灰黃青霉對(duì)瓜列當(dāng)?shù)姆佬Ъ皩?duì)番茄根區(qū)土壤微生物的影響*

陳 杰, 馬永清, 郭振國(guó), 薛泉宏

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灰黃青霉對(duì)瓜列當(dāng)?shù)姆佬Ъ皩?duì)番茄根區(qū)土壤微生物的影響*

陳 杰1,2, 馬永清3**, 郭振國(guó)4, 薛泉宏1

(1. 西北農(nóng)林科技大學(xué)資源環(huán)境學(xué)院 楊凌 712100; 2. 山西農(nóng)業(yè)大學(xué)農(nóng)學(xué)院 太谷 030801; 3. 中國(guó)科學(xué)院水利部水土保持研究所黃土高原土壤侵蝕與旱地農(nóng)業(yè)國(guó)家重點(diǎn)實(shí)驗(yàn)室 楊凌 712100; 4. 西北農(nóng)林科技大學(xué)林學(xué)院 楊凌 712100)

根寄生雜草瓜列當(dāng)()嚴(yán)重危害番茄()等多種經(jīng)濟(jì)作物的產(chǎn)量和品質(zhì)。如何有效防除仍是當(dāng)今瓜列當(dāng)研究重點(diǎn)之一。真菌是列當(dāng)?shù)纳酪蜃又? 但目前對(duì)農(nóng)作物無(wú)致病性的列當(dāng)生防真菌的研究尚少。本研究通過(guò)培養(yǎng)皿試驗(yàn)研究1株灰黃青霉(, CF3)的無(wú)細(xì)胞發(fā)酵濾液對(duì)瓜列當(dāng)種子萌發(fā)和發(fā)芽管生長(zhǎng)的影響, 通過(guò)盆栽試驗(yàn)研究CF3粉狀制劑對(duì)瓜列當(dāng)?shù)姆莱Ч皩?duì)寄主番茄生長(zhǎng)和根區(qū)土壤微生物的影響。結(jié)果表明: 1)培養(yǎng)皿試驗(yàn)中, CF3發(fā)酵液抑制了瓜列當(dāng)種子萌發(fā)和發(fā)芽管生長(zhǎng)。其中, 在放有瓜列當(dāng)種子與番茄幼苗的培養(yǎng)皿中, 加入CF3發(fā)酵液后培養(yǎng)6 d, 瓜列當(dāng)種子的萌發(fā)均被完全抑制; 添加CF3發(fā)酵液與霍格蘭德營(yíng)養(yǎng)液體積比為1∶2、1∶4、1∶6和1∶8的混合液培養(yǎng)8 d后, 瓜列當(dāng)種子的萌發(fā)率與對(duì)照相比分別減少80.26%、70.26%、68.10%和47.51%。CF3發(fā)酵液原液、10倍稀釋液和100倍稀釋液處理后使瓜列當(dāng)發(fā)芽管長(zhǎng)度與對(duì)照相比分別縮短100.00%、68.84%和19.24%。2)盆栽試驗(yàn)中, CF3菌劑抑制了瓜列當(dāng)?shù)某鐾梁蛦沃旯狭挟?dāng)?shù)纳L(zhǎng), 并使番茄增產(chǎn)。施加1.0 g?kg-1CF3菌劑130 d后, 瓜列當(dāng)?shù)某鐾翑?shù)量、出土率和單株瓜列當(dāng)干重分別降低76.19%、85.30%和28.48%, 番茄果實(shí)鮮重增加51.57%。此外, 灰黃青霉菌劑還調(diào)整了番茄根區(qū)土壤的微生物區(qū)系結(jié)構(gòu), 使施加菌劑130 d后番茄根區(qū)土壤中除接入CF3外真菌數(shù)量與對(duì)照相比降低75.60%, 細(xì)菌與真菌的數(shù)量之比增加117.57%。平均來(lái)看, CF3使番茄根區(qū)土壤中除CF3外真菌數(shù)量降低42.81%, 放線菌總數(shù)增加84.15%。本研究表明, 灰黃青霉CF3具有防除番茄上寄生瓜列當(dāng)?shù)哪芰? 適宜作為瓜列當(dāng)?shù)纳勒婢?/p>

瓜列當(dāng); 灰黃青霉; 真菌; 生物防治; 土壤微生物

列當(dāng)(spp.)完全靠從寄主汲取水分和營(yíng)養(yǎng)物質(zhì)來(lái)維持自身生長(zhǎng), 已經(jīng)成為嚴(yán)重影響農(nóng)業(yè)生產(chǎn)的雜草。瓜列當(dāng)()廣泛分布在地中海地區(qū)、非洲、歐洲和亞洲等區(qū)域, 可寄生在茄科(Solanaceae)、葫蘆科(Cucurbitaceae)、豆科(Leguminosae)、傘形科(Umbelliferae)和菊科(Compositae)等多種作物上, 對(duì)農(nóng)業(yè)生產(chǎn)造成嚴(yán)重危害[1-2]。僅在我國(guó)新疆, 受瓜列當(dāng)危害的加工番茄()面積就高達(dá)7 000 hm2, 造成產(chǎn)量損失可達(dá)80%[3]。

