兰科菌根真菌的分离鉴定及其与中国兰属植物共生关系的研究
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摘要
兰科(Orchidaceae)是被子植物中最大科之一,是重要的观赏植物与药用植物。兰科菌根真菌的共生对于兰科植物种子萌发和植物生长是必不可少的,兰科植物的保护与产业化发展都离不开菌根真菌。兰科植物与菌根真菌的共生关系是专一性还是多样性,一直存在争议。菌根真菌的可培养分离与鉴定,可有针对性的收集兰科植物菌根真菌资源。菌根真菌研究非培养手段的开发与利用,有助于解决兰科植物菌根真菌分离鉴定困难的问题。对不同地域不同种类中国兰属植物菌根真菌的研究能为确定兰科植物与菌根真菌的共生关系提供新的论据。本论文主要研究结果归纳如下:
1、利用可培养手段从四川江津兔耳兰(Cymbidium lancifolium)中分离得到一株菌根真菌,经初步鉴定分析其为角担菌属(Ceratobasidium)真菌;从四川江津寒兰(Cymbidium kanran)中分离得到一株菌根真菌,经初步鉴定分析其为胶膜(Tulasnella)属真菌。
2、引物ITS1-OF/ITS4-OF、T2/ITS4、ITS1/ITS4-Tul、CeTh1/CeTh4和S1/ITS4均可用于菌根真菌非培养研究。利用胶膜菌属通用引物T2/ITS4以非培养手段研究浙江兰属植物菌根真菌的侵染率,引物T2/ITS4的扩增片段经测序、比对分析为胶膜菌属真菌rDNA ITS序列,侵染率的统计结果为50.79%,共生关系具属水平专一性与属内多样性。
3、对河南信阳兰属植物根状茎菌根真菌进行可培养研究,3株蕙兰中分离到15株不同内生真菌,从4株春兰中分离得到17株不同内生真菌,未分离得到常见菌根真菌。
4、利用担子菌通用引物ITS1-OF/ITS4-OF,RFLP与测序相结合的手段对河南信阳兰属植物根与根状茎内生担子菌多样性进行非培养研究,蕙兰根状茎中仅分离得到腊壳菌属(Sebacina)菌根真菌;蕙兰根中分离得到4株不同内生真菌;春兰根状茎中仅分离得到腊壳菌属(Sebacina)菌根真菌;春兰根中分离得到8株不同内生真菌,其中包括腊壳菌属(Sebacina)菌根真菌。
5、对湖北、浙江天目山、重庆金佛山、福建武夷山、河南信阳兰属植物进行菌根真菌的非培养研究,发现浙江天目山、重庆金佛山的春兰、蕙兰,湖北的春兰都与胶膜菌属真菌共生;只有河南信阳的春兰、蕙兰与腊壳菌属(Sebacina)菌根真菌共生,由此可见,部分地域的春兰、蕙兰多与胶膜菌属菌根真菌共生,共生关系在属水平上具显著专一性;部分地域春兰、蕙兰与其他菌根真菌共生,共生关系具有多样性。
Orchidaceae is one of the largest families of angiosperms, and it is very famous for its ornamental and medicinal values. Orchids have to rely on the formation of a stable symbiotic relationship with mycorrhizal fungi to complete the normal life cycle. The symbiotic relationship between Orchid and mycorrhizal fungi are specific or diverse have been always controversial. The cultured methods of mycorrhizal fungi can be targeted to collect orchid mycorrhizal fungi. The uncultured methods in the mycorrhizal fungi study can help to solve the difficulties of mycorrhizal fungi separation and identification. The mycorrhizal fungi study of different types and different regions Orchid plants, for determining symbiotic relationship between orchid and mycorrhizal fungi provide new evidence. The major findings are as follows:
1、With the cultured methods, a kind of mycorrhizal fungi was isolated from Sichuan Jiangjin Cymbidium lancifolium. Preliminary identification of the fungi as the Ceratobas-idium; another kind of mycorrhizal fungi was also isolated from the Sichuan Jiangjin C. kanran, and it was preliminary identificated as the Tulasnella.
