葡萄座腔菌Botryosphaeria dothidea侵染及其对苹果果实影响的组织细胞学研究

利用光镜和电镜技术系统研究了苹果轮纹病菌葡萄座腔菌在成熟果实上的侵染扩展过程及其细胞学特征。扫描电镜观察发现,接种后3h位于皮孔处的分生孢子开始萌发,萌发后的孢子从一端或两端产生芽管直接侵入皮孔细胞,接种后9h完成侵入。30d后果面接种部位表现症状,45d后产生子实体。对接种部位取样进行光镜和透射电镜观察发现,病菌菌丝主要存在于寄主细胞壁、细胞内、细胞间隙及细胞壁与细胞膜之间。菌丝呈丝状,分枝,具隔膜。菌丝细胞内含有细胞核、线粒体、液泡等细胞器;菌丝外散发出一些高电子密度的颗粒物质,这些物质以菌丝为中心,呈放射状分布。病菌在果肉细胞生长扩展过程中,果肉细胞发生一系列变化。果肉细胞壁膨胀、变形,胞间层分离、破裂。与菌丝接触或相邻的果肉细胞细胞壁电子致密度降低,被降解成为如散发状的胞壁纤维束丝。果肉细胞的液泡破裂,质壁分离,细胞质凝结坏死并沉积于细胞壁周围,或通过受损的细胞壁胞间连丝从一个细胞转移到另一个细胞。后期菌丝在表皮下聚集生长、发育成分生孢子器。分生孢子器内壁细胞排列紧密,细胞中含有由数条丝状物平行排列而成的细胞器。该细胞器形状多样,周围总是分布着丰富的脂肪粒,推测可能与营养的运输与积累有关。     

小麦雪霉叶枯病菌侵染过程中的细胞学研究

采用电镜技术研究了小麦雪霉叶枯病（Ｇｅｒｌａｃｈｉａｎｉｖａｌｉｓ）侵染过程的细胞学特征,电镜观察发现,分子孢子萌发产生的芽管由孢子细胞壁内层延伸而成,病菌侵入寄主体内后,胞间菌丝先在寄主细胞间扩展,随后胞间菌丝侵入坏死的寄主细胞,形成细胞内菌丝;胞间菌丝和胞内菌丝在形态结构上无明显差异,在病菌扩展过程中,寄生细胞发生了一系列的病理变化,并最终坏死消解,寄主细胞的变化可能与病菌分泌的毒素有关。     

苜蓿假盘菌侵染苜蓿叶片的细胞学研究

采用微分干涉相差显微镜、扫描和透射电镜技术系统研究了苜蓿假盘菌Pseudopeziza medicaginis在苜蓿叶片的侵染过程及超微结构特征。结果表明,接种4h后,子囊孢子萌发产生芽管;12h后,芽管以直接侵入的方式进入表皮细胞形成侵染菌丝;24h后,表皮细胞中侵染菌丝向相邻表皮细胞扩展,同时侵入到叶肉细胞以胞内生长方式扩展;接种72h后,侵染菌丝在表皮细胞下的叶肉组织中形成初始菌落;第5d后,菌丝扩展至整个叶片组织,大量菌丝聚集形成子座组织,并进一步形成子囊盘与子囊。病菌菌丝在侵入寄主细胞初期,并不穿透寄主质膜与原生质,而是被其所包围。但随着菌丝进一步扩展,叶片组织发生了一系列的病理变化,其中包括叶肉细胞肿胀、细胞质消解、叶绿体等细胞器解体以及寄主细胞坏死塌陷,并最终在叶表面产生典型的褐斑病症状。     

尖角突脐孢菌侵染过程及稗草反应超微结构观察*

为了解尖角突脐孢菌侵染稗草植株的致病机理,应用电镜观察了该菌侵染稗草植株过程和寄主细胞的超显微结构变化。结果表明：尖角突脐孢菌分生孢子于接种8h后从突脐孢的基部开始萌发菌丝,接种13h后菌丝直接穿透稗草叶表面细胞连接处或经气孔侵入,接种后24～26h完成侵染过程。被侵染的寄主,膜结构发生变形,同时细胞线粒体的嵴变形膨大,叶绿体内的淀粉粒消失,寄主细胞内有大量酚物质出现。     