有效防除列當(dāng)仍是當(dāng)今農(nóng)業(yè)生產(chǎn)上的難題, 列當(dāng)生防微生物日益受到關(guān)注。真菌是常見的生物防治微生物。目前, 列當(dāng)生防真菌的研究多集中在列當(dāng)病原菌尤其是鐮刀菌(spp.)上[4]。向日葵列當(dāng)()植株上接種尖孢鐮刀菌(fsp.)的分生孢子培養(yǎng)液后, 其死亡率可高達(dá)85%[5]。尖孢鐮刀菌()Foxy Ⅰ和Foxy Ⅱ的孢子懸浮液也可顯著降低鋸齒列當(dāng)()和分枝列當(dāng)()的萌發(fā)率及寄生率[6]?，F(xiàn)有列當(dāng)致病真菌也可能是農(nóng)作物的病原菌, 如輪狀鐮刀菌()可使多種列當(dāng)和農(nóng)作物發(fā)病[7-8]。因此, 利用列當(dāng)病原真菌防除列當(dāng)存在使寄主作物或下茬作物致病的風(fēng)險(xiǎn)。尋找對(duì)農(nóng)作物無(wú)致病性的列當(dāng)生防真菌是解決列當(dāng)危害的可行途徑之一。目前, 已報(bào)道對(duì)農(nóng)作物無(wú)致病性的列當(dāng)生防真菌主要為叢枝菌根真菌[9-10]和疣孢漆斑霉()[11]等少數(shù)真菌。叢枝菌根真菌由于無(wú)法單獨(dú)培養(yǎng)而限制了該類真菌在生產(chǎn)中的應(yīng)用, 疣孢漆斑霉的研究目前僅局限于孢子懸液上[11]。

灰黃青霉()CF3為1株對(duì)多種作物病害病原菌有拮抗作用的生防真菌[8-9]。前期研究表明, 灰黃青霉無(wú)細(xì)胞發(fā)酵濾液具有抑制瓜列當(dāng)種子萌發(fā)的功能[10], 但尚無(wú)該真菌用于瓜列當(dāng)防除的系統(tǒng)研究。本研究通過(guò)瓜列當(dāng)種子與番茄幼苗培養(yǎng)皿內(nèi)共培養(yǎng)試驗(yàn)和培養(yǎng)皿內(nèi)種子萌發(fā)試驗(yàn), 研究灰黃青霉CF3對(duì)瓜列當(dāng)種子萌發(fā)和發(fā)芽管生長(zhǎng)的影響, 并通過(guò)盆栽試驗(yàn)研究在未滅菌土壤條件下灰黃青霉CF3活孢子制劑對(duì)番茄上寄生瓜列當(dāng)?shù)膶?shí)際防除效果及對(duì)寄主番茄生長(zhǎng)和番茄根區(qū)土壤微生物的作用。本研究可為番茄根寄生瓜列當(dāng)?shù)恼婢莱峁┬滤悸芳坝袘?yīng)用價(jià)值的新菌株。

1 材料與方法

1.1 微生物和植物材料

生防真菌: 灰黃青霉CF3由西北農(nóng)林科技大學(xué)資源環(huán)境學(xué)院微生物資源研究室提供, 分離自陜西健康草莓根區(qū)土壤中[12]。

瓜列當(dāng): 瓜列當(dāng)種子采自2011年新疆焉耆縣受瓜列當(dāng)寄生的番茄田中。

番茄: 本研究所用番茄品種為‘白果強(qiáng)豐’, 為易受瓜列當(dāng)寄生的品種, 購(gòu)自陜西楊凌種子公司。

1.2 培養(yǎng)皿共培養(yǎng)試驗(yàn)

瓜列當(dāng)種子表面消毒和預(yù)培養(yǎng): 將瓜列當(dāng)種子先后放于1.0%(/)NaClO溶液和75%的乙醇中分別超聲處理3.0 min。列當(dāng)種子用無(wú)菌水沖洗5次后置于超凈工作臺(tái)中晾干。將滅菌后玻璃纖維濾紙片(直徑8.0 mm)擺放于放有雙層濕潤(rùn)濾紙的培養(yǎng)皿中。隨后, 將30~60粒晾干后的列當(dāng)種子均勻撒于各玻璃纖維濾紙片上。培養(yǎng)皿封口后放置于25 ℃黑暗環(huán)境下培養(yǎng)4 d, 備用。

CF3無(wú)細(xì)胞發(fā)酵濾液(以下簡(jiǎn)稱發(fā)酵液)制備: CF3發(fā)酵液按照Chen等[13]液態(tài)搖床培養(yǎng)的方法進(jìn)行。用不同濃度的霍格蘭德(Hogland)營(yíng)養(yǎng)液[14]將CF3的發(fā)酵液原液稀釋至體積比(CF3∶Hogland)分別為1∶2、1∶4、1∶6和1∶8, 同時(shí)使Hogland營(yíng)養(yǎng)液保持正常濃度。

番茄幼苗的準(zhǔn)備: 將番茄種子播種于裝有蛭石的穴盤中, 每穴6粒。將穴盤放置于光照和黑暗時(shí)間分別為12 h周期的25 ℃培養(yǎng)間內(nèi)培養(yǎng)并按需澆水。待出苗后, 各穴中番茄幼苗僅保留1株。當(dāng)生長(zhǎng)至約10.0 cm高時(shí), 挑選健康生長(zhǎng), 大小一致的番茄幼苗, 小心拔出后用自來(lái)水將根系上蛭石沖洗干凈, 再用無(wú)菌水沖洗3次, 備用。

