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作者简介:

何秀峰(1991-),女,甘肃会宁人,在读硕士研究生,主要从事果树学研究。E-mail:2675749029@qq.com。

通讯作者:

郁松林,E-mail:songlin8900@sina.com。

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目录contents

    摘要

    以一年生“弗雷”葡萄幼苗为试验材料,采用盆栽试验,根据生物炭施用方式与炭土质量比,设置 5 个处理,研究了生物炭不同施用方式及施用量对葡萄幼苗根际土壤养分、酶活性和微生物多样性的影响。结果表明: 与不施生物炭(CK)相比,生物炭混施(HA、HB)和穴施(JA、JB)增加了土壤有机质、有效磷、速效钾含量及蔗糖酶、过氧化氢酶活性,但小幅度降低土壤容重和 pH。同一施用方式下,生物炭施用量越高土壤碱解氮、速效钾含量及蔗糖酶、过氧化氢酶活性越高;同一施用量下,混施处理土壤有机质和速效养分(碱解氮、有效磷、 速效钾)含量及蔗糖酶、过氧化氢酶活性优于穴施处理,其中 HB(混施 5% 生物炭)处理土壤有机质和速效养分含量及蔗糖酶、过氧化氢酶活性增加幅度最大,分别比 CK 高 73.7%、19.2%、42.3%、20.8%、10.5%、8.6%。土壤细菌 Alpha 多样性分析表明 HB 处理可以提高细菌丰度,但对细菌群落多样性影响甚微。穴施处理下硝化螺旋菌属菌群数量高于混施处理,而混施处理下节细菌属和假单胞菌属菌群数量高于穴施处理。UPGMA 聚类分析及 RDA 冗余分析表明混施处理引起根际土壤微生物群落组成和结构发生较大变化,土壤碱解氮、有机质、速效钾及 pH 对细菌群落结构影响较大。综上,生物炭混施对葡萄幼苗根际土壤养分、酶活性及微生物多样性的影响优于穴施,其中 HB 处理效果较优。

    Abstract

    The objectives of this experiment were to study the effects of different application patterns and amounts of biochar on nutrient,enzyme activities and microbial diversity of rhizosphere soil of grape seedlings.The experiment was done by taking 1-year old“Frey”grape seedlings as test material in pot cultivation,and five treatments were set up according to the biochar application pattern and different soil biochar contents.The results showed as follows:Compared with the control (CK),biochar mixed(HA,HB)and hole application(JA,JB)patterns increased the contents of rhizosphere soil organic matter,available phosphorus,available potassium and invertase,catalase activities,but soil bulk density and pH were decreased slightly.Under the same application pattern,the contents of rhizosphere soil alkali hydrolysis nitrogen, available potassium and invertase,catalase activities increased with the increased of the application amount of biochar;under the same application amount,the contents of rhizosphere soil organic matter,available nutrient(alkali hydrolysis nitrogen, available phosphorus,available potassium)and invertase,catalase activities in mixed application pattern was higher than those of hole application pattern,and among the HB treatment(mixed application of 5% biochar),the extent of increasing of rhizosphere soil organic matter,available nutrients contents,invertase and catalase activities were the largest,which were 73.7%,19.2%,42.3%,20.8%,10.5%,8.6% higher than that of CK,respectively.Alpha diversity analysis of soil bacteria showed that HB treatment improved the abundance of bacterial community.However,it had little effect on bacterial community diversity.The number of Nitrospira in hole application pattern was higher than that of mixed application pattern. However,the number of Arthrobacter and Pseudomonas in mixed application pattern was higher than that of hole application pattern.UPGMA clustering and RDA analysis indicated mixed application pattern had larger effects on microbial community composition and structure of rhizosphere soil.The alkali hydrolysis nitrogen,organic matter,available potassium and pH had great influences on the variations of bacterial community structure.In summary,our results indicated that biochar mixed application pattern was better than hole application pattern to influence the rhizosphere soil nutrient,enzyme activities and microbial diversity,among them,HB treatment was better.

