Polygalacturonase-inhibiting protein (PGIP) from Japanese pear: possible involvement in resistance against scab

Venturia nashicola is the causal agent of scab, a fungal disease affecting Asian pears. The Japanese pear cv. ‘Kousui’ is highly susceptible to the race 1 of this fungus whereas the cv. ‘Kinchaku’ and the non-host European pear cv. ‘Flemish Beauty’ are resistant. The aim of this work is to investigate the role of polygalacturonase-inhibiting proteins (PGIPs) of pear during the interactions with V. nashicola leading to susceptibility or resistance. PGIP protein was detected from immature fruit of Kousui and Kinchaku. It showed a molecular mass of 42 kDa that shifted to 35 kDa after chemical deglycosylation. The gene pgip was amplified by PCR using genomic DNA and/or cDNA from young leaves of Kousui, Kinchaku, and European pear cvs. Flemish Beauty, ‘Bartlett’, and an Asian wild pear strain ‘Mamenashi 12’, then sequenced after sub-cloning. Some conserved variations were identified in the sequence indicating that gene family also exists in pgip of Japanese pear and confirmed by Southern blot analysis. The expression of PGIP was studied in scab-inoculated leaves of the susceptible Kousui and the resistant Kinchaku and Flemish Beauty. pgip Gene and its encoding protein were highly and rapidly activated in these resistant plants. In addition, PGIP extracts derived from Kinchaku and Flemish Beauty partially inhibited the activity of polygalacturonase (PG) from V. nashicola suggesting a possible role of PGIP in limiting fungal growth frequently observed in these resistant cultivars.     

Ultrastructural study on scab resistance expressed in epidermal pectin layers of pear leaves

Proliferation and collapse of subcuticular hyphae of Venturia nashicola race 1 were studied ultrastructurally, after inoculation of susceptible Japanese pear cv. Kousui, resistant Japanese pear cv. Kinchaku, resistant Asian pear strain Mamenashi 12 and nonhost European pear cv. Flemish Beauty leaves, to understand the nature of the resistance mechanism. After cuticle penetration by the pathogen, the hyphae were observed at lower frequency in epidermal pectin layers and middle lamellae of leaves of the three resistant plants than in those of susceptible ones. This result suggested that fungal growth was suppressed in the incompatible interaction between pear and V. nashicola race 1. In the pectin layers of all inoculated plants, some hyphae had modifications such as breaks in the plasmalemma with plasmolysis, necrotic cytoplasm and degraded cell walls. More hyphae had collapsed in the leaves of the three resistant plants than in those of the susceptible cv. Kousui. In collapsed hyphae, the polymerized cell walls broke into numerous fibrous and amorphous pieces, showing that the scab resistance might be associated with cell wall-degrading enzymes from pear plants.     

Molecular cloning and expression analysis of genes related to phosphatidic acid synthesis in Japanese pear leaves inoculated with <Emphasis Type="Italic">Venturia nashicola</Emphasis>

Phosphatidic acid, which can be generated by the action of phospholipase D (PLD) and by the combined action of phospholipase C (PLC) and diacylglycerol kinase (DGK), is a plant defense signal. To identify the role of the PLD, PLC, and DGK genes encoding these enzymes in pear resistance to the scab fungus Venturia nashicola, we report the cloning of these genes and analysis of their expression in inoculated pear leaves. The results showed that PLD mRNA increased in the leaves of the susceptible pear cv. Kousui 1 day after inoculation (dai), then decreased to the basal level. In resistant pear cv. Kinchaku, PLD mRNA did not change significantly except for a small increase at 2 and 3 dai. However, the level of PLD mRNA in Kinchaku was higher than in Kousui 2 dai. The level of PLC mRNA significantly increased in cvs. Kousui and Kinchaku by 1 dai, peaked at day 2, then decreased to the basal level. Thus, the gene expression pattern did not differ between the two cultivars. PLC gene expression was also induced by environmental stress. The DGK gene seemed to be constitutively expressed and was not induced by inoculation in either the susceptible or resistant cultivars. Together, this data showed that both PLD and PLC are possibly involved in a defense response to V. nashicola in Japanese pear leaves, and the PLD gene seems to play a more important role than does the PLC gene.     

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。     

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.     

