南京农业大学学报  2018, Vol. 41 Issue (4): 676-684   PDF    
http://dx.doi.org/10.7685/jnau.201801009
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文章信息

李静, 操一凡, 丁佳兴, 孙玉菡, 郑宇, 胡官墨, 沈宗专, 李荣, 沈其荣
LI Jing, CAO Yifan, DING Jiaxing, SUN Yuhan, ZHENG Yu, HU Guanmo, SHEN Zongzhuan, LI Rong, SHEN Qirong
含复合菌群生物育苗基质的研制及其育苗效果
Development of bio-nursery substrates containing PGPR flora and evaluation of their seedlings growth promoting effect
南京农业大学学报, 2018, 41(4): 676-684
Journal of Nanjing Agricultural University, 2018, 41(4): 676-684.
http://dx.doi.org/10.7685/jnau.201801009

文章历史

收稿日期: 2018-01-08
含复合菌群生物育苗基质的研制及其育苗效果
李静 , 操一凡 , 丁佳兴 , 孙玉菡 , 郑宇 , 胡官墨 , 沈宗专 , 李荣 , 沈其荣     
南京农业大学资源与环境科学学院/江苏省固体有机废弃物资源化高技术研究重点实验室/国家有机类肥料工程技术研究中心/江苏省有机固体废弃物资源化协同创新中心, 江苏 南京 210095
摘要[目的]开发含复合菌群生物制品或农业投入品,对于促进作物增产具有重要意义,本文旨在利用分别具有拮抗病原菌、解磷、产生长素能力的菌株,研制含根际促生菌菌群的生物育苗基质,以期为育苗基质产业的发展提供技术支撑。[方法]以本实验室经过广泛田间试验验证,且具有促生及抗土传病害功能的解淀粉芽胞杆菌SQR9为核心菌株,筛选出与之无拮抗效应的产生长素和具解磷能力的菌株;采用Biolog全自动微生物鉴定系统测定菌株对不同碳源利用能力,通过碳源重合关系确定菌种组合;通过育苗和盆栽试验评估含复合菌群生物育苗基质的育苗效果。[结果]经过筛选,与菌株SQR9对峙无拮抗圈的溶磷菌株分别为Y40、Y2和NJAU-3;产吲哚乙酸(IAA)菌株分别为NJAU-5、L-60、NJAU-69和NJAU-84。利用碳源重合关系,构建4种菌株组合2个、3种菌株组合6个和2种菌株组合4个。2季苗盘试验及2季盆栽试验结果均表明,微生物碳源利用重合数与植物促生效果呈显著负相关关系,含菌株SQR9、Y2和L-60与含菌株SQR9、Y2和NJAU-84这2个含复合菌群功能型生物育苗基质,在黄瓜前期育苗效果和后期盆栽效果均优于单菌处理及不添加功能菌普通育苗基质(对照)。[结论]结合拮抗效应测定和碳源利用重合关系,能够有效筛选出高效菌群组合,最终确立含3种菌株的2组复合菌群SQR9+Y2+L-60和SQR9+Y2+NJAU-84为研制生物育苗基质的最佳组合。
关键词根际促生菌菌株   促生   生物育苗基质   溶磷菌   产生长素菌   
Development of bio-nursery substrates containing PGPR flora and evaluation of their seedlings growth promoting effect
LI Jing, CAO Yifan, DING Jiaxing, SUN Yuhan, ZHENG Yu, HU Guanmo, SHEN Zongzhuan, LI Rong , SHEN Qirong    
College of Resources and Environmental Sciences/Jiangsu Provincial Key Laboratory for Solid Organic Waste Utilization/National Engineering Research Center for Organic-based Fertilizers/Jiangsu Collaborative Innovation Center for Solid Organic Waste Resource Utilization, Nanjing Agricultural University, Nanjing 210095, China
Abstract: [Objectives] The development of biocomposite products or agricultural inputs containing compound bacteria is of great significance for the promotion of crop yield. In this paper, we aimed to develop bio-nursery substrates with plant growth promoting rhizobacteria(PGPR)flora by using bacterial strains with antagonistic ability against pathogen, phosphate-solubilizing ability or auxin-producing capability for providing theoretical and technical support for the development of seedling nursery substrate industry. [Methods] Bacillus amyloliquefaciens SQR9, having been demonstrated to efficiently promote plant growth and suppress the soil-borne diseases, was selected as the core, and antagonistic results of different strains with strain SQR9 were first evaluated to selecte the coexistence strains. Then, Biolog Gen Ⅲ plate and automatic microbial identification system were used to evaluate the different carbon sources utilization abilities for strains, and overlapping relationship of arbon sources utilization among different strains were adopted to design the PGPR flora. At last, seedling nursing and pot experiments were performed to determin the plant growth promotion ability of the bio-nursery substrates produced by different PGPR flora. [Results] After screening, strains without antagonistic phenomen to SQR9 were Y40, Y2 and NJAU-3 with phosphate-solubilizing ability, and NJAU-5, L-60, NJAU-69 and NJAU-84 with auxin-producing ability. Via the carbon source overlap relationship, 2 combinations of 4 strains, 6 combinations of 3 strains and 4 combinations of 2 strains were established. The results of two-season seedling nursing and pot experiments showed that there was a significant negative correlationship between the utilization overlap of microbial carbon sources and the plant growth promoting effect. Seedling raising experiments showed that in the substrate inoculated with strains SQR9, Y2 and L-60 and strains SQR9, Y2 and NJAU-84, cucumber seedlings grew much better than those in the ordinary nursery substrate and other bio-nursery substrates. The pot experiments showed that the plants growing in the bio-nursery substrate were significantly higher than those in the ordinary nursery substrate and other bio-nursery substrates. [Conclusions] High efficient bacterial group can be effectively selected by combining the antagonistic effect determination and evaluation of overlapping relationship of arbon sources utilization. In this study, the two groups of bacteria with 3 strains(SQR9+Y2+L-60, SQR9+Y2+NJAU-84)were finally selected for the development of the best bio-nursery substrates.
Key words: PGPR strains    plant growth promotion    bio-nursery substrates    phosphorus solubilizing bacteria    IAA-production bacteria   

