強(qiáng)還原處理所使用有機(jī)物料與其殺菌效果的相互關(guān)系

劉亮亮, 崔慧靈, 孔繼婕, 張金波,2,3,4,5, 蔡祖聰,2,3,4,5, 黃新琦,2,3,4,5*

(1.南京師范大學(xué)地理科學(xué)學(xué)院, 南京 210023; 2. 虛擬地理環(huán)境教育部重點實驗室(南京師范大學(xué)),南京 210023; 3. 江蘇省地理環(huán)境演化國家重點實驗室培育建設(shè)點, 南京 210023; 4. 江蘇省物質(zhì)循環(huán)與污染控制重點實驗室, 南京 210023; 5. 江蘇省地理信息資源開發(fā)與利用協(xié)同創(chuàng)新中心, 南京 210023)

強(qiáng)還原處理所使用有機(jī)物料與其殺菌效果的相互關(guān)系

劉亮亮1, 崔慧靈1, 孔繼婕1, 張金波1,2,3,4,5, 蔡祖聰1,2,3,4,5, 黃新琦1,2,3,4,5*

(1.南京師范大學(xué)地理科學(xué)學(xué)院, 南京 210023; 2. 虛擬地理環(huán)境教育部重點實驗室(南京師范大學(xué)),南京 210023; 3. 江蘇省地理環(huán)境演化國家重點實驗室培育建設(shè)點, 南京 210023; 4. 江蘇省物質(zhì)循環(huán)與污染控制重點實驗室, 南京 210023; 5. 江蘇省地理信息資源開發(fā)與利用協(xié)同創(chuàng)新中心, 南京 210023)

強(qiáng)還原處理; 殺菌效果; 有機(jī)物料; 細(xì)菌多樣性

隨著農(nóng)業(yè)集約化種植模式的不斷發(fā)展,單一作物長期連作引起的土傳病害問題日顯突出。目前采取較多的防治措施主要有物理防治[1](土壤暴曬)、化學(xué)防治[1](殺菌劑)和生物防治[2](生防菌),雖然取得一定的成效,但均有一定的局限性,如土壤暴曬受氣候條件限制，化學(xué)防治破壞生態(tài)環(huán)境，生物防治見效慢和效果不穩(wěn)定等[3]。2000年,荷蘭Blok等[4]和日本Shinmura[5]相繼發(fā)現(xiàn)的土壤強(qiáng)還原法(reductive soil disinfestation,RSD)能夠有效地抑制植物病原菌引起的土傳病害,此方法是由向土壤中添加易降解有機(jī)碳源并灌溉至田間最大持水量以及覆蓋塑料薄膜維持土壤厭氧狀態(tài)組成。RSD過程中的極端厭氧環(huán)境[4]、微生物分解有機(jī)碳源產(chǎn)生的有毒物質(zhì)[6-8](有機(jī)酸、氨、硫化氫以及錳和鐵等低價金屬離子)及微生物群落結(jié)構(gòu)的變化[9]是其主要殺菌機(jī)理。諸多研究[10-12]表明RSD具有殺菌廣譜性和環(huán)保的特征,目前廣泛應(yīng)用于荷蘭、日本及美國等國家。近幾年,蔡祖聰?shù)萚13]研究發(fā)現(xiàn)RSD還可以有效改良因連作障礙引起的設(shè)施蔬菜地退化土壤,改善土壤理化性質(zhì)和恢復(fù)土壤種植生產(chǎn)力。

