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Volume 34 Issue 3
Jun.  2021
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Isolation and Investigation on Properties of Nitrogen-fixing Bacteria from Rhizosphere of Broussonetia papyrifera Hybrid

  • Objective To obtain strains of nitrogen-fixing bacteria from Broussonetia papyrifera (Linnaeus) L’Héritier ex Ventenat hybrid and determine their functional characteristics. Method The Ashby medium was selected as separation condition to isolated nitrogen-fixing bacteria from the roots and rhizosphere soil of B. papyrifera hybrid in Tianshui, Lanzhou and Zhangye experimental areas of Gansu Province. In addition, identification and phylogenetic analysis were carried out on the isolated nitrogen-fixing bacteria. The nitrogenase activity, phosphorus solubility, properties of IAA-producing and inhibition of plant pathogenic fungi of each strain were determined at the same time. Entropy weight method was used for comprehensive evaluation of the features of the strains. Result A total of 10 nitrogen-fixing bacteria was isolated from the soil far from the rhizosphere and topsoil, which belonged to 8 genera, mostly Pseudoxanthomonas. Each strain showed nitrogenase activity, among which the nitrogenase activity of the strains TS4 and HP10 was greater than 180 IU·L−1. Only the strains TS4 and HTZ2 had certain phosphorus solubility. 8 strains could secrete IAA, among which the strain HTZ4 demonstrated the strongest secretion ability, with an IAA increment of 44.62 µg·mL−1. All strains showed no antagonistic effect on Botrytis cinerea Persoon, 7 strains showed inhibitory effects on at least two kinds of plant pathogens. The strains HTZ4, HTZ3, and HP5 showed the strongest inhibitory activities against Alternaria solani (Ellis et G. Martin) Sorauer, Fusarium oxysporum Schlecht and Fusarium solani (Martius) Sacco, respectively, with inhibition rates of 50.00%, 48.78% and 47.37%. To sum up, the strains with strong comprehensive properties were the strains TS4, HP5, ZY9, HTZ4 and HTZ5. Conclusion The results of this study can provide data and references for further developing nitrogen-fixing strains with excellent comprehensive performance, multifunctional nitrogen-fixing bacterial fertilizer for Broussonetia papyrifera hybrid, and promoting the yield of Broussonetia papyrifera hybrid in semi-arid area of Northwest China.
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Isolation and Investigation on Properties of Nitrogen-fixing Bacteria from Rhizosphere of Broussonetia papyrifera Hybrid

    Corresponding author: GUO Qi, guoqi9207@163.com
    Corresponding author: YANG Hui, yanghui43@163.com
  • Institute of Biology, Gansu Academy of Sciences, Key Laboratory of Microbial Resources Exploitation and Application, Gansu International Science and Technology Cooperation Base of Microorganism and Plant Germplasm Resources & Genetic Improvement, Lanzhou 730000, Gansu, China

Abstract:  Objective To obtain strains of nitrogen-fixing bacteria from Broussonetia papyrifera (Linnaeus) L’Héritier ex Ventenat hybrid and determine their functional characteristics. Method The Ashby medium was selected as separation condition to isolated nitrogen-fixing bacteria from the roots and rhizosphere soil of B. papyrifera hybrid in Tianshui, Lanzhou and Zhangye experimental areas of Gansu Province. In addition, identification and phylogenetic analysis were carried out on the isolated nitrogen-fixing bacteria. The nitrogenase activity, phosphorus solubility, properties of IAA-producing and inhibition of plant pathogenic fungi of each strain were determined at the same time. Entropy weight method was used for comprehensive evaluation of the features of the strains. Result A total of 10 nitrogen-fixing bacteria was isolated from the soil far from the rhizosphere and topsoil, which belonged to 8 genera, mostly Pseudoxanthomonas. Each strain showed nitrogenase activity, among which the nitrogenase activity of the strains TS4 and HP10 was greater than 180 IU·L−1. Only the strains TS4 and HTZ2 had certain phosphorus solubility. 8 strains could secrete IAA, among which the strain HTZ4 demonstrated the strongest secretion ability, with an IAA increment of 44.62 µg·mL−1. All strains showed no antagonistic effect on Botrytis cinerea Persoon, 7 strains showed inhibitory effects on at least two kinds of plant pathogens. The strains HTZ4, HTZ3, and HP5 showed the strongest inhibitory activities against Alternaria solani (Ellis et G. Martin) Sorauer, Fusarium oxysporum Schlecht and Fusarium solani (Martius) Sacco, respectively, with inhibition rates of 50.00%, 48.78% and 47.37%. To sum up, the strains with strong comprehensive properties were the strains TS4, HP5, ZY9, HTZ4 and HTZ5. Conclusion The results of this study can provide data and references for further developing nitrogen-fixing strains with excellent comprehensive performance, multifunctional nitrogen-fixing bacterial fertilizer for Broussonetia papyrifera hybrid, and promoting the yield of Broussonetia papyrifera hybrid in semi-arid area of Northwest China.

