亚洲梨黑星病菌致病性及其寄主抗病机制的研究进展

梨黑星病是亚洲梨的主要病害之一。该病是由纳雪黑星病菌(Venturia nashicola)感染所致。V.nashicola主要寄生在亚洲梨叶片表皮细胞壁的果胶质层中。该菌的感染可能主要与分泌的细胞外分泌物质、角质分解酶、过氧化氢和果胶质分解酶有关。而亚洲梨对V.nashicola的抗性可能主要与多聚半乳糖醛酸酶抑制蛋白、多种病程相关蛋白、富亮氨酸重复类受体蛋白激酶等有关。另外,不具直接杀菌能力的系统抗性诱导剂acibenzolar-S-methyl(ASM)在大田试验中对梨黑星病菌有较好控制效果。这与ASM诱导的植物防御反应,包括多聚半乳糖醛酸酶抑制蛋白和几丁质酶等有关。     

瓜黑星病菌、枯萎病菌和蔓枯病菌的三重PCR检测

通过测定黄瓜黑星病菌(Cladosporium cucumerinum)rDNA的ITS序列,比对近缘种及瓜类几种重要病原菌的ITS序列,设计出特异性引物HX-1/HX-2,经过对引物HX-1/HX-2PCR条件的优化,可以扩增出1条190bp的黄瓜黑星病菌特异性DNA条带,灵敏度达到1pg/μL。进一步将引物HX-1/HX-2和瓜类枯萎病菌、瓜类蔓枯病菌特异检测引物Fn-1/Fn-2、Mn-1/Mn-2组合,建立三重PCR体系,可一次检测出瓜类黑星病菌、瓜类枯萎病菌、瓜类蔓枯病菌3种瓜类植物重要的病原菌。建立了可以应用于田间瓜类黑星病菌PCR检测技术和瓜类主要病害三重PCR检测技术,对瓜类病害的诊断和防治具有重要的指导作用。     

梨黑星病菌被哈茨木霉感染后抗氧化系统的变化

测定被哈茨木霉感染后梨黑星病菌抗氧化系统变化的结果表明,被哈茨木霉感染后梨黑星病菌过氧化氢酶、过氧化物酶和超氧化物歧化酶的活性逐渐下降;丙二醛含量和超氧阴离子产生速率上升;还原型谷胱甘肽和抗坏血酸含量降低。初步明确,梨黑星病菌被哈茨木霉感染后,活性氧清除系统等被破坏,膜脂过氧化作用加强,是最终导致菌体受害的原因之一。     

梨黑星病菌寄生部位及致病作用研究

 组织透明及扫描电镜研究结果表明:梨黑星病菌在叶片上主要寄生于表皮细胞和叶脉薄壁组织细胞的表面,沿细胞表面扩展。病原菌并非只在角质层与表皮细胞间生长,而且在表皮细胞和叶脉薄壁组织细胞的细胞间寄生。在大量的观察中,没有发现侵入到细胞内菌丝或菌丝变态结构。梨黑星病菌不寄生叶肉细胞,但能导致叶肉细胞病变。由于黑星病菌的寄生性既不同于专性寄生,也不同于杀生寄生,才使梨黑星病的潜育期和产孢期都长于这两类真菌病害。这是认识和研究梨黑星病发病特点和流行规律的基础。     

黄瓜黑星病菌致病机理的研究Ⅱ细胞壁降解酶及其在致病中的作用

 本文通过活体内外黄瓜黑星病菌(Cladosporium cucumerinum)产生的细胞壁降解酶活性分析,初步明确了Cx-酶(羧甲基纤维素酶)、β-葡萄糖苷酶和PMG(聚甲基半乳糖醛酸酶)、PMTE(果胶甲基反式消除酶)在该菌侵染黄瓜中的作用。瓜条感病的各部位,以病健交界处细胞壁降解酶活性最高;在检测的各类酶中,活性最高的为PMG、PMTE,Cx-酶、β-葡萄糖苷酶和FPA(滤纸酶)活性较低。抗感病黄瓜品种染病后,细胞壁降解酶活性均迅速增强。感病品种染病后,果胶酶(PMG、PMTE)活性远远高于抗病品种,其高峰值是抗病品种的2.72~7.52倍。感病品种接种后纤维素酶(Cx-酶、β-葡萄糖苷酶)活性一直增强;抗病品种接种后β-葡萄糖苷酶随着接种天数的增加酶活性一直增强,Cx-酶在染病后初期活性迅速增强,增至高峰后逐渐降低。     