將8 g滅菌后的蛭石放于側(cè)邊開有1個(gè)約1 cm× 1 cm方孔的直徑為9.0 cm塑料培養(yǎng)皿中。隨后, 依次放入1片滅菌普通濾紙(直徑約8.5 cm)和1片滅菌玻璃纖維濾紙(直徑約8.5 cm)。加入30.0 mL上述各混合溶液后, 向?yàn)V紙上均勻撒入已表面消毒的瓜列當(dāng)種子, 各處理3個(gè)培養(yǎng)皿重復(fù)。以僅加入Hogland營(yíng)養(yǎng)液的處理作為對(duì)照。最后, 各培養(yǎng)皿濾紙上放入1株上述事先準(zhǔn)備好的番茄幼苗。各培養(yǎng)皿用3層封口膜密封后垂直放置于紙盒中并用黑色塑料袋包裹后置于25 ℃、光照和黑暗時(shí)間交替為12 h的光照培養(yǎng)箱中培養(yǎng)。每隔1 d, 從方孔向各培養(yǎng)皿中添加10.0 mL Hogland營(yíng)養(yǎng)液后再次將其封好。于培養(yǎng)6 d、8 d、10 d、12 d和14 d后, 用解剖顯微鏡觀察番茄根系周圍列當(dāng)種子萌發(fā)情況。各培養(yǎng)皿選擇7個(gè)顯微鏡視野統(tǒng)計(jì)瓜列當(dāng)種子的萌發(fā)數(shù)和總數(shù), 并按公式(1)和(2)分別計(jì)算列當(dāng)種子的萌發(fā)率和萌發(fā)抑制率。

G(%)=(G/T)×100 (1)

P(%)=[(G–G)/G]×100 (2)

式中:G為列當(dāng)種子萌發(fā)率,G為萌發(fā)列當(dāng)種子數(shù),T為列當(dāng)種子總數(shù),P為列當(dāng)種子萌發(fā)抑制率,G為對(duì)照中列當(dāng)種子萌發(fā)率,G為CF3發(fā)酵液處理后列當(dāng)種子萌發(fā)率。

1.3 CF3對(duì)瓜列當(dāng)發(fā)芽管生長(zhǎng)影響試驗(yàn)

按1.2中方法制備CF3發(fā)酵液原液并將其分別稀釋10倍和100倍后備用。

將玻璃纖維濾紙片(直徑8.0 mm)擺放于直徑9.0 cm的培養(yǎng)皿中。隨后, 分別向各玻璃纖維濾紙片上先后加入20.0 μL CF3發(fā)酵液、1片預(yù)培養(yǎng)后的瓜列當(dāng)種子片和20.0 μL 0.1 mg?L-1的GR24(人工合成的列當(dāng)種子萌發(fā)誘導(dǎo)物, 由澳大利亞新威爾士州悉尼大學(xué)化學(xué)學(xué)院的Christopher McErlean提供)。以20.0 μL 0.1 mg?L-1的GR24代替CF3發(fā)酵液的處理作為對(duì)照。各處理3個(gè)濾紙片。將各培養(yǎng)皿封口后置于25 ℃黑暗環(huán)境下培養(yǎng)10 d后, 將瓜列當(dāng)種子片放于顯微鏡下拍照。各處理隨機(jī)選擇30粒已經(jīng)萌發(fā)的列當(dāng)種子, 用GetData Graph Digitizer 2.24軟件測(cè)量其發(fā)芽管長(zhǎng)度, 并按公式(3)計(jì)算CF3對(duì)瓜列當(dāng)發(fā)芽管長(zhǎng)度的抑制率:

L(%)=[(C–T)/C]×100 (3)

式中:L為CF3發(fā)酵液對(duì)瓜列當(dāng)發(fā)芽管長(zhǎng)度的抑制率,C為對(duì)照中瓜列當(dāng)發(fā)芽管長(zhǎng)度,T為CF3發(fā)酵液處理后瓜列當(dāng)發(fā)芽管長(zhǎng)度。

1.4 盆栽試驗(yàn)

試驗(yàn)方案: 對(duì)照, 拌土加入瓜列當(dāng)種子; CF3處理, 拌土加入瓜列當(dāng)種子和CF3菌劑(按Chen等[13]的方法制備, 其中灰黃青霉的活孢子含量為109個(gè)?g-1干菌劑)。

盆栽試驗(yàn)于2014年5—9月在寧夏回族自治區(qū)西北農(nóng)林科技大學(xué)水土保持研究所固原生態(tài)試驗(yàn)站進(jìn)行。盆栽試驗(yàn)所用土壤為黑壚土。取田間耕層土壤與細(xì)沙按質(zhì)量比1∶1混合, 加入尿素(20.0 mg?kg-1)、過(guò)磷酸鈣(50.0 mg?kg-1)、瓜列當(dāng)種子(3.4 mg?kg-1)和灰黃青霉菌劑(1.0 g?kg-1)充分混勻后裝入高25.0 cm、直徑為20.0 cm的塑料盆中, 每盆裝土8.0 kg。各處理6盆重復(fù)。