  • 构树(Broussonetia papyrifera (Linnaeus) L’Héritier ex Ventenat)是一种具有重要经济价值的多年生乔木,属于桑科(Moraceae)构属(Broussonetia)。根据Angiosperm Phylogeny Group III分类系统,又属固氮分支[1]。近年来,新品种杂交构树华构1号因抗逆性强、生长速度快、产量高、粗蛋白质含量高的优点,被用作优质的木本饲料来源在全国20多个省(市)推广种植;但在甘肃等西北干旱与半干旱地区,杂交构树的生长存在产量低、翌年返青率低等问题[2]。针对以上问题,研究人员一方面开展了选育适宜甘肃冷凉地区的高产抗寒性品系的工作;另一方面,鉴于研究表明杂交构树高含量的粗蛋白来源和速生可能与其内生和根际共生的共生固氮或联合固氮菌有密切关系[3],可通过从杂交构树根际土壤中筛选本土固氮菌微生物再回接的方式,提高菌肥效果,达到增产的目的。因此,根据土壤环境情况分离筛选杂交构树优良固氮菌株,是研制菌肥最基础的前期工作。本研究旨在从甘肃地区种植的杂交构树根际分离筛选获得固氮菌株,并对其固氮、促生、溶磷、生物防治等功能进行研究,可为进一步开发甘肃地区杂交构树微生物氮肥提供菌种资源。

1.   试验材料
  • 为便于研究,将根际分为远根土(距离根部1~10 cm的土壤)、根表土(距离根部0~1 cm的土壤)与根3个部位,于2019年8月在甘肃的天水(34°5′ N,105°82′ E)、兰州(36°0′ N,103°57′ E)和张掖(38°43′ N,100°40′ E)杂交构树种植试验区,按照蛇型采样法,各取5个点分别采样,现场混合后将根系样品和采用四分法保留的土壤样品装入无菌自封袋中运回实验室进行分离。天水、兰州、张掖采样区0~20 cm土层土壤的全氮含量分别为0.616、0.356、0.284 g·kg−1,硝态氮含量分别为247.00、28.80、26.05 mg·kg−1

  • 灰霉菌(Botrytis cinerea Persoon)(Bc)、立枯丝核菌(Rhizoctonia solani Kuhn)(Rs)、尖孢镰刀菌(Fusarium oxysporum Schlecht)(Fo)、腐皮镰孢菌(Fusarium solani (Martius) Sacco)(Fs)、茄链格孢(Alternaria solani (Elliset G. Martin) Sorauer)(As)由甘肃省农业科学研究院植保所提供。

2.   研究方法
  • 远根土与根表土固氮菌的分离采用常规稀释平板涂布法在Ashby培养基上进行。根系固氮菌的分离:取根径约1~10 mm的根,剪成3~5 cm的段,用自来水冲洗1~2 h,在无菌操作台中用75%酒精浸泡1 min后,无菌水洗涤3~5次,再用0.1%的升汞浸泡3 min后,无菌水洗涤3~5次,吸干表面水分,切成5~8 mm的小段,接种于Ashby培养基。28℃,恒温培养5~7 d,待分离的菌落长出后用平板划线法继续在Ashby培养基上进行纯化。将纯化好的菌株接入LB斜面上,培养3 d后置于冰箱4℃保藏。

