A light and scanning-electron microscopic study of infection of tomato plants by virulent and avirulent races of Cladosporium fulvum

Infection of tomato plants byCladosporium fulvum Cooke was studied using light and scanning-electron microscopy. Races 1.2.3 and 4 ofCladosporium fulvum were used, whereas tomato cultivars, carrying the Cf2 gene (susceptible to race 1.2.3 and immune to race 4) and the Cf4 gene (immune to race 1.2.3 and susceptible to race 4) served as differentials. No differences were observed in growth between compatible and incompatible combinations during germination, subsequent formation of runner hyphae and stomatal penetration. Runner hyphae did not show directional growth towards stomata. Penetration usually occurred on the third or fourth day after inoculation. In compatible combinations the fungus grew intercellularly, often in close contact with spongy mesophyll cells. Under optimal conditions it did not cause visible damage to plant cells during early stages of infection. Under suboptimal conditions in winter, the host cells often reacted with callose deposition, but growth of the fungus did not appear to be inhibited. Ten to twelve days after inoculation conidiophores emerged through the stomata and produced conidia. In incompatible combinations fungal growth was arrested one to two days after penetration and confined to stomata and surrounding cells. Very soon the host cells, in contact with the fungus, deposited extensive amounts of callose. Later these cells turned brown and collapsed. At the surface of the host cells, contacted by fungal hyphae, abundant extracellular material could be observed by scanning-electron microscopy. Removing the epidermis of leaves before inoculation delayed the resistant response. On stripped leaves the rate of fungal growth was equal for both interactions up to ten days after inoculation, but the incompatible combination lacked sporulation.     

Histopathology of compatibility and incompatibility between oilseed rape and Albugo Candida

Cotyledons of one resistant and three susceptible rape lines/cultivars were inoculated with zoospores of Albugo Candida race 7. Samples of whole cotyledons were examined by differential interference contrast microscopy. The time course of the infection process was followed histologically. Germination of zoospore cysts occurred 2-3 h after inoculation. Infection was initiated with germ-tubes penetrating through stomata. Haustorium formation was first observed in the palisade mesophyll cells adjacent to the substomatal chambers 8 h after inoculation.
Only after the establishment of the first haustorium did compatible and incompatible interactions begin to differentiate. In the resistant cultivar, most primary hyphae produced single haustoria. Necrosis of the invaded host cell was first observed 12 h after inoculation followed by cessation of fungal growth. The death of host cells was largely restricted to the penetration site; the adjacent non-penetrated cells remained apparently unaffected. In the susceptible hosts, necrosis of infected cells occurred only infrequently, and hyphal growth continued unabated, resulting in mycelial ramification into the mesophyll. Numerous haustoria were produced.
Histological studies showed that the earliest event distinguishing a compatible from an incompatible interaction occurred after formation of the first haustorium and that resistance was not manifested until the host mesophyll cell had come into contact with the first haustorium. The distinction between compatibility and incompatibility was substantiated by quantitative analysis of white rust development on both resistant and susceptible lines/cultivars.     

A microscopic study of the wheat-powdery mildew relationship after application of the systemic compounds procaine,griseofulvin and 6-azauracil

On wheat seedlings systemically protected by root application of procaine-hydrochloride, griseofulvin or 6-azauracil, germination of oidia ofErysiphe graminis f. sp.tritici and penetration of this fungus into the epidermal cell wall was as high as on control plants. Inhibition of powdery mildew development became apparent only after the penetration process had started. About 85% of the infections were halted within 24 hours after the inoculation, and did not result in the formation of a haustorium. In the other cases usually not more than one haustorium per infection court was formed, which often showed anomalies, characteristic for each compound used. Development of mycelium was scanty or absent and no sporulation occurred. Similarly, on plants of a resistant wheat variety, powdery mildew inhibition became apparent only after penetration of the host had started. There was no development of mycelium or sporulation. A severe reaction of certain epidermal cells to penetration by powdery mildew was observed on resistant as well as on treated and untreated susceptible plants. However, in relation to the total number of infections, the percentage of this type of reaction was low.     

Peroxidase activity in rice leaves infected withHelminthosporium oryzae in relation to lesion maturation

Two-week-old seedlings of two rice varieties, resistant CH13 and susceptible Benibhog, were inoculated with a conidial suspension ofHelminthosporium oryzae. The resistant host exhibited greater peroxidase activity than the susceptible one when infected with the pathogen. Enzyme activities were measured 0, 24, 48, 72, 96 and 120 h after infection. Peroxidase activity in diseased leaves bearing young lesions,i.e., 24 h after inoculation, was not appreciably different from that in non-infected leaves. At the intermediate and mature stages of lesion development,ri.e., 72 and 120 h after inoculation, a marked increase in peroxidase activity was observed. The pattern of increased enzyme activity was similar in both varieties but the whole event proceeded faster in the resistant variety. No marked change in peroxidase activity in healthy leaves of both the varieties was noticed with increase of seedling age.     

