饲用燕麦和小麦根际促生菌特性研究及其生物菌肥的初步研制
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摘要
禾本科牧草及禾谷类作物具有极其重要的经济价值和生态作用，但其均为嗜氮、磷植物。百十年来，化肥一直是提供这类植物氮、磷素的主要途径。然而，随着化肥使用量的不断增大，其副面影响日益明显。因此，探寻新的肥源，尤其是生物肥源，以替代或部分替代化肥的研究倍受关注。本研究利用气相色谱仪(GC)、高压液相色谱仪(HPLC)等先进仪器设备和最可能数法(MPN)、荧光免疫技术(FA)、随机扩增DNA(RAPD)技术、15N同位素稀释等方法，对饲用燕麦和小麦根际PGPR菌进行了分离鉴定和特性研究及其生物菌肥的初步研制。结果表明：
1．燕麦和小麦根际存在PGPR菌。燕麦根际分离获得10个菌株，分属Azotobacter(4个)、Pseudomonas(4个)和Azospirillus(2个)属；小麦根际分离到15个PGPR菌株，分属Azotobacter(5个)、Pseudomonas(4个)、Azospirillus(4个) 、Zoogloea(1个)和Alcaligenes(1个)属。
2．根际不同部位PGPR菌株的种类分布有差异，其种类数量为：RP＞RS＞NRS≥HP。分离到的菌株多数(48％)为根表土壤(RS)与根系表面(RP)共有的，少数(20％)只分离自根系表面(RP，12％)或土壤(NRS，8％)，个别(8％)分离自根内(HP)。也有一些(24％)菌株在土壤、根表土壤和根系表面均有分布。
3．绝大多数(92％)菌株在LB、NFM和CCM培养基，尤其是在LB培养基生长速度较快。所有菌株(除ChW4只在甘露醇外)均可在以甘露醇、糖浆、葡萄糖、蔗糖及1/2甘露醇+1/2蔗糖取代蛋白胨制成的不同碳源培养
基上良好生长，但一些菌株，在以半乳糖或麦芽糖或木糟取代蛋白陈制成的
碳源培养基上不生长或生长不良。
4．多数G2o／o）菌株为产碱菌株，部分（24％）为中性菌抹，其它（24％）
为产田菌株。产陨茵诛中77o／o来自于小麦根际，产酸菌株中6批来自于薪麦
根际。
5．自然状况下（未接种人燕麦和小麦根际各部位均存在着固氮菌类群，
刃 但数量差异较大且相对较少＊乙10‘个ig干土或根）；种群数量分布趋势为：
RP＞ RS＞NRS ＞HP；根际各部位固氮菌占该部位总异氧菌的比例趋势为：HP＞
丫
RP＞RS＞NR。
6．Chwl eons Sp．）菌株是一种相对广谱的菌株，在燕麦和小麦
根际不同部位（除薄麦根内（HP）外）均有分布，但其戮量除在裔麦和小麦
根系表面（RP）较多＊叭旷 饨干土或根）外，其它部位均很少W103个
伯干土或榔。ChW闲仰咖earp．）酉株是一种分布谱相对较窄的菌株，只分
布于小麦的根表上壤（RS）和根系表面（RP），对小麦的专化性相对较强。
7，虽然自然状况下慈麦和小麦根际存在着固氮菌株，但因其数量少＊0－
l炉他干土或榔，固氮量很少，不能满足植物生长发育所需的氮量。因而
应通过使用固氨菌接种剂以提高蒸麦和小麦根际固氮菌种群数量，增强种群
的竟争力，同时槽加根际微生物的固氮总量，满足或基本满足植物生长发育
所需的氮量。
8．分离到的茵株绝大部分0啊具有固氰性能，但固氮酶活性相差较大，
具有较高固氨酶活性的酉株相对较少。分离自慈麦根际菌株，固氮醇活性在
＊．5～l！47．9 nmo p之间，大于 300 if’mt po的菌株有 6个
（6（N入 ChO3的固氨酶活性最高，ChO6固氯醇活性最低；分离自小麦根
际酉株，固氨酌活性在oo．卜七5！石Dmfq闪W血之间，大于300——！Q巳
巾人d的苗株有5个Q3％入ChW的固氮酶活性最高，ChW固氮醇活性最
低．从苗抹固氮酶活住来看，CbO、CbO、CbO6和CbO7等在研制蒸麦
臼回和 CkW6、Cliwl卜 CliWS和 Cllwl等在研制小麦回回方面具有狠大的
v
子
开发潜力。
9．溶磷菌株较少（燕麦3个，小麦2个），其分解磷酸盐的能力差异较
大。接种溶磷菌对燕麦和小麦苗期生长有明显的促进作用。
10．绝大多数菌株具有分泌植物生长素（IAA）的能力，但能力相差较大，
具有较高能力的菌株相对较少。分离自燕麦的菌株均具有分泌植物生长素的
能力，其大小在 2二6叫．3lpg ／nilZ间，大于 spg ／ml的有 4个N0％人 ChO3
【
分泌植物生长素的量最高，而ChOZ最低；分离自小麦菌株中，73．3％具有分
泌植物生长素的能力，其值在 l·40叶·13ug alZ间，大于 SPg ／ml只有 4个 丫
Q6．7％人ChW15分泌植物生长素的量最高，ChW14最低。从菌株分泌植
物生长素（IAA）能力来看，Chwls，ChWS、ChwlZ、ChW6等在研制小
麦菌肥和ChO3、ChO6、ChOS、ChOg等在研制燕麦菌肥方面?