現(xiàn)階段應(yīng)用于RSD處理中的有機(jī)碳源主要包括兩種:1)以乙醇[14]和糖漿[15]為主的易降解液體碳源;2)以纖維素、木質(zhì)素和半纖維素為主的農(nóng)業(yè)固體有機(jī)廢棄物,如玉米秸稈、水稻秸稈和小麥秸稈等[16]。前者應(yīng)用成本較高,后者在我國的處理方法仍以焚燒為主,這不僅浪費資源、造成巨大的經(jīng)濟(jì)損失,還嚴(yán)重地污染大氣環(huán)境,如何處理大量的農(nóng)業(yè)固體有機(jī)廢棄物一直是個難題[17],RSD則可以高效利用這些有機(jī)廢棄物,變廢為寶。然而,目前還少有RSD過程中不同有機(jī)碳源與其殺菌效果之間相互關(guān)系的研究[18]。所以,本試驗在此基礎(chǔ)上通過收集多種不同農(nóng)業(yè)固體有機(jī)廢棄物作為RSD的有機(jī)碳源,比較它們之間殺菌效果的差異性并探討其理化性質(zhì)與殺菌效果之間的關(guān)系,為RSD物料的選擇提供科學(xué)依據(jù)。

1 材料與方法

1.1 供試土壤和有機(jī)物料

試驗土壤采自云南省石屏縣(102.34°E，23.50°N)兩塊多年種植洋桔梗的田地(云南-1和云南-2);供試有機(jī)物料:稻殼、麥麩、蘆葦和甘蔗渣采自當(dāng)?shù)夭⒂梅鬯闄C(jī)粉碎(粒徑<0.5 cm),葡萄糖和纖維素購自南京聚康醫(yī)藥化工有限公司。土壤和物料風(fēng)干后過100目篩并分別測定其理化性質(zhì)(表 1～2)。

表1 土壤理化性質(zhì)

Table 1 Physicochemical properties of the soil

樣地編號Samplingsite總有機(jī)碳/g·kg-1TOC總氮/g·kg-1TNpH電導(dǎo)率/ms·cm-1Ec銨態(tài)氮/mg·kg-1NH+4-N硝態(tài)氮/mg·kg-1NO-3-N云南-1 Yunnan-123.492.637.570.490.39301.17云南-2 Yunnan-233.553.477.480.909.86226.78

表2 有機(jī)物料理化性質(zhì)1)

Table 2 Physicochemical properties of the organic matters

有機(jī)物料Organicmatter總有機(jī)碳/g·kg-1TOC易氧化有機(jī)碳/g·kg-1EOC粒徑<2mm有機(jī)碳/g·kg-1POC水溶性有機(jī)碳/g·kg-1WSOC總氮/g·kg-1TN碳/氮C/N稻殼 Ricehull406.081.8381.65.74.5988.45麥麩 Wheatbran439.3110.0437.838.613.0133.76蘆葦 Reed427.5102.5359.949.26.8062.86甘蔗渣 Sugarcaneresidue442.484.2442.428.14.6694.93葡萄糖 Glucose385.0276.7385.0381.90.00-纖維素 Cellulose432.37.1432.30.960.00-

1) “-”：代表該物料C/N無法計算。 “-”: Unable to be calculated.

1.2 試驗設(shè)計

兩種試驗土壤各設(shè)置5個處理,云南-1:原位土壤(CK1)、土壤灌溉至最大田間持水量(45%V/W)并密封(CK2)、向土壤中分別添加2%(W/W)稻殼、麥麩和蘆葦后灌溉至最大田間持水量并密封(RSD1、RSD2和RSD3);云南-2:原位土壤(CK3)、土壤灌溉至最大田間持水量(45%V/W)并密封(CK4)、 向土壤中分別添加2%(W/W)甘蔗渣、葡萄糖和纖維素后灌溉至最大田間持水量并密封(RSD4、RSD5和RSD6)。每個處理3個重復(fù),每個重復(fù)200 g土,處理周期14 d,處理期間控溫35℃,處理結(jié)束后取樣測定其理化性質(zhì)和生物學(xué)性質(zhì)。

