植物提取物对水果采后致腐真菌的抑制作用研究
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摘要
本研究通过离体(In vitro)抑菌实验从12种具有抗菌活性的植物提取物中筛选出了对水果采后3种致腐真菌——链格孢菌(Alternaria alternata)、褐腐菌(Monilinia fructicola)和青霉菌(Penicillium expansum)有较强抑制作用的提取物；活体(In vivo)实验验证了筛选的提取物在桃品种“瑞光18号”和“黄元帅”苹果上对相应病害发生的防治效果；同时从生化角度初步分析了植物提取物诱导水果产生抗病性的机理。所得结果如下：
1．在In vitro抑菌实验中，采用生长速率法和滤纸片法综合分析得出：植物提取物2、5和12号对A.alternata抑制作用较强。它们对A.alternata的最小抑菌浓度(MIC)依次是6.25、1.65和2.50mg／ml，最小致死浓度(MFC)分别是25.00、6.25和5.00mg／ml。2、3、5和12号四种提取物对M.fructicola表现出了强的抗菌能力，其中3号抑菌效果最好。它们对M.fructicola的MIC分别是6.25、1.65、1.65和1.25mg／ml，MFC都高于MIC两倍，分别是12.50、3.13、3.13、2.50mg／ml。12种提取物中只有2和5号对P.expansum的抑制作用较好。2号对P.expansum的MIC是25.0mg／ml，MFC是MIC的两倍；没有测出提取物5号对P.expansum的MIC和MFC。
2．通过针刺法，在常温下观察了在桃品种“瑞光18号”和“黄元帅”苹果果实上接种提取物对相应病害发生的防治效果。结果表明，除提取物12号外，2、3、5号均能有效防止采后桃褐腐病的发生。2号三级浓度(50.00mg／ml)、5号三级浓度(12.50mg／ml)在培养期间均未发病；提取物3号为一种理想的天然防腐剂成分，其MFC(3.13mg／ml)能完全抑制桃褐腐病的发生。植物提取物2、5、12号(在大于致死浓度的情况下)均能不同程度的抑制A.alternata对采后苹果果实的侵染，并且随着处理浓度升高抑制效果增强。提取物2、3、5、12号能有效防止采后苹果褐腐病的发生。2号二级(25.00mg／ml)和三级浓度(50.00mg／ml)处理和负对照CK~-(0.50mg／ml扑海因)在实验过程中一直没有发病；提取物3号MFC同样能防止苹果褐腐病发生；5号三级浓度(12.50mg／ml)和12号三级浓度(10.00mg／ml)处理也未出现病斑扩展。提取物2号的三个浓度均能推迟P.expansum对果实的侵染，三级浓度(200.00mg／ml)表现最好。CK~-对苹果青霉病的防治效果不如2号的三级浓度。
3．以提取物2号(25.00mg／ml)+M.fructicola为研究对象，从生化角度初步分析了植物提取物诱导水果产生抗病性的机理。无论用2号单独处理还是2号+M.fructicola共同处理苹果果实，均能显著诱导果实中的抗病相关酶苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)活性的升高。过氧化物酶(POD)活性变化没有呈现规律性。总的来说，植物提取物2号发挥对苹果褐腐病的抗性，除其对病原菌本身生长有抑制作用外，还可能与它诱导果实自身抗病酶系统活性，提高果实自然防御机能，进而提高水果采后对病原菌的侵袭能力相关。
Fungitoxic effects of 12 plant extracts were studied against fruit rot-causing fungi, viz. Alternaria alternata, Monilinia fructicola and Penicillium expansion in vitro. The results showed that three kinds of plant extracts had remarkable inhibitory effects not only in vitro experiment but also in vivo test in peach and apple fruits. The enzyme activities of phenylalanine ammonia-lyase (PAL), polyphenoloxidase (PPO) and peroxidase (POD) induced by plant extract No. 2 was also discussed. The main conclusions are as follows:
Firstly, two test methods in vitro showed fungitoxic activities of 12 plant extracts in different degree to the three rot-causing fungi. Plant extracts No.2,5, 12 were more inhibitory against A.AlteRnata than the other ones, and their minimal inhibitory concentrations (MIC) on A.alternata were 6.25,1.65, 2.50 mg/ml, meanwhile their minimal Fungicidal concentrations (MFC) were 25.00, 6.25 and 5.00 mg/ml, respectively; Extracts of No. 2. 3, 5. 12 appeared to have obviously antiseptic activities against M. fructicola. Their MIC on M. fructicola were 6.25. 1.65,1.65 and 1.25 mg/ml, and MFC were 2 times of their MIC; Plant extract No.2 and No.5 appeared antifungal to P. expansum, and its MIC and MFC were 25.00 mg/ml and 50.00 mg/ml respectively, while these of No.5 were not detected. It was found that No.5 only inhibited spores germination rate of P. expansum, but it could not kill them.