梨黑星病菌对氟硅唑的敏感性及与不同杀菌剂之间的交互抗性

采用孢子芽管长度法,测定了采自河北省不同地区的88个梨黑星病菌 Venturia nashicola 菌株对氟硅唑的敏感性,并测定了10个相对敏感菌株和5个相对抗性菌株对甲基硫菌灵、醚菌酯、福美双和苯醚甲环唑的敏感性。结果表明:不同地区黑星病菌对氟硅唑的敏感性存在一定差异,平均EC50值为(1.3821±0.9276)μg/mL;氟硅唑对未使用过麦角甾醇生物合成抑制剂(EBI)类杀菌剂的75个梨黑星病菌菌株的平均EC50值为(1.1676±0.6128)μg/mL,最大值是最小值的16.91倍,其敏感性分布频率呈连续性单峰曲线,且符合正态分布,故可作为敏感基线用于监测田间梨黑星病菌对氟硅唑的敏感性变化。不同菌株对5种药剂的敏感性相互关系分析表明,氟硅唑和苯醚甲环唑之间呈显著正相关,但与醚菌酯、甲基硫菌灵和福美双之间均不存在交互抗性关系。     

黄瓜黑星病菌致病机理的研究Ⅱ细胞壁降解酶及其在致病中的作用

 本文通过活体内外黄瓜黑星病菌(Cladosporium cucumerinum)产生的细胞壁降解酶活性分析,初步明确了Cx-酶(羧甲基纤维素酶)、β-葡萄糖苷酶和PMG(聚甲基半乳糖醛酸酶)、PMTE(果胶甲基反式消除酶)在该菌侵染黄瓜中的作用。瓜条感病的各部位,以病健交界处细胞壁降解酶活性最高;在检测的各类酶中,活性最高的为PMG、PMTE,Cx-酶、β-葡萄糖苷酶和FPA(滤纸酶)活性较低。抗感病黄瓜品种染病后,细胞壁降解酶活性均迅速增强。感病品种染病后,果胶酶(PMG、PMTE)活性远远高于抗病品种,其高峰值是抗病品种的2.72~7.52倍。感病品种接种后纤维素酶(Cx-酶、β-葡萄糖苷酶)活性一直增强;抗病品种接种后β-葡萄糖苷酶随着接种天数的增加酶活性一直增强,Cx-酶在染病后初期活性迅速增强,增至高峰后逐渐降低。     

苹果黑星病菌检疫

苹果黑星病菌是新公布的我国农业植物检疫性有害生物.本文描述了该病在国内的发生历史及病原菌鉴定特征.提出了产地检疫、果品检疫、苗木接穗等繁殖材料的检验方法,并提出了该菌的检疫和防治措施.     

黄瓜黑星病菌致病机理的研究Ⅰ 黄瓜黑星病菌致病毒素的分离和纯化

本文明确了在接种后的黄瓜黄化子叶中能够提取到黄瓜黑星病菌(Cladosporium cucum eri-num ) 的粗毒素,最佳培养时间为接种后4 d。该毒素具有明显的生物活性,能够抑制黄瓜胚根与胚芽的生长,能使扦插的黄瓜水培幼苗致萎。通过硅胶柱层析纯化黄瓜黑星病菌的粗毒素,根据洗脱顺序和洗脱液的种类将其分为4 个洗脱组分。根据硅胶薄层层析紫外显色及生物活性分析可确定,活性物质主要集中在无色的洗脱组分Ⅱ中,该洗脱组分中有 3 组活性物质:CCⅠ:Rf= 0.51; CCⅡ:Rf=0.25; CCⅢ:Rf= 0.06。     

高温高湿对黄瓜黑星病菌孢子萌发及侵染的影响

 采取温湿度相结合的方法来研究高温处理对黄瓜黑星病菌(Cladosporium cucumerinum Ell.and Arth.)侵染的影响,比较研究了35~50℃ 4个温度梯度、50%~90% 5个湿度梯度组合处理对病菌的致死作用。同一相对湿度下,随着温度的上升病菌的致死率增加,病菌致病力降低;在同一温度下随着湿度的不断升高,受处理的孢子萌发率和致病能力都逐渐下降。在RH 80%以上、温度40℃以上时,对孢子的致死率随着处理时间的延长而增高,此高温高湿处理病菌超过2 h,病菌孢子不萌发,也基本没有接种发病率。通过高温控制苗期黄瓜黑星病的研究,初步确定高温高湿防治苗期黄瓜黑星病的最佳温度区间为40℃ 2 h或45℃ 1h (RH 80%)。     