  • 观察菌落形态学特征。根据细菌基因组DNA提取试剂盒(北京索莱宝科技有限公司)说明提取单菌落基因组DNA,送生物工程(上海)股份有限公司16S rDNA测序,结果与NCBI GenBank中的已知序列进行同源性比对后,判定细菌种类并划分到属或种。应用MEGA7.0软件,采用邻接法聚类分析,构建系统发育树,Bootstrap值为1 000。

  • 固氮酶活性按照微生物固氮酶(NITS)ELISA试剂盒(江苏酶免实业有限公司)说明书操作测定;溶磷能力采用定性溶磷圈法[4]和钼锑抗定量比色法[5]测定;分泌IAA 能力采用定性显色法和Salkowski定量比色法[5-6]测定。

  • 采用滤纸条法[7]研究分离到的固氮菌对植物病原真菌的拮抗能力。用LB液体培养基培养分离得到的固氮菌,待菌体浓度达到OD600 = 1时,取菌液备用。在PDA培养基平板中央接种直径为7 mm的植物病原菌菌丝块,分别进行如下3种处理:a对照(无处理);b无菌水滤纸条对照(距菌丝块两侧1.5 cm位置放置用无菌水浸湿的滤纸条);c拮抗试验(距菌丝块两侧1.5 cm位置放置浸湿固氮菌菌液的滤纸条)。3种处理28℃培养7~10 d,观察抑菌效果。待处理b病原菌铺满整个平板时,垂直滤纸条方向测量处理b与处理c病原菌的菌落直径,计算抑制率。抑制率 =(处理b菌落直径−处理c菌落直径)/处理b菌落直径 × 100%。

  • 采用熵权法[8]对分离到菌株性能进行综合评价。

  • 对获得的数据应用SPSS 19.0软件进行单因素方差分析,并用Duncan氏法进行多重比较。

3.   结果与分析
  • 经16S rDNA测序比对发现,从3个试验区杂交构树根际共分离到10株固氮菌,有5株菌为3地共有,分属同1菌株(表1)。与该属模式菌株及最高相似性菌株的16S rDNA基因序列构建的系统发育树(图1)表明:10株菌分属8个属9个种,其中,3地共有菌HTZ1、HTZ2、HTZ3、HTZ4、HTZ5分别被认定为根癌农杆菌(Agrobacterium tumefaciens (Smith et Townsend) Conn)(同源相似性98.16%,下同)、费氏中华根瘤菌(Sinorhizobium fredii (Scholla et Elkan) Chen)(99.82%)、阿拉伯分枝杆菌(Mycolicibacterium arabiense Zhang)(98.88%)、墨西哥假黄单胞菌(Pseudoxanthomonas mexicana Thierry)(97.87%)和日本假黄单胞菌(Pseudoxanthomonas japonensis Thierry)(98.13%)。从天水试验区远根土中另外分离得到的2株菌TS2、TS4,分属纤维弧菌属(Cellvibrio)和固氮菌属(Azotobacter),被认定为Cellvibrio fibrivorans Mergaert(98.17%)和圆褐固氮菌(Azotobacter chroococcum Beijerinck)(99.73%)。从兰州远根土和根表土中另外分离得到的2株菌HP5、HP10分属肠杆菌属(Enterobacter)和鞘氨醇杆菌属(Sphingobium),被认定为烟草肠杆菌(Enterobacter tabaci Duan)(99.53%)和Sphingobium abikonense Kumari(98.70%)。从张掖远根土中分离得的ZY9也属于假黄单胞菌属(Pseudoxanthomonas sp.)(99.38%)。以上菌株在杂交构树根际的分布数量表现为根表土 > 远根土,菌株种类数量最多的为假黄单胞菌属,共3株,优势率为37.5%。3个试验区杂交构树的根内均未分离得到固氮菌。