  • 新疆地处中国西北地区,气候极端干旱,土壤盐碱化逐年加重。盐碱土结构退化、有机质含量、土壤酶和微生物活性低,养分释放慢[1],严重阻碍农业的可持续发展[2]。因此,对新疆盐碱地进行恢复和改良迫在眉睫、刻不容缓。生物炭(biochar)是生物质在无氧或缺氧环境条件下缓慢高温分解得到的富含碳的有机物质[3]。不仅可以减少养分淋失[4],改善土壤结构[5],也可以为土壤微生物提供营养元素和栖居场所[6],减少微生物间的生存竞争,保护土壤有益微生物[7],改善土壤微生物群落结构,影响土壤细菌多样性[8-10], Grossman等[11]认为富含生物炭的亚马逊黑土与相邻未改造土相比土壤细菌种群结构差异显著,Muhammad等[12]研究指出,猪粪炭和果皮炭可通过改变红壤土的化学特性来改变微生物群落结构, Sun等[13]研究发现在东北黑钙土中施入生物炭粒后34 个月分解过程中,细菌群落的物种丰富度增加,但多样性降低。张宏等[14]报道连续施用2 年生物炭后,灰漠土土壤容重降低,土壤有机质、碱解氮、有效磷和速效钾含量增加。土壤理化性质的改变可间接影响土壤酶活性[15],土壤酶作为土壤中各种代谢反应和能量转化的参与者,与微生物间保持着紧密联系[16],其活性变化可反映土壤生物功能与肥力特性。

  • 新疆是中国面积最大的葡萄种植地区,截至2019 年种植面积达到13.3 多万hm2。但由于长期不合理的田间管理,致使土壤板结、次生盐渍化等问题日益突出,亟需采取一些措施对土壤进行改良。 生物炭作为一种新型土壤改良剂,在土壤改良方面发挥重要作用[17-19],近年来受到许多研究者的广泛关注。以往研究主要侧重于不同用量生物炭混施改良土壤。但由于果树根区土壤体积庞大,土壤与生物炭混匀施入在生产中投入的成本较大,果树根区穴施施肥是生产中较为常见的施肥方法,但通过穴施生物炭改良土壤进而提高土壤肥力的研究还鲜见报道。故本研究采用室内盆栽试验,研究生物炭不同施用方式及施用量对葡萄幼苗根际土壤养分、 酶活性及微生物多样性的影响,以期为新疆地区葡萄生产合理使用生物炭提高土壤肥力与维持土壤持续生产力提供理论依据和参考。

  • 1 材料与方法

  • 1.1 试验材料

  • 试验于2018 年5 月至9 月在新疆石河子市石河子大学农学院试验站(44°26′N,85°95′E)日光温室中进行,试验过程中气温保持17 ~ 33℃, 空气相对湿度保持60%~ 80%。供试葡萄品种为一年生“弗雷”葡萄幼苗。供试土壤取自石河子大学农学院试验站葡萄园表层(0 ~ 20 cm),为灰漠土,过0.425 mm筛,其碱解氮为77.28 mg/kg,速效钾261.91 mg/kg, 有效磷42.64 mg/kg, 有机质13.61 g/kg。生物炭购自山东某生物公司,是苹果木经高温裂解制成的,其pH 8.56,有效磷186.67 mg/kg,速效钾376.98 mg/kg,全磷15.3 g/kg,全钾6.6 g/kg,生物质活性炭≥ 50%。

  • 1.2 试验处理

  • 采用盆栽试验,花盆上径29 cm、下径23 cm、 高30 cm,定植前每盆装土8 kg。根据生物炭施入方式与炭土质量比设置5 个处理:不施生物炭(对照,CK);混施2.5%的生物炭(HA);混施5%的生物炭(HB);穴施2.5%的生物炭(JA);穴施5%的生物炭(JB)。每处理3 个区组,每区组6 盆。于2018 年5 月28 日选用长势一致的具4 ~ 5 片叶的一年生“弗雷”葡萄扦插苗栽植到花盆中, 每桶1 株,各处理水肥等管理措施均保持一致。

  • 2018 年9 月18 日取土样,每处理从3 个区组中各随机选取5 株,剪掉地上部分破坏性取出植株,去除根系周围大块土壤,轻轻抖下植株根系表面土壤,混合均匀,过2 mm筛后分为两部分,一部分装入无菌自封袋,置于 −80℃冰箱冷藏保存待进行微生物多样性测定,另一部分自然风干后装袋用于测定土壤基本化学性质与酶活性。

  • 1.3 测定方法

  • 土壤化学性质测定参照Shen等[20]的方法。土壤pH按1∶5 土水比测定;有机质测定采用重铬酸钾外加热法;碱解氮测定采用碱解扩散法;有效磷测定采用碳酸氢钠浸提-钼锑抗比色法;速效钾测定采用醋酸铵浸提-火焰光度法;土壤容重测定采用环刀法。

  • 土壤过氧化氢酶、碱性磷酸酶、蔗糖酶、脲酶活性分别采用高锰酸钾滴定法、磷酸苯二钠比色法、3,5-二硝基水杨酸比色法、苯酚钠-次氯酸钠比色法测定。测定参照关松荫[21]的方法。

