拮抗性大蒜内生细菌的多样性及其促植物生长特性研究
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摘要
本论文综述了植物内生细菌多样性、分类、相关研究技术及植物内生细菌应用方面的研究进展。用浓度梯度法从采自山东金乡、河南杞县和江苏射县三地的健康白皮大蒜中共分离得到174株内生细菌,以稻瘟菌、黄瓜枯萎菌、苹果炭疽菌和苹果腐烂菌作指示菌株,采用平板对峙法,筛选得到62株对4种病原真菌均有拮抗作用的菌株。通过ERIC-PCR指纹图谱分析和16S rDNA PCR-RFLP两种分析方法,采用MVSP3.1非加权平均连锁法(UPGMA)进行聚类分析并构建树状图谱。根据聚类分析结果,选取代表菌株通过16S rDNA序列分析,构建系统发育树。同时对筛选得到的62株供试菌株进行了产IAA、铁载体和溶磷能力测定。
结果显示:62株拮抗性菌株包括8个属:苍白杆菌属,新鞘氨醇杆菌属,短波单胞菌属,假单胞菌属,沙雷菌属,红球菌属,节杆菌属和芽孢杆菌属。不同产地拮抗性大蒜内生细菌多样性呈现明显差异,来自山东金乡的拮抗性内生细菌表型单一,包括3个属:节杆菌属、沙雷菌属和芽胞杆菌属,芽胞杆菌属为其优势菌株,占该产地分离菌总数的84.2%;河南杞县多样性较丰富,包括6个属:假单胞菌属、红球菌属、苍白杆菌属、节杆菌属、沙雷菌属和芽胞杆菌属,芽胞杆菌属仍是其优势菌株,占分离菌总数的47.6%;江苏射县多样性最为丰富,包括8个属:假单胞菌属、红球菌属、苍白杆菌属、节杆菌属、短波单胞菌属、沙雷菌属、芽胞杆菌属和新鞘氨醇杆菌属。苍白杆菌属占分离菌总数的22.8%,节杆菌属占18.2%,沙雷菌属占18.2%,苍白杆菌属为其优势菌株。总体来说假单胞菌属是大蒜内生细菌中最常见的属,与其他植物内生细菌的文献报道一致。
本研究分离获得的内生细菌均有相应的文献报道,且有些已经进行了较为详细深入的研究,如苍白杆菌属,新鞘氨醇杆菌属,短波单胞菌属,沙雷菌属,红球菌属,硫磺节杆菌,嗜尼古丁节杆菌,但这些细菌在之前的报道中大多分离自土壤、水体。本研究从内生细菌角度出发,从大蒜这种本身具有很强抑菌活性的植株中分离获得这些细菌,考察其多样性问题,在以前的研究中尚未见相关的文献报道。研究结果表明不同地域间大蒜内生细菌菌株的多样性呈现出明显差异性。
62株拮抗性菌株产IAA(吲哚乙酸)、铁载体和溶磷能力测定结果表明:62株供试菌株均能产IAA,说明筛选出的拮抗性内生细菌菌株都有促进植物生长的潜力,这些菌株产IAA含量最高可达49.664μg/mL,有5株菌产IAA含量均在20μg/mL以上;苍白杆菌属,红球菌属,芽孢杆菌属均有部分菌株产IAA含量较高;沙雷菌属产IAA含量普遍较高;新鞘氨醇杆菌属,短波单胞菌属,假单胞菌属,节杆菌属产IAA量整体偏低。
62株菌中82.3%(51株)的菌株能产铁载体,8个属均有分布,并且能够产铁载体的菌株其抑菌作用均较强,说明筛选出的大部分拮抗性菌株的抑菌机理很有可能与铁载体的产生有关,其中硫磺节杆菌不产铁载体,嗜尼古丁节杆菌的6株菌都能产生铁载体,表现出种之间的差异。
62个供试菌株中有21株能够溶解有机磷,占供试菌株总数的33.87%, D/d值范围1.13～1.85,这21株菌分别归属于苍白杆菌属(3株),假单胞菌属(4株),沙雷菌属(7株),嗜尼古丁节杆菌(1株),枯草芽孢杆菌(2株),苏云芽孢杆菌(4株);新鞘氨醇杆菌属,短波单胞菌属,红球菌属,硫磺节杆菌的所有供试菌株均不能溶解有机磷;溶解无机磷测定结果表明,62个供试菌株在PKO培养基上均生长良好,但均未形成溶磷圈,D/d值为1.00,因此不能溶解无机磷。
综合供试菌株对病原真菌的拮抗性、产IAA量、产铁载体情况及溶磷效果,认为DSQ5、DSS21、DSQ4、DSJ9、DSJ3、DSS10 6株菌作为生防菌优势明显,作为下一步研究的优良菌株。
174 endophytic bacterial strains were isolated from health garlic in three different sites by the concentration gradient method. Through antagonistic experiments, we obtained 62 endophytic bacterial strains, which have better effect on against 4 pathogenic fungi, including Magnaporthe grisea , Fusarium oxysporum f. sp. Cucumerinum , Colletotrichum gloeosporioides and Valsa mali Miyabe et Yamada. Combined the analysis of ERIC-PCR and PCR-RFLP from 16S rDNA of bacterial with the enzyme type, the built tree plot was constructed based on the cluster analysis with the Unweighted Pair Group Mathematical Average (UPGMA) MVSP3.1. According to the results of cluster analysis, we constructed phylogenetic tree by 11 selected strains with the sequence analysis of their 16S rDNA. The results showed that the 62 tested strains were from eight different genuses, including Ochrobactrum,Novosphingobium,Brevundimonas,Pseudomonas, Serratia,Rhodococcus,Arthrobacter and Bacillus. 62 tested strains from different sites showed significant differences in biodiversity. Strains obtained from Jinxiang were from three genuses, which were respectively Arthrobacter, Serratia and Bacillus. Bacillus was the dominant genus, and accounted for 84.2% of total strains separated in the site; Strains obtained from Qixian were from six genuses, which were Pseudomonas, Rhodococcus, Ochrobactrum, Arthrobacter, Serratia and Bacillus. Bacillus was also the dominant genus, and accounted for 47.6% of total strains separated in the site; Strains obtained from Shexian were from eight genuses: Pseudomonas, Rhodococcus, Ochrobactrum, Arthrobacter, Brevundimonas, Serratia, Bacillus and Novosphingobium.Among Ochrobactrum accounted for 22.8%,Arthrobacter accounted for 18.2%,and Serratia accounted for 18.2% of total strains separated in the site. Overall Pseudomonas was the most common genus in endophytic bacterial from garlic. The result was consistent with report.
