有机肥对香蕉枯萎病及土壤主要理化性质和微生物群落的影响

朱志炎; 梁雪雁; 林凤玲; 马嘉荣; 田志宏; 李建雄

doi:10.19303/j.issn.1008-0384.2021.07.010

有机肥对香蕉枯萎病及土壤主要理化性质和微生物群落的影响

doi: 10.19303/j.issn.1008-0384.2021.07.010

朱志炎^{1, 2,},
梁雪雁²,
林凤玲²,
马嘉荣²,
田志宏¹,
李建雄^2, ,

1.
长江大学生命科学学院，湖北　荆州　434025
2.
中国科学院华南植物园，广东　广州　510650

基金项目: 中国科学院STS区域重点项目（KFJ-STS-QYZX-044）；广东省农业厅委托研究项目（Y434121002）

详细信息

作者简介:
朱志炎（1989−），男，博士研究生，研究方向：植物病理（E-mail：420905220@qq.com）

通讯作者:
李建雄（1969−），男，博士，研究员，研究方向：植物病理（E-mail：jxli@scbg.ac.cn）

中图分类号: S 432.1
计量
- 文章访问数: 756
- HTML全文浏览量: 502
- PDF下载量: 53
- 被引次数: 0
出版历程
- 收稿日期: 2020-11-19
- 修回日期: 2021-03-16
- 网络出版日期: 2021-07-13
- 刊出日期: 2021-07-28

Effects of Bio-organic Fertilizer on Physicochemical Properties and Microflora of Banana Field Infected by Fusarium Wilt Disease

1.
School of Life Sciences, Yangtze University, Jingzhou, Hubei　434025, China
2.
South China Botanical Garden, Chinese Academy of Sciences, Guanzhou, Guandong　510650, China

摘要

摘要: 目的研究香蕉园施用有机肥防治香蕉枯萎病对土壤理化性质及其根际土壤微生物群落的影响。方法香蕉幼苗移栽至病区大田土壤中，处理组植株施用有机肥，并将未施用有机肥的植株设置为对照组。移栽后6个月统计处理组与对照组植株香蕉枯萎病发病率；采集土壤样本，测定根际土壤的土壤肥力；提取根际土壤DNA，采用高通量测序技术，结合生物信息学分析，解析施用有机肥后香蕉根际土壤细菌和真菌群落结构组成及多样性的变化。结果施用有机肥提高了土壤pH值（14.85%）、全氮（25%）和全磷（19.04%）的含量，降低土壤全铁含量（2.62%），香蕉枯萎病发病率下降了75%。和对照相比，子囊菌门（Ascomycota）与壶菌门（Chytridiomycota）的相对丰度分别提高了43.84%和90.64%，变形菌门（Proteobacteria）的相对丰度则降低了18.49%。施用有机肥料提高了青霉菌属（Penicillium）、Gibellulopsis和篮状菌属（Talaromyces）等的相对丰度，比例分别为对照组的2.93倍、2.12倍和11.93倍。施用有机肥料后，香蕉根际土壤真菌群落Chao1指数、ACE指数与香农（Shannon）指数得到提升，分别提升了39.81%、38.43%和86.85%。结论施用有机肥料改善了土壤理化性质，改变了根际土壤微生物群落结构和多样性，降低了香蕉枯萎病发病率。
- 枯萎病 /
- 香蕉 /
- 高通量测序 /
- 细菌群落 /
- 真菌群落 /
- 有机肥
Abstract: Objective Effects of a bio-organic fertilizer on physicochemical properties and microbial community in rhizosphere soil of a banana field infected by Fusarium wilt disease were studied. Methods In a Fusarium wilt infected banana field, an random block design experiment on the application of a bio-organic fertilizer, Biofert, was conducted. Six months after banana seedlings were transplanted to the field, rhizosphere soil samples from lots with and without Biofert application were collected to determine the nutrient contents by chemical analysis and the microbial composition and diversity by high-throughput sequencing and bioinformatics analysis. Results Compared to control, the application of Biofert increased pH by 14.85%, the total N by 25%, and total P by 19.04%, but decreased the total Fe by 2.62% in the soil, while lowered the incidence of Fusarium wilt on the plants by 75%. In the rhizosphere soil, the Biofert-treated lots showed the relative abundance of, other than an 18.49% decrease on Proteobacteria, increases by 43.84% on Ascomycota, 90.64% on Chytridiomycota, 293% on Penicillium, 212% on Gibellulopsis, and 1193% on Talaromyces, 39.81% on fungal Chao1 index, 38.43% on ACE, and 86.85% on Shannon index. Conclusion Biofert application not only improved the soil quality but also significantly altered the structure and diversity of the microbial community in rhizosphere soil and contributed to the reduced incidence of banana wilt disease.
- Fusarium wilt /
- Banana /
- high-throughput sequencing /
- bacterial community /
- fungal community /
- organic fertilizer Biofert

