拮抗酵母控制果蔬采后真菌病害研究進展

王 瑤,姜冬梅,王劉慶,韋迪哲,王 蒙

(北京農(nóng)業(yè)質(zhì)量標準與檢測技術(shù)研究中心,農(nóng)業(yè)部農(nóng)產(chǎn)品質(zhì)量安全風(fēng)險評估實驗室(北京),北京 100097)

新鮮果蔬營養(yǎng)價值很高,是人類平衡膳食的重要組成部分。但果蔬在采后的運輸、貯藏、加工等過程中,水分流失、營養(yǎng)物質(zhì)消耗和抗病能力下降等導(dǎo)致的品質(zhì)損失十分嚴重。果蔬中高營養(yǎng)物質(zhì)、高水分及低pH適宜病原真菌生長,所以,由病原真菌引起的腐爛非常普遍。據(jù)統(tǒng)計,我國采后處理過程中由病原真菌引起的果蔬損失率高達30%,即使在發(fā)達國家,果蔬損失率也可達20%～25%[1]。除了嚴重的經(jīng)濟損失外,果蔬采后病害對人類健康造成的安全隱患也不容忽視,青霉菌(Penicillium)、鏈格孢菌(Alternaria)、鐮刀菌(Fusarium)等病原真菌能夠在引起果蔬病害的同時在腐爛部位積累真菌毒素,從而危害人類健康。例如能夠引起多種果蔬青霉病的擴展青霉(Penicillium expansum),此種病原菌產(chǎn)生的棒曲霉素、桔霉素都是潛在的致癌物質(zhì)[2]。

目前主要采用化學(xué)方法來防治果蔬病害,但長期使用化學(xué)殺菌劑會使病原菌產(chǎn)生抗性,化學(xué)殘留也在威脅著人類健康的同時對環(huán)境造成污染,有關(guān)部門對其使用的監(jiān)管日趨嚴格。另外,隨著人們生活水平的提高,對食品安全和品質(zhì)的要求也越來越高,化學(xué)殺菌劑使用也受到局限。過去的二十幾年,人們一直在研究能夠代替化學(xué)殺菌劑的安全無害的果蔬菜后真菌病害防治方法。對環(huán)境友好的生物防治方法,近年來受到廣泛重視,成為一種發(fā)展趨勢[3]。拮抗酵母是一類使用較為廣泛的生防菌,其主要優(yōu)點是繁殖能力強,次級代謝產(chǎn)物無毒并且能和與化學(xué)殺菌劑共同使用。

本文主要從拮抗酵母的種類及防治效果、作用機制、作用的改良等方面介紹近年來拮抗酵母控制果蔬采后病害研究情況,旨在加深對果蔬菜后真菌病害防控的了解,也可為安全有效地殺菌制劑的開發(fā)提供新思路。

1 拮抗酵母的種類及防治效果

目前大部分拮抗酵母取自果實表面、葉片、根部、土壤和海水等。其中,從果實表面分離到的拮抗酵母最多,一些常見果蔬如番茄、柑橘、蘋果、葡萄、檸檬、無花果、辣椒等表皮上都能分離到對一些常見病原菌拮抗效果比較好的酵母菌[4-20]。例如,Suzzi等[6]報道了關(guān)于葡萄果肉中分離出的若干株天然釀酒酵母的采后防治研究,其中一株釀酒酵母(Saccharomycescerevisiae)和一株接合酵母(Zygosaccharomyces)抑菌譜比較廣泛;蘋果上分離得到的酒清假絲酵母(Candidasake)CPA-1對蘋果采后青霉病、灰霉病及根霉腐病有較好防治效果[8]。除果蔬表皮,葉片、根際、土壤中也存在拮抗酵母菌株[21-24]。例如,從番茄葉片上分離得到的粘紅酵母(Rhodotorulaglutinis)Y-44對番茄葉片和果實的灰霉病有防控作用[21];從柑桔根部分離的檸檬形克勒克酵母(Kloeckeraapiculata)34-9對柑桔和葡萄的青霉病和灰霉病有防控作用[22];從南極土壤中分離得到一株白冬孢酵母(Leucosporidiumscottii)At17,是一種噬冷酵母,能夠很好地防控由擴展青霉和灰霉引起的蘋果青霉病和灰霉病[23];果園土壤中分離到的卡利比克畢赤酵母(Pichiacaribbica)對草莓的根霉腐病和灰霉病有較好防治效果[24]。海洋微生物中也存在拮抗酵母菌株,分離自中國東海的一株海洋紅酵母(Rhodosporidiumpaludigenum),能有效控制由擴展青霉導(dǎo)致的梨采后害和由互各交鏈孢菌導(dǎo)致的冬棗采后病害[25],海洋酵母有較好的抗?jié)B透性,與果實表面分離的酵母相比,更適合應(yīng)用在環(huán)境比較惡劣的條件下。表1列出了部分具有代表性的生防酵母菌株及其防控病害。

