邢衛(wèi)峰等
摘要:通過田間試驗(yàn)發(fā)現(xiàn),生物肥料“寧盾”能夠有效防治甜瓜枯萎病,提高甜瓜的出苗率,促進(jìn)甜瓜的生長,并顯著提高甜瓜的產(chǎn)量和果實(shí)品質(zhì)。在甜瓜連作田中,“寧盾”處理組枯萎病嚴(yán)重度顯著低于對(duì)照組,生防效果高達(dá)8155%。育苗10 d后,“寧盾”處理組出苗率較對(duì)照組高20.66%~61.54%。在甜瓜“新景甜1號(hào)”移栽25 d后,“寧盾”處理組甜瓜的株高、莖粗、最大葉面積分別增加57.50%、8.18%、47.16%,處理組增產(chǎn)達(dá)21.02%;甜瓜“圣姑”移栽到大田45 d后,與對(duì)照組比較,“寧盾”處理組甜瓜株高、莖粗分別增加14.88%、15.15%,增產(chǎn)率高達(dá)57.61%。另外,“寧盾”處理組果實(shí)的硬度、可溶性固形物、可溶性糖含量均顯著高于對(duì)照組,因此“寧盾”對(duì)甜瓜的品質(zhì)具有明顯的改善作用。
關(guān)鍵詞:生物防治;枯萎?。惶鸸?;促生長作用;生物肥料
中圖分類號(hào): S436.5 文獻(xiàn)標(biāo)志碼: A 文章編號(hào):1002-1302(2014)03-0078-03
甜瓜(Cueumis melon)別名香瓜,市場需求量大,隨著栽培技術(shù)的不斷發(fā)展,為了滿足反季節(jié)甜瓜的市場需求,保護(hù)地栽培甜瓜應(yīng)運(yùn)而生,但多年連續(xù)栽培極易產(chǎn)生連作障礙。連作障礙是甜瓜生產(chǎn)中常見的難題之一,連作障礙產(chǎn)生的原因主要是土傳病菌積累、植物自毒作用、土壤鹽漬化和酸化等[1]。連作障礙會(huì)導(dǎo)致作物產(chǎn)量和品質(zhì)的下降,然而由于連作障礙產(chǎn)生的因素非常復(fù)雜,生產(chǎn)上尚缺乏一套行之有效的解決方案或途徑。甜瓜連作田中通常伴隨枯萎病的發(fā)生。溫玲連續(xù)統(tǒng)計(jì)了5年甜瓜連作田枯萎病的發(fā)病情況,5年后甜瓜枯萎病發(fā)病率達(dá)90%,造成了重大的經(jīng)濟(jì)損失[2]。生產(chǎn)上常用的克服連作障礙的技術(shù)有輪作和間套作[3]、選用抗病品種或嫁接[4-5]、無土栽培、合理的土壤管理和生物防治[6-8]等。其中較為有效可行的方法是選育抗病品種或嫁接和生物防治,但選育抗病品種費(fèi)時(shí)費(fèi)力,和嫁接一樣都會(huì)導(dǎo)致甜瓜口感和品質(zhì)下降[9],實(shí)際應(yīng)用受到一定限制;因此生物防治成為目前國內(nèi)外學(xué)者的研究熱點(diǎn),并將逐步成為農(nóng)作物病蟲害防治的重要手段之一。
生物肥料“寧盾”由南京農(nóng)業(yè)大學(xué)生物源農(nóng)藥研發(fā)實(shí)驗(yàn)室研制,主要成分是2種芽孢桿菌和沙雷氏菌。通過前期研究發(fā)現(xiàn),其對(duì)番茄青枯病、辣椒疫病、番茄根結(jié)線蟲病等土傳病害均有較好的防治效果[10-11]?!皩幎堋蓖ㄟ^有效地提高土壤中生物多樣性,提高植物根圍土壤速效氮磷鉀的含量,對(duì)土壤肥力和結(jié)構(gòu)具有良好的改善作用。本研究通過田間試驗(yàn),初次評(píng)價(jià)了生物肥料“寧盾”防治甜瓜枯萎病的效果、對(duì)甜瓜的促生作用和對(duì)果實(shí)品質(zhì)的提高效應(yīng),為甜瓜生產(chǎn)實(shí)踐過程中克服連作障礙、提高綜合效益提供參考。
1 材料與方法
1.1 供試菌劑、甜瓜品種和試驗(yàn)田概況
生物肥料“寧盾”是由南京農(nóng)業(yè)大學(xué)生物源農(nóng)藥研發(fā)實(shí)驗(yàn)室研制、南京農(nóng)大生物源農(nóng)藥創(chuàng)制有限公司開發(fā)的微生物肥產(chǎn)品[登記證號(hào)為微生物肥(2013)準(zhǔn)字(1096)號(hào)],水劑,其中有效活菌含量>108 CFU/mL。
供試甜瓜品種為圣姑,購自農(nóng)友種苗(中國)有限公司;新景甜1號(hào),由黑龍江省景豐良種開發(fā)有限公司育成。
