兰科石斛属植物根部内生真菌多样性研究及应用
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摘要
石斛属(Dendrobium)是兰科植物最大的属,具有重要的观赏药用价值,自然条件下需与真菌共生才能生长。本研究分析了12种石斛属植物根部内生真菌的多样性,并以铁皮石斛为材料,利用组织培养技术获得优质移栽用苗,将3株优良的菌根真菌制成液体单一菌剂,考察不同菌剂对铁皮石斛移栽生长的影响。通过研究主要得到以下结论：
(1)利用3种方法和3种分离培养基对广西雅长自然保护区和贵州兴义温室内的12种野生石斛属植物根部内生真菌进行分离培养,得到内生真菌152株,经形态鉴定和分子鉴定归属为3门9纲22个分类单元。对比不同方法和培养基分离内生真菌的多样性,点样法和FIM培养基分离内生真菌Shannon-Wiener多样性分别为2.79、2.74,远高于其他方法和培养基。对比不同生境及不同附生型的同种石斛内生真菌多样性,显示同种植物广西生境的内生真菌多样性明显高于贵州生境,树附生型明显高于石附生型。说明,地理环境的复杂程度在一定程度上影响着石斛属植物内生真菌的分布。
(2)铁皮石斛生根壮苗及培养基筛选。具有1-2片叶片的铁皮石斛分化苗在MS+0.34g/L KNO3+100g/L土豆提取液+2.4g/L植物凝胶+30g/L蔗糖培养基上生长11周后,平均株高、茎粗、鲜重的增长量分别为3.61cm.4.061mm2.6669g,平均产生新根12.55条、新孽6.20个,平均新根长2.03cm,极显著高于其他两种培养基,是铁皮石斛最佳生根壮苗培养基。
(3)铁皮石斛组培苗的最佳移栽基质筛选。经生根壮苗的铁皮石斛在珍珠岩1cm+仙土1cm+松树皮1cm+活苔藓2cm的基质上移栽90d后,成活率达75.00%,平均鲜重、株高、茎粗增长量分别为108mg、7.50mm、0.260mm,平均新根数1.43条,高于基质1/3花生壳+1/3石灰岩+1/3蛭石+活苔藓2cm,是铁皮石斛移栽的最佳基质。
(4)铁皮石斛组培苗移栽接菌试验。将3株优良兰科菌根真菌制成4g/L的单一液体菌剂,对铁皮石斛进行蘸根、灌根接菌移栽试验。90d后3种菌剂处理的铁皮石斛组培苗移栽成活率、新根、新芽、平均株高、茎粗、鲜重增长量均极显著或显著高于对照,且同一菌剂蘸根处理生长指标均高于灌根处理。说明3株真菌制成的液体菌剂及蘸根方式在铁皮石斛移栽生长中具有良好的应用前景。
Dendrobium is one of the largest genus in Orchid family, which has high economic values as both famous ornamental and medicinal plants. In nature, it needs to associate with fungi for growth and reproduction. In t is study, endophytic fungi from roots of12species of Dendrobium were isolated and identified.Through tissue cultured seedlings, the effects of3strains of orchid mycorrhizal fungi on Dendrobium officinale was studied. The main results are as follows:
(1)152strains of endophytic fungal were isolated from roots of12Dendrobium species in Guangxi and Guizhou habitats with three kinds of separation methods and three kinds of media. Using morphological and ITS rDNA molecular identification method, these fungi were identified into22orders,9classes,3phylum. Comparing the diversity of endophytic fungi isolated by different methods and media, the Shannon-Wiener diversity of spotting method and FIM medium reached2.79and2.54separately. Comparing the endophytic fungi of the same Dendrobium species isolated from different habitats and epiphytic types, the result showed that the diversity of endophytic fungi in Guangxi habitat was much higher than Guizhou, and the in-tree-epiphytic type was higher than that of lithophytic type. The result showed that habitats and epiphytic type can affect the diversity of endophytic fungi of Dendrobium.
(2) Screened the optimum medium for strengthen-seedling and rooting of D. officinale. Seedlings of D. officinale with1-2leaves were cultivated after11weeks in three kinds of media, and the seedlings growed on MS+0.34g/L KNO3+100g/L potato extract+2.4g/L plant gel+30g/L sucrose were better. The average height, stem diameter, fresh weight increased3.61cm,4.06mm,2.67mm respectively. The new roots and new buds were12.55and6.20respectively, the average length of new roots was2.03cm, significantly higher than the other two media.
(3) Screened the optimum medium for transplanting of D. officinalc. After90d, the survival rate of seedlings on the medium which contained perlite1cm, cents soil1cm, pine bark1cm and fresh moss2cm reached75.00%, the average fresh weight, plants'height, stem diameter increased108mg.7.50mm,0.260mm respectively. The average number of new roots was1.43, higher than the medium which contained1/3cm peanut shells,1/3cm limestone,1/3cm vermiculite and fresh moss2cm.
(4) Screened the optimum fungal inoculum and the inoculating method for transplanting of D. officinalc seedling. Three strains of mycorrhizal fungi were made into single liquid fungal inoculum and the concentration was4g/L. There were two inoculating methods for each mycorrhizal fungus:dipping and irrigating. After90d, the transplanting survival rate of tissue culture seedlings, the number of new roots, the rate of shooting, the average of plants'height, stem diameter, fresh weight deal with3kinds of mycorrhizal fungi were extremely higher than the controls. Comparing different inoculating method for the same fungus, dipping treatment was higher than irrigating treatment. The result showed that three strains of fungi with dipping treatment had a good application prospect for transplanting of D. officinale seedling.