苜蓿假盘菌侵染苜蓿叶片的细胞学研究

采用微分干涉相差显微镜、扫描和透射电镜技术系统研究了苜蓿假盘菌Pseudopeziza medicaginis在苜蓿叶片的侵染过程及超微结构特征。结果表明,接种4h后,子囊孢子萌发产生芽管：12h后,芽管以直接侵入的方式进入表皮细胞形成侵染菌丝：24h后,表皮细胞中侵染菌丝向相邻表皮细胞扩展,同时侵入到叶肉细胞以胞内生长方式扩展：接种72h后,侵染菌丝在表皮细胞下的叶肉组织中形成初始菌落;第5d后,菌丝扩展至整个叶片组织,大量菌丝聚集形成子座组织,并进一步形成子囊盘与子囊。病菌菌丝在侵入寄主细胞初期,并不穿透寄主质膜与原生质,而是被其所包围。但随着菌丝进一步扩展,叶片组织发生了一系列的病理变化,其中包括叶肉细胞肿胀、细胞质消解、叶绿体等细胞器解体以及寄主细胞坏死塌陷,并最终在叶表面产生典型的褐斑病症状。     

苜蓿假盘菌侵染苜蓿叶片的细胞学研究

采用微分干涉相差显微镜、扫描和透射电镜技术系统研究了苜蓿假盘菌Pseudopeziza medicaginis在苜蓿叶片的侵染过程及超微结构特征。结果表明,接种4h后,子囊孢子萌发产生芽管;12h后,芽管以直接侵入的方式进入表皮细胞形成侵染菌丝;24h后,表皮细胞中侵染菌丝向相邻表皮细胞扩展,同时侵入到叶肉细胞以胞内生长方式扩展;接种72h后,侵染菌丝在表皮细胞下的叶肉组织中形成初始菌落;第5d后,菌丝扩展至整个叶片组织,大量菌丝聚集形成子座组织,并进一步形成子囊盘与子囊。病菌菌丝在侵入寄主细胞初期,并不     

小麦雪霉叶枯病菌侵染过程的细胞学研究

采用电镜技术研究了小麦雪霉叶枯病菌(Gerlachia nivalis)侵染过程的细胞学特征。电镜观察发现,分生孢子萌发产生的芽管由孢子细胞壁内层延伸而成;病菌侵入寄主体内后,胞间菌丝先在寄主细胞间扩展,随后胞间菌丝侵入坏死的寄主细胞,形成胞内菌丝;胞间菌丝和胞内菌丝在形态结构上无明显差异。在病菌扩展过程中,寄主细胞发生了一系列的病理变化,并最终坏死消解,寄主细胞的变化可能与病菌分泌的毒素有关。     

柑桔叶点霉侵染琯溪蜜柚的超微结构观察及致病机理分析

为探明黑斑病对琯溪蜜柚(Citrus grandis‘Guanximiyou’)果皮细胞的侵染破坏特点及作用机理,人工接种柑橘叶点霉亚洲种(Phyllosticta citriasiana)后,在扫描电镜与透射电镜下观察蜜柚真皮细胞的变化。结果表明,受到侵染后,真皮细胞的液泡膜首先发生褶皱、断裂,最终消失;叶绿体的淀粉粒消失、膜出现褶皱;质膜内陷、局部断裂;类囊体发生膨胀并解体;线粒体数量明显增加,基粒清晰可见,之后线粒体形态改变,双层膜消失,内部空胞化。未观察到细胞间隙及细胞内有菌丝分布,且细胞壁结构始终稳定。P.citriasiana危害蜜柚果皮细胞与毒素作用有关,侵染位点可能是细胞膜,信息通过胞间连丝穿过细胞壁,进入细胞内部。     

小麦叶锈菌侵染过程的显微和超微结构

采用光学显微技术和电子显微技术对小麦叶锈菌的侵染过程进行了研究。发现叶锈菌从气孔侵入后在气孔腔内形成气孔下泡囊,然后分化出圆形的膨大体,由膨大体产生1—2初生菌丝,初生菌丝在寄主细胞间隙延伸扩展,与叶肉细胞壁接触后分化形成吸器母细胞,吸器母细胞进入寄主细胞后形成吸器。初生菌丝在吸器母细胞处产生分枝,形成次生菌丝在叶肉细胞间蔓延。在病原菌侵染早期(接种后8—24h),寄主细胞的超微结构变化并不明显。侵染中、后期(接种48—72h),被侵染叶肉细胞发生严重质壁分离,叶绿体膨胀变形,基粒片层排列疏松。线粒体嵴突退化。     