2、Primers ITS1-OF/ITS4-OF, T2/ITS4, ITS1/ITS4-Tul, CeTh1/CeTh4 and S1/ITS4 all can be used for the uncultured study of mycorrhizal fungi. Using the uncultured meth-ods with universal primers T2/ITS4 to study the infection rate of Zhejiang Orchid Mycorrhizal, products of PCR with primers T2/ITS4 were sequenced and analyed. The results indicated that all these ITS sequences had highly homology with the Tulasnella’s rDNA, and the infection rate was 50.79%.
3、Cultured methods were carried out to analysis the mycorrhizal fungi of the rhizomorph from Henan Xinyang Orchid,and fifteen different endophytic fungus strains were isolated from three C. faberi, and seventeen endophytic fungus strains were isolated from four C. goeringii. All of the experiments did not obtain common mycorrhizal fungi.
4、Using universal Basidiomycetes primers ITS1-OF/ITS4-OF, RFLP and sequen-cing methods to study the diversity of endophytic basidiomycetes fungi in orchid roots and rhizomes from Henan Xinyang, from the rhizome of C. faberi we only got Sebacina mycorrhizal fungi, but from the roots of C. faberi we got four different species of endophytic fungus; from the rhizome of C. goeringii we only isolated Sebacina mycorrhizal fungi, and from the roots of C. goeringii we isolated eight different species of endophytic fungus, including Sebacina mycorrhizal fungi.
5、Uncultured methods were carried out in orchid plants to study mycorrhizal fungi. These plants came from Hubei, Zhejiang Tianmu Mountain, Chongqing Jinfo Mountain, Fujian Wuyi Mountain, Henan Xinyang. It found that C. faberi and C. goeringii from Tianmu Mountain in Zhejiang and Jinfo Mountain in Fujian, and C. goeringii from Hubei had the same symbiotic fungi, Tulasnella; only C. goeringii and C. faberi from Henan Xinyang symbioted with Sebacina. It indicated that most of C. goeringii and C. faberi from different districts symbioted with Tulasnella, and only a few areas of that symbioted with Sebacina. It displayed that symbiotic relationship had a significant specificity at the level of genera.
1、利用可培养手段从四川江津兔耳兰(Cymbidium lancifolium)中分离得到一株菌根真菌,经初步鉴定分析其为角担菌属(Ceratobasidium)真菌;从四川江津寒兰(Cymbidium kanran)中分离得到一株菌根真菌,经初步鉴定分析其为胶膜(Tulasnella)属真菌。
2、引物ITS1-OF/ITS4-OF、T2/ITS4、ITS1/ITS4-Tul、CeTh1/CeTh4和S1/ITS4均可用于菌根真菌非培养研究。利用胶膜菌属通用引物T2/ITS4以非培养手段研究浙江兰属植物菌根真菌的侵染率,引物T2/ITS4的扩增片段经测序、比对分析为胶膜菌属真菌rDNA ITS序列,侵染率的统计结果为50.79%,共生关系具属水平专一性与属内多样性。
3、对河南信阳兰属植物根状茎菌根真菌进行可培养研究,3株蕙兰中分离到15株不同内生真菌,从4株春兰中分离得到17株不同内生真菌,未分离得到常见菌根真菌。
4、利用担子菌通用引物ITS1-OF/ITS4-OF,RFLP与测序相结合的手段对河南信阳兰属植物根与根状茎内生担子菌多样性进行非培养研究,蕙兰根状茎中仅分离得到腊壳菌属(Sebacina)菌根真菌;蕙兰根中分离得到4株不同内生真菌;春兰根状茎中仅分离得到腊壳菌属(Sebacina)菌根真菌;春兰根中分离得到8株不同内生真菌,其中包括腊壳菌属(Sebacina)菌根真菌。
5、对湖北、浙江天目山、重庆金佛山、福建武夷山、河南信阳兰属植物进行菌根真菌的非培养研究,发现浙江天目山、重庆金佛山的春兰、蕙兰,湖北的春兰都与胶膜菌属真菌共生;只有河南信阳的春兰、蕙兰与腊壳菌属(Sebacina)菌根真菌共生,由此可见,部分地域的春兰、蕙兰多与胶膜菌属菌根真菌共生,共生关系在属水平上具显著专一性;部分地域春兰、蕙兰与其他菌根真菌共生,共生关系具有多样性。
