作物杂志, 2018, 34(6): 68-75 doi: 10.16035/j.issn.1001-7283.2018.06.011

遗传育种·种质资源·生物技术

一株甘蔗内生菌鉴定及其溶磷能力的研究

狄义宁1, 刘鲁峰1, 谢林艳1, 李咏梅2, 何鹏飞2, 崔文艳2, 李富生1, 何丽莲1,3

1 云南农业大学农学与生物技术学院,650201,云南昆明

2 云南农业大学植物保护学院,650201,云南昆明

3 云南农业大学甘蔗研究所,650201,云南昆明

Identification and Characterization of a Phosphate-Solubilizing Endophyte from Sugarcane

Di Yining1, Liu Lufeng1, Xie Linyan1, Li Yongmei2, He Pengfei2, Cui Wenyan2, Li Fusheng1, He Lilian1,3

1 College of Agronomy and Biotechnology, Yunnan Agricultural University, Kunming 650201, Yunnan,China

2 College of Plant Protection, Yunnan Agricultural University, Kunming 650201, Yunnan, China

3 Sugarcane Research Institute, Yunnan Agricultural University, Kunming 650201, Yunnan, China

通讯作者: 李富生,教授,主要从事甘蔗资源研究与利用;何丽莲为共同通信作者,高级实验师,主要从事甘蔗资源研究与利用

第一联系人: 刘鲁峰为共同第一作者,博士研究生,研究方向为植物资源评价与利用

收稿日期: 2018-06-22   修回日期: 2018-09-13   网络出版日期: 2018-12-15

基金资助: 云南省现代农业甘蔗产业技术体系建设专项.  2017-2018
福建农林大学国家甘蔗工程技术研究中心开放课题.  NER02018.6.1

Received: 2018-06-22   Revised: 2018-09-13   Online: 2018-12-15

作者简介 About authors

狄义宁,硕士研究生,研究方向为植物资源评价与利用 。

摘要

采用形态观察、生理生化测定以及16S rDNA、gyrB和rpoB基因片段比对,对分离自甘蔗品种粤糖86-368茎部内的一株D5内生菌进行分类鉴定。在此基础上,使用平板溶磷圈法和液体培养法研究其溶磷能力,并通过盆栽试验探究其对玉米幼苗生长的影响。结果表明:D5菌株属于Pseudomonas extremorientalis;D5菌株对不同磷酸盐的溶磷能力表现不同,表现为磷酸三钙>植酸钙>磷酸铁,溶磷量分别为0.939±0.012、0.655±0.016、0.125±0.005mg/mL,同时,在溶磷过程中,液体培养基的pH均有不同程度的降低,活菌数除了在磷酸铁培养基中一直下降外,其他磷源培养基中均是先升后降的趋势。施用菌剂后,处理组(D5+P)比对照组(CK+P)玉米幼苗的总鲜重、总干重、苗株总磷含量分别增加45.92%、32.65%、45.01%。因此,D5菌株具有制备微生物菌肥的潜力。

关键词: 甘蔗 ; 内生菌 ; 分子鉴定 ; 溶磷

Abstract

An endophytic D5 strain was isolated from the stem of Yuetang 86-368 which came from one of the sugarcane cultivars.Colony morphology characteristics, physiological and biochemical tests, 16S rDNA, gyrB and rpoB sequence analyses were carried out. Phosphate-solubilizing capability of the strain was studied by methods of phosphorus hole and liquid culture, and the effect on the growth of maize seedling was studied by a pot experiment. The results showed that the D5 strain belonged to Pseudomonas extremorientalis. Its phosphate-solubilizing capability to different phosphate was different which followed the trend: tricalcium phosphate > phytic acid calcium > iron phosphate. The phosphate-solubilizing capacity was 0.939±0.012, 0.655±0.016, 0.125±0.005mg/mL, respectively. The pH value of the liquid media decreased to different extents and the numbers of living bacterium followed a trend of initial rise and then fall in all phosphorus source in the culture medium except in the iron phosphate cultivation showing a straight decline of dissolved phosphorus. Compared with the control (CK+P), the total fresh weight, total dry weight and total phosphorus content of maize seedlings in the D5 strain were increased by 45.92%, 32.65% and 45.01%, respectively. Therefore, it is an excellent candidate for microbial fertilizer.

Keywords: Sugarcane ; Endophyte ; Molecular identification ; Phosphate solubilizing

PDF (1960KB) 元数据 多维度评价 相关文章 导出 EndNote| Ris| Bibtex  收藏本文

本文引用格式

狄义宁, 刘鲁峰, 谢林艳, 李咏梅, 何鹏飞, 崔文艳, 李富生, 何丽莲. 一株甘蔗内生菌鉴定及其溶磷能力的研究[J]. 作物杂志, 2018, 34(6): 68-75 doi:10.16035/j.issn.1001-7283.2018.06.011

Di Yining, Liu Lufeng, Xie Linyan, Li Yongmei, He Pengfei, Cui Wenyan, Li Fusheng, He Lilian. Identification and Characterization of a Phosphate-Solubilizing Endophyte from Sugarcane[J]. Crops, 2018, 34(6): 68-75 doi:10.16035/j.issn.1001-7283.2018.06.011

农业生产中磷素缺乏已成为增产的主要限制因子。我国大部分土壤不缺磷而缺有效磷,土壤中磷含量达到0.4~1.0g/kg,但这些磷素的95%为无效态磷,施入的磷肥大多被土壤固定形成磷酸铝、磷酸铁、磷酸三钙等无机磷和磷酸肌醇、植酸钙、植酸镁等有机磷形态,植物难以利用[1]。而大量磷肥的施入,不但增加了生产的成本,而且导致环境污染。因此,将难溶性磷盐转化成可溶性磷盐是解决当前磷素缺乏的一个研究热点。