1.3 測定方法

1.3.1 土壤和物料理化性質(zhì)的測定

1.3.2 土壤DNA的提取和尖孢鐮刀菌、細(xì)菌及真菌的定量

土壤DNA的提取采用試劑盒Power SoilTMDNA Isolation Kit(MO BIO Laboratories Inc.,USA),DNA中的尖孢鐮刀菌、細(xì)菌及真菌的定量通過相應(yīng)的特異性引物在定量PCR儀CFX96TMReal-Time System(Bio-Rad Laboratories Inc.,Hercules,CA,USA)上進(jìn)行擴(kuò)增。反應(yīng)體系為:2 μL DNA模板,10 μL SYBR Green premixExTaq(2×,TaKaRa,Japan),正反特異性引物各1 μL(尖孢鐮刀菌:ITS1-F和AFP308;細(xì)菌:Eub338和Eub518;真菌:ITS1-f和5.8 S,表3)及6 μL無菌水。尖孢鐮刀菌定量的反應(yīng)條件:95℃ 2 min預(yù)變性;95℃ 10 s解鏈,58℃ 15 s退火,72℃ 20 s延伸,40個循環(huán);細(xì)菌和真菌的定量反應(yīng)條件:95℃ 2 min預(yù)變性;95℃ 10 s解鏈,53℃ 20 s退火,72℃ 30 s延伸,40個循環(huán)。在每個循環(huán)的延伸階段采集熒光信號,反應(yīng)結(jié)束后繪制熔解曲線。尖孢鐮刀菌、細(xì)菌及真菌的標(biāo)準(zhǔn)曲線參照López-Mondéjar等的方法構(gòu)建[21],斜率分別為-3.358、-3.368和-3.003。

表3 定量PCR和細(xì)菌PCR-DGGE引物列表

Table 3 Primers used in quantitative real-time PCR and bacterial PCR-DGGE

引物Primers序列Sequence(5'-3')參考文獻(xiàn)ReferenceITS1-F(F)CTTGGTCATTTAGAGGAAGTAA[22]AFR308(R)CGAATTAACGCGAGTCCCAAC[23]Eub338(F)ACTCCTACGGGAGGCAGCAG[24]Eub518(R)ATTACCGCGGCTGCTGG[25]ITS1-f(F)TCCGTAGGTGAACCTGCGG[26]5.8S(R)CGCTGCGTTCTTCATCG[27]GC-U968(F)L1401(R)GC-AACGCGAAGAACCTTACGCGTGTGTACAAGACCC[28][28]GC(Bacteria)CGCCCGGGGCGCGCCCCGGGCGGGGCGGGGGCACGGGGGG[29]

1.3.3 變性梯度凝膠電泳檢測土壤細(xì)菌微生物多樣性

細(xì)菌PCR擴(kuò)增所使用的通用引物為GC-U968(40 bp GC夾子)和L1401(表 3);反應(yīng)體系為:1 μL DNA模板,12.5 μL MixTaq(2×),正反引物各1 μL,9.5 μL無菌水;反應(yīng)條件:94℃ 5 min預(yù)變性;94℃ 10 s解鏈,52℃ 20 s退火,72℃ 30 s延伸,32個循環(huán)后72℃ 10 min再延伸,擴(kuò)增效果通過瓊脂糖凝膠電泳檢測。

采用D-GENE System(Bio-Rad Laboratories Inc.,Hercules,CA,USA)進(jìn)行變性梯度凝膠電泳(Denaturing gradient gel electrophoresis,DGGE)。在梯度為40%～60%的聚丙烯酰胺凝膠[6% (W/V),40% acrylamide/bis-acrylamide, 37.5:1,Bio-Rad]中加入PCR產(chǎn)物,然后以60℃、80 V電泳16 h。結(jié)束后通過成像儀檢測電泳效果,并使用Quantity One 4.6.3 軟件對采集圖像進(jìn)行多樣性分析。

