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.
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喜树Camptotheca acuminata Decne为喜树属蓝果树科植物，树干高大通直、树内含丰富的喜树碱，因而是国内外优良的木材、化工以及城市绿化观赏树种，同时也是我国第一批国家重点保护野生植物。生产上喜树以播种、扦插繁殖为主[1]。但播种育苗周期长、繁殖系数低、易变异。而扦插繁殖繁殖速度快、纯度高、根系发达、移植成活率高、生长快。但喜树嫩枝扦操作复杂，技术含量较高，若技术环节掌握不当，就会造成喜树育苗成活率低。
近年来，关于树种嫩枝扦插的研究文献较多[2]，其中吲哚丁酸(IBA)与生根粉(ABT)在植物扦插生根中的应用较为广泛[3-6]。许多研究表明插穗内源激素的动态平衡，共同调控不定根的发生[7-8]。用IAA/ABA比值可用来表示高山杜鹃的生根能力[9]。金雀花硬枝在扦插生根过程中，内源IAA含量、IAA/ZT、IAA/ABA、ZT/ABA均高于CK，ABA含量低于CK[10]。插穗ABA和IBA含量与嫩枝插穗愈伤组织诱导率无显著相关性[11]。激素处理过的兴安圆柏插穗，高IAA/ABA和低浓度的GA含量，利于愈伤和根源基的形成[12]。
本课题组前期研究表明，促进喜树扦插生根效果较好的生长调节剂主要有IBA和ABT，两者生根效应各有优长，IBA诱导的不定根细而长，纤维质；ABT诱导的不定根少而粗呈刷状。若将两者混用比单独使用效果好。但关于IBA和ABT对喜树扦插内源激素的影响方面的研究国内外尚属空白。因此本试验采用IBA与ABT不同浓度组合处理喜树插穗，来研究其对喜树扦插成活的影响，分析扦插过程中内源激素变化的规律，阐明扦插生根的调控机理，以期为喜树大面积繁育苗木提供理论依据和生产指导。
1. 材料与方法
1.1 试验材料
试验于2015年3~6月河南科技大学进行，选均匀一致、生长健壮、无病虫害的2年生喜树嫩枝，剪成上平下斜15 cm左右为插穗。
1.2 试验设计及方法
插穗基部用0.1%的多菌灵溶液消毒0.5 h，然后在不同浓度组合IBA、ABT处理液中浸蘸20 s。选对喜树扦插生根效果较好的IBA、ABT质量浓度[1]组合为：(1) IBA 600 mg·L-1+ABT 600 mg·L-1；(2) IBA 800 mg·L-1+ABT 800 mg·L-1；(3) IBA 1 000 mg·L-1+ABT 1 000 mg·L-1。以清水为对照，每个处理50株，设3个重复。扦插基质为草炭:珍珠岩=1:1。扦插52 d统计插穗成活率、新生叶片数量。
1.3 扦插管理
扦插时保持插穗间距均匀，扦插深度5~8 cm，蜡封切口。扦插后每10 d取1次样，进行各项激素指标测定。取样时，随机取幼叶及插条基部2 cm，蒸馏水冲洗干净后所取鲜样迅速用液氮固定, 保存于超低温冰箱中。
1.4 激素测定
称取1.0 g样品, 加入4 mL内含100 mg PVP(聚乙烯吡咯烷酮)的80%甲醇提取液, 弱光冰浴研磨，匀浆, 转入10 mL离心管，4℃下提取4 h，1 000 r·min-1离心15 min, 取上清，Sep-PakC18柱纯化2次，滤液定容至1.5 mL。采用酶联免疫法(ELISA)测定样品中脱落酸(ABA)、赤霉素(GA3)、生长素(IAA)和玉米素(ZR)含量。试剂盒购于中国农业大学。
1.5 数据处理
试验数据采用Excel及DPS 7.05进行统计与处理。
2. 结果与分析
2.1 不同处理对喜树插穗生长状况的影响
从表 1可以看出，52 d后，对照插穗平均每株为3片叶片，而经过IBA和ABT处理后，可显著促进喜树插穗的新生叶片数量，数量维持在4~6片，为对照的1.3~2.0倍。从表 1还可以看出，
表 1 不同处理对喜树插穗新叶数量及生根率的影响Table 1. Effect of treatments on number of new leaves and rooting rateIBA+ABT
/(mg·L-1)新生叶片数量
/个生根率
/%600+600 6±0.22 b 43.33±1.2 b 600+800 5±0.16 b 51.67±1.8 bc 600+1000 6±0.18 b 48.33±1.5 b 800+600 4±0.11 b 50.00±2.0 bc 800+800 4±0.13 b 55.00±1.7 c 800+1000 6±0.17 b 55.00±2.1 c 1000+600 6±0.31 b 58.33±1.6 c 1000+800 5±0.26 b 65.00±2.1 d 1000+1000 5±0.22 b 75.00±1.8 e 清水对照 3±0.12 a 33.00±0.8 a 注：同列数据后不同小写字母为差异显著(P < 0.05)。下表同。 IBA和ABT组合中，质量浓度高于600 mg·L-1时，能显著提高喜树生根率。为此，本试验选对喜树扦插生根效果较好且差异显著的组合：(1) IBA 600 mg·L-1+ABT 600 mg·L-1；(2) IBA 800 mg·L-1+ABT 800 mg·L-1；(3) IBA 1 000 mg·L-1+ABT 1 000 mg·L-1。
