Changes in the structure of Arabidopsis thaliana roots induced during development of males of the plant parasitic nematode Heterodera schachtii

Plant parasitic nematodes of the genus Heterodera show a high degree of sexual dimorphism, which is reflected by different nutritional demands and differences in the structure of the induced specific syncytial feeding site in the plant. The determination of the sex of the nematode Heterodera schachtii and other related species was repeatedly reported to be dependent on trophic factors, which are provided by the induced syncytia. The structural differences of syncytia induced by H. schachtii in roots of Arabidopsis thaliana were analysed at the anatomical and ultrastructural level. Syncytia of males were induced in the root pericycle. The developing syncytium then expanded into procambial or cambial cells of the vascular cylinder. Differentiated vascular elements were not included. The expansion of the syncytium triggered the proliferation of cambial and peridermal tissues, in a manner similar to secondary growth, and the formation of additional xylem and phloem elements. In comparison to syncytia associated with females, syncytia associated with males were less hypertrophied and were composed of more cells. Distinct cell wall openings were mostly found between the few strongly hypertrophied syncytial elements at the actual feeding site in the pericycle. The ultrastructure was very similar to female-associated syncytia but showed conspicuous differences in the structure and localization of cell wall ingrowths. These ingrowths were rare and weakly developed and occurred not only at the interface with xylem elements but also at the internal and external walls of the syncytia. After feeding had ceased at the end of the third developmental stage the syncytia degenerated.     

Feeding behavior of sedentary plant parasitic nematodes

With the aid of video-enhanced contrast light microscopy the feeding behavior of the cyst nematodeHeterodera schachtii and the root-knot nematodeMeloidogyne incognita has been studied in roots of various cruciferous plants, especially inArabidopsis thaliana, a model plant for molecular studies. The juvenile stage 2 (J2) nematodes ofH. schachtii migrate intracellularly with destructive stylet thrusts before establishing their permanent feeding site (syncytium) within the vascular cylinder. After the selection of the initial syncytial cell (ISC) they undergo a preparation period before they start feeding from the ISC. During all developmental stages feeding occurs in cycles, composed of three distinct phases. In contrast, the J2 ofMeloidogyne incognita reach their permanent feeding site by intercellular migration, first towards the apex of the root, where they turn round, and move towards the differentiating vascular cylinder. A characteristic behavioral pattern, composed of continuous head and stylet movements, interspersed by periods of stylet-tip protrusion and metacorpal bulb pumping is maintained during all phases of parasitism, i.e., root invasion, intercellular migration and feeding from the giant-cells.     

Defence responses ofArabidopsis thalianaduring invasion and feeding site induction by the plant–parasitic nematodeHeterodera glycines

The nature of local defence responses occurring during the infection ofArabidopsis thalianaby the soybean cyst nematodeHeterodera glycineswas analysed by light and electron microscopy.Arabidopsis thalianais not a regular host of this nematode, but invasion, feeding site induction and in rare cases development of juveniles was observed. Compared toHeterodera schachtii, which interacts compatibly withA. thaliana, the period of invasion and migration was prolonged. During migration a strong hypersensitive response, characterized by extensive necroses, browning, autofluorescence and cell wall depositions, occurred in cells that were not in direct contact with the nematodes. Cells selected as initial syncytial cells were pierced with the stylet and, after having passed a preparation phase, secretions were released. The cells responded with rapid vacuolation and deposition of callose-like material around the stylet and at the cell walls, and subsequent necrosis. In a very few cases the initial syncytial cells and neighbouring cells deposited callose-like material on their walls, but these cells remained intact so that functional syncytia developed, which allowed occasional nematode development.     