1.4 數(shù)據(jù)分析

采用SPSS 19.0(SPSS Inc.,Chicago,USA)中的單因素方差、Excel 2013和Origin 9.0統(tǒng)計軟件對土壤和物料的理化性質(zhì)與生物學(xué)性質(zhì)進(jìn)行處理分析,并使用LSD最小顯著差數(shù)法檢驗各處理間的差異顯著性(P<0.05);采用SPSS 19.0統(tǒng)計軟件中的雙變量分析法分別檢測物料理化性質(zhì)與殺菌效果、細(xì)菌數(shù)量及細(xì)菌群落結(jié)構(gòu)之間的相關(guān)性。

2 結(jié)果與分析

處理結(jié)束后,稻殼、麥麩和蘆葦RSD處理土壤pH與CK1相比差異不顯著(P>0.05),CK2土壤pH顯著低于CK1和稻殼、麥麩及蘆葦RSD處理(P<0.05),稻殼RSD處理土壤pH顯著高于麥麩RSD處理;CK4和甘蔗渣RSD處理土壤pH與CK3相比差異不顯著,但葡萄糖和纖維素RSD處理土壤pH較CK3顯著下降,分別下降了0.63和0.26。

CK2和稻殼、麥麩及蘆葦RSD處理土壤電導(dǎo)率與CK1相比均顯著下降,且RSD處理土壤電導(dǎo)率顯著低于CK2;CK4和甘蔗渣、葡萄糖及纖維素RSD處理土壤電導(dǎo)率與CK3相比均顯著下降,且葡萄糖RSD處理土壤電導(dǎo)率顯著高于CK4和甘蔗渣及纖維素RSD處理(表4)。

表4 各處理結(jié)束后土壤理化性質(zhì)分析表1)

Table 4 Physicochemical properties of the soil at the end of treatments

樣地編號Samplingsite處理TreatmentpH電導(dǎo)率/ms·cm-1Ec銨態(tài)氮/mg·kg-1NH+4-N硝態(tài)氮/mg·kg-1NO-3-N云南-1 Yunnan-1CK1(7.57±0.01)ab(0.49±0.00)a(0.39±0.01)d(301.17±6.63)aCK2(7.36±0.07)c(0.40±0.01)b(0.44±0.06)d(248.53±11.00)bRSD1(7.60±0.02)a(0.29±0.00)c(3.40±0.08)c(4.54±0.39)cRSD2(7.50±0.01)b(0.23±0.02)d(64.23±1.58)a(2.86±0.14)cRSD3(7.54±0.03)ab(0.26±0.00)d(9.50±0.15)b(2.63±0.07)c云南-2 Yunnan-2CK3(7.47±0.00)a(0.90±0.00)a(9.86±0.01)d(226.78±0.01)aCK4(7.50±0.03)a(0.57±0.01)c(11.57±0.48)d(121.29±2.81)bRSD4(7.55±0.05)a(0.54±0.01)c(18.24±0.07)c(3.49±1.07)cRSD5(6.84±0.22)b(0.63±0.04)b(66.85±2.06)a(4.57±0.41)cRSD6(7.21±0.05)b(0.55±0.02)c(44.61±0.06)b(4.64±0.34)c

1) CK1和CK3為原位土壤;CK2和CK4為土壤灌溉至田間最大持水量(45%V/W)并密封;RSD1～RSD6: 200 g土壤中分別添加2%(W/W)有機(jī)物后灌溉至田間最大持水量(45%V/W)并密封,RSD1: 稻殼; RSD2: 麥麩; RSD3: 蘆葦; RSD4: 甘蔗渣; RSD5: 葡萄糖; RSD6: 纖維素。表中同列數(shù)據(jù)后不同字母表示相同采樣點不同處理經(jīng)LSD最小顯著差數(shù)法檢驗差異顯著(P<0.05),下同。 CK1 and CK3: Untreated soil; CK2 and CK4: 200 g of soil flooded (45%) to saturate and sealed with valve bag;RSD1-RSD6: 200 g of soil incorporated with 2% (W/W) organic matter and flooded to saturate following sealed with valve bag. RSD1: Rice hull, RSD2: Wheat bran, RSD3: Reed, RSD4: Sugarcane residue, RSD5: Glucose, RSD6: Cellulose. Different letters are statistically different between the different treatments of the same sampling site following LSD tests (P<0.05); The same below.