Coffee resistance to Colletotrichum kahawae is associated with lignification, accumulation of phenols and cell death at infection sites

Histological and ultrastructural studies were undertaken to compare Colletotrichum kahawae growth and the sequence of responses it induced in resistant and susceptible coffee genotypes. Coffee resistance was characterized by a restricted fungal growth associated with hypersensitive-like cell death and early accumulation of phenolic compounds, such as flavonoids (cytoplasmic contents) and hydroxycinnamic acid derivatives (cell walls). This accumulation of phenols in the cell walls preceded their lignification and thickening. In the susceptible genotype, a late accumulation of hydroxycinnamic acid derivatives in a number of cell walls and the encasement of some intracellular hyphae were also observed, but these delayed host responses did not prevent fungal growth and sporulation.     

The Course of Colonization of Two Different Vitis Genotypes by Plasmopara viticola Indicates Compatible and Incompatible Host-Pathogen Interactions

ABSTRACT The course of colonization of leaf mesophyll by the causal agent of grapevine downy mildew, Plasmopara viticola, in a susceptible and a resistant grapevine genotype was examined in order to characterize the development of the pathogen in compatible and incompatible host-pathogen interactions. Within a few hours after inoculation, the pathogen was established in the susceptible Vitis vinifera cv. Müller-Thurgau and formed primary hyphae with a first haustorium. No further development occurred in the following 10 to 18 h. The next step, in which the hyphae grew and branched to colonize the intercellular space of the host tissue, was observed 1.5 days after inoculation. After 3 days, the intercostal fields were entirely filled with mycelium and sporulation was abundant under favorable environmental conditions. The first infection steps were essentially the same in the resistant V. rupestris. However, the invasive growth of P. viticola was delayed, and further development ceased before the intercostal fields were filled with mycelium.     

多堆柄锈菌侵染玉米的细胞学及超微结构特征

为明确玉米对多堆柄锈菌Puccinia polysora侵染后病理反应的细胞学特征,利用扫描和透射电镜技术分析了玉米自交系与多堆柄锈菌互作中二者的细胞变化过程。多堆柄锈菌对玉米的侵染主要以直接穿透叶片表皮侵入为主,少量可从气孔和细胞间隙侵入。接种后,病菌夏孢子在感病自交系叶片上快速并大量萌发,在叶表生长蔓延并侵入表皮组织细胞,7 d后形成夏孢子堆;在抗病自交系上,病菌萌发、菌丝生长均受到明显抑制,少量入侵的病菌也由于寄主细胞死亡而导致菌丝和夏孢子干瘪死亡。侵染早期在感病寄主细胞间隙出现菌丝并穿透细胞壁,在胞内产生分枝菌丝,此时寄主细胞结构正常;随着菌丝进一步扩展,叶绿体等结构发生紊乱,被侵染细胞逐渐死亡。在抗病自交系上,接菌24 h后寄主即出现过敏性坏死反应,侵入位点与周围细胞快速坏死,抑制菌丝生长蔓延;叶绿体中清晰可见深色颗粒状物质;72 h后细胞壁外侧产生大量致密的深色结晶体,应为与抗病反应相关的酚类物质。表明抗多堆柄锈菌的玉米材料可能存在2种抗病途径,即寄主与病菌互作中由分子识别引起的免疫反应和病菌侵入后的系统防卫反应。     

The haustorial interface in a resistant interaction of Erysiphe pisi with Pisum sativum

The interface between Erysiphe pisi and pea cv. JI 1049 was studied at the ultrastructural and cytochemical levels and compared with those in two susceptible cultivars. Haustorial efficiencies, as indexed by the length of mycelium associated with each haustorium, and growth rates on the resistant and one susceptible cultivar were also compared.The interaction in the resistant cultivar differed from those in the susceptible cultivars in the following ways: (i) there was no contact between the host plasmalemma and the A neckband region; (ii) papillae were contiguous with the surface of the neck and thus were probably formed before haustoria and (iii) there appeared to be less polysaccharide in the extrahaustorial membrane. The extrahaustorial membrane in the resistant cultivar lacked ATPase activity, whereas the rest of the host plasmamembrane had normal activity.Each haustorium supported a significantly greater total hyphal length in the resistant than in the susceptible cultivars. Growth rates of superficial hyphae were very similar on the susceptible and resistant cultivars but there was a delay in the onset on hyphal growth on the resistant cultivar which correlated with the previously reported delay in formation of the first haustorium. In contrast to hyphal growth rates, the rate of haustorium production was significantly less in the resistant cultivar. It is proposed that resistance in cv. JI 1049 operates at the stages prior to haustorium formation, similarly to that in some non-host and partial resistance systems. Once formed, however, the function of the haustorium seems to be unimpaired, despite the observed interfacial differences.     