按1.2中方法于穴盤中培育番茄幼苗。待長(zhǎng)至約10.0 cm高后, 將番茄幼苗拔出移栽至裝有上述混合土壤的塑料盆中, 每盆1株。所有處理移栽后均按需澆水和除草。

移栽番茄幼苗后70 d, 從各處理中分別選取3盆采樣, 另外3盆于移栽后130 d采樣。于兩次采樣時(shí)期, 統(tǒng)計(jì)列當(dāng)出土數(shù)和寄生總數(shù), 稱取列當(dāng)總干重, 并根據(jù)公式(4)和(5)分別計(jì)算列當(dāng)?shù)某鐾谅屎蛦沃炅挟?dāng)干重; 測(cè)量番茄株高, 統(tǒng)計(jì)番茄果實(shí)個(gè)數(shù), 并稱量番茄莖葉干重和果實(shí)鮮重。此外, 在移栽后130 d采樣時(shí)采集番茄根系并稱取干重。

E(%)=(E/T)×100 (4)

(g)=T/T(5)

式中:E為瓜列當(dāng)出土率,E為瓜列當(dāng)出土數(shù)量,T為瓜列當(dāng)寄生總數(shù),為瓜列當(dāng)單株干重,T為瓜列當(dāng)總干重,T為瓜列當(dāng)寄生總數(shù)。

在兩采樣時(shí)期均采集番茄根區(qū)土壤, 采用稀釋涂平皿法[15]對(duì)其中細(xì)菌、放線菌和真菌進(jìn)行分離并統(tǒng)計(jì)相應(yīng)數(shù)量, 計(jì)算細(xì)菌與真菌及放線菌與真菌的數(shù)量比。同時(shí), 統(tǒng)計(jì)CF3的數(shù)量, 計(jì)算CF3在真菌總數(shù)中的定殖率及除CF3外真菌的數(shù)量。各指標(biāo)處理與對(duì)照相比的增加率按照公式(6)計(jì)算。

I(%) =[(-)/]×100 (6)

式中:I為處理與對(duì)照相比的增加率,為處理的數(shù)值,為對(duì)照的數(shù)值。

1.5 統(tǒng)計(jì)分析

數(shù)據(jù)分析采用Microsoft Excel 2010和SPSS 18.0軟件進(jìn)行。試驗(yàn)數(shù)據(jù)均進(jìn)行單因素方差分析。CF3抑制瓜列當(dāng)種子萌發(fā)和發(fā)芽管長(zhǎng)度的數(shù)據(jù)采用Tukey法進(jìn)行分析(<0.05)。其中, 對(duì)瓜列當(dāng)種子萌發(fā)率進(jìn)行多重比較時(shí), 先進(jìn)行反正弦轉(zhuǎn)換, 再進(jìn)行多重比較。盆栽試驗(yàn)數(shù)據(jù)均采用兩獨(dú)立樣本的檢驗(yàn)方法進(jìn)行分析(<0.05)。

2 結(jié)果與分析

2.1 CF3對(duì)番茄誘導(dǎo)瓜列當(dāng)種子萌發(fā)的影響

將列當(dāng)種子與番茄幼苗于培養(yǎng)皿內(nèi)共培養(yǎng)6 d后, 不添加CF3發(fā)酵液的對(duì)照中, 番茄根系周圍瓜列當(dāng)種子的萌發(fā)率為23.48%; 而添加不同比例CF3發(fā)酵液的處理中, 番茄根系周圍瓜列當(dāng)種子的萌發(fā)均被完全抑制。培養(yǎng)8 d后, CF3發(fā)酵液對(duì)瓜列當(dāng)種子萌發(fā)的抑制率為47.51%~80.26%。培養(yǎng)10 d后, 在添加CF3發(fā)酵液與Hogland營(yíng)養(yǎng)液體積比(CF3∶Hogland)為1∶2、1∶4和1∶6的處理中, 瓜列當(dāng)種子的萌發(fā)仍被抑制, 抑制率分別為45.58%、24.89%和24.80%。繼續(xù)培養(yǎng)至12 d和14 d后, 僅添加CF3∶Hogland為1∶2溶液的處理在培養(yǎng)14 d后瓜列當(dāng)種子萌發(fā)率減少了19.23%。上述指標(biāo)對(duì)照與處理間差異顯著(<0.05)。CF3發(fā)酵液能夠抑制番茄根系誘導(dǎo)的瓜列當(dāng)種子的萌發(fā)。隨添加比例降低和培養(yǎng)時(shí)間延長(zhǎng), CF3發(fā)酵液對(duì)瓜列當(dāng)種子萌發(fā)的抑制作用減弱(表1)。

表1 灰黃青霉CF3發(fā)酵液對(duì)番茄誘導(dǎo)瓜列當(dāng)種子萌發(fā)的抑制作用

表中數(shù)據(jù)為平均值±標(biāo)準(zhǔn)差(=3)。同列數(shù)據(jù)后不同小寫字母表示處理間數(shù)值經(jīng)反正弦轉(zhuǎn)換再經(jīng)Tukey檢驗(yàn)后差異顯著(<0.05)。CF3: volume of cell-free culture filtrate ofCF3;Hogland: Hogland solution volume;G: germination rate;P: percent reduction. Values are means ± standard deviation (= 3). Different lowercase letters in the same column indicate significant differences (arcsine transformed) among treatments (< 0.05) based on the Tukey’s test.