  • 土壤微生物DNA的提取使用试剂盒Soil DNA Kit(D5625-01)。抽提完成的DNA,使用琼脂糖凝胶电泳和紫外分光光度计检测质量和浓度。随后取适量样品于离心管中,用无菌水稀释。为确保扩增效率和准确性,使用520F(5’-AYTGGGYDTAAAGNG-3’) 和802R(5’-TACNVGGGTATCTAATCC-3’)引物及NEB Q5 DNA高保真聚合酶进行V3、V4 区基因片段的PCR。PCR扩增体系见表1。PCR反应程序为:98℃预变性30 s;27 个循环包括(98℃变性15 s;50℃ 退火30 s;72℃延伸30 s),72℃延伸5 min,4℃保存。之后进行PCR产物回收、定量和混样,混样后进行文库的构建和质检,对合格文库上机测序。

  • 表1 PCR扩增体系

  • 1.4 生物信息分析及数据处理

  • 下机数据中存在一定比例的干扰数据,为使分析结果更加准确、可靠,对原始数据使用FLASH[22] 和QIIME[23]进行拼接和过滤,得到有效数据。然后将有效序列中相似度为97%的序列聚类获得OTUs,随后参考Greengene数据库[24]进行物种注释,开展多样性指数、物种分类及RDA冗余分析, 并对Unweighted的Unifrac距离矩阵进行NMDS分析和UPGMA聚类分析。

  • 应用Excel 2007 进行数据统计与计算,采用SPSS 19.0 进行数据方差分析,Duncan多重比较法进行差异显著性检测,显著性水平设定为 α=0.05。

  • 2 结果与分析

  • 2.1 不同生物炭处理对土壤有机质及养分的影响

  • 由表2 可以看出,生物炭混施(HA、HB)方式下,土壤有机质、速效钾含量随生物炭施用量的增加而增加,其中HA处理土壤有机质、速效钾含量显著高于CK(P < 0.05),分别比CK高53.7%、 19.0%。HB处理土壤有机质、速效钾含量亦显著高于CK,分别比CK高73.7%、20.8%,但HA与HB处理间土壤有机质、速效钾含量差异不显著(P >0.05)。土壤碱解氮、有效磷含量也随生物炭施加量的增加而增加,其中HB处理土壤碱解氮、有效磷含量显著高于CK(P < 0.05),分别比CK高19.2%、42.3%,HA处理土壤碱解氮、有效磷含量与CK间无显著差异。穴施(JA、JB)方式下,有机质及各养分物质含量均低于混施处理,除碱解氮外都高于CK,其中碱解氮、速效钾含量表现出随施炭量的增加而增加,有机质、有效磷含量则表现出相反的趋势。同一施用量下混施处理土壤有机质、速效钾、碱解氮、有效磷含量均高于穴施处理,生物炭对土壤pH和容重影响较小。

  • 表2 不同生物炭处理对土壤有机质及养分的影响

  • 注:表中数据为平均值 ± 标准误;同列数据后不同字母表示处理间在0.05 水平上差异显著,下同。

  • 2.2 不同生物炭处理对土壤酶活性的影响

  • 由表3 可知,不同生物炭处理对土壤酶活性的影响不同,混施(HA、HB)方式下,土壤蔗糖酶和过氧化氢酶活性随生物炭施用量的增加而增加, 其中HA处理土壤蔗糖酶和过氧化氢酶活性显著高于CK(P < 0.05), 分别比CK高8.2%、6.7%,HB处理土壤蔗糖酶和过氧化氢酶活性亦显著高于CK, 分别比CK高10.5%、8.6%;穴施(JA、JB)方式下,土壤蔗糖酶和过氧化氢酶活性也随施炭量的增加而增加,与CK相比,JA处理土壤蔗糖酶和过氧化氢酶活性显著增加6.6%、1.9%(P < 0.05),JB处理土壤蔗糖酶和过氧化氢酶活性显著增加7.0%、 3.8%。同一施用量下,混施处理土壤蔗糖酶和过氧化氢酶活性高于穴施处理。各处理中脲酶活性变化表现为HB处理脲酶活性显著低于CK 18.8%(P < 0.05),HA、JA处理脲酶活性高于CK,但与CK间无显著差异,而JB处理脲酶活性显著高于CK 6.3%(P < 0.05),生物炭对酸性磷酸酶影响较小。