根际促生菌(PGPR)能够有效定殖于作物根际, 发挥兼具促生与生防的功能[1]。然而, 单一菌种普遍存在功能多样性缺乏和抵抗负荷差等缺点。由于作物的生产需要多种营养元素, 单一PGPR菌株的产品已很难完全满足作物的需求。复合菌群菌种多样性的特点使其能够适应各种生境[2], 相互协调[3]。在农业上, 复合菌群能提高土壤肥力, 加速土壤养分的分解转化, 从而达到节约肥料, 促进植物根生长的功效[4]; 另外, 有些复合菌群对杂草危害和病虫害具有防治作用, 同时能够提高作物的抗病、抗寒、抗旱能力[5]。因此, 开发含复合菌群生物制品或农业投入品, 对于促进作物增产具有重要意义。

随着设施农业的迅速发展, 无土栽培和穴盘育苗得到广泛推广, 工厂化育苗越来越受到现代化农业的重视, 加速了固体栽培基质的开发研究[6]。目前, 进口基质的质量明显高于国产基质, 因此提升我国育苗基质的整体水平尤为迫切[7]。本实验室将特定的根际促生功能微生物在育苗时保活添加到基质中, 研制成的活性生物育苗基质可以育出根际带有大量活性功能微生物的优质种苗, 从而发挥微生物的促生作用, 提高作物在大田种植中的产量[8-9]。但由于菌种单一, 限制了该产品的发展, 因此, 含复合菌群功能型生物基质的研发尤为迫切。

为增加生物育苗基质功能多样性, 本研究以本实验室已经证实能够促进多种作物生长和防控土传病害的解淀粉芽胞杆菌(Bacillus amyloliquefaciens)SQR9[10]为核心菌株, 基于菌株间相互作用和碳源重合度筛选复合菌群, 采用含复合菌群的生物基质育苗, 评估所研制的复合菌生物育苗基质的育苗效果, 为含多功能复合菌群生物育苗基质的产业化提供技术支撑。

1 材料与方法 1.1 供试材料

供试黄瓜品种为‘露丰4号’。无土栽培基质及其基本性质参见文献[9]。供试PGPR菌株:海水芽胞杆菌(Bacillus aquimaris)L-60[11]、弯曲芽胞杆菌(Bacillus flexus)NJAU-57和巨大芽胞杆菌(Bacillus megaterium)NJAU-69为本实验室分离保存并鉴定的具有产IAA能力的根际促生菌; NJAU-5和NJAU-84为本实验室分离保存未鉴定的具有产IAA能力的根际促生菌。不动杆菌属(Acinetobacter sp.)Y40、假单胞菌属(Pseudomonas sp.)Y2和不动杆菌属(Acinetobacter sp.)NJAU-3, 为本实验室分离保存并鉴定的具有解磷功能的根际促生菌[12-13]。解淀粉芽胞杆菌(Bacillus amyloliquefaciens)SQR9为本实验室分离保存的具有防控土传病害和促进植物生长的根际促生菌, 该菌株为革兰氏阳性菌, 保存于中国微生物菌种保存管理委员会普通微生物中心, 菌种保藏号为CGMCC NO.5808。

1.2 菌株SQR9与其他菌株拮抗性能的测定

接种菌株SQR9于100 mL液体NA培养基(葡糖糖10 g, 蛋白胨5 g, 酵母粉0.5 g, 牛肉膏3 g, 蒸馏水1 000 mL, pH7.0, 115 ℃灭菌30 min)中, 于37 ℃、175 r·min-1摇床中培养24 h, 取出菌液用无菌水稀释至D600为1, 装入无菌的小喷壶(小喷壶用体积分数为75%的乙醇浸泡, 并置于超净台紫外灯下照射24 h以上, 用前于超净工作台中用无菌水冲洗干净)。同时在NA平板上点接待测菌株, 于30 ℃恒温培养箱中培养24 h后, 将稀释好的SQR9菌液喷于点接有待测菌株的NA平板上, 于30 ℃恒温培养箱中再培养24 h, 观察有无拮抗圈。

1.3 不同细菌碳源代谢活性测定

采用Biolog全自动微生物鉴定系统测定不同细菌碳源代谢活性, 步骤[14-15]如下:将待鉴定菌株在NA培养基中连续培养转接2~3次, 选择处于对数期且生长良好的单菌落, 用无菌棉签挑取少量菌体, 接种于专用的管内, 振荡, 使接种管内的菌悬液均一混合, 并与标准菌悬液进行浊度对比, 其浊度误差控制在2%;将菌悬液接种到Biolog Gen Ⅲ板, 每孔接种菌悬液量为200 μL; 将接种好的鉴定板编号并置于铁盒内(铁盒提前灭菌)于培养箱中30 ℃培养, 在培养4~8 h和12~24 h时用读数仪读取菌株的特征性碳源代谢指纹。

1.4 含复合菌群生物育苗基质的研制

将各功能菌菌悬液按5%(体积质量比, 下同)添加至普通育苗基质中, 混合均匀后放置1~2 d后, 涂布计数每种含单一菌株生物育苗基质的功能菌数, 确保每种单菌生物育苗基质中功能菌数量均能达到107 CFU·g-1以上, 获得含单一菌株生物育苗基质; 然后按相同比例分别将含单一菌株生物育苗基质混合均匀, 研制含复合菌群生物育苗基质[16-17]。如含2种菌株生物育苗基质, 即是将含该2种菌株的单菌生物育苗基质1:1(体积比)混合而得。

1.5 育苗试验

两季育苗试验于2016年7月8日—9月10日在南京农业大学资源与环境科学学院温室内进行。按照碳源重合利用情况, 将所有处理分为碳源重合利用相对较少和相对较多组合进行分析。碳源重合利用相对较少组合含10个处理:6个含复合菌群SQR9+Y2+L-60+NJAU-84、SQR9+Y2+L-60、SQR9+L-60+NJAU-84、SQR9+Y2+NJAU-84、SQR9+Y2和SQR9+L-60处理, 4个含单菌SQR9、Y2、L-60和NJAU-84处理(该4株菌株用于以上组合菌群); 碳源重合利用相对较多组合为10个处理:6个含复合菌群SQR9+Y40+NJAU-69+NJAU-84、SQR9+Y40+NJAU-69、SQR9+NJAU-69+NJAU-84、SQR9+Y40+NJAU-84、SQR9+Y40和SQR9+NJAU-69处理, 4个含单菌SQR9、Y40、NJAU-69和NJAU-84处理(该4株菌株用于以上组合菌群)。设添加无菌水的普通育苗基质育苗为对照(CK)。2组同时具备的含单菌育苗基质处理(分别为含菌株SQR9和NJAU-84)每季只做1次试验, 因此, 育苗试验共有18个处理, 1个对照。每个处理18个重复, 6个重复为1组, 每组分别种于不同区域, 于试验开始20 d后测定各处理的株高、茎粗、地上部鲜质量和地上部干质量。

