不同生态区苜蓿根瘤菌对宿主竞争结瘤能力变异的研究
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摘要
本研究从新疆、重庆和北京3个地区收集了7个苜蓿品种，其中新疆3个，分别是新牧1号(MⅠ)、新牧2号(MⅡ)和新疆野生苜蓿(MX)，重庆3个，分别是西南农业大学育成的西农1号(MC)、耐湿苜蓿品系(MH)、耐酸苜蓿品系(MA)，北京1个(地方品种，MB)。从每个品种的植株上各取一个根瘤，分离出7个纯化的根瘤菌菌株，其菌株分别称为野生型Ⅰ、Ⅱ、X、C、H、A和B菌株。然后，对这7个菌株进行耐药性检测，用抗生素对其标记，筛选并选取了抗链霉素、新霉素、壮观霉素和卡那霉素的突变菌株4个，分别是H~+Str、A~+Neo、I~+Spe和X~+Kan。经检测，突变菌株的竞争结瘤能力和固氮能力与相应的野生型根瘤菌菌株相同，它们能够代替野生型菌株来做竞争结瘤和固氮能力试验。本研究共进行了2个试验，其一是竞争结瘤试验，即把4个菌株配制成的菌液等数量混合后接种到七个不同品种的苜蓿上，在4个不同培养条件下培养40d后，取出苜蓿植株，每株分别取根瘤5个，用于鉴定根瘤内根瘤菌菌株的种类，计算各菌株的占瘤率和双感染或多感染的发生率；其二是固氮能力试验，即把四个菌株分别单独接种到7个苜蓿品种上，培养40d后，收获植株，烘干至恒重，称量单株苜蓿总干重。试验结果如下：
在常规、高湿和酸性培养条件下，根瘤菌本身的基因型是影响竞争结瘤能力的主要因素，而苜蓿基因型以及苜蓿品种与根瘤菌菌株相互作用对竞争结瘤能力几乎没有影响。各供试菌株的竞争结瘤能力在这三种培养条件下表现一致，但菌株间存在显著差异，其中X~+Kan的竞争结瘤能力显著小于其它三个菌株。高湿和酸性培养条件不能改变影响根瘤菌竞争结瘤能力的因素。但在干旱培养条件下，根瘤菌的基因型和苜蓿品种与根瘤菌菌株互作影响根瘤菌的竞争结瘤能力，而苜蓿基因型对其没有影响。也就是说只有干旱胁迫能改变影响根瘤菌竞争结瘤的因素，并且显著地提高I~+Spe的竞争结瘤能力，使I~+Spe的竞争结瘤能力显著高于其它三个菌株。培养条件对竞争结瘤能力也没有作用。
在试验一中同时发现苜蓿根瘤菌有较高的双感染或多感染发生频率；各种逆境培养条件均能提高双感染或多感染的发生率。在常规培养条件和高湿培养条件下，影响根瘤菌双感染或多感染发生率的因素主要是苜蓿基因型和根瘤菌菌株，与苜蓿品种与根瘤菌菌株相互作用没有关系；在干旱条件下，只有苜蓿基因型才能影响根瘤菌的双感染或多感染的发生率；在酸性条件下，苜蓿基因型、根瘤菌以及苜蓿品种与根瘤菌菌株相互作用对根瘤菌的双感染或多感染的发生率都没有影响，此时双感染或多感染起作用的可能是培养条件。培养条件是否影响苜蓿根瘤菌的双感染或多感染的发生率依苜蓿品种而定，在苜蓿MB和MI两个品种上，生态条件能改变根瘤菌的双感染或多感染的发生率；在其它5个品种苜蓿上，培养条件不能改变根瘤菌的双感染或多感染的发生率。
试验二的研究结果表明，苜蓿品种、根瘤菌菌株以及苜蓿品种×根瘤菌菌株相互作用都能影响根瘤菌的固氮能力。在试验条件下，所有的菌株在MC植株上都表现出强的固氮能力；
西南农业人学缺{学位论文摘要
................
菌株XK*m在不卜刁的汀怡品种卜都表现出了较强的固集吃能力，但是在较好的苗楷「、甘，种MC卜表
现出的囚氮能力却不是最好;在品种与lyl株的红_作效应中，尤火ISI，e织.合的}占}氮能力比其
’自夕}{含都高，M 1 xXK:、;:约介的11钊氮能力同样较.岛，这两个组合的亲和性比较好，按种同源
根瘤菌的首讼样t株其}占{氮能力井不一定强，1).1此认为1个具有较强】占1氮能)]的科妙如翔钧株){
不一定源自同源的首信品种。
菌株XK;。rl的}古}氮能力显著高丁·菌株ANe。和H Str，也高一于菌株工Sp。们芳异不显著，
菌株A Ne。、H众:和ISpe之间的固氮能力没有显著差异;但是菌株XK二竟争结瘤能力最补，
该菌株在各酉拾，W，种上的!If瘤率显著小于菌株ANeo、HSt和1 Spe，而菌株A Noo、日出和!