利用解磷微生物能将植物难以吸收的磷元素转化为可利用的状态是解决农业生产中磷素缺乏的关键一环。目前,大多数研究集中在从土壤、各种水体等环境中分离解磷细菌(phosphate-solubilizing bacteria,PSB),报道的解磷细菌主要为芽孢杆菌属(Bacillus)、欧文氏菌属(Erwinia)、假单孢菌属(Pseudomonas)、土壤杆菌属(Agrobacterium)、肠细菌属(Enterbacter)等[2]。而鲜有从植物体内分离解磷细菌的报道,其实内生菌普遍存在于植物体内,它们与植物宿主之间存在的特殊关系使得它们更有利于占领生态位,在促进植物生长和生防功能方面表现出较明显的优势。甘蔗是我国重要经济作物,甘蔗内生菌最早发现于1961年,迄今为止已有10种以上的内生菌种属被发现[3],这些内生菌大多具有固氮功能,与甘蔗相关的解磷菌大多分离自甘蔗根际土壤[4,5]

研究溶磷微生物的溶磷能力的方法通常有平板溶磷圈法、液体培养法和土壤培养法3种[6]。平板溶磷圈法:将溶磷菌接种在含难溶性磷酸盐的固体培养基上培养,测定菌落周围产生透明圈的大小。液体培养法:将溶磷菌接种到不溶性磷化物的液体培养液中进行培养,测定培养液中可溶性磷的含量。土壤培养法:将溶磷菌进行土培或砂培,测定土壤或沙子中的有效磷含量。目前对溶磷微生物的溶磷机理的探究,特别是对无机磷的溶解方面,认为是通过有机酸螯合作用实现的。细菌在生长过程中可以产生各种酶类,如核酸酶、磷酸酯酶、植酸酶等,这些酶分泌到细胞外,从而使土壤中有机磷被分解、矿化,起到溶磷作用。

云南农业大学甘蔗资源圃内保育有千余份甘蔗野生种质和品种资源,在对其鉴定评价的基础上[7,8]从其根、茎等部位分离到大量的有益内生菌(待发表)。本文通过平板溶磷圈法和液体培养法对前期研究中筛选到的甘蔗内生菌D5进行溶磷能力和溶磷特性研究,并通过玉米幼苗盆栽试验研究其促生长的作用,同时从菌株的形态特征、生理生化特性和16S rDNA、gyrB和rpoB基因序列分析,确定其分类地位,从而为后续试验研究和开发应用提供理论依据和优良菌株。

1 材料与方法

1.1 供试菌株

内生菌株D5(GenBank ID:MG385660)为分离自云南农业大学甘蔗资源圃保育的甘蔗栽培品种“粤糖86-368”茎部;对照菌株为高效溶磷细菌洋葱伯克霍尔德氏菌(Burkholderia cepacia),代号为1-2,玉米材料为“云禾单2号”。

1.2 试验方法

1.2.1 平板溶磷圈法测定溶磷能力 将菌株D5、1-2(对照)接种在改良的Pikovskaya[9](简称PVK)、NBRIP[10]和植酸钙[11]固体培养基上,置于28℃恒温培养箱中倒置培养。7d后测量菌落直径(d)和溶磷圈直径(D),计算溶磷圈与菌落直径比(D/d)来初步确定供试菌株的解磷能力。

1.2.2 摇瓶测定解磷菌溶磷量 试验以不接菌摇瓶为对照(CK),处理组以1%的接种量将D5、1-2目标菌株接入已灭菌(1MPa,121℃高压蒸汽灭菌30min)的PVK、NBRIP、植酸钙、磷酸铁液体培养基中[磷酸铁培养基是将NBRIP中的Ca3(PO42置换为FePO4,其余成分不变],每个处理设置3个重复,于28℃、160r/min恒温摇床培养。在接菌后1、3、5、7、9d吸取各处理发酵液进行稀释涂板,在CK培养基中无菌生长的情况下,记录各处理组发酵液中目标菌的活菌数;使用雷磁PHS-3C测定发酵液pH;采用钼蓝法[12]测定可溶性磷含量,发酵液经10 000r/min离心5min后加入等体积钼蓝试剂[12],反应时间30min,于波长850nm读取吸光度A,用不同浓度K2HPO4溶液与钼蓝试剂等体积反应绘制标准曲线。

1.2.3 D5菌对玉米幼苗生长的影响及磷素吸收盆栽试验 玉米幼苗准备:选择饱满健康的玉米种子,用有效氯2%的次氯酸钠2min,75%酒精5min表面消毒,然后用无菌水冲洗2~3次。浇施无菌水放置于光照培养箱内催芽,待其幼芽生长到约0.5cm时进行移栽。

苗株移栽及栽培基质准备:选取长势一致的玉米苗,将其移栽到黑色营养钵(高10cm,直径12cm)中,基质体积为50mL。基质分为加磷与不加磷处理,加磷处理为草泥炭:珍珠岩:蛭石(体积比2:1:1),每100g混合基质中拌入6%的磷矿粉,磷矿粉来源为市场选购,可溶性磷含量为(0.90±2.72)g/kg(钼锑抗法测量),无磷基质组则不混磷矿粉。

菌液处理:D5菌经过LB培养48h后,于8 000r/min离心10min,用无菌水重悬,采用稀释涂布平板法计数,菌液于4℃保存备用。

营养液配制:采用Hoagland营养液与改良的Hoagland无磷培养液。无磷培养液是将含磷物质等摩尔置换制备,将磷酸铵置换为硝酸铵,磷酸二氢钾置换为硫酸钾。

试验设置5个处理。处理1:浇施0.5倍去磷改良Hoagland营养液+无磷基质(CK);处理2:浇施0.5倍去磷改良Hoagland营养液+含磷基质(CK+P);处理3:浇施0.5倍Hoagland全液+含磷基质(CK2);处理4:浇施0.5倍去磷改良Hoagland营养液+D5菌+含磷基质(D5+P);处理5:浇施0.5倍无磷源改良Hoagland营养液+D5菌+无磷基质(D5)。每盆种植2株玉米苗,重复5次,每个处理共10株,采用灌根法,每周灌根浇水1次,每次25mL,共两次。菌液灌根密度设置为107cfu/g。

