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。     

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.     

抗黑星病芽变鸭梨组织结构、生理生化与抗病性关系研究

对一株抗黑星病芽变‘鸭梨’的叶片组织结构和生理生化特性进行了研究,发现其叶片蜡质含量高、栅栏组织双层出现时间早、厚度大、海绵组织结构致密、气孔数目少,推断这些特性决定黑星病菌分生孢子不易侵入叶片内部;叶绿素含量高为植株抗病性提供了生理基础,可溶性糖含量低有可能抑制黑星病营养菌丝在细胞间的生长和扩展。通过研究该抗黑星病芽变‘鸭梨’的抗病机理,为‘鸭梨’抗黑星病育种提供了理论依据。     

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.     

不同处理及pH变化对果胶酶诱导抗病性的影响

从不同处理方法、酶液pH变化等方面对果胶酶诱导黄瓜抗黑星病效能的影响进行了研究。结果表明，在果胶酶的4种不同处理试验中，以全株表面喷雾法处理的黄瓜黄化子叶黑星病的病情指数降低最多，浓度100和200U／ml的诱导防病效果分别为56．34％和64．13％；下胚轴注射、灌根和漫种等3种处理方法的诱导抗病效果均不显著。经果胶酶诱导处理的黄瓜绿苗叶片病情明显降低，而处理叶上部的未处理叶片发病情况与相应对照差别不大。该结果表明，果胶酶诱导黄瓜绿苗抗黑星病的作用属于局部诱导。果胶酶处理次数的增加可增强其诱导抗病效果，并延长诱导抗病时间。酶液pH变化对果胶酶的诱导抗病作用影响显著，pH5．5时，2个浓度的诱导效果最好，分别为51．87％和66．42％。     

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.     

Some studies on the systemic activity of the thiophanate fungicides in plants

Three closely related fungicides, thiophanate (NF 35), thiophanate methyl (NF 44) and NF 48 have been found to be active against a wide range of diseases including the powdery mildews of apple, cucumber and barley; apple and pear scab and grey mould of soft fruit. This paper deals with the preliminary experiments to compare their relative mobilities and biological persistence in crop plants, using the powdery mildews as indicator infections. NF 44 and NF 48 in particular showed a high degree of systemic activity by root uptake and persistence of action against barley and cucumber mildew but very little systemic activity against apple mildew on potted rootstocks. They have also displayed trans-laminar and localised movement towards the leaf apex in sprayed cucumber and barley leaves but movement was not so marked in sprayed apple leaves. NF 48 was also active as a seed dressing against loose smut of barley.     

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。     

草酸青霉菌果胶酶诱导黄瓜抗黑星病研究

 本文就草酸青霉菌(Penicillium oxalicum)的固态发酵提取产物--果胶酶粗酶液诱导黄瓜对黑星病(Cladosporium cucumerinum)的抗性进行了研究。以果胶酶粗酶液喷雾处理4个感黑星病黄瓜品种的黄化苗,48 h后挑战接种黑星病菌孢子悬液,其中"中农5号"黄瓜品种表现的诱导抗病效果最好,诱抗效果达62.51%。不同浓度果胶酶诱导处理黄瓜,发现果胶酶浓度在20 U/mL时,可导致黄瓜发病略高于对照;在40~200 U/mL浓度范围内,诱导效果较为明显。通过研究果胶酶诱导抗病的时效性,表明诱导处理前接种病菌或诱导处理后0、6 h接种的各处理病情指数与对照间没有差别,而诱导处理12~72 h后接种病菌的,果胶酶的诱导抗病效果均很明显,诱抗效果达29.64%~60.02%。实验还表明,随着挑战接种压力的增大,果胶酶的诱导抗病效果降低。果胶酶不能抑制黑星病菌孢子萌发,相反可以促进孢子萌发和芽管生长。     

Effect of acibenzolar-S-methyl, strobilurins and other fungicide treatments on phylloplane fungi of crops

Strobilurins, broad-spectrum fungicides used to control various diseases, may also inhibit the growth of nontarget fungi in the natural environment. Acibenzolar-S-methyl (ASM) is a plant activator; however, the effect of ASM on the growth of microflora on the leaf surface is not clear yet. ASM, strobilurins and other fungicides were sprayed on Japanese pear and cucumber plants in the field. Variations in the morphological characteristics and rDNA-ITS nucleotide sequence of isolated phylloplane fungi were evaluated. The isolation frequency of Alternaria spp., Aureobasidium spp., Cladosporium spp. and Myrothecium spp. on Japanese pear leaves treated with kresoxim-methyl and polycarbamate was lower than on nontreated pear leaves. Moreover, the frequency of Alternaria spp., Cladosporium spp., Epicoccum spp. and Fusarium spp. on leaves of two cucumber cultivars treated with azoxystrobin, triflumizole and mancozeb was lower than the frequency of fungal isolation on nontreated cucumber leaves. The effect of ASM on fungal isolation frequency was not significantly different from that on nontreated pear or cucumber leaves. The fungal genera observed and isolation frequency on the leaves treated with polycarbamate, azoxystrobin, triflumizole and mancozeb tended to be reduced compared to the no-spray treatment.     

