三重PCR检测黄瓜炭疽病菌、菌核病菌和细菌性萎蔫病菌

 本试验建立一种可同时检测黄瓜炭疽病(Colletotrichum orbiculare)、黄瓜菌核病(Sclerotinia sclerotiorum(Lib.)de Bary)和黄瓜细菌性萎蔫病(Erwinia tracheiphila)等黄瓜主要病害病原菌的三重PCR检测体系。采用正交试验设计方法, 对三重PCR的影响因素分析研究, 进行退火温度优化, 并以3个引物组、Taq DNA聚合酶、dNTP和Mg²⁺ 共6因素3水平进行多重PCR体系优化, 成功建立了适合黄瓜主要病害的三重PCR最佳检测体系, 即25 μL的反应体系中含有0.24 μmol·L^-1 CY1/CY2;0.72 μmol·L^-1 SSFWD/SSREV;0.336 μmol·L^-1 ET-P1/ ET-P2;1 U Taq聚合酶;0.15 mmol·L^-1 dNTP;1 mmol·L^-1 MgCl₂, 最适退火温度为63℃。该方法能够快速从田间黄瓜发病植株和根围土壤中将黄瓜炭疽病菌、黄瓜菌核病菌和黄瓜细菌性萎蔫病菌检测出来, 灵敏度可以达到10 pg·μL^-1。     

黄瓜炭疽病菌代谢物中抑菌活性成分研究

采用大孔吸附树脂提取和硅胶柱层析分离技术,从黄瓜炭疽病菌Colletotrichum orbiculare代谢物中获得一个抗真菌化合物,采用高分辨质谱、核磁共振波谱和红外光谱等技术鉴定了其化学结构,并评价了其对番茄灰霉病菌、西瓜枯萎病菌、烟草赤星病菌、苹果炭疽病菌和玉米弯孢病菌的孢子萌发和菌丝生长的抑制作用。结果表明:该化合物为间二羟基苯甲酸大环内酯类抗生素——根赤壳菌素;其对所有供试真菌的孢子萌发均未表现出明显的抑制活性,但对番茄灰霉病菌Botrytis cinerea的菌丝生长表现出很强的抑制作用。     

不同辣椒炭疽病菌对唑菌酯的敏感性差异

辣椒炭疽病是辣椒生产上的重要病害,严重制约辣椒生产,唑菌酯防治辣椒炭疽病的相关研究未见报道。为此,本研究利用菌丝生长速率法测定了4种不同炭疽病菌对唑菌酯的敏感性,并对其作用靶标基因细胞色素b基因(Cytb)进行比较分析。结果表明:唑菌酯对Colletotrichum brevisporum YYGXZ07,C.truncatum CZHP03,C.acutatum HHBY48和C.gloeosporioides CSLL11的EC50分别为0.098、3.363、10.156和31.982μg/mL,而且供试菌株在含药培养基上的生长速率排序为C.truncatum CZHP03C.brevisporum YYGXZ07C.acutatum HHBY48C.gloeosporioides CSLL11;不同菌株的作用靶标基因细胞色素b氨基酸比对分析发现C.acutatum HHBY48、C.truncatum CZHP03和C.brevisporum YYGXZ07与C.gloeosporioides CSLL11的Cytb氨基酸序列同源性分别为82.98%、88.30%和87.77%,推测6个氨基酸位点可能与菌株对唑菌酯的敏感性存在关联。     

黄瓜炭疽病菌对杀菌剂的敏感性的优化测定方法研究

本文以黄瓜炭疽病菌(GloeosporiumorbiculareArs)为研究对象,采用随机试验设计,比较了不同培养基、孢子萌发方法对病菌孢子萌发的影响,建立了一套测定炭疽病菌分生孢子对杀菌剂敏感性的简易方法。结果表明:不同培养基培养的分生孢子,均以使用玻片水琼胶表面萌发法的萌发效果最好,且以PDA培养基培养的分生孢子在玻片水琼胶表面萌发率最高、最稳定。此外使用该方法测定了黄瓜炭疽病菌对福美双、百菌清的敏感性。     

爵床提取物对辣椒炭疽病菌的抑制作用及其苗期防效

爵床(J usticia procumbens L.)是一种传统的中药材.为探明爵床提取物对辣椒炭疽病菌的抑制作用,采用生长速率法和孢子萌发法分别测定了爵床提取物对辣椒炭疽病菌菌丝生长、孢子产生和孢子萌发的抑制作用,采用苗期盆栽试验观察爵床提取物对辣椒炭疽病的防治效果.结果表明,爵床甲醇提取物可抑制辣椒炭疽病菌菌丝生长、孢子产生和孢子萌发,其活性随着浓度的提高而增强,抑制中浓度分别为4.23、2.16和2.98 mg/mL.通过对爵床提取物处理后病菌孢子萌发形态显微观察,发现病菌孢子芽管长度受到明显抑制甚至不能萌发.当提取物浓度为16mg/mL时对苗期辣椒炭疽病的防效达73.08％.     

