作物杂志, 2020, 36(6): 163-169 doi: 10.16035/j.issn.1001-7283.2020.06.024

生理生化·植物营养·栽培耕作

链霉菌株Streptomyces sp. FXP04对水稻种子萌发和幼苗生长的影响

付学鹏,1,2, 沈童飞1, 孙晓波3, 刘晓涵1, 杨晓杰,1,2

1齐齐哈尔大学生命科学与农林学院,161006,黑龙江齐齐哈尔

2黑龙江省抗性基因工程与寒地生物多样性保护重点实验室,161006,黑龙江齐齐哈尔

3泰来县农业技术推广中心,162400,黑龙江泰来

Effects of Streptomyces sp. FXP04 on Seed Germination and Seedling Growth of Rice

Fu Xuepeng,1,2, Shen Tongfei1, Sun Xiaobo3, Liu Xiaohan1, Yang Xiaojie,1,2

1Department of Life Sciences and Agroforestry, Qiqihar University, Qiqihar 161006, Heilongjiang, China

2Heilongjiang Key Laboratory of Resistance Gene Engineering and Preservation of Biodiversity in Cold Areas, Qiqihar 161006, Heilongjiang, China

3Agricultural Technology Extension Center of Tailai County, Tailai 162400, Heilongjiang, China

通讯作者: 杨晓杰,从事天然产物开发与应用,E-mail: yangxiaojie1980@163.com

收稿日期: 2020-05-2   修回日期: 2020-10-14   网络出版日期: 2020-12-15

基金资助: 黑龙江省自然科学基金(C2017068)
黑龙江省教育厅基本业务专项(135109254)
植物性食品加工技术特色学科专项(YSTSXK201888)

Received: 2020-05-2   Revised: 2020-10-14   Online: 2020-12-15

作者简介 About authors

付学鹏,研究方向为作物病虫害生物防控,E-mail: 02383@qqhru.edu.cn

摘要

为了明确链霉菌株Streptomyces sp. FXP04对水稻的促生效果,以前期筛选到的链霉菌株Streptomyces sp. FXP04为供试菌株,采用发酵液浸种和菌液喷洒土壤的方法,研究了该菌株生长稳定期的发酵液和菌液对水稻种子萌发和幼苗生长的影响。结果如下,培养皿试验中,Streptomyces sp. FXP04发酵液原液和LB培养液原液(对照)都抑制种子萌发,1/50倍发酵液显著促进种子萌发,而1/10倍和1/100倍发酵液对种子萌发没有显著影响。盆栽试验中,1/50倍和1/100倍的Streptomyces sp. FXP04菌液都促进了水稻种子萌发,增加了水稻幼苗株高、主根长、干重、根总长度、须根数、根表面积和根总体积,并且1/50倍菌液的促生效果和1/100倍菌液无显著差异。研究结果表明,链霉菌株Streptomyces sp. FXP04对水稻种子萌发和幼苗生长有促进作用,可作为水稻的候选促生菌株开展后续研究和应用。

关键词: 链霉菌 ; Streptomyces sp. FXP04 ; 水稻 ; 种子萌发 ; 幼苗生长

Abstract

In order to determine the growth promoting effects of Streptomyces sp. FXP0404 on rice growth, the Streptomyces sp. FXP04 isolated in our previous experiments was selected as the test strain to study the effects of fermented liquid and cell suspension of Streptomyces sp. FXP04 on the seed germination and seedling growth of rice, using the methods of soaking seeds with fermented liquid and spraying soil with cell suspension. The results showed that both the original fermented liquid and LB liquid (control) inhibited seeds germination in the culture dish. Compared to water treatment, the rice seed germination was significantly increased in the treatment of 1/50 times fermented liquid, however, there were no significant effect on seed germination in the 1/10 and 1/100 times fermented liquid treatments. In the pot experiment, both 1/50 and 1/100 times cell suspension of Streptomyces sp. FXP04 promoted the germination of rice seeds, increased the plant height, main root length, dry weight, total root length, fibrous root number, root surface area and total root volume of rice seedlings, however, there was no significant difference between 1/50 and 1/100 times of Streptomyces sp. FXP04 cell suspension. The results demonstrated that Streptomyces sp. FXP04 could promote the seed germination and seedling growth of rice, and could be used as a candidate plant growth-promoting rhizobacteria on rice for further research and application.