2.2 不同处理对叶、插穗IAA含量变化的影响
IAA主要是控制植物的营养生长，其含量多少直接影响植物扦插不定根的形成。本试验结果(表 2)表明，总体而言经IBA和ABT处理后，可有效增加喜树扦插过程中插穗叶及基部茎段内源激素IAA含量，且IBA和ABT质量浓度越大，内源IAA含量越高。其中，叶及基部茎都在扦插后第40 d增幅最大。IBA 1 000 mg·L-1+ABT 1 000 mg·L-1处理时，叶、茎内源IAA含量分别比对照增加57.5%、77.4%。各处理间的不同之处在于，处理B、处理C叶IAA含量处理前期升高，在第20~30 d时降低，之后有显著增加，这可能与这一时期2处理发生新生叶、根较多，需要消耗更多的IAA有关。
表 2 不同处理对叶、插穗内IAA含量变化的影响Table 2. Changes on IAA in camptotheca cuttingsby treatments of IBA and ABT[单位/(ng·g-1FW)] 处理 扦插后天数/d 10 20 30 40 50 幼叶 CK 20.2±1.2 a 28.1±1.7 a 35.5±1.3 a 40.1±0.5 a 60.2±1.8 a 处理1 21.3±1.1 a 36.5±2.1 b 48.3±1.1 b 55.1±1.4 b 66.3±1.9 ab 处理2 21.5±0.7 a 39.3±1.8 b 36.1±1.7 a 62.2±1.6 c 69.5±2.1 b 处理3 22.4±0.9 a 45.2±1.2 c 35.2±1.6 a 63.1±2.1 c 86.4±2.4 c 插穗 CK 0 9.3±0.5 a 23.3±1.4 a 31.0±0.6 a 49.5±1.5 a 处理1 0 22.1±1.1 b 32.2±1.1 b 39.6±0.5 b 51.2±1.7 a 处理2 0 24.3±0.9 b 30.5±0.8 b 46.5±1.3 c 60.1±1.9 b 处理3 0 26.4±0.6 b 32.6±0.6 b 55.0±2.3 d 75.3±1.8 c 2.3 不同处理对叶、插穗GA3含量变化的影响
表 3表明，经IBA和ABT处理后，喜树扦插过程中插穗叶及基部茎段内源激素GA3含量随扦插时间延长而增加趋势。但各处理中，IBA和ABT质量浓度越大GA3含量越低，且插穗茎段内GA3含量低于相应叶内GA3含量。其中，处理C叶GA3含量第30 d显著低于对照24.8%；茎段GA3含量第40 d显著低于对照35.0%。
表 3 不同处理对叶、插穗内GA3含量变化的影响Table 3. Changes on GA3in camptotheca cuttings by treatments of IBA and ABT[单位/(ng·g-1FW)] 处理 扦插后天数/d 10 20 30 40 50 幼叶 CK 351.1±15.8 c 507.5±25.1 c 638.5±10.4 c 901.2±41.4 c 1050.6±66.0 b 处理1 303.5±20.1 b 431.4±19.6 b 550.3±15.0 b 752.3±33.3 b 982.4±52.3 a 处理2 260.4±16.7 b 407.2±31.2 ab 501.2±12.6 ab 723.4±23.6 ab 1002.6±55.5 b 处理3 135.6±10.6 a 383.5±910.7 a 480.2±31.6 a 700.6±31.1 a 981.4±22.7 a 插穗 CK 151.2±9.4 b 182.1±10.1 c 221.5±10.3 b 250.7±12.6 b 331.5±22.1 c 处理1 120.5±10.1 a 153.5±8.6 b 166.1±12.1 a 182.5±10.7 a 232.2±11.5 b 处理2 115.3±10.0 a 133.7±7.4 a 151.4±11.6 a 177.7±6.7 a 215.0±12.3 b 处理3 123.1±8.7 a 138.0±6.6 a 143.6±10.2 a 162.9±5.5 a 186.4±9.4 a 2.4 不同处理对叶、插穗ZR含量变化的影响
表 4表明，经IBA和ABT处理后，喜树扦插过程中插穗叶及基部茎段内源激素ZR含量随扦插时间的延长表现为不同的变化趋势。叶内源激素ZR含量随扦插时间的延长逐渐增加，第20 d时各处理ZR含量均高于对照，但第30 d时处理B、C显著低于对照，分别比对照降低了11.3%、15%。可能与处理B、C新生叶、根数量较多，需要消耗较多的ZR有关。基部茎段内源激素ZR含量随扦插时间的延长呈前30 d降低后期增加的趋势。第30 d时，处理C最低，比对照降低了68%，说明处理C用于生根消耗的激素ZR较其他处理高。
