T-Space · 27 Morchella spp. (morel) is a prized edible and nutritional fungus and its fruit-body (ascocarp) ... 33 outdoor cultivation of morels has