甜瓜圣姑試驗(yàn)安排在江蘇省東??h白塔埠鎮(zhèn)前營村甜瓜連作田進(jìn)行,面積0.28 hm2;試驗(yàn)田連續(xù)5年種植甜瓜,甜瓜枯萎病發(fā)生嚴(yán)重。甜瓜新景甜1號(hào)試驗(yàn)安排在江蘇省東??h白塔埠鎮(zhèn)前營村甜瓜非連作田進(jìn)行,面積0.28 hm2。
1.2 試驗(yàn)設(shè)計(jì)
本試驗(yàn)共設(shè)2個(gè)處理:處理組為生物肥料“寧盾” 120 L/hm2;對(duì)照組為清水對(duì)照。每處理設(shè)3小區(qū)重復(fù),每個(gè)小區(qū)面積為223.5 m2,各處理隨機(jī)區(qū)組排列。小區(qū)之間以保護(hù)行隔離,試驗(yàn)田按常規(guī)管理。甜瓜移栽到田間大棚時(shí),用濃度 1×107 CFU/mL 的“寧盾”澆灌根部,“寧盾”使用量為 120 L/hm2。
1.3 調(diào)查內(nèi)容與方法
1.3.1 出苗率統(tǒng)計(jì) 隨機(jī)選擇顆粒飽滿程度一致、健康的甜瓜種子,分組裝入小燒杯中,編號(hào),種子表面用3%次氯酸鈉溶液消毒10 min,用無菌水沖洗3次,分別以“寧盾”菌液或清水浸種5 min;之后分別置于無菌紗布上,28 ℃催芽24 h,其間適量補(bǔ)水,將催芽的種子放于苗床中,10 d后統(tǒng)計(jì)每個(gè)品種各處理組種子的出苗情況,計(jì)算出苗率:出苗率=出苗的種子數(shù)/供檢測的種子數(shù)×100%。
1.3.2 促生作用調(diào)查 在調(diào)查甜瓜生長指標(biāo)時(shí),每小區(qū)取24株甜瓜植株測量株高、莖粗、葉片數(shù)和最大葉面積。
1.3.3 品質(zhì)檢測方法 硬度和可溶性固形物的檢測方法參見Miccolis等的方法[12];可溶性糖的測定采用李合生等的蒽酮比色法[13];可溶性蛋白測定采用考馬斯亮藍(lán)比色法[13];維生素C測定采用2,6-二氯靛酚滴定法[13]。
1.3.4 生物防效統(tǒng)計(jì) 用5點(diǎn)取樣法,調(diào)查統(tǒng)計(jì)病株數(shù),并計(jì)算病情指數(shù)和防治效果。病害的嚴(yán)重度分級(jí)標(biāo)準(zhǔn)[14]如下:0級(jí),全株無病,外部無癥狀;1級(jí),全株葉片總數(shù)的25%以下葉片發(fā)病,或莖內(nèi)維管束25%以下變褐色;2級(jí),全株葉片總數(shù)的26%~50%葉片發(fā)病,或莖內(nèi)維管束26%~50%變褐色;3級(jí),全株葉片總數(shù)的51%~75%葉片發(fā)病,或莖內(nèi)維管束51%~75%變褐,部分葉片萎蔫;4級(jí),全株葉片總數(shù)的76%~100%葉片發(fā)病,或莖內(nèi)維管束75%~100%變褐,或整株因病萎蔫枯死。病害嚴(yán)重度和生防效果的計(jì)算公式如下:
病害嚴(yán)重度=[∑(發(fā)病植株數(shù)×病級(jí)數(shù))/(總植株數(shù)×最高病級(jí)數(shù))]×100%;
生防效果=[(對(duì)照病害嚴(yán)重度-處理病害嚴(yán)重度)/對(duì)照病害嚴(yán)重度]×100%。
1.3.5 數(shù)據(jù)統(tǒng)計(jì)分析 生長指標(biāo)以及品質(zhì)的相關(guān)數(shù)據(jù)分析通過軟件DPS 7.05完成。
2 結(jié)果與分析
2.1 生物肥料“寧盾”對(duì)甜瓜出苗的影響
10 d后統(tǒng)計(jì)每個(gè)品種各處理組種子的出苗情況,結(jié)果顯示,寧盾處理組甜瓜圣姑、新景甜1號(hào)出苗率較對(duì)照組出苗率分別提高61.54%、20.66%(表1),表明生物肥料“寧盾”有利于甜瓜種子的萌發(fā)和幼苗的生長(圖1)。
3 結(jié)論與討論