辣椒炭疽病菌对嘧菌酯的敏感性测定

 从江苏和海南省随机采集分离获得45个辣椒炭疽病菌单孢菌株,根据孢子形态鉴定其病原菌为Colletotrichum gloeosporioides和C.capsici,其中C.gloeosporioides占总菌株数的64.4%.筛选出甘油琼脂(AEA)培养基和水琼脂(WA)培养基,分别作为产孢法和孢子萌发法测定辣椒炭疽病菌对嘧菌酯敏感性的适宜培养基.通过孢子萌发法测定2种病原菌45个菌株对嘧菌酯的敏感性范围在0.009~0.091μg/mL之间,平均EC₅₀为(0.047±0.040)μg/mL.其中29个C.gloeosporioides菌株和16个C.capsici菌株的平均EC₅₀值分别为(0.051±0.047)μg/mL和(0.041±0.024)μg/mL.研究发现旁路氧化酶抑制剂水杨肟酸(SHAM)对嘧菌酯抑制分生孢子萌发有协同增效作用,且嘧菌酯抑制辣椒炭疽病菌菌丝生长的能力较弱.     

细胞壁降解酶在油茶炭疽病菌致病过程中的作用研究

为明确细胞壁降解酶在油茶炭疽病菌致病过程中的作用,本文研究了活体内外炭疽病菌产生的细胞壁降解酶活性及其对叶片的降解情况。结果表明,活体外以羧甲基纤维素钠(CMCNa)为诱导底物,羧甲基纤维素酶(Cx酶)和漆酶活性最高;以柑橘果胶为诱导底物,果胶酶活性最高;以油茶叶为诱导底物,纤维素酶、果胶酶和漆酶可产生较高活力;并且经5种诱导物诱导的酶液对叶片均有降解作用。发病叶片的各部位,以病健交界处细胞壁降解酶活性最高。接种4d后开始发病,其细胞壁降解酶活性迅速增强;6d后滤纸酶(FPA)、β-葡萄糖苷酶和漆酶活性达最大值,分别为4.53、7.44、1.21U/mg;而Cx酶和果胶酶在第8天时酶活性最高,分别为15.79和25.49U/mg;接种10~16d,酶活性比较稳定。上述结果表明,纤维素酶、果胶酶和漆酶在油茶炭疽病菌致病过程中起重要作用。     

枯草芽孢杆菌B006对黄瓜枯萎病菌和辣椒疫霉病菌的抑制作用及其抗菌组分分析

枯草芽孢杆菌菌株B006发酵液的甲醇提取物对黄瓜枯萎病菌Fusarium oxysporum f. sp. cucumerinum菌丝的抑制作用大于对辣椒疫霉病菌Phytophthora capsici菌丝的抑制作用;但对P. capsici游动孢子萌发的抑制作用明显大于对黄瓜枯萎病菌孢子萌发的抑制作用,提取物原液在5 min内可使辣椒疫霉的游动孢子全部崩解。提取物经60、90和120℃加热处理10 min后仍保持抑菌活性,其盐酸水解液在TLC层析板显现橙红色斑点,说明提取物具有环肽结构。HPLC-ESI-MS分析可得到m/z为995、1009、1023、1037、1051和1436、1450、1478和1492的质谱峰,与抗菌物质surfactin和fengycin的分子量大小一致。以终浓度为10^6 cfu·g^-1的接种量将B006菌剂施入无菌育苗基质中,在黄瓜和辣椒4～6片叶期时用固相萃取法提取根际的抗菌物质,并进行HPLC-ESI-MS分析,可从根际检测到surfactin和fengycin,但未从对照处理根上检测到脂肽类抗生素。本研究对理解枯草芽孢杆菌在田间状况下的防病机理有一定意义。     