Grasses are the most important plants in the world because of their economic and ecological function. However, the plants need more nitrogen and phosphorus in their life. To provide more nitrogen and phosphorus for plant growth, chemical fertilizers play an important role. Unfortunately, chemical fertilizers produce more pollution and other disadvantage as well. Therefore, exploiting other source to supply part or all nitrogen and phosphorus for plant is more important.
The present study tries to isolate and characterize PGPR strains that can be used as biofertilizer for oat and wheat by using advanced equipment and facilities like GC & HPLC as well as reliable method such as NPM, FA, RAPD and I5N-isotope dilution. The results obtained are as follows:
1. There are PGPR strains colonized on root rhizosphere and in histoplant of oat and wheat. Total lOstrains isolated from oat, and they locate to Azotobacter (4strains), Pseudomonas (4 strains) and Azosprillus (2 strains). Total 15 PGPR strains obtained from wheat as well, and all of them belong to Azotobacter (5 strains), Pseudomonas (4 strains), Azosprillus (4 strains), Alcaligems (1 strain) and Tjjoglea (1 strain).
2. Strains distribution vary with fractions (NRS, RS, RP and HP), and the amount tendency is as RP>RS>NRS > HP. Most of them (48%) attached on the
rhizosphere (RS & RP); Some of them (20%) only exist in soil (NRS, 8%) or on the root surface (RP, 12%); Few of them (8%) colonize in histoplan (HP); And still some of them (24%) lie hi soil (MRS) as well as rhizosphere (RS & RP).
3. Most of strains (92%) obtained fast grow on LB, NFM and CCM media, especially on LB medium. All the strains (except ChW4) can growth well on different LB medium which peptone was replaced with any one of other carbon source, that is mannitol, molasses, glucose, sucrose, and l/2mannitol +l/2sucrose. While some strains are poor growth on LB medium which peptone was replaced with xylose or galactose or maltose.
4. Out of 25 strains, there are 13 alkaline producer strains (77% from wheat), 6 acid producer strains (67% from oat), and 6 neutral strains.
5. In natural condition (without inoculate), there are diazotrophs exist in root -soil system, but the amount is significant difference and rather low (02~106 bacteria /g dry soil or root) with fractions. Tendency of diazotrophs quantity and nitrogen fixer percent is as RP>RS>NRS>HP and HP >RP>RS>NRS respectively.
6. Generally, ChWl (Pseudomonas sp.) is widely distribution strains compared with ChW6 (Zoogloea sp.). ChWl was easily isolated from different fractions of oat and wheat (except HP), but ChW6 only be isolated from the fractions (RS and RP) of wheat. It means that ChW6 has certain special combined with wheat.
7. In natural condition, although there are diazotrophs exist hi root-soil system, the amount is rather low (lOMO6bacteria /g dry soil or root). Therefore, it is necessary to inoculate proper inoculum to increase diazotrophs quantity so as to improve competition of diazotrophs as well as quantity of fixing nitrogen.
8. Most (80%) of strains show positive nitrogenaes activity though only a few of diem show high value by using GO Nitrogenase activity is 112.5-1147.9 nmol C2H4/h/ml and 124.6~651.6 nmol C2H4 / h / ml of wheat strains and oat strains
respectively. There are 6 and 5 strains, which nitrogenase activity are higher than 300 nmol C2H4/h/mls, hi oat and wheat respectively. Strains ChO3, ChO5, Ch09, ChO7 and ChW6, ChWll, ChW5, ChWl show more potential used as producing biofertilizer for oat and wheat respectively according to their nitrogenase activity.