生物菌劑“寧盾”是新研發(fā)的一種復(fù)合菌劑,具有防治多種土傳病害、促進(jìn)植物生長、改善果實(shí)品質(zhì)的效果,在實(shí)際生產(chǎn)應(yīng)用中有很大的防病促生長潛力。本試驗(yàn)田連作障礙的主要產(chǎn)生原因是甜瓜枯萎病的發(fā)生,試驗(yàn)結(jié)果顯示,生物源農(nóng)藥“寧盾”能夠有效防治枯萎病,防效高達(dá)81.55%。生物源農(nóng)藥“寧盾”的主效成分是芽孢桿菌,關(guān)于芽孢桿菌的防病機(jī)理已經(jīng)有了較深入的探討。曾有研究發(fā)現(xiàn),Bacillus sp. AR156可以同時(shí)激發(fā)水楊酸介導(dǎo)的信號(hào)通路和JA/ET介導(dǎo)的信號(hào)通路,誘導(dǎo)植物產(chǎn)生ISR從而抵抗病原物的侵害[15];除此之外,陳云等對(duì)Bacillus subtilis 防病機(jī)理做了進(jìn)一步研究,發(fā)現(xiàn)Bacillus subtilis可以通過在番茄根圍形成穩(wěn)定的生物膜,增強(qiáng)對(duì)番茄青枯病的防治效果,同時(shí)發(fā)現(xiàn)Bacillus subtilis對(duì)多種病原物有較強(qiáng)的拮抗作用[16]。
“寧盾”不僅有良好的防治枯萎病的能力,試驗(yàn)結(jié)果還顯示,“寧盾”處理組甜瓜的出苗率較對(duì)照組提高了21.66%~61.65%,且顯著促進(jìn)了甜瓜的生長,株高、莖粗和葉面積均有不同程度的增加。其他報(bào)道顯示,PGPR能夠改變根構(gòu)型,抑制主根的生長,促進(jìn)側(cè)根的生長,根圍促生菌能夠顯著增加根重,增強(qiáng)植物從土壤中吸收養(yǎng)分的能力[17];伯霍爾德桿菌屬PsJN接種擬南芥野生型Col-0顯著增加了擬南芥葉面積,但沒有顯著增加葉片數(shù)[18]。前期研究發(fā)現(xiàn),“寧盾”的單菌成分能夠產(chǎn)生IAA、嗜鐵素、產(chǎn)生VOCs,且具有解磷的作用,能夠體外降解有機(jī)磷和無機(jī)磷。而據(jù)有關(guān)報(bào)道,許多根圍促生菌能夠產(chǎn)生IAA,可以直接或間接促進(jìn)植物幼苗的生長和提高其產(chǎn)量[19]。次生代謝產(chǎn)物嗜鐵素競爭其他菌類的鐵離子,抑制有害微生物的生長。大多數(shù)根圍促生菌具有解磷固氮作用,分解土壤中難以利用的有機(jī)磷或無機(jī)磷成為可利用的磷,以利于植物營養(yǎng)吸收。最近Meldau等報(bào)道芽孢桿菌B55產(chǎn)生的揮發(fā)性物質(zhì)DMDS(二甲基二硫)直接參與硫代謝促進(jìn)煙草的生長[20]。而這些特性確保給作物提供充足的營養(yǎng),從而提高作物的產(chǎn)量和品質(zhì)。
Yamaguehi等認(rèn)為,甜瓜果實(shí)中糖含量的高低是衡量其品質(zhì)的主要依據(jù)[21]。試驗(yàn)結(jié)果顯示,“寧盾”處理組甜瓜的含糖量顯著高于對(duì)照組,提高了10.78%;同樣,其他品質(zhì)指標(biāo),如可溶性蛋白、維生素C、可溶性固形物都有一定程度的增加;因此“寧盾”對(duì)甜瓜的品質(zhì)有很好的改善,克服了嫁接、選育抗病品種等方法引起的品質(zhì)下降,成為克服連作障礙更為有效和可行的方法。
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[12]Miccolis V,Saltveit M E. Influence of storage period and temperature on the postharvest characteristics of six melon(Cucumis melo L.,Inodorus Group)cultivars[J]. Postharvest Biology and Technology,1995(5):211-219.
[13]李合生,孫 群,趙世杰,等. 植物生理生化實(shí)驗(yàn)原理和技術(shù)[M]. 北京:高等教育出版社,2000.
[14]李瑞琴. 甜瓜枯萎病病原學(xué)及防治技術(shù)研究[D]. 楊凌:西北農(nóng)林科技大學(xué),2004:13-16.
