摘要
本论文首先对安徽铜陵地区牡丹根腐病原菌进行了分离与鉴定,并从生物学特性、生物学性状的遗传稳定性、营养体亲和性、同工酶电泳分析及有效杀菌剂筛选等方面对牡丹根腐病原菌进行了较为系统的研究,主要结果如下:
1牡丹根腐病原菌的鉴定
从铜陵地区发病的牡丹上分离到12株根腐病原菌,通过对其形态特征与培养性状观察,并结合其生物学特性研究,根据C.Booth、Nelson的镰刀菌分类系统,将该菌确定为茄病镰刀菌[Fusarium solani(Mart.)Sacc.]。
2牡丹根腐病菌生物学特性的研究
对牡丹根腐病菌(Fusarium solani)的研究结果表明,该菌多数对单糖、双糖、多糖等碳源及有机氮和无机氮均能够利用。其中葡萄糖、麦芽糖、甘油为适宜碳源,产孢量以果糖最高。以有机脲为氮源培养基上产孢量最高。蛋白胨为最适宜的氮源培养基,病原菌在其上生长速率最大。全黑暗条件下气生菌丝较多,产孢量也最高。PSA、淀粉、PDA和茄汁是适合病原菌菌丝生长的培养基。该菌在10-35℃条件下均能生长,最适生长温度为25-30℃,温度低于5℃或高于40℃菌丝不能生长,在25-35℃之间孢子萌发率达到最大;孢子致死温度为55℃。病原菌在pH4.1-11.5之间均能生长,其中在pH5.5-7.0之间病原菌丝生长最好;pH在7.0时分生孢子的萌发率最高。
3生物学性状遗传稳定性
试验结果表明,牡丹根腐病菌(F solani)的菌落形态和和菌丝生长速率在无性单孢后代可以稳定遗传,单孢后代之间差异不显著。菌株MD-1-5和GH-1-1的各代单分生孢子株在菌落形态上与其对应亲本之间基本相同,其菌落近圆形,边缘规则整齐,质地均匀,气生菌丝较少。菌株MD-1-5和GH-1-1的各代单孢株在生长速率上较为相似。菌株F-3各无性单孢株后代的菌落形态和生长速率与亲本是相似的,F-3各单孢株间与亲本在生长速率上均无显著差异。
4营养亲和性测定
利用氯酸盐培养基对分离得到12个牡丹根腐病菌(F solani)进行诱变,共获得99个nit突变体,利用不同氮源培养基对突变体进行鉴定,得到nit1型64株,nit3型19株,nitM型16株。来源于同一野生菌株的不同类型nit突变体间可产生互补反应而形成异核体,其中以nitM型突变株互补性最好,将各菌株的nit突变株和测试菌株的nitM配对,结果12个菌株归为3个不同的营养体亲和群(VCGs)。将诱变获得的99个nit突变株分别在PDA斜面上转管培养4次(80 d)后,除4株恢复成野生菌株外,其余多数nit突变菌株表现稳定。
5.酯酶同工酶及可溶性蛋白电泳分析
采用同工酶电泳技术,对采自安徽铜陵的12株牡丹根腐病菌(Fusarium solani)菌株进行了酯酶同工酶和可溶性蛋白分析。结果表明,供试的12个牡丹根腐病菌菌株的酯酶同工酶在R伪0.78处各有1条酶带,但各个菌株酶带的深浅、浓淡均有差异,根据酶带的强弱可以将其分为3种类型。12个菌株的可溶性蛋白图谱存在较大的差异,根据各菌株的主谱带数可将其分为4种类型:A型具有8条主谱带,共2个菌株,占供试菌株的16.7%;B型具有7条主谱带,共8个菌株,占供试菌株的66.7%;C型和D型分别具有6条和5条主谱带,各有1个菌株,均占供试菌株的8.3%。从谱带迁移的位置可以看出,12个供试菌株的可溶性蛋白图谱主要分布在3个区域内:区域ⅠRf为0.24～0.36;区域ⅡRf为0.42～0.67;区域ⅢRf伪0.70～0.79。在Rf为0.24,Rf为0.36,Rf为0.42处的3条谱带为安徽丹皮根腐病菌的特征性谱带。酯酶同工酶和可溶性蛋白电泳均表明各供试菌株在遗传上具有相似性且种内变异程度较小。
6牡丹根腐病菌有效杀菌剂筛选
分别以菌丝生长速率法和孢子萌发法测定了6种杀菌剂对牡丹根腐病菌的毒力。菌丝生长速率法测定毒力的结果表明,6种杀菌剂对牡丹根腐病菌的EC_(50)值大小顺序为:嘧菌酯＞百菌清＞恶醚唑＞代森锰锌＞多菌灵＞咪鲜胺;咪鲜胺对牡丹根腐病原菌具有十分显著的抑制效果。孢子萌发法测定毒力的结果表明,百菌清对牡丹根腐病原菌孢子萌发的毒力最强,EC_(50)值为0.343 mg/L;咪鲜胺、恶醚唑、多菌灵、代森锰锌毒力依次减弱;嘧菌酯的毒力最小。
The causal organism of tree peony root rot was identified and a systematic research was done on biological characteristics, inheritance stability of biological characters, vegetative compatibility, electrophoresis analysis on esterase isozymes and soluble protein of Fusarium solani and on screening of effective fungicides controlling causal organism of tree peony root rot. The test results were as follows:
1. Identification of the Causal Organism of Tree Peony Root Rot
According to the classification system of C. Booth and Nelson, 12 strains isolated from tree peony root rot in Tongling, Anhui were identified as Fusarium solani through its morphology and culture characteristics and biological characteristics.