1.6 盆栽试验

2季盆栽试验于2016年9—11月在南京农业大学资源与环境科学学院温室内进行。根据含复合菌群生物育苗基质的育苗结果, 选择碳源重合利用相对较少组合中, 除含SQR9+L-60+NJAU-84菌群生物育苗基质所育种苗(2季种苗长势均明显差于其他2个含3菌株生物育苗基质所育种苗)外, 其余9个处理及普通育苗基质所育种苗进行试验。处理1(CK), 移栽添加无菌水普通育苗基质所育种苗; 处理2(SQR9+Y2+L-60+NJAU-84), 移栽含SQR9+Y2+L-60+NJAU-84复合菌群生物育苗基质所育种苗; 处理3(SQR9+Y2+NJAU-84), 移栽含SQR9+Y2+NJAU-84复合菌群生物育苗基质所育种苗; 处理4(SQR9+Y2+L-60), 移栽含SQR9+Y2+L-60复合菌群生物育苗基质所育种苗; 处理5(SQR9+L-60), 移栽含SQR9+L-60复合菌群生物育苗基质所育种苗; 处理6(SQR9+Y2), 移栽含SQR9+Y2复合菌群生物育苗基质所育种苗; 处理7(SQR9), 移栽含SQR9单一菌株生物育苗基质所育种苗; 处理8(L-60), 移栽含L-60单一菌株生物育苗基质所育种苗; 处理9(Y2), 移栽含Y2单一菌株生物育苗基质所育种苗; 处理10(NJAU-84), 移栽含NJAU-84单一菌株生物育苗基质所育种苗。待幼苗长到2叶1心期, 选取长势较好且均一的种苗移栽, 每个杯子装土800 g, 移栽1棵苗为1个重复, 每个处理9个重复; 每3 d浇1次霍格兰营养液, 于移栽20 d后测定各处理的株高、茎粗、地上部鲜质量和地上部干质量。

1.7 菌株的鉴定

对菌群最终组合中未鉴定菌株进行鉴定, 菌落形态及生理生化特征鉴定参照《伯杰细菌鉴定手册》(第8版)。基于16S rDNA序列的分子生物学鉴定参考文献[18]。

1.8 数据分析

采用Excel 2013和SPSS 22.0软件进行数据统计分析, 使用最小显著差异法(LSD)检验进行多重比较(P < 0.05)。

2 结果与分析 2.1 与菌株SQR9无拮抗作用的菌株筛选及其碳源利用能力

平板对峙试验结果表明, 与菌株SQR9无拮抗圈产生的溶磷菌株分别为菌株Y40、Y2和NJAU-3, 无拮抗圈产生的产IAA菌株分别为菌株NJAU-5、L-60、NJAU-69和NJAU-84, 有拮抗圈产生的菌株为NJAU-57。在Biolog Gen Ⅲ鉴定板上的显色反应结果表明:这8株菌对71种碳源利用存在差异。

2.2 菌群组合的设计

根据菌株对碳源利用能力的不同, 分析每种功能菌(溶磷或产IAA)与菌株SQR9碳源重合利用关系(图 1), 得到菌株SQR9与具解磷能力菌株碳源重合利用最多和最少的2种菌组合分别是SQR9+Y40和SQR9+Y2;与具产IAA能力菌株之间碳源重合利用最多和最少的2种菌组合分别是SQR9+NJAU-69和SQR9+L-60;与2类菌之间碳源重合利用最多和最少的组合分别是SQR9+NJAU-69+Y40和SQR9+L-60+Y2;与3种菌之间碳源重合利用最多和最少的组合分别是SQR9+Y40+NJAU-69+NJAU-84和SQR9+Y2+L-60+NJAU-84。

图 1 以菌株SQR9为核心的各功能菌之间碳源重合利用关系 Figure 1 Overlapping utilization of carbon sources between strain SQR9and various functional bacteria
2.3 碳源重合利用相对较少组合生物育苗基质的育苗效果

表 1可知:第1季苗盘育苗试验中, 与不接菌对照(CK)相比, 含SQR9+Y2+L-60菌群生物育苗基质所育种苗的株高、茎粗、地上部鲜质量和地上部干质量分别显著增加123.77%、54.40%、164.71%和142.86%(P < 0.05);与单菌生物育苗基质(SQR9)所育种苗相比分别显著增加40.19%、57.07%、69.81%和70.00%;与含2种菌生物育苗基质(SQR9+Y2)所育种苗相比, 株高显著增加38.55%。第2季苗盘育苗试验中, 与不接菌对照(CK)相比, 含SQR9+Y2+L-60菌群生物育苗基质所育种苗的株高、茎粗、地上部鲜质量和地上部干质量, 分别显著增加62.26%、61.43%、121.28%和162.25%;与单菌生物育苗基质(SQR9)所育种苗相比, 分别显著增加17.81%、38.65%、95.00%和110.00%;与含2种菌生物育苗基质(SQR9+Y2)所育种苗相比, 茎粗和地上部干质量分别显著增加35.74%和40.00%。