Keywords: Streptomyces ; Streptomyces sp. FXP04 ; Rice ; Seed germination ; Seedling growth

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本文引用格式

付学鹏, 沈童飞, 孙晓波, 刘晓涵, 杨晓杰. 链霉菌株Streptomyces sp. FXP04对水稻种子萌发和幼苗生长的影响[J]. 作物杂志, 2020, 36(6): 163-169 doi:10.16035/j.issn.1001-7283.2020.06.024

Fu Xuepeng, Shen Tongfei, Sun Xiaobo, Liu Xiaohan, Yang Xiaojie. Effects of Streptomyces sp. FXP04 on Seed Germination and Seedling Growth of Rice[J]. Crops, 2020, 36(6): 163-169 doi:10.16035/j.issn.1001-7283.2020.06.024

开放科学(资源服务)标识码(OSID):

水稻是我国主要的粮食作物之一,在我国北方地区,水稻育苗时常因温度、湿度、光照和病虫害等原因出现种子萌发率低、根系生长缓慢、苗弱、抗逆性差等现象,从而影响水稻苗后期的生长及产量。在提倡“减肥减药”的现阶段,植物根际促生菌(PGPR)在作物促生、抗逆、防病中的作用越来越受到人们的重视[1,2]。前人已筛选出了许多对水稻有促生和防病功能的促生菌[3],如芽孢杆菌属(Bacillus[4]、固氮菌属(Azotobacter[5]、肠杆菌属(Enterobacter[6]、节杆菌属(Arthrobacter[6]和链霉菌属(Streptomyces[7]等。在众多根际促生菌中,链霉菌属是革兰氏阳性放线菌中种类最多的一个属(超过500种),已经发现很多种链霉菌能够提高植物的抗病和抗逆性,促进植物生长[7]。在水稻促生菌筛选方面,链霉菌株702[8]和链霉菌株JD211[9,10,11,12]都能促进水稻种子萌发及其幼苗生长;从蚯蚓粪中筛选到的链霉菌株CAI-85和CAI-93能够使水稻分蘖数、花序数、根长、根体积及根干重增加[13]。由于受到宿主种属范围、根际定殖、作用方式、根际生态和接种方式等因素的影响,链霉菌在不同地区、土壤和作物中的促生和抗病效果存在差异[7]。为了进一步促进更多链霉菌在农业中的应用,需要尝试从更多介质中采用更多方法筛选更优质高效的促生菌株。黑龙江省是我国水稻主产区之一,其高寒气候和黑土特性也亟待开发更多适应性强的促生菌株。

本课题组在前期研究中从黑龙江省齐齐哈尔市马铃薯根际土中筛选到1株链霉菌,经形态学和16S rRNA鉴定,命名为Streptomyces sp. FXP04,该菌株能促进马铃薯幼苗生长,降低晚疫病病情指数,但其对水稻种子萌发及幼苗生长的影响尚不清楚。本研究用不同浓度的Streptomyces sp. FXP04发酵液和菌液分别对“中组4号”水稻种子进行处理,研究该菌株对水稻种子萌发及幼苗生长的影响,明确该菌株对水稻的促生作用及适宜接种方式,研究结果可为该菌株作为生物肥的开发利用提供理论基础和技术支持。

1 材料与方法

1.1 试验材料

试验用土为黑龙江省泰来县水稻田土,土壤基本理化性质:碱解氮343mg/kg,有效磷258.34mg/kg,速效钾90.2mg/kg,有机质29.78g/kg,pH值7.03,电导率1.22mS/cm。水稻品种为“中组4号”,菌株为Streptomyces sp. FXP04。