HTML全文

图 1 不同处理香蕉发病情况

注：A，香蕉幼苗根未施有机肥料；B，香蕉幼苗根施有机肥料；C，香蕉幼苗（未施用有机肥料）球茎；D，香蕉幼苗（施用有机肥料）球茎。

Figure 1. Disease symptoms on banana plants

Note: A: plants applied with no Biofert; B: plants applied with Biofert; C: protocorm of plants applied with no Biofert; D: protocorm of plants applied with Biofert.

下载: 全尺寸图片幻灯片

图 2 香蕉植株种植土壤中枯萎病致病菌株分离与检测

注：A，菌株形态特征；B，TR4菌株特异性条带扩增。

Figure 2. Isolation and detection of pathogens of Fusarium wilt from banana plants

Note: A: Morphological characteristics of strain; B: Specific bands of TR4.

下载: 全尺寸图片幻灯片

图 3 不同处理土壤在3%差异水平的稀释曲线

注：（1）曲线趋于平缓，表明样品测定充分。（2）CK：未施加有机肥料土壤；BIO，施加有机肥料土壤。（3）A：细菌；B：真菌。

Figure 3. Rarefaction curves at 3% dissimilarity level of treatment soils

Note:（1）Curves tend to flatten, and samples are fully sequenced.（2） CK: plants applied with no Biofert; BIO: plants applied with Biofert.（３）A: bacteria; B: fungi.

下载: 全尺寸图片幻灯片

图 4 不同处理根际土壤基于Bray-Curtis算法进行的主坐标分析（PCoA）

注：CK，未施加有机肥料的植株根际土壤；BIO，施加有机肥料的植株根际土壤；A，细菌；B，真菌。

Figure 4. Principal co-ordinates analysis based on distance matrix calculated using Bray-Curtis algorithm for treatment soils

Note: CK: plants applied with no Biofert; BIO: plants applied with Biofert. A: bacteria; B, fungi.

下载: 全尺寸图片幻灯片

图 5 不同处理样品间细菌与真菌丰度最高的10个门类

注：（1）CK：未施加有机肥料的植株根际土壤。（2）BIO：施加有机肥料的植株根际土壤。（3）A：细菌；B：真菌。（4）处理后面的不同数字（1，2，3）代表3个重复。

Figure 5. Relative abundance of top 10 bacteria and fungi phyla in treatment soils

Note: （1）CK: plants applied with no Biofert.（2）BIO: plants applied with Biofert.（3） A: bacteria; B: fungi. （4）Numeric numbers after letters on treatments (1, 2, 3) represent 3 replicates.

下载: 全尺寸图片幻灯片

图 6 不同处理间根际土壤中的LEfSe分析

Figure 6. Microbial variations based on LEfSe analysis in treatment soils

下载: 全尺寸图片幻灯片

图 7 不同处理对根际土壤微生物群落功能影响

Figure 7. Metagenomes predicted by PICRUSt showing significant differences on functionality in treatment soils

下载: 全尺寸图片幻灯片

表 1 各处理根际土壤部分理化性质以及植株发病率

Table 1. Physiochemical properties of rhizosphere soil and disease incidence on plants

处理　　 Treatment	对照 CK	有机肥 BIO
pH	5.05±0.04	5.81±0.01*
含水量 Water content/%	24.51±0.37	23.61±2.18
全氮 Total nitrogen/%	0.16±0.01	0.20±0.01*
全磷 Total phosphorus/（%）	0.84±0.01	0.10±0.01*
全铁 Total iron/（mg·kg⁻¹）	78363±1344.51	76304±803.05*
发病率 Disease incidence rate/（%）	80%	20%*
注：同一行中“”表示差异显著（P＜0.05）。 Note: “” in same line indicates significant difference (P＜0.05).