表1 部分具有代表性的生防效果較好的酵母菌株及其防控病害Table 1 Partially representative antagonistic yeasts used for management ofpostharvest diseases

2 拮抗酵母生防作用機制

采后生物防治系統(tǒng)十分復(fù)雜并且易受諸多因素影響,溫度、pH、水分活度、氧化壓力等都能對其產(chǎn)生影響。拮抗酵母通常是通過噴灑或浸涂在果蔬表面起到采后防治作用,若果蔬表面有傷口,拮抗酵母就會在24 h內(nèi)迅速在傷口上生長,占據(jù)傷口空間并消耗營養(yǎng)物質(zhì)。因此,在第一個24 h內(nèi),拮抗酵母的防控機制主要是與病原真菌產(chǎn)生空間與營養(yǎng)上的競爭關(guān)系,從而抑制病原菌生長[26-27]。24 h后,其他作用機制逐漸發(fā)揮作用,與營養(yǎng)、空間競爭共同防治采后腐爛。傷口處大量繁殖的酵母菌能夠誘導(dǎo)寄主的防御機制,通過改變傷口組織pH和一些氧化物的產(chǎn)生來調(diào)解酵母菌群密度并改變酵母形態(tài)[28-29]。隨之拮抗酵母可在果實損傷處形成生物膜,分泌病原菌細胞壁降解酶并消耗病原菌生長所需的鐵離子。一些拮抗酵母的作用機理如表2所示。

表2 果蔬采后拮抗酵母生物防控作用機制Table 2 Yeast strains,mechanism of actionagainst postharvest pathogenic molds on different fruits and vegetables

2.1 營養(yǎng)及空間競爭

營養(yǎng)及空間的競爭是拮抗酵母控制真菌病害最主要的作用機理,尤其是針對水果采后真菌病害的防治。采后病害的發(fā)生多由病原微生物引起,果實有傷口時,果皮表面的拮抗菌和病原菌孢子同時開始搶占傷口的營養(yǎng),以營養(yǎng)與空間競爭為拮抗機理的拮抗菌能夠在相當短的時間內(nèi)利用傷口營養(yǎng)大量繁殖,盡可能快的消耗傷口營養(yǎng)、并占領(lǐng)全部空間,使得病原菌得不到合適的營養(yǎng)與空間,不能生棲繁衍,從而抑制病害的發(fā)生。拮抗菌和病原菌競爭的營養(yǎng)物質(zhì)主要是碳水化合物、氮源等[30-36]。Li等[19]研究表明拮抗酵母SporidioboluspararoseusY16能夠在葡萄傷口處及表面快速生長繁殖,以抑制病原菌黑曲霉對果實的侵染。將季也蒙假絲酵母(Candidaguilliermondii)接種到桃果實的傷口上,在有病原菌存在的情況下該酵母菌的數(shù)量一天內(nèi)可以增加200多倍,這種高速的繁殖活動反映出拮抗菌與病原菌之間的營養(yǎng)競爭[37]。Filnow[38]用14C標記的果糖、葡萄糖、蔗糖來檢驗在蘋果傷口上的隱球酵母(Candidalaurentii)和擲孢酵母(Sporobolomycesroseus)與Botrytiscinerea病原菌孢子在營養(yǎng)利用能力上的差別,發(fā)現(xiàn)拮抗菌比病原菌孢子消耗了更多的糖類物質(zhì)。

對氮源的競爭作用也是拮抗酵母的作用機理之一,尤其是在碳源充足的果實傷口處。季也蒙假絲酵母(C.guilliermondii)[34]對擴展青霉的拮抗作用就是通過氮源的競爭實現(xiàn)的。

2.2 直接寄生病原菌

許多酵母能夠附著在果蔬腐爛部位和病原真菌菌絲上,形成一層生物膜,并且能夠分泌一些酶類,如幾丁質(zhì)酶和β-1,3-葡聚糖酶,從而分解病原菌的細胞壁或菌絲體[39-46]。這類拮抗酵母又被稱作“killer yeast”。1991年,Wisniewski[47]首次發(fā)現(xiàn)了這種防控機制,奧默畢赤酵母(Pichiaguilliermondii)能夠緊緊地附著在灰霉的菌絲體上并能夠分泌降解病原菌細胞壁的β-1,3-葡聚糖酶。Wan等[48]發(fā)現(xiàn),膜醭畢赤酵母(Pichiamembranaefaciens)對桃軟腐病的防控作用也是相同的機理。此外,附著在病原菌上的酵母菌大量消耗病原菌生長所需的鐵離子,使病原菌不能生長而起到抑制病害的作用。Saravanakumar等[40]研究表明,拮抗酵母MetschnikowiapulcherrimaMACH1通過消耗病原菌灰霉、交鏈孢菌、青霉生長所必需的鐵離子而抑制了其對蘋果的侵染。

