Effects of Bio-organic Fertilizer on Physicochemical Properties and Microflora of Banana Field Infected by Fusarium Wilt Disease
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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。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.
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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.
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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.
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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.
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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.
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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.
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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
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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
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
BIOpH 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).
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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 communityACE 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).
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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 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
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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.
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