[15]Niu D D,Liu H X,Jiang C H,et al. The plant growth-promoting rhizobacterium Bacillus cereus AR156 induces systemic resistance in Arabidopsis thaliana by simultaneously activating salicylate-and jasmonate/ethylene-dependent signaling pathways[J]. Molecular Plant-Microbe Interactions,2011,24(5):533-542.
[16]Chen Y,Yan F,Chai Y R,et al. Biocontrol of tomato wilt disease by Bacillus subtilis isolates from natural environments depends on conserved genes mediating biofilm formation[J]. Environmental Microbiology,2013,15(3):848-864.
[17]López-Bucio J,Campos-Cuevas J C,Hernández-Calderón E,et al. Bacillus megaterium rhizobacteria promote growth and alter root-system architecture through an auxin-and ethylene-independent signaling mechanism in Arabidopsis thaliana[J]. Molecular Plant-Microbe Interactions,2007,20(2):207-217.
[18]Poupin M J,Timmermann T,Vega A,et al. Effects of the plant growth-promoting bacterium Burkholderia phytofirmans PsJN throughout the life cycle of Arabidopsis thaliana[J]. PLoS One,2013,8(7):e69435.
[19]Tsavkelova E A,Cherdyntseva T A,Klimova S Y,et al. Orchid-associated bacteria produce indole-3-acetic acid,promote seed germination,and increase their microbial yield in response to exogenous auxin[J]. Archives of Microbiology,2007,188(6):655-664.
[20]Meldau D G,Meldau S,Hoang L H,et al. Dimethyl disulfide produced by the naturally associated Bacterium bacillus sp.B55 promotes growth by enhancing sulfur nutrition nicotiana attenuate[J]. The Plant Cell,2013,25(7):2731-2747.
[21]Yamaguchi M,Hughes D M. Quality of cantaloupe muskmelons:variability and attributes[J]. Scientia Horticuhurae,1977,6(1):59-70.
[10]Wei L H,Xue Q Y,Wei B Q,et al. Screening of antagonistic bacterial strains against Meloidogyne incognita using protease activity[J]. Biocontrol Science and Technology,2010,20(7):739-750.
[11]Jiang Z Q,Guo Y H,Li S M,et al. Evaluation of biocontrol efficiencies of different Bacillus preparations and different field application methods against Phytophthora bight of bell pepper[J]. Biological Control,2006,36(2):216-223.
[12]Miccolis V,Saltveit M E. Influence of storage period and temperature on the postharvest characteristics of six melon(Cucumis melo L.,Inodorus Group)cultivars[J]. Postharvest Biology and Technology,1995(5):211-219.
[13]李合生,孫 群,趙世杰,等. 植物生理生化實(shí)驗(yàn)原理和技術(shù)[M]. 北京:高等教育出版社,2000.
[14]李瑞琴. 甜瓜枯萎病病原學(xué)及防治技術(shù)研究[D]. 楊凌:西北農(nóng)林科技大學(xué),2004:13-16.
[15]Niu D D,Liu H X,Jiang C H,et al. The plant growth-promoting rhizobacterium Bacillus cereus AR156 induces systemic resistance in Arabidopsis thaliana by simultaneously activating salicylate-and jasmonate/ethylene-dependent signaling pathways[J]. Molecular Plant-Microbe Interactions,2011,24(5):533-542.
[16]Chen Y,Yan F,Chai Y R,et al. Biocontrol of tomato wilt disease by Bacillus subtilis isolates from natural environments depends on conserved genes mediating biofilm formation[J]. Environmental Microbiology,2013,15(3):848-864.
[17]López-Bucio J,Campos-Cuevas J C,Hernández-Calderón E,et al. Bacillus megaterium rhizobacteria promote growth and alter root-system architecture through an auxin-and ethylene-independent signaling mechanism in Arabidopsis thaliana[J]. Molecular Plant-Microbe Interactions,2007,20(2):207-217.
[18]Poupin M J,Timmermann T,Vega A,et al. Effects of the plant growth-promoting bacterium Burkholderia phytofirmans PsJN throughout the life cycle of Arabidopsis thaliana[J]. PLoS One,2013,8(7):e69435.
[19]Tsavkelova E A,Cherdyntseva T A,Klimova S Y,et al. Orchid-associated bacteria produce indole-3-acetic acid,promote seed germination,and increase their microbial yield in response to exogenous auxin[J]. Archives of Microbiology,2007,188(6):655-664.
[20]Meldau D G,Meldau S,Hoang L H,et al. Dimethyl disulfide produced by the naturally associated Bacterium bacillus sp.B55 promotes growth by enhancing sulfur nutrition nicotiana attenuate[J]. The Plant Cell,2013,25(7):2731-2747.
[21]Yamaguchi M,Hughes D M. Quality of cantaloupe muskmelons:variability and attributes[J]. Scientia Horticuhurae,1977,6(1):59-70.