Fungal Community and C, N, P, and S Functional Genes in Rhizosphere Soil of Cassava Field Treated with a Slow-release Fertilizer
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摘要:
目的 研究木薯缓释肥对根际土壤真菌和C、N、P、S功能基因动态的影响。 方法 开展木薯田间试验,设置3个处理:不施肥(T1)、双膜缓释肥C2基施(T2)和植后34 d追施(T3)。在植后77、104、134 d采集根际和非根际土样,测定高通量(Illumina Miseq PE300)真菌(ITS rRNA)多样性,C、N、P、S共72个(含总DNA)功能基因的copies(基因芯片技术)和土壤速效养分(仅用于相关分析)。 结果 (1)植后104 d根际土壤被孢霉纲、银耳纲、圆盘菌纲相对丰度均为T2<T1;植后134 d,根际土壤散囊菌纲为T2>T1。T2散囊菌纲(134 d)、T3丛赤壳科(77 d)和T3粪壳菌纲(104 d)在根际相对富集。T1根际被孢霉纲相对丰度的时间大小顺序为134 d<104 d;T2根际散囊菌纲和粪壳菌纲均为134 d>77 d;T3根际的球囊菌纲为104 d>77 d。(2)Sobs、ACE、Chao1指数在T1(104 d)、T2(104 d)和T3(134 d)根际分别显著或极显著大于非根际。根际土壤Shannon指数在植后77 d为T1<T2和T3,T1和T2的时间大小顺序分别为104 d>77 d和104 d<77 d。土壤Simpson指数的大小顺序为T1根际(77 d)大于T1非根际(77 d)、T1根际(104 d)、T2根际(77 d)和T3根际(77 d)。(3)LEfSe分析结果表明,处理间根际相对富集1个纲、1个目和2个科。对比非根际,植后77 d根际相对富集2个种,植后104 d相对富集3个目、1个科、1个属,植后134 d相对富集各1个门、目、科和属。时间比较中,104 d和134 d根际分别相对富集2个目和1个纲。(4)134 d,lig等9个功能基因在T1非根际土壤中的丰度显著高于根际土壤。在T1根际土壤中,chiA和aclB的丰度均为77 d高于104 d和134 d。(5)AK在104 d与31个功能基因显著相关。银耳纲、肉座菌目、丛赤壳科和球囊菌纲分别和其他40个、15个、14个、9个功能基因显著相关。 结论 缓释肥基施和追施可提高木薯根际真菌群落的多样性和丰度,施肥、时间、根际等均对真菌群落结构和少数功能基因有显著的影响,相关性分析结果暗示木薯根际真菌可能参与土壤速效养分的循环和功能基因的作用,为进一步了解木薯根际微生态过程提供科学依据。 Abstract:Objective A slow-release fertilizer was applied on a cassava lot to analyze the responses of the fungal community and C, N, P, and S functional genes in the rhizosphere soil. Method A field experiment was conducted with treatments of no fertilization (T1), basal application of double-coating slow-release fertilizer C2 (T2), and C2 applied 34 d after planting (DAP) (T3). Rhizosphere and bulk soil samples were collected at 77, 104, and 134 DAP to determine fungal diversity according to ITS rRNA sequenced by a high-throughput Illumina Miseq PE300, copies of 72 functional genes of C, N, P, and S cycles (including total DNA) by the gene chip technology, and available nutrients by chemical analysis for a correlation analysis. Result (1) Significant differences on the relative abundance (RA) of Mortierellomycetes, Tremellomycetes, and Orbiliomycetes were found in the rhizosphere soils on 104 DAP showing T2<T1, while that of Scatterocysts on 134 DAP indicating T2>T1. Fungal class Scatterycetes under T2 on 134 DAP, Rubiaceae under T3 on 77 DAP, and Coprochetes under T3 on 104 DAP were relatively enriched in the rhizosphere than in the bulk soil. The RAs of the rhizosphere fungi also differed significantly on time of sampling and under different treatments. They were 134 DAP<104 DAP for Mortieromycetes under T1, 134 DAP>77 DAP for Scatterocystae and Coprochestae under T2, and 104 DAP>77 DAP for Sphaeromycetes under T3. (2) The Sobs, ACE, and Chao1 indexes under T1 on 104 DAP, T2 on 104 DAP, and T3 on 134 DAP were significantly higher in the rhizosphere than in the bulk soil (P<0.05 or P<0.01). The Shannon index of rhizosphere soil was lower under T1 than under T2 or T3 on 77 DAP. Under T1, the index was 104 DAP>77 DAP; and under T2, it was the opposite. The Simpson indexes ranked in the order of the rhizosphere soil under T1 on 77 DAP>the bulk soil under T1 on 77 DAP>the rhizosphere soil under T1 on 104 DAP>the rhizosphere soil under T2 on 77 DAP>the rhizosphere soil under T3 on 77 DAP. (3) The LEfSe analysis indicated the fertilizer applications enriched one class, one order, and two