Molecular Identification of a Sexual Interloper: The Pear Pathogen, Venturia pirina, has Sex on Apple

ABSTRACT Venturia pirina (the pear scab pathogen) and V. inaequalis (the apple scab pathogen) were detected as ascospores discharged from apple leaf litter in New Zealand (spring 1998). Pseudothecia of both species were located on dead apple leaves; however, only those of V. inaequalis were associated with scab lesions. V. pirina was identified by rDNA sequence analyses, because morphological characters could not distinguish this fungus from V. asperata (a rare saprophyte on apple) and other Venturia spp. pathogenic on rosaceous fruit trees. Species-specific polymerase chain reaction primers designed to the 18S end of the internal transcribed spacer 1 region differentiated Venturia fruit tree pathogens reliably. V. pirina field isolates were pathogenic on pear, but only weak saprophytes on apple. In rare instances, when appressoria of V. pirina appeared to penetrate the cuticle of apple leaves, epidermal cells responded with a localized hypersensitive response (HR). To our knowledge, this is the first report of induction of HR-like events by V. pirina on its nonhost, apple, and also the first record of sexual reproduction of V. pirina on apple. It is assumed that V. pirina pseudothecia formed from saprophytic lesions in senescing apple leaves when active defense mechanisms such as HR were no longer induced.     

Specific Polymerase Chain Reaction Identification of Venturia nashicola Using Internally Transcribed Spacer Region in the Ribosomal DNA

ABSTRACT A technique based on the polymerase chain reaction (PCR) was developed for the identification of Venturia nashicola using nucleotide sequence information of the ribosomal DNA region. The complete internal transcribed spacer (ITS) region of V. nashicola strains and phylo-genetically related species was amplified with the two universal ITS1 and ITS4 primers, sequenced, and digested with five restriction enzymes. The alignment of nucleotide sequences and analyses of digestion patterns indicated constant polymorphisms between V. nashicola and related species at nucleotides 126 and 127, which overlapped a TaqI restriction site. An oligonucleotide primer named A126 was designed for identifying this variable region. A primer set (A126 and ITS4) that allowed the amplification of a 391-bp DNA fragment within the ITS region by PCR was specific to V. nashicola when it was checked against fungal genomic DNAs of related fungi. This primer set was a good candidate for a species-specific reagent in a procedure for identification of V. nashicola by PCR.     

Characterization of Ribosomal DNA from Venturia inaequalis and Its Phylogenetic Relationship to rDNA from Other Tree-Fruit Venturia Species

ABSTRACT A portion of the 18S ribosomal DNA (rDNA) gene, the internal transcribed spacers (ITS1 and ITS2), and the 5.8S rDNA gene were polymerase chain reaction-amplified from strains and field populations of Venturia inaequalis and assessed for genetic variation. A previously reported optional group I intron in the 18S rDNA gene of V. inaequalis was detected in 75.0% of 92 strains collected worldwide and in 61.1 and 71.2% of 54 and 59 strains from two Michigan orchards, respectively. Sequence and restriction analysis of rDNA revealed four intron alleles, three of which were present both in worldwide strains and in each field population. Two ITS1 alleles were detected and found to be linked to specific intron alleles. The ITS1-5.8S-ITS2 sequences from V. asperata V. carpophila, V. cerasi, V. inaequalis, V. nashicola, V. pyrina, and Cladosporium caryigenum were compared using phylogenetic analysis. Strains of the Venturia species were placed in three distinct monophyletic groups in a phylogenetic tree. The first group comprised V. inaequalis; the second, V. pyrina and V. nashicola; and the third, V. cerasi, V. carpophila, and V. asperata. The described intron and ITS1 alleles in V. inaequalis provide genetic markers for subdividing populations of V. inaequalis, and the ITS1-5.8S-ITS2 sequences are valuable in determining the relationship of the species from tree-fruit crops with other Venturia species.     