Ultrastructural study on acibenzolar-S-methyl-induced scab resistance in epidermal pectin layers of Japanese pear leaves

The infection behavior of Japanese pear scab pathogen Venturia nashicola race 1 was studied ultrastructurally in acibenzolar-S-methyl (ASM)-pretreated susceptible Japanese pear (cv. Kousui) leaves to determine the mechanism of ASM-induced scab resistance. On ASM-pretreated leaf surfaces, the infection behavior (conidial germination and appressorial formation) was similar to that on distilled water (DW)-pretreated leaves prior to cuticle penetration by the pathogen. However, after penetration, differentiated behavior was found in epidermal pectin layers and middle lamellae of the ASM-pretreated leaves. Subcuticular hyphae in epidermal pectin layers and middle lamellae of ASM-pretreated pear leaves were observed at lower frequency than in DW-treated leaves. The results indicated that fungal growth was suppressed in ASM-pretreated pear leaves. In the pectin layers of ASM- and DW-pretreated leaves, some hyphae showed morphological modifications, which were used as criteria to judge collapse of hyphal cells, including plasmolysis, necrotic cytoplasm, and cell wall destruction. More hyphae had collapsed in ASM-pretreated leaves than in DW-treated ones. In addition, the cell walls of collapsed hyphae broke into numerous fibrous and amorphous pieces, suggesting that ASM-induced scab resistance might be associated with cell-wall-degrading enzymes from pear plants. In addition, results from morphometrical analysis suggested that the activity or production of pectin-degrading enzyme from hyphae were inhibited by ASM application when compared with DW treatment.     

Identification of putative defense-related genes in Japanese pear against <Emphasis Type="Italic">Alternaria alternata</Emphasis> infection using suppression subtractive hybridization and expression analysis

The Japanese pear pathotype of Alternaria alternata, a toxin-dependent necrotrophic pathogen, causes black spot of Japanese pear by producing the host-specific AK-toxin. Pre-inoculation with nonpathogenic A. alternata or pretreatment with an elicitor prepared from A. alternata reduced disease symptoms caused by the pathogen. Salicylic acid- and jasmonic acid-dependent signaling pathways are not involved in the induced resistance to infection by the pathogen. The expression of multiple defense-related genes in Japanese pear leaves inoculated with nonpathogenic A. alternata was examined using suppression subtractive hybridization. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank database as accessions DC993229–DC993535.     

Reduced sensitivity to fenarimol in Japanese field strains of Venturia nashicola

Monoconidial strains of Venturia nashicola Tanaka et Yamamoto were isolated from Japanese or Chinese white pear trees which had never been treated with sterol demethylation inhibitors (DMIs) and their baseline sensitivities to fenarimol were determined by mycelial growth tests on fungicide-amended culture media. Strains were also obtained from Japanese pear orchards, which had been intensively treated with DMIs for several years and monitored for the shifts of fenarimol sensitivity in comparison with the baseline sensitivity. Results suggested slight shifts to lower fenarimol sensitivity in strains isolated from DMI-treated Japanese pear orchards. However, in inoculation tests on pear seedlings, fenarimol still provided adequate control of V. nashicola strains with reduced sensitivity to fenarimol in vitro, suggesting that the performance of this fungicide will still be maintained in the field. © 1998 Society of Chemical Industry     

梨叶叶龄与梨黑星病菌侵染发病的关系

田间试验结果表明，鸭梨叶片在生长发育期抗病性差，易感染黑星病；发育成熟后，抗病性逐渐增强。自然条件下，受叶片抗病性和孢子着落量的综合影响，鸭梨成熟叶片很少感染黑星病。鸭梨叶片从展叶到发育成熟，在梨树生长发育前期（盛花期后60d内）约需40d，后期（盛花期60d后）约需30d。正处在生长发育期的鸭梨叶片受侵染，潜育期为18－25d，发病后，病斑大，产孢期短；成熟叶片受侵染，潜育期多在40d以上，发病后，病斑小，产孢期长。梨树生长前期梨黑星病的流行主要由于大部分叶片正处在生长发育期抗病性较差；后期果实发病则主要源于成熟叶片内长期潜育和长期产孢的黑星病菌。     

Resistance of wheat to Mycosphaerella graminicola involves early and late peaks of gene expression