一株辣椒尖孢炭疽病菌拮抗菌株的分离鉴定与发酵条件优化

 为了探寻辣椒尖孢炭疽病的生防药剂,从而控制该病在辣椒生产上的扩散。以辣椒尖孢炭疽病菌（Colletotrichum acutata）为指示菌,从浏阳河风光带土样中分离筛选出6株拮抗放线菌株,其中菌株ND045对指示菌的拮抗效果最好,菌丝生长抑制率高达71.6%。经形态观察、培养性状、生理生化鉴定,并结合16S rDNA序列分析,将ND045菌株鉴定为吸水链霉菌（Streptomyces hygroscopicus）。抗菌谱研究表明,该菌株对稻瘟病菌(Magnaporthe oryzae)、辣椒疫霉病菌(Phytophthora capsici)、黄瓜枯萎病菌（Fusarium omysporum）和辣椒白绢病菌（Sclerotium rolfsii）都具有较强的拮抗作用,抑菌率达40.58%～72.00%。通过单因子变量法和均匀设计法对菌株ND045进行发酵条件研究,结果表明,其最佳的发酵条件为：15 g大米粉、5 g大豆粉、0.5 g MgSO₄、0.01 g FeSO₄·7H₂O、1.5 g NaCl、水1 000 mL、30℃、pH 8。经优化后,菌丝生长抑制率最高达到82.61%,比优化前提高了11.01%。该研究结果为辣椒尖孢炭疽病的生物防治提供科学依据。     

B型烟粉虱对辣椒、芹菜、黄瓜寄主选择作用的研究

为探讨作物间信息化学物质对烟粉虱的寄主选择作用,进行了模拟田间条件的黄瓜植株喷施芹菜汁、芹菜植株喷施黄瓜汁,黄瓜与芹菜间作以及田间辣椒与黄瓜间作等系列试验。结果表明:在黄瓜上喷施芹菜汁液对烟粉虱有显著的驱避作用;在芹菜上喷施黄瓜汁对烟粉虱有显著的吸引作用;在黄瓜行内间作芹菜对烟粉虱有显著的驱避作用。黄瓜地里间作辣椒,在黄瓜生长期能有效地控制烟粉虱。该研究为烟粉虱寄主选择机理深入研究及非化学防治方法提供了依据。     

实时荧光PCR检测瓜炭疽病菌

采用实时荧光PCR技术建立了瓜炭疽病菌(Colletotrichum orbiculare)的检测方法。根据瓜炭疽病菌甘油醛-3-磷酸脱氢酶(GAPDH)基因和谷氨酰胺合成酶(GS)基因序列,设计了该病菌特异性引物和TaqMan探针,并对所设计的引物和探针的反应条件进行了优化。采用本试验建立的实时荧光PCR方法对瓜上的其他菌株及近似菌株进行检测,可将瓜炭疽病菌与其他病原菌区分开。灵敏度试验表明,25μL体系中只要有39.6pg的核酸量就可以被检测到,检测灵敏度达到1.584pg/μL,比普通PCR检测灵敏度高100倍。同时对田间采集的病株和未知样品进行的检测证明了引物和TaqMan探针的特异性。     

9种杀菌剂对西瓜炭疽病菌的室内毒力测定及配比试验

为筛选防治西瓜炭疽病的高效低毒杀菌剂, 缓解和治理生产中病菌对药剂的抗性, 在室内离体条件下采用生长速率抑制法及孢子萌发抑制法测定了9种杀菌剂对西瓜炭疽病菌( Colletotrichum orbiculare )的毒力。结果表明, 嘧菌酯、咪鲜胺和甲基硫菌灵对病原菌菌丝生长的EC50在0.093 3~0.118 2 mg/L之间, 均小于1 mg/L, 表明西瓜炭疽病菌对上述杀菌剂比较敏感; 百菌清、烯肟菌酯和戊菌唑的EC50在2.310 1~5.925 9 mg/L, 病菌对药剂的敏感程度相对较低; 代森锰锌、恶霉灵和多菌灵的EC50分别为36.876 3、74.466 6和99.898 5 mg/L, 抑菌活性较差。孢子萌发试验中, 嘧菌酯、咪鲜胺和甲基硫菌灵对病菌孢子萌发的抑制活性最高, EC50在0.069 4~0.167 2 mg/L之间; 百菌清、烯肟菌酯、代森锰锌的抑制活性次之, EC50在1.853 0~9.503 9 mg/L之间; 多菌灵的抑制活性相对最低, EC50为99.335 3 mg/L。将两种不同作用机理的杀菌剂嘧菌酯与咪鲜胺按照2∶1的比例混配, 联合毒力测定和评价结果表明两者混配对抑制西瓜炭疽病菌具有增效作用。     