表 1 功能菌间碳源重合利用相对较少组合研制的生物育苗基质对黄瓜的育苗效果 Table 1 Effects of bio-nursery substrate produced by strain groups with less overlapping utilization of carbon sources on the growth of cucumber seedlings
处理
Treatment
株高/cm
Plant height
茎粗/mm
Stem diameter
地上部鲜质量/g
Fresh weight of shoot
地上部干质量/g
Dry weight of shoot
第1季
The firstreason
第2季
The secondreason
第1季
The firstreason
第2季
The secondreason
第1季
The firstreason
第2季
The secondreason
第1季
The firstreason
第2季
The secondreason
SQR9+Y2+L-60+NJAU-84 4.50±0.22cd 4.84±0.50ab 3.19±0.97abc 2.94±0.57cde 1.67±0.15cd 2.45±0.37abc 0.12±0.02bcd 0.15±0.03bc
SQR9+Y2+L-60 5.93±0.39a 5.16±0.11a 3.86±0.59a 4.52±0.24a 2.70±0.79ab 3.12±0.13a 0.17±0.05abc 0.21±0.01a
SQR9+L-60+NJAU-84 3.92±0.10d 4.56±0.37abcd 3.12±0.39abc 3.24±0.32bcd 1.38±0.27de 1.72±0.05cde 0.11±0.01cd 0.12±0.02cde
SQR9+Y2+NJAU-84 5.40±0.63ab 4.72±0.56abc 3.86±0.21a 3.78±0.64b 2.65±0.19ab 2.44±0.84abc 0.18±0.01ab 0.15±0.02bc
SQR9+Y2 4.28±0.10cd 4.92±0.26ab 3.50±0.24ab 3.33±0.43bcd 2.78±0.48a 2.41±0.62abc 0.19±0.05a 0.15±0.04b
SQR9+L-60 4.75±0.76bc 4.46±0.33bcd 3.21±0.64abc 3.46±0.22bc 2.14±0.42bc 3.02±0.66ab 0.13±0.03abcd 0.14±0.01bc
SQR9 4.23±0.84cd 4.38±0.39bcd 2.46±0.84c 3.26±0.33bcd 1.59±0.49cde 1.60±0.20de 0.10±0.03d 0.10±0.01def
Y2 3.88±0.19d 4.04±0.53d 2.25±0.13c 2.68±0.26e 1.31±0.42de 2.33±0.79bc 0.07±0.03d 0.10±0.01ef
L-60 3.75±0.41d 3.96±0.50d 2.90±0.46bc 2.87±0.32de 1.33±0.23de 1.54±0.62e 0.07±0.02d 0.10±0.02ef
NJAU-84 4.08±0.60cd 4.20±0.7cd 2.65±0.91bc 2.81±0.14de 1.33±0.28de 2.28±0.24bcd 0.08±0.02d 0.13±0.01bcd
CK 2.65±0.29e 3.18±0.20e 2.50±0.09c 2.80±0.46de 1.02±0.14e 1.41±0.30e 0.07±0.02d 0.08±0.02f
注:1)SQR9+Y2+L-60+NJAU-84为含SQR9+Y2+L-60+NJAU-84菌群生物育苗基质; SQR9+Y2+L-60为含SQR9+Y2+L-60菌群生物育苗基质; SQR9+L-60+NJAU-84为含SQR9+L-60+NJAU-84菌群生物育苗基质; SQR9+Y2+NJAU-84为含SQR9+Y2+NJAU-84菌群生物育苗基质; SQR9+Y2为含SQR9+Y2菌群生物育苗基质; SQR9+L-60为含SQR9+L-60菌群生物育苗基质; SQR9为含SQR9单一菌株生物育苗基质; Y2为含Y2单一菌株生物育苗基质; L-60为含L-60单一菌株生物育苗基质; NJAU-84为含NJAU-84单一菌株生物育苗基质; CK为添加无菌水普通育苗基质。2)不同字母表示同一季处理在0.05水平上差异显著。下同。
Note:1)SQR9+Y2+L-60+NJAU-84 indicates the bio-nursery substrates produced by SQR9+Y2+L-60+NJAU-84;SQR9+Y2+L-60 indicates the bio-nursery substrates produced by SQR9+Y2+L-60;SQR9+L-60+NJAU-84 indicate the bio-nursery substrates produced by SQR9+L-60+NJAU-84;SQR9+Y2+NJAU-84 indicates the bio-nursery substrates produced by SQR9+Y2+NJAU-84;SQR9+Y2 nidicates the bio-nursery substrates produced by SQR9+Y2;SQR9+L-60 indicates the bio-nursery substrates produced by SQR9+L-60;SQR9 indicates the bio-nursery substrates produced by SQR9;Y2 indicates the bio-nursery substrates produced by Y2;L-60 indicates the bio-nursery substrates produced by L-60;NJAU-84 indicate the bio-nursery substrates produced by NJAU-84;CK indicates adding sterile water to common nursery substrate. 2)Different letters in one column represent significant difference at 0.05 level. The same as follows.

两季苗盘育苗试验结果表明, 添加功能菌的生物育苗基质均对黄瓜种苗具有促生效果; 含组合菌群与单一菌株生物育苗基质所育种苗生长性状间存在差异, 其中含SQR9+Y2+L-60菌群生物育苗基质各项生长指标均显著高于对照及相应含单菌生物育苗基质所育种苗; 含SQR9+Y2+L-60+NJAU-84菌群生物育苗基质高于含4株菌和含2株菌菌群生物育苗基所育种苗的各项指标值, 部分指标差异显著。

2.4 碳源重合利用相对较多组合生物育苗基质的育苗效果

表 2可知:含组合菌株与单一菌株生物育苗基质所育种苗生长性状间存着差异。含2种菌(SQR9+Y40和SQR9+NJAU-69)、含3种菌(SQR9+Y40+NJAU-69、SQR9+NJAU-69+NJAU-84和SQR9+Y40+NJAU-84)和含4种菌(SQR9+Y40+NJAU-69+NJAU-84)生物育苗基质所育种苗大部分生长指标均高于对照及相应含单菌生物育苗基质所育种苗; 含单一菌株生物育苗基质所育种苗优于对照所育种苗, 但含2种菌、3种菌和4种菌生物育苗基质所育种苗各项指标之间差异不显著。