表 4 不同处理对叶、插穗内ZR含量变化的影响Table 4. Changes on ZR in camptotheca cuttings by treatments of IBA and ABT[单位/(ng·g-1FW)] 处理 扦插后天数/d 10 20 30 40 50 幼叶 CK 31.2±10.3 a 250.2±19.4 a 600.1±20.5 b 752.3±30.5 b 815.7±22.5 a 处理1 33.3±20.4 a 362.3±20.0 b 712.5±22.6 c 780.7±32.7 b 802.3±16.7 a 处理2 35.2±9.8 a 402.1±21.5 c 532.1±12.0 a 653.1±21.1 a 810.8±14.3 a 处理3 43.0±12.2 b 472.3±22.9 d 510.2±11.6 a 634.6±20.5 a 798.6±25.5 a 插穗 CK 15.5±1.1 a 12.0±0.4 c 10.0±0.7 c 28.2±1.3 c 33.4±1.6 b 处理1 12.1±0.9 a 11.3±0.2 ab 8.6±0.1 b 15.1±1.1 a 28.2±2.2 ab 处理2 13.5±1.3 a 9.6±0.1 b 7.5±0.1 b 16.4±0.5 a 24.0±1.4 a 处理3 14.6±0.7 a 5.1±0.0 a 3.2±0 a 18.3±0.1 b 26.7±0.9 a 2.5 不同处理对叶、插穗ABA含量变化的影响
植物ABA具有抑制茎叶生长的作用。表 5表明，经IBA和ABT处理后，喜树扦插过程中插穗叶、茎段内ABA含量显著升高，且低于对照值。各处理间ABA含量随IBA和ABT质量浓度增加而降低，叶、茎段均在扦插第30 d时，显著低于对照值，其中，处理C叶、茎段ABA含量分别比对照降低19.2%、32.2%。可见，经IBA和ABT处理可显著抑制喜树扦插ABA生成，降低ABA对插穗叶、根的抑制作用。
表 5 不同处理对叶、插穗内ABA含量变化的影响Table 5. Changes on ABA in camptotheca cuttingsby treatments of IBA and ABT[单位/(ng·g-1FW)] 处理 扦插后天数/d 10 20 30 40 50 幼叶 CK 202.1±10.5 b 253.3±12.8 c 521.7±33.5 b 501.4±22.1 b 488.8±17.6 c 处理1 183.2±9.5 b 231.1±11.6 b 510.4±34.8 b 493.2±24.6 b 478.5±13.5 bc 处理2 165.0±6.6 a 210.5±22.2 a 499.6±44.1 b 471.5±18.8 ab 452.1±26.5 b 处理3 153.5±10.4 a 193.1±14.3 a 421.6±15.7 a 451.6±16.7 a 411.6±22.2 a 插穗 CK 133.6±10.0 b 199.5±15.0 c 230.1±16.7 c 302.1±15.7 c 330.5±23.1 c 处理1 132.7±9.1 b 175.9±13.7 b 188.2±10.3 b 268.0±9.4 b 303.1±10.5 b 处理2 121.4±13.3 ab 163.7±12.8 b 173.4±11.4 ab 243.2±6.5 a 287.3±10.0 ab 处理3 110.1±10.2 a 135.2±9.4 a 156.0±9.4 a 220.1±7.8 a 268.1±7.7 a 2.6 不同处理对叶、插穗IAA/ABA、ZR/ABA、GA3/ABA变化的影响
植物内IAA、GA3、ZR具有延缓衰老促进生长发育的生理效应，但ABA促进植物衰老、抑制生长。高比值的IAA/ABA、ZR/ABA、GA3/ABA利于延缓植物衰老促进生长。本试验结果(表 6)表明，IBA和ABT处理后，可显著提高喜树扦插过程中插穗叶、茎段内IAA/ABA，且IBA和ABT浓度越大，IAA/ABA比值越高。插穗叶、茎段内IAA/ABA最高值分别出现在第20、30 d，处理C最大比值分别比对照提高114.0%、150.0%。插穗叶、茎段内ZR/ABA变化与IAA/ABA变化类似，最高值分别出现在第20、30 d(表 7)，处理C最大比值分别比对照提高151.4%、39.9%。这种情况也正好与本试验中，处理C插穗第20 d为新生叶集中期、第30 d为新生根系集中期的现象一致。