培养20d后以10株玉米苗为一组测量鲜重和干重,根据《土壤农业化学分析方法》[13]测量植株全磷含量。

1.2.4 形态特征观察及生理生化鉴定 将D5菌株在LB平板上培养48h,观察其形态特征,进行革兰氏染色;按照常见细菌系统鉴定手册方法[14,15]分析其生理生化特性(柠檬酸盐的利用、淀粉水解、硝酸盐还原、吲哚产生、明胶液化、硫化氢试验等)。

1.2.5 菌株16S rDNA、gyrB、rpoB片段的分子鉴定 根据Cheng等[16]的方法提取细菌DNA。gyrB基因片段采用通用引物UP-1S:GAA GTC ATC ATG ACC GTT CTG CA,UP-2Sr":AGC AGG GTA CGG ATG TGC GAG CC;16S rDNA采用通用引物P0:GAG AGT TTG ATC CTG GCT CAG,P6:CTA CGG CTA CCT TGT TAC GA;rpoB基因片段采用通用引物M13 forward (-20):GTA AAA CGA CGG CCA GT,M13 reverse (-48):AGC GGA TAA CAA TTT CAC ACA GGA。PCR反应程序:94℃ 4min 30s;94℃ 45s,53℃5s,72℃ 1min,30个循环;72℃ 10min。PCR产物交擎科公司进行测序。将测序所得结果用DNAMAN软件拼接成完整的序列,用于NCBI nucleotide Blast在基因库中完成比对。

1.2.6 菌株系统发育树的构建 参照常见细菌系统鉴定手册方法[17],通过MEGA 6软件,采用最大似然法对16S rDNA、gyrB、rpoB基因片段构建系统发育树,模型为Tamura-Nei,自展值统一设置为1000。

2 结果与分析

2.1 D5菌株的溶磷效果

2.1.1 D5在不同磷源固体培养基上的溶磷效果溶磷圈直径(D)与菌落直径(d)的比值(D/d)是表征解磷菌相对溶磷能力的一个指标,可初步确定菌株的溶磷能力。D5菌株在PVK、NBRIP和植酸钙培养基上溶磷效果较好,D/d值分别达到1.50、2.88、2.44(表1)。

表1   平板溶磷圈法测量的菌株溶磷能力(7d)

Table 1  Halo of phosphate-solubilization microorganism in solid media (7d) cm

培养基类型Medium type菌株Strain菌落颜色Colony color菌落直径Colony diameter (d)溶磷圈直径Phosphorus ring diameter (D)D/d
PVK (Pikovskaya)1-2褐红0.4751.5443.25
D5砖红1.1001.6501.50
NBRIP1-2黑蓝0.8501.8002.12
D50.4901.1502.88
植酸钙Calcium phytate1-2淡紫0.6241.2241.96
D5淡黄0.5081.2402.44

新窗口打开| 下载CSV


与对照菌株洋葱伯克霍尔德氏菌1-2相比,D5菌株在NBRIP无机磷培养基和植酸钙有机磷培养基上D/d值均大于1-2,在PVK培养基上虽然溶磷圈与菌落直径最大,达到1.650cm和1.100cm,但D/d值却小于1-2。上述结果表明,D5菌株在固体培养基上对无机磷和有机磷均表现出了一定的解磷潜力。

2.1.2 可溶性磷含量标准曲线的建立 选取5个不同浓度梯度的K2HPO4溶液,建立标准曲线。回归方程为y=0.038+1.777x(r2=0.9992)。

2.1.3 D5菌在不同磷源液体培养基下的溶磷量、活细胞数及pH的动态变化 平板溶磷圈法只可定性测定其溶磷能力。采用液体培养法可对菌株的溶磷能力进行定量测定,对其溶磷能力进行具体描述。在PVK、NBRIP、磷酸铁以及植酸钙的液体培养基中,D5菌株对不同磷酸盐的溶磷能力表现不同,其强弱表现为磷酸钙>植酸钙>磷酸铁。除了对磷酸铁溶磷效果不太明显,D5菌株溶磷趋势均是先升高后下降再逐渐趋于稳定,比阴性对照(CK)和阳性对照1-2菌株表现更优,解磷效果明显(图1)。

图1

图1   培养液中可溶性磷含量随时间的变化

PVK(A)、NBRIP(B)、磷酸铁(C)、植酸钙(D),下同

Fig.1   Variation available phosphorus content in culture medium during solubilizing period

PVK (A), NBRIP (B), ironic phosphate (C) and calcium phytate (D), the same below


图1-A可以看出,在PVK培养基中,D5菌在前3d与对照菌溶磷量基本持平;3d后,对照菌株1-2溶磷量开始下降,而D5菌持续增加,第7天时达到最大溶磷量(0.939±0.012mg/mL)。从图1-B看出,在NBRIP培养基中,菌株D5溶磷量与对照菌株1-2相似,在第7天达到最大值,可溶性磷含量为(0.752±0.009)mg/mL。而从图1-C可看出,在磷酸铁培养基中,D5和对照菌株1-2对磷酸铁的溶磷效果均不明显。从图1-D可以看出,在植酸钙培养基中,D5在第5天时解磷量达到最大值,解磷量为(0.655±0.016)mg/mL。由液体培养试验可知,D5菌株在以Ca3(PO42和植酸钙为磷源环境时,具有良好的解磷效果。这也进一步印证了其他研究者的研究结果,不同的解磷菌对不同的磷源具有特异性。

