【作者】 陈桂梅；

【导师】 唐明；

【作者基本信息】 西北农林科技大学 ， 微生物学， 2009， 硕士

【摘要】 本文采用富集培养技术从油松菌根分离鉴定菌根伴生真菌,通过光学和扫描电子显微镜对外生菌根真菌与菌根伴生真菌在平板对峙培养和对油松苗接种后根际上的互作情况进行观察,研究菌根伴生真菌对外生菌根真菌产酶的影响以及接种外生菌根真菌和菌根伴生真菌油松根系活力,硝酸还原酶活性等生理指标的变化,揭示菌根伴生真菌与外生菌根真菌的互作机制。得出以下主要结果:1.菌根伴生真菌的分离和鉴定从油松菌根分离到4株菌根伴生真菌(Mycorrhizal-associated Fungi),经形态特征和菌落特征观察及分子生物学鉴定,分别属于半知菌类的丝孢纲、丛梗孢目、丛梗孢科、木霉属中的哈茨木霉Trichoderma harzianum HDTP-1和T.harzianum HDTP-3(简称HDTP-1和HDTP-3);藻状菌纲、毛霉目、毛霉科、毛霉属中的冻土毛霉Mucor hiemalisSA10-6 HDTP-4和M.hiemalis XSD-98 HDTP-5(简称HDTP-4和HDTP-5)。2.外生菌根真菌与菌根伴生真菌的互作对峙培养结果表明褐黄牛肝菌(Boletus luridus)、粘盖牛肝菌(Suillus bovinus、褐环粘盖牛肝菌(Suillus luteus)和铆钉菇(Gomphidius viscidus)对4株菌根伴生真菌均有抑制作用,互作区域外生菌根真菌有缠绕或侵入菌根伴生真菌菌丝体的现象,菌根伴生真菌菌丝体分支增多或降解,短支顶端膨大。4株外生菌根真菌在含有菌根伴生真菌菌丝体培养液中均能生长,与对照相比有显著促进作用。铆钉菇侵染油松形成菌根2个月后,接种菌根伴生真菌菌丝和孢子悬液2、3、5和7d观察其互作情况。接种2或3 d后,菌根伴生真菌菌丝在非菌根化的油松根表大量生长,但在菌根化油松根表受到抑制。接种5 d后菌根化油松根表有断裂的伴生真菌菌丝体和粘液物质形成,偶尔可见孢子附着于菌根表面,而接种7 d后,菌根表面就只能观察到伴生真菌菌丝碎片和原生质状物质。3.菌根伴生真菌对外生菌根真菌产酶的影响①菌根伴生真菌均能诱导褐黄牛肝菌和粘盖牛肝菌产生几丁质酶;除HDTP-3外,其余3株能诱导褐环粘盖牛肝菌产生几丁质酶,但产酶能力较低;HDTP-4诱导铆钉菇产几丁质酶能力最高。②4株菌根伴生真菌中,只有HDTP-5能诱导褐环粘盖牛肝菌产β-1,3-葡聚糖酶;除HDTP-4外,其它3株均能诱导粘盖牛肝菌和铆钉菇产生β-1,3-葡聚糖酶;4株菌根伴生真菌均能诱导褐黄牛肝菌产β-1,3-葡聚糖酶。③4株菌根伴生真菌均能诱导褐环粘盖牛肝菌、粘盖牛肝菌和褐黄牛肝菌产β-葡萄糖苷酶;而铆钉菇只被HDTP-1和HDTP-3诱导,且酶活性较低。④4株菌根伴生真菌诱导的外生菌根真菌所产的中性蛋白酶活性,均以褐黄牛肝菌最高,铆钉菇最低。⑤菌根伴生真菌极显著诱导褐环粘盖牛肝菌和粘盖牛肝菌的漆酶活性;显著诱导褐黄牛肝菌和铆钉菇漆酶活性。⑥菌根伴生真菌极显著诱导3种牛肝菌、显著诱导铆钉菇多酚氧化酶活性;除粘盖牛肝菌外,菌根伴生真菌诱导其它3种外生菌根真菌产多酚氧化酶活性无显著差异。4.外生菌根真菌和菌根伴生真菌对油松的接种效应外生菌根真菌铆钉菇和菌根伴生真菌能够通过影响油松根系活力、根系质膜相对透性、提高硝酸还原酶活性、增加叶绿素含量等方式,使地上与地下部分协同作用,促进油松生长。菌根化油松(无论只接铆钉菇或铆钉菇与菌根伴生真菌双接种)在总干重、根系活力、根系硝酸还原酶活性和叶绿素含量上显著高于非菌根化油松苗。但接种铆钉菇的油松和铆钉菇与菌根伴生真菌双接种的油松之间无差异,对照株和直接菌根伴生真菌植株之间也无差异。对促进油松苗木生长的不同外生菌根真菌和菌根伴生真菌最佳组合有待于进一步探讨。更多还原

