韓 梅,劉 蕊,李正鵬,嚴(yán)清彪,孫小鳳

種植綠肥對作物產(chǎn)量和細(xì)菌群落穩(wěn)定性的影響

韓 梅,劉 蕊,李正鵬,嚴(yán)清彪,孫小鳳*

(青海大學(xué)農(nóng)林科學(xué)院,青海 西寧 810016)

為深入研究青海高原地區(qū)長期種植綠肥,減施化肥條件下小麥/油菜產(chǎn)量及土壤理化性狀及微生物群落的變化,通過2011年建立的定位試驗(yàn),設(shè)GF0(毛苕子作為綠肥,不施用化肥)、GF60(毛葉苕子配施60%化肥)、GF70(毛葉苕子配施70%化肥)、GF80(毛葉苕子配施80%化肥)、GF90(毛葉苕子配施90%化肥)、GF100(毛葉苕子配施100%化肥)、F0(休耕并且不施化肥)、F100(休耕并且施用100%化肥)共8個處理,利用高通量測序等技術(shù),研究了種植利用綠肥減施化肥條件下小麥和油菜的產(chǎn)量、土壤性質(zhì)和微生物群落結(jié)構(gòu)的變化特征.結(jié)果表明,在保證小麥、油菜不減產(chǎn)的基礎(chǔ)上,種植綠肥后茬作物減施化肥達(dá)到30%左右.綠肥配施減量化肥對土壤肥力有顯著的提升作用,特別是土壤有機(jī)碳和全氮含量有明顯的提高,分別提高1.34%～7.46%、2.16%～7.48%.化肥與綠肥配施增強(qiáng)了土壤微生物群落多樣性,其中酸桿菌門和變形菌門是本研究土壤中的優(yōu)勢菌群.共生網(wǎng)絡(luò)分析表明,綠肥處理提高了細(xì)菌群落的穩(wěn)定性和抗干擾能力,同時(shí)綠肥應(yīng)用增加了細(xì)菌群落中的關(guān)鍵物種.本研究表明,不同土壤微生物豐度受種植模式、施肥量影響很大,長期種植綠肥、減施化肥提高了土壤微生物豐度和多樣性,增強(qiáng)微生物群落的穩(wěn)定性.種植綠肥條件下化肥減肥量30%左右保證作物穩(wěn)產(chǎn)可為當(dāng)?shù)氐氖┓使芾硖峁﹨⒖?