1.2 培养基配方及发酵培养

LB培养液(1L):酵母浸出汁5.0g,NaCl 8.0g,蛋白胨10g,pH值7.2,经121℃高压灭菌20min,在无菌条件下接种Streptomyces sp. FXP04菌株,置于恒温摇床,28℃、120r/min黑暗条件培养。待发酵液浑浊(4~6d,视接种量而定)后,使用分光光度计测定发酵液在600nm处的OD值,测定后放于4℃冰箱保存备用。

1.3 试验设计及指标测定

挑选饱满的水稻种子,于30℃条件下用无菌水浸种催芽24h。将菌液经5 000r/min离心15min后弃沉淀,保留上清液,将上清液抽滤,再经0.22μm滤膜过滤,得到不含Streptomyces sp. FXP04菌体的发酵液。

1.3.1 发酵液浸种处理对水稻种子萌发的影响 用发酵液原液及其无菌去离子水稀释的10倍(1/10)、50倍(1/50)和100倍(1/100)溶液分别对催芽后的水稻种子进行浸种处理;以无菌去离子水对LB培养液作上述倍数稀释作为对照(CK1);以无菌去离子水处理作为两者的共同对照(CK2)。每个处理3次重复,每次重复50粒种子。将种子放入培养皿中,培养皿中分别加入各处理溶液10mL,每隔2d更换1次对应的浸种溶液(各溶液不用时于4℃保存),28℃黑暗条件下培养,自种子浸种当日起每天观察种子发芽情况,以种子露白作为发芽标准。

1.3.2 菌液喷洒土壤对水稻种子萌发和幼苗生长的影响 将菌液用无菌去离子水按照1/50和1/100的倍数稀释,分别取100mL均匀喷撒在土壤表面,然后将100粒种子均匀撒播在土壤表面,覆土,用喷壶洒水浸湿土壤。室内培养(光照/黑暗:16h/8h),室温25℃±2℃。记录种子萌发情况及幼苗生长情况;播种20d后取样,测定幼苗干重及根冠比。使用根系扫描仪分析根系生长情况。

1.4 数据处理

采用 Excel 2007对数据进行记录和整理,使用SPSS 16.0软件和Turkey HSD检验对数据进行one-way ANOVA分析(P<0.05)。

2 结果与分析

2.1 发酵液浸种对水稻种子萌发的影响

3次重复试验的发酵液原液OD600值分别为1.689、1.986和1.859,经生长曲线分析,3次发酵都处于稳定期,且结果相似,因此本文展示其中1次(OD600=1.859)的数据。

表1图1可以看出,发酵液原液和LB培养液处理组都没有种子萌发。在所有稀释倍数中,1/50倍发酵液处理的种子萌发率最高,并显著高于1/50倍LB培养液处理(P<0.05)。1/10倍发酵液在处理初期和后期(6d和10d)显著高于1/10倍LB培养液(P<0.05);1/100倍发酵液处理与1/100倍LB培养液处理的种子萌发率无显著差异(P≥0.05)。在所有稀释倍数中,在处理初期和后期(6d和10d)只有1/50倍发酵液处理高于清水处理的萌发率(P<0.05)。

表1   发酵液对水稻种子萌发率的影响

Table 1  Effect of fermentation liquid on rice seed germination percentage %

处理Treatment处理后天数Days after treating (d)
6810
LB培养液原液Original LB liquid0.00±0.00d0.00±0.00c0.00±0.00d
发酵液原液Original fermentation liquid0.00±0.00d0.00±0.00c0.00±0.00d
1/10倍LB培养液1/10 times LB liquid62.60±4.42c83.34±5.21b86.66±5.64c
1/10倍发酵液1/10 times fermentation liquid74.00±6.32a89.34±4.58b93.34±8.54b
1/50倍LB培养液1/50 times LB liquid68.00±5.20b86.66±6.35b88.46±6.52bc
1/50倍发酵液1/50 times fermentation liquid78.66±4.33a94.00±8.25a96.60±8.25a
1/100倍LB培养液1/100 times LB liquid69.34±5.24b85.34±7.53b89.44±7.97bc
1/100倍发酵液1/100 times fermentation liquid72.66±5.27b90.00±8.25ab90.00±8.25c
H2O72.00±6.33b92.00±9.62a93.34±8.54b

