隋鹏祥(1990—),男,辽宁丹东人,博士研究生,助理研究员,主要从事旱地耕层调控技术研究。E-mail:
为揭示长期耕作对农田黑土几丁质降解菌群及酶活性的影响及其驱动因子,以黑土区耕法长期定位试验为平台,采用荧光定量和高通量测序技术研究不同耕作措施(灭茬起垄、免耕、间隔深松和翻耕)下0~40 cm土层
This study aimed to illustrate the impacts of different tillage practices on chitin degrading microbial communities and chitinase activity in farmland black soil, and to explore the main environmental factors in driving a change in chitin degrading microbial communities and chitinase activity.
Based on the long-term positioning platform of different tillage practices in black soil and the combination of fluorescence quantification and high throughput sequencing technology, this research intends to study the effects of long-term different tillage practices (Conventional tillage, No-tillage, Sub-soiling tillage, Moldboard plowing tillage) on chitin degrading bacteria harboring
Results showed that no-tillage increased
These results provide a theoretical basis for understanding the effect of different tillage practices on soil chitin degradation in black soil areas.
几丁质是自然界中含量仅次于纤维素的第二大生物聚合物,是无脊椎动物外骨骼和真菌细胞壁的主要成分,广泛存在于土壤中[
土壤环境中存在多种几丁质降解菌,国内外对几丁质降解菌的分离、纯化和鉴定进行了大量研究,其菌群主要以细菌为主,常见的包括放线菌、厚壁菌和变形菌等,在农业生防和培肥中作为生物防治剂、土壤改良剂等得到广泛应用[
不同耕作方式通过改变耕层土壤结构和作物残茬在土壤中的空间分布,影响土壤微生物群落和酶活性[
定位试验位于吉林省农业科学院公主岭院内试验田(43°52.23′N,124°81.18′E),该地区是中温带大陆性季风气候,年均气温4.5℃,有效积温2 800℃,太阳有效辐射4 800 MJ·m–2,年均降雨量567 mm。土壤类型为中层黑土、壤质黏土。
耕法长期定位试验始于1983年,设灭茬起垄(CT)、免耕(NT)、间隔深松(ST)和翻耕(MP)4种耕作措施。具体田间机械操作如下:灭茬起垄为秋季将秸秆移出农田后旋耕灭茬起垄,春季播种机等行距65 cm播种;免耕为秋季收获后留茬40 cm,春季免耕播种机等行距65 cm播种,苗带行间交替休闲,生育期内不进行任何田间机械作业;间隔深松为秋季收获后留茬40 cm,春季免耕播种机宽窄行播种,宽行90 cm,窄行40 cm,宽窄行交替休闲,并在玉米拔节期采用刀踵式深松铲进行宽行深松35 cm;翻耕为秋季秸秆移除后深翻35 cm埋茬后耙平镇压,春季播种机等行距65 cm机械播种。各小区面积1 950 m2,3次重复,20行区,150 m行长,供试玉米品种为翔玉998。每年5月1日左右播种,10月1日左右收获。种植密度为60 000株·hm–2。施纯N 225 kg·hm–2、P2O5 90 kg·hm–2和K2O 80 kg·hm–2,全部的磷、钾肥和1/3氮肥作基肥,2/3氮肥在玉米拔节前追施(6月中下旬)。耕法定位试验除耕整地和播种为机械作业外,打药、除草、追肥、收获及秸秆移除均为人工操作,避免其他环节机械压实对耕层产生影响。
土壤样品于2020年10月中旬玉米收获后秋整地前采集,每个处理3次重复,各小区使用S型采样法分别采集5个苗带和5个行间0~20 cm和20~40 cm土层混合样品2 kg、原状土2 kg和环刀样品。混合样品分成两份,一份存储在–80℃下用于荧光定量PCR测定和DNA提取。一份自然风干,过筛研磨后用于土壤理化性质和几丁质酶活性测定。原状土带回实验室后将大土块按自然裂痕剥离为1 cm3左右小土块,待自然风干后再挑除粗根系和小石块。
将风干原状土样分别过孔径为5 mm和2 mm的筛子,土样被分为 > 5 mm、5~2 mm、 < 2 mm三个级别。然后计算三个级别土样在原状土中所占比例,取混合土样50 g,慢慢放入振荡式土壤团粒筛分仪(DM200-Ⅳ型,中国),筛网没入水面的套筛(孔径依次分别为2,0.25及0.053 mm)顶部,浸泡10 min,再以3 cm振幅、20次·min–1的运动频率振荡2 min后,从上到下依次取下套筛,收集筛上土样于60℃烘干称重保存。土壤基本理化性质按照《土壤农化分析》的方法测定[