下载: 导出CSV

表 2 不同处理间根际土壤在97%相似水平下的ACE、Chao 1和香农指数

Table 2. ACE, Chao 1, and Shannon indices of rhizosphere soil in treatment soils at 97% similarity

处理 Treatment	群落特征 Community characteristics
	细菌群落 Bacterial community			真菌群落 Fungal community
	ACE	Chao1	Shannon	ACE	Chao1	Shannon
对照 CK	14903.14±58.67	1508.36±61.57	5.28±0.28	248.69±20.88	258.18±15.02	2.13±0.04
有机肥 BIO	1392.29±14.31	1414.43±9.99	5.02±0.57	344.28±23.56*	360.97±27.02*	3.98±0.16*
注：同一列中“”代表2个数据之间的差异水平显著（P＜0.05）。 Note: “” on same column indicates statistically significant differences based on Duncan’s test (P ＜ 0.05).

下载: 导出CSV

表 3 不同处理间根际土壤中微生物相关性网络分析

Table 3. Sparcc’s correlation network analyses on microbial communities in treatment soils

处理 Treatment	细菌群落 Bacterial community		真菌群落 Fungi community
处理 Treatment	有机肥 BIO	对照 CK	有机肥 BIO	对照 CK
菌属负相关数 Number of negative correlation	37	23	35	23
菌属正相关数 Number of positive correlation	42	34	23	33
菌属1 Genus1	—	—	镰刀菌属 Fusarium	镰刀菌属 Fusarium
菌属2 Genus2	—	—	木霉属 Trichoderma	葡萄穗霉属 Stachybotrys
相关系数 Correlation	—	—	0.93017	1
相关性 Orientation	—	—	负 Negative	正 Positive

下载: 导出CSV

表 4 根际土壤中丰度前20的细菌属和真菌属与植株发病率之间的相关性分析

Table 4. Sparcc’s correlation coefficients between top 20 bacteria and fungi genera and disease index

细菌 Bacteria		真菌 Fungi
属 Genera	相关系数 Disease index	属 Genera	相关系数 Disease index
芽孢杆菌属 Bacillus	−0.138	曲霉菌属 Aspergillus	−0.908
苔藓杆菌属 Bryobacter	0.321	离蠕孢属 Bipolaris	−0.01
伯克氏菌属 Burkholderi	−0.047	Condenascus	−0.533
待鉴定酸杆菌 Candidatus_Solibacter	−0.279	杯梗孢属 Cyphellophora	0.752
侏囊菌属 Haliangium	−0.539	Dimorphiseta	0.411
罗丹杆菌 Rhodanobacter	−0.11	镰刀菌属 Fusarium	0.939*
醇单胞菌属 Sphingomonas	0.207	Gibellulopsis	−0.178
链霉菌属 Streptomyces	0.14	腐质霉属 Humicola	−0.429
Subgroup_6	0.089	小羊蹄菌属 Microdochium	0.443
放线菌属 Acidobacteriaceae_Subgroup_1	−0.249	被孢霉属 Mortierella	−0.466
束鞘藻 Coleofasciculaceae	0.354	黑孢霉属 Nigrospora	−0.028
芽单胞菌 Gemmatimonadaceae	−0.417	青霉菌属 Penicillium	−0.918*
SC-I-84	0.136	小不整球壳属 Plectosphaerella	−0.215
黄色杆菌 Xanthobacteraceae	−1*	拟棘壳孢属 Pyrenochaetopsis	0.831
酸杆菌 Acidobacteriales	−0.5	沙蜥属 Saitozyma	−0.64
Chloroplast	0.704	壳多胞菌属 Stagonospora	−0.293
Elsterales	−0.415	圆孢霉属 Staphylotrichum	−0.591
盖勒氏菌 Gaiellales	−0.041	篮状菌属 Talaromyces	−0.635
变形菌 Gammaproteobacteria_Incertae_Sedis	−0.651	木霉属 Trichoderma	−0.037
Subgroup_2	−0.291	Xenomyrothecium	−0.204
注：“^”代表在数据之间差异达到显著水平（P＜0.05）。 Note: “” indicating statistically significant differences at the 0.05 probability level.