Orchidaceae is one of the largest families of angiosperms, and it is very famous for its ornamental and medicinal values. Orchids have to rely on the formation of a stable symbiotic relationship with mycorrhizal fungi to complete the normal life cycle. The symbiotic relationship between Orchid and mycorrhizal fungi are specific or diverse have been always controversial. The cultured methods of mycorrhizal fungi can be targeted to collect orchid mycorrhizal fungi. The uncultured methods in the mycorrhizal fungi study can help to solve the difficulties of mycorrhizal fungi separation and identification. The mycorrhizal fungi study of different types and different regions Orchid plants, for determining symbiotic relationship between orchid and mycorrhizal fungi provide new evidence. The major findings are as follows:
1、With the cultured methods, a kind of mycorrhizal fungi was isolated from Sichuan Jiangjin Cymbidium lancifolium. Preliminary identification of the fungi as the Ceratobas-idium; another kind of mycorrhizal fungi was also isolated from the Sichuan Jiangjin C. kanran, and it was preliminary identificated as the Tulasnella.
2、Primers ITS1-OF/ITS4-OF, T2/ITS4, ITS1/ITS4-Tul, CeTh1/CeTh4 and S1/ITS4 all can be used for the uncultured study of mycorrhizal fungi. Using the uncultured meth-ods with universal primers T2/ITS4 to study the infection rate of Zhejiang Orchid Mycorrhizal, products of PCR with primers T2/ITS4 were sequenced and analyed. The results indicated that all these ITS sequences had highly homology with the Tulasnella’s rDNA, and the infection rate was 50.79%.
3、Cultured methods were carried out to analysis the mycorrhizal fungi of the rhizomorph from Henan Xinyang Orchid,and fifteen different endophytic fungus strains were isolated from three C. faberi, and seventeen endophytic fungus strains were isolated from four C. goeringii. All of the experiments did not obtain common mycorrhizal fungi.
4、Using universal Basidiomycetes primers ITS1-OF/ITS4-OF, RFLP and sequen-cing methods to study the diversity of endophytic basidiomycetes fungi in orchid roots and rhizomes from Henan Xinyang, from the rhizome of C. faberi we only got Sebacina mycorrhizal fungi, but from the roots of C. faberi we got four different species of endophytic fungus; from the rhizome of C. goeringii we only isolated Sebacina mycorrhizal fungi, and from the roots of C. goeringii we isolated eight different species of endophytic fungus, including Sebacina mycorrhizal fungi.
5、Uncultured methods were carried out in orchid plants to study mycorrhizal fungi. These plants came from Hubei, Zhejiang Tianmu Mountain, Chongqing Jinfo Mountain, Fujian Wuyi Mountain, Henan Xinyang. It found that C. faberi and C. goeringii from Tianmu Mountain in Zhejiang and Jinfo Mountain in Fujian, and C. goeringii from Hubei had the same symbiotic fungi, Tulasnella; only C. goeringii and C. faberi from Henan Xinyang symbioted with Sebacina. It indicated that most of C. goeringii and C. faberi from different districts symbioted with Tulasnella, and only a few areas of that symbioted with Sebacina. It displayed that symbiotic relationship had a significant specificity at the level of genera.
引文
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