毛葉苕子；減肥；微生物群落；共發(fā)生網(wǎng)絡(luò)；關(guān)鍵物種

種植綠肥是減少化肥投入、提高作物產(chǎn)量的傳統(tǒng)有效方法[1-3].在許多農(nóng)業(yè)系統(tǒng)中,使用綠肥可以降低土壤侵蝕風(fēng)險(xiǎn)、改善土壤特性、保證土壤健康、增加土壤微生物的豐度和活性[4-8].豆科植物因其生物固氮能力而成為農(nóng)田綠肥中最常用的綠肥作物.毛葉苕子(Roth)因其適應(yīng)性廣、新鮮生物量大、固氮能力強(qiáng)等特點(diǎn),被廣泛應(yīng)用為北方綠肥作物.在旱地地區(qū),種植豆科綠肥比休耕更能提高土壤肥力,促進(jìn)作物可持續(xù)生產(chǎn)[9-10].我國各種耕作制度下存在著大量季節(jié)性閑置耕地,并且不同區(qū)域有著多樣的氣候環(huán)境,適宜間作、輪作、混作等多種種植方式,發(fā)展綠肥具有很大潛力[11].青海省河湟地區(qū)位于青海高原的東北部屬典型的兩季不足、一季有余.為充分利用光熱土資源,在青海高原建立了豆科綠肥-小麥/油菜輪作技術(shù)體系.毛葉苕子的種植不僅解決了青海高原休耕和裸地問題,而且改善了高原環(huán)境.經(jīng)過多年的實(shí)踐證明,該技術(shù)在優(yōu)化作物栽培結(jié)構(gòu)、提高復(fù)種指數(shù)和土壤質(zhì)量、緩解牧區(qū)冬季牧草短缺問題等方面是一項(xiàng)有效的實(shí)踐.種植綠肥有利于土壤微環(huán)境的改良、提升作物產(chǎn)量和品質(zhì)等[12];翻壓還田對后茬作物的生長與產(chǎn)量、養(yǎng)分吸收利用以及土壤物理、化學(xué)、生物特性等同樣存在一定的影響[13].其在養(yǎng)分供應(yīng)、培肥土壤、生物固氮、涵養(yǎng)水源等[14]方面均體現(xiàn)了其為農(nóng)田提供的有價(jià)值的生態(tài)系統(tǒng)服務(wù)功能.綠肥作物在飼用價(jià)值方面也發(fā)揮著重要的作用,尤其在夏季飼料不足、冬季飼料缺乏的青藏高原地區(qū).畜牧業(yè)的發(fā)展最重要的是飼料及飼草的質(zhì)量,飼料短缺問題是畜牧業(yè)快速發(fā)展的阻礙[15].土壤微生物群落結(jié)構(gòu)可以作為土壤肥力和土壤質(zhì)量的有效指標(biāo).前人研究發(fā)現(xiàn),綠肥可以改變微生物群落,從而影響土壤微生物誘導(dǎo)的土壤養(yǎng)分循環(huán)過程[16-17].持續(xù)種植綠肥可提高土壤微生物量碳、氮和土壤酶活性[18-19]. 而且種植翻壓綠肥可以增加土壤微生物的數(shù)量和多樣性,改變土壤微生物的組分和結(jié)構(gòu)[20].土壤微生物群落結(jié)構(gòu)的變化可能與長期種植綠肥改善土壤肥力、環(huán)境等有關(guān)[21]本研究在2011年建立的長期試驗(yàn)基礎(chǔ)上,利用高通量測序技術(shù),對小麥和油菜產(chǎn)量、土壤性質(zhì)和微生物群落進(jìn)行了研究.本研究在種植綠肥條件下減施化肥,調(diào)查毛葉苕子-小麥/油菜輪作系統(tǒng)作物產(chǎn)量、土壤性狀及微生物群落結(jié)構(gòu)和穩(wěn)定性的影響.

1 材料與方法

1.1 試驗(yàn)地點(diǎn)

試驗(yàn)地點(diǎn)位于青海省西寧市青海省農(nóng)林科學(xué)院(36°62′N,101°77′E, 海拔2300m),大陸性半干旱氣候,年平均氣溫5.9℃,年平均降水量367.5mm,年平均蒸發(fā)量1729.8mm.土壤類型為栗鈣土.2011年試驗(yàn)前,土壤有機(jī)碳、全氮、全磷和全鉀含量分別為9670,1110,2180,26330mg/kg,速效氮、速效磷和速效鉀分別為98,13.1,139mg /kg,土壤pH值為8.31.

1.2 試驗(yàn)設(shè)計(jì)

于2011年布置長期定位試驗(yàn).該試驗(yàn)采用完全隨機(jī)區(qū)組設(shè)計(jì)(表1、圖1),共8個處理,(1)GF0,毛苕子作為綠肥,不施用化肥;(2)GF60,毛葉苕子配施60%化肥;(3)GF70,毛葉苕子配施70%化肥;(4)GF80,毛葉苕子配施80%化肥;(5)GF90,毛葉苕子配施90%化肥;(6)GF100,毛葉苕子配施100%化肥;(7)F0,休耕并且不施化肥;(8)F100,休耕并且施用100%化肥.休耕代表不種綠肥.各處理重復(fù)4次,試驗(yàn)中,100%化肥用量為當(dāng)?shù)剞r(nóng)田的推薦用量,施N量為225kg/hm2,施P2O5量為112.5kg/hm2,不施鉀肥.尿素(N 46%)和過磷酸鈣(P2O512%)作為氮磷肥.

表1 試驗(yàn)方案

圖1 大田照片

青海省的種植制度是一年一熟.本試驗(yàn)采用毛葉苕子-小麥/油菜輪作制度.小麥、油菜和毛葉苕子品種分別為“青春38號”、“浩油11號”和“土庫曼毛苕”.毛葉苕子干物質(zhì)中碳(C)、氮(N)、磷(P)、鉀(K)平均含量分別為46.32%、2.45%、0.31%和1.82%.小麥或油菜在3月中旬施肥后播種,7月下旬收獲,小麥、油菜播種量分別為600kg/hm2,7.5kg/hm2.小麥、油菜收獲后,以105kg/hm2播種毛葉苕子.十月中旬,收割毛葉苕子,地上部分作為飼料移出田地,根茬還田.