2.2 土壤尖孢鐮刀菌、細(xì)菌和真菌數(shù)量變化

處理結(jié)束后,CK2土壤尖孢鐮刀菌數(shù)量(1.86×107copies/g)與CK1(2.73×107copies/g)相比差異不顯著(P>0.05),但稻殼(3.39×106copies/g)、麥麩(2.75×106copies/g)及蘆葦 (4.20×106copies/g)RSD處理尖孢鐮刀菌數(shù)量與CK1相比分別顯著下降了88.60%、90.76%及85.91% (P<0.05);CK4(1.64×107copies/g)和纖維素(1.61×107copies/g)RSD處理土壤尖孢鐮刀菌數(shù)量與CK3(2.32×107copies/g)相比分別下降了36.7%和30.65%,但差異不顯著,而甘蔗渣(6.42×106copies/g)和葡萄糖(3.29×106copies/g)RSD處理土壤尖孢鐮刀菌數(shù)量與CK3相比分別顯著下降了72.3%和85.82%,并且甘蔗渣、葡萄糖和纖維素RSD處理間的殺菌效果差異顯著(圖1)。

處理結(jié)束后,CK2土壤細(xì)菌數(shù)量(6.18×1010copies/g)降低至CK1(8.22×1010copies/g)的75%,稻殼(9.58×1010copies/g)和蘆葦(1.07×1011copies/g)土壤細(xì)菌數(shù)量分別增加至CK1的1.16和1.30倍,但差異不顯著(P>0.05),而麥麩(1.35×1011copies/g)RSD處理土壤細(xì)菌數(shù)量顯著增加至CK1的1.64倍;CK4(2.30×1011copies/g)和甘蔗渣(2.67×1011copies/g)、葡萄糖(2.67×1011copies/g)及纖維素(1.42×1011copies/g)RSD處理土壤細(xì)菌數(shù)量分別顯著增加至CK3(7.69×1010copies/g)的2.99、3.47、3.48和1.84倍(圖1)。

圖1 不同有機(jī)物料強(qiáng)還原處理對土壤微生物數(shù)量的影響Fig.1 Effects of various organic matters on soil microbial quantity during reductive soil disinfestation

CK2土壤真菌數(shù)量(8.50×108copies/g)與CK1 (8.57×108copies/g)差異不顯著(P>0.05),但稻殼(1.33×109copies/g)、麥麩(2.24×109copies/g)和蘆葦(1.29×108copies/g)RSD處理分別顯著增加至CK1的1.55、2.61和1.50倍,且麥麩RSD土壤真菌數(shù)量顯著高于其他厭氧處理;除纖維素(1.51×109copies/g)RSD處理土壤真菌數(shù)量顯著增加至CK3(1.14×109copies/g)的1.03倍,其他厭氧處理土壤真菌數(shù)量較CK3差異不顯著(圖1)。