All the endophytic bacterias isolated in this study had been reported in literatures, and some strains even had a more in-depth study, such as Ochrobactrum, Novosphingobium, Brevundimonas, Serratia, Rhodococcus and Arthrobacter nicotinovorans et al.But these bacterias were all isolated from soil and water before. In this study, we obtainded these endophytic bacteria in garlic, which itself has a strong inhibitory activity, and furthermore we studied their biodiversity. The research in this area was the first time. On the whole, the strains obtaind from different sites showed different diversity in genus and amount.The strains from different sites and different variants showed obvious differences on their biodiversity.
We also tested that all the 62 strains have the capacity on produce acetic acid (IAA), the highest content of IAA, which be produced by one strain, is about 49.664μg.mL-1. Likewise, we also found that the content of IAA have reached 20μg/mL, produced by 5 strains, from all the strains. And above 82.3 %( 51 strains) strains belonged to eight different genuses but except Arthrobacter sulfureus could produce siderophore.
Moreover, 33.87%(21 strains) strains could dissolve organic phosphorus and the range of D/d was from 1.13 to 1.85. 21 strains are attributed to Ochrobactrum(4 strains),Pseudomonas(4 strains),Serratia(7 strains),Arthrobacter nicotinovorans(1 strain),Bacillus subtilis(2 strains)and Bacillus thuringiensis(4 strains);all remaining strains belonged to Novosphingobium, Brevundimonas, Rhodococcus and Arthrobacter sulfureus could not dissolve organic phosphorus. And all tested strains (62strains) could not dissolve Inorganic phosphorus.
From the results of 62 tested strains producing IAA, siderophores and phosphate solubilization, we could see that some strains belong to Ochrobactrum, Rhodococcus and Bacillus and all strains belong to Serratia could produce higher levels of IAA. Meanwhile strains from Novosphingobium,Brevundimonas,Pseudomonas and Arthrobacter had a lower ability to produce IAA.
82.3% (51) strains were distributed eight different genuses could produce siderophore.The results showed that the antagonistic inhibition mechanism of the majority of selected strains is likely due to the production of siderophore .We also could see that Arthrobacter sulfurous was different from Arthrobacter nicotinovorans in ability to produce siderophore, and strains belonged to Arthrobacter sulfurous could produce siderophore,in contrast strains belong to Arthrobacter nicotinovorans could not. All tested strains could not dissolve inorganic phosphorus and showed different ability in dissolving organic phosphorus. Some strains belonged to Arthrobacter or Bacillus and all strains belonged to Serratia or Pseudomonas had the ability of dissolving organic phosphorus; But all srains belonged to Rhodococcus could not.