在对溶磷过程的活菌数统计时发现,在PVK培养基(图2-A)中,菌株D5在第3天时活菌数量达到最大峰值,随后开始逐渐降低,同时,对照菌株1-2活菌数量也在第3天时达到最大值,而后开始缓慢降低。从图2-B可得,D5菌在3d后开始死亡,活菌数量持续衰减。从图2-D可得,在植酸钙培养基中,D5菌在第3天时活菌数达到峰值,而3d后活菌数量急剧降低。在以上3种磷源环境中,菌株D5活菌数均呈现先上升后下降的规律,唯有在图2-C中,D5菌的活菌数量在接入后便开始急剧下降,在5d时,培养基上已无活菌,故试验在第5天时停止。

图2

图2   培养液中活菌数随时间的变化

Fig.2   Active bacteria number in culture medium during solubilizing period


在每一段时期,试验对各个处理的发酵液pH进行统计时发现,接种功能菌后,接菌培养基的pH(4~5)均比对照CK低(图3)。

图3

图3   培养液中pH随时间的变化

Fig.3   pH in culture medium during solubilizing period


2.1.4 接种D5菌对玉米幼苗生长和植株磷含量的影响 接种溶磷菌D5可以显著促进玉米苗株的生长,由表2可知,在含磷基质中,接种D5处理(D5+P)的总鲜重比未接种处理(CK+P)高45.92%,总干重增加32.65%。仅比全营养液处理(CK2)低9.09%。

表2   接种D5菌对玉米幼苗生长影响

Table 2  Effects of D5 inoculation on maize seedling growth and plant phosphorus content

处理
Treatment
地上鲜重(g)
Aboveground fresh weight
地上干重(g)
Aboveground dry weight
地下鲜重(g)
Underground fresh weight
地下干重(g)
Underground dry weight
总鲜重(g)
Total fresh weight
总干重(g)
Total dry weight
D5+P16.781.6510.640.9527.422.60
D58.730.948.590.7417.321.68
CK+P11.971.126.820.8418.791.96
CK6.770.737.430.6514.201.38
CK220.181.7710.231.0930.412.86

新窗口打开| 下载CSV


图4可知,接种D5对苗株总磷含量起到增加作用,接种D5比未接种总磷含量高出45.01%,与CK2相差6.92%。

图4

图4   盆栽试验各处理10株苗全磷总含量与盆栽长势

Fig.4   Total phosphorus content and pot growth of 10 seedlings in pot experiment


2.2 D5菌株的鉴定

2.2.1 形态观察,生理生化测定及16S rDNA、gyrB、rpoB基因同源性比对 将D5菌株在LB平板上划线后,放置于28℃恒温培养箱中培养48h,通过观察可知,菌落为圆形,较小,突起,表面光滑,直径0.1~0.2cm,无菌丝并且易挑起,符合细菌菌落形态。在甲基红试验,革兰氏染色和V.P试验中均呈阴性,具有氧化酶及水解淀粉和液化明胶的能力,不产生H2S和吲哚,不能利用乳糖,基本符合假单胞菌属生理特性。基因片段在NCBI中进行Blast比对,结果见表3,D5菌株在16S rDNA、gyrB、rpoB基因水平上均归类于假单胞菌属(Pseudomonas),16S rDNA基因片段比对时发现,与Pseudomonas extremorientalis CNU082017同源一致性达到100%,rpoB基因片段与模式菌株Pseudomonas extremorientalis DSM15824T比对结果为99%。

表3   D5菌株16S rDNA,gyrB,rpoB基因片段在NCBI数据库中的比对结果

Table 3  Comparison of 16S rDNA, gyrB and rpoB gene fragments of strain D5 in NCBI database

菌株Strain类群Species属Genus长度Length (bp)同源菌株Homologous bacteria序列号GenBank一致性Indentities(%)
D5Pseudomonas16S rDNA1 436Pseudomonas extremorientalisCNU082017KF979139.1100
gyrB1 171Pseudomonas extremorientalis BS2774LT629708.199
rpoB1 552Pseudomonas extremorientalisDSM15824TKX186863.199

新窗口打开| 下载CSV


2.2.2 系统发育树的构建 以16S rDNA、gyrB、rpoB基因构建系统发育树(图5、6、7),D5菌株与假单胞菌属Pseudomonas extremorientalis处在同一分支,在16S rDNA基因水平上,D5与Pseudomonas extremorientalis KMM 3447T可信度达到94%;在gyrB基因上,与模式菌株Pseudomonas extremorientalis LMG 19695T可信度达100%;在rpoB基因水平上,与Pseudomonas extremorientalis LMG 19695T可信度达到92%。D5被鉴定为Pseudomonas extremorientalis。

图5

图5   溶磷性内生菌株16S rDNA 序列系统发育树

系统发育树分支上的数值表示大于50%的自举值,标尺指核苷酸替代数,下同

Fig.5   Phylogenetic tree generated by the neighbor-joining method based on 16S rDNA sequences of selected strains

Bootstrap values (1000 replicates) above 50% are indicated above the branches. Scale bar indicates 0.05% substitution of nucleotide. The same below


图6

图6   溶磷性内生菌株gyrB序列系统发育树

Fig.6   Phylogenetic tree generated by the neighbor-joining method based on gyrB sequences of selected strains


图7

图7   溶磷性内生菌株rpoB序列系统发育树

Fig.7   Phylogenetic tree generated by the neighbor-joining method based on rpoB sequences of selected strains


3 讨论

目前对甘蔗内生解磷菌种类以及菌株解磷功能的研究比较少,Gangwar等[18]曾在8株甘蔗内生菌中筛选出一株具有解磷能力的细菌并将其鉴定为肠杆菌属(Enterobacter)。而在甘蔗内生假单胞菌属(Pseudomonas)的研究中,荧光假单胞菌(P. fluorescence)与恶臭假单胞菌(P. putida)较为常见[19,20]。本研究从甘蔗茎内分离到的P. extremorientalis菌株尚未见报道。