2. Biological characteristics of Fusarium solani caused tree peony root rot
The studies on the biological characteristics of Fusariun solani showed that the pathogen could use many carbohydrate and nitrogen sources, but the optimum carbon sources were glucose, maltose and mannose. The optimum medium for conidial production was sucrose. Fructose and maltose, as sources of carbon, were more optimum for mycelial growth of Fusariun solani inTiquid medium. The optimum nitrogen sources were (NH_4)_2HPO_4, KNO_3 and NH_4NO_3 respectively. The optimum medium for conidial production was NH_4NO_3, and the growth of Fusariun solani in liquid medium showed that NaNO_3, as a source of nitrogen, was optimum for mycelial growth. The optimum media for mycelial growth of Fusariun solani were potato I and potato II, and the conidial production in the soluble starch medium was the highest one. The hyphae could grow in 10-35℃, the optimum temperature was in the range of 25-30℃, and the temperature lower than 5℃or higher than 40℃was not fit for the mycelial growth, the lethal temperature of conidial was 52℃for ten minutes. The mycylia could grow under the condition of pH 4.1-12.5, and the optimum were pH 5.5 and pH 10.3, the optimum pH for conidia spore germination was between 3.99 and 7.0.
3. Inheritance stability of biological character of Fusarium solani isolates inconidium progeny
The test result showed that the colony morphology and mycelial growth rate could be stably inherited in macroconidia progeny, no significant difference on morphology and mycelial growth rate among spores in later generation. The colony morphology of MD-1-5 and GH-1-1, with nearly circular and neat colony, was identical with its parental strains in each generation. The mycelial growth rates of strains MD-1-5 and GH-1-1 were nearly the same in each macroconidia generation. The strains of asexual spores of F-3 were nearly the same to its parental strain on colony morphology and mycelial growth rates, and no significant differences were existed in strains from F-3 on mycelial growth rate.
4. Studies on the vegetative compatibility of Fusarium solani causing treepeony root rot
Among 12 Fusarium solani isolates, we totally got 99 nit mutants by using chlorate medium. All of the nit mutants were then transferred into the media containing different nitrate as single nitrogen source to find which nit type they belonged to. Among the 99 nit mutants, 64 nit mutants were nit1,19 nit mutants were nit3, and 16 nit mutants were nitM. Complementation occurred between the different nit mutantphenotypes derived from the same parental strain. Complementation occurred readily and reliably in nitM which was used to identify the vegetative compatibility of other nit mutants, and the test results showed 12 isolates belongs to 3 vegetative compatibility groups. The nit mutants were all transferred on the PDA medium, and the most majority of nit mutants were stable except 4 nit mutants.
5. Analysis on esterase isozymes and soluble protein electrophoresis
The soluble protein and esterase isozyme of 12 isolates of Fusarium solani, the causal organism of tree peony root rot in Tongling, were analyzed by the technique of electrophoresis. The results showed that there was only one main band at Rf 0.78 in esterase isozyme electrophoresis of the 12 isolates. The soluble protein patterns were obviously different among the 12 isolates tested, and they can be classified into 4 types. Of 12 isolates, 2 isolates belonged to Type A having 8 main bands, occupying 16.7% of the total. Eight isolates belonged to Type B having 7 main bands, occupying 66.7%. Type C and Type D had 6 and 5 main bands respectively, including 1 isolate (occupying 8.3%) each. The soluble protein bands were mainly distributed in 3 areas: the Rf in Area I was from 0.24 to 0.36, Rf in Area II was from 0.42 to 0.67, and Rf in Area IIIwas from 0.70 to 0.79. The three bands at Rf 0.24, Rf 0.36 and Rf 0.42 were the specific bands of the pathogenic fungus. Both esterase isozyme and soluble protein electrophoresis showed similarity among the isolates tested and low variation in species.
