摘要
果蔬采后由致腐性真菌引起的经济损失是巨大的。利用拮抗微生物取代化学杀菌剂,或降低化学杀菌剂的使用量,具有巨大的应用前景,这一领域的研究也是当前采后病害控制的热点。但目前既使是已商业化的生物防治微生物制剂,其效力与化学杀菌剂相比,仍有较大差距。因此,筛选新型高效的酵母拮抗菌,研究其对果蔬采后病害生物防治的效果及其机理,具有重要的理论和现实意义。
本课题从中国东海近海海域中分离筛选到一株对果蔬采后病害具有较好抑制效果的海洋酵母,经鉴定为Rhodosporidium paludigenum Fell&Tallman(1984)。本文对海洋酵母R.paludigenum的生长性能、安全性、分离干燥方法及对果蔬采后的主要致病菌和果蔬自然腐烂的抑制效果进行了研究;探讨了该酵母与其它防治手段结合使用对果蔬采后病害抑制效果的影响;初步研究了该酵母对果蔬致病菌的抑制机理及对果蔬贮藏后品质的影响,并将其作为生防制剂用于樱桃番茄采后保鲜中试实验。其主要研究结果如下:
(1)从中国东海近海海域中分离筛选到一株对果蔬采后病害具有较好抑制效果的海洋酵母,经英国国际微生物所菌种保存和鉴定中心(CABI Bioscience Identification Services)鉴定为Rhodosporidium paludigenum Fell&Tallman(1984)。
(2)对R.paludigenum耐盐性和生长曲线的测定结果表明:R.paludigenum与其它陆地生防酵母相比,具有更高的耐盐性;当氯化钠浓度达到16%时,菌体生长才完全受到抑制,说明该菌株是一株兼性海洋酵母;而且试验结果表明,R.paludigenum在NYDB培养基中28℃下培养时,酵母的生长速度很快,没有明显的延滞期(Lag phase),到27 h时,酵母细胞数目达到最高值,符合单细胞生长曲线的规律。
(3)用R.paludigenum干粉制剂进行了食品级小鼠急性经口毒性实验,初步确定该制剂属于实际无毒类。
(4)探讨了不同干燥方法、贮藏条件对R.paludigenum生物活性的影响。试验表明:采用10%脱脂乳作为冷冻干燥保护剂制能提高活性干酵母的存活率;并且在4℃密封保存条件下,活性干酵母的活性能在贮藏的前4个月时间内得到较好的保持。
(5)利用R.paludigenum对6种果实的5种主要采后致病菌进行了生防试验:试验表明该酵母可显著抑制链格孢、灰葡萄孢在番茄果实上的侵染;抑制指状青霉、扩展青霉引起的柑橘腐烂;抑制由链格孢引起的冬枣腐烂;抑制灰葡萄孢引起的梨和苹果腐烂;抑制由多毛孢引起的柿子腐烂。同时试验表明,R.paludigenum对病原真菌的抑制效力与其细胞悬浮液的浓度有关,浓度越高,对致病菌的抑制作用也越强。以上结果说明R.paludigenum可能是具有一定广谱抗菌活性的新型生物拮抗菌。
(6)对R.paludigenum拮抗机理的研究结果表明,营养与空间的竞争可能是R.paludigenum抑制致病霉菌的主要方式;此外,R.paludigenum能激发与果实抗性相关的酶活性,如POD、PPO等,因此诱导果实的自身抗性可能也是R.paludigenum抑菌机制的一部分。
(7) R.paludigenum能和多种防治手段相结合,显著增强对果实采后病害的防治效果:R.paludigenum和适当浓度金属盐类(氯化钠、氯化钙)、多种食品添加剂(碳酸盐、CMC-Na,尼泊金酯钠盐等)结合使用,能降低樱桃番茄、冬枣等果实的腐烂率;该酵母和低剂量杀菌剂嘧霉胺结合使用时,对樱桃番茄灰霉病发病率的控制效果与常规剂量嘧霉胺单独使用时接近;与热处理结合,也能降低对樱桃番茄黑斑病的发病率;此外,试验还表明,1×10~7 cells/mL R.paludigenum和热处理、尼泊金丁酯钠三种方法结合使用时,对樱桃番茄黑斑病的控制显著低于1×10~8 cells/mL R.paludigenum单独处理的水平。
(8) R.paludigenum单独作用或结合其他防治手段,对樱桃番茄、柑橘、冬枣等果实贮藏时自然腐烂均具有显著的抑制作用;经R.paludigenum处理的樱桃番茄、冬枣和柑橘等果实,其在贮藏期间的品质指标与对照果实相比,没有明显的差异。
(9) R.paludigenum对番茄采后病害生物防治中试结果表明,该酵母对樱桃番茄采后灰霉病和黑斑病的防治具有显著的效果,当1×10~8 cells/mL R.paludigenum和2%氯化钙结合使用,或1×10~7cells/mL R.paludigenum和低剂量杀菌剂嘧霉胺结合使用时,樱桃番茄黑斑病发病率分别降低了35%和40%,显示了R.paludigenum作为采后生防拮抗菌的潜力。
Considerable amount of fruits and vegetables is lost due.to decay after harvest.Microbial biocontroi agents have shown great potential as an alternative to synthetic fungicides for the control of postharvest decay of fruits and vegetables.Unfortunately,at present,the effectiveness and stability of yeast antagonists have not been reached a sufficient control level Wben compared with the synthetic fungicides.Meanwhile,although screening of large numbers of organisms has been reported, a serious shortcoming of biological control as currently tested and studied is that too few strains are examined.Thus,there is an increasing need for isolation of yeasts from new source,selection,testing of the yeast on the surfaces of fruits and studying the biocontrol mechanism of the yeast.