2.3 誘導(dǎo)抗病作用

采后果蔬在貯藏過程中,由于自身的后熟和衰老,果蔬的抗病能力逐漸下降,易受到病原菌的侵染,加速腐爛。1994年,Droby和Chalutz首次發(fā)現(xiàn)了拮抗酵母的誘導(dǎo)抗病性[49]。張璐等[50]研究膜醭畢赤酵母對草莓采后灰霉病抗病性的誘導(dǎo)作用時發(fā)現(xiàn),膜醭畢赤酵母通過誘導(dǎo)草莓果實β-1,3-葡聚糖酶、過氧化物酶、苯丙氨酸解氨酶等抗性相關(guān)酶活性的增強,提高了果實對灰霉病的抵抗能力。隨著DNA芯片和高通量測序技術(shù)的進步,有關(guān)寄主組織和拮抗酵母的基因表達研究也得到了極大發(fā)展,這為更好地理解酵母菌誘導(dǎo)抗病機理提供了幫助。Jiang等[51]用微陣列分析的方法分析了羅倫隱球酵母(Cryptococcuslaurentii)誘導(dǎo)圣女果抗病性的機制,研究結(jié)果表明,與代謝、信號轉(zhuǎn)導(dǎo)和應(yīng)激反應(yīng)相關(guān)的基因表達量均上調(diào),與光合作用和能量代謝相關(guān)的基因表達量均降低,圣女果抗病性的增強應(yīng)該與這些相關(guān)基因表達量的變化密切關(guān)系。Hershkovitz等[52]研究Metschnikowiafructicola誘導(dǎo)葡萄抗病機理時發(fā)現(xiàn),病程相關(guān)基因和防御信號相關(guān)基因表達量均上調(diào),這些基因與誘導(dǎo)抗性反應(yīng)相關(guān)。蛋白質(zhì)組學(xué)的研究也指出膜醭畢赤酵母誘導(dǎo)了桃果實抗氧化蛋白和病程相關(guān)蛋白的表達,驗證了這類蛋白在此防控機制中起到的重要作用[53]。卡利比克畢赤酵母[54]和S.pararoseusY16[19]在防治桃和葡萄的采后病害時也是通過誘導(dǎo)果實自身抗病性這一機理來實現(xiàn)的。

2.4 分泌揮發(fā)性有機化合物(VOCs)抑菌

揮發(fā)性有機化合物是一類低分子量、低極性且飽和蒸氣壓高的化合物[55],許多拮抗酵母都能產(chǎn)生具有抗真菌性的揮發(fā)性有機化合物。Fiori等[56]采用平板對峙法研究了體外實驗拮抗酵母產(chǎn)生的VOCs對炭黑曲霉生長的影響,結(jié)果表明中間假絲酵母(Candidaintermedia)235和Lachanceathermotolerans751產(chǎn)生的VOCs對炭黑曲霉生長有明顯抑制作用,并且對炭黑曲霉引起的葡萄采后腐爛也有較好的抑制作用。Hua等[57]研究表明異常畢赤酵母(Pichiaanomala)WRL-076產(chǎn)生的VOCs能夠顯著抑制樹生堅果上黃曲霉菌孢子的萌發(fā)與菌絲的生長,并抑制毒素的合成,經(jīng)SPME-GC/MS鑒定發(fā)現(xiàn)有效物質(zhì)為2-苯乙醇。Parafati[58]研究了4種酵母Wickerhamomycesanomalus,M.pulcherrima,Aureobasidiumpullulans和釀酒酵母(Saccharomycescerevisiae)在體外實驗和體內(nèi)實驗中對灰葡萄孢菌和青霉生長的影響,結(jié)果表明,體外實驗中,4種酵母都能顯著抑制病原菌分生孢子的萌發(fā)和菌絲的生長,其中W.anomalus和A.pullulans的效果最為明顯;體內(nèi)實驗中,W.anomalus產(chǎn)生的VOCs能夠完全抑制人為接種引起的草莓灰霉病,顯著抑制柑橘的青霉病。