有研究者认为,单一的溶磷圈直径或溶磷圈直径与菌落直径的比值并不能真实反映菌株在溶磷培养基中所释放的可溶性磷量,因为有些溶磷细菌虽然在固体平板上无法形成水解圈,但液体培养时可以利用多种难溶性无机磷[21]。本研究首先通过平板透明圈的大小来推测菌株溶磷能力,而后对培养液中可溶性磷的含量进行定量分析,再利用盆栽试验测定植株全磷含量,3项指标综合分析,验证了D5菌株溶磷与供磷的能力,结果更具说服力。另外,在对接种菌株后的培养液pH进行测定时,发现接种溶磷菌处理的pH低于不接菌处理,推测D5菌株溶磷机制符合有机酸理论[22]。赵小蓉等[23]发现培养介质pH的降低并不是细菌溶磷的必要条件,而且具体到某个菌株,也不严格遵循pH越低溶磷量越高这个规律。所以,D5菌株溶磷机制与pH的关系仍有待进一步研究。最后,本研究仅在玉米苗期进行了促生长的相关试验,而对于浇施D5菌后是否有利于甘蔗幼苗磷元素含量的增加则有待研究。

The authors have declared that no competing interests exist.
作者已声明无竞争性利益关系。

参考文献

张宝贵, 李贵桐 .

土壤生物在土壤磷有效化中的作用

土壤学报, 1998,35(1):102-111.

[本文引用: 1]

黄静 .

植物内生解磷细菌的分离筛选及其生物多样性

南京:南京农业大学, 2009.

DOI:10.7666/d.Y1762551      URL     [本文引用: 1]

磷是植物生长必需的矿质元素之一。尽管土壤磷素含量丰富,但其中大部分不能被植物直接吸收利用。具有解磷能力的微生物在土壤磷的生物循环系统中担任着重要的角色,它可以将难溶无机磷转化为可以被植物吸收利用的可溶性磷。目前研究者们都是从土壤、各种水体等环境中分离解磷细菌,还没有人从植物体内分离解磷细菌。因此本文从微生物解磷资源的开发入手,利用解磷细菌筛选原理,从健康生长的常见作物体内分离筛选植物内生解磷细菌,研究其生物多样性和促生特性,拓宽了解磷细菌的来源,为解磷细菌应用于农业生产提供理论依据。 本试验共分离到61株内生解磷细菌。用NBRIP液体摇瓶法研究这些解磷细菌对磷酸钙的溶解能力,培养5天后发现菌株均能不同程度地溶解磷酸钙释放出有效磷,解磷量最大的达到537.6 mg·L-1。菌株对磷酸钙的解磷能力与pH呈一定的负相关性(r2=-0.46),所有接菌处理均使培养液pH不同程度地降低,最大降幅达到2.79个pH单位。选择其中的部分代表性菌株进行磷矿粉的分解实验,培养5天后,供试菌株释放出的有效磷含量为1.8-231.0 mg·L-1,是对照的3.2-42倍。菌株对磷矿粉的分解能力与培养液pH值的负相关性达到-0.67。 对供试菌株进行促生相关的生物学特性的测定,结果表明分离的解磷内生细菌均不同程度的具有促生特性,80%的供试菌株能产生IAA,79%的供试菌株能产生铁载体,37%的供试菌株能利用ACC为唯一氮源生长。 利用细菌通用引物对供试菌株16S核糖体DNA进行扩增获得约1500 bp片段,分别用内切酶HhaⅠ和HaeⅢ对扩增产物进行限制性酶切,产生不同的酶切图谱。根据酶切图谱聚类分析结果,供试菌株在76%相似性水平上被归为13类,显示出分离的内生解磷细菌种群具有一定的多样性。结合解磷能力、促生特性及酶切聚类分析结果等指标,挑选具有代表性的20株内生解磷细菌菌株进行16Sr DNA序列系统发育分析及鉴定,共鉴定出9个属的内生解磷细菌,分别为假单胞菌属(Pseudomonas)不动杆菌属(Acinetobacter)、伯克霍尔德氏菌属(Burkholderia)、泛菌属(Pantoea)、肠杆菌属(Enterobacter)、嗜酸菌属(Acidovorax)、罗尔斯顿菌属(Ralstonia)、芽孢杆菌属(Bacillus)、克雷伯氏菌属(Klebsiella),其中假单胞菌属、泛菌属、伯克霍尔德氏菌属为优势种属。结果表明植物内生解磷细菌种类比较丰富,而且大多数均是常见的单独作为内生细菌或解磷细菌报道的菌属,其中嗜酸菌属和罗尔斯顿菌属作为内生细菌或解磷细菌都很少见报道。 从分离物中选择3株菌(Pantoea agglomerans U2-22、Pantoea dispersa M1R4、Burkholderia cepacia M2S2)以利福平标记,浸种法回接于玉米,在添加不同磷源的蛭石中进行盆栽试验。结果表明在以磷酸钙为磷源时,接菌处理(U2-22、M1R4、M2S2)使植株总干重比对照分别增加11.6%、25.5%、7.57%,其中接M1R4、M2S2显著促进玉米对磷的吸收积累,全植株中磷积累总量比对照分别增加43.1%、40.8%;在磷矿粉为磷源时,M1R4、M2S2使植株总干重比对照分别增加10.5%、3.37%,但不影响植株对磷的吸收积累,U2-22没有增加玉米生物量,但是显著提高了植株地上部及根部对磷的吸收积累量,分别比对照增加35.0%和36.3%。

Dobereiner J .

Nitrogen-fixing bacteria of the genus Beijerinck Derx in the rhizosphere of sugar cane

Plant and Soil, 1961,15(3):211-216.

