柑橘采后蒂腐病菌生物学特性及其拮抗菌研究
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摘要
柑橘采后病害会导致巨大的经济损失，传统上控制柑橘采后病害的主要手段是使用化学药剂。但随着人们健康环保意识的不断增强，对化学杀菌剂造成的环境污染、农药残留、危害健康等一系列问题的日益重视以及病原菌对化学药剂抗药性的增强，因此迫切需要一种新的高效、环保、无毒的生物保鲜剂，以取代化学杀菌剂在柑橘采后保鲜中的使用。
本研究以我国重要水果椪柑为试材，分离得到了柑橘采后蒂腐病的两种病原菌——黑色蒂腐病菌(Diplodia natalensis Evans)和褐色蒂腐病菌(Phomopsis cytosporella Penz．et Sacc．)，并对其生物学特性及拮抗菌的筛选进行了初步研究，得到了两株对柑橘采后蒂腐病有很好防治效果的的拮抗酵母菌株Pg2-5和H-20，主要研究结果如下：
1．分离得到了两种柑橘采后蒂腐病的病原菌(D．natalensis和P．cytosporella)，其生物学特性研究结果表明：不同培养基对菌落形态、菌丝生长速率及产孢量都有很大影响，其中D．natalensis在马铃薯蔗糖培养基菌丝生长最为旺盛，生长速度最快，产孢量最大，可达9.08×10~7个·mL~(-1)；P．cytosporella在察氏+苜蓿培养基中产孢量最大，产孢量为3.26×10~8个·mL~(-1)。病原菌适宜的生长为25-30℃，最适温度为30℃；最适产孢温度为28℃。光照条件对病菌的生长及产孢没有显著的影响；D．natalensis的分生孢子致死温度约为75℃处理10min，P．cytosporella的分生孢子致死温度为70℃左右处理10min。药敏性实验结果显示，在测试的几种常用化学药剂中，以咪鲜胺对两种病菌的抑制效果最好。
2．筛选出能有效防治D．natalensis和P．cytosporella的酵母菌各一株：在离休初筛和复筛分别得到对两种病菌有拮抗作用的酵母菌9株和7株。进一步通过果实挑战接种，筛选出能有效抑制D．natalensis的拮抗酵母菌株Pg2-5和抑制P．cytosporella的酵母菌株H-20，果实发病率分别为20.00％和30.00％，与对照76.67％和80.00％相比，分别降低了56.67和50个百分点。
3．测定不同浓度拮抗菌对两种病菌的抑制效果：结果表明，在一定浓度范围内，拈抗酵母菌株Pg2-5和H-20的抑菌效果均与悬浮液的浓度成正相关，与培养时间呈负相关。在病菌浓度为10~7个·mL~(-1)，拮抗菌株浓度为10~8个·mL~(-1)时，接种培养8d后，接种Pg2-5菌株的果实发病率在23.33％，接种H-20菌株的果实发病率为26.67％，与对照76.67％和76.67％相比，有明显的抑制效果。而拮抗菌株浓度为10~7个·mL~(-1)，10~6个·mL~(-1)。时，虽然果实的发病率上升，但也明显低于对照。
4．鉴定筛选出的拮抗酵母菌：经形态和生理生化指标对Pg2-5和H-20进行了鉴定。结果表明：Pg2-5为假丝酵母属(Candida)欧诺比假丝酵母(Candida ernobii)。H-20为毕赤氏酵母属(Pichia)异常毕赤氏酵母(Pichia anomala)。
Postharvest disease may cause great losses, which was controlled mainly by synthetic fungicides traditionally. With public's growing concern of the human health conditions and attention to the environmental pollution, pesticide residues, harmful to health and increased systemic resistance associated with pesticides usage in orchards, there is a great need for alternative approaches. Biological control with antagonists, has been proved to be one of safe, toxicless biological agent as an alternative to the pestcides.