(1)利用3种方法和3种分离培养基对广西雅长自然保护区和贵州兴义温室内的12种野生石斛属植物根部内生真菌进行分离培养,得到内生真菌152株,经形态鉴定和分子鉴定归属为3门9纲22个分类单元。对比不同方法和培养基分离内生真菌的多样性,点样法和FIM培养基分离内生真菌Shannon-Wiener多样性分别为2.79、2.74,远高于其他方法和培养基。对比不同生境及不同附生型的同种石斛内生真菌多样性,显示同种植物广西生境的内生真菌多样性明显高于贵州生境,树附生型明显高于石附生型。说明,地理环境的复杂程度在一定程度上影响着石斛属植物内生真菌的分布。
(2)铁皮石斛生根壮苗及培养基筛选。具有1-2片叶片的铁皮石斛分化苗在MS+0.34g/L KNO3+100g/L土豆提取液+2.4g/L植物凝胶+30g/L蔗糖培养基上生长11周后,平均株高、茎粗、鲜重的增长量分别为3.61cm.4.061mm2.6669g,平均产生新根12.55条、新孽6.20个,平均新根长2.03cm,极显著高于其他两种培养基,是铁皮石斛最佳生根壮苗培养基。
(3)铁皮石斛组培苗的最佳移栽基质筛选。经生根壮苗的铁皮石斛在珍珠岩1cm+仙土1cm+松树皮1cm+活苔藓2cm的基质上移栽90d后,成活率达75.00%,平均鲜重、株高、茎粗增长量分别为108mg、7.50mm、0.260mm,平均新根数1.43条,高于基质1/3花生壳+1/3石灰岩+1/3蛭石+活苔藓2cm,是铁皮石斛移栽的最佳基质。
(4)铁皮石斛组培苗移栽接菌试验。将3株优良兰科菌根真菌制成4g/L的单一液体菌剂,对铁皮石斛进行蘸根、灌根接菌移栽试验。90d后3种菌剂处理的铁皮石斛组培苗移栽成活率、新根、新芽、平均株高、茎粗、鲜重增长量均极显著或显著高于对照,且同一菌剂蘸根处理生长指标均高于灌根处理。说明3株真菌制成的液体菌剂及蘸根方式在铁皮石斛移栽生长中具有良好的应用前景。
Dendrobium is one of the largest genus in Orchid family, which has high economic values as both famous ornamental and medicinal plants. In nature, it needs to associate with fungi for growth and reproduction. In t is study, endophytic fungi from roots of12species of Dendrobium were isolated and identified.Through tissue cultured seedlings, the effects of3strains of orchid mycorrhizal fungi on Dendrobium officinale was studied. The main results are as follows:
(1)152strains of endophytic fungal were isolated from roots of12Dendrobium species in Guangxi and Guizhou habitats with three kinds of separation methods and three kinds of media. Using morphological and ITS rDNA molecular identification method, these fungi were identified into22orders,9classes,3phylum. Comparing the diversity of endophytic fungi isolated by different methods and media, the Shannon-Wiener diversity of spotting method and FIM medium reached2.79and2.54separately. Comparing the endophytic fungi of the same Dendrobium species isolated from different habitats and epiphytic types, the result showed that the diversity of endophytic fungi in Guangxi habitat was much higher than Guizhou, and the in-tree-epiphytic type was higher than that of lithophytic type. The result showed that habitats and epiphytic type can affect the diversity of endophytic fungi of Dendrobium.
(2) Screened the optimum medium for strengthen-seedling and rooting of D. officinale. Seedlings of D. officinale with1-2leaves were cultivated after11weeks in three kinds of media, and the seedlings growed on MS+0.34g/L KNO3+100g/L potato extract+2.4g/L plant gel+30g/L sucrose were better. The average height, stem diameter, fresh weight increased3.61cm,4.06mm,2.67mm respectively. The new roots and new buds were12.55and6.20respectively, the average length of new roots was2.03cm, significantly higher than the other two media.
(3) Screened the optimum medium for transplanting of D. officinalc. After90d, the survival rate of seedlings on the medium which contained perlite1cm, cents soil1cm, pine bark1cm and fresh moss2cm reached75.00%, the average fresh weight, plants'height, stem diameter increased108mg.7.50mm,0.260mm respectively. The average number of new roots was1.43, higher than the medium which contained1/3cm peanut shells,1/3cm limestone,1/3cm vermiculite and fresh moss2cm.
(4) Screened the optimum fungal inoculum and the inoculating method for transplanting of D. officinalc seedling. Three strains of mycorrhizal fungi were made into single liquid fungal inoculum and the concentration was4g/L. There were two inoculating methods for each mycorrhizal fungus:dipping and irrigating. After90d, the transplanting survival rate of tissue culture seedlings, the number of new roots, the rate of shooting, the average of plants'height, stem diameter, fresh weight deal with3kinds of mycorrhizal fungi were extremely higher than the controls. Comparing different inoculating method for the same fungus, dipping treatment was higher than irrigating treatment. The result showed that three strains of fungi with dipping treatment had a good application prospect for transplanting of D. officinale seedling.
引文
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