林木促生抗逆优良菌根真菌的作用机制及应用技术
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菌根是自然界中一种植物-菌物共生现象，很多树种缺少菌根就不能成活或生长很差。松树和杨树是我国的主要造林树种，亦为菌根依赖型树种。本项目自2004年立项以来，针对我国大面积栽植的松树和及杨树在自然条件下因菌根形成少，不能充分利用和发挥土壤肥力而导致长势弱、易发生病虫害这一生产难题，开展了相关研究工作，取得了以下研究成果。 1、从江苏、云南和我国北方等地调查收集了松树和杨树外生菌根真菌，建立了林木外生菌根真菌种质资源库。筛选出了对松树和杨树具促生抗逆作用的优良菌根真菌6种和5种，其平均促生效果分别为18％和18%。 2、通过探讨菌根化根系构型及其解剖结构、养分吸收及代谢、光合效率、激素分泌等与促生作用的关系，揭示了优良外生菌根真菌对松树杨树的促生机理。促生效果显著的菌根化松树具有较小的中柱、较厚的皮层和密集的哈氏网。菌套可形成拟薄壁组织，有利于菌根共生体功能的发挥。菌根中真菌的管状液泡系统及其活力与对黑松的促生作用相关。菌根真菌通过提高叶片实际光化学效能促进植株碳水化合物积累。优良外生菌根真菌分泌的玉米素与杨树生长呈显著正相关。 3、通过研究细胞膜保护酶、内源多胺、根部差异表达蛋白等变化状况，阐明了外生菌根真菌对松树杨树的抗旱机理。菌根真菌提高了马尾松相对膜透性的耐受阈值 降低了MDA含量 POD活性及变化与植株抗旱能力成正相关。菌根能提高马尾松植株内源Spm含量和Put的降解能力，调节各种内源多胺的分配比例，维持较高的（Spd+Spm）/Put值，进而提高松树抗旱能力。菌根化马尾松根部差异表达蛋白种类变化明显，质谱分析表明，Pschi4、1，4-苯醌还原酶、核糖体L16蛋白等在松树抗旱机制中可能起关键作用。 4、通过分析根系离子吸收平衡及解毒机制、离子微域分布与抗盐作用的关系，明确了外生菌根真菌对松树杨树的抗盐机理。采用离子发射光谱测定发现外生菌根真菌提高杨树耐盐性与植株提高K、P含量，降低Na+含量，从而减少Na+的毒害作用有关。运用能谱分析仪及扫描电镜发现盐胁迫下根尖横切面P元素含量从外到内呈先升后降趋势，在皮层和中柱内含量最高，这有助于促进Na+和Cl-向植物体各部位运输，通过老叶脱落减少体内的盐分含量。 5、建立了林木外生菌根真菌扩繁、发酵工艺与保存技术。
Mycorrhiza is a ubiquitous plant-fungus symbiosis in nature; many tree species cannot survive or grow poorly without mycorrhizae. Pinus and Populus are major afforestation tree species in China, and they are also mycorrhiza-dependent tree species. Since the project was initiated in 2004, we have conducted targeted research to address the practical production challenge that large-scale planted Pinus and Populus in China grow weakly and are prone to pests and diseases due to scarce natural mycorrhizal formation, which prevents them from fully utilizing soil fertility. The following research achievements have been obtained. 1. We surveyed and collected ectomycorrhizal fungi associated with Pinus and Populus from regions including Jiangsu, Yunnan and northern China, and established a germplasm resource bank of forest tree ectomycorrhizal fungi. We screened out 6 and 5 excellent mycorrhizal fungal strains with growth-promoting and stress-resistant effects for Pinus and Populus respectively, with an average growth-promoting effect of 18% for both. 2. We revealed the growth-promoting mechanism of excellent ectomycorrhizal fungi on Pinus and Populus by exploring the correlations between mycorrhizal root system architecture, its anatomical structure, nutrient uptake and metabolism, photosynthetic efficiency, hormone secretion and other traits, and the growth-promoting effect. Mycorrhizal Pinus with significant growth-promoting effects has a smaller stele, thicker cortex and dense Hartig net. The fungal mantle can form pseudoparenchyma, which facilitates the function of the mycorrhizal symbiont. The tubular vacuole system and its activity of fungi in mycorrhizae are correlated with the growth-promoting effect on Pinus thunbergii. Mycorrhizal fungi promote plant carbohydrate accumulation by improving the actual photochemical efficiency of leaves. The zeatin secreted by excellent ectomycorrhizal fungi shows a significant positive correlation with Populus growth. 3. We clarified the drought resistance mechanism of ectomycorrhizal fungi on Pinus and Populus by studying the changes in cell membrane protective enzymes, endogenous polyamines, root differentially expressed proteins and other indicators. Mycorrhizal fungi increased the tolerance threshold of relative membrane permeability in Pinus massoniana and reduced the malondialdehyde (MDA) content. Peroxidase (POD) activity and its changes are positively correlated with plant drought resistance. Mycorrhizae can increase the endogenous spermine (Spm) content and putrescine (Put) degradation ability of Pinus massoniana plants, regulate the distribution ratio of various endogenous polyamines, maintain a high (spermidine (Spd) + spermine (Spm))/putrescine (Put) ratio, thereby improving the drought resistance of Pinus. The types of differentially expressed proteins in the roots of mycorrhizal Pinus massoniana changed significantly. Mass spectrometry analysis showed that Pschi4, 1,4-benzoquinone reductase, ribosomal L16 protein and other proteins may play key roles in the drought resistance mechanism of Pinus. 4. We clarified the salt resistance mechanism of ectomycorrhizal fungi on Pinus and Populus by analyzing the correlations between root ion absorption balance and detoxification mechanism, ionic microdomain distribution and salt resistance effect. Using ion emission spectrometry, we found that the improved salt tolerance of Populus by ectomycorrhizal fungi is related to the plant's increased K and P contents and reduced Na+ content, thereby reducing the toxic effect of Na+. Using energy dispersive spectrometer (EDS) and scanning electron microscope (SEM), we found that under salt stress, the P content in the cross-section of root tips showed a trend of first increasing and then decreasing from the outside to the inside, with the highest content in the cortex and stele, which helps promote the transport of Na+ and Cl- to various parts of the plant, and reduces the salt content in the plant through the abscission of old leaves. 5. We established the propagation, fermentation process and preservation technology of forest tree ectomycorrhizal fungi.