下载: 导出CSV

参考文献(36)

[1]	LIN Y H, LIN Y J, CHANG T D, et al. Development of a TaqMan Probe-Based Insulated Isothermal Polymerase Chain Reaction (iiPCR) Assay for Detection of Fusarium oxysporum f. sp. cubense Race 4 [J]. PLoS One, 2016, 11(7): 1−13.
[2]	LI X J, LI K, ZHOU D B, et al. Biological control of banana wilt disease caused by Fusarium oxyspoum f. sp. Cubense using Streptomyces sp. H4 [J]. Biological Control, 2021, 155(10): 1−9.
[3]	LIN Y J, LIN H K, LIN Y H. Construction of Raman spectroscopic fingerprints for the detection of Fusarium wilt of banana in Taiwan [J]. PLoS One, 2020, 15(3): 1−14.
[4]	WARMAN N M, AITKEN E A B. The Movement of Fusarium oxysporum f. sp. cubense (Sub-Tropical Race 4) in Susceptible Cultivars of Banana [J]. Frontiers in Plant Science, 2018, 9: 1748−1757. doi: 10.3389/fpls.2018.01748
[5]	李进, 张立丹, 刘芳, 等. 碱性肥料对香蕉枯萎病发生及土壤微生物群落的影响 [J]. 植物营养与肥料学报, 2016, 22(2):429−436. doi: 10.11674/zwyf.14460 LI J, ZHANG L D, LIU F, et al. Effects of alkaline fertilizer on occurrence of banana wilt disease and soil microbial community [J]. Journal of Plant Nutrition and Fertilizer, 2016, 22(2): 429−436.(in Chinese) doi: 10.11674/zwyf.14460
[6]	王荣, 刘吉青, 周海霞, 等. 生物有机肥与保水剂对设施连作黄瓜生长和土壤肥力的影响 [J]. 河南农业科学, 2018, 47(8):45−53. WANG R, LIU J Q, ZHOU H X, et al. Effects of bio-organic fertilizer and water-retaining agent on cucumber growth and soil fertility under continuous cropping in greenhouse [J]. Journal of Henan Agricultural Sciences, 2018, 47(8): 45−53.(in Chinese)
[7]	ZHU Z Y, TIAN Z H, LI J X. A Streptomyces morookaensis strain promotes plant growth and suppresses Fusarium wilt of banana [J]. Tropical Plant Pathology, 2021, 46(2): 175−185. doi: 10.1007/s40858-020-00396-z
[8]	CHENG C Z, LI D, QI Q, et al. The root endophytic fungus Serendipita indica improves resistance of Banana to Fusarium oxysporum f. sp. cubense tropical race 4 [J]. European Journal of Plant Pathology, 2020, 156(1): 87−100. doi: 10.1007/s10658-019-01863-3
[9]	LUO G W, LI L, FRIMAN V P, et al. Organic amendments increase crop yields by improving microbe-mediated soil functioning of agroecosystems: A meta-analysis [J]. Soil Biology and Biochemistry, 2018, 124: 105−115. doi: 10.1016/j.soilbio.2018.06.002
[10]	CHENG H Y, ZHANG D Q, HUANG B, et al. Organic fertilizer improves soil fertility and restores the bacterial community after 1, 3-dichloropropene fumigation [J]. The Science of the Total Environment, 2020, 738(6): 1−42.
[11]	陶成圆. 含解淀粉芽孢杆菌NJN-6的生物有机肥防控香蕉枯萎病研究[D]. 南京: 南京农业大学, 2016. TAO C Y. Effect of the combination of bio-organic fertilizer with Bacillus amyloliquefaciens NJN-6 on the control of banana Fusarium wilt disease[D]. Nanjing: Nanjing Agricultural University, 2016. (in Chinese)
[12]	TAO C Y, LI R, XIONG W, et al. Bio-organic fertilizers stimulate indigenous soil Pseudomonas populations to enhance plant disease suppression [J]. Microbiome, 2020, 8(1): 137. doi: 10.1186/s40168-020-00892-z
