摘要
本文首次从作物根际土壤中分离、筛选出对几种作物病原菌具有稳定拮抗作用并产聚-γ-谷氨酸的芽胞杆菌B6-1,对菌种进行鉴定,确定其拮抗谱,分离、纯化并鉴定抗菌物质,用红薯渣和豆渣作为固态发酵的主要基质,获得同时高产拮抗物质和聚-γ-谷氨酸,并对该固态发酵产物的生防和肥料增效作用进行了评价。结果如下:
1.运用先富集再加热的方法,从作物根际土壤中分离得到313株芽胞杆菌。平板共培养筛选到37株对至少一种指示病原真菌表现出稳定的拮抗效果。其中菌株B6-1表现拮抗谱最广、拮抗能力最强。根据其生理生化特征和16S rDNA序列分析,被确定为枯草芽胞杆菌(Bacillus subtilis)。
2.菌株B6-1产抗真菌活性物质。其发酵液具有油取代、液滴坍塌、红细胞溶血、水溶液中分子之间发生凝聚等典型的表面活性剂特性。所产拮抗物质能被酸沉淀,并能抵抗一定的高温、较宽的pH范围和紫外等条件。以上特性说明枯草芽胞杆菌菌株B6-1所产的拮抗物质应该属于脂肽类抗生素。
3.采用超滤、酸沉淀、固相萃取相结合的方法来分离纯化菌株B6-1所产的抗生素。经过高效液相色谱(HPLC)分部收集并检测各峰拮抗活性,再经过LC-MS分析,主要活性物质的分子量分别为1450、1464、1478、1506,它们之间分子量差异为14的整数倍,即(CH_2)_n。部分分子量与已经报道的C15、C16、C17、C19脂肽类表面活性剂抗生素fengycins相吻合,但其拮抗谱不同,而且fengycins不具备溶血活性,所以可能还存在其它具溶血活性的脂肽。该fengycins具体的结构还有待进一步工作确定。
4.在NB液体培养基发酵中,B6-1产生脂肽抗生素却不产γ-PGA,而在经过优化的高产γ-PGA的液体培养基中,γ-PGA的产量达到34.7 g/L,却不能检测到脂肽抗生素。采用红薯渣、豆渣作主要基质的固态发酵,γ-PGA的产量达到36.3g/(kg干物质)。同时脂肽抗生素的相对产量达到70个单位,而NB液体发酵中为20个单位。同时证实γ-PGA无抗菌活性。
5.当施用B6-1采用红薯渣、豆渣作主要基质的固态发酵产物后,黄瓜枯萎病发病率降低至17%。在全营养水平下,添加B6-1的高产γ-PGA的固态发酵产物时,地下、地上部分干重和根冠比分别为0.447 g、2.448 g、0.183,而对照组分别为0.334 g、2.183g、0.153,地下、地上部分干重和根冠比各自分别比对照增加33.8%、12.2%、18.3%。在1/2营养水平下,添加B6-1的高产γ-PGA的固态发酵产物时,地下、地上部分干重和根冠比分别为0.339 g、1.405 g、0.242,而对照组分别为0.230 g、1.111 g、0.201,地下、地上部分干重和根冠比各自分别比对照增加47.4%、26.4%、20.4%。在1/3营养水平下,添加B6-1的高产γ-PGA的固态发酵产物时,地下、地上部分干重和根冠比分别为0.344 g、0.956 g、0.351,而对照组分别为0.203 g、0.814 g、0.251。结果说明,在各肥料水平下,根部、地上部干重和根冠比都较对照增加,随着肥料水平的下降,根部、地上部干重和根冠比较对照的增加幅度提高,特别是在低肥料浓度下,对根冠比的影响尤为明显。这一方面说明B6-1固态发酵产物中的γ-PGA可以促进作物的生长,另一方面由于在肥料较缺乏的时候,γ-PGA的增产效果尤为明显,也说明B6-1固态发酵产物中的γ-PGA具有肥料增效剂的作用,特别是更加有利于作物根对营养的吸收。
6.在低温下γ-PGA具有提高菌体存活率的作用,特别是随着冻藏时间的延长,保护作用越显著。γ-PGA添加浓度以1.5%为适宜。经过冷冻干燥和冻藏一定时期后,添加γ-PGA的乳酸菌产酸活力下降较少,且基本没有迟滞期,都能在更短时间内产酸。
For the first time,the Bacillus strain B6-1,isolated from the rhizosphere of crops, co-produced the antifungal substances and poly-γ-glutamic acid,and was identified as Bacillus subtilis.The co-productions of antagonistic substances and poly-γ-glutamic acid were obtained when using soybean and sweet potato residues with perlites cultivated with B6-1 in solid-state fermentation.The antifungal compounds were purified and identified as lipopeptides fengycins.Suppression effect of residues cultivated with B6-1 on cucumber wilts and fertilizer synergistic effect were studied.The results were followed as:
1.313 distinct presumptive Bacillus isolates were obtained from the rhizosphere of crops.37 of them displayed distinct and stable antagonism against at least one of indicators tested through dual culture assays.B6-1 showed the broadest antifungal spectrum,strongest antagonistic activity.Based on the physiological,biochemical charactcristics and 16S rDNA data,the strain B6-1 can be designated as Bacillus subtilis.
2.The results from antagonistic mechanism demonstrated the production of antifungal metabolites.The culture filtrate of Bacillus subtilis B6-1 displayed oil displacement,droplet collapsc,hemolytic activity and aggregational behavior.The antifungal compounds could bc precipitated by acid and remain its activities against heat, pH,UV.All the results above implied the antifungal compounds belong to lipopeptides.
3.Using a procedure including ultrafiltration,acid precipitation and solid-phase extraction,the antifungal compounds were purified.The major antifungal compounds showed samc UV spectra in methanol and a homologous[M+H]~+ ion peak at m/z 1450, 1464,1478,1506,respectively.These mass data correspond well to those determined by other authors,indicating the presence of C15,C16,C17,C19 fengycins,corroborating the deduction above.But there are no reports about hemolytic activity of fengycins.So other lipopeptides with hemolytic activity may be present.Further studies would be done in future.
4.In the nutrient broth fermentation,B6-1 produced the lipopeptides but noγ-PGA. And in the optimized medium for production ofγ-PGA,the yield ofγ-PGA reached 3.47%but the lipopeptides could not be detected.The averageγ-PGA yield in SSF was 36.3 g/(kg dry substrate) in 250 ml flask.At the same time,the production of lipopeptides was verified.The maximum dilution in SSF that can be detected was 70 folds,remarkably higher compared with 20 folds in NB.
5.When the residue mixtures cultivated with B6-1were introduced,the disease occurrence was effectively reduced to 17%.At full nutrition solution,when residue cultures were introduced,the dry weight of shoots and roots and the roots to shoots ratio increased by 33.8%,12.2%,18.3%,respectively.At 1/2 nutrition solution,they increased by 47.4%,26.4%,20.4%respectively,and at 1/3 nutrition solution,increased 69.5%, 17.4%,43.8%,respectively.The addition of residue cultures significantly increased the dry weight of both roots and shoots and the roots to shoots ratio(R/S),at all levels of nutrients.The increases were more significant at lower nutrient levels.
6.The survival ratio of lactic acid bacteria was improved by adding more than 1.5% PGA during freezing and frozen-storage and the effect was better as the frozen-storage was prolonged.The acidification activity remained strong and the lag phase of lactic acid bacteria was shorter.
引文
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