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外生菌根真菌与宿主植物共生过程中,外生菌根真菌可以为宿主植物提供水分以及氮、磷等必要的矿质元素,来促进宿主植物的生长和提高其抵抗不良环境的能力[1];而宿主植物以脂质或糖的形式为外生菌根真菌提供碳水化合物供菌丝的生长[2]。在森林生态系统中,宿主植物-外生菌根真菌-根际土壤微生物之间存在相互依存、协同进化的关系,根际土壤微生物不仅能够提高外生菌根真菌的接种率[3],还能促进外生菌根真菌与宿主植物发生共生关系[4],这类根际土壤微生物称之为菌根辅助菌。大量研究表明,菌根辅助菌不仅能通过降解植物细胞壁等促进外生菌根真菌定殖,提高外生菌根真菌菌丝的存活率,还能通过降解细胞壁获得碳源供外生菌根真菌生长,诱导外生菌根真菌孢子萌发,抑制病原菌的产生[5]。菌根辅助菌还能分泌生长因子、次级代谢物等供外生菌根真菌生长,因此,筛选外生菌根真菌生长所需的菌根辅助菌对于提高外生菌根菌的产量十分重要[6]。
灰肉红菇(Russula griseocarnosa X.H. Wang, Zhu L. Yang & Knudsen 2009)又名“正红菇”、“真红菇”、“大红菌”等,味道鲜美,干品价格为800~1 000 元·kg−1,常见于广东、广西、云南、福建、贵州等地[7];玫瑰红菇(Russula rosea Persoon 1796)别称苦红菇、红辣子等,其菌肉硬,子实体中存在苦红菇酸A和苦红菇酸B四环三萜酸化合物,稍带苦辣味[8],很容易与灰肉红菇混淆,价格为200~500 元·kg−1,分布地方比较广,在全国各地均有分布。灰肉红菇和玫瑰红菇作为红菇属中常见的野生食药用外生菌根菌,富含氨基酸、脂肪酸和矿质元素等营养物质,不仅具有高蛋白、低脂肪、粗纤维等功能,还具有抗氧化、抗肿瘤、抗癌等功效,是非常重要的林下非木质林产品资源[9-10]。李国杰等[11]报道了我国82种可食用红菇和22种可药用红菇,其中,灰肉红菇和玫瑰红菇均具有重要的食药用价值。灰肉红菇与玫瑰红菇不仅在味道上有显著差异,而且亲缘关系较远[12-13],目前均不能进行人工栽培。在野外考察中,发现灰肉红菇和玫瑰红菇共存于同一片区域。
本研究以同一生态条件下2种外生菌根真菌玫瑰红菇和灰肉红菇的根际和非根际土壤微生物(细菌、真菌)作为研究对象,采用Illumina MiSeq二代双端测序的方法进行测序分析,通过比较2种红菇根际与非根际土壤微生物的群落结构差异,筛选相应的菌根辅助菌,结合土壤理化性质和2种红菇的生境,为2种红菇的保育促繁、开发利用提供理论依据。
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2种红菇根际与非根际土壤理化性质表明:灰肉红菇和玫瑰红菇根际土壤的有机碳(p=0.047)和有效钾(p=0.046)显著高于非根际土壤,在土壤pH、有效氮和有效磷中差异不显著(表1)。
表 1 2种红菇根际与非根际土壤理化性质
Table 1. Physicochemical properties of mycosphere and bulk soil of two species of Russula
样品
SamplepH 有机碳
SOC/(g·kg−1)有效氮
AN/(mg·kg−1)有效磷
AP/(mg·kg−1)有效钾
AK/(mg·kg−1)RR 4.22±0.11 a 38.80±3.69 a 327.98±30.38 a 10.40±3.38 a 241.52±42.44 a RG 4.25±0.06 a 35.39±6.07 a 281.06±52.77 a 6.97±1.15 a 307.30±32.35 a BK 4.46±0.08 a 12.88±7.81 b 132.48±86.95 a 2.44±2.03 a 112.14±51.54 b 注:平均值±标准误差。RR为玫瑰红菇根际土壤;RG为灰肉红菇根际土壤;BK为非根际土壤。下同。
Notes: Means±standard error. RR stands for the mycosphere soil of Russula rosea, RG stands for the mycosphere soil of Russula griseocarnosa, BK stands for bulk soil.The same below. -
通过对2种红菇根际与非根际土壤细菌、真菌的高通量测序分析,数据经过过滤、质控后,玫瑰红菇根际、灰肉红菇根际和非根际土壤细菌分别获得39 685、40 681、33 688条有效数据;玫瑰红菇根际、灰肉红菇根际和非根际土壤真菌分别获得46 151、52 073、65 339条有效数据。2种红菇根际与非根际土壤的细菌、真菌实际测量出的OTU数目的稀释性曲线均趋于平坦(图1),说明测序的结果合理可靠,更多的测序数据量发现新的OTU贡献率较低。在本研究中,土壤细菌共获得2 914个OTU,土壤真菌共获得536个OTU。
图 1 2种红菇根际与非根际土壤微生物的稀释性曲线