Pathology of cortical invasion by Plasmodiophora brassicae in clubroot resistant and susceptible Brassica oleracea hosts

The differences in physiological response of Brassica oleracea to infection and growth of Plasmodiophora brassicae in infected tissue were studied in clubroot resistant and susceptible hosts, grown in sand solution culture artificially inoculated with spores of P. brassicae (10⁸ spores mL⁻¹). Primary (root hair) and secondary (cortical) stages of P. brassicae occurred in both resistant and susceptible hosts. Symptoms of cortical invasion by P. brassicae in resistant and susceptible hosts included cell wall breaks, presence of vesicles or inclusion bodies within the cell walls, cell wall thickening in association with plasmodesmata and enlarged and/or disorganized host nuclei. The main difference between the resistant and susceptible host reaction was the absence of degradation of the secondary thickening and cell walls of the xylem in the resistant host. This study supports the existence of an amoeboid form of the pathogen in addition to the recognized two-phase life history of P. brassicae. Furthermore, it suggests that resistance in B. oleracea does not prevent the development of this amoeboid form. A reduced number of cell wall breakages suggests movement of the amoeboid form, may have been restricted but not prevented in the resistant host.     

Ultrastructural localization of chitin in cell walls of Rigidoporus lignosus, the white-rot fungus of rubber tree roots

The distribution of N-acetylglucosamine residues in the cell wall of the white-rot pathogenic fungus, Rigidoporus lignosus, was studied by using gold labelled wheatgerm agglutinin bound to ovomucoid-colloidal gold. Ultrastructural investigation of R. lignosus-infected root tissues of Hevea brasiliensis showed a modification of the fungal cell wall throughout the infection process. Gold particles were found to occur on both thick- and thin-walled hyphae of R. lignosus rhizomorphs at the root surface. Walls of hyphae that had penetrated the roots were only labelled when they were out of the host cell, suggesting that modification of chitin molecules may be related to the excretion of host cell wall degrading enzymes. Variation in the distribution of gold particles was observed over hyphal walls of both colonized phellem and xylem cells. The observation that N-acetylglucosamine residues were released in the host cell cytoplasm suggests that lytic enzymes alter the fungal cell walls. Released chitin oligosaccharides may play a role in the induction of the root's defence system against fungal attack.     

中国小黑豆抗源对大豆孢囊线虫4号生理小种抗性机制的研究Ⅱ.抗感品种根部合胞体超微结构的比较

 接种大豆孢囊线虫4号生理小种后4、9、14天,对我国小黑豆抗源灰皮支黑豆和元钵黑豆及感病对照鲁豆1号根部形成的合胞体组织的超微结构进行了研究。结果发现:感病品种的合胞体细胞较大、靠近木质部导管一侧有内生长,各种细胞器丰富,内质网数量多形体长、多为光滑型;抗病品种合胞体细胞较小,核糖体较多,内质网小而少、多为粗糙型,细胞内出现较多的类脂肪体,在侵染早期,细胞质快速降解,有时发现细胞质膜与细胞壁发生分离。     

中国小黑豆抗源对大豆孢囊线虫4号生理小种抗性机制的研究Ⅱ.抗感品种根部合胞体超微结构的比较

 接种大豆孢囊线虫4号生理小种后4、9、14天,对我国小黑豆抗源灰皮支黑豆和元钵黑豆及感病对照鲁豆1号根部形成的合胞体组织的超微结构进行了研究。结果发现:感病品种的合胞体细胞较大、靠近木质部导管一侧有内生长,各种细胞器丰富,内质网数量多形体长、多为光滑型;抗病品种合胞体细胞较小,核糖体较多,内质网小而少、多为粗糙型,细胞内出现较多的类脂肪体,在侵染早期,细胞质快速降解,有时发现细胞质膜与细胞壁发生分离。     