使用FastDNA® SPIN kit试剂盒提取土壤总DNA,称取–80℃下保存的新鲜土壤样品0.5 g,按照说明书步骤,使用NanoDropND-1000光谱仪测定260 nm的OD值,检测提取DNA的浓度和纯度。使用ABI7500实时PCR系统对
不同耕作措施0~40 cm土层理化性质
Soil physicochemical properties of 0~40 cm soil layer under different tillage practices
土壤深度 |
处理 |
pH | BD/(g·cm–3) | MWD/mm | SOC/(g·kg–1) | TN/(g·kg–1) | TP/(g·kg–1) | AN/(mg·kg–1) | AP/(mg·kg–1) |
注:CT,灭茬起垄;NT,免耕;ST,间隔深松;MP,翻耕;BD,土壤容重;MWD,平均重量直径;SOC,土壤有机碳;TN,全氮;TP,全磷;AN,有效氮;AP,有效磷。下同。Note:CT,conventional tillage;NT,no-tillage;ST,sub-soiling tillage;MP,moldboard plowing tillage;BD,soil bulk density;MWD,mean weight diameter;SOC,soil organic carbon;TN,total nitrogen;TP,total phosphorus;AN,available nitrogen;AP,available phosphorus. The same below. | |||||||||
0~20 cm | CT | 5.48 | 1.36 | 0.98 | 13.31 | 1.51 | 0.54 | 135.6 | 24.69 |
NT | 5.45 | 1.43 | 1.19 | 17.10 | 1.49 | 0.58 | 141.2 | 31.83 | |
ST | 5.15 | 1.30 | 1.17 | 14.52 | 1.64 | 0.48 | 158.7 | 19.95 | |
MP | 5.48 | 1.32 | 1.06 | 15.55 | 1.48 | 0.55 | 135.1 | 27.17 | |
20~40 cm | CT | 5.96 | 1.56 | 1.01 | 7.67 | 0.94 | 0.37 | 83.2 | 3.98 |
NT | 6.31 | 1.55 | 1.04 | 6.72 | 0.76 | 0.32 | 64.6 | 3.59 | |
ST | 5.67 | 1.44 | 1.05 | 9.21 | 1.21 | 0.42 | 106.7 | 8.44 | |
MP | 6.13 | 1.43 | 1.06 | 15.13 | 1.08 | 0.43 | 99.7 | 6.17 |
对上述引物进一步进行扩增子测序测定
水稳性团聚体稳定性指标使用平均重量直径(MWD)[
式中,
在SPSS 25.0软件中利用单因素方差分析
由
不同耕作措施下
Gene abundance of
不同耕作措施几丁质降解菌群的Chao 1丰富度和Shannon多样性指数见
不同耕作措施下
Alpha diversity of
几丁质降解菌的主坐标分析
Principal coordinates analysis(PCoA)ordinations of microbial community for chitin-degrading bacteria
相关分析表明(
土壤理化性质与
Pearson correlation of soil physicochemical properties and
指标 |
Chao1丰富度指数 |
Shannon多样性指数 |
β多样性 |
|
**, |
||||
BD | –0.159 | –0.210 | –0.256 | 0.808** |
MWD | 0.701** | 0.342 | –0.292 | –0.168 |
SOC | 0.599** | 0.138 | 0.248 | –0.800** |
TN | 0.443* | 0.138 | 0.167 | –0.731** |
TP | 0.505* | –0.115 | 0.194 | –0.747** |
AN | 0.493* | 0.131 | 0.170 | –0.728** |
AP | 0.498* | –0.163 | –0.032 | –0.725** |
pH | –0.481* | –0.153 | –0.167 | 0.661** |
不同耕作措施和采样深度下土壤几丁质降解菌群中总共检测出优势菌群(相对丰度 > 1%)为2个菌门,2个菌纲,6个菌目,5个菌科和3个菌属(
不同耕作措施下土壤几丁质降解菌群落组成(a)和ASV/OTU韦恩图(b)
Microbial community(a)and venn diagram(b)of chitin-degrading bacteria under different tillage practices