    分离地
    Collection sites
    菌株
    Strain
    分离部位
    Separated position
    菌落直径
    Colony dia-meter/mm
    生长速度
    Growth rate
    菌落形态
    Colonial morphology
    ①②③ HTZ1 TS 2.0~3.0 ++ 无色透明,表面凸起,边缘整齐,后期有流动性
    Colorless and transparent, convex surface, neat edge, fluidity in later period
    TS2 FRS 2.0~3.0 ++ 无色半透明,表面凸起,边缘不规则,后期有流动性
    Colorless translucent, convex surface, irregular edge, fluidity in later period
    ①②③ HTZ2 TS 0.5~1.0 ++ 乳白半透明,表面凸起,边缘整齐
    Milky white translucent, convex surface, neat edges
    TS4 FRS 0.5~1.0 ++ 乳白不透明,表面凹陷,边缘不规则
    Opalescent opacity, concave surface, irregular edge
    HP5 FRS 1.0~2.0 + 橘黄不透明,表面凸起,边缘整齐,后期生长非常缓慢
    Orange and opaque, raised surface, neat edges, very slow growth later
    ①②③ HTZ3 TS 1.0~2.0 +++ 淡黄半透明,表面凸起,有粘性,边缘不规则,后期流动性强
    Pale yellow and translucent, convex surface, viscous, irregular edge, strong fluidity in later period
    ①②③ HTZ4 TS 0.5~1.0 +++ 淡黄半透明,表面凸起,有粘性,边缘整齐,后期流动性强
    Pale yellow and translucent, convex surface, viscous, neat edge, strong fluidity in later period
    ①②③ HTZ5 TS 0.5~1.0 +++ 淡黄不透明,表面凹陷,有粘性,边缘不规则,后期流动性强
    Pale yellow and opaque, sunken surface, viscous, irregular edge, strong fluidity in later period
    ZY9 FRS 0.5~1.0 ++ 乳白不透明,表面凹陷,边缘不规则,后期棕褐
    Opalescent opaque, surface sunken, margin irregular, brown in later period
    HP10 TS 0.5~1.0 ++ 乳白不透明,表面褶皱,边缘不规则,后期棕褐
    Opalescent opacity, surface pleated, margin irregular,brown in later period
      注:①、②、③分别代表天水、兰州、张掖试验区;分离部位中TS、FRS分别代表根表土、远根土;生长速度中“+、++、+++”分别代表菌株前期生长较慢、中等、较快,48 h内的为生长速度较快,72 h内为中等,96 h内较慢。
      Notes: ①, ② and ③ represent Tianshui, Lanzhou and Zhangye experimental areas respectively. TS and FRS represent root topsoil and far rhizosphere soil, respectively."+, ++, +++" respectively represent the slow, medium and fast growth in the early stage of the strain, the fast growth within 48 h, the medium growth within 72 h, and the slow growth within 96 h.

    Table 1.  Results and colony morphology of root-bound nitrogen-fixing bacteria from hybrid Broussonetia papyrifera

    Figure 1.  Phylogenetic trees of nitrogen fixing strains from rhizosphere constructed based on 16S rDNA gene sequence

  • 表2表明:分离得到的10株固氮菌固氮酶活性存在差异,其中,HTZ4的固氮酶活性最低(130.41 IU·L−1),TS4的活性最高(184.51 IU·L−1)。所有固氮菌均无溶解无机磷的能力,仅HTZ2和TS4在蒙金娜有机磷培养上出现溶磷圈,说明这2株菌具有一定溶解有机磷(卵磷脂)的能力(图2表2),但二者差异不显著,而在发酵液中有机磷的增量表现为TS4显著大于HTZ2。能够分泌IAA的固氮菌有8株,占分离获得菌株数量的80%。HTZ4和HTZ1产IAA显色反应最明显,说明具有较强的分泌IAA的能力,分泌量分别达44.62、36.52 µg·mL−1,与其他菌株差异极显著;HTZ4菌液IAA的分泌量比HP10的高出约20倍(表2)。