Evidence of Two Formae Speciales in Venturia inaequalis, Responsible for Apple and Pyracantha Scab

ABSTRACT Genetic relationships, mating crosses, and host specificity of Venturia inaequalis isolates from Malus spp. and of Spilocaea pyracanthae isolates from Pyracantha spp. were evaluated. Sequence analysis of the complete internal transcribed spacer (ITS) region (ITS1-5.8S to ITS2) revealed a total similarity between these two putative species. ITS restriction fragment length polymorphism carried out with five restriction enzymes on a collection of 28 isolates confirmed a lack of diversity in this region between and within these two populations. Additional isolates from three related species (V. pirina, V. nashicola, and S. eriobotryae) were divided into two distinct monophyletic groups in a phylogenetic tree using ITS sequence comparison. These groups were related to their anamorph (i.e., Spilocaea or Fusicladium). When inoculated on their host of origin, fields isolates caused typical symptoms of scab disease, and a host specificity was demonstrated by cross pathogenicity of isolates from Malus x domestica and Pyracantha spp. Mating on dried leaves in vitro between one isolate of each putative species led to production of numerous perithecia. Ninety-six sporulating monoascosporic progenies were isolated from this cross. Based on these genetic and pathogenic data, we proposed that pathogens responsible for scab on Malus spp. and Pyracantha spp. are considered as two formae speciales belonging to V. inaequalis.     

Limitations in the exploitation of N-phenylcarbamates and N-phenylformamidoximes to control benzimidazole-resistant Venturia nashicola on Japanese pear

The efficacy of N -phenylcarbamates and N -phenylformamidoximes against benzimidazole-resistant strains of Venturia nashicola was tested experimentally in Japanese pear orchards. Both compounds gave effective control when applied in an orchard where highly benzimidazole-resistant strains were predominant. However, unsatisfactory control was observed in an orchard where the fungal population was dominated by fungal strains with intermediate or weak benzimidazole resistance. Strains resistant to N -phenylcarbamates and N -phenylformamidoximes were widely distributed in Japanese pear orchards, suggesting that effective use of these compounds to control benzimidazole-resistant strains of V. nashicola would not be practicable. Increased sensitivity to N -phenylformamidoximes in highly benzimidazole-resistant isolates was shown to be controlled by a single chromosomal gene, but progenies that were highly resistant to both a benzimidazole fungicide and a N -phenylformamidoximes appeared in crosses between parents resistant to each fungicide alone. Similar strains were also found in pear orchards, and this'double resistance'was shown to be heritable.     

Infection Behavior of Venturia nashicola, the Cause of Scab on Asian Pears

ABSTRACT The infection of Japanese pear by Venturia nashicola, the cause of scab on Asian pears (Japanese pear, Pyrus pylifolia var. culta; Chinese pear, P. ussuriensis), was examined using light and electron microscopy to determine the mechanism of resistance in pears. Early stages of infection were similar on the susceptible cv. Kosui, the resistant cv. Kinchaku, and the nonhost European pear (P. communis) cv. Flemish Beauty. V. nashicola penetrated only the cuticle layer on pear leaves and formed subcuticular hyphae on all three cultivars. Hyphae were localized in the pectin layer of pear leaves and never penetrated into the cytoplasm of epidermal cells. This restriction of fungal growth suggested that pectinases released by infection hyphae or subcuticular hyphae may be important in infection. Subcuticular hyphae were modified ultrastructurally in the pectin layer of resistant pear cultivars accompanied by fungal cell death. In contrast, fungal cells appeared intact in susceptible pear cultivars, suggesting the existence of resistance mechanisms.     

Control efficacy of the systemic acquired resistance (SAR) inducer acibenzolar-S-methyl against Venturia nashicola in Japanese pear orchards

Journal of General Plant Pathology - Scab caused by Venturia nashicola is one of the most serious diseases of Asian pears, and conventional fungicides used to control scab can lose their efficacy...     

Resistance of Venturia nashicola to thiophanate-methyl and benomyl: build-up and decline of resistance in the field

Changes in the proportion of benzimidazole-resistant strains in populations of Venturia nashicola were monitored in pear orchards. Successive applications of thiophanate-methyl increased the level of resistance in the populations. When the application of benzimidazoles was stopped and other fungicides were applied, the proportion of highly resistant strains gradually decreased and the proportion of intermediately resistant, weakly resistant and sensitive strains increased. It is suggested that this phenomenon is an example of genetic homeostasis within microbial populations.