  • 表3 不同生物炭处理对土壤酶活性的影响

  • 2.3 不同生物炭处理对土壤微生物多样性的影响

  • 2.3.1 细菌OTUs丰度

  • 细菌OTUs丰度稀疏曲线表明随着测序量的上升,稀疏曲线斜率逐渐降低,渐趋平坦但并未达到平台期,说明测序量已足够反映样品的物种组成特征,但依然有小部分低丰度类群未被覆盖(图1)。

  • 图1 细菌OTUs丰度稀疏曲线

  • 2.3.2 不同生物炭处理对土壤细菌Alpha多样性指数的影响

  • 土壤细菌丰富度指数Chao1、ACE分析表明,同一施炭量下Chao1 和ACE均表现出HB>JB、 HA>JA, 其中HB处理Chao1 和ACE指数高于CK,分别比CK高1.06%,1.02%,但差异不显著(P>0.05), 而HA、JA、JB处理Chao1 和ACE指数不同程度地低于CK;同一施用方式下,Chao1 和ACE随施炭量的增加而增加。 多样性指数Shannon分析表明,混施处理与穴施处理间土壤细菌群落多样性无显著差异(表4)。

  • 表4 不同生物炭处理对土壤细菌Alpha多样性指数的影响

  • 2.3.3 细菌群落组成的NMDS分析及UPGMA聚类分析

  • NMDS分析表明,CK与HA、HB、JA、JB间距离远近表现为HA>JA>HB>JB,其中JA与JB相接近,而HA与HB相接近(图2)。UPGMA聚类分析表明,CK与JA、JB被聚为一个分支,而HA、 HB被聚为一个分支(图3)。说明CK与JA、JB间的物种组成相似性高于CK与HA、HB间的物种组成相似性。

  • 图2 土壤中细菌群落的NMDS分析

  • 图3 门水平上的细菌相对丰度聚类树

  • 2.3.4 氮磷钾代谢相关菌属对生物炭的响应

  • 穴施(JA、JB)方式下,与氮代谢相关菌属硝化螺旋菌属(Nitrospira)随生物炭施用量的增加而增加,而混施(HA、HB)方式下与之相反,其中HB处理硝化螺旋菌属不同程度地低于CK及其它处理, 比CK低20.1%;HA、JA、JB处理硝化螺旋菌属高于CK,但差异不显著(P>0.05)。与磷代谢相关菌属节细菌属(Arthrobacter)在两种施用方式下均表现出随生物炭施用量的增加而增加;同一施用量下,混施处理节细菌属高于穴施处理,其中HA、HB、JB处理节细菌属高于CK, 分别比CK高2.6%、10.8%、9.8%, 各处理间及与CK间差异不显著,JA处理节细菌属显著低于CK 32.6%(P< 0.05)。与钾代谢相关菌属假单胞菌属(Pseudomonas)在同一施用方式下大小排序为HA>HB、JB>JA,同一施用量下,混施处理假单胞菌属高于穴施处理,其中HA、HB、JB处理假单胞菌属高于CK,分别比CK高160.2%、73.2%、 16.8%,HA与JB、CK处理间差异显著(P< 0.05),但与HB处理间差异不显著(P>0.05),JA处理假单胞菌属比CK低34.7%,但差异不显著。各处理间鞘氨醇单胞菌属(Sphingomonas)、链霉菌属(Streptomyces)均无显著差异(表5)。

  • 表5 氮磷钾代谢相关菌属对生物炭处理的响应

  • 2.3.5 RDA冗余分析

  • RDA冗余分析图直观地给出环境因子对研究对象的解释程度,影响因素之间的夹角代表各因素之间的相关性大小,其中锐角表示两个因素正相关,直角表示不相关,钝角表示负相关,而射线的长短代表环境因子对研究对象的解释度高低,射线越长,解释度越高;反之越低。由图4 可知,土壤碱解氮(AN)、有机质(OM)、速效钾(AK)及pH是影响土壤细菌群落结构的关键参数。

  • 图4 土壤细菌群落结构与环境因子的RDA分析

  • 3 讨论

  • 生物炭孔隙结构发达、表面积大[25],同时还有很强的吸附性和蓄集能力。唐光木等[26]研究得出,施用生物炭后新疆灰漠土土壤有机质含量显著提高。本研究发现施入生物炭后土壤有机质含量增加,一方面,生物炭本身是一种富含有机碳的物质,另一方面,生物炭可以提高土壤对养分的吸持能力。本研究还发现,施用生物炭后土壤容重和pH呈下降趋势,可能与生物炭疏松多孔及灰漠土盐基饱和度较高有关。