辽宁省西瓜炭疽病病原菌鉴定及生物学特性研究

针对西瓜炭疽病病原菌进行了鉴定及其生物学特性研究。通过病原菌形态学鉴定,柯赫氏法则证病,rDNA ITS测序,鉴定该病致病菌为瓜类炭疽菌（Colletotrichumorbiculare）。生物学特性测定结果表明：病菌菌丝生长最适培养基为PSA; 最适碳、氮源分别为蔗糖和NH4Cl; 最适温度为25℃; 最适pH为6; 黑暗条件利于病菌生长。病菌产孢最适培养基为西瓜茎叶煎汁培养基; 最适碳、氮源分别为蔗糖和NH42SO4; 最适温度为25℃; 最适pH为6; 黑暗条件利于孢子产生。菌丝致死温度为55℃,10min; 分生孢子致死温度为52℃,10min。     

In vivo control and in vitro antifungal activity of rhamnolipid B,a glycolipid antibiotic,against Phytophthora capsici and Colletotrichum orbiculare

The glycolipid antibiotic rhamnolipid B isolated from Pseudomonas aeruginosa strain B5 was evaluated for in vitro antifungal activity and in vivo control against phytophthora blight and anthracnose under glasshouse conditions. Rhamnolipid B showed antifungal activity against Cercospora kikuchii, Cladosporium cucumerinum, Colletotrichum orbiculare, Cylindrocarpon destructans, Magnaporthe grisea and Phytophthora capsici. Microscopic observation revealed that the high level of antifungal activity (10 µg ml ⁻¹) against P capsici was mainly due to a lytic effect on zoospores. Zoospore lysis began in the presence of 10 µg ml ⁻¹ of rhamnolipid B and most of the zoospores were collapsed at 25 µg ml ⁻¹. Rhamnolipid B showed inhibitory activity against the germination of zoospores and hyphal growth of P capsici at concentrations of 50 µg ml ⁻¹. Spore germination of the anthracnose plant pathogen C orbiculare was also inhibited in the presence of 50 µg ml ⁻¹ of rhamnolipid B, although hyphal growth was not affected at this concentration. In the glasshouse, the efficacy of rhamnolipid B against phytophthora blight was similar to that of metalaxyl on pepper plants when treated just before inoculation with P capsici. Treatment with either at 500 µg ml ⁻¹ completely protected pepper plants from phytophthora blight. Rhamnolipid B also suppressed the development of C orbiculare infection on leaves of cucumber plants. © 2000 Society of Chemical Industry     

Australian Uromyces viciae-fabae: Host and nonhost interaction among cultivated grain legumes

Uromyces viciae-fabae, rust of faba bean, parasitizes other legume crops such as lentils (Lens culinaris) and field peas (Pisum sativum) in some environments. In this study we examined the host range of two Australian isolates of U. viciae-fabae collected and purified from a faba bean crop and classified as U. viciae-fabae ex V. faba. Field pea (P. sativum), chickpea (Cicer arientinum), lupin (Lupinus spp.), lentil (L. culinaris), and mung bean (Vigna radiata) genotypes were tested with these isolates, as well as resistant and susceptible genotypes of the faba bean host. Race specificity for these two pathogen isolates was observed on Vicia faba, with two faba bean genotypes showing partial resistance. Both U. viciae-fabae isolates also colonized field pea seedlings and successfully produced uredinia under glasshouse conditions, despite this fungus not being known as a pathogen of Australian field pea crops. No sporulation of either isolate of U. viciae-fabae ex V. faba was observed on any of the remaining legume species tested. However, obvious differences in fungal growth were observed, ranging from small infection sites with very rare haustorium formation in mung bean to more extensive growth and the development of potential uredinial structures in chickpea. These observations are discussed in relation to the phylogenetic relationship of these host and nonhost species.     

Isolation and in vitro and in vivo activity against Phytophthora capsici and Colletotrichum orbiculare of phenazine-1-carboxylic acid from Pseudomonas aeruginosa strain GC-B26

The bacterial strain GC-B26, which showed strong antifungal and anti-oomycete activity against some plant pathogens, was isolated from a grassland soil in Korea. Based on morphological, physiological and biochemical characteristics, GC-B26 was identical to Pseudomonas aeruginosa (Schroeter) Migula. The antibiotic G26A, active against Phytophthora capsici Leonian and Colletotrichum orbiculare (Berk & Mont) van Arx, was isolated from the culture filtrates of Ps aeruginosa strain GC-B26 using various chromatographic procedures. The EI mass and UV spectral results indicated that G26A is an analogue of phenazines, having molecular formula C13H8N2O2 (M+, m/z 224.0664). On the basis of NMR spectral data, G26A was confirmed as phenazine-1-carboxylic acid. C orbiculare, P capsici and Pythium ultimum Trow were most sensitive to G26A, with MIC values of approximately 5 microg ml(-1). However, no antimicrobial activity was found against yeasts and bacteria, even at a concentration of over 100 microg ml(-1). Treatment with the antibiotic gave highly significant protective activity against the development of Phytophthora disease on pepper and anthracnose on cucumber plants. The disease control efficacy was only slightly less than that of the commercial fungicides metalaxyl and chlorothalonil.     