表 2 碳源重合利用相对较多功能菌组合研制的生物育苗基质对黄瓜的育苗效果 Table 2 Effects of bio-nursery substrate produced by strain groups with more overlapping utilization of carbon sources on the growth of cucumber seedlings
处理
Treatment
株高/cm
Plant height
茎粗/mm
Stem diameter
地上部鲜质量/g
Fresh weight of shoot
地上部干质量/g
Dry weight of shoot
第1季
The firstreason
第2季
The secondreason
第1季
The firstreason
第2季
The secondreason
第1季
The firstreason
第2季
The secondreason
第1季
The firstreason
第2季
The secondreason
SQR9+Y40+NJAU-69+NJAU-84 5.00±0.60a 5.04±0.60ab 2.91±0.50ab 2.80±0.29ab 1.48±0.35abcd 2.20±0.57ab 0.07±0.00b 0.10±0.01cde
SQR9+Y40+NJAU-69 4.93±0.43ab 4.96±0.32ab 3.27±0.78ab 3.22±0.29ab 1.76±0.49abc 1.84±0.37abc 0.08±0.04b 0.10±0.03cde
SQR9+NJAU-69+NJAU-84 4.65±0.39abcd 4.46±0.53abc 3.35±0.27a 4.21±0.27a 2.00±0.50a 2.46±0.28a 0.12±0.03a 0.12±0.03abcd
SQR9+Y40+NJAU-84 4.58±0.38abcd 5.16±0.68a 3.34±0.48a 2.83±0.45ab 1.44±0.20abcd 2.31±1.09ab 0.14±0.01b 0.09±0.03de
SQR9+Y40 4.93±0.29ab 4.52±0.63abc 2.40±0.25b 2.62±0.45c 1.04±0.11bc 2.16±0.59abc 0.07±0.00b 0.14±0.04ab
SQR9+NJAU-69 4.82±0.62abc 5.16±0.09a 2.54±0.44ab 3.35±0.93b 1.78±0.49abc 2.27±0.77ab 0.13±0.03a 0.15±0.02a
SQR9 4.23±0.98abcd 4.38±0.39bc 2.46±0.84ab 3.26±0.33ab 1.59±0.49abcd 1.60±0.19bc 0.10±0.03ab 0.10±0.02cde
Y40 3.93±0.10d 4.98±0.45ab 3.21±0.50ab 2.95±0.25ab 1.84±0.55ab 1.97±0.36abc 0.10±0.02ab 0.11±0.02bcde
NJAU-69 4.03±0.56cd 4.44±0.63abc 2.61±0.23ab 3.32±0.44b 1.21±0.22bcd 2.09±0.43abc 0.07±0.00b 0.12±0.01abcd
NJAU-84 4.08±0.60bcd 4.20±0.70c 2.65±0.91ab 2.81±0.14ab 1.33±0.28abcd 2.28±0.24ab 0.08±0.02b 0.13±0.01abc
CK 2.65±0.29e 3.18±0.20d 2.50±0.09ab 2.80±0.46ab 1.02±0.14d 1.41±0.30c 0.07±0.01b 0.08±0.02e
注:SQR9+Y40+NJAU-69+NJAU-84为含SQR9+Y40+NJAU-69+NJAU-84菌群生物育苗基质; SQR9+Y40+NJAU-69为含SQR9+Y40+NJAU-69菌群生物育苗基质; SQR9+NJAU-69+NJAU-84为含SQR9+NJAU-69+NJAU-84菌群生物育苗基质; SQR9+Y40+NJAU-84为含SQR9+Y40+NJAU-84菌群生物育苗基质; SQR9+Y40为含SQR9+Y40菌群生物育苗基质; SQR9+NJAU-69为含SQR9+NJAU-69菌群生物育苗基质; SQR9为含SQR9单一菌株生物育苗基质; Y40为含Y40单一菌株生物育苗基质; NJAU-69为含NJAU-69单一菌株生物育苗基质; NJAU-84为含NJAU-84单一菌株生物育苗基质; CK为添加无菌水普通育苗基质。
Note:SQR9+Y40+NJAU-69+NJAU-84 indicates the bio-nursery substrates produced by SQR9+Y40+NJAU-69+NJAU-84;SQR9+Y40+NJAU-69 indicates the bio-nursery substrates produced by SQR9+Y40+NJAU-69;SQR9+NJAU-69+NJAU-84 indicates the bio-nursery substrates produced by SQR9+NJAU-69+NJAU-84;SQR9+Y40+NJAU-84 indicates the bio-nursery substrates produced by SQR9+Y40+NJAU-84;SQR9+Y40 indicates the bio-nursery substrates produced by SQR9+Y40;SQR9+NJAU-69 indicates the bio-nursery substrates produced by SQR9+NJAU-69;Y40 indicates the bio-nursery substrates produced by Y40;NJAU-69 indicates the bio-nursery substrates produced by NJAU-69;NJAU-84 indicates the bio-nursery substrates produced by NJAU-84;CK indicates adding sterile water to common nursery substrate.
2.5 微生物碳源利用重合数与植物促生效果的关系

图 2所示:微生物碳源利用重合数与植物促生效果的斯皮尔曼相关分析表明, 第1季(图 2-A)和第2季(图 2-B), 微生物碳源利用重合数与植物促生效果均呈显著负相关关系。

图 2 第1季(A)和第2季(B)微生物碳源利用重合数与植物促生效果的斯皮尔曼相关分析 Figure 2 Spielman correlation analysis between the overlapping number of carbon sources utilization and plant growth promoting effect in the first(A)and the second(B)season
2.6 盆栽试验结果

根据以上育苗结果, 选择含SQR9+Y2+L-60+NJAU-84菌群、SQR9+Y2+L-60菌群、SQR9+Y2+NJAU-84菌群、SQR9+Y2菌群、SQR9+L-60菌群、SQR9菌株、Y2菌株、L-60菌株和NJAU-84菌株生物育苗基质和对照(CK)所育种苗进行盆栽试验。2季盆栽效果基本一致(表 3)。第1季盆栽试验中, 株高、茎粗、地上部鲜质量和地上部干质量, 含SQR9+Y2+L-60菌群生物育苗基质所移栽种苗与含单菌生物育苗基质所移栽种苗(SQR9)相比, 分别显著增加了96.86%、45.83%、37.20%和95.45%;与含2种菌生物育苗基质所移栽种苗(SQR9+Y2)相比, 分别显著增加38.19%、33.97%、33.19%和59.26%;与含4种菌生物育苗基质所移栽种苗(SQR9+Y2+L-60+NJAU-84)相比, 分别显著增加86.87%、44.33%、31.28%和59.26%。