1.3 樣品采集與指標(biāo)測定

2017年土壤采樣共3次,分別為播種前(3月15日)、抽穗期(6月15日)和收獲后(7月25日).采樣后的土壤分取部分保存于4℃用于測定銨、硝酸鹽氮,部分自然風(fēng)干進(jìn)行用于測定其他理化性狀,部分保存于-80℃用于提取DNA.

采用重鉻酸鉀外加熱法測定土壤有機(jī)碳(soil organic carbon,SOC).凱氏定氮法測定土壤全氮(total nitrogen,TN).用0.01mol/L CaCl2浸提NH4+-N和NO3--N,然后用連續(xù)流分析儀(AA3, SEAL, 德國)測定.采用堿解擴(kuò)散法測定土壤速效氮(available N).采用電位法測定土壤 pH值(水土比 2.5:1)[22].

1.4 土壤微生物DNA提取及測序

采用E.Z.N.ATMMag-Bind Soil DNA Kit (OMEGA,美國)提取土壤微生物DNA.使用Nanodrop 2000分光光度計(jì)測定DNA含量.16S rRNA基因v3-v4序列引物為341F/805R (CCCT ACAC GACG CTCT TCCG ATCTG / GACT GGAG TTCC TTGG CACC CGAG AATT CCA)[23].利用Illumina MiSeq PE300平臺進(jìn)行測序[24].

1.5 生物信息學(xué)和統(tǒng)計(jì)分析

測序獲得的原始reads使用FLASH (version 1.2.7, http://ccb.jhu.edu/software/FLASH/)進(jìn)行提取、裁剪和質(zhì)量篩選.利用Mothur軟件(http://www.mothur.org/ wiki/Schloss_SOP)進(jìn)行各處理Alpha多樣性分析.使用Usearch(7.1版http://drive5.com/ uparse/)將剩余的高質(zhì)量reads以97%的相似性進(jìn)行操作分類單元(operational taxonomic units,OTUs)聚類[25].使用RDP分類器將OTUs的代表性序列與Sliva數(shù)據(jù)庫進(jìn)行比較[26].計(jì)算Good’s coverage[27]來估計(jì)樣本的多樣性和豐度.原始數(shù)據(jù)上傳至國家基因組科學(xué)數(shù)據(jù)中心,序列號為SAMC392370- SAMC392373.

利用分子生態(tài)網(wǎng)絡(luò)分析(Molecular Ecological Network Analyses pipeline,http://ieg2.ou.edu/MENA/)[28]對處理F0、F100、GF0和GF100進(jìn)行共生網(wǎng)絡(luò)分析..通過Gephi(http://gephi.github.io/)使生成可視化網(wǎng)絡(luò)圖.使用-score和-score定義關(guān)鍵物種(keystones),將網(wǎng)絡(luò)中心點(diǎn)、模塊中心點(diǎn)和連接節(jié)點(diǎn)作為分子生態(tài)網(wǎng)絡(luò)中的關(guān)鍵物種[29-30].

2 結(jié)果與分析

2.1 綠肥配施化肥對小麥和油菜產(chǎn)量的影響

圖2 2012～2018年小麥和油菜產(chǎn)量

由圖2可見,綠肥配施化肥顯著提高了油菜和小麥的產(chǎn)量.油菜GF100處理4a平均產(chǎn)量最高,達(dá)到1934kg/hm2,顯著高于F100處理(1766kg/hm2).不同字母表示差異有統(tǒng)計(jì)學(xué)意義(<0.05).GF90、GF80和GF70處理的油菜產(chǎn)量與F100處理無顯著差異,說明綠肥還田后減少10%～30%的化肥用量,而且油菜產(chǎn)量不減少.小麥GF100處理(6645kg/hm2) 3a平均產(chǎn)量顯著高于F100處理(5873kg/hm2).GF90、GF80、GF70和GF60處理小麥產(chǎn)量均與F100處理無差異,說明保證小麥產(chǎn)量的前提下,綠肥根茬還田可減少小麥季化肥用量的40%.綠肥對提高作物高產(chǎn)和減少化肥的影響已得到廣泛證實(shí)[2,31].在我國黃土高原建立的長期研究發(fā)現(xiàn),施用豆科綠肥平均替代了31%的化學(xué)氮肥[32].在瑞士的田間研究發(fā)現(xiàn),覆蓋作物使玉米平均產(chǎn)量增加12%,種植毛葉苕子平均使玉米氮吸收增加79kg/ hm2[33].