DOI:10.1007/BF01400455      URL     [本文引用: 1]

The rhizosphere effect of sugar cane on nitrogen fixing bacteria of the genus Beijerinckia Derx was studied under field conditions, during two growing periods of the cane. Counts of Beijerinckia in soil samples from the rhizosphere and from the rhizoplan (soil from root surface) showed an increase in this nitrogen fixer of up to 20 times in the rhizosphere and up to 50 times in the rhizoplan. Bacteria, actinomyces, and fungi developing on egg-albumin agar decreased in the rhizosphere of sugar cane.

陈炫, 林希昊 .

甘蔗根际土壤解磷细菌的筛选及培养条件优化

热带农业科学, 2017,37(12):61-69.

URL     [本文引用: 1]

利用平板溶磷圈法和钼锑抗比色法,从甘蔗根际土壤筛选出2株溶磷能力较强的解磷细菌(N1-1-3和H4-3-1),并通过单因素试验和多因素正交试验对其进行培养条件优化。结果表明,N1-1-3菌株以乳糖为碳源、氯化铵为氮源、培养温度30℃、初始pH为6.5为最佳培养条件,溶磷量为190.57 mg/L;H4-3-1菌株以葡萄糖为碳源、硝酸钾为氮源、培养温度30℃、初始pH为7.5为最佳培养条件,溶磷量为167.82 mg/L。由于N1-1-3和H4-3-1解磷菌株溶磷能力较强,可作为盆栽试验或田间试验的应用材料。

史国英, 莫燕梅, 岑贞陆 , .

一株高效解无机磷细菌BS06的鉴定及其解磷能力分析

微生物学报, 2015,42(7):1271-1278.

DOI:10.13344/j.microbiol.china.140721      URL     [本文引用: 1]

[Objective] The purpose of the present study was to investigate the phosphate-solubilizing ability of a bacterial stain BS06, for developing and applying effectively phosphate-solubilizing microbes in sugarcane production in Guangxi. [Methods] 16S rRNA gene sequence homology comparison, recA gene analysis as well as morphological, physiological and biochemical analyses were carried out to identify the bacterial strain BS06. The phosphate-solubilizing ability of BS06 was investigated under the conditions of changing the carbon and nitrogen source, separately in inorganic phosphate medium. [Results] The strain BS06 was identified as Burkholderia cepacia. The strain had high and efficient phosphate-solubilizing ability under the condition of carbon source with lactose and nitrogen source with sodium nitrate separately, and the concentration of water soluble phosphorus in fermentation solution was 262.71 mg/L and 305.85 mg/L, respectively. Compared with the uninoculated controls, BS06 significantly increased dry weights and phosphorus contents of micropropagated sugarcane seedlings. [Conclusion] The bacterial strain BS06 had high potential of exploitation.

赵小蓉, 林启美, 孙焱鑫 .

细菌解磷能力的测定方法的研究

微生物学报, 2001,28(1):1-4.

[本文引用: 1]

曹哲群, 肖芙荣, 陈疏影 , .

7个蔗茅野生种及其后代材料苗期耐寒性鉴定

作物杂志, 2017(5):43-48.

DOI:10.16035/j.issn.1001-7283.2017.05.008      URL     [本文引用: 1]

以7个蔗茅野生种及其与甘蔗杂交的3个后代材料[滇蔗品种(系)]为试验材料,在苗期进行低温(3℃)胁迫,测定与耐寒性相关的各项生理生化指标。结果表明:(1)通过极点排序法计算得到电导率,可溶性糖、丙二醛、脯氨酸、可溶性蛋白、叶绿素含量,超氧化物歧化酶活性的权重系数分别为0.109、0.194、0.148、0.133、0.108、0.183、0.124,其中可溶性糖与叶绿素含量的权重系数最大,说明这两项生理指标对甘蔗耐寒性影响较大;(2)在低温(3℃)胁迫下,10个供试材料的耐寒性强弱为蔗茅99-3〉蔗茅99-2〉蔗茅Ⅱ91-2〉蔗茅99-4〉蔗茅99-1〉蔗茅90-29〉蔗茅Ⅰ91-8〉滇蔗04-14〉滇蔗01-58〉滇蔗02-39,表明蔗茅野生种的耐寒性均强于滇蔗品种(系)。

石景雨, 何丽莲, 王先宏 , .

不同甘蔗品种叶片中总黄酮含量与提取工艺的优化研究

作物杂志, 2016(5):19-24.

DOI:10.16035/j.issn.1001-7283.2016.05.004      URL     [本文引用: 1]

以云南农业大学自育的甘蔗品种滇蔗01-58为试验材料,以芦丁为标准品,采用分光光度计法研究滇蔗01-58叶片中总黄酮的最佳提取条件。以提取率作为指标,测定不同类型甘蔗[野生种、栽培品种(系)]叶片中黄酮含量。结果表明,基于单因素和正交试验的最佳提取条件为乙醇浓度70%、提取温度70℃、提取时间90min;不同类型甘蔗叶片的黄酮提取率有明显差异,栽培种黄酮含量最高的为滇蔗09-38,野生种黄酮含量最高的为五节芒Ι91-27,总体表现为野生种叶片中黄酮含量高于栽培品种(系)。

叶劲松, 吴克, 俞志敏 .

一株无机磷细菌筛选及溶磷能力的测定

江苏农业科学, 2013,41(6):333-335.

DOI:10.3969/j.issn.1002-1302.2013.06.120      URL     [本文引用: 1]

以Ca3(PO4)2为唯一磷源,从云南土壤样品中筛选到1株无机磷溶解能力较强的细菌。形 态特征为乳黄色,短杆、产荚膜、革兰氏阴性。溶磷试验结果表明:经固体平板培养,溶磷圈培养5d时达到10.7mm,溶磷圈和菌落直径比达到1.6;经 7d液体摇床培养,分别以磷酸钙和磷矿粉为唯一磷源的培养液中有效磷总量最高达161.32、18.12mg,/L,相应的溶磷率为16.14%、 2.49%。此菌溶磷活性较高且稳定,在微生物肥料等研制中有较大应用潜力。

Johri J K, Surange S, Nautiyal C S .