Two pathogen strains Diplodia natalensis Evans and Phomopsis cytosporella Penz. et Sacc. which caused stem-end rot of Citrus were obtained from surface of the natural rotted Citrus fruits. And the biological characters of them were studied. Two antagonist strains Pg2-5 and H-20 showed well inhibitory efficacy against postharvest stem-end rot of Citrus. The main results were as below:
1. D. natalensis and P. cytosporella were separated from surface of the natural rotted Citrus fruits. The results of biological characteristics showed that, there was significant difference in colony form, growth rate of mycelia and yield of conidia in different culture media, among which potato sucrose agar played the best role in D. natalensis. Yield of conidia of D. natalensis can reach 9.08×10~7 cells·mL~(-1), while that of P. cytosporella was 3.26×10~8 cells·mL~(-1) in the Czapack and clover medium. Suitable temperature for the growth of mycelia was 25℃～30℃, optimum temperature was 30℃, and optimum temperature for producing of conidia was 28℃. Lighting did not effect the growth of the pathogen and yield of conidia evidently. The lethal temperature of conidia of D. natalensis and P. cytosporella were about 75℃and 70℃maintaining 10 min separately, and the inhibition effect of 25% imidazole was the best among the tested pestcides.
2. Isolates which could control Diplodia and Phomopsis stem-end rot were screened separately. 9 strains exhibited inhibitory activity to D. natalensis Evans and 7 strains exhibited inhibitory activitory to P. cytosporella by primary and further screening in vitro. It was found that isolate Pg2-5 was most effective against D. natalensis and H-20 was the best one against P. cytosporella by in vivo challenge inoculation. The disease incidence rates were 20.00% and 30.00% separately, which were 56.67%, 50% lower than the control correspondingly.
3. Inhibition effects of antagonists with different concentrations were tested. The results showed that: the biocontrol activity was positively correlated to the concentration of antagonistic yeast, negatively correlated to the time of incubation at an extent. When concentration of pathogen was 10~7 cells·mL~(-1) and antagonist concentration was 10~8 cells·mL~(-1), the disease incidence rate of Pg2-5 against Diplodia stem-end rot was 20.00%, and that of H-20 was 30.00%, while the control of that of two diseases were both 76.67%. When concentration of pathogen was 10~7 cells·mL~(-1) and antagonist concentrations were 10~7 cells·mL~(-1) and 10~6 cells·mL~(-1) separately, though the disease incidence rates went climb, they were significantly lower than the control.
4.Antagonistic isolates were identified. Isolates Pg2-5 and H-20 were identified by morphological and physiological testing. Pg2-5 belonged to Candida, Candida ernobii and H-20 was Pichia Pichia anomala.
本研究以我国重要水果椪柑为试材，分离得到了柑橘采后蒂腐病的两种病原菌——黑色蒂腐病菌(Diplodia natalensis Evans)和褐色蒂腐病菌(Phomopsis cytosporella Penz．et Sacc．)，并对其生物学特性及拮抗菌的筛选进行了初步研究，得到了两株对柑橘采后蒂腐病有很好防治效果的的拮抗酵母菌株Pg2-5和H-20，主要研究结果如下：
1．分离得到了两种柑橘采后蒂腐病的病原菌(D．natalensis和P．cytosporella)，其生物学特性研究结果表明：不同培养基对菌落形态、菌丝生长速率及产孢量都有很大影响，其中D．natalensis在马铃薯蔗糖培养基菌丝生长最为旺盛，生长速度最快，产孢量最大，可达9.08×10~7个·mL~(-1)；P．cytosporella在察氏+苜蓿培养基中产孢量最大，产孢量为3.26×10~8个·mL~(-1)。病原菌适宜的生长为25-30℃，最适温度为30℃；最适产孢温度为28℃。光照条件对病菌的生长及产孢没有显著的影响；D．natalensis的分生孢子致死温度约为75℃处理10min，P．cytosporella的分生孢子致死温度为70℃左右处理10min。药敏性实验结果显示，在测试的几种常用化学药剂中，以咪鲜胺对两种病菌的抑制效果最好。
2．筛选出能有效防治D．natalensis和P．cytosporella的酵母菌各一株：在离休初筛和复筛分别得到对两种病菌有拮抗作用的酵母菌9株和7株。进一步通过果实挑战接种，筛选出能有效抑制D．natalensis的拮抗酵母菌株Pg2-5和抑制P．cytosporella的酵母菌株H-20，果实发病率分别为20.00％和30.00％，与对照76.67％和80.00％相比，分别降低了56.67和50个百分点。
3．测定不同浓度拮抗菌对两种病菌的抑制效果：结果表明，在一定浓度范围内，拈抗酵母菌株Pg2-5和H-20的抑菌效果均与悬浮液的浓度成正相关，与培养时间呈负相关。在病菌浓度为10~7个·mL~(-1)，拮抗菌株浓度为10~8个·mL~(-1)时，接种培养8d后，接种Pg2-5菌株的果实发病率在23.33％，接种H-20菌株的果实发病率为26.67％，与对照76.67％和76.67％相比，有明显的抑制效果。而拮抗菌株浓度为10~7个·mL~(-1)，10~6个·mL~(-1)。时，虽然果实的发病率上升，但也明显低于对照。
4．鉴定筛选出的拮抗酵母菌：经形态和生理生化指标对Pg2-5和H-20进行了鉴定。结果表明：Pg2-5为假丝酵母属(Candida)欧诺比假丝酵母(Candida ernobii)。H-20为毕赤氏酵母属(Pichia)异常毕赤氏酵母(Pichia anomala)。
Postharvest disease may cause great losses, which was controlled mainly by synthetic fungicides traditionally. With public's growing concern of the human health conditions and attention to the environmental pollution, pesticide residues, harmful to health and increased systemic resistance associated with pesticides usage in orchards, there is a great need for alternative approaches. Biological control with antagonists, has been proved to be one of safe, toxicless biological agent as an alternative to the pestcides.