2.2 CF3對(duì)瓜列當(dāng)發(fā)芽管生長(zhǎng)及形態(tài)的影響

培養(yǎng)皿內(nèi)種子萌發(fā)試驗(yàn)中, CF3發(fā)酵液原液完全抑制了瓜列當(dāng)發(fā)芽管的生長(zhǎng)。經(jīng)CF3發(fā)酵液10倍和100倍稀釋液處理后的瓜列當(dāng)發(fā)芽管長(zhǎng)度分別為0.36 mm和0.94 mm, 與對(duì)照(1.16 mm)相比分別縮短68.84%和19.24%(0.05)(圖1)。GR24處理的對(duì)照中, 瓜列當(dāng)發(fā)芽管表面光滑, 呈透明或半透明狀。經(jīng)CF3發(fā)酵液處理后的瓜列當(dāng)盡管發(fā)芽管長(zhǎng)度縮短, 但在形態(tài)上與對(duì)照相比無(wú)明顯差異。CF3發(fā)酵液對(duì)萌發(fā)后瓜列當(dāng)發(fā)芽管生長(zhǎng)的抑制作用隨稀釋倍數(shù)的增加而逐漸減弱(圖2)。

圖1 灰黃青霉CF3發(fā)酵液對(duì)瓜列當(dāng)發(fā)芽管長(zhǎng)度的抑制作用

CK: 0.1 mg?L-1GR24處理。圖中誤差線為標(biāo)準(zhǔn)差(=30)。圖中不同小寫字母表示處理間數(shù)值經(jīng)Tukey檢驗(yàn)差異顯著(0.05)。CK: treated by 0.1 mg?L-1GR24. Bars in the figure mean standard deviation (= 30). Different lowercase letters in the figure indicate significant differences among treatments (< 0.05) based on the Tukey’s test.

圖2 灰黃青霉CF3發(fā)酵液對(duì)瓜列當(dāng)發(fā)芽管形態(tài)的影響

a、b、c和CK分別為CF3發(fā)酵液原液、10倍、100倍稀釋液和0.1 mg?L-1GR24處理10 d后的瓜列當(dāng)發(fā)芽管形態(tài)。a, b, c and CK are the forms ofgerm-tubes treated by the un-diluted, 10 fold, 100 fold diluted cell-free culture filtrates ofCF3, and GR24 at 0.1 mg?L-1for 10 days, respectively.

2.3 CF3菌劑對(duì)番茄根寄生瓜列當(dāng)?shù)姆莱饔?/h3>

2.3.1 對(duì)番茄根寄生瓜列當(dāng)?shù)囊种谱饔?/p>

移栽番茄后130 d, 施加CF3菌劑處理的瓜列當(dāng)出土數(shù)量為每盆1.67個(gè), 瓜列當(dāng)出土率為12.09%, 單株瓜列當(dāng)干重為0.70 g, 與對(duì)照相比分別降低了76.19%、85.30%和28.48%。移栽70 d 后, CF3菌劑使單株瓜列當(dāng)干重與對(duì)照相比也減少83.28%。兩次采樣的平均值中, 施加CF3菌劑使瓜列當(dāng)出土率和單株瓜列當(dāng)干重與對(duì)照相比分別降低90.19%和55.64%。上述指標(biāo)處理與對(duì)照間差異均顯著(0.05)。此外, CF3菌劑還使移栽70 d后瓜列當(dāng)?shù)某鐾翑?shù)量和出土率與對(duì)照相比分別減少50.00%和87.01%, 但對(duì)照與處理間差異均不顯著。兩次采樣時(shí)期, CF3菌劑對(duì)瓜列當(dāng)總干重均無(wú)顯著影響。然而, 施加灰黃青霉菌劑130 d 后, 瓜列當(dāng)?shù)募纳倲?shù)與對(duì)照相比增加57.69%(0.05); 但在移栽后70 d和兩次采樣平均值中, CF3對(duì)瓜列當(dāng)?shù)募纳倲?shù)均無(wú)顯著影響?？傮w來(lái)看, 施加CF3活菌制劑對(duì)番茄生長(zhǎng)后期瓜列當(dāng)?shù)某鐾良皢沃旯狭挟?dāng)?shù)纳L(zhǎng)有較強(qiáng)抑制作用(表2)。

2.3.2 對(duì)寄主番茄生長(zhǎng)的影響

移栽130 d后, 施加灰黃青霉CF3菌劑的處理中, 番茄果實(shí)鮮重為每株346.81 g, 與對(duì)照(228.82 g)相比顯著增加51.57%(0.05)。施加CF3菌劑也使同一采樣時(shí)期番茄根系干重與對(duì)照相比增加了12.46%, 但對(duì)照與處理間差異不顯著。移栽70 d后, CF3菌劑使番茄果實(shí)個(gè)數(shù)與對(duì)照相比顯著增加100.00%(0.05)。平均來(lái)看, CF3菌劑使番茄果實(shí)個(gè)數(shù)與不施加菌劑的對(duì)照相比顯著增加66.67%(0.05); 使番茄果實(shí)鮮重增加24.91%, 但對(duì)照與處理間差異不顯著。CF3菌劑在兩采樣時(shí)期對(duì)番茄株高和莖葉干重均無(wú)顯著影響?？傮w來(lái)看, CF3菌劑對(duì)番茄植株生長(zhǎng)影響不大, 但對(duì)番茄果實(shí)個(gè)數(shù)和鮮重有增加作用(表3)。

