中文摘要

天山雪莲（Saussurea involucrata）是菊科风毛菊属多年生草本植物，被列为国家二级保护植物，野生资源濒临枯竭，目前还不能规模化人工栽培。本论文用筛选出的2株天山雪莲幼苗促生菌，研究它们在多种实验条件下对幼苗根系的侵染情况；研究与促生菌共生对幼苗生长和生理的影响，以及对幼苗响应低温胁迫的影响，研究促生菌的产酶和产激素能力。本研究为2株促生菌在天山雪莲种苗培育和微生物肥料开发两个方面的应用提供了科学依据。

论文的主要研究结果：

（1）从144株内生真菌中筛选出2株天山雪莲幼苗促生菌，5#和16#；它们都是深色有隔内生真菌（Dark Septate Endophytes,DSE）。5#和16#在4~25 ℃均能正常生长，15 ℃生长最快，生长速率分别是5.46±2.21 mm/d和1.88±0.67 mm/d。经分子鉴定，5#和16#分别为Juxtiphoma sp.和 Leptodophora sp.。

（2）5#和16#在15 ℃、20 ℃和25 ℃，以及在20 ℃/10 ℃的条件下均能在天山雪莲幼苗根组织内定殖。5#在20 ℃和20 ℃/10 ℃培养8周的定殖率大于90%，16#在4种温度培养6周的定殖率均大于90%。

（3）幼苗分别与2株促生菌在20 ℃共生培养27d，与幼苗单独培养组（CK）相比，除均能显著提高全株鲜重外，与5#共生幼苗丙二醛含量28.74±3.44 nmol/g，是CK的85.2%（p<0.05），SOD和CAT活性分别是CK的61.9%和2.2倍（p<0.05）；与16#共生幼苗可溶性糖含量40.38±28.30 mg/g，是CK的1.9倍（p<0.05），POD和CAT活性分别是CK的1.4倍和3.1倍（p<0.05）。幼苗分别与2株促生菌在20 ℃/10 ℃共生培养40d，与CK相比，除均能显著提高根鲜重，显著降低脯氨酸和可溶性糖含量外，与5#共生幼苗SOD和CAT活性分别是CK的1.5倍和74.8%（p<0.05）；与16#共生幼苗SOD、POD和CAT活性分别是CK的3.1倍、1.5倍和1.5倍（p<0.05）。以上结果表明，5#和16#在20 ℃/10 ℃对幼苗生理的影响更明显，促生作用与降低幼苗渗透调节物质的积累相关。此外，16#促生作用于其提高幼苗抗氧化能力相关。

（4）将在20 ℃单独培养（CK）和与5#共生培养39d的幼苗，先在0 ℃放置12h（胁迫），再在20 ℃放置2d（恢复），2组幼苗都没有出现死亡。2个组POD活性在胁迫后均上升，变化趋势一致，但与5#共生幼苗在胁迫后POD活性比胁迫前的提高了42.2%（p<0.05），而CK组幼苗仅提高了5.9%（p>0.05）。此外，与5#共生幼苗在胁迫后MAD和可溶性糖含量表现为提高的趋势，而CK组的表现为下降的趋势。以上结果表明，与5#共生使幼苗能更快地响应低温胁迫。转录组分析显示，5#促生菌共生下天山雪莲幼苗中脱水蛋白基因（DHNs）整体表达水平下降。低温胁迫显著诱导DHNs上调，其中2个基因在加菌组中的上调幅度高于对照组，表明5#调控了DHNs的表达及其低温响应模式。

（5）分别在基质中单独培养促生菌（20 ℃，27d），与空白基质（CK）相比，2株促生菌均能显著提高基质的ACP、AKP、SC、UE和CAT活性（p<0.05），显著提高基质的IAAs、GAs和CTKs等类激素含量（p <0.05），表明2株活性菌能产生多种土壤酶和植物激素。与幼苗单独培养组（P）基质相比，与5#和16#共生幼苗组基质的5种土壤酶含量提幅度分别在0.6~16.1倍和0.6~10倍（p<0.05）；基质的IAAs和CTKs含量仍有显著提高（p<0.05），但具体的激素种类有所不同。在IAAs中，与5#和16#共生分别显著提高基质的IBA和EMIAA及IAA含量。在CTKs中，与2株促生菌共生均能显著提高基质的IP、IPP和CZ含量，此外与5#和16#共生还分别能显著提高基质的DZR和TZR含量。