families of fungi in the rhizosphere soil, whereas the bulk soil was more abundant in two species on 77 DAP, in 3 orders, one family, and one genus on 104 DAP, and in one phylum, one order, one family, and one genus on 134 DAP. Two orders on 104 DAP and one class on 134 DAP were enriched in the rhizosphere soil. (4) On 134 DAP, the 9 functional genes, such as lig in the bulk soil under T1, had significantly more copies than in the rhizosphere soil. In the rhizosphere soil, the RAs of chiA and aclB under T1 on 77 DAP were higher than those on 104 or 134 DAP. (5) AK significantly correlated with 31 functional genes on 104 DAP. Some fungal classes, such as Tremella, Sarcoidales, Claviculaceae, and Sphaeromycetes, significantly correlated with 40, 15, 14, and 9 other functional genes, respectively. Conclusion Fertilization by ways of T2 or T3 enriched the diversity and abundance of cassava rhizosphere fungal community. Fertilizer used, application time, and rhizosphere could all significantly affect the fungal community structure and some functional genes in the soil. The correlations might lead to further studies to unveil the intricate ecosystem. -
图 1 真菌纲和部分主要目和科的heatmap图
(1)第一个/之前的字母表示处理间的差异,两个/之间的字母表示根际与非根际之间的差异,第二个/后的字母表示采样时间之间的差异;(2)处理间/根际与非根际/时间比较,其中一个因素的比较均排除另外两个因素的差异进行。如处理间比较仅限同一采样时间和同一土壤类型(根际或非根际)的不同处理比较。不同的小写字母表示差异显著(P<0.05),不同的大写字母表示极显著(P<0.01)。(2)处理编号中,R表示根际土壤,N表示非根际土壤。
Figure 1. Heatmap of fungal classes and dominant orders and families
(1) Letters before first “/” indicate significant difference between treatments; those between two “/” indicate difference between rhizosphere and bulk soil; and those after second “/” indicate difference between sampling times. (2) Mutually exclusive comparisons between intertreatment/rhizosphere and bulk soil/time, e.g., a comparison between treatments is limited to treatments on either rhizosphere or bulk soil at same sampling time. Data with different lowercase letters indicate significant differences at P<0.05; those with different uppercase letters, extremely significant differences at P<0.01. (2) In the treatmet name, R: rhizosphere soil; N: bulk soil.
图 2 功能基因丰度(对数转化)
第一个/之前的数字是表示非零值的样品个数,第一个/之前的字母表示处理间是否有显著差异,两个/之间的字母表示根际与非根际之间的差异,第二个/后的字母表示采样时间之间的差异。不同小写字母表示差异显著(P<0.05)。
Figure 2. Logarithmic transformed abundance of functional genes
Number before first "/" indicates count of non-zero values, while letter, significant difference between treatments; letter between two "/" indicates difference between rhizosphere and bulk soil; and letter after second "/"indicates difference between sampling times. Data with different lowercase letters indicate significant differences at P<0.05.
表 1 真菌属水平α-多样性
Table 1. α-diversity of fungi at family level
土壤类型和时间
Soil types and time处理
TreatmentSobs Shannon Simpson ACE Chao1 T1 211.8±65.4a/a/a 3.241±0.083b/a/b 0.083±0.007a/a/A 222.6±72.9a/a/a 223.5±73.3a/a/a R77d T2 227.5±28.8a/a/a 3.562±0.205a/a/a 0.059±0.016b/a/a 232.4±31.4a/a/a 234.6±33.7a/a/a T3 239.3±29.8a/a/a 3.584±0.115a/a/a 0.057±0.009b/a/a 245.1±32.5a/a/a 249.0±34.3a/a/a T1 197.5±28.5a/a/a 3.528±0.188a/a/a 0.057±0.012a/b/a 203.5±33.9a/a/a 207.7±39.8a/a/a N77d T2 188.5±26.8a/a/a 3.559±0.131a/a/a 0.055±0.009a/a/a 191.8±27.9a/a/a 193.4±29.7a/a/a T3 206.3±18.1a/a/a 3.491±0.079a/a/a 0.066±0.016a/a/a 209.8±20.4a/a/a 211.3±19.5a/a/a T1 