渝。〔、的}}.习留率没子J’显著斧异。竟争结瘤能力差的根瘤菌菌株的固氮能力强，这，，丁能’、根瘤菌
携带的}占{氮能力川)c1和竟争结瘤华因是连锁的有关。
Seven alfalfa varieties in the study were collected from three kind of different ecological region, in which 3 varieties from Xinjing, respectively Xinmu No. 1 (MI), Xinmu No.2 (MII) and Xinjiang wild variety (MX) , 3 from Chongqing, Xinong No. 1 (MC) , a variety of resistant humid (MH) and a variety of resistant acid (MA), and one local variety (MB) from Beijing. Seven rhizobium meliloti strains were separated and purified from the plant of these varieties. These wild type strains were marked as I , II, X, C, H, A and B strains. And after the antibiotic resistance were examined, four antibiotic mutant strains from corresponding ones, H+Str, A+Neo, II +Spe and X+Kan, were obtained by the way of experiment of strains labeled with 4 kinds of antibiotics. The mutant strains were employed to carry out all of experiments because they and their corresponding wild strains had the same capacity of nodulation and fixation nitrogen representing their wilding strains. Two experiments in the study were carried out. The
first was the research of nodulation competition of rhizobium meliloti strains. The strains solutions, mixed equally with amount of the four mutant strains, were inoculated in seven alfalfa varieties. The plants were cultured in four different culture conditions. After 40d, five nodules from per plant were extracted to identify the kind of rhizobia and their occupancy in the same nodule, while the nodule occupancy frequency and double infection or excessive infection frequency of each strain were calculated. Another one was studied for the nitrogen fixation capability of different combination of host plant varieties × rhizobium strain. Seven alfalfa varieties were separately inoculated four rhizobium strains. The nitrogen fixation capability was measured by the dry weight of per plant after 40d. The results were as follows.
Rhizobium's genotype is the key factor of competitive nodulation under experimental condition of general culture, high humid culture and acid culture, while both alfalfa's genotype and alfalfa-rhizobium reciprocity had hardly any influences on competitive nodulation ability of rhizobium. Every experimental rhizobium strains had the same trend of competitive nodulation ability in the three culture conditions. But the competitive nodulation ability of the four strains was different significantly, in which the ability of X+Kan was feebler than other three strains. Both high humid culture condition and acid culture condition didn't alter affecting factors of competitive nodulation ability, but the rhizobium's genotype and the alfalfa varieties × rhizobium strains reciprocity could influence competitive nodulation ability, while the alfalfa's genotype doesn't affect the competitive nodulation ability under drought condition. That is to say, the drought resistance could alter affecting factors of competitive nodulation ability. And the stress could enhance
significantly the competitive nodulation ability of I +Spe strain compared with other strains. Culture conditions didn't influence competitive nodulation ability.
From the first experiment, the result also showed that the alfalfa plant infected by rhizobium meliloti appeared the characteristic of double infection or more infection. The conditions of adversity could enhance double infection or more infection frequency. Alfalfa genotype and R. meliloti 's genotype were the main factor of affecting the frequency of double infection or more infection under the general culture and high humid culture condition, but it had not any relation with the alfalfa varieties × rhizobium strains reciprocity. Alfalfa genotypic was the unique factor influenced the frequency of double infection or more infection under drought stress condition. Yet under the acid condition, both Rhizobium's genotype and alfalfa varieties x rhizobium strains reciprocity didn't influence the frequency, and the culture condition might be the affected factor. Culture conditions altered double or more infection frequency according to the different varieties. Culture con
在常规、高湿和酸性培养条件下，根瘤菌本身的基因型是影响竞争结瘤能力的主要因素，而苜蓿基因型以及苜蓿品种与根瘤菌菌株相互作用对竞争结瘤能力几乎没有影响。各供试菌株的竞争结瘤能力在这三种培养条件下表现一致，但菌株间存在显著差异，其中X~+Kan的竞争结瘤能力显著小于其它三个菌株。高湿和酸性培养条件不能改变影响根瘤菌竞争结瘤能力的因素。但在干旱培养条件下，根瘤菌的基因型和苜蓿品种与根瘤菌菌株互作影响根瘤菌的竞争结瘤能力，而苜蓿基因型对其没有影响。也就是说只有干旱胁迫能改变影响根瘤菌竞争结瘤的因素，并且显著地提高I~+Spe的竞争结瘤能力，使I~+Spe的竞争结瘤能力显著高于其它三个菌株。培养条件对竞争结瘤能力也没有作用。
在试验一中同时发现苜蓿根瘤菌有较高的双感染或多感染发生频率；各种逆境培养条件均能提高双感染或多感染的发生率。在常规培养条件和高湿培养条件下，影响根瘤菌双感染或多感染发生率的因素主要是苜蓿基因型和根瘤菌菌株，与苜蓿品种与根瘤菌菌株相互作用没有关系；在干旱条件下，只有苜蓿基因型才能影响根瘤菌的双感染或多感染的发生率；在酸性条件下，苜蓿基因型、根瘤菌以及苜蓿品种与根瘤菌菌株相互作用对根瘤菌的双感染或多感染的发生率都没有影响，此时双感染或多感染起作用的可能是培养条件。培养条件是否影响苜蓿根瘤菌的双感染或多感染的发生率依苜蓿品种而定，在苜蓿MB和MI两个品种上，生态条件能改变根瘤菌的双感染或多感染的发生率；在其它5个品种苜蓿上，培养条件不能改变根瘤菌的双感染或多感染的发生率。
试验二的研究结果表明，苜蓿品种、根瘤菌菌株以及苜蓿品种×根瘤菌菌株相互作用都能影响根瘤菌的固氮能力。在试验条件下，所有的菌株在MC植株上都表现出强的固氮能力；
西南农业人学缺{学位论文摘要
................