2.2 綠肥配施化肥對土壤理化性狀的影響

種植綠肥7年后,由于綠肥的生長和根茬的分解,土壤性狀發(fā)生了改變(表3).在小麥播種,抽穗和收獲階段,土壤pH值的變化趨勢是相似的,與F0相比,F100處理顯著增加土壤pH值,而綠肥降低了土壤pH值.綠肥和化肥配施提高了土壤有機(jī)碳和總氮含量,GF70、GF60和GF80處理有機(jī)碳含量分別在播種、抽穗期和收獲期最高.播期,GF60、GF100和F100處理土壤銨態(tài)氮含量顯著高于其他處理. GF100和F100處理在抽穗期和收獲期最高.綠肥處理降低了3個生育期的硝態(tài)氮含量.速效氮以播種期GF100處理最高,抽穗期和收獲期F100處理最高.持續(xù)施用綠肥增加了土壤有機(jī)碳和易氧化碳的含量[10].西北地區(qū)夏季綠肥耕作優(yōu)于休耕[10,32].將綠肥納入農(nóng)業(yè)輪作可以影響土壤碳庫,并有助于土壤有機(jī)碳儲量[34].

表3 三個生育階段不同處理中的土壤理化性狀

注:同一時(shí)期同一列內(nèi)不同字母表示處理間有顯著差異(<0.05).

2.3 綠肥配施化肥對細(xì)菌群落結(jié)構(gòu)的影響

由圖3可見,在屬水平上,三個采樣階段Gp6、鞘單胞菌屬()和芽單胞菌屬()的豐度最高,分別被劃分為酸桿菌門(Acidobacteria)、變形菌門(Proteobacteria和芽單胞菌門(Gemmatimonadetes).Gp6在播種、抽穗期和收獲期分別占8.19%、8.08%和7.89%.鞘氨單胞菌的豐度在播種期高于芽單胞菌,而芽單胞菌的豐度在抽穗期和采收期高于芽單胞菌.在門水平上,變形菌門(Proteobacteria)最為豐富,在3個采樣階段占總reads的36%以上.酸桿菌門(Acidobacteria)、擬桿菌門(Bacteroidetes)和芽單胞菌(Gemmatimonadetes)的平均豐度分別為20.52%、8.25%和7.00%.在播種期,浮霉菌門(Planctomycetes)的豐度低于厚壁菌門(Firmicutes)和疣菌門(Verrucomicrobia),而在抽穗期和采收期則高于厚壁菌門和疣菌門.

為了進(jìn)一步研究不同處理下的群落結(jié)構(gòu)變化,分析了處理間豐度較高的8個菌門(相對豐度> 4%)的分布(圖4).播種期,GF0處理的浮霉菌門豐度顯著高于GF70和GF80處理;GF70處理的疣菌門豐度顯著高于GF60和F100處理;F100處理的厚壁菌門豐度顯著高于GF90處理.抽穗期不同處理改變了變形菌門、擬桿菌門、疣菌門、厚壁菌門和放線菌門(Actinobacteria)的分布.與GF90和F100處理相比, GF100處理顯著增加了變形菌門的豐度;與GF0和GF60處理相比,GF90處理顯著增加擬桿菌門的豐度;與GF60和F0處理相比,GF90處理顯著增加了疣菌門的豐度;與GF70、GF80、GF90和GF100處理相比,F0處理顯著增加了厚壁菌門的豐度;與GF0和GF90處理相比,GF80和F100處理顯著提高了放線菌的豐度.收獲期8個豐度較高的菌群在不同處理間差異不顯著.綠肥的種植可以有效地提高土壤酶和土壤生物活性[19,35-37].土壤微生物驅(qū)動生物地球化學(xué)循環(huán),特別是土壤碳、氮、硫等的轉(zhuǎn)化[8,38].在藏西北地區(qū)的研究發(fā)現(xiàn),在高海拔土壤中,酸桿菌門、綠桿菌門和變形菌門最為豐富,土壤總碳和碳/氮比值等土壤性質(zhì)是該研究區(qū)域微生物分布的主要驅(qū)動因素[39].在本研究中,最豐富的門是酸桿菌門和變形菌門,這與鄰近地區(qū)的研究一致.