    固氮菌
    Nitrogen fixing
    bacteria
    固氮酶活性
    Nitrogenase activity/
    (IU·L−1)
    溶解有机磷
    Soluble phosphorus
    reaction/(mm·mm−1
    有机磷增量
    Organophosphorus increment/
    (µg·mL−1
    产IAA显色反应
    IAA chromogenic
    reaction
    IAA增量
    IAA Increment/
    (µg·mL−1
    HTZ1 139.53 ± 1.09 E 0.00 ± 0.02 B 0.00 ± 0.12 C +++ 36.52 ± 0.69 B
    TS2 137.40 ± 2.05 E 0.00 ± 0.05 B 0.00 ± 0.02 C 0.00 ± 0.00 E
    HTZ2 163.08 ± 1.06 C 1.18 ± 0.02 A 1.60 ± 0.91 B ++ 8.47 ± 0.29 C
    TS4 184.51 ± 0.66 A 1.37 ± 0.06 A 3.15 ± 0.31 A + 3.84 ± 0.24 DE
    HP5 153.05 ± 0.60 D 0.00 ± 0.01 B 0.00 ± 0.22 C + 1.73 ± 0.08 E
    HTZ3 177.52 ± 0.91 B 0.00 ± 0.02 B 0.00 ± 0.17 C 0.00 ± 0.09 E
    HTZ4 130.41 ± 1.61F 0.00 ± 0.05 B 0.00 ± 0.04 C +++ 44.62 ± 0.70 A
    HTZ5 166.58 ± 1.32 C 0.00 ± 0.11 B 0.00 ± 0.02 C ++ 7.74 ± 0.31 C
    ZY9 155.64 ± 0.52 D 0.00 ± 0.08 B 0.00 ± 0.00 C + 5.26 ± 0.17 D
    HP10 182.84 ± 0.55 AB 0.00 ± 0.10 B 0.00 ± 0.00 C + 2.26 ± 0.25 E
      注:表中数据为平均值 ± 标准误。同列不同大写字母表示差异极显著(p < 0.01) ;产IAA显色反应中“−、+、+ +、+ + +”分别代表不变色、浅红色、粉红色、红色。
      Notes: Data in the table are mean ± standard error. Different capital letters in the same column indicated significant difference (p < 0.01);In IAA reaction, "−, +, + +, + + +" respectively represent non-discoloration, light red, pink, and red.

    Table 2.  Results of activity, dissolved phosphorus and IAA production capacity of nitrogen-fixing bacteria

    Figure 2.  Phosphorolysis ability of strains HTZ2(A)and TS4(B)on Montana organophosphate medium

    10株固氮菌对不同植物病原菌的拮抗作用不同(表3图34)。所有菌株对Bc均无拮抗作用,除HTZ2、TS4和TS2对另外4种供试病原菌也无抑制效果或效果微弱外,其余7株菌分别对至少2种以上病原菌有一定的抑制作用,其中,HP5、HP10、HTZ4、ZY9对AsFsFo均有拮抗作用,HTZ4、HP5分别对AsFs的抑菌率最大,分别为50.00%、47.37%,其次为HP10,对AsFs抑制率分别为45.83%、44.44%;HP5、ZY9对Rs也有拮抗作用。

    Figure 3.  Antagonistic test results of the HP5 strain against some pathogenic fungi