2.3.3 對(duì)寄主根區(qū)土壤微生物區(qū)系的影響

移栽番茄70 d后, 灰黃青霉CF3菌劑使番茄根區(qū)土壤中細(xì)菌和放線菌的數(shù)量與不施加菌劑對(duì)照相比分別增加142.35%和126.26%。移栽番茄130 d后, CF3菌劑使番茄根區(qū)土壤中除CF3外真菌數(shù)量比對(duì)照降低75.60%, 細(xì)菌與真菌的數(shù)量之比與對(duì)照相比增加117.57%。平均來(lái)看, CF3使番茄根區(qū)土壤中除CF3外真菌數(shù)量降低42.81%, 放線菌總數(shù)增加84.15%。上述指標(biāo)對(duì)照與處理間差異顯著(0.05)。CF3對(duì)番茄根區(qū)土壤中放線菌與真菌的數(shù)量之比無(wú)顯著影響。移栽番茄70 d、130 d后及兩次采樣平均值中, 灰黃青霉CF3在番茄根區(qū)土壤真菌中的定殖率分別為54.23%、46.34%及50.29%(表4)。

表2 盆栽試驗(yàn)中灰黃青霉CF3菌劑對(duì)瓜列當(dāng)寄生數(shù)量和生物量的影響

CF3: 灰黃青霉。表中數(shù)據(jù)為平均值±標(biāo)準(zhǔn)差(=3或6)。*表示處理與對(duì)照經(jīng)檢驗(yàn)差異顯著(0.05)。CF3:. Values are means ± standard deviation (=3 or 6). * in the table indicates significant difference (< 0.05) between CF3 and CK treatments based on thetest.

表3 盆栽試驗(yàn)中灰黃青霉CF3菌劑對(duì)番茄生長(zhǎng)的影響

CF3: 灰黃青霉。表中數(shù)據(jù)為平均值±標(biāo)準(zhǔn)差(=3或6)。*表示處理數(shù)值與相應(yīng)對(duì)照相比經(jīng)檢驗(yàn)差異顯著(0.05)。CF3:. Values are means ± standard deviation (= 3 or 6). * in the table indicates significant difference (< 0.05) between CF3 and CK treatments based on thetest.

表4 盆栽試驗(yàn)中灰黃青霉CF3菌劑對(duì)番茄根區(qū)土壤微生物區(qū)系的影響

CF3: 灰黃青霉表中數(shù)據(jù)為平均值±標(biāo)準(zhǔn)差(=3或6)。*表示處理數(shù)值與相應(yīng)對(duì)照相比經(jīng)檢驗(yàn)差異顯著(0.05)。CF3:. Values are means ± standard deviation (= 3 or 6). * indicates significant difference (< 0.05) between CF3 and CK treatments based on thetest.

3 討論

本研究中, 灰黃青霉CF3的無(wú)細(xì)胞發(fā)酵濾液在培養(yǎng)皿試驗(yàn)中抑制了瓜列當(dāng)?shù)姆N子萌發(fā)及萌發(fā)后瓜列當(dāng)發(fā)芽管的生長(zhǎng)。盆栽試驗(yàn)中, CF3活菌制劑降低了番茄生長(zhǎng)后期瓜列當(dāng)?shù)某鐾翑?shù)量和出土率, 減少了單株瓜列當(dāng)?shù)纳锪? 最終減輕了瓜列當(dāng)對(duì)寄主番茄的危害, 使番茄增產(chǎn)。

本研究中, CF3發(fā)酵液對(duì)瓜列當(dāng)種子萌發(fā)和發(fā)芽管生長(zhǎng)均有抑制作用。已有研究表明, 假單胞菌(spp.)和芽孢桿菌(spp.)能夠抑制列當(dāng)發(fā)芽管的伸長(zhǎng)[16]。來(lái)源于真菌的球香豆榴素和細(xì)胞松弛素也能夠抑制列當(dāng)發(fā)芽管的生長(zhǎng)[17]。本團(tuán)隊(duì)前期研究表明, 灰黃青霉CF3通過(guò)產(chǎn)生展青霉素抑制了GR24誘導(dǎo)的瓜列當(dāng)種子萌發(fā)[18]。本研究進(jìn)一步表明, CF3發(fā)酵液還能夠抑制番茄幼苗誘導(dǎo)的瓜列當(dāng)種子萌發(fā), 并對(duì)GR24誘導(dǎo)萌發(fā)后瓜列當(dāng)發(fā)芽管的生長(zhǎng)也有強(qiáng)烈抑制作用。一些化合物作用于列當(dāng)后會(huì)破壞列當(dāng)發(fā)芽管的正常生長(zhǎng)。-sphaeropsidone處理后的列當(dāng)發(fā)芽管頂端有異狀突起, 而由-sphaeropsidone或sphaeropsidin A處理后的列當(dāng)發(fā)芽管出現(xiàn)壞死現(xiàn)象[17]。然而, 在本研究中, CF3發(fā)酵液對(duì)瓜列當(dāng)發(fā)芽管形態(tài)無(wú)影響。

