天山雪莲（Saussurea involucrata Kar. et kir.）和鼠曲雪兔子（Saussurea gnaphalodes (Royle) Sch.-Bip.）是雪莲类药材的来源植物，属于菊科风毛菊属多生长在高海拔地区，鼠曲雪兔子是现存海拔最高的雪莲原植物，恶劣的生境和较少的适宜生长区域导致雪莲类资源匮乏。目前，天山雪莲尚不能规模化种植，促进天山雪莲幼苗生长的根内生真菌未在低温下发挥作用，寻找有益的冷适应微生物将有利于天山雪莲的人工种植。

本研究首先采用高通量测序技术基于细菌16S序列和真菌的ITS序列，分析天山雪莲与鼠曲雪兔子根内和根际微生物多样性，结合土壤理化性质分析环境适应微生物群落。然后采用低温分离法在两种植物的根组织块中分离获得可培养的根内生真菌，并结合形态学方法和分子生物学方法对内生真菌进行鉴定。在不同温度下进行根内生真菌的生长速度和共生培养考察，筛选在低温条件下能促进天山雪莲幼苗生长的冷适应菌株。主要研究结果如下：

1、天山雪莲与鼠曲雪兔子的根内和根际细菌多样性

基于高通量测序技术，天山雪莲与鼠曲雪兔子根内和根际细菌共获得有效序列675883条，聚类为1970个细菌OTUs，28门480属，其中505个共有细菌OTUs，也存在相对丰度具有显著差异的细菌类群。天山雪莲根内和根际细菌隶属28门475属，以变形菌门Proteobacteria（25.05%-86.58%）为优势门。鼠曲雪兔子根内和根际细菌隶属28门466属，以变形菌门（21.20%-50.05%）和放线菌门（20.54%-36.56%）为优势门。鼠曲雪兔子根内细菌多样性高于天山雪莲根内细菌多样性，植物对根际细菌发生了选择性富集，特别是变形菌门和拟杆菌门Bacteroidota。拟杆菌门细菌在鼠曲雪兔子根中相对丰度是根际的2.87倍；研究发现其丰度与土壤营养含量呈负相关，可能与该植物适应贫瘠环境有关。

2、天山雪莲与鼠曲雪兔子的根内和根际真菌多样性

基于高通量测序技术，天山雪莲与鼠曲雪兔子根内和根际真菌共获得有效序列544354条，聚类为651个真菌OTUs，属于11门238属，其中14个共有真菌OTUs，也存在相对丰度具有显著差异的真菌类群。天山雪莲根内和根际真菌隶属11门230属，以子囊菌门Ascomycota（60.58%-98.82%）为优势门，以Tetracladium（5.81%-7.05%）和链格孢属Alternaria（3.16%-3.82%）相对丰度较高。鼠曲雪兔子根内和根际真菌隶属9门157属，以子囊菌门（91.22%-95.97%）和担子菌门（3.63%-5.31%）为优势门，以Tetracladium（2.40%-16.52%）、背芽突霉属Cadophora（1.12%-4.13%）和异茎点霉属Paraphoma（2.17%-2.22%）相对丰度较高。

鼠曲雪兔子根内真菌多样性高于天山雪莲根内真菌多样性，植物对根际真菌发生了选择性富集，背芽突霉属真菌在天山雪莲和鼠曲雪兔子根中富集，是根际真菌相对丰度的3.69-22.84倍。小菇属和链格孢属真菌在天山雪莲根中富集，Tetracladium和外瓶柄霉属在鼠曲雪兔子根中富集。

3、天山雪莲与鼠曲雪兔子根内生真菌的低温分离与鉴定

基于低温分离法，天山雪莲与鼠曲雪兔子根内共分离内生真菌242株，经过形态学和分子鉴定隶属2门5纲7目11科14属，共有属是链格孢属、背芽突霉属和Alpinaria，其中Alpinaria为稀有属。从天山雪莲根内分离获得了142株真菌，属于2门9属。4 ℃和15 ℃分离分别获得了8属和7属，4 ℃分离获得3个特有属，优势属是链格孢属。15 ℃分离获得2个特有属，优势属是Rhexocercosporidium。XLS2和XLS5分离分别获得了7属和10属，XLS2分离获得3个特有属，优势属是背芽突霉属。XLS5分离获得有4个特有属，优势属是链格孢属和Rhexocercosporidium。从鼠曲雪兔子根内分离获得了100株真菌，属于1门8属。4 ℃分离群落有1个特有属Alpinaria，优势属是背芽突霉属和Ophiobolus。15 ℃分离群落有3个特有属，优势属是背芽突霉属、Ophiobolus和异茎点霉属。在28个代表菌株中，天山雪莲与鼠曲雪兔子根内DSEs共20株，以链格孢属、Rhexocercosporidium和背芽突霉属为优势属。嗜冷菌株共5株，耐冷菌株共9株，最适生长温度为15 ℃，背芽突霉属为两种植物根内冷适应真菌的共有属。