249.8±29.1a/a/a 3.604±0.213a/a/a 0.051±0.008a/a/B 257.9±32.3a/a/a 259.4±32.7a/a/a R104d T2 232.5±21.4a/A/a 3.412±0.217a/a/b 0.068±0.018a/a/a 241.1±23.3a/A/a 242.8±23.1a/A/a T3 254.5±39.7a/a/a 3.439±0.264a/a/a 0.084±0.058a/a/a 265.0±43.4a/a/a 265.7±44.1a/a/a T1 204.5±27.1a/b/a 3.516±0.102a/a/a 0.060±0.005a/a/a 207.5±28.3a/b/a 208.1±28.4a/b/a N104d T2 189.5±33.7a/B/a 3.141±0.619a/a/a 0.121±0.117a/a/a 195.3±35.9a/B/a 195.1±36.5a/B/a T3 192.8±28.2a/a/a 3.343±0.265a/a/a 0.073±0.020a/a/a 195.6±29.1a/a/a 196.6±29.9a/a/a T1 223.8±25.6a/a/a 3.258±0.464a/a/ab 0.110±0.067a/a/AB 236.6±32.7a/a/a 237.1±32.6a/a/a R134d T2 219.0±34.9a/a/a 3.392±0.353a/a/ab 0.069±0.027a/a/a 231.2±41.4a/a/a 237.5±46.8a/a/a T3 249.5±19.8a/a/a 3.491±0.167a/a/a 0.062±0.016a/a/a 264.7±25.8a/a/a 272.0±28.1a/a/a T1 204.8±28.1a/a/a 2.811±0.883a/a/a 0.193±0.227a/a/a 218.6±24.4a/a/a 220.0±21.4a/a/a N134d T2 180.8±32.3a/a/a 3.391±0.178a/a/a 0.065±0.014a/a/a 184.0±33.8a/a/a 184.9±34.1a/a/a T3 202.8±26.4a/b/a 3.441±0.303a/a/a 0.065±0.031a/a/a 206.8±28.4a/b/a 208.5±29.2a/b/a ①R表示根际土壤,N表示非根际土壤。R77d和N77d分别表示植后77 d的根际土壤和植后77 d的非根际土壤。②“//”及其前后的字母含义与图1相同。
①R: rhizosphere soil; N: bulk soil. R77d and N77d: rhizosphere soil and bulk soil on 77 DAP, respectively. ② Letters mean as Fig. 1.表 2 土壤真菌LEfSe分析信息
Table 2. LEfSe analysis information of soil fungi
处理对及优势处理
The treatments for comparison
and the advantageous one生物标记物
Biomarker处理对及优势处理
The treatments for comparison
and the advantageous one生物标记物
Biomarker77 d
T1R-T2R-T3Rf_Sordariales_fam_Incertae_sedis, g_Ramophialophora, s_Ramophialophora_sp. 104 d
T2-T2Rg_Cephalotrichum, s_unclassified_g_Cephalotrichum 104 d
T1R-T2R-T3Rf_Bionectriaceae, g_Clonostachys, s_Clonostachys_sp.; o_Chaetosphaeriales, f_Chaetosphaeriaceae, g_Codinaea, s_unclassfied_g_Codinaea 134 d
T2-T2RAscomycota, c_Eurotiomycetes; o_Microascales, f_Microascaceae, g_Cephalotrichum, s_unclassified_g_cephalotrichum 134 d
T1R-T2R-T3Rc_Eurotiomycetes, o_Microascales, f_Microascaceae 134 d
T3-T3Rg_Fusarium, s_unclassified_g_Fusarium; f_Bionectraceae g_Clonostachys, s_Clonostachys_sp. 77 d
T1-T1Rs_Clonostachys_sp. 104 d
T3-T3Rf_Stachybotryaceae 77 d
T2R-T2s_Chaetomium_sp. T1R
77d-104d-134do_Branch06, f_unclassified_o_Branch06, g_unclassified_o_branch06, s_Branch06_sp.; o_Capnodiales, f_Mycosphaerellaceae 104 d
T1-T1Ro_Branch06, f_unclassified_o_Branch06, g_unclassified_o_branch06, s_Branch06_sp.; o_Capnodiales; o_Chaetosphaeriales, f_Chaetosphaeriaceae, g_Codinaea, s_unclassified_g_Codinaea T2R
77d-104d-134dc_Eurotiomycetes ①T1R-T2R-T3R单元格内的上下两行分别表示共同和异同的处理,如77d T1R-T2R-T3R表示77d的T1、T2、T3根际比较,且粗体加下划线格式表示右侧列出的真菌门类为该处理的Biomarker。R表示根际土。②真菌门类名称前的c_表示纲(class),o_表示目(order),f_表示科(family),g_表示属(genus),s_表示种(species),不标记表示门水平;“,”表示后者包含于前者的门类,“;”表示后者与前者不属于同一门类。
(1) Upper and lower rows in cells of same type represent common and different treatments, respectively. For example, 77d T1R-T2R-T3R is comparison of rhizosphere soils under T1, T2, and T3 on 77 DAP. Name with underlined bold fonts indicates fungal phyla listed on the right to be biomarker of treatment, and R, rhizosphere soil. (2) Before a fungus name, c_ represents at class level, o_, order, f_, family, g_, genus, s_, species, and unmarked, phylum. "," indicates two in same phylum; and ";" two are in different phyla. -
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