盆栽試驗(yàn)中, 施加CF3活菌制劑抑制了瓜列當(dāng)?shù)某鐾梁蛦沃旯狭挟?dāng)?shù)纳L(zhǎng), 并使番茄增產(chǎn)。列當(dāng)出土數(shù)量和生物量是評(píng)價(jià)生防菌防除列當(dāng)效果的重要指標(biāo)。盆栽試驗(yàn)中施加列當(dāng)病原菌尖孢鐮刀菌和腐皮鐮刀菌()均降低了分枝列當(dāng)?shù)某鐾翑?shù)量和生物量[19-20]; 熒光假單胞菌()也能夠降低和分枝列當(dāng)?shù)某鐾翑?shù)量及干重[21]。本研究中, 將CF3菌劑施加于盆栽土壤后, 阻礙了寄生后瓜列當(dāng)?shù)某鐾敛⒁种屏思纳髥沃旯狭挟?dāng)?shù)纳L(zhǎng)?；尹S青霉在土壤中通過(guò)作用于寄生后瓜列當(dāng), 抑制了寄生于番茄根系上單株瓜列當(dāng)?shù)纳L(zhǎng), 使其出土慢, 個(gè)體小, 從而減少了瓜列當(dāng)對(duì)寄主番茄養(yǎng)分的掠奪, 減輕了瓜列當(dāng)對(duì)寄主番茄造成的危害, 最終使番茄增產(chǎn)。

此外, 值得注意的是, 盆栽施加CF3菌劑130 d后, 在降低瓜列當(dāng)出土率和單株生物量的同時(shí)也增加了瓜列當(dāng)?shù)募纳倲?shù), 但列當(dāng)總干重并未增加, 表明CF3菌劑可抑制單株瓜列當(dāng)生長(zhǎng)。在土壤中, 列當(dāng)在寄主根系分泌特殊化學(xué)物質(zhì)的誘導(dǎo)下萌發(fā)并寄生于周圍的根系上[22]。多種生防菌施加于土壤后對(duì)作物根系有促生作用[23-25]。本試驗(yàn)中, 施加CF3菌劑使番茄根系生物量與不施加菌劑的對(duì)照相比在數(shù)值上增加了12.46%, 表明CF3具有促進(jìn)番茄根系生長(zhǎng)的潛能。CF3菌劑一方面可能通過(guò)促進(jìn)番茄根系發(fā)育來(lái)增加列當(dāng)寄生的位點(diǎn); 另一方面也可能通過(guò)促進(jìn)番茄根系生長(zhǎng), 刺激番茄根系產(chǎn)生更多根系分泌物, 進(jìn)而誘導(dǎo)更多的瓜列當(dāng)種子萌發(fā), 最終增加了瓜列當(dāng)成功寄生的概率。

灰黃青霉CF3通過(guò)調(diào)整寄主番茄根區(qū)土壤的微生物結(jié)構(gòu)來(lái)減輕瓜列當(dāng)對(duì)番茄的危害。列當(dāng)寄生會(huì)使對(duì)植物生長(zhǎng)有益的細(xì)菌和放線菌的數(shù)量減少[26]。來(lái)源于土壤中的有益微生物具有修復(fù)土壤微生物結(jié)構(gòu)的功能[27-29]。盆栽施加淡紫褐鏈霉菌()活孢子制劑后, 在增加寄主向日葵根區(qū)土壤中細(xì)菌數(shù)量及比例的同時(shí)也降低了向日葵列當(dāng)?shù)某鐾翑?shù)量[13]。本研究中, 施加灰黃青霉CF3活孢子菌劑增加了細(xì)菌與真菌的數(shù)量之比, 降低了除CF3外真菌的數(shù)量; 同時(shí)降低了瓜列當(dāng)?shù)某鐾翑?shù)量和出土率。通過(guò)將活菌制劑施加于土壤中來(lái)調(diào)整番茄根區(qū)土壤中微生物結(jié)構(gòu)可能是灰黃青霉CF3減輕瓜列當(dāng)對(duì)番茄危害的主要機(jī)制之一。施加CF3菌劑后, 番茄上瓜列當(dāng)出土數(shù)量和出土率與寄主番茄根區(qū)土壤中微生物變化之間的關(guān)系仍待進(jìn)一步詳細(xì)研究。

利用灰黃青霉CF3防除列當(dāng)具有對(duì)寄主及下茬作物無(wú)害, 能夠在土壤中良好定殖等優(yōu)點(diǎn)。目前, 關(guān)于列當(dāng)生防真菌的研究多集中在列當(dāng)病原菌上, 大多是通過(guò)將列當(dāng)病原真菌施用于土壤或已經(jīng)出土的列當(dāng)植株使其發(fā)病來(lái)減輕列當(dāng)造成的危害[5-6]。然而, 列當(dāng)病原真菌也可能是農(nóng)作物的病原菌, 利用列當(dāng)病原菌防除列當(dāng)存在使寄主或下茬作物患病的風(fēng)險(xiǎn)。本文所用灰黃青霉CF3來(lái)源于健康農(nóng)田土壤, 對(duì)多種作物病原菌有拮抗作用, 對(duì)農(nóng)作物無(wú)致病性。因此, 將CF3施加到有列當(dāng)?shù)耐寥乐? 既能夠防除列當(dāng)、提高寄主作物的抗病性, 又對(duì)寄主及下茬作物無(wú)害。CF3還能夠有效定殖于寄主根區(qū)土壤中而不污染土壤環(huán)境。因此, 灰黃青霉CF3適宜作為番茄上寄生瓜列當(dāng)?shù)纳锓莱?。田間條件下灰黃青霉對(duì)番茄上寄生瓜列當(dāng)?shù)姆莱Ч孕柽M(jìn)一步試驗(yàn)研究。