Figure 1. Rarefaction curve of mycosphere and bulk soil in two species of Russula
2种红菇根际土壤细菌Chao指数显著高于非根际土壤(p=0.03),Shannon指数低于非根际土壤,2种红菇的根际土壤细菌多样性差异不显著(表2)。在土壤真菌多样性中发现,灰肉红菇的根际土壤真菌Chao指数(p=0.034)和Shannon指数(p=0.01)显著高于玫瑰红菇根际土壤(表2)。
表 2 2种红菇根际与非根际土壤微生物多样性指数的比较分析
Table 2. Diversity index of mycosphere and bulk soil microorganism of two species of Russula
样品
Sample细菌 Bacterial 真菌 Fungal Chao指数 Chao index Shannon指数 Shannon index Chao指数 Chao index Shannon指数 Shannon index RR 1 724.65±11.97 a 5.76±0.04 ab 140.75±10.45 b 1.58±0.22 b RG 1 650.96±47.92 a 5.63±0.14 b 215.57±23.31 a 2.95±0.17 a BK 1 457.40±27.87 b 5.96±0.05 a 169.08±5.73 ab 1.80±0.27 b -
2种红菇的根际和非根际土壤共注释到24个细菌门,5个真菌门,其中,变形菌门(Proteobacteria)、酸杆菌门(Acidobacteria)、绿弯菌门(Chloroflexi)和放线菌门(Actinobacteria)为主要的细菌门(图2A);子囊菌门(Ascomycota)、担子菌门(Basidiomycota)和接合菌门(Zygomycota)为主要的真菌门(图2B)。灰肉红菇和玫瑰红菇的根际土壤细菌中变形菌门(p=0.017)、酸杆菌门(p<0.01)和土壤真菌中担子菌门(p=0.002)均显著高于非根际土壤(图2)。
图 2 2种红菇根际与非根际土壤微生物在门水平上的群落组成 (A:细菌 B:真菌)
Figure 2. Community composition of mycosphere and bulk soil in two species of Russula at phylum level ( A: Bacterial B: Fungal)
在细菌属共计注释到445个属,变异杆菌属(Variibacter)、热酸菌属(Acidothermus)、慢生根瘤菌属(Bradyrhizobium)、Candidatus_Solibacter属和Burkholderia−Paraburkholderia属等13个属的相对丰度大于0.1%,为主要的细菌属。在前50个属中,玫瑰红菇和灰肉红菇根际土壤细菌的变异杆菌属(Variibacter)(p<0.01)、Candidatus_Solibacter属(p=0.033)、堆囊菌属(Sorangium)(p=0.029)、分枝杆菌属(Mycobacterium)(p<0.01)、Singulisphaera属(p=0.023)、Isosphaera属(p=0.001)、蛭弧菌属(Bdellovibrio)(p=0.034)和类芽孢杆菌属(Paenibacillus)(p=0.005)显著高于非根际土壤,这些可能是红菇属生长的主要菌根辅助细菌;丛生放线菌属(Actinospica)(p=0.016)、鞘氨醇单胞菌属(Sphingomonas)(p=0.023)、Chthonomonas属(p=0.001)和Vicinamibacter属(p<0.01)等显著低于非根际土壤(图3A)。在土壤细菌的群落结构发现,灰肉红菇和玫瑰红菇处于同一支,说明二者之间的菌群结构相似。进一步对灰肉红菇和玫瑰红菇的根际土壤细菌进行比较发现,灰肉红菇根际土壤细菌中变异杆菌属(Variibacter)(p=0.022)、Bryobacter属(p=0.023)和芽生绿菌属(Blastochloris)(p=0.008 9)显著高于玫瑰红菇根际土壤细菌,而热酸菌属(Acidothermus)(p=0.013)、分枝杆菌属(Mycobacterium)(p=0.044)、Reyranella属(p=0.009)、纤线杆菌属(Ktedonobacter)(p=0.013)和罗思河小杆菌属(Rhodanobacter)(p=0.013)显著低于玫瑰红菇根际土壤细菌(图4A),这说明不同红菇中的土壤细菌优势辅助菌略有差异。
图 3 2种红菇根际与非根际土壤微生物在前50个属水平上的群落组成(A:细菌 B:真菌)
Figure 3. Community composition of mycosphere and bulk soil in two species of Russula at the top 50 genera (A: Bacterial B: Fungal)