Abstract

Saussurea involucrata, a perennial herbaceous plant in the Asteraceae family, is classified as a nationally protected Class II species in China. Due to the depletion of wild populations, large-scale artificial cultivation has not yet been achieved. In this study, two endophytic strains with growth-promoting effects on S. involucrata seedlings were selected to investigate: (1) their root colonization ability under different conditions; (2) their effects on seedling growth and physiological responses, as well as the seedlings' responses to low-temperature stress. (3) their influence on enzyme production and phytohormone synthesis. This research provides a scientific basis for the potential application of these strains in seedling propagation and microbial fertilizer development for S. involucrata.

The main findings are as follows:

(1) Two dark septate endophytes (DSE), strains 5# and 16#, were selected from 144 isolates. Both strains grew well between 4 °C and 25 °C, with the fastest growth at 15 °C, at rates of 5.46 ± 2.21 mm/d (5#) and 1.88 ± 0.67 mm/d (16#). Molecular identification showed that strains 5# and 16# belonged to Juxtiphoma sp. and Leptodophora sp., respectively.

(2) Both strains could successfully colonize the root tissues of S. involucrata seedlings at 15 °C, 20°C, 25 °C, and 20 °C/10 °C. The colonization rate of strain 5# exceeded 90% after 8 weeks at 20 °Cand 20 °C /10 °C, while strain 16# achieved over 90% colonization after 6 weeks under all four temperature conditions.

(3) After 27 days of co-cultivation with strains 5# or 16# at 20 °C, compared with the uninoculated control (CK), both strains significantly increased the fresh weight of the whole plant. In the 5# group, the malondialdehyde (MDA) content was 28.74 ± 3.44 nmol/g, which was 85.2% of the CK value (p < 0.05), and the SOD and CAT activities were 61.9% and 2.2 times those of CK (p < 0.05), respectively. In the 16# group, soluble sugar content was 40.38 ± 28.30 mg/g (1.9 times the CK level, p < 0.05), and POD and CAT activities were 1.4 and 3.1 times those of CK, respectively (p < 0.05). After 40 days of co-cultivation under 20 °C/10 °C, both strains significantly increased root fresh weight, and decreased proline and soluble sugar contents. In the 5# group, SOD and CAT activities were 1.5 times and 74.8% of those in CK (p < 0.05), respectively; in the 16# group, SOD, POD, and CAT activities were 3.1, 1.5, and 1.5 times those in CK, respectively (p < 0.05). These results suggest that both strains exhibited more pronounced growth-promoting effects at 20 °C/10 °C, likely related to reduced accumulation of osmolytes. The growth-promoting effect of 16# was also associated with enhanced antioxidant capacity in seedlings.

(4) Seedlings co-cultured with 5# and CK seedlings (cultured at 20 °C for 39 days) were subjected to 0 °C for 12 hours (stress) and then returned to 20 °C for 2 days (recovery). No seedling death was observed in either group. POD activity increased in both groups after stress, but the increase in the 5# group was 42.2% (p < 0.05), compared to only 5.9% in CK (p > 0.05). Additionally, MDA and soluble sugar contents in the 5# group tended to increase after stress, while they decreased in CK. These results suggest that strain 5# facilitated a faster response to low-temperature stress. Transcriptome analysis showed that DHNs (dehydrins) in seedlings co-cultured with 5# had lower basal expression levels compared with CK. Cold stress significantly induced DHNs upregulation in both groups, with two DHNs showing greater fold changes in the 5# group than in CK, indicating that 5# modulated the expression and cold response patterns of DHN genes.

(5) When the strains were cultured alone in substrate at 20 °C for 27 days, compared with blank control (CK), both strains significantly increased the activities of soil enzymes (ACP, AKP, SC, UE, CAT) and the contents of hormones such as IAA, GAs, and CTKs (p < 0.05). Compared with the seedling-only group (P), the co-culture groups (with 5# and 16#) showed increases in the five soil enzyme activities by 0.6–16.1 times and 0.6–10 times, respectively (p < 0.05). IAA and CTK contents also increased significantly, though with some differences in specific types. In the IAA category, co-culture with 5# and 16# significantly increased IBA, EMIAA, and IAA levels. In the CTK category, both strains significantly increased IP, IPP, and CZ levels, and 5# and 16# additionally enhanced DZR and TZR levels, respectively.