小麦纹枯病菌侵染过程的组织学研究

本文报道了小麦纹枯病菌侵染小麦的过程,病菌在穿透寄主之前产生侵染热 丝圈以及形态简单的单附着胞等侵染结构,由染垫基部丝或附产丰胞产生的侵染菌丝直接或通过气孔侵入寄主,也可见菌丝直接侵入寄主;菌丝侵入寄主表皮后,迅速在受侵细胞内呈网状产扩展,并直接穿透毗邻细胞壁,向其它细胞纵横扩展,受病组织出现细胞变形,变空;接近菌丝的质膜发生质壁分离,质膜断裂,叶绿体有,变小或接近消失,类本被破坏,叶绿体内嗜颗粒     

Products Released from Enzymically Active Cell Wall Stimulate Ethylene Production and Ripening in Preclimacteric Tomato (Lycopersicon esculentum Mill.) Fruit

Enzymically active cell wall from ripe tomato (Lycopersicon esculentum Mill.) fruit pericarp release uronic acids through the action of wall-bound polygalacturonase. The potential involvement of products of wall hydrolysis in the induction of ethylene synthesis during tomato ripening was investigated by vacuum infiltrating preclimacteric (green) fruit with solutions containing pectin fragments enzymically released from cell wall from ripe fruit. Ripening initiation was accelerated in pectin-infiltrated fruit compared to control (buffer-infiltrated) fruit as measured by initiation of climacteric CO₂ and ethylene production and appearance of red color. The response to infiltration was maximum at a concentration of 25 micrograms pectin per fruit; higher concentrations (up to 125 micrograms per fruit) had no additional effect. When products released from isolated cell wall from ripe pericarp were separated on Bio-Gel P-2 and specific size classes infiltrated into preclimacteric fruit, ripening-promotive activity was found only in the larger (degree of polymerization >8) fragments. Products released from pectin derived from preclimacteric pericarp upon treatment with polygalacturonase from ripe pericarp did not stimulate ripening when infiltrated into preclimacteric fruit.     

Changes in the distribution of cell wall polysaccharides in early fruit pericarp and ovule, from fruit set to early fruit development, in tomato (Solanum lycopersicum)

During fruit development in tomato (Solanum lycopersicum), cell proliferation and rapid cell expansion occur after pollination. Cell wall synthesis, alteration, and degradation play important roles during early fruit formation, but cell wall composition and the extent of cell wall synthesis/degradation are poorly understood. In this study, we used immunolocalization with a range of specific monoclonal antibodies to examine the changes in cell wall composition during early fruit development in tomato. In exploring early fruit development, the ?1 day post-anthesis (DPA) ovary and fruits at 1, 3, and 5 DPA were sampled. Paraffin sections were prepared for staining and immunolabeling. The 5 DPA fruit showed rapid growth in size and an increase in both methyl-esterified pectin and de-methyl-esterified pectin content in the pericarp, suggesting rapid synthesis and de-methyl esterification of pectin during this growth period. Labeling of pectic arabinan with LM6 antibody and galactan with LM5 antibody revealed abundant amounts of both, with unique distribution patterns in the ovule and premature pericarp. These results suggest the presence of rapid pectin metabolism during the early stages of fruit development and indicate a unique distribution of pectic galactan and arabinan within the ovule, where they may be involved in embryogenesis.     

Silencing of SlPL,which encodes a pectate lyase in tomato,confers enhanced fruit firmness,prolonged shelf‐life and reduced susceptibility to grey mould

Pectate lyase genes have been documented as excellent candidates for improvement of fruit firmness. However, implementation of pectate lyase in regulating fruit postharvest deterioration has not been fully explored. In this report, 22 individual pectate lyase genes in tomato were identified, and one pectate lyase gene SlPL (Solyc03g111690) showed dominant expression during fruit maturation. RNA interference of SlPL resulted in enhanced fruit firmness and changes in pericarp cells. More importantly, the SlPL‐RNAi fruit demonstrated greater antirotting and pathogen‐resisting ability. Compared to wild‐type, SlPL‐RNAi fruit had higher levels of cellulose and hemicellulose, whereas the level of water‐soluble pectin was lower. Consistent with this, the activities of peroxidase, superoxide dismutase and catalase were higher in SlPL‐RNAi fruit, and the malondialdehyde concentration was lower. RNA‐Seq results showed large amounts of differentially expressed genes involved in hormone signalling, cell wall modification, oxidative stress and pathogen resistance. Collectively, these data demonstrate that pectate lyase plays an important role in both fruit softening and pathogen resistance. This may advance knowledge of postharvest fruit preservation in tomato and other fleshy fruit.     