图 4 2种红菇根际土壤微生物在属水平上的比较分析 (A:细菌 B:真菌)
Figure 4. Comparative analysis of mycosphere soil microorganisms of two species Russula at genus level (A: Bacterial B: Fungal)
在土壤真菌属共计注释到146个属,木霉属(Trichoderma)、被孢霉属(Mortierella)、青霉属(Penicillium)、棉革菌属(Tomentella)和粘头束霉属(Gliocephalotrichum)为主要的真菌属。在前50个属中,灰肉红菇和玫瑰红菇根际土壤真菌中木霉属(Trichoderma)(p=0.023)、青霉属(Penicillium)(p=0.038)和Hypomyces属(p=0.038)显著高于非根际土壤,而豆马勃属(Pisolithus)(p=0.023)、枝孢属(Cladosporium)(p=0.030)和拟棘壳孢属(Pyrenochaetopsis)(p=0.003)显著低于非根际土壤(图3B)。进一步对灰肉红菇和玫瑰红菇的根际土壤真菌进行比较,发现玫瑰红菇根际土壤真菌的Oidiodendron属(p=0.018)和木霉属(Trichoderma)(p=0.018)显著高于灰肉红菇根际土壤真菌,灰肉红菇根际土壤真菌的Cladophialophora属(p=0.049)、拟盘多毛孢属(Pestalotiopsis)(p=0.023)、拟青霉属(Paecilomyces)(p=0.020)和节菱孢属(Arthrinium)(p=0.044)显著高于玫瑰红菇根际土壤真菌(图4B),这些结果表明不同红菇中主要的优势真菌菌群略有差异。
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RDA结果分析可以反映菌群、样本和环境之间的关系。通过对2种红菇根际与非根际土壤(细菌、真菌)在97%水平上的OTU进行RDA分析,结果表明:2两种红菇根际土壤样品与非根际土壤样品相距较远,菌群结构差异明显,而2种红菇样品相距较近,菌群结构相似,重复样地之间测序结果合理(图5)。通过对样地菌群与环境因子进行分析,发现pH与土壤细菌(p=0.020)、真菌(p=0.048)群落之间的相关性最大,说明pH是影响土壤微生物群落结构的主要因素。
图 5 样地与土壤理化性质的RDA冗余分析 (A:细菌 B:真菌)
Figure 5. Redundancy analysis (RDA) between soil physicochemical properties and sample( A: Bacterial B: Fungal)
通过对前50个属水平的菌群和环境因子进行Spearman相关性分析,结果显示:有效氮(AN)、有效磷(AP)、有机碳(SOC)、有效钾(AK)为一类,pH为单独的一类,说明pH对菌群的影响较大(图6)。在土壤细菌中,变异杆菌属(Variibacter)(p=0.049)、堆囊菌属(Sorangium)(p=0.025)、分枝杆菌属(Mycobacterium)(p=0.038)、根微菌属(Rhizomicrobium)(p=0.005)、芽生绿菌属(Blastochloris)(p=0.030)、Reyranella属(p=0.036)、蛭弧菌属(Bdellovibrio)(p=0.020)和北里孢菌属(Kitasatospora)(p=0.049)与pH呈显著负相关,Chthonomonas属(p=0.007)、Aquicella属(p=0.013)和玫瑰弯菌属(Roseiflexus)(p=0.015)与pH呈显著正相关;Candidatus_Solibacter属、堆囊菌属(Sorangium)和Mucilaginibacter属与土壤SOC、AN、AP和AK呈显著正相关,Chthonomonas则与土壤SOC、AN、AP和AK呈显著负相关(图6A)。在土壤真菌中,木霉属(Trichoderma)、被孢霉属(Mortierella)、粘头束霉属(Gliocephalotrichum)、茎霉属(Chaunopycnis)和瘤孢属(Sepedonium)与pH呈显著负相关,与有机碳呈显著正相关;Arnium属、豆马勃属(Pisolithus)和隐囊菌属(Aphanoascus)与pH呈显著正相关,与有机碳、AK呈显著负相关(图6B)。