4、促进天山雪莲幼苗生长的菌株

菌株Sg03（Alternaria sp.4）、S11（Juxtiphoma sp.）和S12（Lophiostoma sp.）在15 ℃和20 ℃都有促生作用，菌株S01（Alpinaria sp.）、S13（Rhexocercosporidium sp.1）、S14（Rhexocercosporidium sp.2）、Sg01（Alpinaria sp.2）和Sg08（Gaeumannomyces sp.）只在15 ℃有促生作用。其中S01、S11、S12、Sg03和Sg08在15 ℃低温下对天山雪莲幼苗的生长有显著积极促进作用。来自鼠曲雪兔子根内的菌株Sg03，15℃条件下可显著促进天山雪莲生长。根总长度、叶干重、根干重和根冠比分别比CK提高了11.55倍、2.83倍、6.20倍和0.81倍（P<0.05）。

本研究基于高通量测序技术对两种植物根内和根际微生物进行研究，样品类型、宿主植物种类和土壤理化性质对微生物群落结构和多样性都有影响。通过低温分离并鉴定其内生真菌，并研究根内可培养内生真菌的物种多样性，获得了高通量测序解析的核心菌属背芽突霉属。再通过共生培养实验考察确定S01、S11、S12、S13、S14、Sg01、Sg03、Sg08和Sg13等菌株在15 ℃对天山雪莲幼苗生长的促进作用。本研究为解析雪莲寒冷生境的冷适应机理、天山雪莲人工种植和保护天山雪莲资源奠定基础。

Saussurea involucrata Kar. et kir. and Saussurea gnaphalodes (Royal) Sch.-Bip. are the sources plants of Saussureae Involucratae Herba, belong to the Compositae in the Saussurea, often grow at high altitudes. The harsh habitat and few suitable growing areas lead to the scarcity of Saussurea resources. At present, S. involucrata cannot be cultivated on a large scale, The root endophytic fungi which could promote S. involucrata seedlings growth didn’t take effect at low temperature. Searching for beneficial cold-adaptable microorganisms of Saussurea will help the artificial cultivation of S. involucrata.

This study firstly applied High-throughput sequencing technology based on the 16S sequences of bacteria and ITS sequences of fungi was used to analyze the diversity of endophytic and rhizospheric microorganisms associated with the roots of the S. involucrata and S. gnaphalodes, and analyzed environmental adaptable microbial communities, combined with soil physical and chemical properties. Then using low-temperature separation method to obtain cultivable root endophytic fungi from the root tissue blocks of these two plants, and identifing the endophytic fungi by morphological methods combined with molecular biology methods. At different temperatures, Investigating the root endophytic fungi growth rate and symbiosis culture with the S. involucrata seedlings, the cold-adaptable strains which could promote the growth of S. involucrata seedlings at low temperatures were selected. The main results are as follows:

Diversity of endophytic and rhizospheric bacteria associated with the roots of the S. involucrata and S. gnaphalodes

Based on the high-throughput sequencing technology, the endophytic and rhizospheric bacteria associated with the roots of the S. involucrata and S. gnaphalodes obtained 675,883 effective bacterial sequences, clustered into 1,970 bacterial OTUs, 28 phyla and 480 genera, of which 505 common bacterial OTUs, there were bacterial taxa with significant differences in their relative abundance. Endophytic and rhizospheric bacteria associated with the roots of the S. involucrata belonged to 28 phyla and 475 genera, had the dominant phylum as Proteobacteria (25.05% - 86.58%). Endophytic and rhizospheric bacteria associated with the roots of the S. gnaphalodes belonged to 28 phyla and 466 genera, the dominant phyla were Proteobacteria (21.20% - 50.05%) and Actinobacteriota (20.54% - 36.56%). The endophytic bacterial diversity in S. gnaphalodes higher than that in S. involucrata, plants experienced selective enrichment of bacteria in the rhizospheric bacteria, especially Proteobacteria and Bacteroidetes. The relative abundance of Bacteroides in the S. gnaphalodes root was 2.87 times of that in its rhizosphere. It was found that its abundance was negatively correlated with soil nutrient content, which may be related to the adaptation of the plant to poor environment.