Large-scale cDNA-AFLP profiling identified numerous genes with increased expression during the resistance response of wheat to the Septoria tritici blotch fungus, Mycosphaerella graminicola. To test whether these genes were associated with resistance responses, primers were designed for the 14 that were most strongly up-regulated, and their levels of expression were measured at 12 time points from 0 to 27 days after inoculation (DAI) in two resistant and two susceptible cultivars of wheat by real-time quantitative polymerase chain reaction. None of these genes was expressed constitutively in the resistant wheat cultivars. Instead, infection of wheat by M. graminicola induced changes in expression of each gene in both resistant and susceptible cultivars over time. The four genes chitinase, phenylalanine ammonia lyase, pathogenesis-related protein PR-1, and peroxidase were induced from about 10- to 60-fold at early stages (3 h–1 DAI) during the incompatible interactions but were not expressed at later time points. Nine other genes (ATPase, brassinosteroid-6-oxidase, peptidylprolyl isomerase, peroxidase 2, 40S ribosomal protein, ADP-glucose pyrophosphorylase, putative protease inhibitor, methionine sulfoxide reductase, and an RNase S-like protein precursor) had bimodal patterns with both early (1–3 DAI) and late (12–24 DAI) peaks of expression in at least one of the resistant cultivars, but low if any induction in the two susceptible cultivars. The remaining gene (a serine carboxypeptidase) had a trimodal pattern of expression in the resistant cultivar Tadinia. These results indicate that the resistance response of wheat to M. graminicola is not completed during the first 24 h after contact with the pathogen, as thought previously, but instead can extend into the period from 18 to 24 DAI when fungal growth increases dramatically in compatible interactions. Many of these genes have a possible function in signal transduction or possibly as regulatory elements. Expression of the PR-1 gene at 12 h after inoculation was much higher in resistant compared to susceptible recombinant-inbred lines (RILs) segregating for the Stb4 and Stb8 genes for resistance. Therefore, analysis of gene expression could provide a faster method for separating resistant from susceptible lines in research programs. Significant differential expression patterns of the defense-related genes between the resistant and susceptible wheat cultivars and RILs after inoculation with M. graminicola suggest that these genes may play a major role in the resistance mechanisms of wheat.     

Induced Resistance of Acibenzolar-S-methyl (CGA 245704) to Cucumber and Japanese Pear Diseases

Antifungal activity of the novel compound acibenzolar-S-methyl (CGA245704: benzo[1,2,3]thiadiazole-7-carbothioic acid S-methyl ester) was examined in vitro. No remarkable activity was observed on mycelial growth and conidial germination of almost all fungi tested. Only melon isolates of Didymella bryoniae were sensitive to this compound. On potted plants, acibenzolar-S-methyl showed control efficacy on anthracnose and scab of cucumber and rust of Japanese pear but not on Fusarium wilt of cucumber. In field trials, the occurrence of both rust and scab on Japanese pear was suppressed with this compound. Based on these experiments, it was suggested that acibenzolar-S-methyl induced resistance to some but not all diseases on cucumber and Japanese pear. Induction of disease resistance in cucumber was rapidly triggered after treatment with acibenzolar-S-methyl.     

广东省甘薯疮痂病病原菌鉴定及国内主要菜用甘薯种质抗性评价

为明确广东省甘薯疮痂病病原菌种类及当前国内主要菜用甘薯种质对疮痂病的抗性,结合形态学特征和rDNA-ITS序列分析对病原菌进行鉴定,并通过病圃诱发结合人工喷雾接种法对来自9个省区30个菜用甘薯种质进行连续2年的抗性鉴定。结果显示,共获得15株形态学特征及培养性状相似的菌株,其中代表菌株CRI-CJ的形态学特征与甘薯痂圆孢菌Elsinoe batatas基本一致,且在基于rDNA-ITS序列的系统发育树中与甘薯痂圆孢菌聚在一支,表明甘薯疮痂病病原菌为甘薯痂圆孢菌。30个菜用甘薯品种(系)中,抗性、中抗、中感和感病品种(系)分别为7、7、5和11个,占总数的23.3%、23.3%、16.7%和36.7%。其中抗性品种(系)有广菜薯11-52、广菜薯15-6、广菜薯16-1、广菜薯16-19、广菜薯17-23、广菜薯17-9和广菜薯18-6,中抗品种(系)有EC01、广薯菜2号、广菜薯3号、广菜薯6号、广菜薯7号、广菜薯17-25和广菜薯18-3。不同地理来源的品种(系)对甘薯疮痂病的抗性水平存在差异,11个非广东省品种(系)中感病、中感和中抗品种(系)分别为8、2和1个,占比分别为72.7...     