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通过对2种红菇根际与非根际土壤细菌在KEGG代谢通路注释结果进行单因素方差分析,发现相对丰度超过0.5%的代谢通路共有71条,其中具有显著性差异的代谢通路有33条(图7)。玫瑰红菇和灰肉红菇根际土壤细菌在信号转导[双组分调节系统(Two-component system)(p<0.001)]、氨基酸代谢[甘氨酸、丝氨酸和苏氨酸代谢(Glycine, serine and threonine metabolism)(p<0.001)、半胱氨酸和蛋氨酸代谢(Cysteine and methionine metabolism)(p=0.026)、组氨酸代谢(Histidine metabolism)(p=0.001)、酪氨酸代谢(Tyrosine metabolism)(p=0.001)]、能量代谢[氮代谢(Nitrogen metabolism)(p<0.001)]、膜运输[分泌系统(Secretion system)(p<0.001)、细菌分泌系统(Bacterial secretion system)(p<0.001)]、细胞迁移[细菌趋化性(Bacterial chemotaxis)(p<0.001)]、聚糖生物合成与代谢[脂多糖生物合成蛋白(Lipopolysaccharide biosynthesis proteins)(p=0.014)]显著高于非根际土壤(图7),其中,灰肉红菇的分泌系统(Secretion system)和细菌分泌系统(Bacterial secretion system)代谢途径显著高于玫瑰红菇(图7),而甘氨酸、丝氨酸和苏氨酸代谢(Glycine, serine and threonine metabolism)和组氨酸代谢(Histidine metabolism)显著低于玫瑰红菇(图7)。
玫瑰红菇和灰肉红菇对根际土壤微生物群落结构的影响
The Effect of Russula rosea and Russula griseocarnosa on Microorganism Structure of Mycosphere Soil
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摘要:
目的 通过分析玫瑰红菇和灰肉红菇根际土壤微生物群落,筛选促进2种红菇生长的土壤微生物,为2种红菇的保育促繁以及人工栽培的实现提供理论依据。 方法 以同一生态条件下2种野生食药用外生菌根真菌(玫瑰红菇和灰肉红菇)的根际与非根际土壤作为研究对象,采用高通量Illumina MiSeq测序技术分析2种红菇根际与非根际土壤微生物(真菌、细菌)的群落结构,并结合土壤理化性质进行综合分析。 结果 2种红菇根际土壤微生物略有差异,玫瑰红菇的根际土壤微生物优势菌群为热酸菌属、分枝杆菌属、Reyranella属、纤线杆菌属、罗思河小杆菌属和Oidiodendron属;灰肉红菇的根际土壤微生物优势菌群为变异杆菌属、Bryobacter属,芽生绿菌属、拟青霉属和节菱孢属;细菌(变异杆菌属、Candidatus_Solibacter属、堆囊菌属、分枝杆菌属、Singulisphaera属、Isosphaera属、蛭弧菌属、类芽孢杆菌属)和真菌(木霉属、青霉属和Hypomyces属)为2种红菇共有的菌根辅助菌。pH是影响根际土壤微生物群落结构的关键因素。 结论 2种红菇会根据生长需要特异性地选择对自己生长有益的土壤微生物,形成不同的微生物群落。本研究筛选出的红菇根际菌根辅助菌可为制备红菇微生物专用菌肥以及为红菇的人工促繁提供理论依据,这对红菇的可持续利用,扩大林下食用菌规模,提高经济效益具有重要意义。 Abstract:Objective To provide a theoretical basis for the conservation and propagation of the two species of Russula and the realization of artificial cultivation, the mycosphere soil microbial communities of Russula rosea and Russula griseocarnosa was analyzed , and the soil microorganisms that promoting the growth of the two species of Russula were screened. Method The community structure of mycosphere and bulk soil microorganisms (fungi and bacteria) of two species of Russula were analyzed by Illumina MiSeq sequencing technology, and evaluated comprehensively based on soil physicochemical property. Result The mycosphere soil microorganisms of the two species of Russula were slightly different. The dominant mycosphere soil microorganisms of R. rosea were Acidothermus, Mycobacterium, Reyranella, Ktedonobacter, Rhodanobacter and