2.4 細(xì)菌群落的相互作用關(guān)系和網(wǎng)絡(luò)拓?fù)涮卣?/h3>

表4 細(xì)菌共生的網(wǎng)絡(luò)特性

由表4和圖5可見,F100的網(wǎng)絡(luò)最復(fù)雜,其次是GF0和GF100,F0的網(wǎng)絡(luò)最簡單.F100處理的節(jié)點(diǎn)數(shù)和邊數(shù)最高(分別為778和2383),F0處理的節(jié)點(diǎn)數(shù)和邊數(shù)最低(分別為53和124).GF0和GF100處理的節(jié)點(diǎn)數(shù)和邊數(shù)相似.綠肥處理(GF0和GF100)與無綠肥處理(F0和F100)呈負(fù)相關(guān).GF0和GF100處理的平均連通性和平均聚類系數(shù)均低于F0和F100處理.GF0和GF100的網(wǎng)絡(luò)模塊度和模塊數(shù)均高于F0和F100,說明綠肥的施用增加了網(wǎng)絡(luò)模塊度.通過構(gòu)建并比較了添加綠肥和不添加綠肥處理下土壤細(xì)菌網(wǎng)絡(luò)的結(jié)構(gòu),以評價(jià)物種間的相互作用.

圖5 細(xì)菌群落的相互作用網(wǎng)絡(luò)

不同顏色的節(jié)點(diǎn)代表不同的模塊,節(jié)點(diǎn)大小代表節(jié)點(diǎn)的度,紅色連接線代表節(jié)點(diǎn)間正相互作用,藍(lán)色連接線代表節(jié)點(diǎn)間負(fù)相互作用

網(wǎng)絡(luò)的模塊化值在0.794到0.879之間,高于建議的閾值0.4,說明本研究構(gòu)建的網(wǎng)絡(luò)遵循模塊化結(jié)構(gòu)[21,40].節(jié)點(diǎn)(特定OTUs)和邊(正或負(fù)鏈接)的數(shù)量是各種生物相互作用的表征[41].這些相互作用創(chuàng)造了復(fù)雜的微生物群落,并建立了特定生態(tài)系統(tǒng)中的群落特征[42].處理中沒有綠肥(F0和F100)大多是正相關(guān)性,而施用綠肥不論是否添加化肥,都大大增加了網(wǎng)絡(luò)的負(fù)相關(guān)比.這些結(jié)果結(jié)合節(jié)點(diǎn)和邊的數(shù)目,表明應(yīng)用毛葉苕子隨著綠肥增加細(xì)菌群落的穩(wěn)定性和抗干擾能力.更高的平均度(avgK)意味著更復(fù)雜的網(wǎng)絡(luò)[28].平均度以F100處理最高,F0處理次之,均高于綠肥處理.化肥使細(xì)菌群落變得更加復(fù)雜而不穩(wěn)定.環(huán)境特征,如溫度和土壤pH值在構(gòu)建網(wǎng)絡(luò)交互作用中起重要作用[28].

進(jìn)一步研究OTU的拓?fù)渥饔?以確定關(guān)鍵物種[30].從關(guān)系定義的各節(jié)點(diǎn)的網(wǎng)絡(luò)角色可以看出,綠肥和化肥不同處理共生網(wǎng)絡(luò)的拓?fù)湫再|(zhì)和關(guān)鍵物種不同(圖6).在F0處理中,沒有找到關(guān)鍵物種,所有節(jié)點(diǎn)都被定義為外圍節(jié)點(diǎn).F100處理中, 0.77%被定義為模塊中心點(diǎn),0.26%的被定義為連接節(jié)點(diǎn).在GF0處理中,定義為模塊中心點(diǎn)和連接節(jié)點(diǎn)比例分別為1.32%和0.73%.在GF100中,0.76%被定義為模塊模塊中心點(diǎn),沒有連接節(jié)點(diǎn).而GF0處理中模塊集線器和連接器的比例最大,說明綠肥的利用增加了細(xì)菌群落的關(guān)鍵物種.與特定土壤過程相關(guān)的生物在微生物相互作用中發(fā)揮著更重要的作用[43],更多的關(guān)鍵物種在土壤地球化學(xué)過程中發(fā)揮著更重要的作用.綠肥的利用可以通過改變關(guān)鍵物種的分布來改善土壤質(zhì)量.OTUs網(wǎng)絡(luò)中關(guān)鍵物種間互作仍需進(jìn)一步研究,以確定與綠肥和施肥管理相關(guān)的生理特征.