Comparison of ripening processes in intact tomato fruit and excised pericarp discs

Physiological processes characteristic of ripening in tissues of intact tomato fruit (Lycopersicon esculentum Mill.) were examined in excised pericarp discs. Pericarp discs were prepared from mature-green tomato fruit and stored in 24-well culture plates, in which individual discs could be monitored for color change, ethylene biosynthesis, and respiration, and selected for cell wall analysis. Within the context of these preparation and handling procedures, most whole fruit ripening processes were maintained in pericarp discs. Pericarp discs and matched intact fruit passed through the same skin color stages at similar rates, as expressed in the L^*a^*b^* color space, changing from green (a^* < −5) to red (a^* > 15) in about 6 days. Individual tissues of the pericarp discs changed color in the same sequence seen in intact fruit (exocarp, endocarp, then vascular parenchyma). Discs from different areas changed in the same spatial sequence seen in intact fruit (bottom, middle, top). Pericarp discs exhibited climacteric increases in ethylene biosynthesis and CO₂ production comparable with those seen in intact fruit, but these were more tightly linked to rate of color change, reaching a peak around a^* = 5. Tomato pericarp discs decreased in firmness as color changed. Cell wall carbohydrate composition changed with color as in intact fruit: the quantity of water-soluble pectin eluted from the starch-free alcohol insoluble substances steadily increased and more tightly bound, water-insoluble, pectin decreased in inverse relationship. The cell wall content of the neutral sugars arabinose, rhamnose, and galactose steadily decreased as color changed. The extractable activity of specific cell wall hydrolases changed as in intact fruit: polygalacturonase activity, not detectable in green discs (a^* = −5), appeared as discs turned yellow-red (a^* = 5), and increased another eight-fold as discs became full red (a^* value +20). Carboxymethyl-cellulase activity, low in extracts from green discs, increased about six-fold as discs changed from yellow (a^* = 0) to red.     

Down-regulation of an Auxin Response Factor in the tomato induces modification of fine pectin structure and tissue architecture

It has previously been shown that down-regulation of an auxin response factor gene (DR12) results in pleiotropic phenotypes including enhanced fruit firmness in antisense transgenic tomato (AS-DR12). To uncover the nature of the ripening-associated modifications affecting fruit texture, comparative analyses were performed of pectin composition and structure in cell wall pericarp tissue of wild-type and AS-DR12 fruit at mature green (MG) and red-ripe (RR) stages. Throughout ripening, pectin showed a decrease in methyl esterification and in the content of galactan side chains in both genotypes. At mature green stage, pectin content in methyl ester groups was slightly higher in AS-DR12 fruit than in wild type, but this ratio was reversed at the red-ripe stage. The amount of water- and oxalate-soluble pectins increased at the red-ripe stage in the wild type, but decreased in AS-DR12. The distribution of methyl ester groups on the homogalaturonan backbone differed between the two genotypes. There was no evidence of more calcium cross-linked homogalacturan involved in cell-to-cell adhesion in AS-DR12 compared with wild-type fruit. Furthermore, the outer pericarp contains higher proportion of small cells in AS-DR12 fruit than in wild type and higher occurrence of (1-->5) alpha-L-arabinan epitope at the RR stage. It is concluded that the increased firmness of transgenic fruit does not result from a major impairment of ripening-related pectin metabolism, but rather involves differences in pectin fine structure associated with changes in tissue architecture.     