Oidiodendron, while the dominant mycosphere soil microorganisms of R. griseocarnosa were Variibacter, Bryobacter, Blastochloris, Cladophialophora, Paecilomyces and Arthrinium. Bacteria (Variibacter, Candidatus_Solibacter, Sorangium, Mycobacterium, Singulisphaera, Isosphaera, Bdellovibrio and Paenibacillus) and fungi (Trichoderma, Penicillium, and Hypomyces) were the common growth promoting microbes of Russula rosea and R. griseocarnosa. pH was the key factor affecting mycosphere soil microorganisms. Conclusion The two species of Russula can specifically select soil microorganisms that are beneficial to their growth according to their growth needs and form different microbial community structures. The mycosphere mycorrhizal auxiliary bacteria that were screened out in this study can provide theoretical basis for the preparation of special microbial fertilizer and artificial propagation promotion of Russula, which has great promotion for the sustainable utilization of Russula, the expansion of understory edible fungi scale and the improvement of economic benefits. -
表 1 2种红菇根际与非根际土壤理化性质
Table 1. Physicochemical properties of mycosphere and bulk soil of two species of Russula
样品
SamplepH 有机碳
SOC/(g·kg−1)有效氮
AN/(mg·kg−1)有效磷
AP/(mg·kg−1)有效钾
AK/(mg·kg−1)RR 4.22±0.11 a 38.80±3.69 a 327.98±30.38 a 10.40±3.38 a 241.52±42.44 a RG 4.25±0.06 a 35.39±6.07 a 281.06±52.77 a 6.97±1.15 a 307.30±32.35 a BK 4.46±0.08 a 12.88±7.81 b 132.48±86.95 a 2.44±2.03 a 112.14±51.54 b 注:平均值±标准误差。RR为玫瑰红菇根际土壤;RG为灰肉红菇根际土壤;BK为非根际土壤。下同。
Notes: Means±standard error. RR stands for the mycosphere soil of Russula rosea, RG stands for the mycosphere soil of Russula griseocarnosa, BK stands for bulk soil.The same below.表 2 2种红菇根际与非根际土壤微生物多样性指数的比较分析
Table 2. Diversity index of mycosphere and bulk soil microorganism of two species of Russula
样品
Sample细菌 Bacterial 真菌 Fungal Chao指数 Chao index Shannon指数 Shannon index Chao指数 Chao index Shannon指数 Shannon index RR 1 724.65±11.97 a 5.76±0.04 ab 140.75±10.45 b 1.58±0.22 b RG 1 650.96±47.92 a 5.63±0.14 b 215.57±23.31 a 2.95±0.17 a BK 1 457.40±27.87 b 5.96±0.05 a 169.08±5.73 ab 1.80±0.27 b -
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