Diversity of endophytic and rhizospheric fungi associated with the roots of the S. involucrata and S. gnaphalodes 

Based on the high-throughput sequencing technology, the endophytic and rhizospheric fungi associated with the roots of the S. involucrata and S. gnaphalodes obtained 544,354 effective fungal sequences, clustered into 651 fungal OTUs, 11 phyla and 238 genera, of which 14 common fungal OTUs, there were fungal taxa with significant differences in their relative abundance. Endophytic and rhizospheric fungi associated with the roots of the S. involucrata belonged to 11 phyla and 230 genera, and had the dominant phyla Ascomycota (60.58% -98.82%), the relative abundance of Tetracladium (5.81%-7.05%) and Alternaria (3.16%-3.82%) were higher. Endophytic and rhizospheric fungi associated with the roots of the S. gnaphalodes belonged to 9 phyla and 157 genera, Ascomycota (91% -95.22%) and Basidiomycota (5.33% -5.31%) were the dominant phylum, the relative abundance of Tetracladium (2.40%-16.52%), Cadophora (1.12%-4.13%) and Paraphoma (2.17%-2.22%) were higher.

The endophytic fungal diversity in S. gnaphalodes higher than that in S. involucrata, plants experienced selective enrichment of fungi in the rhizosphere soil, Cadophora was enriched in the roots of S. involucrata and  S. gnaphalodes, which was 3.69-22.84 times the relative abundance of rhizosphere fungi. Mycena and Alternaria were enriched in the roots of S. involucrata. Tetracladium and Exophiala were enriched in the roots of S. gnaphalodes.

Low-temperature separation and identification the endophytic fungi isolated from the roots of the S. involucrata and S. gnaphalodes

Based on the low-temperature separation method, 242 endophytic fungi were isolated from the S. involucrata roots and the S. gnaphalodes. According to morphological and molecular identification, The 242 strains were classified to 2 phyla, 5 classes, 7 orders, 11 families and 14 genera, there were common genera Alternaria, Cadophora and Alpinaria, the genus Alpinaria was unfamiliar. 142 endophytic fungi were isolated from the S. involucrata roots, classified to 2 phyla and 9 genera. There were 8 and 7 genera were obtained from 4 ℃ and 15 ℃, respectively. The community gotten at 4 ℃ included 3 endemic genera, and the dominant genus was Alternaria. The community gotten at 15 ℃ included 2 endemic genera, and the dominant genus was Rhexocercosporidium. There were 7 and 10 genera were obtained from XLS2 and XLS5, respectively. The community gotten at XLS2 included 3 endemic genera, and the dominant genus was Cadophora. The community gotten at XLS5 included 4 endemic genera, and the dominant genera were Alternaria and Rhexocercosporidium. 100 endophytic fungi were isolated from the S. gnaphalodes roots, classified to 1 phyla and 8 genera. there were 5 and 7 genera were obtained from 4 ℃ and 15 ℃, respectively. The community gotten at 4 ℃ included 1 endemic genus Alpinaria, and the dominant genera were Cadophora and Ophiobolus. The community gotten at 15 ℃ included 3 endemic genera, and the dominant genera were Alternaria, Rhexocercosporidium and Cadophora. In the 28 representative strains, DSEs were 20 strains, Alternaria, Rhexocercosporidium and Cadophora were the dominant genera. There were 5 psychrophiles strains and 9 psychrotrophs strains, and the optimum growth temperature was 15 ℃. Cadophora was a common genus of cold-adaptable fungi in the roots of the two plants.

Endophytic fungi promote the growth of S. involucrata seedlings

Strains Sg03 (Alternaria sp.4), S11 (Juxtiphoma sp.) and S12 (Lophiostoma sp.) could promote the growth of S. involucrata seedlings both at 15 ℃ and 20 ℃, while S01 (Alpinaria sp.), S13 (Rhexocercosporidium sp.1), S14 (Rhexocercosporidium sp.2), Sg01 (Alpinaria sp.2) and Sg08 (Gaeumannomyces sp.) could do only at 15 ℃. Among them, S01, S11, S12, Sg03 and Sg08 significantly and actively promoted the growth of the S. involucrata seedlings at 15 ℃. Strain Sg03 from the root of the S. gnaphalodes was significantly promoted at 15 ℃, which could increase the S. involucrata seedlings total root length, leaf dry weight, root dry weight, and root crown ratio by 11.55, 2.83, 6.20, and 0.81 times over CK, respectively (P <0.05)

In this study, Based on the high-throughput sequencing method the diversity of endophytic and rhizospheric microorganisms associated with the roots of the two plants were studied. sample type, host plant species and soil physiochemical properties had effects on microbial community structure and diversity. Through the low-temperature separation method to isolate endophytes from roots and they were identified, the species diversity of culturable endophytic fungi isolated from roots was studied, obtained the high-throughput sequencing core fungi genus Cadophora. Then the symbiotic culture experiment was conducted to determine strains S01, S11, S12, S13, S14, Sg01, Sg03, Sg08 and Sg13 can promote the S. involucrata seedlings growth at different temperatures. This study laid a foundation for the analysis of the cold-adaptation mechanism of S. involucrata to cold habitat, the artificial cultivation of S. involucrata and the protection of Saussurea resources.