2.3 細(xì)菌多樣性變化

通過Quantity one 軟件對圖譜進(jìn)行分析,選用香農(nóng)-維納指數(shù)(Shannon-Wiener index,H′)和豐富度(Abundance,S)評價各處理間細(xì)菌群落結(jié)構(gòu)的變化。從圖2細(xì)菌DGGE圖譜的條帶亮度、位置及數(shù)量可見各處理間細(xì)菌多樣性差異較明顯。CK2細(xì)菌多樣性與CK1相比,H′和S均顯著下降(P<0.05),稻殼RSD處理的H′與S與CK1相比差異不顯著,麥麩和蘆葦RSD處理的H′與S均顯著高于CK1,且麥麩RSD處理的H′與S值最高,表明添加麥麩的RSD處理細(xì)菌多樣性最豐富。CK4和甘蔗渣、葡萄糖及纖維素RSD處理與CK3相比,H′均顯著下降,且葡萄糖和纖維素RSD處理的H′顯著低于CK4和甘蔗渣RSD處理;CK4的S值與CK3相比差異不顯著,甘蔗渣RSD處理的S值顯著低于CK3,但葡萄糖和纖維素RSD處理的S值顯著低于CK3、CK4和甘蔗渣RSD處理,表明添加葡萄糖和纖維素的RSD處理細(xì)菌多樣性最低(表 5)。

圖2 處理結(jié)束后細(xì)菌DGGE圖Fig.2 DGGE profiles of bacteria at the end of treatments

樣地編號Samplingsite處理Treatment細(xì)菌Bacteria香農(nóng)-維納指數(shù)H'Shannon-Wienerindex豐富度SAbundance云南-1Yunnan-1CK1(3.14±0.04)c(29.67±0.58)cCK2(2.80±0.03)d(20.67±0.58)dRSD1(3.18±0.08)c(29.67±2.08)cRSD2(3.51±0.03)a(41.33±0.58)aRSD3(3.36±0.02)b(35.00±1.00)b云南-2Yunnan-2CK3(2.95±0.01)a(22.00±0.00)aCK4(2.82±0.01)b(21.67±0.57)abRSD4(2.84±0.01)b(21.00±0.01)bRSD5(2.67±0.06)c(17.33±1.15)cRSD6(2.71±0.01)c(17.00±0.00)c

2.4 物料理化性質(zhì)與殺菌效果、細(xì)菌數(shù)量及細(xì)菌多樣性間相關(guān)性分析

當(dāng)雙變量檢驗不包含CK2和CK4時,有機(jī)物料的TOC、EOC、POC(<2 mm)和TN與殺菌效果呈極顯著正相關(guān)性(P<0.01),C/N與殺菌效果呈顯著負(fù)相關(guān)性(P<0.05);包含CK2和CK4時,TOC和POC(<2 mm)與殺菌效果無顯著相關(guān)性(P>0.05),而EOC和TN仍與殺菌效果呈顯著正相關(guān)以及C/N與殺菌效果呈顯著負(fù)相關(guān)。C/N始終與細(xì)菌數(shù)量呈極顯著正相關(guān)性,EOC在無CK2和CK4時與其呈顯著正相關(guān),而其他理化性質(zhì)與細(xì)菌數(shù)量都無顯著相關(guān)性。無論是否包含CK2和CK4,物料理化性質(zhì)中的TN始終與細(xì)菌H′和S呈極顯著正相關(guān)性,C/N始終與H′和S呈顯著負(fù)相關(guān)(表6)。

表6 物料理化性質(zhì)與殺菌效果、細(xì)菌數(shù)量及細(xì)菌多樣性相關(guān)性1)

Table 6 Correlations between physicochemical properties of the types of organic matter and disinfestation effects,bacterial populations and bacterial diversities

物料性質(zhì)Propertiesoforganicmatters殺菌效果DisinfestationeffectIW細(xì)菌BacteriumIW細(xì)菌多樣性BacterialdiversityH'IWSIW總有機(jī)碳 TOC0.63**NNNNNNN易氧化有機(jī)碳 EOC0.68**0.57*N0.49**NNNN總氮 TN0.63**0.54*NN0.91**0.91**0.955**0.962**碳/氮 C/N-0.47*-0.59*0.54**0.61**-0.95**-0.98**-0.927**-0.951**粒徑<2mm有機(jī)碳POC(d<2mm)0.599**NNNNNNN水溶性有機(jī)碳 WSOCNN0.48*0.55*NNNN

1) C/N=(土壤TOC+2%物料TOC)/(土壤TN+2%物料TN); “I” 和“W”分別表示包含CK2和CK4與不包含CK2和CK4,“*”、“**”和“N”分別表示物料性質(zhì)與殺菌效果間的相關(guān)性經(jīng)雙變量檢驗為顯著、極顯著和無相關(guān)性。 C/N was calculated using the following formula: (soil TOC+2% organic matter TOC)/(soil TN+2% organic matter TN). I and W: the treatments with and without CK2 and CK4, respectively;*,**and N represent significant correlation, highly significant correlation, and no correlation following bivariate test.