Two pathogen strains Diplodia natalensis Evans and Phomopsis cytosporella Penz. et Sacc. which caused stem-end rot of Citrus were obtained from surface of the natural rotted Citrus fruits. And the biological characters of them were studied. Two antagonist strains Pg2-5 and H-20 showed well inhibitory efficacy against postharvest stem-end rot of Citrus. The main results were as below:
1. D. natalensis and P. cytosporella were separated from surface of the natural rotted Citrus fruits. The results of biological characteristics showed that, there was significant difference in colony form, growth rate of mycelia and yield of conidia in different culture media, among which potato sucrose agar played the best role in D. natalensis. Yield of conidia of D. natalensis can reach 9.08×10~7 cells·mL~(-1), while that of P. cytosporella was 3.26×10~8 cells·mL~(-1) in the Czapack and clover medium. Suitable temperature for the growth of mycelia was 25℃～30℃, optimum temperature was 30℃, and optimum temperature for producing of conidia was 28℃. Lighting did not effect the growth of the pathogen and yield of conidia evidently. The lethal temperature of conidia of D. natalensis and P. cytosporella were about 75℃and 70℃maintaining 10 min separately, and the inhibition effect of 25% imidazole was the best among the tested pestcides.
2. Isolates which could control Diplodia and Phomopsis stem-end rot were screened separately. 9 strains exhibited inhibitory activity to D. natalensis Evans and 7 strains exhibited inhibitory activitory to P. cytosporella by primary and further screening in vitro. It was found that isolate Pg2-5 was most effective against D. natalensis and H-20 was the best one against P. cytosporella by in vivo challenge inoculation. The disease incidence rates were 20.00% and 30.00% separately, which were 56.67%, 50% lower than the control correspondingly.
3. Inhibition effects of antagonists with different concentrations were tested. The results showed that: the biocontrol activity was positively correlated to the concentration of antagonistic yeast, negatively correlated to the time of incubation at an extent. When concentration of pathogen was 10~7 cells·mL~(-1) and antagonist concentration was 10~8 cells·mL~(-1), the disease incidence rate of Pg2-5 against Diplodia stem-end rot was 20.00%, and that of H-20 was 30.00%, while the control of that of two diseases were both 76.67%. When concentration of pathogen was 10~7 cells·mL~(-1) and antagonist concentrations were 10~7 cells·mL~(-1) and 10~6 cells·mL~(-1) separately, though the disease incidence rates went climb, they were significantly lower than the control.
4.Antagonistic isolates were identified. Isolates Pg2-5 and H-20 were identified by morphological and physiological testing. Pg2-5 belonged to Candida, Candida ernobii and H-20 was Pichia Pichia anomala.
引文
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56. Prabakar K., Muthulakshimi P., Raguchandet T., et al. Influence of temperature and relative humidity on anthracnose pathogen growth and disease development in mango under in vitro[J]. The Madras Agricultural Journal, 2003, 90 (7/9): 495-501.
57. Qin G. Z., Tian S. P., Liu H. B., Xu Y.. Biocontrol efficacy of three antagonistic yeasts against Penicillium expansum in harvested apple fruits[J]. Acta Botanica Sinica, 2003, 45 (4): 417-421.