ASV/OTU韦恩图(
采用冗余分析进一步明确土壤理化性质对
几丁质降解菌群与土壤理化性质的冗余分析(RDA)
Redundancy analysis(RDA)of chitin-degrading bacteria community with soil physicochemical properties
不同耕作措施下几丁质酶活性
Chitinase activity in different tillage practices
如
几丁质降解菌群与几丁质酶活性的结构方程模型分析(a)及标准化效应(b)
Chitin-degrading bacteria community and chitinase using structural equation modeling(a)and standardized effects(b)
长期耕作会对农田耕层和亚耕层土壤养分循环和土壤肥力维持造成不同的影响,该过程首先是调控土壤微生物活性,再进一步作用于土壤生物化学循环[
本研究中,对比传统耕作,长期免耕后显著降低几丁质降解菌群Shannon多样性(
本研究中,放线菌门相对丰度与土壤容重和pH显著正相关,与土壤养分含量显著负相关(
土壤几丁质酶水解几丁质,参与几丁质转化氨基糖的过程,氨基糖是可矿化氮的主要来源,占土壤有机氮的5%~10%,因此几丁质酶分解几丁质是控制氮循环速率的关键步骤[
土壤碳氮的可用性取决于土壤聚合物(例如木质素、几丁质等)的分解情况,这些聚合物受微生物所分泌的分解酶活性调控,以优化土壤碳氮供需平衡,当土壤碳利用率高,而氮利用率低时,则增加几丁质等富氮聚合物的分解,这是微生物氮开采(Microbial N mining)理论[
东北黑土区不同耕作措施的长期应用使农田土壤几丁质降解菌群和酶活性发生显著分异。免耕促进效果仅表现在耕层土壤,显著降低亚耕层几丁质降解菌群数量及多样性,而间隔深松和翻耕则增加亚耕层几丁质降解菌数量,并维持其群落多样性,提高几丁质酶活性。几丁质降解菌群和土壤理化性质密切相关。耕作方式和土壤深度直接影响几丁质酶活性,也通过调控土壤结构稳定性、全量养分含量和几丁质降解菌群间接影响几丁质酶活性。
LeCleir G R, Buchan A, Maurer J, et al. Comparison of chitinolytic enzymes from an alkaline, hypersaline lake and an estuary[J]. Environmental Microbiology, 2007, 9(1): 197—205.
Wieczorek A S, Hetz S A, Kolb S. Microbial responses to chitin and chitosan in oxic and anoxic agricultural soil slurries[J]. Biogeosciences, 2014, 11(12): 3339—3352.
Sharp R. A review of the applications of chitin and its derivatives in agriculture to modify plant-microbial interactions and improve crop yields[J]. Agronomy, 2013, 3(4): 757—793.
Kielak A M, Cretoiu M S, Semenov A V, et al. Bacterial chitinolytic communities respond to chitin and pH alteration in soil[J]. Applied and Environmental Microbiology, 2013, 79(1): 263—272.
Cretoiu M S, Kielak A M, Schluter A, et al. Bacterial communities in chitin-amended soil as revealed by 16S rRNA gene based pyrosequencing[J]. Soil Biology & Biochemistry, 2014, 76: 5—11.
Brzezinska M S, Jankiewicz U, Walczak M. Biodegradation of chitinous substances and chitinase production by the soil actinomycete
Patil R S, Ghormade V, Deshpande M V. Chitinolytic enzymes: An exploration[J]. Enzyme and Microbial Technology, 2000, 26(7): 473—483.
Adrangi S, Faramarzi M A. From bacteria to human: A journey into the world of chitinases[J]. Biotechnology Advances, 2013, 31(8): 1786—1795.