Combined the capacity of the strains, including antifungal activity, IAA production, siderophore production and the organic phosphorus dissolutions, we selected 6 better strains and numbered them as DSQ5, DSS21, DSQ4, DSJ9, DSJ3 and DSS10.The acquisition of these strains could be a foundation on the develop new Biopesticides from the endophytic bacteria of garlic later.
结果显示:62株拮抗性菌株包括8个属:苍白杆菌属,新鞘氨醇杆菌属,短波单胞菌属,假单胞菌属,沙雷菌属,红球菌属,节杆菌属和芽孢杆菌属。不同产地拮抗性大蒜内生细菌多样性呈现明显差异,来自山东金乡的拮抗性内生细菌表型单一,包括3个属:节杆菌属、沙雷菌属和芽胞杆菌属,芽胞杆菌属为其优势菌株,占该产地分离菌总数的84.2%;河南杞县多样性较丰富,包括6个属:假单胞菌属、红球菌属、苍白杆菌属、节杆菌属、沙雷菌属和芽胞杆菌属,芽胞杆菌属仍是其优势菌株,占分离菌总数的47.6%;江苏射县多样性最为丰富,包括8个属:假单胞菌属、红球菌属、苍白杆菌属、节杆菌属、短波单胞菌属、沙雷菌属、芽胞杆菌属和新鞘氨醇杆菌属。苍白杆菌属占分离菌总数的22.8%,节杆菌属占18.2%,沙雷菌属占18.2%,苍白杆菌属为其优势菌株。总体来说假单胞菌属是大蒜内生细菌中最常见的属,与其他植物内生细菌的文献报道一致。
本研究分离获得的内生细菌均有相应的文献报道,且有些已经进行了较为详细深入的研究,如苍白杆菌属,新鞘氨醇杆菌属,短波单胞菌属,沙雷菌属,红球菌属,硫磺节杆菌,嗜尼古丁节杆菌,但这些细菌在之前的报道中大多分离自土壤、水体。本研究从内生细菌角度出发,从大蒜这种本身具有很强抑菌活性的植株中分离获得这些细菌,考察其多样性问题,在以前的研究中尚未见相关的文献报道。研究结果表明不同地域间大蒜内生细菌菌株的多样性呈现出明显差异性。
62株拮抗性菌株产IAA(吲哚乙酸)、铁载体和溶磷能力测定结果表明:62株供试菌株均能产IAA,说明筛选出的拮抗性内生细菌菌株都有促进植物生长的潜力,这些菌株产IAA含量最高可达49.664μg/mL,有5株菌产IAA含量均在20μg/mL以上;苍白杆菌属,红球菌属,芽孢杆菌属均有部分菌株产IAA含量较高;沙雷菌属产IAA含量普遍较高;新鞘氨醇杆菌属,短波单胞菌属,假单胞菌属,节杆菌属产IAA量整体偏低。
62株菌中82.3%(51株)的菌株能产铁载体,8个属均有分布,并且能够产铁载体的菌株其抑菌作用均较强,说明筛选出的大部分拮抗性菌株的抑菌机理很有可能与铁载体的产生有关,其中硫磺节杆菌不产铁载体,嗜尼古丁节杆菌的6株菌都能产生铁载体,表现出种之间的差异。
62个供试菌株中有21株能够溶解有机磷,占供试菌株总数的33.87%, D/d值范围1.13～1.85,这21株菌分别归属于苍白杆菌属(3株),假单胞菌属(4株),沙雷菌属(7株),嗜尼古丁节杆菌(1株),枯草芽孢杆菌(2株),苏云芽孢杆菌(4株);新鞘氨醇杆菌属,短波单胞菌属,红球菌属,硫磺节杆菌的所有供试菌株均不能溶解有机磷;溶解无机磷测定结果表明,62个供试菌株在PKO培养基上均生长良好,但均未形成溶磷圈,D/d值为1.00,因此不能溶解无机磷。
综合供试菌株对病原真菌的拮抗性、产IAA量、产铁载体情况及溶磷效果,认为DSQ5、DSS21、DSQ4、DSJ9、DSJ3、DSS10 6株菌作为生防菌优势明显,作为下一步研究的优良菌株。
174 endophytic bacterial strains were isolated from health garlic in three different sites by the concentration gradient method. Through antagonistic experiments, we obtained 62 endophytic bacterial strains, which have better effect on against 4 pathogenic fungi, including Magnaporthe grisea , Fusarium oxysporum f. sp. Cucumerinum , Colletotrichum gloeosporioides and Valsa mali Miyabe et Yamada. Combined the analysis of ERIC-PCR and PCR-RFLP from 16S rDNA of bacterial with the enzyme type, the built tree plot was constructed based on the cluster analysis with the Unweighted Pair Group Mathematical Average (UPGMA) MVSP3.1. According to the results of cluster analysis, we constructed phylogenetic tree by 11 selected strains with the sequence analysis of their 16S rDNA. The results showed that the 62 tested strains were from eight different genuses, including Ochrobactrum,Novosphingobium,Brevundimonas,Pseudomonas, Serratia,Rhodococcus,Arthrobacter and Bacillus. 62 tested strains from different sites showed significant differences in biodiversity. Strains obtained from Jinxiang were from three genuses, which were respectively Arthrobacter, Serratia and Bacillus. Bacillus was the dominant genus, and accounted for 84.2% of total strains separated in the site; Strains obtained from Qixian were from six genuses, which were Pseudomonas, Rhodococcus, Ochrobactrum, Arthrobacter, Serratia and Bacillus. Bacillus was also the dominant genus, and accounted for 47.6% of total strains separated in the site; Strains obtained from Shexian were from eight genuses: Pseudomonas, Rhodococcus, Ochrobactrum, Arthrobacter, Brevundimonas, Serratia, Bacillus and Novosphingobium.Among Ochrobactrum accounted for 22.8%,Arthrobacter accounted for 18.2%,and Serratia accounted for 18.2% of total strains separated in the site. Overall Pseudomonas was the most common genus in endophytic bacterial from garlic. The result was consistent with report.