Antioxidant,ethylene and membrane leakage responses to powdery mildew infection of near-isogenic barley lines with various types of resistance

Leaves of powdery mildew-susceptible barley (Hordeum vulgare cv. Ingrid) and related near-isogenic lines bearing various resistance genes (Mla12, Mlg or mlo5) were inoculated with Blumeria graminis f. sp. hordei race A6. Fungal attack induced several-fold increases in ethylene emission and electrolyte leakage in leaves of susceptible Ingrid beginning 3 days after inoculation. Activities of peroxidase, superoxide dismutase, glutathione S-transferase, ascorbate peroxidase and glutathione reductase enzymes were induced markedly in susceptible leaves 5–7 days after inoculation. Similar, but less pronounced pathogen-induced changes were detected in inoculated leaves of Mla-type resistant plants that show hypersensitive cell death upon inoculation, and, to an even lesser extent, in the Mlg and mlo lines, where no visible symptoms accompanied the incompatible interaction. Glutathione content increased only in susceptible barley 7 days after inoculation. Catalase activity, total ascorbate content and redox state were not influenced by inoculation in any of the genotypes. The activity of dehydroascorbate reductase was significantly reduced 3–5 days after inoculation in the susceptible parental plants and after 5 days in Mla and Mlg lines, while it was stable in the mlo barley. Slightly elevated levels of H₂O₂ were observed in the inoculated resistant plants. In contrast, H₂O₂ content decreased in the susceptible line 7 days after pathogen attack. These data indicate that high levels of antioxidants are involved in the compatible interaction of susceptible barley and powdery mildew by protecting the pathogen from oxidative damage.     

Microscopical studies of resistance to powdery mildew disease in the hop cultivar Wye Target

Microscopical studies of fungal development and host responses during infection of the hop cultivars Northern Brewer (susceptible) and Wye Target (resistant) with a Zenith isolate of Sphaerotheca humuli are described. Resistance to powdery mildew disease in cv. Wye Target is principally determined by the R₂ major gene. Fungal growth was typically restricted in cv. Wye Target following formation of a single haustorial initial or haustorium which failed to develop the characteristic lobes found in susceptible cells. The hypersensitive reaction of penetrated epidermal cells was associated with death of haustorial initials but the associated appressorium remained alive during the first 2 days after inoculation. In leaves expressing resistance, histochemical staining revealed deposition of lignin-like material and callose in penetrated cells and to a lesser extent in underlying palisade mesophyll cells. Transmission electron microscopy and enzymic digestion clearly demonstrated extensive paramural deposition of β-1.3 glucans (callose) in reacting mesophyll cells. Plant cell death, lignification and widespread callose deposition were rarely observed at infection sites in cv. Northern Brewer but collars of callose were deposited around the necks of all haustoria formed.     

Proteomic analysis of Colletotrichum kahawae-resistant and susceptible coffee fruit pericarps

Coffee berry disease (CBD) is caused by the fungus Colletotrichum kahawae and is restricted to the African continent, where it generates losses of up to 80 % of coffee production. Weather conditions in certain growing areas at high altitudes in Colombia appear to be very favourable for the development of this disease. Certain genotypes of Coffee arabica are resistant to this pathogen, such as the Timor Hybrid and some Ethiopian accessions. It is important to identify the proteins in these coffee genotypes that are associated with resistance to this fungus. Therefore, we compared the proteomes of two genotypes that are resistant to different isolates of C. kahawae with the proteome of the susceptible coffee genotype Caturra. We optimized the methodology applied for the extraction, cleaning and purification of proteins from the green fruit pericarp at 150 to 170 days after flowering. Through two-dimensional differential gel electrophoresis, proteomic map images were obtained for the resistant and susceptible genotypes. Fifty-two protein spots that were significantly different between the resistant and susceptible genotypes were detected. These protein spots were isolated and sequenced via mass spectrometry. The sequence analysis identified 14 proteins in the Timor Hybrid and 14 in CCC1147 that were associated with resistance and pathogen defence.     