注:发酵液原液的OD600值为1.859。同列数据后不同小写字母表示组间差异显著(P<0.05),下同

Note: The OD600 value of fermentation is 1.859. Different lowercase letters after the same column of data indicate significant difference between groups (P < 0.05). The same below

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图1

图1   发酵液对水稻种子萌发的影响

Fig.1   Effect of fermentation liquid on rice seeds germination


2.2 菌液对土培水稻种子萌发的影响

表2可以看出,播种后7d时,1/100倍菌液和LB培养液处理的种子萌发率较高,但是两者间无显著差异;1/50倍菌液和1/100倍菌液处理的种子萌发率无显著差异(P≥0.05),但显著高于1/50倍LB培养液处理的种子(P<0.05)。播种18d后时,1/50倍菌液处理的种子萌发率最高,显著高于1/50倍LB培养液处理的种子(P<0.05);1/100倍菌液处理的种子萌发率显著高于1/100倍培养液处理的种子。

表2   土培条件下菌液对水稻种子发芽率的影响

Table 2  Effect of cell suspension on rice seed germination percentage in soil %

处理Treatment处理后天数
Days after treating (d)
718
1/50倍菌液1/50 times cell suspension20.00±2.00ab79.00±3.00a
1/50倍LB培养液1/50 times LB liquid12.00±1.00b51.33±8.02c
1/100倍菌液1/100 times cell suspension26.67±3.51a76.33±4.51a
1/100倍LB培养液1/100 times LB liquid25.67±5.03a66.00±2.65b

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2.3 菌液对土培水稻幼苗株高和根长的影响

播种20d时,选择长势一致的水稻幼苗20株测量株高和主根长。从表3图2可以看出,1/50倍和1/100倍菌液处理的水稻幼苗株高和主根长显著高于1/50倍和1/100倍LB培养液处理的幼苗(P<0.05),1/50倍和1/100倍菌液处理间幼苗株高和主根长无显著差异(P≥0.05)。

表3   土培条件下发酵液对水稻幼苗生长的影响

Table 3  Effects of cell suspension on rice seedlings growth in soil

处理Treatment株高
Plant height (cm)
主根长
Main root length (cm)
1/50倍菌液
1/50 times cell suspension
15.88±1.36a13.88±1.26a
1/50倍LB培养液
1/50 times LB liquid
12.50±0.64b9.325±1.00b
1/100倍菌液
1/100 times cell suspension
15.20±0.87a12.50±1.66a
1/100倍LB培养液
1/100 times LB liquid
13.20±1.30b8.60±0.99b

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图2

图2   土培条件下菌液对水稻幼苗生长的影响

Fig.2   Effect of cell suspension on rice seedling growth in soil


2.4 菌液对土培水稻幼苗根系生长的影响

播种20d时,使用根系扫描仪分析水稻幼苗根总长度、须根数、根表面积和根总体积。由图3可知,与1/50倍LB培养液相比,经1/50倍菌液处理后水稻根直径≤0.5mm的根总长度、根总体积、根表面积和须根数都显著升高(P<0.05);1/100倍菌液处理后的水稻根总长度、根总体积、根表面积和须根数也呈现出相同趋势;与1/50倍菌液相比,1/100倍菌液处理显著提高了水稻根总长度和根表面积,但对根总体积和须根数无显著影响。

图3

图3   菌液对水稻根系生长的影响

CP50和CP100分别表示1/50和1/100倍的菌液;LB50和LB100分别表示1/50和1/100倍的LB培养液;柱上方的不同小写字母表示组间差异显著(P<0.05),下同。D表示根直径(mm)