    固氮菌
    Nitrogen-fixing bacteria
    抑菌率 Antibacterial rate/%
    FsAsFoBcRs
    HTZ1 0.00 ± 0.12 F 18.37 ± 0.02 F 28.57 ± 0.02 C 0.00 0.00 ± 0.11 D
    TS2 0.00 ± 0.02 F 6.25 ± 0.02 G 0.00 ± 0.11 E 0.00 0.00 ± 0.19 D
    HTZ2 0.00 ± 0.11 F 0.00 ± 0.10 H 0.00 ± 0.05 E 0.00 0.00 ± 0.10 D
    TS4 0.00 ± 0.08 F 0.00 ± 0.02 H 0.00 ± 0.13 E 0.00 0.00 ± 0.01 D
    HP5 47.37 ± 0.76 A 40.00 ± 0.44 C 11.73 ± 0.25 D 0.00 37.58 ± 0.29 B
    HTZ3 0.00 ± 0.05 F 21.55 ± 0.32 E 48.78 ± 1.01 A 0.00 0.00 ± 0.02 D
    HTZ4 42.85 ± 0.31 C 50.00 ± 0.54 A 11.76 ± 0.12 D 0.00 0.00 ± 0.04 D
    HTZ5 25.19 ± 0.45 E 0.00 ± 0.05 H 0.00 ± 0.08 E 0.00 40.74 ± 0.13 A
    ZY9 27.27 ± 0.18 D 37.50 ± 1.65 D 30.30 ± 0.09 B 0.00 34.61 ± 0.96 C
    HP10 44.44 ± 0.77 B 45.83 ± 0.76 B 26.82 ± 1.33 C 0.00 0.00 ± 0.05 D

    Table 3.  Results of antagonism between nitrogen-fixing bacteria and plant pathogenic fungi

    Figure 4.  Antagonistic test results of the HP10 strain against some pathogenic fungi

    通过对分离得到的10株固氮菌进行固氮、溶磷、分泌生长素、抑菌特性测试结果表明:各菌株对不同测试指标表现各异,其中,TS4、HTZ2 具有固氮、溶磷、分泌IAA的能力,但不具备抑制供试病原菌的能力;HP5、HTZ4、HTZ5、ZY9、HP10除无溶磷能力外,具有不同程度的固氮、分泌IAA、抑制病原菌FsAsFoRs的作用,而TS2具有较强的固氮能力。为此,对10株固氮菌的上述4个特性进行了综合评价(表4),排名前5位的菌株为TS4、HP5、ZY9、HTZ4和HTZ5。

    菌株
    Bacterial strain
    各指标比重值 Specific gravity value of each index得分
    Score
    排名
    Ranking
    固氮
    Nitrogen fixation
    有机磷增量
    Organophosphorus increment
    IAA增量
    IAA increment
    抑菌 Bacteriostatic activity
    FsAsFoRs
    HTZ1 0.031 8 0.000 0 0.330 7 0.000 0 0.083 7 0.180 9 0.000 0 0.078 8 8
    TS2 0.024 4 0.000 0 0.000 0 0.000 0 0.028 5 0.000 0 0.000 0 0.004 1 10
    HTZ2 0.114 0 0.336 8 0.076 7 0.000 0 0.000 0 0.000 0 0.000 0 0.104 2 6
    TS4 0.188 9 0.663 2 0.034 8 0.000 0 0.000 0 0.000 0 0.000 0 0.186 2 1
    HP5 0.079 0 0.000 0 0.015 7 0.253 2 0.182 2 0.074 3 0.332 8 0.131 7 2
    HTZ3 0.164 5 0.000 0 0.000 0 0.000 0 0.098 2 0.308 8 0.000 0 0.055 5 9
    HTZ4 0.000 0 0.000 0 0.404 0 0.229 0 0.227 8 0.074 4 0.000 0 0.118 3 4
    HTZ5 0.126 3 0.000 0 0.070 1 0.134 6 0.000 0 0.000 0 0.360 8 0.106 3 5
    ZY9 0.088 1 0.000 0 0.047 6 0.145 7 0.170 8 0.191 8 0.306 5 0.130 5 3
    HP10 0.183 0 0.000 0 0.020 5 0.237 5 0.208 8 0.169 8 0.000 0 0.084 4 7
    熵值 Entropy 0.868 8 0.425 2 0.677 7 0.709 0 0.787 3 0.735 3 0.557 0
    权重 Weight 0.058 6 0.256 6 0.143 9 0.129 9 0.095 0 0.118 2 0.197 8