9. A few (5 strains) of phosphorus-solubilizing strains were obtained. Seedling growth was promoting by inoculate the phosphate solubilizing strains.
10. Most of strains have ability to produce phytohormone (IAA) although a few of strains show high IAA concentration by HPLC. IAA concentration is 2.16~17.31ug /ml andl.40~15.13ug/ml of oat and wheat strains respectively. There are 4 strains from oat and 4 strains
1．燕麦和小麦根际存在PGPR菌。燕麦根际分离获得10个菌株，分属Azotobacter(4个)、Pseudomonas(4个)和Azospirillus(2个)属；小麦根际分离到15个PGPR菌株，分属Azotobacter(5个)、Pseudomonas(4个)、Azospirillus(4个) 、Zoogloea(1个)和Alcaligenes(1个)属。
2．根际不同部位PGPR菌株的种类分布有差异，其种类数量为：RP＞RS＞NRS≥HP。分离到的菌株多数(48％)为根表土壤(RS)与根系表面(RP)共有的，少数(20％)只分离自根系表面(RP，12％)或土壤(NRS，8％)，个别(8％)分离自根内(HP)。也有一些(24％)菌株在土壤、根表土壤和根系表面均有分布。
3．绝大多数(92％)菌株在LB、NFM和CCM培养基，尤其是在LB培养基生长速度较快。所有菌株(除ChW4只在甘露醇外)均可在以甘露醇、糖浆、葡萄糖、蔗糖及1/2甘露醇+1/2蔗糖取代蛋白胨制成的不同碳源培养
基上良好生长，但一些菌株，在以半乳糖或麦芽糖或木糟取代蛋白陈制成的
碳源培养基上不生长或生长不良。
4．多数G2o／o）菌株为产碱菌株，部分（24％）为中性菌抹，其它（24％）
为产田菌株。产陨茵诛中77o／o来自于小麦根际，产酸菌株中6批来自于薪麦
根际。
5．自然状况下（未接种人燕麦和小麦根际各部位均存在着固氮菌类群，
刃 但数量差异较大且相对较少＊乙10‘个ig干土或根）；种群数量分布趋势为：
RP＞ RS＞NRS ＞HP；根际各部位固氮菌占该部位总异氧菌的比例趋势为：HP＞
丫
RP＞RS＞NR。
6．Chwl eons Sp．）菌株是一种相对广谱的菌株，在燕麦和小麦
根际不同部位（除薄麦根内（HP）外）均有分布，但其戮量除在裔麦和小麦
根系表面（RP）较多＊叭旷 饨干土或根）外，其它部位均很少W103个
伯干土或榔。ChW闲仰咖earp．）酉株是一种分布谱相对较窄的菌株，只分
布于小麦的根表上壤（RS）和根系表面（RP），对小麦的专化性相对较强。
7，虽然自然状况下慈麦和小麦根际存在着固氮菌株，但因其数量少＊0－
l炉他干土或榔，固氮量很少，不能满足植物生长发育所需的氮量。因而
应通过使用固氨菌接种剂以提高蒸麦和小麦根际固氮菌种群数量，增强种群
的竟争力，同时槽加根际微生物的固氮总量，满足或基本满足植物生长发育
所需的氮量。
8．分离到的茵株绝大部分0啊具有固氰性能，但固氮酶活性相差较大，
具有较高固氨酶活性的酉株相对较少。分离自慈麦根际菌株，固氮醇活性在
＊．5～l！47．9 nmo p之间，大于 300 if’mt po的菌株有 6个
（6（N入 ChO3的固氨酶活性最高，ChO6固氯醇活性最低；分离自小麦根
际酉株，固氨酌活性在oo．卜七5！石Dmfq闪W血之间，大于300——！Q巳
巾人d的苗株有5个Q3％入ChW的固氮酶活性最高，ChW固氮醇活性最
低．从苗抹固氮酶活住来看，CbO、CbO、CbO6和CbO7等在研制蒸麦
臼回和 CkW6、Cliwl卜 CliWS和 Cllwl等在研制小麦回回方面具有狠大的
v
子
开发潜力。
9．溶磷菌株较少（燕麦3个，小麦2个），其分解磷酸盐的能力差异较
大。接种溶磷菌对燕麦和小麦苗期生长有明显的促进作用。
10．绝大多数菌株具有分泌植物生长素（IAA）的能力，但能力相差较大，
具有较高能力的菌株相对较少。分离自燕麦的菌株均具有分泌植物生长素的
能力，其大小在 2二6叫．3lpg ／nilZ间，大于 spg ／ml的有 4个N0％人 ChO3
【
分泌植物生长素的量最高，而ChOZ最低；分离自小麦菌株中，73．3％具有分
泌植物生长素的能力，其值在 l·40叶·13ug alZ间，大于 SPg ／ml只有 4个 丫
Q6．7％人ChW15分泌植物生长素的量最高，ChW14最低。从菌株分泌植
物生长素（IAA）能力来看，Chwls，ChWS、ChwlZ、ChW6等在研制小
麦菌肥和ChO3、ChO6、ChOS、ChOg等在研制燕麦菌肥方面?