All the endophytic bacterias isolated in this study had been reported in literatures, and some strains even had a more in-depth study, such as Ochrobactrum, Novosphingobium, Brevundimonas, Serratia, Rhodococcus and Arthrobacter nicotinovorans et al.But these bacterias were all isolated from soil and water before. In this study, we obtainded these endophytic bacteria in garlic, which itself has a strong inhibitory activity, and furthermore we studied their biodiversity. The research in this area was the first time. On the whole, the strains obtaind from different sites showed different diversity in genus and amount.The strains from different sites and different variants showed obvious differences on their biodiversity.
We also tested that all the 62 strains have the capacity on produce acetic acid (IAA), the highest content of IAA, which be produced by one strain, is about 49.664μg.mL-1. Likewise, we also found that the content of IAA have reached 20μg/mL, produced by 5 strains, from all the strains. And above 82.3 %( 51 strains) strains belonged to eight different genuses but except Arthrobacter sulfureus could produce siderophore.
Moreover, 33.87%(21 strains) strains could dissolve organic phosphorus and the range of D/d was from 1.13 to 1.85. 21 strains are attributed to Ochrobactrum(4 strains),Pseudomonas(4 strains),Serratia(7 strains),Arthrobacter nicotinovorans(1 strain),Bacillus subtilis(2 strains)and Bacillus thuringiensis(4 strains);all remaining strains belonged to Novosphingobium, Brevundimonas, Rhodococcus and Arthrobacter sulfureus could not dissolve organic phosphorus. And all tested strains (62strains) could not dissolve Inorganic phosphorus.
From the results of 62 tested strains producing IAA, siderophores and phosphate solubilization, we could see that some strains belong to Ochrobactrum, Rhodococcus and Bacillus and all strains belong to Serratia could produce higher levels of IAA. Meanwhile strains from Novosphingobium,Brevundimonas,Pseudomonas and Arthrobacter had a lower ability to produce IAA.
82.3% (51) strains were distributed eight different genuses could produce siderophore.The results showed that the antagonistic inhibition mechanism of the majority of selected strains is likely due to the production of siderophore .We also could see that Arthrobacter sulfurous was different from Arthrobacter nicotinovorans in ability to produce siderophore, and strains belonged to Arthrobacter sulfurous could produce siderophore,in contrast strains belong to Arthrobacter nicotinovorans could not. All tested strains could not dissolve inorganic phosphorus and showed different ability in dissolving organic phosphorus. Some strains belonged to Arthrobacter or Bacillus and all strains belonged to Serratia or Pseudomonas had the ability of dissolving organic phosphorus; But all srains belonged to Rhodococcus could not.
Combined the capacity of the strains, including antifungal activity, IAA production, siderophore production and the organic phosphorus dissolutions, we selected 6 better strains and numbered them as DSQ5, DSS21, DSQ4, DSJ9, DSJ3 and DSS10.The acquisition of these strains could be a foundation on the develop new Biopesticides from the endophytic bacteria of garlic later.
引文
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