表 3 黄瓜盆栽试验结果 Table 3 The results of cucumber pot experiment
处理
Treatment
株高/cm
Plant height
茎粗/mm
Stem diameter
地上部鲜质量/g
Fresh weight of shoot
地上部干质量/g
Dry weight of shoot
第1季
The firstreason
第2季
The secondreason
第1季
The firstreason
第2季
The secondreason
第1季
The firstreason
第2季
The secondreason
第1季
The firstreason
第2季
The secondreason
SQR9+Y2+L-60+NJAU-84 3.35±0.66d 3.25±0.44d 3.88±0.68b 4.00±0.09b 4.86±0.98bc 4.05±0.66c 0.27±010b 0.38±0.13ab
SQR9+Y2+NJAU-84 5.28±0.39b 5.50±0.08a 5.47±0.41a 4.72±0.16a 6.41±0.29a 5.36±0.42b 0.40±0.03a 0.53±0.09a
SQR9+L-60+Y2 6.26±0.19a 6.43±0.28b 5.60±0.24a 5.18±0.35a 6.38±0.28a 6.64±0.49a 0.43±0.01a 0.40±0.09ab
SQR9+L-60 3.25±0.29d 3.33±0.42d 4.29±0.39b 3.85±0.15b 4.89±0.97bc 4.19±0.75c 0.27±0.03b 0.36±0.12ab
SQR9+Y2 4.53±0.68c 4.20±0.22c 4.18±0.67b 3.03±0.32b 4.79±0.55bc 4.34±0.95bc 0.27±0.04b 0.40±0.06ab
SQR9 3.18±0.50d 2.90±0.54d 3.84±0.36b 3.81±0.28b 4.65±0.45bc 3.94±0.50cd 0.22±0.08b 0.31±0.03b
L-60 2.85±0.31de 2.95±0.77d 3.84±0.53b 3.64±0.44b 4.69±0.80bc 2.93±0.87de 0.26±0.07b 0.36±0.02b
Y2 2.85±0.31de 3.10±0.96d 3.85±0.60b 3.53±0.28b 3.29±0.91de 2.79±0.84e 0.22±0.02b 0.33±0.08ab
NJAU-84 2.35±0.24e 2.93±0.88d 3.56±0.21bc 3.54±0.42b 3.80±0.85cd 3.23±0.58cde 0.26±0.11b 0.28±0.11b
CK 2.23±0.26e 2.63±0.19d 2.96±0.36c 2.99±0.68c 2.35±0.82e 2.45±0.84e 0.12±0.01c 0.26±0.08b
注:SQR9+Y2+L-60+NJAU-84为移栽含SQR9+Y2+L-60+NJAU-84复合菌群生物育苗基质所育种苗; SQR9+NJAU-84+Y2为移栽含SQR9+NJAU-84+Y2复合菌群生物育苗基质所育种苗; SQR9+L-60+Y2为移栽含SQR9+L-60+Y2复合菌群生物育苗基质所育种苗; SQR9+L-60为移栽含SQR9+L-60复合菌群生物育苗基质所育种苗; SQR9+Y2为移栽含SQR9+Y2复合菌群生物育苗基质所育种苗; SQR9, 移栽含SQR9单一菌株生物育苗基质所育种苗; L-60为移栽含L-60单一菌株生物育苗基质所育种苗; Y2为移栽含Y2单一菌株生物育苗基质所育种苗; NJAU-84为移栽含NJAU-84单一菌株生物育苗基质所育种苗; CK为添加无菌水普通育苗基质。
Note:SQR9+Y2+L-60+NJAU-84 indicates treatment transplanted with seedlings from bio-nursery substrates produced by SQR9+Y2+L-60+NJAU-84;SQR9+NJAU-84+Y2 indicates treatment transplanted with seedlings from bio-nursery substrates produced by SQR9+NJAU-84+Y2;SQR9+L-60+Y2 indicates treatment transplanted with seedlings from bio-nursery substrates produced by SQR9+L-60+Y2;SQR9+L-60 indicates treatment transplanted with seedlings from bio-nursery substrates produced by SQR9+L-60;SQR9+Y2 indicates treatment transplanted with seedlings from bio-nursery substrates produced by SQR9+Y2;SQR9, treatment transplanted with seedlings from bio-nursery substrates produced by SQR9;L-60 indicates treatment transplanted with seedlings from bio-nursery substrates produced by L-60;Y2 indicates treatment transplanted with seedlings from bio-nursery substrates produced by Y2;NJAU-84 indicates treatment transplanted with seedlings from bio-nursery substrates produced by NJAU-84;CK indicates treatment transplanted with seedlings from adding common nursery substrate.

第2季盆栽试验中, 含SQR9+Y2+NJAU-84菌群生物育苗基质所移栽种苗, 与含单菌生物育苗基质(SQR9)所移栽种苗相比, 株高、茎粗、地上部鲜质量和地上部干质量, 分别显著增加89.66%、23.88%、36.04%和70.97%;与含2种菌生物育苗基质(SQR9+L-60)所移栽种苗相比, 株高、茎粗和地上部鲜质量分别显著增加了65.17%、22.60%和27.92%;与含4种菌生物育苗基质(SQR9+Y2+L-60+NJAU-84)所移栽种苗相比, 株高、茎粗和地上部鲜质量分别显著增加了9.23%、18.00%和32.35%。

2季盆栽试验结果表明, 含SQR9+Y2+L-60菌群和含SQR9+Y2+NJAU-84菌群生物育苗基质所移栽种苗的株高、茎粗和地上部鲜质量分别显著高于CK(对照), 含SQR9、Y2、L-60与NJAU-84单一菌株生物育苗基质, 含SQR9+Y2与SQR9+L-60菌群生物育苗基质和SQR9+Y2+L-60+NJAU-84菌群生物育苗基质处理所移栽育种苗。同时, 含2种菌株生物育苗基质和4种菌株生物育苗基质所移栽种苗各项生长指标均高于对照及相应含各单菌生物育苗基质所移栽种苗, 但三者之间无显著差异。因此, 最终确立生物育苗基质菌群最佳组合为SQR9+Y2+L-60和SQR9+Y2+NJAU-84。

2.7 菌株的鉴定

菌株NJAU-84在LB平板上的菌落呈淡黄色, 呈饱满突起规则圆形, 表面光滑, 具有一定的黏性, 易挑起。革兰氏染色阳性, 能够产生芽胞, 能够利用D-麦芽糖、D-麦芽糖、D-海藻糖、D-纤维二糖、龙胆二糖、蔗糖、D-松二糖、水苏糖、蜜三糖、棉子糖、β-甲酰-D-葡糖苷、N-乙酰-D-葡糖胺、α-D-葡糖、D-甘露糖、D-果糖、D-甘露醇和D-葡糖酸。所构建发育树如图 3所示, 菌株NJAU-84与Bacillus aryabhattai同源性达99.8%, 结合菌株的形态特征、理化特性和16S rDNA序列分析, 将菌株NJAU-84初步鉴定为芽胞杆菌。

图 3 菌株NJAU-84的系统发育树 Figure 3 The phylogenetic tree of strain NJAU-84
3 讨论

有益菌群的多样性能够促进菌群功能的发挥[19], 生态系统的营养丰富度能够调节生态系统的功能[20], 加之根际微生物主要通过根际营养而存活于根际发挥功能[21], 因此, 通过功能菌之间的碳源利用能够互补并有效调节复合菌群的营养利用竞争, 从而构建出最优的菌群组合。本研究基于此, 构建了2类复合菌群, 一类为碳源重合少的, 一类为碳源重合多的作为对照。