Secondly, the controlling effects of plant extracts screened previously on fruit rots were determined in harvested 'Rui Guang No. 18' peach and 'Golden Delicious' apple fruits during storage. Fruits were wounded and artificially inoculated with spores (5 ×104 spores /ml) of A. alternata, M. fructicola or P. expansum following the application of plant extracts . All the fruits were stored at 25℃ after treatments. The results indicated that M. fructicola rot of peach fruits could be controlled by plant extracts No. 2,3, 5 obviously besides No. 12.
Treatments of plant extracts No.2, 5 or 12 all significantly reduced disease incidence and severity of A.alternata rot in apple fruits compared with the control, the higher the concentration, the better the effects showed.
M. fructicola rot of apple fruits could be remarkably suppressed by plant extracts No.2 , 3, 5, 12.
All treatments with 50 mg/ml of extract No.2 , 12.50 mg/ml of No.5 and 10.00 mg/ml of No.12 resulted
in better disease control and showed the similar inhibitory effects as the treatment with iprodione (CK-)
(P < 0.01). Plant extract No.3 was a perfectly natural preservative and made zero disease incidence for
fruits treated with its MFC.
In addition, the apple fruits treated with three concentrations of plant extract No.2 had lower infection rates and smaller lesion diameters than CK for P. expansum disease. The concentration of 200.00 mg/ml was the most suppressant to P. expansum .
Thirdly, activities of PAL, PPO and POD induced by inoculation with plant extract No.2 (25.00 mg/ml) alone or No.2 + M. fructicola were studied in postharvest apple fruits. The activities of PAL and PPO in fruits after inoculation with No.2 maintained at a high level throughout the experiment. PAL and PPO could also be increased by infection with artificial wound and M. fructicola, but the levels of
activity were lower than those above-mentioned. This result was in accordance with in vivo experiment in which there was no rot fruit after treatment with 25.00 mg/ml of No.2, but CK showed a disease incidence of 100% and higher lesion diameter. However, fruit inoculated with No.2 alone or No.2 + M fructicola did not stimulated POD activity. In a word, the results suggested that activation of these defense enzymes might also involve in the action of plant extract against M. fructicola, in addition to its fungitoxicity.
1．在In vitro抑菌实验中，采用生长速率法和滤纸片法综合分析得出：植物提取物2、5和12号对A.alternata抑制作用较强。它们对A.alternata的最小抑菌浓度(MIC)依次是6.25、1.65和2.50mg／ml，最小致死浓度(MFC)分别是25.00、6.25和5.00mg／ml。2、3、5和12号四种提取物对M.fructicola表现出了强的抗菌能力，其中3号抑菌效果最好。它们对M.fructicola的MIC分别是6.25、1.65、1.65和1.25mg／ml，MFC都高于MIC两倍，分别是12.50、3.13、3.13、2.50mg／ml。12种提取物中只有2和5号对P.expansum的抑制作用较好。2号对P.expansum的MIC是25.0mg／ml，MFC是MIC的两倍；没有测出提取物5号对P.expansum的MIC和MFC。
2．通过针刺法，在常温下观察了在桃品种“瑞光18号”和“黄元帅”苹果果实上接种提取物对相应病害发生的防治效果。结果表明，除提取物12号外，2、3、5号均能有效防止采后桃褐腐病的发生。2号三级浓度(50.00mg／ml)、5号三级浓度(12.50mg／ml)在培养期间均未发病；提取物3号为一种理想的天然防腐剂成分，其MFC(3.13mg／ml)能完全抑制桃褐腐病的发生。植物提取物2、5、12号(在大于致死浓度的情况下)均能不同程度的抑制A.alternata对采后苹果果实的侵染，并且随着处理浓度升高抑制效果增强。提取物2、3、5、12号能有效防止采后苹果褐腐病的发生。2号二级(25.00mg／ml)和三级浓度(50.00mg／ml)处理和负对照CK~-(0.50mg／ml扑海因)在实验过程中一直没有发病；提取物3号MFC同样能防止苹果褐腐病发生；5号三级浓度(12.50mg／ml)和12号三级浓度(10.00mg／ml)处理也未出现病斑扩展。提取物2号的三个浓度均能推迟P.expansum对果实的侵染，三级浓度(200.00mg／ml)表现最好。CK~-对苹果青霉病的防治效果不如2号的三级浓度。
3．以提取物2号(25.00mg／ml)+M.fructicola为研究对象，从生化角度初步分析了植物提取物诱导水果产生抗病性的机理。无论用2号单独处理还是2号+M.fructicola共同处理苹果果实，均能显著诱导果实中的抗病相关酶苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)活性的升高。过氧化物酶(POD)活性变化没有呈现规律性。总的来说，植物提取物2号发挥对苹果褐腐病的抗性，除其对病原菌本身生长有抑制作用外，还可能与它诱导果实自身抗病酶系统活性，提高果实自然防御机能，进而提高水果采后对病原菌的侵袭能力相关。