According to the experiment design,one of the marine yeasts isolated from the south of East China Sea was identified as Rhodosporidium paludigenum Fell & Tallman(1984),which showed great biocontrol efficiency in inhibiting postharvest diseases of fruits and vegetables.The capability of growth of R.paludigenum,the separation and dryness methods,the safety of R.paludigenum,the efficacy of R.paludigenum alone or in combination with Other control means for controlling postharvest disease of cherry tomatoes,Chinese jujubes,oranges,pears,apples and persimmons were tested.Meanwhile,the efficacy of R.paludigenum for reducing natural decay development and quality parameters and the middle model application experiments was done.In addition,the mechanisms of R.paludigenum were studies.
The results were shown as below:
1.One of the marine yeasts isolated from the south of East China Sea was identified as Rhodosporidium paludigenum Fell & Tallman by CABI Bioscience Identification Services,which was shown great biocontrol efficiency in inhibiting the black and gray rots of cherry tomato.
2.On the salt endurance and growth curve test,the marine yeast R.paludigenum showed more tolerance to osmotical stress than Rhodotorula glutinis or Cryptococcus laurentii in the nutrient yeast dextrose broth(NYDB).R.paludigenum stoped growing in the liquid medium(NYDB) added sodium chloride up to 16%(w/v),whereas R.glutinis or C.laurentii could not grow in the presence of sodium chloride over 12%(w/v).
3.On the safety test,the results of acute toxicity to mice(food standard) showed the dried powder ofR.paludigenum is safe.
4.On the test of manipulation of formulations,the activity of R.paludigenum was affected by the skim milk.The better protection from freeze-drying injury to R.paludigenum was provided by 10%skim milk.
5.R.paludigenum showed great biocontrol activity in reducing the development of postharvest diseases of fruits and vegetables,including:black rot of cherry tomato and Chinese jujube,caused by Alternaria alternata;gray mold decay of cherry tomato,pear and apple;caused by Botrytis cinerea; green mold of orange caused by and Penicillium digitatum and Penicillium expansum;postharvest rot .of persimmon caused by Pestalotis diospyri.The concentrations of R.paludigenum had significant effects on biocontrol effectiveness to postharvest diseases of fruits:the higher the concentrations of R. paludigenum,the better the control efficacy.
6.The main mode of action of R.paludigenum is competitive for nutrients and space.R. paludigenum rapidly colonized fruits wounds at ambient temperature so as to control pathogens of fruits.R.paludigenum induced the production of resistance enzymes of fruits,including polyphenol oxidase(PP0) and peroxidase(POD).Enhancing the resistance activity of fruits to pathogens may be another mechanism of control postharvest disease of fruits and vegetables of R.paludigenum.
7.The efficacy of R.paludigenum on the control of postharvest decay of cherry tomato,Chinese jujube and.pear was enhanced significantly by combining with metal salts(NaCl and CaCl_2),food additives(carbonate,CMC-Na and sodium paraben) in vivo test.Meanwhile,the combination of 1x10~7 cells/mL R.paludigenum and low doses of pesticide(0.1mg/g pyrimethanil) may enhance the inhibition of gray mold of cherry tomato.The efficacy of R.paludigenum for control of black mold of cherry tomato was improved when combined with heat treatment.Moreover,1x10~7cells/mL R. paludigenum in combination with SBP and heat treatment showed great inhibition against of black rot of cherry tomato caused by A.alternata.
8.R.paludigenum alone or in combination with CaCl_2,CMC-Na and sodium paraben significantly reduced the natural development of decay of cherry tomato,Chinese jujube and citrus, which suggested that R.paludigenum could be an alternative to chemicals for control of postharvest rots on these fruits.The quality parameters of fruits treated with R.paludigenum alone or in combination with CaCl_2,CMC-Na and sodium paraben were similar to those of control fruit,which suggested that R.paludigenum did not impair quality parameters of fruits.
9.On pilot-scale experiment tests,R.paiudigenum had great biocontrol activity against gray mold and black mold of cherry tomatoes,1x10~8 cells/mL R.paludigenum in combinationwith 2% CaCl_2 or 1x10~7 cells/mL R.paludigenum in combination with low doses pesticide(0.1mg/g pyrimethanil) showed great biocontrol activity,which showed the great potential of R.paludigenum to control the postharvest decay of fruits and vegetables.
引文
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