两季育苗试验结果表明, 复合菌群的促生效果明显高于不添加菌的对照和添加单菌的处理, 体现出了复合菌群的优越性。沈永红等[22]研究发现, 将菌株两两混合培养后, 其发酵液对高岭土悬浊液的絮凝率较单一菌株培养时明显提高, 并且具有较好的热稳定性; 赵硕伟等[23]对7株菌进行不同的组合来研究复合菌群对石油的降解, 发现Gordonia sp.和Pesudomonas sp.组成的复合菌群降解石油的能力超过任何单一菌株。本研究在育苗基质领域具有创新性, 验证了复合菌群的高效促生效果。

微生物碳源利用重合数与植物促生效果呈显著负相关关系, 这可能与各功能菌株之间碳源重合利用多有关。PGPR菌株对营养进行竞争, 尤其是对碳源的竞争。在营养缺乏的环境中, PGPR菌株抑制真菌生长, 这种机制可以抑制土体中真菌孢子的萌发[24]

盆栽试验结果表明, 利用SQR9+Y2+L-60和SQR9+Y2+NJAU-84复合菌群研发的生物基质所育黄瓜种苗移栽后, 生长指标均高于对照和单菌育苗基质所育种苗。复合菌群中解淀粉芽胞杆菌SQR9、假单胞菌Y2和海水芽胞杆菌L-60均为根际有益菌, 促生效应均已被报道, 与芽胞杆菌属细菌NJAU-84接近的巨大芽胞杆菌同样是有益菌, 因此推测苗期功能菌的定殖, 是促进盆栽作物生长的重要原因, 这也与张杨等[9]的研究结果一致。另有研究表明, 移栽后作物生长指标的提高与苗期相应作物生长量有一定的关系, 一般而言苗期作物生长量与移栽后作物产量成正比[25-27]

综上所述, 结合苗盘试验及2季盆栽试验结果, 复合菌群SQR9+Y2+L-60和SQR9+Y2+NJAU-84为最佳菌群组合, 利用其能够有效研制出促生性能优异的新型含复合菌群的生物育苗基质。

