中华羊茅内生真菌及其对宿主抗寒性的影响
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摘要
本研究以我国高寒草原优良牧草中华羊茅(Festuca sinensis Keng)为研究对象,通过实验室、盆栽与田间试验相结合的方法,对中华羊茅内生真菌的形态多样性、系统发育分析及内生真菌对中华羊茅抗寒性的影响等方面进行了研究。主要结果如下：
1.以采自甘肃夏河桑科草原和甘加草原的中华羊茅为材料,通过对种子和茎髓部内生真菌带菌的检测,并在PDA培养基上进行分离培养,共得到48个内生真菌菌株。经水琼脂培养基次级培养以促进产孢,依据菌落生长形态、分生孢子、分生孢子梗等形态学特征的观察与测定,可将其分为Neotyphodium属内生真菌的三个形态群,其中：13个菌株属ZHYM-I形态群,20个菌株属ZHYM-II形态群,15个菌株属ZHYM-III形态群。分离自中华羊茅的48个内生真菌菌株表现出了丰富的形态学多样性。
2.对代表性的中华羊茅内生真菌菌株的tefA和tubB基因序列进行了克隆测序,并对比已建立的种进行了系统发育学分析,结果表明分离得到的中华羊茅内生真菌均为Neotyphodium属内生真菌,在分子学角度印证了形态学结果。
3.通过不同低温胁迫条件下中华羊茅种子的萌发试验,明确了内生真菌可以提高中华羊茅种子的发芽率、发芽速度、促进胚根、胚芽的生长,在5℃和10℃低温胁迫条件下,中华羊茅带菌(E+)植株的发芽率、发芽指数、胚芽和胚根长显著高于不带菌(E-)植株(p<0.05)。
4.通过对中华羊茅幼苗的低温胁迫试验,明确了内生真菌能够显著地增加植株的分蘖和地下生物量的积累(p<0.05),增强植株的抗寒能力。发现其可溶性糖含量、可溶性蛋白含量、脯氨酸、POD、SOD、CAT酶活性、Fv/Fm值也高于E-植株(p<0.05)。
5.通过对中华羊茅成株的田间自然越冬试验,明确了内生真菌能够明显促进E+植株的越冬存活率和返青再生能力。
Chinese fescue (Festuca sinensis Keng), a good quality forage in alpine region of Qinghai-Tibeten Plateau of China was studied, mainly including morphological diversity, phylogenetic analysis and effects of Neotyphodium endophyte infection on cold tolerance to Chinese fescue. The results summarized as followings:
1. All of the Chinese fescue samples tested in the study were collected from Sangke and Ganga grassland of Gansu provience. A total of 48 endophytic fungal strains were isolated and purified from seeds. Based on the observation and measurement of the morphological characteristics of appearance of colony, conidia and conidiophores, all isolates were classified into 3 types, i.e.:13 strains of them were belonged to the morphological Neotyphodium group ZHYM-Ⅰ,20 were ZHYM-Ⅱ,15 were ZHYM-Ⅲ. A significant diversity were observed among these 48 strains in morphology.
2. DNA fragment of translation elongation factor 1-αgene (tefA) introns andβ-tubulin gene (tubB) introns were cloned and sequenced. Sequences from representative strains of Neotyphodium spp. obtained from GenBank were aligned with the sequences found in this study. The preliminary results show that the isolates obtained from Chinese fescue were belonged to Neotyphodium.
3. Germination testing indicated that endophyte infection could increase the germination rate, accelerate germination speed and promote growth of radicle and embryo of seeds. Especially under 10℃stress, the germination rate, germination index and length of radicle and embryo of endophyte infected F. sinensis were significantly higher than those of endophyte free seedlings (p<0.05).
4. Stress experiment at low temperature indicated that, compared with the endopyte-free seedlings, endophyte infection could significantly increase tiller number and underground biomass accumulation of seedling (p<0.05), could significantly increase soluble sugar content, protein content, praline, the POD, SOD, CAT activity and maximal photochemical efficiency of PSⅡ(p<0.05), performed strong cold tolerance.