小麦新抗源一粒葡抗条锈病的组织学和超微结构研究

 采用荧光显微镜、微分干涉显微镜和电子显微镜技术,系统研究了小麦新抗源一粒葡抗小麦条锈病的组织学和超微结构特征。结果表明:相对于感病品种铭贤169,一粒葡对条锈菌的侵染,在组织学和超微结构上均表现出明显的抗性特征。在组织学水平,表现为菌丝生长受抑,菌落发育延迟或败育,吸器母细胞和吸器数目明显减少;同时,侵染点的寄主细胞表现出不同程度的过敏性坏死症状。电镜观察发现,在一粒葡和感病品种中,条锈菌均可由芽管顶端直接进入或通过形成附着胞进入小麦气孔。其后,在一粒葡上,病菌胞间菌丝、吸器母细胞、吸器在细胞和亚细胞水平均发生了一系列异常变化,表现为原生质染色逐渐加深,液泡增多变大,逐渐消解原生质;胞间菌丝、吸器母细胞细胞壁不规则增厚;胞间菌丝线粒体肿胀,数目增多,逐渐解体;吸器母细胞细胞质逐渐空泡化后丧失其生理功能;吸器外质膜皱褶;吸器外间质加宽并有丝状或颗粒状物质形成,吸器体壁逐渐消解出现孔洞,吸器体最终畸形坏死。同时,寄主细胞产生一系列显著的结构防卫反应:形成胞壁沉积物、乳突、吸器鞘等结构,以及发生坏死,阻碍并抑制病菌的发育及扩展。     

Silicon-induced cell wall fortification of rice leaves: a possible cellular mechanism of enhanced host resistance to blast

ABSTRACT Locations of silicon accumulation in rice leaves and its possible association with resistance to rice blast were investigated by electron microscopy and X-ray microanalysis. A blast-susceptible cultivar, Jinmi, and a partially resistant cultivar, Hwaseong, were grown under a hydroponic culture system with modified Yoshida's nutrient solution containing 0, 50, 100, and 200 ppm of silicon. Electron-dense silicon layers were frequently found beneath the cuticle in epidermal cell walls of silicon-treated plants. Increasing levels of silicon were detected in the outer regions of epidermal cell walls. Silicon was present mainly in epidermal cell walls, middle lamellae, and intercellular spaces within subepidermal tissues. Furthermore, silicon was prevalent throughout the leaf surface, with relatively small deposition on stomatal guard cells in silicon-treated plants. Silicon accumulation and epidermal cell wall thickness in leaves were greater in cv. Jinmi than in cv. Hwaseong. However, the thickness ratios of the silicon layers to epidermal cell walls were greater in cv. Hwaseong (53.25 to 93.28%) than in cv. Jinmi (36.58 to 66.54%). Leaf blast severity was lower in cv. Hwaseong than in cv. Jinmi and was significantly reduced in silicon-treated plants of both cultivars. These results suggest that silicon-induced cell wall fortification of rice leaves may be closely associated with enhanced host resistance to blast.     

Panama Disease: Cell Wall Reinforcement in Banana Roots in Response to Elicitors from Fusarium oxysporum f. sp. cubense Race Four

ABSTRACT The biochemical basis of tolerance in banana to Fusarium wilt, caused by the pathogen Fusarium oxysporum f. sp. cubense race four, was investigated. Tissue culture banana plants from tolerant cv. Goldfinger and susceptible cv. Williams were maintained in a hydroponic system and inoculated with conidial suspensions to evaluate the degree of tolerance to susceptibility between the two clones and to investigate the effectiveness of this technique as a potential tool for early screening for resistance in breeding programs. Similarly, defense responses were induced by treatment of the plants with an elicitor preparation from the mycelial cell walls of the pathogen. Differences in the induction of lignin and callose deposition, phenolics, and the enzymes involved in cell wall strengthening; phenylalanine ammonia lyase, cinnamyl alcohol dehydrogenase, peroxidase, and polyphenol oxidase were determined. Root tissue of the tolerant cv. Goldfinger responded to the fungal elicitor through the strong deposition of lignin, preceded by the induction or activation of the enzyme activities involved in the synthesis and polymerization thereof, whereas only slight increases were observed for the susceptible cv. Williams. No increase in callose content was observed for either clone. These results indicate an important role for cell wall strengthening due to the deposition of lignin as an inducible defense mechanism of banana roots against F. oxysporum f. sp. cubense race four.     