    Table 4.  Comprehensive evaluation results of nitrogen-fixing bacteria characteristics

4.   讨论
  • 联合固氮菌不同于具有专一宿主植物(如豆科)的共生固氮菌(如根瘤菌),大多聚集在植物根表而在自然生态系统中广泛存在[9],除具有固氮能力外,还可通过分泌生长激素、溶磷、增强抗病性和抗逆性等间接作用促进植物生长[10]。目前,利用联合固氮菌调控植物微生态环境已应用在玉米[11]、甘蔗[12]等作物上,其生态和经济效益潜力前景广阔。本研究对杂交构树根际联合固氮菌的分离属首次。从甘肃3个杂交构树种植试验区共分离到分属8个属的固氮菌,以假黄单胞菌属居多,与通过高通量测序得出构树根际微生物以假单胞菌属数量最多的结果不一致[3],这可能与杂交构树生长年限和取材地域的差异有关。分离得到的固氮菌菌株在根际的分布数量表现为根表土 > 远根土 > 根,这一规律与从玉米和禾草根际分离促生菌[13-14]得到的结果一致;但3地根中没有分离到固氮菌,又与前二者能够在根中获得菌株不一致,这可能与各自的繁殖方式不同有关,前二者主要进行种子繁殖,存在一定内源微生物,杂交构树为无性繁殖组培苗,受微生物侵染的几率较小,炼苗后移植大田的种植年限也较短(3地种植期均为2 a)。另外,黄酮类化合物是杂交构树的主要药用成分,也是微生物向杂交构树根际定殖的重要信号物质[3],幼龄期杂交构树根中黄酮类物质的积累明显不足,有可能造成微生物向根表移动而尚未侵入内根际;但也不能排除分离方法的局限性是根内未分离出菌株的原因之一。上述推测需后续开展试验进一步证实。

    本研究中,5株3地共有菌均来自根表土,说明离杂交构树根系较近土壤中的微生物种类可能只与根中信号传导物质有关;而3地特有种,除HP10以外均来自远根土,说明较远土壤中的微生物种类可能受3地气候、土壤理化性质、类型及耕作措施等因素的影响较大。有研究表明,土壤含氮量是影响植物固氮菌种类和数量的重要因素[15-16],高水平的氮素不利于固氮菌菌群的生长。本研究中,从天水试验区根际土壤中获得的固氮菌种类与数量并未因土壤中全氮和硝态氮水平与兰州、张掖试验区差异较大而呈明显差异,差异仅表现为特有种,可能原因是3地试验区土壤含氮量整体偏低而不足以产生“氮阻遏”效应。杂交构树根部虽不形成根瘤,但从根际分离到了根瘤菌,说明根瘤菌也可定殖于非豆科植物根际固氮[17]

    从菌株特性看,本研究中固氮、溶磷、分泌IAA和抑制植物病原菌能力较强的菌株在属种的分布无较强规律性,具有一定的普遍性,且大多分离自根表土,离根系较近,表明这些菌株的活性与杂交构树根际分泌物的相互作用也有一定相关性[18]。本研究应用熵权法筛选出了综合性能较强的5株固氮菌,但排名靠后的菌株在某些特性方面的表现也较优异,如鞘氨醇杆菌属菌株HP10,在本研究中表现为较高的固氮酶活性和抑制腐皮镰孢菌、茄链格孢的能力,该属菌株在降解芳香族化合物、耐盐碱、青贮中蛋白水解[19-21]等方面都有应用,具有多种生物学功能。因此,可进一步发掘其应用范围或研究与其他菌株的联合应用。另外,杂交构树抗逆性强,耐盐碱[22],后续可在盐碱地种植的杂交构树根际发掘耐盐、耐碱固氮菌株。

5.   结论
  • 从甘肃境内3个种植区的杂交构树根际分离获得10株固氮菌,并分析了其固氮、溶磷、分泌IAA和抑制植物病原菌的特性,为西北半干旱地区杂交构树根际微生物深入研发与利用奠定了基础。

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