Fig.3   Effects of cell suspension on rice root growth

CP50 and CP100 indicate 1/50 and 1/100 times of cell suspension, respectively; LB50 and LB100 indicate 1/50 and 1/100 times of LB liquid, respectively; Different lowercase letters above column indicate significant difference between groups (P < 0.05), the same below. D indicates the root diameter (mm)


与1/50倍LB培养液相比,经1/50倍菌液处理后水稻0.5mm<根直径≤1.0mm的根总长度和须根数显著升高;与1/100倍LB培养液相比,经1/100倍菌液处理后水稻0.5mm<根直径≤1.0mm的根总体积和根表面积显著升高,根总长度和须根数无显著差异。与1/50倍菌液相比,1/100倍菌液处理显著提高了水稻幼苗根总体积和根表面积,根总长度和须根数无显著差异。

对于直径≥1.5mm的根来说,相对于1/50倍LB培养液,1/50倍菌液处理使得根总长度、根总体积、根表面积和须根数都显著升高(P<0.05);对于1/100的LB培养液和菌液处理,对根总长度、根总体积和根表面积都表现出相同的影响效果;与1/100倍菌液相比,1/50倍菌液处理的根总长度和须根数显著提高(P<0.05),根体积和根表面积没有显著差异。

2.5 菌液对土培水稻幼苗干重和根冠比的影响

图4可以看出,与1/50和1/100倍LB培养液相比,1/50和1/100倍菌液处理的水稻幼苗地上部干重、地下部干重和总干重都显著提高(P<0.05)。1/50菌液和1/100倍菌液对水稻幼苗干重和根冠比的影响无显著差异(P≥0.05)。

图4

图4   菌液对水稻幼苗干重和根冠比的影响

Fig.4   Effects of cell suspension on dry weight and root-shoot ratio of rice seedlings


3 讨论

植物根际促生菌是一类能够促进植物生长、防治病害、诱导抗性、增加产量的一类有益微生物[1,2]。根际促生菌种类很多,其中很多种链霉菌对水稻均具有促生和提高抗逆抗病的效果[7,8,9,10,11,12,13]。在棉花田土壤及珙桐植株内分离出对水稻种子萌发和幼苗生长有促进作用的促生菌[8,9,10,11,12];在蚯蚓粪中筛选出对水稻分蘖数、花序数、根长、根体积及根干重有显著促进作用的链霉菌株[13]。目前,尚未见在我国东北高寒黑土中分离出对水稻具有促生作用的链霉菌的报道。

本研究利用前期从黑龙江省马铃薯根际土中筛选到的能够促进马铃薯幼苗生长和降低马铃薯晚疫病病情指数的链霉菌-Streptomyces sp. FXP04菌株,通过3次独立试验明确了该菌株对水稻种子萌发和幼苗生长的促进作用。在水稻种子培养皿萌发试验中,将处于生长稳定期的Streptomyces sp. FXP04发酵液稀释不同倍数后浸种,发现其中发酵液原液及LB培养液原液(未接种Streptomyces sp. FXP04的对照)完全抑制了种子萌发,说明二者对水稻种子的萌发有抑制作用。发酵液经无菌水稀释后表现出对种子萌发的促进作用,其中1/50倍发酵液的促萌发效果显著高于1/10和1/100倍的发酵液,并且只有1/50倍发酵液的促萌发效果显著高于清水处理的种子。李张等[9]用筛选到的链霉菌JD211菌株滤液处理水稻种子,在稀释1/1000倍的条件下对种子萌发有促进作用。本研究中,发酵液中没有Streptomyces sp. FXP04菌体,发酵液对水稻种子有促萌发作用,说明Streptomyces sp. FXP04可能产生了具有促进种子萌发的次生代谢物,如生长素(IAA)和细胞分裂素等[7]。Gopalakrishnan等[13]在蚯蚓粪中分离到的6株链霉菌都能产生并分泌IAA;麻金金[14]在玫瑰黄链霉菌NKZ-259菌株的次生代谢物中检测到IAA;Goudjal等[15]从阿尔及利亚贫瘠沙漠中生存的植物内筛选到的内生链霉菌Streptomyces sp. PT2也能够分泌IAA。这些研究证明,链霉菌具有分泌IAA的潜能。值得注意的是,这些研究是在体外试验中检测到链霉菌分泌的IAA,至于链霉菌在定殖植物的根际能否产生IAA还需要通过突变体进行更深入的研究[7,16-17]