Characterisation of a protein from Venturia inaequalis that induces necrosis in Malus carrying the Vm resistance gene

Apple scab (black spot) is caused by the fungus, Venturia inaequalis. Race 1 isolates of this fungus are avirulent on Malus hosts carrying the resistance gene V_m. Detached leaves from a V_m host (resistant, differential host 5) and ‘Royal Gala’ (susceptible, host 1) were inoculated with a conidial suspension of V. inaequalis. In the resistant reaction, a hypersensitive response (HR), characterised by necrosis and the accumulation of autofluorescent materials in epidermal and mesophyll cells, was observed at the site of fungal penetration. No HR was observed in the susceptible host. V. inaequalis grown in vitro produced an elicitor that induced necrosis, similar to the HR, when infiltrated into leaves of the resistant V_m host. No response, however, was observed in the susceptible host. The elicitor was proteinaceous and a fraction with elicitor activity was isolated using ultra-filtration, acetone precipitation and ion-exchange chromatography. The elicitor activity was resistant to boiling but it was abolished by digestion with proteinase K. The protein fraction contained three major proteins all with low isoelectric points (pI 3·0–4·5). The fraction also elicited necrosis in the differential host 4, but not in any of the other resistant hosts tested, including differential hosts 2, 3, and 6. Therefore, the fraction may contain elicitors with more than one host specificity.     

Encasement of Erysiphe graminis haustoria after treatment with bromuconazole

Preventive application of bromuconazole caused reduction in size and increased encasement rate of haustoria of Erysiphe graminis DC. For example, seven days after inoculation, 60 and 70% of haustoria had been encased in leaves treated with 8 mg litre⁻¹ and 16 mg litre⁻¹ respectively; the average length of the digitations was 8–10 μm in treated cells compared to 24 μm in untreated cells. The encasement process extended from the neck region to the whole haustorium. Haustorial bodies from treated plants had electron-dense cytoplasm and their organelles were more difficult to identify than in control plants. Extrahaustorial matrix was reduced to an unusually thin, osmiophilic pellicle, surrounded by abundant heterogeneous encasement material. Curative treatment induced similar changes, especially in the margin of the colonies. In the centre of the colony, haustoria were less affected by the fungicide; deposition of collar-like material, modification of extrahaustorial matrix and membrane and accumulation of plant cytoplasm around the digitations resulted in an intermediate, ‘swollen’ state of digitate haustoria. The possible pathway of encasement events is discussed.     

大丽轮枝菌侵染抗感棉种的组织学过程观察

 棉花黄萎病是一种极难防治的土传性真菌病害,研究病原菌侵染棉花的组织学过程对致病机理解析和抗病资源利用具有重要意义。本研究利用绿色荧光蛋白标记的大丽轮枝菌系统研究了其对抗病棉种海岛棉7124和三裂棉、感病棉种军棉1号和戴维逊棉的侵染过程。结果表明,大丽轮枝菌对抗/感棉种的初始侵染没有明显差异,接菌5 h后,分生孢子均能吸附在感病和抗病棉种的根表面。但侵染过程存在显著差异,侵染感病棉种中病原菌3~5 d到达皮层,5~7 d达到维管束,随后迅速扩展繁殖,侵染14 d后即完成系统侵染,并开始产生黄萎病症状;而病原菌侵染抗病棉种,5~7 d才侵入皮层,7~10 d到达维管束,14 d后仍无法扩展,病原菌的定殖与发展受到限制,无法形成系统侵染,较少形成黄萎病症状。本研究通过绿色荧光蛋白标记大丽轮枝菌对抗/感棉种的侵染过程研究,为大丽轮枝菌致病机理研究和抗性资源利用提供了强有力的理论依据。     

In vivo growth and pathotoxin production by the maize pathogen Cochliobolus carbonum

Conidia of Cochliobolus carbonum secrete a toxin (HC-toxin) during appressorium formation on maize leaves. Plasma desorption mass spectrometry revealed that approximately 70 ng of toxin per 10⁶ conidia were secreted during the first 16 h of morphogenesis. Growth of the fungus was monitored microscopically. Extensive fungal growth occurred in the susceptible interaction by 24 h. In spite of a substantial amount of HC-toxin, the fungus failed to become established in the resistant host even after 36 h. Results suggest that the resistance conditioned by Hm1, which encodes a toxin reductase, causes inactivation of the toxin early in the interaction.     

Early vascular defense reactions of cotton roots infected with a defoliating mutant strain of Verticillium dahliae

Susceptible and resistant cotton lines were cytologically and histochemically investigated for their defense reactions to a highly aggressive and defoliating strain of Verticillium dahliae, a fungus responsible for vascular wilt. Cytochemistry showed that early responses consisted of reinforcement in structural barriers with polysaccharides, including callose and cellulose. Ultrastructural modifications of parenchyma cells of the vascular tissues were associated with strong production of terpenoids and phenolics. These defense reactions were detected early in roots of the resistant line, one to four days after inoculation, while they were seen later in roots of the susceptible line.     

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。