58. Raymond G., Mcguire and Robert D. Hagenmaier S.. Coatings for grapefruits that favor biological control of Penicillium digitatum by Candida oleophila[J]. Biological control, 1996,7: 100-106.
59. Roberts R. G.. Postharvest biological control of gray mold of apple by Cryptococcus laurentii[J]. Phytopathology, 1990, 80: 526-530.
60. Saligkarias I. D., Gravanis F. T., Epton H. A. S.. Biological control of Botrytis cinerea on tomato plants by the use of epiphytic yeasts Candida guilliermondii strains 101 and US 7 and Candida oleophila strain 1-192: in vivo sudies[J]. Biological Control, 2002, 25: 143-150.
61. Singh V., Deverall B. J.. Bacillus subitlis as a control agent against fungal pathogens of citrus fruits[J]. Trans Br Mycol Soc, 1984, 83: 487-490.
62. Smilanick J. L., Denis-Arrue R.. Control of green mold of lemons with Pesudomonas species[J]. Plant Disease.1992, 76: 481-485.
63. Smilanick J. L., Mansour M. F., Margosan D. A., et al. Influence of pH and NaHCO_3 on the effectiveness of imazalil to inhibit germination of spores of Penicillium digitatum and to control postharvest green mold on citrus fruit[J]. Plant Disease, 2005, 89: 640-648.
64. Sommer N. F.. Role of controlled environments in supression of postharvest diseases[J]. Can. J. Plant Pathol, 1985, 7:331-336.
65. Spadaro D., Gullino M. L.. State of the art and future prospects of the biological control of postharvest fruit diseases[J]. International Journal of Food Microbiology, 2004, 185-194.
66. Veloshinie G., Lise K., Dharini S.. Semi-commercial evaluation of Bicallus licheniformis to control mango postharvest diseases in South Africa[J]. Postharvest Biology and Technology, 2005, 38: 57-65.
67. William S. C, Wojciech J. J., Britta L., Robert A. S., Mary J. C. Control of blue mold of apple by combining controlled atmosphere, an antagonist mixture, and sodium bicarbonate[J]. Postharvest Biology and Technology, 2007, in press.
68. Wilson C. L., Chalutz E.. Postharvest biological-control of Penicillium rots of citrus with antagonistic yeasts and bacteria[J]. Scientia Hortic, 1989,40: 105-112.
69. Wilson C. L., Wisniewski M. E., Droby S. and Chalutz E.. A selection of strategy for microbial antagonists to control postharvest diseases of fruits and vegetables[J]. Scientia Horticulture, 1993,53: 183-189.
70. Wisniewski M. E, Droby S., Chaultz E., Eilam Y.. Effects of Ca~(2+) and Mg~(2+) on Botrytis cinerea and Penicillium expansum in vitro and on the biocontrol activity of Candida oleophila [J]. Plant Pathology, 1995,44: 1016-1024.
71. Wisniewski M. E., Wilson C.L.. Biological control of postharvest disease of fruits and vegetables: recent advances[J]. HortScience, 1992, 27: 94-98.
72. Wojciech J., Janisiewicz, Lise K.. Biological control of postharvest diseases of fruits[J]. Annu.Rev.Phytopathol, 2002,40: 411-441.
73. Yu T., Wu P. G, Qi J. J., Zheng X. D., Jiang F., Zhan X.. Improved control of postharvest blue mold rot in pear fruit by a combination of Cryptococcus laurentii and gibberellic acid[J]. Biological Control, 2006, 39: 128-134.
74. Zahavi T., Cohen L., Weiss B., Schena L., Daus A., Kaplunov P., Zutkhi J., Ben-Arie R., Droby S.. Biological control of Botrytis, Aspergillus and Rhizopus rots on table and wine grapes in Israel[J]. Postharvest Biology and Technology, 2000, 20: 115-124.
75. Zhang H. Y, Zheng X. D., Yu T. Biological control of postharvest diseases of peach with Cryptococcus laurentii[J]. Food Control, 2007, 18: 287-291.
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67. William S. C, Wojciech J. J., Britta L., Robert A. S., Mary J. C. Control of blue mold of apple by combining controlled atmosphere, an antagonist mixture, and sodium bicarbonate[J]. Postharvest Biology and Technology, 2007, in press.
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72. Wojciech J., Janisiewicz, Lise K.. Biological control of postharvest diseases of fruits[J]. Annu.Rev.Phytopathol, 2002,40: 411-441.
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