3 結(jié)論

3.1 綠肥提高作物產(chǎn)量和土壤肥力

在保持油菜、小麥產(chǎn)量的前提下,長期翻壓毛葉苕子根茬能減施10%～40%化肥.綠肥配施減量氮肥可以化肥增加土壤有機(jī)碳和全氮含量.

3.2 綠肥配施化肥對細(xì)菌群落結(jié)構(gòu)的影響

播種期,綠肥配施70%的化肥處理的疣菌門豐度顯著高于GF60和F100處理;抽穗期綠肥配施化肥處理顯著增加了變形菌門、擬桿菌門、疣菌門、厚壁菌門、放線菌的豐度.

3.3 綠肥增加細(xì)菌群落的網(wǎng)絡(luò)穩(wěn)定性

綠肥不論是否添加化肥,都大大增加了網(wǎng)絡(luò)的負(fù)相關(guān)比.綠肥增加細(xì)菌群落的穩(wěn)定性和抗干擾能力，增加了細(xì)菌群落的關(guān)鍵物種.

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Green manuring increased crop yields and the stability of bacterial community.

HAN Mei, LIU Rui, LI Zheng-peng, YAN Qing-biao, SUN Xiao-feng*

(Academy of Agriculture and Forestry, Qinghai University, Xining 810016, China)., 2021,41(11)：5382～5390

In order to provide theoretical support for the effects of reducedfertilizer in hairy vetch -wheat/oilseed rape rotation system on crop production and soil fertility in Qinghai Plateau. a location field experiment about hairy vetch -wheat/oilseed rape rotation established in 2011 was used in this study. The experiment including eight treatments, e.g., only green manure (GF0), green manure and 60% of chemical fertilizer (GF60), green manure and 70% of chemical fertilizer (GF70), green manure and 80% of chemical fertilizer (GF80), green manure and 90% of chemical fertilizer (GF90), green manure and 100% of chemical fertilizer (GF100), only 100% of chemical fertilizer (F100), and the no-fertilizer control (F0). The wheat and oilseed rape yields, soil properties and microbial community structures were measured. Results showed that the green manure -wheat/oilseed rape rotation system could reduce about 30% chemical fertilizer without reduction of wheat and oilseed rape yields, when compared with F100. Green manure combined with reduced chemical fertilizer improved soil fertility, especially increasingthe contents of soil organic C and total N by 1.34%～7.46% and 2.16%～7.48%, respectively.Green manure combined with reduced chemical fertilizer increased the diversity of soil microbial, and the most abundant phyla were Acidobacteria and Proteobacteria. The results of co-occurrence networks showed that green manuring increased the percentages of negative correlations andmodularity of the networks, indicating that application of hairy vetch as green manure increased the stability and anti-jamming capability of the bacterial communities. The network roles suggested that utilization of green manure increased the keystones in the bacterial community. It was concluded that the microbial diversity and abundance were affected by the cropping system and fertilization amounts. The utilization of green manure can reduce 30% of chemical fertilizer without reduction of wheat and oilseed rape yields. The reduced fertilizer amount could be a reference to the local fertilizer management practices and will give a theoretical support for this agricultural system.

hairy vetch；reduced fertilizer；microbial community；co-occurrence networks；keystones

X171.5,X172

A

1000-6923(2021)11-5382-09

韓 梅(1973-),女,青海湟中人,副研究員,碩士,主要從事綠肥方面研究.發(fā)表論文40余篇.

2021-04-02

財(cái)政部和農(nóng)業(yè)農(nóng)村部:國家現(xiàn)代農(nóng)業(yè)產(chǎn)業(yè)技術(shù)體系資助(綠肥, CARS-22);青海省農(nóng)科院基金項(xiàng)目(2019-NKY-06)

* 責(zé)任作者, 研究員, 610193056@qq.com