[13]	剧虹伶. 辣椒-香蕉轮作联合生物有机肥减轻高发枯萎病蕉园连作障碍机制研究[D]. 海口: 海南大学, 2017. JU H L. Research on the mechanisms of mitigate continuous obstacle of banana orchard with serious wilt disease by combined pepper-banana rotation together with application of bio-organic fertilizer[D]. Haikou: Hainan University, 2017. (in Chinese)
[14]	吉福桑, 杨振, 徐亚, 等. 盐胁迫下巴西蕉叶片的转录组和蛋白质组关联分析 [J]. 分子植物育种, 2020, 18(23):7671−7678. JI F S, YANG Z, XU Y, et al. Association analysis on transcriptomics and proteomics of Musa paradisiaca banana leaf under salt stress [J]. Molecular Plant Breeding, 2020, 18(23): 7671−7678.(in Chinese)
[15]	GUO L J, YANG L Y, LIANG C C, et al. Differential Colonization Patterns of Bananas (Musa spp.) by Physiological Race 1 and Race 4 Isolates of Fusarium oxysporum f. sp. cubense [J]. Journal of Phytopathology, 2015, 163(10): 807−817. doi: 10.1111/jph.12378
[16]	FAN H Y, LEI Z X, DONG H H, et al. Immune responses in Brazilian banana determining the pathogenic differences between the physiological races 1 and 4 of Fusarium oxysporum f. sp. cubense [J]. Journal of Plant Pathology, 2019, 101(2): 225−234. doi: 10.1007/s42161-018-0165-0
[17]	刘光崧. 土壤理化分析与剖面描述[M]. 北京: 中国标准出版社, 1996: 50−81.
[18]	MOSTERT D, MOLINA A B, DANIELLS J, et al. The distribution and host range of the banana Fusarium wilt fungus, Fusarium oxysporum f. sp. cubense, in Asia [J]. PLoS One, 2017, 12(7): 1−10.
[19]	DITA M A, WAALWIJK C, BUDDENHAGEN I W, et al. A molecular diagnostic for tropical race 4 of the banana Fusarium wilt pathogen [J]. Plant Pathology, 2010, 59(2): 348−357. doi: 10.1111/j.1365-3059.2009.02221.x
[20]	MAGOČ T, SALZBERG S L. FLASH: fast length adjustment of short reads to improve genome assemblies [J]. Bioinformatics, 2011, 27(21): 2957−2963. doi: 10.1093/bioinformatics/btr507
[21]	BOLGER A M, LOHSE M, USADEL B. Trimmomatic: a flexible trimmer for Illumina sequence data [J]. Bioinformatics (Oxford, England), 2014, 30(15): 2114−2120. doi: 10.1093/bioinformatics/btu170
[22]	EDGAR R C. UPARSE: highly accurate OTU sequences from microbial amplicon reads [J]. Nature Methods, 2013, 10(10): 996−998. doi: 10.1038/nmeth.2604
[23]	CHEN D, LIU X, LI C Y, et al. Isolation of Bacillus amyloliquefaciens S20 and its application in control of eggplant bacterial wilt [J]. Journal of Environmental Management, 2014, 137(1): 120−127.
[24]	ZUO C W, LI C Y, LI B, et al. The toxic mechanism and bioactive components of Chinese leek root exudates acting against Fusarium oxysporum f. sp. cubense tropical race 4 [J]. European Journal of Plant Pathology, 2015, 143(3): 447−460. doi: 10.1007/s10658-015-0697-5
[25]	CHOWDHURY S P, HARTMANN A, GAO X W, et al. Biocontrol mechanism by root-associated Bacillus amyloliquefaciens FZB42 - a review [J]. Frontiers in Microbiology, 2015, 6(7): 780−790.