Grasses are the most important plants in the world because of their economic and ecological function. However, the plants need more nitrogen and phosphorus in their life. To provide more nitrogen and phosphorus for plant growth, chemical fertilizers play an important role. Unfortunately, chemical fertilizers produce more pollution and other disadvantage as well. Therefore, exploiting other source to supply part or all nitrogen and phosphorus for plant is more important.
The present study tries to isolate and characterize PGPR strains that can be used as biofertilizer for oat and wheat by using advanced equipment and facilities like GC & HPLC as well as reliable method such as NPM, FA, RAPD and I5N-isotope dilution. The results obtained are as follows:
1. There are PGPR strains colonized on root rhizosphere and in histoplant of oat and wheat. Total lOstrains isolated from oat, and they locate to Azotobacter (4strains), Pseudomonas (4 strains) and Azosprillus (2 strains). Total 15 PGPR strains obtained from wheat as well, and all of them belong to Azotobacter (5 strains), Pseudomonas (4 strains), Azosprillus (4 strains), Alcaligems (1 strain) and Tjjoglea (1 strain).
2. Strains distribution vary with fractions (NRS, RS, RP and HP), and the amount tendency is as RP>RS>NRS > HP. Most of them (48%) attached on the
rhizosphere (RS & RP); Some of them (20%) only exist in soil (NRS, 8%) or on the root surface (RP, 12%); Few of them (8%) colonize in histoplan (HP); And still some of them (24%) lie hi soil (MRS) as well as rhizosphere (RS & RP).
3. Most of strains (92%) obtained fast grow on LB, NFM and CCM media, especially on LB medium. All the strains (except ChW4) can growth well on different LB medium which peptone was replaced with any one of other carbon source, that is mannitol, molasses, glucose, sucrose, and l/2mannitol +l/2sucrose. While some strains are poor growth on LB medium which peptone was replaced with xylose or galactose or maltose.
4. Out of 25 strains, there are 13 alkaline producer strains (77% from wheat), 6 acid producer strains (67% from oat), and 6 neutral strains.
5. In natural condition (without inoculate), there are diazotrophs exist in root -soil system, but the amount is significant difference and rather low (02~106 bacteria /g dry soil or root) with fractions. Tendency of diazotrophs quantity and nitrogen fixer percent is as RP>RS>NRS>HP and HP >RP>RS>NRS respectively.
6. Generally, ChWl (Pseudomonas sp.) is widely distribution strains compared with ChW6 (Zoogloea sp.). ChWl was easily isolated from different fractions of oat and wheat (except HP), but ChW6 only be isolated from the fractions (RS and RP) of wheat. It means that ChW6 has certain special combined with wheat.
7. In natural condition, although there are diazotrophs exist hi root-soil system, the amount is rather low (lOMO6bacteria /g dry soil or root). Therefore, it is necessary to inoculate proper inoculum to increase diazotrophs quantity so as to improve competition of diazotrophs as well as quantity of fixing nitrogen.
8. Most (80%) of strains show positive nitrogenaes activity though only a few of diem show high value by using GO Nitrogenase activity is 112.5-1147.9 nmol C2H4/h/ml and 124.6~651.6 nmol C2H4 / h / ml of wheat strains and oat strains
respectively. There are 6 and 5 strains, which nitrogenase activity are higher than 300 nmol C2H4/h/mls, hi oat and wheat respectively. Strains ChO3, ChO5, Ch09, ChO7 and ChW6, ChWll, ChW5, ChWl show more potential used as producing biofertilizer for oat and wheat respectively according to their nitrogenase activity.
9. A few (5 strains) of phosphorus-solubilizing strains were obtained. Seedling growth was promoting by inoculate the phosphate solubilizing strains.
10. Most of strains have ability to produce phytohormone (IAA) although a few of strains show high IAA concentration by HPLC. IAA concentration is 2.16~17.31ug /ml andl.40~15.13ug/ml of oat and wheat strains respectively. There are 4 strains from oat and 4 strains
引文
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