3 拮抗酵母生防作用的改良

一般生物防治中,都使用單一的生物控制劑。但經(jīng)實驗研究,諸多手段聯(lián)用能夠提高一些生防酵母的拮抗作用,并使其效果更佳穩(wěn)定。

3.1 與其他拮抗微生物結(jié)合

單一生防酵母的防控作用有時并不理想,但與其他拮抗微生物結(jié)合使用時往往可以提高其生防效力。Etebarian等[59]的研究表明,使用2種拮抗菌枝頂孢屬(Acremoniumbreve)和假單孢菌屬(Pseudomonassp.)的混合物可完全抑制蘋果灰霉病和青霉病的發(fā)生。Janisiewicz[60]的研究表明美極梅奇酵母和羅倫隱球酵母混合使用比單獨使用控制采后蘋果青霉菌腐爛效果更好。

3.2 與化學(xué)物質(zhì)結(jié)合

在商業(yè)菌制劑產(chǎn)品的大規(guī)模使用過程中,酵母暴露于諸如高溫、冷凍干燥、噴霧干燥、氧化壓力等各種各樣的環(huán)境壓力下。因此,在商品化制備中,增強環(huán)境耐受能力是提高拮抗的酵母的生存能力和性能的一種有效策略。Teixidó等[61]研究表明,通過向培養(yǎng)基中添加甘油、葡萄糖或海藻糖,能夠增加清酒假絲酵母胞內(nèi)總多元醇和糖的含量,并且提高其對水分脅迫的耐受性,從而提高其在擴展青霉對蘋果侵害時的防治效果。同樣,將羅倫隱球酵母在含海藻糖的培養(yǎng)基中培養(yǎng)可以增加其胞內(nèi)海藻糖含量,該菌經(jīng)冷凍干燥,應(yīng)用于低溫和控制大氣環(huán)境條件下后,發(fā)現(xiàn)其適應(yīng)性和生防能力有所增強[62]。一個菌制劑產(chǎn)品的商業(yè)化需要足夠的保質(zhì)期,干制劑或液體制劑已被廣泛用于制備酵母生物防治產(chǎn)品[40]。干制劑的優(yōu)點是能夠在非冷藏條件下長期保存,產(chǎn)品在存儲過程中免受污染,便于產(chǎn)品的運輸、配送和存儲[63]。液體制劑優(yōu)點是制造該產(chǎn)品的成本較低,脫水和復(fù)水過程往往導(dǎo)致拮抗菌部分死亡,液體制劑不需要干燥,使用時也可免去復(fù)水過程[64]。兩種類型的菌制劑往往需要外源保護劑來改善環(huán)境壓力對細胞適應(yīng)性和防效的影響。5%或10%的外源海藻糖的添加顯著提高了凍干后假絲酵母和粘紅酵母的生存能力[26]。在液體制劑中抗氧化劑L-抗壞血酸能夠增強糖保護劑(海藻糖和半乳糖)對羅倫隱球酵母和膜醭畢赤酵母的活力保護作用[65]。

酵母和其他抗菌化合物結(jié)合也能夠有效提升生防效果。Qin等[66]報道稱,水楊酸能夠增強黏紅酵母防治擴展青霉和鏈格孢對甜櫻桃的侵害。低濃度的水楊酸不影響酵母和致病菌的生長,但是能夠誘導(dǎo)諸如多酚氧化酶、苯丙氨酸脫氨酶和β-1,3-葡聚糖酶等與防控致病菌有關(guān)的酶的活性,推測水楊酸是通過誘導(dǎo)宿主果蔬的生化防御反應(yīng)而非對致病菌產(chǎn)生毒害效應(yīng)來增強拮抗酵母的生防效果,諸多研究能夠證明此結(jié)論[67-69]。

在多種水果,如蘋果[70]、梨[71]、桃[72]和枇杷[73]中,植物生長調(diào)節(jié)和防御劑——茉莉酸甲酯也能夠用來增強拮抗酵母的生防效果。殼聚糖和其衍生物,以其抗真菌和誘導(dǎo)宿主防御反應(yīng)的特點,能夠防治采后病害。基于這些特性,殼聚糖可作為有效添加劑用以提高諸如羅倫隱球酵母的生防效果[74]。幾丁質(zhì)能夠提高膠紅酵母的抗真菌作用[75]。植酸能夠提高粘紅酵母對草莓采后灰霉病和自然腐病的生防效率[76]。

另外一類與生防制劑結(jié)合使用防治采后病害的化合物是無機鹽和礦物質(zhì),如氯化鈣[77]、鉬酸銨[78]、碳酸氫鈉[79]、硅膠[80]和硼酸鹽[81]。在一定濃度下,這些物質(zhì)能夠直接抑制病原菌生長,但是對拮抗酵母的活性沒有影響。一些可增強拮抗酵母生防效力的化學(xué)物質(zhì)如表3所示。