参考文献(References)
[1] 崔晓双, 王伟, 张如, 等. 基于根际营养竞争的植物根际促生菌的筛选及促生效应研究[J]. 南京农业大学学报, 2015, 38(6): 958-966.
Cui X S, Wang W, Zhang R, et al. Screening of plant growth-promoting rhizobacteria based on rhizosphere nutrition competiveness and investigation of their promoting effects[J]. Journal of Nanjing Agricultural University, 2015, 38(6): 958-966. DOI: 10.7685/j.issn.1000-2030.2015.06.013 (in Chinese with English abstract)
[2] 郭良栋. 中国微生物物种多样性研究进展[J]. 生物多样性, 2012, 20(5): 572-580.
Guo L D. Progress of microbial species diversity research in China[J]. Biodiversity Science, 2012, 20(5): 572-580. (in Chinese with English abstract)
[3] 李鸣雷, 商鸿生, 谷洁, 等. 促进农业废弃物腐解的复合微生物菌剂的筛选[J]. 西北农业学报, 2005, 14(2): 101-104.
Li M L, Shang H S, Gu J, et al. Screening of the composite microorganism for promoting the decomposition of agriculture residue[J]. Acta Agriculturae Boreali-occidentalis Sinica, 2005, 14(2): 101-104. (in Chinese with English abstract)
[4] 秦韵婷. 红枣根际复合功能菌的研制及肥效研究[D]. 乌鲁木齐: 新疆农业大学, 2015.
Qin Y T. The production of composite functional rhizobacteria for Jujube and study on fertilizing effect[D]. Urumqi: Xinjiang Agricultural University, 2015(in Chinese with English abstract). http://cdmd.cnki.com.cn/Article/CDMD-10758-1015645747.htm
[5] 周先锋. 复合菌群处理高浓度城市生活污水的技术研究[D]. 天津: 天津大学, 2005.
Zhou X F. Technology study on disposing municipal sewage of high concentration in complex flora[D]. Tianjing: Tianjin University, 2005(in Chinese with English abstract). http://cdmd.cnki.com.cn/article/cdmd-10056-2006052863.htm
[6] 张杨, 文春燕, 赵买琼, 等.辣椒根际促生菌的分离筛选及生物育苗基质研制[J].南京农业大学学报, 2015, 38(6):950-957. DOI:10.7685.j.issn.1000-2030.2015.06.012.
Zhang Y, Wen C Y, Zhao M Q, et al. Isolation of plant growth promoting rhizobacteria from pepper and development of bio-nursery substrates[J]. Journal of Nanjing Agricultural University, 2015, 38(6):950-957(in Chinese with English abstract). http://nauxb.njau.edu.cn/oa/darticle.aspx?type=view&id=201506012
[7] 杜林峰, 孙向阳, 陈建武, 等. 国内外泥炭基质养分比较研究[J]. 北方园艺, 2008(7): 55-57.
Du L F, Sun X Y, Chen J W, et al. Comparison of nutrient of peat growing mediums by domestic and foreign manufacturers[J]. Northern Horticulture, 2008(7): 55-57. (in Chinese with English abstract)
[8] 文春燕, 高琦, 张杨, 等. 含PGPR菌株LZ-8生物育苗基质的研制与促生效应研究[J]. 土壤, 2016, 48(2): 414-417.
Wen C Y, Gao Q, Zhang Y, et al. Development of biological matrix produced by PGPR strain LZ-8 and analysis for its growth promoting effect[J]. Soils, 2016, 48(2): 414-417. (in Chinese with English abstract)
[9] 张杨, 王甜甜, 孙玉涵, 等. 西瓜根际促生菌筛选及生物育苗基质研制[J]. 土壤学报, 2017, 54(3): 704-714.
Zhang Y, Wang T T, Sun Y H, et al. Screening of plant growth-promoting rhizobacteria from watermelon and development of bio-nursery substrates[J]. Acta Pedologica Sinica, 2017, 54(3): 704-714. (in Chinese with English abstract)
[10] Cao Y, Ling N, Yang X M, et al. Bacillus subtilis SQR9 can control Fusarium wilt in cucumber by colonizing plant roots[J]. Biology and Fertility of Soils, 2011, 47(5): 495-506. DOI: 10.1007/s00374-011-0556-2
[11] 沈其荣, 李荣, 刘小玉, 等. 一株有效促进作物生长的海水芽孢杆菌L-60及其应用: 201510982083. 3[P]. 2015-12-24.
Shen Q R, Li R, Liu X Y, et al. Bacillus aquimaris strain L-60 that effectively promotes plant growth and its application: 201510982083. 3[P]. 2015-12-24(in Chinese).
[12] 沈其荣, 李荣, 赵买琼, 等. 一株假单胞菌属解磷细菌Y2及其制备的生物有机肥和应用: 201510399056. 3[P]. 2015-07-08.
Shen Q R, Li R, Zhao M Q, et al. Phosphate-solubilizing bacterium Pseudomonas sp. Y2 and development and application of bio-organic fertilizer produced by this strain: 201510399056. 3[P]. 2015-07-08(in Chinese).
[13] 沈其荣, 李荣, 赵买琼, 等. 一株不动杆菌属解磷促生细菌Y40及其应用: 201510399215. X[P]. 2015-07-09.
Shen Q R, Li R, Zhao M Q, et al. Phosphate-solubilizing bacterium Acinetobacter sp. Y40 and its application: 201510399215. X[P]. 2015-07-09(in Chinese).
[14] 刘江. 无机磷细菌的筛选及其Biolog和分子生物学鉴定[D]. 杨凌: 西北农林科技大学, 2011.
Liu J. Isolation and identification of inorganic phosphorus-solobiliaing by Biolog and molecular method[D]. Yangling: Northwest A & F University, 2011(in Chinese with English abstract). http://cdmd.cnki.com.cn/Article/CDMD-10712-1011403456.htm
[15] 姬广海, 张世光, 许志刚. 植物病原细菌的BIOLOG系统鉴定及其多元统计初析[J]. 应用与环境生物学报, 2001, 7(3): 288-290.
Ji R H, Zhang S G, Xu Z G. Identification and multivariate statistical analysis of BIOLOG system of plant pathogenic bacteria[J]. Chinese Journal of Applied and Environmental Biology, 2001, 7(3): 288-290. (in Chinese with English abstract)
[16] Xu Z, Zhang R, Wang D, et al. Enhanced control of cucumber wilt disease by Bacillus amyloliquefaciens SQR9 by altering the regulation of its DegU phosphorylation[J]. Applied and Environmental Microbiology, 2014, 80(9): 2941-2950. DOI: 10.1128/AEM.03943-13
[17] Qiu M, Xu Z, Li X, et al. Comparative proteomics analysis of Bacillus amyloliquefaciens SQR9 revealed the key proteins involved in situ root colonization[J]. Journal of Proteome Research, 2014, 13(12): 5581-5591. DOI: 10.1021/pr500565m
[18] Yang C L, Li R, Song Y, et al. Identification of the biochemical degradation pathway of triazophos and its intermediate in Diaphorobacter sp. TPD-1[J]. Current Microbiology, 2011, 62(4): 1294-1301. DOI: 10.1007/s00284-010-9859-z
[19] Hu J, Wei Z, Gu S H, et al. Probiotic diversity enhances rhizosphere microbiome function and plant disease suppression[J]. mBio, 2016, 7(6): e01790-16.
[20] Yang T, Wei Z, Friman V P, et al. Resource availability modulates biodiversity-invasion relationships by altering competitive interactions[J]. Environmental Microbiology, 2017, 19(8): 2984-2991. DOI: 10.1111/emi.2017.19.issue-8
[21] Liu H, Xiong W, Zhang R, et al. Continuous application of different organic additives can suppress tomato disease by inducing the healthy rhizospheric microbiota through alterations to the bulk soil microflora[J]. Plant and Soil, 2017, 423(1): 229-240.
[22] 沈永红, 薛玉, 刘宇鹏, 等. 高效微生物絮凝剂产生菌的筛选及复合菌群培养研究[J]. 河南大学学报(自然科学版), 2015, 45(3): 334-338.
Shen Y H, Xue Y, Liu Y P, et al. Isolation of strains producing high efficient microbial flocculant and culture of multiple microorganisms[J]. Journal of Henan University(Natural Science Edition), 2015, 45(3): 334-338. (in Chinese with English abstract)
[23] 赵硕伟, 沈嘉澍, 沈标, 等. 复合菌群的构建及其对石油污染土壤修复的研究[J]. 农业环境科学学报, 2011, 30(8): 1567-1572.
Zhao S W, Shen J S, Shen B, et al. Construction of multiple bacterial consortium and its application in bioremediation of petroleum-contaminated soil[J]. Journal of Agro-Environment Science, 2011, 30(8): 1567-1572. (in Chinese with English abstract)
[24] Qiu M, Li S, Zhou X, et al. De-coupling of root-microbiome associations followed by antagonist inoculation improves rhizosphere soil suppressiveness[J]. Biology Fertilization Soils, 2013, 50(2): 217-224.
[25] 张慧, 杨兴明, 冉炜, 等. 土传棉花黄萎病拮抗菌的筛选及其生物效应[J]. 土壤学报, 2008, 45(6): 1095-1101.
Zhang H, Yang X M, Ran W, et al. Screening of bacteria antagonistic against soil-borne cotton verticillium wilt and their biological effects on the soil-cotton system[J]. Acta Pedologica Sinica, 2008, 45(6): 1095-1101. (in Chinese with English abstract)
[26] 江欢欢, 程凯, 杨兴明, 等. 辣椒青枯病拮抗菌的筛选及其生物防治效应[J]. 土壤学报, 2010, 47(6): 1225-1231.
Jiang H H, Cheng K, Yang X M, et al. Isolation and biological effect of capsicum wilt antagonist[J]. Acta Pedologica Sinica, 2010, 47(6): 1225-1231. DOI: 10.11766/trxb200911110503 (in Chinese with English abstract)
[27] 韩晓玲, 张乃文, 贾敬芬. 生物有机无机复混肥对番茄产量、品质及土壤的影响[J]. 土壤肥料, 2005(3): 51-53.
Han X L, Zhang N W, Jia J F. Effects of biological organic-inorganic compound fertilizers on yield, quality of tomato and soil[J]. Soils and Fertilizers, 2005(3): 51-53. (in Chinese with English abstract)