黄瓜炭疽病拮抗细菌的筛选及其抑制效果

从连作多年的黄瓜根际土样中筛选得到1株对黄瓜炭疽病菌Colletotrichum orbiculare具有较强拮抗活性的菌株WF-3。通过菌落形态观察、生理生化特性及16S rDNA序列分析,该菌株鉴定为甲基营养型芽孢杆菌Bacillus methylotrophicus。活体盆栽条件下,该菌株对黄瓜炭疽病的防治效果为66.48%。两年的田间试验表明拮抗菌株WF-3培养物对黄瓜炭疽病具有良好的防治效果,防效分别为68.14%和73.70%。     

稻瘟病菌激发子诱导非寄主植物的抗病效果

 Hyphal cell wall crude elicitor(CWE)of rice blast pathogen could induce hypersensitive response in tobacco and induce other nonhost plants to be resistant to other fungal pathogens.When corn was treated with CWE,they inhibited the infection of Exserohilum turcicum and Curvularia lunata,and when plants of capsicum and cucumber were treated with CWE,they inhibited the infection of Colletotrichum gloeospori-oides.CWE solution showed no bioactivity on spore germination and hyphal growth of the experiment fungi in vitro.Nonhost resistance induced by CWE to other fungal pathogens was not complete resistance.The induced resistant effect(IRE) increased as CWE concentration increased,however,IRE had somehow satura-ted concentration of CWE.Induced nonhost resistance by incompatible pathogen was quantitative to other compatible pathogens.The induced resistance was best at 2 or 3 d after CWE treatment,and then decreased.IRE was about 20 percent in 10 d after CWE treatment.     

Development of the infection strategy of the hemibiotrophic plant pathogen,Colletotrichum orbiculare,and plant immunity

The hemibiotrophic fungus Colletotrichum orbiculare forms appressoria as infection structures and primarily establishes biotrophic infection in cucumber epidermal cells. Subsequently, it develops necrotrophic infection. In the pre-invasion stage, morphogenesis of appressoria of C. orbiculare is triggered by signals from the plant surface. We found that C. orbiculare PAG1 (Perish-in-the-Absence-of-GYP1), a component of MOR [morphogenesis-related NDR (nuclear Dbf2-related) kinase network] plays an essential role as a key component of the plant-specific signaling pathway and that hydrolysis of cutin by a spore surface esterase creates a cutin monomer that constitutes a key plant-derived signal. Development of the infection structure of C. orbiculare is strictly regulated by the cell cycle and we found that proper regulation of G1/S progression via two-component GAP genes, consisting of BUB2 (Budding-Uninhibited-by-Benomyl-2) and BFA1 (Byr-Four-Alike-1) is essential for the establishment of successful infection. In the post-invasion stage, the establishment of the biotrophic phase of hemibiotrophic fungi is crucial for successful infection. We found that C. orbiculare WHI2 (WHIsky-2), an Saccharomyces cerevisiae stress regulator homolog, is involved in the phase transition from biotrophy to necrotrophy through TOR (Target of Rapamycin) signaling, and is thus essential for full pathogenesis.     

Isolation and antifungal activity of kakuol, a propiophenone derivative from Asarum sieboldii rhizome

An antifungal substance active against Colletotrichum orbiculare (Berk & Mont) Arx was isolated from the methanol extracts of Asarum sieboldii (Miq) Maek rhizomes. High-resolution MS, NMR and UV spectral data confirmed that the antifungal substance is kakuol, 2-hydroxy-4,5-methylenedioxypropiophenone. Colletotrichum orbiculare was most sensitive to kakuol, with MIC of 10 microg ml(-1). Kakuol also completely inhibited the mycelial growth of Botrytis cinerea Pers ex Fr and Cladosporium cucumerinum Ellis & Arthur at 50 microg ml(-1) and 30 microg ml(-1), respectively. However, no antimicrobial activity was found against yeast and bacteria even at 100 microg ml(-1). Kakuol exhibited a protective activity against the development of anthracnose disease on cucumber plants. The control efficacy of kakuol against the anthracnose disease was in general somewhat less than that of the commercial fungicide chlorothalonil. This is the first report to demonstrate in vitro and in vivo antifungal activity of kakuol against C. orbiculare infection.