表3 提高拮抗酵母生防效果的添加物Table 3 Effect of additive for improving biocontrol on antagonistic yeasts

3.3 與物理方法結(jié)合

拮抗酵母與一些物理方法結(jié)合使用也可提高其生防效力。Zhang等[82]研究表明,季也蒙畢赤酵母與熱處理結(jié)合可以更好控制采后櫻桃番茄灰霉腐爛黑點腐爛及根霉腐爛。微波處理可以增強羅倫隱球酵母對匍枝根霉引起的梨采后病害的防治效果[83]。Chun等[84]的研究表明,紫外線照射處理(UV-C)與熱帶假絲酵母(Candidatropicalis)結(jié)合使用對菠蘿采后黑腐病的防治效果優(yōu)于單獨使用熱帶假絲酵母。

4 應(yīng)用前景

一個能夠成功應(yīng)用的拮抗菌需要具備在果蔬加工貯藏條件下遺傳穩(wěn)定性高、低濃度有效、適應(yīng)性強、抗菌譜廣、生產(chǎn)成本低、便于售賣以及安全無害等特點。但由于果蔬種類、加工貯藏條件不盡相同,結(jié)抗酵母生防效果可能會有很大差異[3]。因此在推廣一種生防菌制劑之前需要對其進行試點研究和大規(guī)模的商業(yè)測試并且需要選擇不同地點的多個試點進行測試,以獲得大量數(shù)據(jù)對結(jié)抗菌劑的防控效果進行評估。盡管目前全球范圍的研究中,已有不少拮抗酵母對果蔬采后病害防控有較好效果,但能夠?qū)⑵渲瞥赊卓咕鷦┎⒊晒ν度胧袌龅娜匀挥邢?比較常見的有三種:ShemerTM、CandifruitTM和Boni-ProtectTM。這幾種菌制劑能夠?qū)Σ煌N類果蔬的多種采后病害進行防控,這也是為什么這幾種生防菌劑具有商業(yè)可行性的原因,其中基于梅奇酵母NRRL Y-27328研制而成的Shemer,能夠?qū)ζ咸焰邔僬婢⑶嗝咕⒏埂⑶箤僬婢鸬牟葺⑵咸选⒏适怼⒑}卜和柑橘等果蔬的采后腐爛有較好的防控作用[85]。

為能研發(fā)出更好的生防酵母菌劑,今后還需要加強以下幾方面的研究：a. 繼續(xù)加強酵母菌生防機理的研究,從分子水平揭示采后病原菌與酵母菌相互作用的機制;b. 進一步分離和篩選能有效抑制采后病害的拮抗酵母菌;c. 加強酵母菌作為保鮮劑在果蔬上使用方式的研究,特別是研究酵母菌在采前使用對采后病害控制的效果;d. 構(gòu)建拮抗微生物工程菌株,將拮抗性能強的拮抗菌基因轉(zhuǎn)移到另一種在果蔬表面更具適應(yīng)性的酵母菌中,從而提高生防效果。

5 展望

新鮮果蔬中的農(nóng)藥殘留將持續(xù)成為監(jiān)管部門和消費者十分關(guān)心的問題之一。拮抗酵母防治相對化學(xué)合成殺菌劑更為健康環(huán)保,符合現(xiàn)代消費者的消費理念和需求,具有廣闊的發(fā)展前景。拮抗酵母防治有望成為取代化學(xué)殺菌劑防治的有效方法,但大規(guī)模的商業(yè)化使用是一個漫長且成本高的過程。能夠?qū)崿F(xiàn)商業(yè)應(yīng)用的拮抗酵母需具備遺傳穩(wěn)定性高、適應(yīng)力強、成本低等諸多特性,雖然目前人們已經(jīng)研究出很多有防治效果的菌株,但能夠真正投入使用的仍然很有限,需要克服的問題有:a. 提高商業(yè)條件下的生防效果;b. 保證防效的基礎(chǔ)上,降低成本;c. 保持拮抗產(chǎn)品的有效期;d. 產(chǎn)品應(yīng)具有廣譜性;e. 應(yīng)具備良好的營銷策略。總之,拮抗酵母抗菌劑的研發(fā)還需大量探索,現(xiàn)代生物技術(shù),如基因組學(xué)、蛋白組學(xué)等的快速發(fā)展和先進儀器設(shè)備的應(yīng)用都會為拮抗酵母抗菌劑的研發(fā)帶來機遇。

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