Occurrence of salt,pH,and temperature-tolerant,phosphate-solubilizing bacteria in alkaline soils

Current Microbiology, 1999,39(2):89-93.

DOI:10.1007/s002849900424      URL     PMID:10398833      [本文引用: 1]

An ecological survey was conducted to characterize 4800 bacterial strains isolated from the root-free soil, rhizosphere, and rhizoplane of Prosopis juliflora growing in alkaline soils. Of the 4800 bacteria, 857 strains were able to solubilize phosphate on plates. The incidence of phosphate-solubilizing bacteria (PSB) in the rhizoplane was highest, followed by rhizosphere and root-free soil. Eighteen bacterial strains out of 857 PSB were able to produce halo at 30°C in a plate assay in the presence of 5% salt (NaCl) and solubilize tricalcium phosphate in National Botanical Research Institute's phosphate growth medium (NBRIP) broth, in the presence of various salts, pHs, and temperatures. Among the various bacteria tested, NBRI4 and NBRI7 did not produced halo in a plate assay at 30°C in the absence of salt. Contrary to indirect measurement of phosphate solubilization by plate assay, the direct measurement of phosphate solubilization in NBRIP broth assay always resulted in reliable results. The phosphate solubilization ability of NBRI4 was higher than in the control in the presence of salts (NaCl, CaCl 2 , and KCl) at 30°C. Phosphate solubilization further increased in the presence of salts at 37°C as compared with 30°C. At 37°C, CaCl 2 reduced phosphate solubilization ability of NBRI4 compared with the control. The results indicated the role of calcium salt in the phosphate solubilization ability of NBRI4.

邵佳慧 .

解淀粉芽孢杆菌SQR9吲哚乙酸合成途径

南京:南京农业大学, 2014.

[本文引用: 1]

Pei-Chun (Lisa) Hsu .

Determination of genes involved in bacterial phosphate solubilization.

Lincoln University, New Zealand, 2014.

[本文引用: 2]

鲁如坤 . 土壤农业化学分析方法. 北京: 中国农业科技出版社, 2000: 312-314.

[本文引用: 1]

Buchanan R E, Gibbons N E. 伯杰细菌鉴定手册. 8版. 北京: 科学出版社, 1984: 274-278.

[本文引用: 1]

Holt J G, Krieg N R, Sneath P H A ,et al.

Bergey’s Manual of Determinative Bacteriology (9th ed)

Baltimore:Williams and Wilkins, 2004.

URL     [本文引用: 1]

Cheng H R, Jiang N .

Extremely rapid extraction of DNA from bacteria and yeasts

Biotechnology Letters, 2006,28(1):55-59.

DOI:10.1007/s10529-005-4688-z      URL     PMID:16369876      [本文引用: 1]

A very simple and rapid method for extracting genomic DNA from Gram-negative bacteria, Gram-positive bacteria and yeasts is presented. In this method, bacteria or yeasts are lysed directly by phenol and the supernatant is extracted with chloroform to remove traces of phenol. The supernatant contains DNA that is suitable for molecular analyses, such as PCR, restriction enzyme digestion and genomic library construction. This method is reproducible and simple for the routine DNA extraction from bacteria and yeasts.

Kim B J, Kim C J, Chun J , et al.

Phylogenetic analysis of the genera Streptomyces and Kitasatospora based on partial RNA polymerase β-subunit gene (rpoB) sequence

International Journal of Systematic and Evolutionary Microbiology, 2004,54(2):593-598.

DOI:10.1109/7.705893      URL     PMID:15023980      [本文引用: 1]

The RNA polymerase beta-subunit genes (rpoB) of 67 Streptomyces strains, representing 57 species, five Kitasatospora strains and Micromonospora echinospora KCTC 9549 were partially sequenced using a pair of rpoB PCR primers. Among the streptomycetes, 99.7-100% similarity within the same species and 90.2-99.3% similarity at the interspecific level were observed by analysis of the determined rpoB sequences. The topology of the phylogenetic tree based on rpoB sequences was similar to that of 16S rDNA. The five Kitasatospora strains formed a stable monophyletic clade and a sister group to the clade comprising all Streptomyces species. Although there were several discrepancies in the details, considerable agreement was found between the results of rpoB analysis and those of numerical phenetic classification. This study demonstrates that analysis of rpoB can be used as an alternative genetic method in parallel to conventional taxonomic methods, including numerical phenetic and 16S rDNA analyses, for the phylogenetic analyses of the genera Streptomyces and Kitasatospora.

Gangwar M, Kaur G .

Isolation and characterization of endophytic bacteria from endorhizosphere of sugarcane and ryegrass

Internet Journal of Microbiology, 2008,7(1):1-7.

[本文引用: 1]

Viswanathan R, Samiyappan R .

Induction of systemic resistance by plant growth promoting rhizobacteria against red rot disease in sugarcane

Sugar Tech, 1999,1(3):67-76.

DOI:10.1007/BF02945166      URL     [本文引用: 1]

Plant growth promoting rhizobacteria (PGPR) belonging to fluorescent pseudomonads group were isolated from sugarcane rhizosphere. Selected strains were studied for the induced systemic resistance (ISR) against Colletotrichum falcatum Went causing red rot disease in the sugarcane stalks by three different resistance evaluation methods. The talc based formulations of the PGPR strains were prepared and applied through different methods and in different growth phases of the crop in the field. The pathogen was inoculated artificially later in the upper internodes and ISR was assessed. All the tested PGPR strains have significantly reduced the disease development in the stalks. ISR effect was more pronounced when the resistance was evaluated by nodal method of testing. In another trial the PGPR strains were applied to the basal stalk region in the marcotting mixture. When the pathogen was inoculated later on the upper internodes, disease development was significantly reduced in the PGPR treated canes as compared to the untreated controls. The induced resistance was maintained upto 90 days in the host after the PGPR treatment. In all these studies disease development was contained in a highly susceptible sugarcane cultivar CoC 671. When the pathogen load in the diseased canes was assessed by ELISA it was found that the PGPR treated canes recorded low titer than the control treatments. Biochemical studies indicated enhanced levels in the defense related enzymes like chitinase and peroxidase in the treated cane tissues. The pathogen inoculation resulted in lower brix, purity and commercial cane sugar percent in the PGPR untreated canes compared to the PGPR treated ones. Moreover acid and neutral invertase levels were significantly higher in the former than the latter. The induced resistance in sugarcane especially in a highly susceptible variety offers a possible alternative approach to tackle the disease under field situations and to enhance sugarcane yield.