Horn S J, Sørbotten A, Synstad B, et al. Endo/exo mechanism and processivity of family 18 chitinases produced by
Kawase T, Saito A, Sato T, et al. Distribution and phylogenetic analysis of family 19 chitinases in Actinobacteria[J]. Applied and Environmental Microbiology, 2004, 70(2): 1135—1144.
Cretoiu M S, Kielak A M, Abu Al-Soud W, et al. Mining of unexplored habitats for novel chitinases—
Veliz E A, Martínez-Hidalgo P, Hirsch A M. Chitinase- producing bacteria and their role in biocontrol[J]. AIMS Microbiology, 2017, 3: 689—705.
Bai Y, Eijsink V G H, Kielak A M, et al. Genomic comparison of chitinolytic enzyme systems from terrestrial and aquatic bacteria[J]. Environmental Microbiology, 2016, 18(1): 38—49.
闫海洋, 金荣德, 朴光一, 等. 不同肥力土壤中分解几丁质微生物代谢产物对玉米的促生效果研究[J]. 玉米科学, 2015, 23(3): 119—123.
Yan H Y, Jin R D, Piao G Y, et al. Effect of decomposition chitin microbial metabolites on maize growth-promoting under different fertility soils[J]. Journal of Maize Sciences, 2015, 23(3): 119—123
Cronin D, Moënne-Loccoz Y, Dunne C, et al. Inhibition of egg hatch of the potato cyst nematode
Sindhu S S, Dadarwal K R. Chitinolytic and cellulolytic
张路, 王杰, 王向涛, 等. 不同恢复方式对退化高寒草甸土壤
Zhang L, Wang J, Wang X T, et al. Effect of restoration types on the community structure of microbes harboring
Anderson C, Beare M, Buckley H L, et al. Bacterial and fungal communities respond differently to varying tillage depth in agricultural soils[J]. PeerJ, 2017, 5: e3930.
宋霄君, 吴会军, 武雪萍, 等. 长期保护性耕作可提高表层土壤碳氮含量和根际土壤酶活性[J]. 植物营养与肥料学报, 2018, 24(6): 1588—1597.
Song X J, Wu H J, Wu X P, et al. Long-term conservation tillage improves surface soil carbon and nitrogen content and rhizosphere soil enzyme activities[J]. Journal of Plant Nutrition and Fertilizers, 2018, 24(6): 1588—1597
Li M, He P, Guo X L, et al. Fifteen-year no tillage of a Mollisol with residue retention indirectly affects topsoil bacterial community by altering soil properties[J]. Soil & Tillage Research, 2021, 205: 104804.
Li Y, Song D P, Liang S H, et al. Effect of no-tillage on soil bacterial and fungal community diversity: A meta- analysis[J]. Soil and Tillage Research, 2020, 204: 104721.
Liu H W, Carvalhais L C, Crawford M, et al. Strategic tillage increased the relative abundance of Acidobacteria but did not impact on overall soil microbial properties of a 19-year no-till Solonetz[J]. Biology and Fertility of Soils, 2016, 52(7): 1021—1035.
Chaudhary M, Naresh R K, Vek V, et al. Soil organic carbon fractions, soil microbial biomass carbon, and enzyme activities impacted by crop rotational diversity and conservation tillage in north west IGP: A review[J]. International Journal of Current Microbiology and Applied Sciences, 2018, 7(11): 3573—3600.
鲍士旦. 土壤农化分析[M]. 3版. 北京: 中国农业出版社, 2000: 42—108.
Bao S D. Soil and agricultural chemistry analysis[M]. 3rd ed. Beijing: China Agriculture Press, 2000: 42—108.
Xiao X, Yin X B, Lin J, et al. Chitinase genes in lake sediments of Ardley Island, Antarctica[J]. Applied and Environmental Microbiology, 2005, 71(12): 7904—7909.
Zheng H B, Liu W R, Zheng J Y, et al. Effect of long-term tillage on soil aggregates and aggregate- associated carbon in black soil of Northeast China[J]. PLoS One, 2018, 13(6): e0199523.
Sui P X, Tian P, Lian H L, et al. Straw incorporation management affects maize grain yield through regulating nitrogen uptake, water use efficiency, and root distribution[J]. Agronomy, 2020, 10(3): 324.