  • 磷、钾元素作为植物生长必须的营养元素,在土壤中主要以难溶性和矿化态形式存在[27],Vanek等[28]研究得出,施入土壤中的磷大部分以难溶态形式存在,植物难以吸收利用。21 世纪初,余海英等[29]调查山东寿光设施蔬菜地养分用量时发现,该地区每年随化肥投入的磷、钾养分各占养分总量的61%和66%,然而磷、钾养分的利用率仅为8%和46%。本试验研究得出,施入生物炭后, 与CK相比,HA、HB、JB处理不同程度的提高了与磷、钾代谢相关菌属节细菌属(Arthrobacter)和假单胞菌属(Pseudomonas)的菌群数量,也提高了土壤有效磷、速效钾的含量。说明解磷、解钾菌数量上升可以将土壤固定态磷、钾分解转化为作物可直接吸收利用的有效磷、钾。碱解氮含量表现为HB>HA>CK>JB>JA,混施处理优于穴施处理,且生物炭含量越高效果越佳,这与王卫民等[30]的研究结果不一致,可能是不同的原材料和制备条件产生了不同种类的生物炭[31],也可能是生物炭施加浓度、土壤理化性质及植物种类不同。

  • 生物炭具有吸附性,既能够吸附反应底物促进酶促反应,提高酶活性;也能够吸附保护酶促反应结合位点,抑制酶促反应,降低酶活性[32]。本研究发现施加生物炭显著提高了土壤过氧化氢酶和蔗糖酶活性,与本研究结果相似,顾美英等[33]研究表明沙土中施加生物炭后土壤蔗糖酶、过氧化氢酶活性显著提高,可能是因为生物炭的添加提高了土壤有机质,既为微生物活动提供了能源物质,也为酶促反应提供了丰富的底物,从而提高了土壤过氧化氢酶和蔗糖酶活性。脲酶活性变化与硝化螺旋菌属变化相似,JB处理下脲酶活性和硝化螺旋菌属数量最高,HB处理下两者最低,土壤酶作为土壤微生物的代谢产物,随土壤微生物的变化而变化, 生物炭的施用方式影响了它在土层中的空间分布, 混施处理下土壤保水性好,通气性差,从而使硝化螺旋菌属(Nitrospira)作为一类好养细菌在穴施处理下高于混施处理。

  • 有研究表明生物炭的多孔性及对水肥的吸附性为微生物的生长与繁殖创造了良好的环境[34]。 Shannon指数用来描述细菌群落的多样性, 而Chao1 和ACE指数用来估计细菌群落中物种的丰富度。本研究得出,只有JA处理chao1 和ACE指数显著低于CK处理, 其余处理chao1、ACE和shannon指数与CK相比均无显著差异,但在同一施用方式下Chao1、ACE指数随生物炭施用量的增加而增加,高量生物炭具有更多的表面功能基团可以有效吸附易矿化有机碳和NH4 +,为微生物提供充足的食物来源,促进微生物丰度的增加[35]。韩光明[36]研究得出添加生物炭提高了灰漠土土壤微生物的种群结构多样性。本研究进行NMDS分析及UPGMA聚类分析得出,CK与JA、JB间的相似性高于CK与HA、HB间的相似性,说明生物炭混施可较大程度地改变土壤细菌群落结构,可能是由于混施条件下,土壤保水性好,影响了土壤的透气性,使得一些厌氧微生物滋生,由于土壤细菌种类繁多,结构复杂,具体原因有待进一步研究。

  • 细菌作为土壤中数量最多的微生物类群,能够及时感知土壤养分、pH等外界条件的变化,从而反映土壤质量的变化[37]。施加生物炭可以引起土壤中细菌群落结构发生变化,并且细菌群落结构与土壤pH及养分显著相关[938]。本研究发现土壤碱解氮、有机质、速效钾及pH与土壤细菌群落结构变化密切相关,是土壤细菌群落结构发生变化的主要控制因子。

  • 4 结论

  • 施加生物炭增加了土壤有机质、有效磷、速效钾含量及蔗糖酶、过氧化氢酶活性,降低了土壤容重和pH。综合比较生物炭施用方式和施用量得出, HB(混施5%生物炭)处理土壤有机质和速效养分(碱解氮、有效磷、速效钾)含量及蔗糖酶、过氧化氢酶活性不同程度的高于其它处理。HB处理可以提高土壤细菌丰度,但对多样性影响较小。硝化螺旋菌属在穴施处理下菌群数量高于混施处理,而节细菌属和假单胞菌属菌群数量均表现出混施处理高于穴施处理。UPGMA聚类分析及RDA冗余分析表明混施生物炭后根际土壤微生物群落组成和结构发生较为明显的变化,土壤碱解氮、有机质、速效钾及pH与细菌群落结构变化密切相关。

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