链霉菌的接种方式影响促生效果[7]。Bonaldi等[18]研究表明,生菜种子用Streptomyces ZEA17L菌株包衣比在基质中直接接种更能促进该菌株在生菜根际的定殖;Gopalakrishnan等[13]采用定殖前菌液蘸根,定殖后定期浇灌菌液的方式处理水稻幼苗,能够促进水稻分蘖数、花序数、根长、根体积及根干重,提高产量等;用固体发酵和土壤拌菌的方式分别将链霉菌株702[8]和JD211[10,11]接种到水稻种子,也促进了水稻种子萌发和幼苗生长。本研究使用育苗盘模拟田间水稻育苗,将链霉菌液喷洒在苗床上,使种子能够接触到链霉菌株。结果表明,在播种后18d时,1/50和1/100倍菌液处理的种子萌发率显著高于对应的LB培养液及清水处理,说明采用菌液喷洒的方式在1/50和1/100倍浓度下该菌株能促进水稻种子萌发,此种菌液施用方式及浓度可为该菌应用于水稻田间育苗提供理论参考。

在幼苗生长时期,1/50倍和1/100倍的Streptomyces sp. FXP04菌液处理都能促进水稻幼苗株高、根长和幼苗干重提高,但两者间无显著差异。链霉菌株JD211对水稻幼苗生长有极显著的促进作用[11],且能促进水稻幼苗的根数、根长及根体积[12]。根系的生长对于水稻吸收水分和矿质营养具有重要的作用[19],本研究中Streptomyces sp. FXP04菌液对水稻株高和干重的促进效应可能是通过改善水稻根系生长实现的,如根长、根表面积和根体积。土壤中施入Streptomyces sp. FXP04促进水稻种子萌发和幼苗生长的原因,除了Streptomyces sp. FXP04可能分泌IAA,还可能与Streptomyces sp. FXP04会溶磷、解钾或产铁载体有关[7]。有研究表明,白网链霉菌株PSPSA1能够溶磷,提高牡丹根际土壤有效磷含量[20];淡紫灰链霉菌菌株DC-A有较强的产IAA、溶磷和产铁载体的促生活性[21];申枚灵等[22]从光果甘草(Glycyrrhiza glabra)分离的多株内生链霉菌株能够分泌IAA、具有产铁载体的能力和溶磷能力;陈宇丰等[23]从香蕉根际分离到的链霉菌株M3-4解钾率能达到20%。本研究中Streptomyces sp. FXP04能否溶磷、解钾或产铁载体有待进一步研究。

从盆栽效果看,在生长处于稳定期的Streptomyces sp. FXP04菌液稀释50倍和100倍对水稻种子萌发和幼苗生长具有相似的促进作用,说明该链霉菌株对水稻幼苗的生长有促进作用,可以作为潜在的生物菌肥。如果用该菌株制成生物菌肥,稀释100倍有利于降低生产成本,但实践应用中采用的稀释倍数还需要进一步试验。

4 结论

链霉菌株Streptomyces sp. FXP04发酵液和菌液在合适的浓度下可提高水稻种子萌发率,其菌液在合适的浓度下能够提高水稻幼苗的株高、干重、主根长、根总长度、须根数、根表面积和根总体积,说明Streptomyces sp. FXP04对水稻种子萌发和幼苗生长有促进作用,可作为水稻的候选促生菌株开展后续研究和应用。

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