5. The result of the over-wintering experiment showed that Neotyphodium endopyte infection could enhance the winter survival rate and spring re-growth capability of the plant.
1.以采自甘肃夏河桑科草原和甘加草原的中华羊茅为材料,通过对种子和茎髓部内生真菌带菌的检测,并在PDA培养基上进行分离培养,共得到48个内生真菌菌株。经水琼脂培养基次级培养以促进产孢,依据菌落生长形态、分生孢子、分生孢子梗等形态学特征的观察与测定,可将其分为Neotyphodium属内生真菌的三个形态群,其中：13个菌株属ZHYM-I形态群,20个菌株属ZHYM-II形态群,15个菌株属ZHYM-III形态群。分离自中华羊茅的48个内生真菌菌株表现出了丰富的形态学多样性。
2.对代表性的中华羊茅内生真菌菌株的tefA和tubB基因序列进行了克隆测序,并对比已建立的种进行了系统发育学分析,结果表明分离得到的中华羊茅内生真菌均为Neotyphodium属内生真菌,在分子学角度印证了形态学结果。
3.通过不同低温胁迫条件下中华羊茅种子的萌发试验,明确了内生真菌可以提高中华羊茅种子的发芽率、发芽速度、促进胚根、胚芽的生长,在5℃和10℃低温胁迫条件下,中华羊茅带菌(E+)植株的发芽率、发芽指数、胚芽和胚根长显著高于不带菌(E-)植株(p<0.05)。
4.通过对中华羊茅幼苗的低温胁迫试验,明确了内生真菌能够显著地增加植株的分蘖和地下生物量的积累(p<0.05),增强植株的抗寒能力。发现其可溶性糖含量、可溶性蛋白含量、脯氨酸、POD、SOD、CAT酶活性、Fv/Fm值也高于E-植株(p<0.05)。
5.通过对中华羊茅成株的田间自然越冬试验,明确了内生真菌能够明显促进E+植株的越冬存活率和返青再生能力。
Chinese fescue (Festuca sinensis Keng), a good quality forage in alpine region of Qinghai-Tibeten Plateau of China was studied, mainly including morphological diversity, phylogenetic analysis and effects of Neotyphodium endophyte infection on cold tolerance to Chinese fescue. The results summarized as followings:
1. All of the Chinese fescue samples tested in the study were collected from Sangke and Ganga grassland of Gansu provience. A total of 48 endophytic fungal strains were isolated and purified from seeds. Based on the observation and measurement of the morphological characteristics of appearance of colony, conidia and conidiophores, all isolates were classified into 3 types, i.e.:13 strains of them were belonged to the morphological Neotyphodium group ZHYM-Ⅰ,20 were ZHYM-Ⅱ,15 were ZHYM-Ⅲ. A significant diversity were observed among these 48 strains in morphology.
2. DNA fragment of translation elongation factor 1-αgene (tefA) introns andβ-tubulin gene (tubB) introns were cloned and sequenced. Sequences from representative strains of Neotyphodium spp. obtained from GenBank were aligned with the sequences found in this study. The preliminary results show that the isolates obtained from Chinese fescue were belonged to Neotyphodium.
3. Germination testing indicated that endophyte infection could increase the germination rate, accelerate germination speed and promote growth of radicle and embryo of seeds. Especially under 10℃stress, the germination rate, germination index and length of radicle and embryo of endophyte infected F. sinensis were significantly higher than those of endophyte free seedlings (p<0.05).
4. Stress experiment at low temperature indicated that, compared with the endopyte-free seedlings, endophyte infection could significantly increase tiller number and underground biomass accumulation of seedling (p<0.05), could significantly increase soluble sugar content, protein content, praline, the POD, SOD, CAT activity and maximal photochemical efficiency of PSⅡ(p<0.05), performed strong cold tolerance.
5. The result of the over-wintering experiment showed that Neotyphodium endopyte infection could enhance the winter survival rate and spring re-growth capability of the plant.
引文
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