【Abstract】 In this paper, Mycorrhizal-associated fungi were isolated by enrichment culture techniques from Pinus tabulaeformis and identificated, then the interaction between mycorrhizal-associated fungi and ectomycorrhizal fungi in co-culture and in the rhizosphere of P. tabulaeformis were observed by optical and electron microscopy techniques, the influence of autoclaved mycelia of mycorrhizal- associated fungi on the production of enzymes by four ECM fungal species , as well as the root activity, nitrate reductase activity etc. physiological indicators of P. tabulaeformis inoculated mycorrhizal-associated fungi and ectomycorrhizal fungi was grown in pots were determined. Mechanism of the interaction between mycorrhizal-associated fungi and ectomycorrhizal fungi was revealed, so as to provide a scientific basis for the guidance of agricultural production and analysis of interactions among ecosystem species. The main results were as follows:1 Identify the mycorrhiza-associated fungi of P. tabulaeformis Through morphology and molecular biology techniques, The two strains HDTP-1 and HDTP-3 were identified as Trichoderma harzianum in Trichoderma sp. of Moniliaceae family in Moniliales, Hyphomycetales, Deuteromycotina, While HDTP-4 and HDTP-5 belong to Mucor sp. of Mucorales family in Mucorales, Phycomycetes, Fungi, were Mucor hiemalis SA10-6, Mucor hiemalis XSD-98, respectively.2 The interaction between mycorrhizal-associated fungi and ectomycorrhizal fungi in co-culture and the rhizosphere of P. tabulaeformis Four kinds of ectomycorrhizal fungi could inhibited the growth of mycorrhizal-associated fungi, Moreover, ectomycorrhizal fungi coiled around and penetration of the hyphae into mycorrhizal-associated fungi, the hyphae of mycorrhizal-associated fungi were more branched or deformed or apical swelling. Four kinds of ectomycorrhizal fungi could grow in culture medium contain mycelia of mycorrhizal-associated fungi,Two month-old mycorrhizae of P. tabulaeformis/G. viscidus were inoculated with a conidial suspension of mycorrhiza-associated fungi and examined at intervals of 2, 3, 5, and 7 days post-inoculation (p.i.). On non-mycorrhizal roots, conidia germination was high and long hyphae formed 2 or 3 days p.i., whereas their germination was totally inhibited on mycorrhizal roots. At 5 days after inoculation, only sporangia were seen with mycelial mats firmLy attached to the roots by the mantle hyphae, whereas some remnants of sporangiophores, ruptured sporangial walls and degraded hyphae of mycorrhiza-associated fungi were overgrown by the mantle hyphae. During the next 2 days, On mycorrhizal roots, only mycelial debris and protoplast-like material were observed 7 days p.i.3 Influence of autoclaved mycorrhizal-associated fungal mycelia on enzymes in ectomycorrhizal fungi①Mycelia of 4 strains of mycorrhizal-associated fungi could inducechitinase in B. luridus and S. bovines. For the synthesis of chitinolytic enzyme by S. luteus, lower activity was observed in T. harzianum HDTP-1、M. hiemalis SA10-6 HDTP-4 and M. hiemalis XSD-98 HDTP-5, whereas M. hiemalis SA10-6 HDTP-4 turned out to be the best substrate for the induction of chitinase for G. viscidus.②For the synthesis ofβ-1, 3-glucanases by S. luteus in only M. hiemalis XSD-98 HDTP-5 was observed. Noβ-1, 3-glucanases was noted for S. bovines in the presence of the mycelium of M. hiemalis SA10-6 HDTP-4. Mycelia of 4 strains of mycorrhizal-associated fungi could induceβ-1, 3-glucanases in B. luridus③The activity ofβ-glucosidases was observed for S. luteus, B. luridus and S. bovines in the presence of mycelium of 4 strains of mycorrhizal- associated fungi. For the synthesis ofβ-glucosidases by G. viscidus, lower activity was observed in T. harzianum HDTP-1 and harzianum HDTP-3.④Among several strains of ectomycorrhizal fungi, B. luridus was characterized as the highest neutral proteolytic activity when induced by the mycelia of the four strains of mycorrhiza-associated fungi, while G. viscidus was the lowest.⑤For the synthesis of laccase by S. luridus and S. bovines in 4 strains of mycorrhiza-associated fungi were greatly higher than control, whereas higher than control for B. luridus and G. viscidus.⑥For the synthesis of polyphenol Oxidase by S. luridus and S. bovines in 4 strains of mycorrhiza-associated fungi were greatly higher than control, whereas higher than control for B. luridus and G. viscidus.4 The inoculation effect of ectomycorrhizal fungi and mycorrhizal-associated fungi on P. tabulaeformis Ectomycorrhizal fungi and mycorrhizal-associated fungi could activate soil nutrient and promote plant growth through improving root activity and nitrate reductase activity, changing root plasma membrane permeability, increasing total content of chlorophyll. these indicated that the promoting growth effect of ectomycorrhizal fungi and mycorrhizal-associated fungi on P. tabulaeformis was a comprehensive influence by above-ground and under-ground’s synergistic effect. Both mycorrhizal plants and mycorrhizal plants inoculated with mycorrhizal-associated fungi produced a significantly higher total biomass, root activity, nitrate reductase activity and total content of chlorophyll than non-mycorrhizal plants. However, there was no difference in the growth of mycorrhizal plants inoculated or uninoculated with mycorrhizal-associated fungi. There was also no significant difference in non-mycorrhizal plants grown in the presence of mycorrhizal-associated fungi and that of the control plants.更多还原