菌株XK*m在不卜刁的汀怡品种卜都表现出了较强的固集吃能力，但是在较好的苗楷「、甘，种MC卜表
现出的囚氮能力却不是最好;在品种与lyl株的红_作效应中，尤火ISI，e织.合的}占}氮能力比其
’自夕}{含都高，M 1 xXK:、;:约介的11钊氮能力同样较.岛，这两个组合的亲和性比较好，按种同源
根瘤菌的首讼样t株其}占{氮能力井不一定强，1).1此认为1个具有较强】占1氮能)]的科妙如翔钧株){
不一定源自同源的首信品种。
菌株XK;。rl的}古}氮能力显著高丁·菌株ANe。和H Str，也高一于菌株工Sp。们芳异不显著，
菌株A Ne。、H众:和ISpe之间的固氮能力没有显著差异;但是菌株XK二竟争结瘤能力最补，
该菌株在各酉拾，W，种上的!If瘤率显著小于菌株ANeo、HSt和1 Spe，而菌株A Noo、日出和!
渝。〔、的}}.习留率没子J’显著斧异。竟争结瘤能力差的根瘤菌菌株的固氮能力强，这，，丁能’、根瘤菌
携带的}占{氮能力川)c1和竟争结瘤华因是连锁的有关。
Seven alfalfa varieties in the study were collected from three kind of different ecological region, in which 3 varieties from Xinjing, respectively Xinmu No. 1 (MI), Xinmu No.2 (MII) and Xinjiang wild variety (MX) , 3 from Chongqing, Xinong No. 1 (MC) , a variety of resistant humid (MH) and a variety of resistant acid (MA), and one local variety (MB) from Beijing. Seven rhizobium meliloti strains were separated and purified from the plant of these varieties. These wild type strains were marked as I , II, X, C, H, A and B strains. And after the antibiotic resistance were examined, four antibiotic mutant strains from corresponding ones, H+Str, A+Neo, II +Spe and X+Kan, were obtained by the way of experiment of strains labeled with 4 kinds of antibiotics. The mutant strains were employed to carry out all of experiments because they and their corresponding wild strains had the same capacity of nodulation and fixation nitrogen representing their wilding strains. Two experiments in the study were carried out. The
first was the research of nodulation competition of rhizobium meliloti strains. The strains solutions, mixed equally with amount of the four mutant strains, were inoculated in seven alfalfa varieties. The plants were cultured in four different culture conditions. After 40d, five nodules from per plant were extracted to identify the kind of rhizobia and their occupancy in the same nodule, while the nodule occupancy frequency and double infection or excessive infection frequency of each strain were calculated. Another one was studied for the nitrogen fixation capability of different combination of host plant varieties × rhizobium strain. Seven alfalfa varieties were separately inoculated four rhizobium strains. The nitrogen fixation capability was measured by the dry weight of per plant after 40d. The results were as follows.
Rhizobium's genotype is the key factor of competitive nodulation under experimental condition of general culture, high humid culture and acid culture, while both alfalfa's genotype and alfalfa-rhizobium reciprocity had hardly any influences on competitive nodulation ability of rhizobium. Every experimental rhizobium strains had the same trend of competitive nodulation ability in the three culture conditions. But the competitive nodulation ability of the four strains was different significantly, in which the ability of X+Kan was feebler than other three strains. Both high humid culture condition and acid culture condition didn't alter affecting factors of competitive nodulation ability, but the rhizobium's genotype and the alfalfa varieties × rhizobium strains reciprocity could influence competitive nodulation ability, while the alfalfa's genotype doesn't affect the competitive nodulation ability under drought condition. That is to say, the drought resistance could alter affecting factors of competitive nodulation ability. And the stress could enhance
significantly the competitive nodulation ability of I +Spe strain compared with other strains. Culture conditions didn't influence competitive nodulation ability.
From the first experiment, the result also showed that the alfalfa plant infected by rhizobium meliloti appeared the characteristic of double infection or more infection. The conditions of adversity could enhance double infection or more infection frequency. Alfalfa genotype and R. meliloti 's genotype were the main factor of affecting the frequency of double infection or more infection under the general culture and high humid culture condition, but it had not any relation with the alfalfa varieties × rhizobium strains reciprocity. Alfalfa genotypic was the unique factor influenced the frequency of double infection or more infection under drought stress condition. Yet under the acid condition, both Rhizobium's genotype and alfalfa varieties x rhizobium strains reciprocity didn't influence the frequency, and the culture condition might be the affected factor. Culture conditions altered double or more infection frequency according to the different varieties. Culture con
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