Fungitoxic effects of 12 plant extracts were studied against fruit rot-causing fungi, viz. Alternaria alternata, Monilinia fructicola and Penicillium expansion in vitro. The results showed that three kinds of plant extracts had remarkable inhibitory effects not only in vitro experiment but also in vivo test in peach and apple fruits. The enzyme activities of phenylalanine ammonia-lyase (PAL), polyphenoloxidase (PPO) and peroxidase (POD) induced by plant extract No. 2 was also discussed. The main conclusions are as follows:
Firstly, two test methods in vitro showed fungitoxic activities of 12 plant extracts in different degree to the three rot-causing fungi. Plant extracts No.2,5, 12 were more inhibitory against A.AlteRnata than the other ones, and their minimal inhibitory concentrations (MIC) on A.alternata were 6.25,1.65, 2.50 mg/ml, meanwhile their minimal Fungicidal concentrations (MFC) were 25.00, 6.25 and 5.00 mg/ml, respectively; Extracts of No. 2. 3, 5. 12 appeared to have obviously antiseptic activities against M. fructicola. Their MIC on M. fructicola were 6.25. 1.65,1.65 and 1.25 mg/ml, and MFC were 2 times of their MIC; Plant extract No.2 and No.5 appeared antifungal to P. expansum, and its MIC and MFC were 25.00 mg/ml and 50.00 mg/ml respectively, while these of No.5 were not detected. It was found that No.5 only inhibited spores germination rate of P. expansum, but it could not kill them.
Secondly, the controlling effects of plant extracts screened previously on fruit rots were determined in harvested 'Rui Guang No. 18' peach and 'Golden Delicious' apple fruits during storage. Fruits were wounded and artificially inoculated with spores (5 ×104 spores /ml) of A. alternata, M. fructicola or P. expansum following the application of plant extracts . All the fruits were stored at 25℃ after treatments. The results indicated that M. fructicola rot of peach fruits could be controlled by plant extracts No. 2,3, 5 obviously besides No. 12.
Treatments of plant extracts No.2, 5 or 12 all significantly reduced disease incidence and severity of A.alternata rot in apple fruits compared with the control, the higher the concentration, the better the effects showed.
M. fructicola rot of apple fruits could be remarkably suppressed by plant extracts No.2 , 3, 5, 12.
All treatments with 50 mg/ml of extract No.2 , 12.50 mg/ml of No.5 and 10.00 mg/ml of No.12 resulted
in better disease control and showed the similar inhibitory effects as the treatment with iprodione (CK-)
(P < 0.01). Plant extract No.3 was a perfectly natural preservative and made zero disease incidence for
fruits treated with its MFC.
In addition, the apple fruits treated with three concentrations of plant extract No.2 had lower infection rates and smaller lesion diameters than CK for P. expansum disease. The concentration of 200.00 mg/ml was the most suppressant to P. expansum .
Thirdly, activities of PAL, PPO and POD induced by inoculation with plant extract No.2 (25.00 mg/ml) alone or No.2 + M. fructicola were studied in postharvest apple fruits. The activities of PAL and PPO in fruits after inoculation with No.2 maintained at a high level throughout the experiment. PAL and PPO could also be increased by infection with artificial wound and M. fructicola, but the levels of
activity were lower than those above-mentioned. This result was in accordance with in vivo experiment in which there was no rot fruit after treatment with 25.00 mg/ml of No.2, but CK showed a disease incidence of 100% and higher lesion diameter. However, fruit inoculated with No.2 alone or No.2 + M fructicola did not stimulated POD activity. In a word, the results suggested that activation of these defense enzymes might also involve in the action of plant extract against M. fructicola, in addition to its fungitoxicity.
引文
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