梨抗黑星病AFLP标记筛选

 Pear scab caused by Venturia nashicola is one of the most destructive diseases of pears. Molecular markers linked to scab resistance gene is expected to be useful for improving pear. In this study, the F₁ population derived from the cross of ‘Huangguan’ and ‘Yali’ was analyzed genetically. The resistance of pear to scab was proved to be controlled by a single gene in a dominant manner. Bulked segregant analysis (BSA) was conducted to screen 64 fluorescent AFLP primer pairs. A marker designated as D3-365 was found to be linked to the resistant locus. Selective genotype linkage analysis showed that the genetic distance between the marker and the resistant locus was 14.9 cM.     

玫瑰黄链霉菌Men-myco-93-63诱抗粗蛋白对黄瓜的诱抗作用

玫瑰黄链霉菌Strempomyces roseoflavus Men-myco-93-63是分离自马铃薯疮痂病自然衰退土壤中的一株拮抗链霉菌,该菌株及其代谢产物对多种重要的植物病原菌都具有较强的抑制作用,为了探明该生防菌产生的诱抗粗蛋白对黄瓜抗病性的诱导作用,采用离体叶片接种的方法,发现诱抗粗蛋白诱导黄瓜叶片灰霉病发病直径显著小于对照;经组织染色法和紫外分光光度计法测定,发现诱抗粗蛋白可以诱导黄瓜叶片中活性氧（ROS）的积累和超氧化物歧化酶（SOD）、过氧化物酶（POD）和多酚氧化酶（PPO）等抗病相关酶活性的显著提高;通过实时荧光定量PCR（qRT-PCR）测定,发现诱抗粗蛋白还可以诱导黄瓜叶片中PR-1a、PR-3、PR-9和NPR1等抗病相关基因表达的上调,试验结果表明Men-myco-93-63产生的诱抗粗蛋白能够诱导黄瓜抗病性的提升。     

持久抗条锈小麦品种抗病性特点分析

利用我国１５个小麦条锈菌主要流行小种和致病类型，测定了国内外７个持久抗条锈小麦品种的抗病性。结果表明，尽管不同持久抗病品种苗期抗病谱有很大差异，但成株期对许多供试菌系均表现有效抗性。抗病温度敏感性试验显示，随着培养温度由低温、常温向高温提高，持久抗性品种抗性逐渐增强，表现为反应型和严重度降低。抗病基因分析表明，持久抗病品种不仅含有温敏型微效抗病基因，而且含有成株抗病基因，它们共同对品种持久抗条锈性起作用     

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.     

小麦抗赤霉病性田间自然鉴定与抗赤霉病育种中的问题

１９８４年以来,承接了全国小麦育种攻关中的小麦抗赤霉病性鉴定任务,对来自全国１０个麦区１２６４８份小麦育种材料料霉病性进行田间自然鉴定。结果表明,至今仍未发现免疫的小麦材料。长江中下游冬麦区,华南冬麦区及东北春麦区的供鉴小麦抗性较强,全国Ｒ级小麦材料均分布在这些赤病频繁流行的麦区;西南冬麦区,黄淮冬麦区及北部春麦区的供鉴小麦抗性最差。我国现有小麦材料的抗病性与丰产性仍存在较大矛盾,至今仍缺乏可应用     

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.     

Cross-resistance ofAphis gossypii to selected insecticides on cotton and cucumber

Cross-resistance of a fenvalerate-resistant (R-fenvalerate) strain and an imidacloprid-resistant (R-imidacloprid) strain of the cotton aphidAphis gossypii Glover to methomyl, endosulfan and omethoate was determined on cotton and cucumber in the laboratory. The R-fenvalerate strain had been selected for 16 consecutive generations on cotton, and the R-imidacloprid strain for 12. Compared with the susceptible (S) strains, the resistance in the 17th generation of the R-fenvalerate strain to fenvalerate was 29,035-fold on cotton and 700-fold on cucumber, whereas the resistance in the 13th generation of the R-imidacioprid strain to imidacloprid was 4.7-fold on cotton and 3.6-fold on cucumber. The R-fenvalerate strain did not show any cross-resistance to imidacloprid, methomyl or omethoate on cotton and cucumber, but became slightly cross-resistant to endosulfan (RR [resistance ratio] = 5.8 and 7.4 on cotton and cucumber, respectively). On cotton the R-imidacloprid strain became highly cross-resistant to fenvalerate (RR = 108.9) and slightly cross-resistant to methomyl, endosulfan and omethoate (RR = 6.3, 3.0 and 8.5, respectively). In contrast, on cucumber the R-imidacloprid strain became highly cross-resistant to fenvalerate (RR = 33.5) and slightly cross-resistant to methomyl and omethoate (RR = 7.0 and 3.3, respectively), but did not show any cross-resistance to endosulfan. It appears thatA. gossypii of both resistant strains was more resistant to the tested insecticides on cotton than on cucumber, except for imidacloprid —where the resistance on cotton was not significantly different from that on cucumber.