Bioluminescence reporter assay system to monitor Arabidopsis MPK3 gene expression in response to infection by Botrytis cinerea

The Arabidopsis MPK3 gene product participates in disease resistance mediated by the MAP kinase cascade. The expression of the MPK3 gene is induced by pathogen inoculation and treatment with chemicals such as salicylic acid (SA) and methyl jasmonate (JA), but the detailed expression pattern of the MPK3 gene has been largely unknown. To investigate MPK3 gene expression in response to disease stress, we fused the MPK3 promoter to the firefly luciferase gene to create a real-time monitoring system for regulated gene expression in planta. The results of an in vivo reporter assay using transgenic Arabidopsis plants harboring MPK3::Fluc showed that the MPK3 promoter activity was induced by treatment with chemicals such as SA and benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH), that induce defense gene expression. Inoculation with the fungal pathogen Botrytis cinerea resulted in systemic induction of MPK3::Fluc.     

马铃薯疮痂病高效生防芽孢杆菌的筛选及发酵条件优化

利用已鉴定的5株拮抗马铃薯黑痣病病原菌的生防芽孢杆菌（Bacillus）,筛选其对马铃薯疮痂病病原菌的抑制作用,通过检测生防菌脂肽类抗生素合成相关基因研究其生防机制。对峙试验结果表明,5株生防菌中以QHZ-3对马铃薯疮痂病病原菌的抑制效果最佳,抑菌率为44%;抗生素合成基因检测结果显示,QHZ-1和QHZ-2具有丰原素（Fengycin）和伊枯草菌素（Iturin）合成酶基因;QHZ-4仅具有Fengycin合成酶基因;而QHZ-3与QHZ-5菌株同时具有表面活性素（surfactin）、伊枯草菌素（Iturin）和丰原素（fengycin）3种脂肽类抗生素合成酶基因。选择最具生防潜力的QHZ-3进行进一步试验,发现该菌株发酵滤液对疮痂链霉菌的抑菌圈直径达到30.4 mm,且在高温、紫外照射以及强酸强碱的条件下抑菌圈直径仍然可保持在27 mm以上。单因素试验结果表明,每瓶（250 mL）装液量60 mL,接种量6%,摇床转速180 r·min^-1,发酵时间60 h的条件下,菌株QHZ-3的生长量最高。正交试验结果显示,装液量每瓶40 mL、摇床转速170 r·min^-1、接种量6%、发酵时间48 h的组合下,QHZ-3生长状态最佳,活菌数可达到1.31×10¹⁰ cfu·mL^-1。二次固态发酵条件优化结果显示,以腐熟的牛粪有机肥为载体,在接种量为15%时,活菌数最高,达到1.14×10⁹ cfu·mL^-1。     

Ascospores of the Japanese pear scab fungus (<Emphasis Type="Italic">Venturia nashicola</Emphasis> Tanaka &; Yamamoto) are discharged during the day

We investigated the diurnal pattern of ascospore discharge of the Japanese pear scab fungus (Venturia nashicola Tanaka & Yamamoto) in an orchard. Ascospores of V. nashicola were mainly discharged during the day. Most ascospores were discharged from 7:00 to 19:00: 99.6% in 2001, 99.3% in 2002, and 93.8% in 2005. Because the ascospores were discharged only when the fallen diseased leaves were wet from precipitation, the wetness of these leaves is probably imperative for spore discharge. Ascospore discharge began immediately after precipitation in the daytime. When it rained at night, however, ascospore discharge did not begin until the following morning and never began immediately after precipitation. We also investigated other meteorological factors. When fallen diseased leaves were wet, the percentage of ascospore discharge was positively correlated with the amount of solar radiation and atmospheric temperature and negatively correlated with relative humidity. Ascospore discharge was interrupted by a decrease in solar radiation and atmospheric temperature and by increased relative humidity at night. This report is the first that V. nashicola discharges ascospores primarily during the day.     

Gummy stem blight of balsam pear caused by <Emphasis Type="Italic">Didymella bryoniae</Emphasis> and its anamorph <Emphasis Type="Italic">Phoma cucurbitacearum</Emphasis>

Gummy stem blight of balsam pear found in the Kanto district and in the Hokkaido Prefecture was demonstrated to be caused by Didymella bryoniae (Auerswald) Rehm based on inoculation experiments, molecular analysis, and morphological identification of the pathogenic fungus. This fungus was also pathogenic to related plants belonging to Cucurbitaceae. The imperfect stage of the fungus was identified as Phoma cucurbitacearum (Fr.: Fr.) Sacc. based on morphological similarities.