6. Screening tests of the effective fungicides controlling tree peony root rot
Toxicity of 6 fungicides to Fusarium solani which caused tree peony root rot were tested by the means of mycelial growth method and spore germination method under the condition of laboratory, respectively. The toxicity result by the way of mycelial growth method showed that the order of EC50 values of 6 fungicides was as following: Azoxystrobin>Chlorothalonil>Difenoconazole>Mancozeb> Carbendazin> Prochloraz; the inhibition effect of Prochloraz against the pathogen was significant among those fungicides being tested. The toxicity result by mean of spore germination method showed that Chlorothalonil, with EC_(50) value as 0.343 mg·L~(-1), has the highest toxicity on the fungus. Toxicity of Prochloraz, Difenoconazole, Carbendazin, Mancozeb were gradually weakened in turn, with the toxicity of Azoxystrobin being the lowest one.
引文
[1]李方军.牡牡丹化学成分及药理作用研究进展[J].安徽医药,2004,8(1):9-10.
[2]马玉玲,刘建勋,孙卫,等.牡牡丹心对犬心肌缺血及心脏血流动力学影响的研究.山西医药杂志,1984,13(4):212-214.
[3]王斌,葛志东,周爱武,等.牡丹总苷体外对三类免疫细胞功能的影响.中国药理学通报[J],1999,15(1):63-65.
[4]梅俏,魏伟,许建明,等.牡丹总苷对化学性肝损伤保护作用机制[J].中国药理学通报,1999,15(2):176-178.
[5]梅俏,魏伟,许建明,等.牡丹总苷对小鼠化学性肝损伤的影响[J].安徽医科大学学报,1999,34(2):86-88.
[6]汤文路,李俊,徐叔云.牡丹总苷抗炎作用的研究[J].安徽医科大学学报,1999,34(1):11-13.
[7]巫冠中,杭秉茜,杭静霞,等.牡丹的抗炎作用.中国药科大学学报[J],1990,21(4):222-225.
[8]刘超,陈光亮,赵帜平,等.牡丹多糖对正常及高血糖小鼠的降血糖作用[J].安徽中医学院学报,1998,17(6):45-47.
[9]孙文姬,简桂良,刘秀兰,等.山东菏泽地区牡丹根腐病病原真菌的分离鉴定[J].植物病理学报,1995,29(2):176-180.
[10]陈让廉.铜陵牡丹[M].北京:中国林业出版社,2004,81-82.
[11]高智谋,程家高,凌云波,等.牡丹(牡丹)病害的发生规律及其综合防治措施[J].安徽农业科学,2005,33(4):585-586,590.
[12]韩金声.花卉病害防治[M].昆明:云南科技出版社,1987.63.
[13]方中达.中国农业百科全书,植物病理学卷[M].北京:中国农业出版社,1996.69,73-74,153,318,374,464,468.
[14]方中达.中国农业植物病害[M].北京:中国农业出版社,1996.133,610,689,692.
[15]吴玉柱,季延平,王海明,等.6种生防真菌、细菌防治牡丹根腐病的研究[J].山东林业科技2004,155(6):39-40.
[16]孔健,赵白鸽,王文夕,等.枯草芽孢杆菌抗菌物质对镰刀菌抑制机理的镜下研究[J].植物病理学报,1998,28(4):337-340.
[17]朱茂山,赵奎华,刘长远,等.枯草芽孢杆菌B18菌株对黄瓜枯萎病菌抑菌作用研究[J].辽宁农业科学,2003(3):39.
[18]张维强,等编著.同工酶与植物遗传育种[M].北京:北京农业大学出版社,1993.
[19]Markert C L,et al.Multiples forms of enzymes:tissue,ontogenetic,and species specific pattems[J].Procd.Nat.Acad.Soc.USA,1959,45:753-763.
[20]刘刚.真菌分类技术进展[J].微生学通报,1995,22(6):362-365.
[21]边银丙,罗信昌,周启,等.木耳栽培菌株酯酶同工酶的酶谱多样性研究[J],菌物系统,2000,19(1):89-90.