Immunohistochemical changes in methyl-ester distribution of homogalacturonan and side chain composition of rhamnogalacturonan I as possible components of basal resistance in tomato inoculated with Ralstonia solanacearum

The composition and structure of pectic cell wall polysaccharides of stem sections were investigated in healthy and Ralstonia solanacearum-inoculated tomato genotypes L390 and Hawaii 7996, susceptible and resistant to bacterial wilt, respectively, by immunohistochemical analysis. Constitutive differences between genotypes manifested in methyl-ester distribution of homogalacturonan (HG), arabinan and galactan side chain composition of rhamnogalacturonan I (RG I) and arabinogalactan-protein (AGP) in the xylem parenchyma and in vessel cell walls. After inoculation increased labeling was observed with all the antibodies (JIM5, JIM7, LM2, LM5, LM6, LM7) specific for HG, RG I and AGP epitopes, in the xylem parenchyma and around xylem vessels of stem sections of L390, but not of Hawaii 7996. Also vessel cell walls were stronger stained after inoculation in L390, particularly for the non-blockwise de-esterification of HG, possibly indicating for the first time the non-blockwise action pattern of bacterial pectin methyl esterase. In genotype Hawaii 7996 a reaction to inoculation was observed only in vessel walls, with a significantly increased number of stained vessels—five- and nine-fold for arabinan and galactan epitopes of RG I, respectively. Differences in xylem cell wall structure may play a role as a constitutive resistance mechanism in the multigenic resistance of tomato against bacterial wilt, while changes after inoculation may contribute to induced basal resistance on cell wall level.     

The composition of wall alterations and appositions (reaction material) and their role in the resistance of onion bulb scale epidermis to colonization by Botrytis allii

Ultrastructural examination of deposits of reaction material (RM) showed them to be composed of paramural granules (osmiophilic to varying degrees) lying within amorphous electron–dense material. Epidermal cell walls per se became increasingly electron–dense around the short infection hyphae produced by Botrytis allii. Fluorescence microscopy, histochemical tests and microautoradiography showed the accumulation of phenolic compounds at infection sites. Use of phenylalanine and cinnamate for E.M. autoradiography demonstrated the accumulation of phenolics within paramural granules, amorphous aggregates and within the penetrated cell wall, Paramural deposits were soluble in organic solvents and probably represented reservoirs of low MW phenolics which were progressively incorporated into insoluble polymers within the cell wall. Initial synthesis or accumulation of phenolics appeared to take place in rough endoplasmic reticulum during the first 2 h after cuticle penetration. Suppression of RM deposition and wall alterations was achieved by treatment of bulb scales with cycloheximide. Tissues treated with the protein synthesis inhibitor became susceptible to colonization by B. allii. Accumulation of phenolics at infection sites rendered the onion cell walls resistant to attack by a mixture of cell wall–degrading enzymes, Methanolic extracts of epidermal strips containing RM deposits contained very weakly active inhibitors of B. allii germ–tube growth. The role of RM deposits and wall alterations in the resistance of onion epidermis to colonization by Botrytis is discussed.     

Histopathology of roots of three tomato cultivars infected with two separate isolates of the false root-knot nematode <Emphasis Type="Italic">Nacobbus aberrans</Emphasis>

The plant-parasitic nematode Nacobbus aberrans attacks weeds and cultivated plants, causing drastic crop yield losses. Several tomato cultivars, such as Superman and Mykonos, are produced by Seminis® as nematode resistant and are widely used in Argentina; their specific resistance response to different nematode species, however, is still unknown. In this study we explored the response of tomato cultivars to the infection of N. aberrans isolates, and determined the host status by performing histological analyses and estimating egg mass index (EMI). Two Argentina isolates (from Lules-Tucumán and Río Cuarto-Córdoba) were tested separately in plants of Superman, Mykonos and Platense cultivars. Plants were maintained in a greenhouse for 90 days; then EMI was estimated in root systems and the material was processed to prepare histological slides and for histochemical test. Infected roots exhibited galls with females and a syncytium (feeding site) developed inside them. The vascular tissues were disorganized and displaced to the periphery; xylem percentage was lower than that in the control plants. All the cultivars were susceptible and developed a close plant-parasite relationship, with Mykonos and Platense cultivars infected with the Lules isolate being the most highly affected, as indicated by their highest EMI values. Superman was the least susceptible cultivar, as evidenced by its lowest EMI values, the amount of starch observed, the presence of thickened cell walls around the nematode and the egg mass, and the low percentage of gall occupied by syncytium.     