Pectin Modification in Cell Walls of Ripening Tomatoes Occurs in Distinct Domains

The class of cell wall polysaccharides that undergoes the most extensive modification during tomato (Lycopersicon esculentum) fruit ripening is pectin. De-esterification of the polygalacturonic acid backbone by pectin methylesterase facilitates the depolymerization of pectins by polygalacturonase II (PGII). To investigate the spatial aspects of the de-esterification of cell wall pectins and the subsequent deposition of PGII, we have used antibodies to relatively methylesterified and nonesterified pectic epitopes and to the PGII protein on thin sections of pericarp tissue at different developmental stages. De-esterification of pectins and deposition of PGII protein occur in block-like domains within the cell wall. The boundaries of these domains are distinct and persistent, implying strict, spatial regulation of enzymic activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins strongly associated with cell walls of pericarp tissue at each stage of fruit development show ripening-related changes in this protein population. Western blots of these gels with anti-PGII antiserum demonstrate that PGII expression is ripening-related. The PGII co-extracts with specific pectic fractions extracted with imidazole or with Na2CO3 at 0[deg]C from the walls of red-ripe pericarp tissue, indicating that the strong association between PGII and the cell wall involves binding to particular pectic polysaccharides.     

Effect of silencing the two major tomato fruit pectin methylesterase isoforms on cell wall pectin metabolism

Post‐harvest storage is largely limited by fruit softening, a result of cell wall degradation. Pectin methylesterase (PE) (EC 3.1.1.11) is a major hydrolase responsible for pectin de‐esterification in the cell wall, a response to fruit ripening. Two major PE isoforms, PE1 and PE2, have been isolated from tomato (Solanum lycopersicon) pericarp tissue and both have previously been down‐regulated using antisense suppression. In this paper, PE1 and PE2 double antisense tomato plants were successfully generated through crossing the two single antisense lines. In the double antisense fruit, approximately 10% of normal PE activity remained and ripening associated pectin de‐esterification was almost completely blocked. However, double antisense fruit softened normally during ripening. In tomato fruit, the PE1 isoform was found to contribute little to total PE activity and have little effect on the degree of esterification of pectin. In contrast, the other dominant fruit isoform, PE2, has a major impact on de‐esterification of total pectin. PE2 appears to act on non‐CDTA‐soluble pectin during ripening and on CDTA‐soluble pectin before the start of ripening in a potentially block‐wise fashion.     

Macromolecular components of tomato fruit pectin

Chelate and alkaline-soluble pectin extracted from cell walls of pericarp tissue from mature green, turning, and red ripe (Lycopersicon esculentum Mill.) fruit (cv. Rutgers), were studied by high-performance size-exclusion chromatography. Computer-aided curve fitting of the chromatograms to a series of Gaussian-shaped components revealed that pectin from all fractions was composed of a linear combination of five macromolecular-sized species. The relative sizes of these macromolecules as obtained from their radii of gyration were 1:2:4:8:16. Dialysis against 0.05 M NaCl induced partial dissociation of the biopolymers. Apparently, the weight fraction of smaller sized species increased at the expense of larger ones. Also, the dissociation produced low-molecular-weight fragments. Behavior in the presence of 0.05 M NaCl led to the conclusion that cell wall pectin acted as if it were an aggregated mosaic, held together at least partially through noncovalent interactions.     

裂果易发性不同的荔枝品种果皮中细胞壁代谢酶活性的比较

“糯米糍”荔枝裂果率极显著高于“淮枝”,前者果皮中的果胶酶、纤维素酶和果胶甲酯酶的活性高于后者,其中以果胶酶活性差异最明显,其次是纤维素酶,果胶甲酯酶差异最小;“糯米糍”细胞壁结合型的过氧化物酶(POD)和多酚氧化酶(PP0)活性明显高于“淮枝”,而水溶性POD和PP0的活性则两个品种间无明显差异。据此认为,果皮细胞壁水解酶活性以及细胞壁结合型的POD和PPO的活性高的荔枝品种,其裂果率也高。文章对细胞壁代谢相关酶类在果皮抗裂性形成中的作用进行了讨论。     

Putative virulence factors of Botrytis cinerea acting as a wound pathogen

Abstract The grey mold fungus, Botrytis cinerea , is a wound pathogen of worldwide distribution, and causes rots of almost all fruits and vegetables. The fungus can also penetrate directly from appressoria to cause severe losses to growers of flowers. B. cinerea secretes a number of inducible attack enzymes which can degrade host cell walls to widen the infection, including β-glucosidase, pectin methylesterase, the polygalacturonases, and aspartate proteinase; laccase and benzyl alcohol oxidase appear to have roles of detoxifying compounds derived from the host during pathogenesis, a function that we believe enhances the virulence of the pathogen. With additional research, several oxidases may also become a part of the virulence group. Much more work is needed, in particular the analysis of mutants, to assign formally the roles suggested here.