3 討論

已有的研究表明,添加不同有機(jī)物料的RSD處理殺菌效果存在差異性[16],本試驗中雖然大部分RSD處理都取得了較好的殺菌效果,但也呈現(xiàn)出顯著差異性,尤其是添加纖維素RSD處理的殺菌效果僅為30.65%(圖1),所以研究物料理化性質(zhì)與殺菌效果間的關(guān)系至關(guān)重要。

黃新琦等[6]的研究結(jié)果表明:單獨厭氧處理并不能取得較好的殺菌效果,有機(jī)碳源的添加是RSD取得成功的關(guān)鍵所在,這與本試驗物料TOC與殺菌效果間的顯著正相關(guān)(含CK2和CK4)結(jié)果一致。但在不包含CK2和CK4時TOC與殺菌效果無顯著相關(guān)性,主要是由于各物料中的TOC含量大致相同(40%左右)。物料中的TOC可大致分為易降解(如EOC)和難降解(纖維素、木質(zhì)素等)兩種形態(tài)[30-31],Mowlick等[9]通過PCR-DGGE及克隆文庫發(fā)現(xiàn)RSD處理前期厚壁菌門Firmicutes利用有機(jī)質(zhì)大量繁殖并成為優(yōu)勢種群,其中優(yōu)勢屬中碳源分解者瘤胃球菌屬Ruminococcus和產(chǎn)酸菌(如Clostridia和Coprococcus)[32]居多,且Rui 等[33]推測RSD處理前期碳源分解者能夠快速分解物料中的易降解有機(jī)碳并協(xié)同產(chǎn)酸菌產(chǎn)生有機(jī)酸,后期有機(jī)質(zhì)中剩余部分為難降解有機(jī)碳源,微生物分解緩慢,產(chǎn)酸能力也較弱,從而有機(jī)酸迅速下降,上述RSD殺菌機(jī)理中有機(jī)酸(乙酸、丙酸、丁酸和異戊酸)的殺菌作用也最為關(guān)鍵;此外,RSD處理過程中產(chǎn)生的有益微生物,如芽胞桿菌Bacillus可利用易降解有機(jī)碳源大量繁殖,這些有益微生物的拮抗作用、競爭作用及寄生作用能夠較好地抑制病原菌[2];本試驗中物料添加以EOC為主的葡萄糖RSD處理的殺菌效果(85.82%)顯著高于添加纖維素處理的殺菌效果(30.65%),推測EOC與細(xì)菌及殺菌效果間的顯著正相關(guān)性可能與生防菌的繁殖及有機(jī)酸的產(chǎn)生有關(guān)。