Superoxide generation in extracts from isolated plant cell walls is regulated by fungal signal molecules

ABSTRACT Fractions solubilized with NaCl from cell walls of pea and cowpea plants catalyzed the formation of blue formazan from nitroblue tetrazolium. Because superoxide dismutase decreased formazan production by over 90%, superoxide anion (O(2) ) may participate in the formation of formazan in the solubilized cell wall fractions. The formazan formation in the fractions solubilized from pea and cowpea cell walls was markedly reduced by exclusion of NAD(P)H, manganese ion, or p-coumaric acid from the reaction mixture. The formazan formation was severely inhibited by salicylhydroxamic acid and catalase, but not by imidazole, pyridine, quinacrine, and diphenyleneiodonium. An elicitor preparation from the pea pathogen Mycosphaerella pinodes enhanced the activities of formazan formation nonspecifically in both pea and cowpea fractions. The suppressor preparation from M. pinodes inhibited the activity in the pea fraction in the presence or absence of the elicitor. In the cowpea fraction, however, the suppressor did not inhibit the elicitor-enhanced activity, and the suppressor alone stimulated formazan formation. These results indicated that O(2) generation in the fractions solubilized from pea and cowpea cell walls seems to be catalyzed by cell wall-bound peroxidase(s) and that the plant cell walls alone are able to respond to the elicitor non-specifically and to the suppressor in a species-specific manner, suggesting the plant cell walls may play an important role in determination of plant-fungal pathogen specificity.     

Cytology of Infection of Maize Seedlings by Fusarium moniliforme and Immunolocalization of the Pathogenesis-Related PRms Protein

ABSTRACT We have investigated the histology of infection of maize seedlings by Fusarium moniliforme in association with a biochemical host defense response, the accumulation of the PRms (pathogenesis-related maize seed) protein. Light microscopy of trypan blue-stained sections and scanning electron microscopy revealed direct penetration by F. moniliforme hyphae through the epidermal cells of the seedling and colonization of the host tissue by inter- and intracellular modes of growth. Pathogen ingress into the infected tissue was associated with the induction of defense-related ultrastructural modifications, as exemplified by the formation of appositions on the outer host cell wall surface, the occlusion of intercellular spaces, and the formation of papillae. Cellular and subcellular immunolocalization studies revealed that PRms accumulated at very high levels in those cells types that represent the first barrier for fungal penetration such as the aleurone layer of germinating seeds and the scutellar epithelial cells of isolated germinating embryos. A highly localized accumulation of PRms within papillae of the inner scutellar parenchyma cells also occurred, suggesting that signaling mechanisms that lead to the accumulation of PRms in papillae of cell types that are distant from the invading pathogen must operate in the infected maize tissues. Our study also revealed the presence of a large number of fungal cells with an abnormal shape that showed PRms-specific labeling. PRms was found to accumulate in clusters over the fungal cell wall. Taken together, the occurrence of PRms in cell types that first establish contact with the pathogen, as well as in papillae, and in association with fungal cell walls suggests that PRms may have a function in the plant defense response.     