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Effect ofon control ofand microorganisms in rhizosphere soils of tomato*

CHEN Jie1,2, MA Yongqing3**, GUO Zhenguo4, XUE Quanhong1

(1. College of Natural Resources and Environment, Northwest A&F University, Yangling 712100, China; 2. College of Agriculture, Shanxi Agricultural University, Taigu 030801, China; 3. The State Key Laboratory of Soil Erosion and Dryland Farming, Institute of Soil and Water Conservation, Chinese Academy of Sciences and Ministry of Water Resources, Yangling 712100, China; 4. College of Forestry, Northwest A&F University, Yangling 712100, China)

Rootparasitic weedadversely affects yield and quality of tomato (). The means of effective control is still the focus inresearch. Fungus is one of the biocontrol agents ofspp.. However, few studies have been done on the use of non-pathogentic fungi to controlspp. weed. In this study, the effects of cell-free culture filtrate of, a non-pathogentic fungus strain of, onseed germination and germ tube growth were investigated in a petri-dish experiment. In addition, a pot experiment was conducted to explore the effect of powderedinoculum on weedycontrol. The effects ofinoculum on the growth of host tomato plants and the change in microflora in rhizosphere soils of tomato plants were also investigated. Results showed that: 1) in the petri-dish experiment, cell-free culture filtrate ofsignificantly inhibited bothseed germination and germ tube growth. Whenseeds and tomato seedlings were co-cultured for 6 days,seed germination was completely inhibited (100.0%) in treatments withcell-free culture filtrate. After co-culturing for 6 days,seed germination rates reducedby 80.26%, 70.26%, 68.10% and 47.51%, respectively in treatments with volume ratios ofcell-free culture filtrate and Hogland nutrient solution ratios of 1∶2, 1∶4, 1∶6 and 1∶8. The lengths ofgerm tubes significantly reduced by 100.00%, 68.84% and 19.24%, respectively when treated by undiluted, 10-fold diluted and 100-fold dilutedcell-free culture filtrate. 2) In the pot experiment,inoculum inhibited the emergence oftubercles and the growth of individualtubercle, but simultaneously increased tomato fruit yield. The number of epigealtubercles, epigeal rate oftubercles and dry weight of individualtubercles all significantly reduced after the application of powderedinoculum at 1.0 g?kg-1for 130 days respectively by 76.19%, 85.30% and 28.48% than the control. After the application ofinoculum for 130 days, tomato fruit yield was 346.8 g per plant (51.57%) more than the control (228.8 g). In addition,also adjusted microflora structure in rhizosphere soils of tomato plants. The application ofinoculum reduced fungi population (excluding CF3) and significantly increased population ratio of bacteria to fungi in rhizosphere soils of tomato plants by 75.60% and 117.57%, respectively, compared with the control. On average, application ofinoculant reduced fungi population (excluding) and increased actinomycetes population in rhizosphere soils of tomato plants respectively by 42.81% and 84.15% over the control. In conclusion,had the ability to controlinfection of tomato plant with fungus as suitable biological agent to control.

;; Fungus; Biocontrol; Soil microorganism

, E-mail: mayongqing@ms.iswc.ac.cn

Oct. 10, 2018;

Dec. 20, 2018

Q939.96

A

2096-6237(2019)05-0766-08

10.13930/j.cnki.cjea.180903

陳杰, 馬永清, 郭振國(guó), 薛泉宏. 灰黃青霉對(duì)瓜列當(dāng)?shù)姆佬Ъ皩?duì)番茄根區(qū)土壤微生物的影響[J]. 中國(guó)生態(tài)農(nóng)業(yè)學(xué)報(bào)(中英文), 2019, 27(5): 766-773

CHEN J, MA Y Q, GUO Z G, XUE Q H. Effect ofon control ofand microorganisms in rhizosphere soils of tomato[J]. Chinese Journal of Eco-Agriculture, 2019, 27(5): 766-773

* 新疆生產(chǎn)建設(shè)兵團(tuán)農(nóng)業(yè)與社會(huì)發(fā)展領(lǐng)域科技計(jì)劃項(xiàng)目(2016AC007)資助

馬永清, 主要研究方向?yàn)榧纳参锱c植物化感作用。E-mail: mayongqing@ms.iswc.ac.cn

陳杰, 主要研究方向?yàn)橥寥牢⑸?。E-mail: chenjie03306@163.com

2018-10-10

2018-12-20

* This study was supported by the Science and Technology Plan for the Field of Agriculture and Social Development by the Xinjiang Production and Construction Corps (2016AC007).