Morugán-Coronado A, Pérez-Rodríguez P, Insolia E, et al. The impact of crop diversification, tillage and fertilization type on soil total microbial, fungal and bacterial abundance: A worldwide meta-analysis of agricultural sites[J]. Agriculture, Ecosystems & Environment, 2022, 329: 107867.
Mu X Y, Zhao Y L, Liu K, et al. Responses of soil properties, root growth and crop yield to tillage and crop residue management in a wheat-maize cropping system on the North China Plain[J]. European Journal of Agronomy, 2016, 78: 32—43.
Turner S, Mikutta R, Guggenberger G, et al. Distinct pattern of nitrogen functional gene abundances in top- and subsoils along a 120, 000-year ecosystem development gradient[J]. Soil Biology & Biochemistry, 2019, 132: 111—119.
邓超超, 李玲玲, 谢军红, 等. 耕作措施对陇中旱农区土壤细菌群落的影响[J]. 土壤学报, 2019, 56(1): 207—216.
Deng C C, Li L L, Xie J H, et al. Effects of tillage on soil bacterial community in the dryland farming area of central Gansu[J]. Acta Pedologica Sinica, 2019, 56(1): 207—216
Wang X B, Zhou B Y, Sun X F, et al. Soil tillage management affects maize grain yield by regulating spatial distribution coordination of roots, soil moisture and nitrogen status[J]. PLoS One, 2015, 10(6): e0129231.
Li C H, Ma B L, Zhang T Q. Soil bulk density effects on soil microbial populations and enzyme activities during the growth of maize(
Lauber C L, Hamady M, Knight R, et al. Pyrosequencing-based assessment of soil pH as a predictor of soil bacterial community structure at the continental scale[J]. Applied and Environmental Microbiology, 2009, 75(15): 5111—5120.
洪艳华. 长期耕作对黑土理化性质及微生物群落结构的影响[D]. 黑龙江大庆: 黑龙江八一农垦大学, 2021.
Hong Y H. Effects of long-term tillage on physicochemical properties and microbial community structure in black soil[D]. Daqing, Heilongjiang: Heilongjiang Bayi Agricultural University, 2021.
张淼, 刘俊杰, 刘株秀, 等. 黑土区农田土壤氮循环关键过程微生物基因丰度的分布特征[J]. 土壤学报, 2022, 59(5): 1258—1269.
Zhang M, Liu J J, Liu Z X, et al. Distribution characteristics of microbial gene abundance in key processes of soil nitrogen cycling in black soil zone[J]. Acta Pedologica Sinica, 2022, 59(5): 1258—1269
Li H, Zhang Y Y, Yang S, et al. Variations in soil bacterial taxonomic profiles and putative functions in response to straw incorporation combined with N fertilization during the maize growing season[J]. Agriculture, Ecosystems & Environment, 2019, 283: 106578.
张威, 张明, 白震, 等. 土壤中几丁质酶的研究进展[J]. 土壤通报, 2007, 38(3): 569—575.
Zhang W, Zhang M, Bai Z, et al. Research advances in soil chitinase[J]. Chinese Journal of Soil Science, 2007, 38(3): 569—575
Chen R R, Senbayram M, Blagodatsky S, et al. Soil C and N availability determine the priming effect: Microbial N mining and stoichiometric decomposition theories[J]. Global Change Biology, 2014, 20(7): 2356—2367.
白伟, 孙占祥, 张立祯, 等. 耕层构造对土壤三相比和春玉米根系形态的影响[J]. 作物学报, 2020, 46(5): 759—771.
Bai W, Sun Z X, Zhang L Z, et al. Effects of plough layer construction on soil three phase rate and root morphology of spring maize in northeast China[J]. Acta Agronomica Sinica, 2020, 46(5): 759—771
Kumeta Y, Inami K, Ishimaru K, et al. Thermogravimetric evaluation of chitin degradation in soil: Implication for the enhancement of ammonification of native organic nitrogen by chitin addition[J]. Soil Science and Plant Nutrition, 2018, 64(4): 512—519.
Wild B, Li J, Pihlblad J, et al. Decoupling of priming and microbial N mining during a short-term soil incubation[J]. Soil Biology & Biochemistry, 2019, 129: 71—79.