Importance of Cell Wall Degrading Enzymes Produced by Fusarium graminearum during Infection of Wheat Heads

Cytological studies were carried out to elucidate the importance of cell wall degrading enzymes (CWDE) during infection of wheat spikes by Fusarium graminearum. Scanning electron micrographs revealed that at 6–24 hours after inoculation (hai) of single spikelets with macroconidia of F. graminearum, the fungus germinated by forming several germ tubes and developed a dense hyphal network in the cavity of the spikelet. At 24–36hai, the fungus formed infection hyphae which invaded the ovary and inner surface of the lemma and palea. Transmission electron microscopical studies revealed that the fungus extended inter- and intracellularly in the ovary, lemma and rachis and caused considerable damage and alterations to the host cell walls. In different tissues of healthy and F. graminearum-infected wheat spikes the cell wall components cellulose, xylan and pectin were localized by means of enzyme-gold and immuno-gold labelling techniques. Localization of cellulose, xylan and pectin showed that host cell walls which were in direct contact with the pathogen surface had reduced gold labelling compared to considerable higher labelling densities of walls distant from the pathogen–host interface or in non-colonized tissues. The reduced gold labelling densities in the infected host cell walls indicate that these polysaccharide degrading enzymes might be important pathogenicity factors of F. graminearum during infection of wheat spikes. The results revealed that, infection and colonization of wheat spikes by F. graminearum and reactions of infected host tissue were similar to those reported for F. culmorum.     

葡萄扇叶病毒引起的寄主细胞病变研究

 电镜观察了葡萄扇叶病毒杭州分离物(Grapevine fanleaf virus Hangzhou isolate,GFLV-H)侵染苋色藜(Chenopodium amaranticolor)和昆诺藜(C.quinoa)的细胞超微结构变化。病毒粒子存在于叶肉组织薄壁细胞的细胞质内,常成单纵列排列在小管结构中,小管形成聚集体。在系统感染的昆诺藜细胞中位于管状结构中的病毒粒子穿过胞间连丝。2种感病寄主细胞质内膜结构增生,在核周围形成含细纤维状物质的小囊泡。液泡边缘存在少许小泡,有多泡体和髓鞘样结构伸入液泡,叶绿体和线粒体等细胞器也发生不同程度的变化。     

Abscission Layer Formation as a Resistance Response of Peruvian Apple Cactus Against Glomerella cingulata

ABSTRACT Stem disks from 2-year-old cacti Cereus tetragonus (susceptible) and C. peruvianus (resistant) were inoculated in the center (pith) with Glomerella cingulata isolated from Colletotrichum stem rot in three-angled cacti. The susceptible cactus became extensively colonized, whereas colonization was limited to a small area in the resistant cactus. The resistant cactus formed prominent abscission layers (ALs) in parenchyma internal to the inoculation site. Ethanol extracts of the fungal culture also stimulated AL formation in the resistant cactus. Initial cell division followed at 2 to 4 days after treatment, and layering of multiple cells at 7 days after treatment. After 10 days, the outer layers were sometimes sloughed from the inner layers. No AL formation was induced in susceptible C. tetragonus treated with ethanol extract or in untreated control cacti. Light and electron microscopy revealed that initial cell division occurred by cell wall formation, and that an additional cell wall was layered in pre-existing parenchyma cells without ordinary cell division. Later, separation layers formed in ALs where inner cell walls appeared to be thickened secondarily, and the cell walls and middle lamella within the layer dissolved. These results suggest that AL formation in the resistant cactus is induced by fungal metabolites, and that it serves as a histological barrier against anthracnose pathogens.     

抗感枯萎病黄瓜品种的病理组织结构学研究

选取不同抗性黄瓜品种中农13(抗)和中农6号(感)为试材,采用蘸根法接种,利用电镜技术和石蜡切片方法观察了病菌侵染前后根茎组织结构的变化。结果表明,抗病品种有环纹和网纹两种导管类型,有较厚的角质层,感病品种有环纹导管一种类型,无角质层。抗病品种在接种后细胞壁加厚、导管腔内出现褐色物、胞壁覆盖物以及侵填体。感病品种仅出现胞壁加厚和胼胝体。对于病原菌侵染,抗感品种具有不同程度的反应,抗病品种反应程度早于且强于感病品种。