[22]褚西宁,白玉明,袁静明,等.九株根霉可溶性蛋白和三种酶的电泳图谱比较研究[J],真菌学报,1994,13(2):121-126.
[23]刑旺兴,宓鹤鸣,陈士景,等.中药红曲基真菌的酯酶同工酶分析[J],微生物学通报,2000,27(61:437-440.
[24]Hall R.Proteins and catalase isoenzymes from Fusarium solani and their taxonomic significance [J].Aust.J.Biol.Sci.1967,20:419-428.
[25]Bonde M.R,Peterson G.L,Dowler W Met al.A comparison ofisozymes of Phakopsora pachyrhizi from the Eastern Hemisphere and the New World[J].Phytopathology,1988,78:1491-1494.
[26]Royse D J,May B.Use ofisozyme vanatiionto identity genotypic classes of Agarics brunnescens[J].Mycologia,1992,74(1):93-102.
[27]Chang L O,Srb A.M,Steward EC et al.Electrophoresis separation of the soluble proteins of Neurospora[J].Nature,1962,193:756-759.
[28]Garces-de-G-E,Orozco-de-A M,Sinisterra-X,et al.Soluble protein contents,isozyme characterization,benomyl response,vegetative compatibility and mycelial growth of Fusarium oxysporum f.sp.dianthi populations[J].International society for horticultural science,1992,307:73-82.
[29]Baayen R P,van-Dreven F,Krijger M C,et al.Genetic diversity in Fusarium oxysporum f.sp.dianthi and Fusarium redolens f.sp.dianthi[J].European Journal Plant Patho 1 ogy.1997,103:395-408.
[30]Leslie J F.Fungal vegetative compatibility[J].Annual Review of Phytopathology,1993,31:127-125.
[31]龚国淑,衬希芹,郑艳,等.胶孢炭疽菌的营养体亲和性.中国食用菌[J],2005,24(增刊):22-25.
[32]Rizwana R,et al.Ultra violet light-induced instability of vegetative compatibility groups of Cryphonectria parasitica[J].Phytopathology,1992,82:1206-1211.
[33]王冬梅,王雪薇,王纯利,等.新疆西瓜枯萎病菌营养体亲和性的初步研究[J].新疆农业大学学报.1998,21(2):119-123.
[34]KISTLER H C.Genetic diversity in the plant pathogenic fungus ofFusarium oxysporum[J].Phytopathology,1997,87(4):474-479.
[35]KISTLER H C,ALBOUVETT E C,BAAYEN R P,et al.Systematic numbering of vegetative compatibility groups in the plant pathogenic fungus Fusarium oxysporum[J].Phytopathology, 1998,88(1):30-32.
[36]鲍建荣,王拱辰,郑重.尖孢镰孢(Fusanum oxysporum)酸盐营养突变株及其营养体亲和性[J],植物病理学报,1992,22(4):329-333.
[37]VAKALOUNAKIS D J,FRAGKIADAKIS G A.Genetic diversity ofFusarium oxysporum isolates from cucumber:differentiation by pathogenicity,vegetative compatibility,and RAPD fingerprinting[J].Phytopathology,1999,89(2):161-168.
[38]王克荣,孟爱中,步正庚.棉花枯萎病菌的营养体亲和性[J],南京农业大学学报,1996,19(1):30-33.
[39]丁之铨,孙文姬,陈其瑛.我国棉枯萎镰刀菌小种间的营养体亲和性[J],棉花学报,1992,4(2):85-91.
[40]Correll J C,et al.The relationship between formae speciales,races and vegetative compatibility groups in Fusarium oxysporum[J].Phytopathology,1991,81:1061-1064.
[41]Katan T,et al.Vegetative compatibility grouping ofFusarium oxysporum f.sp.vasinfectum from tissue and the rhizosphere of cotton plants[J].Phytopathology,1988,78:852-855.
[42]Correll J C,et al,Identification ofFusarium oxysporium f.sp.apii on the basis of colony size,virulence and vegetative compatibility[J].Phytopathology,1986,76:396-400.
[43]孔琼,王云月,朱有勇.香荚兰根腐病菌硝酸盐营养突变类型及营养亲和性[J],江西农业大学学报.2005,27(2):254-256.
[44]邹庆道,陈捷,高增贵,等.玉米穗、茎腐病串珠镰孢菌nit突变体的筛选及营养亲和性测定[J].菌物系统,2003,22(3):504-507.
[45]李明,钱月珍,杨雪静,等.串珠镰刀菌的营养体亲和性[J],南京农业大学学报,2000,23(2):31-34.