表 6 不同处理对叶、插穗内IAA/ABA含量变化的影响Table 6. Changes on IAA/ABA in camptotheca cuttingsby treatments of IBA and ABT[单位/(ng·g-1FW)] 处理 扦插后天数/d 10 20 30 40 50 幼叶 CK 0.10±0.003 a 0.11±0.002 a 0.07±0.006 a 0.08±0.005 a 0.12±0.002 a 处理1 0.12±0.02 ab 0.16±0.002 b 0.09±0.002 b 0.10±0.003 b 0.14±0.003 b 处理2 0.13±0.002 b 0.19±0.001 c 0.09±0.001 b 0.13±0.003 c 0.15±0.005 b 处理3 0.14±0.008 b 0.24±0.001 d 0.11±0.002 c 0.14±0.003 c 0.18±0.006 c 插穗 CK 0.00 0.05±0.002 a 0.10±0.001 a 0.10±0.001 a 0.15±0.002 a 处理1 0.00 0.13±0.001 b 0.17±0.002 b 0.15±0.002 b 0.17±0.003 b 处理2 0.00 0.15±0.002 b 0.17±0.002 b 0.19±0.002 c 0.21±0.012 c 处理3 0.00 0.19±0.001 c 0.25±0.002 c 0.23±0.002 d 0.21±0.015 c 表 7 不同处理对叶、插穗内ZR/ABA含量变化的影响Table 7. Changes on ZR/ABA in camptotheca cuttingsby treatments of IBA and ABT[单位/(ng·g-1FW)] 处理 扦插后天数/d 10 20 30 40 50 幼叶 CK 0.15±0.003 a 0.99±0.008 a 1.15±0.008 a 1.30±0.006 a 1.67±0.012 a 处理1 0.18±0.005 a 1.57±0.009 b 1.29±0.017 b 1.47±0.005 c 1.68±0.013 a 处理2 0.21±0.006 b 1.91±0.072 b 1.30±0.006 b 1.39±0.008 ab 1.80±0.021 b 处理3 0.28±0.009 c 2.48±0.093 c 1.50±0.007 c 1.41±0.016 b 1.95±0.018 c 插穗 CK 0.04±0.002 a 0.06±0.001 a 0.09±0.006 a 0.04±0.003 a 0.07±0.002 a 处理1 0.05±0.005 b 0.06±0.002 a 0.09±0.005 a 0.06±0.005 b 0.09±0.003 b 处理2 0.04±0.005 a 0.05±0.001 a 0.11±0.004 b 0.07±0.006 b 0.09±0.002 b 处理3 0.05±0.006 b 0.06±0.002 a 0.12±0.002 c 0.08±0.007 c 0.10±0.005 b 本试验中IBA和ABT处理后，喜树插穗叶、茎段内GA3/ABA比值变化并不如ZR/ABA、IAA/ABA那样显著。各处理插穗叶、茎段内GA3/ABA比值对各自对照无显著差异(表 8)。
表 8 不同处理对叶、插穗内GA3/ABA含量变化的影响Table 8. Changes on GA3/ABA in camptotheca cuttingsby treatments of IBA and ABT[单位/(ng·g-1FW)] 处理 扦插后天数/d 10 20 30 40 50 幼叶 CK 1.75±0.12 c 1.76±0.09 a 0.60±0.09 a 1.30±0.06 a 1.90±0.07 a 处理1 1.67±0.13 b 1.86±0.08 a 1.00±0.08 b 1.41±0.02 ab 2.05±0.20 a 处理2 1.58±0.09 b 1.90±0.13 b 1.00±0.03 b 1.54±0.03 b 2.22±0.02 b 处理3 0.85±0.06 a 2.00±0.14 b 1.00±0.02 b 1.56±0.03 b 2.39±0.03 b 插穗 CK 0.90±0.07 a 0.81±0.07 a 0.82±0.06 a 0.61±0.03 a 0.66±0.01 a 处理1 0.91±0.05 a 0.85±0.06 a 0.88±0.05 a 0.68±0.04 ab 0.76±0.02 b 处理2 0.95±0.04 a 0.79±0.05 a 0.87±0.03 a 0.73±0.02 ab 0.73±0.03 b 处理3 1.09±0.08 b 1.02±0.07 b 0.92±0.01 b 0.76±0.03 b 0.69±0.04 a 可见，高量的IBA和ABT组合主要是通过增大IAA/ABA、ZR/ABA比值，来促进喜树插穗多生根叶、提高成活率的。因而IAA/ABA、ZR/ABA比值可作为衡量喜树插穗成活质量的2个检验指标，而非GA3/ABA。