[26]	曹云. SQR 9微生物有机肥防治黄瓜土传枯萎病的效应与机制研究[D]. 南京: 南京农业大学, 2011. CAO Y. Control of Fusarium wilt disease of cucumber by application of bio-organic fertilizer and its working mechanism[D]. Nanjing: Nanjing Agricultural University, 2011. (in Chinese)
[27]	KAI T, TAMAKI M. Effect of organic and chemical fertilizer application on growth, yield, and soil biochemical properties of Landrace Brassica napus L. leaf-and-stem vegetable and Landrace (norabona) [J]. Journal of Agricultural Chemistry and Environment, 2020, 9(4): 314−330. doi: 10.4236/jacen.2020.94023
[28]	袁听, 覃金兰, 何珊, 等. 印度梨形孢对植物生长调节剂在水稻上过量使用的缓解作用 [J]. 福建农业学报, 2020, 35(4):398−405. YUAN T, QIN J L, HE S, et al. Mitigating ill-effect of plant growth regulator overuse on rice plants by Piriformospora indica [J]. Fujian Journal of Agricultural Sciences, 2020, 35(4): 398−405.(in Chinese)
[29]	GU S H, WEI Z, SHAO Z Y, et al. Competition for iron drives phytopathogen control by natural rhizosphere microbiomes [J]. Nature Microbiology, 2020, 5(8): 1002−1010. doi: 10.1038/s41564-020-0719-8
[30]	LI C Y, ZUO C W, DENG G M, et al. Contamination of bananas with beauvericin and fusaric acid produced by Fusarium oxysporum f. sp. cubense [J]. PLos One, 2013, 8(7): 1−11.
[31]	彭双, 王一明, 叶旭红, 等. 土壤环境因素对致病性尖孢镰刀菌生长的影响 [J]. 土壤, 2014, 46(5):845−850. PENG S, WANG Y M, YE X H, et al. Effects of Soil Habitat Factors on Growth of Fusarium oxysporum f. sp. niveum and Fusarium oxysporum f. sp. cucumerinum [J]. Soils, 2014, 46(5): 845−850.(in Chinese)
[32]	马国斌, 林德佩, 王叶筠, 等. 培养条件对西瓜枯萎病菌镰刀菌酸产生的影响 [J]. 植物病理学报, 1996, 26(2):92−96. MA G B, LIN D P, WANG Y J, et al. Effect of cultural conditions on production of fusaric acid of watermelon Fusarium wilt fungus [J]. Acta Phytopathologica Sinica, 1996, 26(2): 92−96.(in Chinese)
[33]	薛超. 香蕉根际土壤微生物区系特征与土传枯萎病防控研究[D]. 南京: 南京农业大学, 2015. XUE C. Manipulation of microbial community in banana rhizospherer to suppress Fusarium wilt of banana[D]. Nanjing: Nanjing Agricultural University, 2015. (in Chinese)
[34]	柳凯, 季倩茹, 陈静, 等. 施用Streptomyces alfalfae XY25^T对根肿病土壤性质及微生物群落的影响 [J]. 微生物学通报, 2020, 47(1):97−108. LIU K, JI Q R, CHEN J, et al. Effect of Streptomyces alfalfae XY25^T on soil properties and microflora in clubroot-diseased soil [J]. Microbiology China, 2020, 47(1): 97−108.(in Chinese)
[35]	CHEN Z T, AO J Q, YANG W C, et al. Purification and characterization of a novel antifungal protein secreted by Penicillium chrysogenum from an Arctic sediment [J]. Applied Microbiology and Biotechnology, 2013, 97(24): 10381−10390. doi: 10.1007/s00253-013-4800-6
[36]	NGUVO K J, GAO X Q. Weapons hidden underneath: Bio-control agents and their potentials to activate plant induced systemic resistance in controlling crop Fusarium diseases [J]. Journal of Plant Diseases and Protection, 2019, 126(3): 177−190. doi: 10.1007/s41348-019-00222-y