[46]方中达.植病研究方法(第三版)[M].北京:中国农业出版社,1998.
[47]王拱辰,刘文军,陈辉珍.镰孢属李瑟组研究-促进李瑟组产孢的VBC培养基[J].浙江农业大学学报,1995,21(4):353-356.
[48]杨曙东,梁训义,茹水江,等.浙江省番茄早疫病菌生物学特性研究[J].浙江农业学报,2002,14(6):320-325.
[49]Correll J C,et al,Identification of Fusarium oxysporium f.sp.apii on the basis of colony size,virulence and vegetative compatibility[J].Phytopathology,1986,76:396-400.
[50]毛盛贤,向华.同工酶遗传学引论[M].北京:首都师范大学出版社,2000.
[51]Wilson C M.In Methods in Enzymology[M],New York:Academic Press,1983.
[52]Sneath P.H,Sokal R.R.Numerical taxonomy:The principles and practice of numerical classification[M].San Francisco:WH Freeman and Co.1973.
[53]唐启义,冯明光著.实用统计分析及其计算机处理平台[M].北京:中国农业出版社,1997.
[54]方中达,植病研究方法.北京:中国农业出版社,1999.
[55]赵善欢主编.植物化学保护[M],北京:中国农业出版社,2000.
[56]黄圣明,刘秀娟,谢艺贤,等.咪鲜安等八种杀尚剂对芒果炭疽菌的毒性比较[J],热带作物学报,1992,13(1):67-69.
[57]潘劲松,谢标洪.不同杀菌剂对棉花枯萎病病原菌室内毒力的研究[J],湖北植保,2000,3(1)10-21
[58]Nelson P E,Toussoun T A,Marasaa W F O.Fusarium species:An illustrated manual for identification[M].The Pennsylvania state University Press,1983,1-193.
[59]Booth C.The Genus ofFusarium[M].Kew,England:CMI,1971,14- 15,27-31,44-58,122-154.
[60]王勇,杨秀荣,杨依军,等.茄根腐病致病病原一茄病镰刀菌及其蓝色变种的生物学特性研究[J].天津农学院学报,2002,2(9):21-25.
[61]洪瑞芬,季延平.刺槐溃疡病病原菌的研究[J].林业科学研究,1995,6(9):616-619.
[62]支月娥,顾振芳,朱拙安,等.金瓜贮藏期炭疽病和湿腐病的生物学特性研究[J].上海农学院学报,1997,15(3):199-203.
[63]梁英梅,田呈明,高爱琴,等.银鹊树溃疡病病原菌的研究[J].西北林学院学报,1999,14(4):53-58.
[64]赖传雅,赖传碧,曾凡凯,等.茶扦插苗根腐性苗枯病菌腐皮镰孢生物学特性研究[J].植物病理学报,2002,1(32):79-83.
[65]Joaquim T R,Rowe R C.Reassessment of vegetative compatibility relationships among strains of Verticillium dahliae using nitrate-no-nutilizing mutants[J]Phytopathology,1990,80:1160-1166.
[66]鲍建荣.西瓜枯萎病菌营养体亲和群研究.浙江农业大学学报[J],1992,18(4):43-47.
[67]Larkin R P,et al.Vegetative compatibility within Fusarium oxysporum f.sp.niveum and its relationship to virulence,aggressiveness and race[J].Canadian Journal of Microbiology,1990,36(5):353-358.
[68]Puhalla J E,et al.Classification of strains of Fusarium oxysporum on the basis of vegetative compatibility[J].Canadian Journal of Botany,1985,63:79-183.
[69]张莉,李辉,李国英,等.新疆棉花枯萎病菌酯酶同工酶的研究[J],石河子大学学报.2000,4(2):87-91.
[70]朱培良,沈瑛,袁筱萍,等.稻瘟菌不同小种间可溶性蛋白和酯酶同工酶多态性[J],植物保护.1995,21(6):15-16.
[71]潘月敏,高智谋,汪敬鑫,等.几种杀菌剂复配对棉花红腐病菌的联合毒力研究[J].棉花学报,2007,19(2):93-97.
[72]蒋家珍,吴学名,赵美琦,等.19种杀菌剂对镰刀菌的毒力测定及应用分析[J].广东农业科学,2004(2):35-36.
[73]赵善欢.植物化学保护[M].北京:中国农业出版社,2000.
[74]李宝笃,沈崇光.植物病原真菌对杀菌剂的抗性及对策[J].植物病理学报,1994,24(3):193-196.