本試驗除了物料的碳源(TOC和EOC)與殺菌效果呈顯著正相關(guān)外,TN也與殺菌效果呈顯著正相關(guān),換言之,在物料TOC大致相同的情況下,TN越高,C/N越低,殺菌效果越好。Rodriguez-Kabana[34]和Oka[35]等的研究結(jié)果也表明物料中的C/N與對根結(jié)線蟲的抑制呈負(fù)相關(guān),這與本試驗的結(jié)果一致。主要原因可能與氨化作用有關(guān),即微生物分解有機(jī)氮過程中產(chǎn)生的有毒物質(zhì)氨具有殺菌作用,TN越高氨化作用強(qiáng)度越大,產(chǎn)生的氨也越多,從而殺菌效果也越好[36];Shrestha等[37]認(rèn)為物料C/N還與RSD處理過程中的厭氧條件呈顯著負(fù)相關(guān),即RSD處理中添加的物料C/N越低,厭氧強(qiáng)度越劇烈,而諸多研究表明RSD過程中的厭氧條件也是殺菌機(jī)理之一[4-5];此外,可能與本試驗中C/N和細(xì)菌數(shù)量及細(xì)菌多樣性分別呈顯著正相關(guān)和負(fù)相關(guān)有關(guān),由于RSD過程中優(yōu)勢種群厚壁菌門大量繁殖,導(dǎo)致細(xì)菌多樣性減少,而低C/N有利于刺激細(xì)菌的繁殖和提高它們的活性,從而利于抑制病原菌[38]。

總之,RSD方法不僅可以有效殺滅土傳病原菌,改善土壤理化性質(zhì),修復(fù)退化設(shè)施蔬菜地土壤,恢復(fù)土壤種植生產(chǎn)力,還可為高效利用農(nóng)業(yè)固體有機(jī)廢棄物提供有效途徑。本研究結(jié)果表明,高EOC和低C/N的農(nóng)業(yè)固體廢棄有機(jī)物所參與的RSD處理殺菌效果更好。

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(責(zé)任編輯：田 喆)

Relationships between different types of organic matters and disinfestation effects during reductive soil disinfestation

Liu Liangliang1, Cui Huiling1, Kong Jijie1, Zhang Jinbo1,2,3,4,5, Cai Zucong1,2,3,4,5, Huang Xinqi1,2,3,4,5

(1.CollegeofGeography,NanjingNormalUniversity,Nanjing210023,China; 2.KeyLaboratoryofVirtualGeographicEnvironment(NanjingNormalUniversity),MinistryofEducation,Nanjing210023,China; 3.StateKeyLaboratoryCultivationBaseofGeographicalEnvironmentalEvolution(JiangsuProvince),Nanjing210023,China; 4.JiangsuProvincialKeyLaboratoryofMaterialCyclingandPollutionControl,Nanjing210023,China; 5.JiangsuCenterforCollaborativeInnovationinGeographicalInformationResourceDevelopmentandApplication,Nanjing210023,China)

Although reductive soil disinfestation (RSD) through creating anaerobic condition and incorporating soil with large amounts of organic matters is an effective method for soil disinfestation, there is limited information about the relationships between the type of organic matter and disinfestation effects (DEs). Hence, in this study, various RSD treatments incorporated with rice hull (RSD1), wheat bran (RSD2), and reed (RSD3), sugarcane residue (RSD4), glucose (RSD5), and cellulose (RSD6) were respectively conducted, and quantitative real-time PCR and denaturing gradient gel electrophoresis (DGGE) were performed to investigate the relationships between organic matters and DEs. The results showed that the content of soil NO-3-N and electrical conductivity (Ec) significantly decreased in the RSD treatments, whereas the content of NH+4-N significantly increased. The DEs of these RSD treatments (RSD1-6) were 88.60%, 90.76%, 85.91%, 72.3%, 85.87%, and 30.65%, respectively. Total organic carbon (TOC), easily oxidized organic carbon (EOC), and total nitrogen (TN) in the organic matter had a significant positive correlation with DE and bacterial population (P<0.05). Besides, the initial soil C/N after incorporating the organic matters had a significant negative correlation with DE, and had a significant positive correlation with bacterial population. Therefore, the RSD treatments incorporated with the organic matters with higher EOC, TN and lower C/N would obtain a better disinfestation effect.

reductive soil disinfestation; disinfestation effect; organic matter; bacterial diversity

2016-04-14

2016-05-25

國家自然科學(xué)基金(41301335)

S 432.4

A

10.3969/j.issn.0529-1542.2017.02.012

* 通信作者 E-mail:xqhuang@njnu.edu.cn