Magnani G S, Didonet C M, Cruz L M , et al.

Diversity of endophytic bacteria in Brazilian sugarcane

Genetics & Molecular Research, 2010,9(1):250-258.

DOI:10.4238/vol9-1gmr703      URL     PMID:20198580      [本文引用: 1]

Endophytic bacteria live inside plant tissues without causing disease. Studies of endophytes in sugarcane have focused on the isolation of diazotrophic bacteria. We examined the diversity of endophytic bacteria in the internal tissues of sugarcane stems and leaves, using molecular and biochemical methods. Potato-agar medium was used to cultivate the endophytes; 32 isolates were selected for analysis. DNA was extracted and the 16S rRNA gene was partially sequenced and used for molecular identification. Gram staining, catalase and oxidase tests, and the API-20E system were used to characterize the isolates. The strains were divided into five groups, based on the 16S rRNA sequences. Group I comprised 14 representatives of the Enterobacteriaceae; group II was composed of Bacilli; group III contained one representative, Curtobacterium sp; group IV contained representatives of the Pseudomonadaceae family, and group V had one isolate with an uncultured bacterium. Four isolates were able to reduce acetylene to ethylene. Most of the bacteria isolated from the sugarcane stem and leaf tissues belonged to Enterobacteriaceae and Pseudomonaceae, respectively, demonstrating niche specificity. Overall, we found the endophytic bacteria in sugarcane to be more diverse than previously reported.

刘文干, 何圆球, 张坤 , .

一株红壤溶磷菌的分离、鉴定及溶磷特性

微生物学报, 2012,52(3):326-333.

URL     Magsci     [本文引用: 1]

【目的】为了提高红壤磷素利用率,探讨溶磷菌溶磷机理。【方法】利用难溶性无机盐培养基从花生根际土壤样品中分离到一株溶磷菌C5-A,结合菌落形态特征、生理生化和16S rRNA 序列确定该菌株的系统发育地位;通过菌株C5-A 在NBRIP 液体培养基培养过程中培养液pH 变化确定其溶磷能力;利用液体发酵实验测定不同的碳源、氮源对菌株C5-A 溶磷的影响;通过高效液相色谱检测C5-A 在不同氮源培养液中有机酸的种类和浓度。【结果】菌株C5-A 鉴定为洋葱伯克霍尔德氏菌(Burkholderia cepacia),遗传稳定性较好。在FePO4和AlPO4培养液中,菌株C5-A 的溶磷量和pH 变化呈显著负相关;菌株C5-A对磷酸三钙、磷酸铝、磷酸铁、磷矿粉均有较强的溶解能力,最高溶磷量分别为125. 79、227.34、60.02 和321.15 mg/L;菌株C5-A对不同浓度的两种磷矿粉有较强的溶解能力;分别以麦芽糖和草酸铵为碳源和氮源时溶磷量最高。高效液相色谱检测出10 种有机酸,分别为草酸(葡萄糖酸)、乙酸、苹果酸、琥珀酸和5 种未知有机酸,然而,乙酸而非草酸似乎是影响C5-A 溶磷的重要有机酸。【结论】从红壤花生根际土壤中筛选到一株对难溶性无机盐具有较强溶解能力溶的菌株C5-A,有望为开发高效红壤微生物磷肥提供种质资源。

Illmer P, Schinner F .

Solubilization of inorganic calcium phosphates-solubilization mechanisms

Soil Biology and Biochemistry, 1995,27(3):257-263.

DOI:10.1016/0038-0717(94)00190-C      URL     [本文引用: 1]

Two species [ Penicillium aurantiogriseum and Pseudomonas sp. (PI1889)] having high abilities in solubilizing inorganic phosphates (hydroxylapatite and brushite) were used to examine solubilization mechanisms. No direct contact between microorganisms and calcium phosphates (Ca-Ps) were necessary for effective solubilization. The P-concentration in solution did not increase according to a sigmoid curve type. Therefore, sampling time is of particular importance for estimating P mobilization. The widespread theory that solubilization is always caused by the release of organic acids may prove wrong for our two organisms. In our opinion the most probable reason for solubilization without acid production is the release of protons accompanying respiration or NH 4 + assimilation. It was shown that solubilization of Ca-Ps with two different solubility products may lead to a short-term increase of the amount of at least one Ca . Precipitation and subsequent resolubilization of different organic or inorganic phosphates result in hardly predictable P-concentrations in culture solution.

赵小蓉, 林启美, 孙众焱 , .

小麦根际与非根际解磷细菌的分布

华北农学报, 2001,16(1):111-115.

DOI:10.3321/j.issn:1000-7091.2001.01.021      URL     Magsci     [本文引用: 1]

在冬小麦苗期,利用根际和非根际土壤测定分解卵磷脂和溶解磷酸三钙的细菌,发现根际土壤解磷细菌的数量大大高于非根际土壤,但无论是根际还是非根际土壤,有机磷细菌比无机磷细菌多。根际土壤解磷细菌种类较多,而非根际土壤解磷细菌种类较少。根际土壤的有机磷细菌主要为假单胞菌属,无机磷细菌主要是假单胞菌属和欧文氏菌属,而非根际土壤的优势解磷菌属不明显。

/