3. 讨论与结论
本试验研究结果表明，经IBA和ABT处理后，可有效增加喜树扦插过程中插穗叶及基部茎段内源激素IAA含量。IAA和插穗生根潜力密切相关，插穗内较高浓度的IAA有利于根原基分化形成和皮部生根，是促进不定根形成的主要激素[13]，且IAA与生根率关系最密切[14]。结果与华北五角枫扦插生根过程中内源IAA增加类似。
GA对细胞分裂和伸长有促进作用，且GA含量的增加与插条愈伤组织的形成和不定根的发生呈正相关性[15]，但扦插时并非插穗内GA含量越高越好，相反高量内源GA3(高于25 ng·mg-1·g-1FW)会抑制插穗不定根的形成[14]，本试验结果表明，喜树扦插过程中插穗叶及基部茎段内源激素GA3在扦插期间含量增加，但经IBA和ABT处理后，各处理插穗叶及基部茎段内源激素GA3含量均低于对照值，说明处理后更有利于喜树插条愈伤组织形成和不定根的发生。
经IBA和ABT处理后，叶内源激素ZR含量随扦插时间的延长逐渐增加，第20 d时各处理ZR含量均高于对照，但第30 d时处理B、C显著低于对照，可能与处理B、C新生叶、根数量较多，需要消耗较多的ZR有关。喜树基部茎段内源激素ZR含量随扦插时间的延长呈前30 d降低，后期增加的趋势，这中前期低浓度的内源ZR适宜于促进根原基分化形成，而后期较高量的内源ZR有利于根原基生长发育[14]。
经IBA和ABT处理后，喜树扦插过程中插穗叶、茎段内ABA含量显著升高，且低于对照值。这与波叶金桂插穗ABA变化趋势结论一致[16]，表明喜树扦插成活率与插穗内ABA质量浓度呈负相关，且高量的ABA是影响生根效果的重要因素[17]，但经IBA和ABT处理后低量的内源ABA更有助于喜树扦插生根。
研究表明，高的IAA/ABA值有利于扦插过程中根原基的分化与形成，低的IAA/ABA值促进扦插过程中不定根的伸长；高水平的IAA/ZR值有助于根原基分化形成，较低的IAA/ZR值有利于不定根的伸长；低水平的GA3/ABA值有利于根原基的分化形成，较高的GA3/ABA值促进不定根的伸长[18-19]。本试验表明，IBA和ABT处理后，可显著提高喜树扦插过程中插穗叶、茎段内IAA/ABA和ZR/ABA(表 7)，且IBA和ABT浓度越大，比值越高。但喜树插穗叶、茎段内GA3/ABA比值变化不如ZR/ABA、IAA/ABA变化显著。可见，高量的IBA和ABT组合主要是通过增大IAA/ABA、ZR/ABA比值，来促进喜树插穗多生根叶、提高成活率的。因而可将IAA/ABA、ZR/ABA比值作为衡量喜树插穗成活质量的2个重要检验指标。
此外，植物成活难易程度除了和插穗内源激素含量有关，还和扦插内可溶性糖含量、淀粉含量、POD、IAAO、木质素含量等活性密切相关[20-22]，影响喜树插穗成活的内在生理因素，尚待进一步研究。
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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.
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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.
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图 3 功能基因之间及其与速效养分的相关性（仅列出有显著相关）
Figure 3. Correlation between functional genes and available nutrients (only significant correlations are listed)
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图 4 真菌纲和部分主要目和科和功能基因的相关性
Figure 4. Correlation between functional genes and some major orders and families of fungi
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.
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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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