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.     

大豆疫霉菌对大豆下胚轴侵染过程的细胞学研究

 接种后1.5~24h,用光镜和电镜研究了2个大豆品种与大豆疫霉菌Ps411的亲和性和非亲和性互作。观察结果表明,大豆疫霉菌对大豆下胚轴的侵染过程可分为侵入前、侵入、皮层组织中的扩展和进入维管束组织4个连续阶段。大豆下胚轴接种后在25℃保湿培养,1.5h后游动孢子即形成休止孢并萌发产生附着孢,3h后侵入表皮细胞,6h后进入皮层组织,24h后进入维管束组织。病原菌主要以侵染菌丝直接侵入表皮,表皮细胞间隙是主要侵入部位。皮层细胞是病原菌定殖和发展的主要场所,胞间菌丝侵入皮层细胞并形成吸器。在菌丝与寄主细胞接触部位的寄主细胞壁与质膜之间常有胞壁沉积物的形成。在抗病品种上病菌的侵染事件与感病品种基本一致,但不能形成正常的吸器,胞壁沉积物明显多于感病品种,菌丝在寄主组织内的扩展明显受到抑制。利用β-1,3-葡聚糖免疫金标记单克隆抗体进行的免疫细胞化学的研究表明,胞壁沉积物内含有大量的β-1,3-葡聚糖,在大豆疫霉菌菌丝壁中也存在β-1,3-葡聚糖。以上结果表明,病原菌的侵染可诱导抗病寄主细胞内β-1,3-葡聚糖迅速的合成与积累、并形成胞壁沉积物,以抵御病菌的侵染与扩展。     

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.     

Interaction between powdery mildew and barley with mlo5 mildew resistance

Powdery mildew infection of barley with the mlo5 barley powdery mildew resistance gene was examined, using near-isogenic barley lines, with and without mlo5 resistance, and two near-isogenic powdery mildew isolates, HL3/5 and GE3 with high (virulent) or low (avirulent) penetration efficiency on the resistant barley line. In all isolate–host combinations (except GE3 on the resistant barley line), frequency of haustorium formation increased significantly from zero at 11 h after inoculation to a maximum by 13 h, and there was no subsequent increase up to 24 h. In the susceptible barley line, 27% of appressoria from both isolates formed haustoria. Although this was significantly higher than the frequency of haustorium formation (18%) of HL3/5 on the resistant barley line, HL3/5 was much more successful than GE3 (frequency of haustorium formation less than 1%). The fact that HL3/5 did not possess a generally higher ability to penetrate successfully to form haustoria on the susceptible barley line, indicates that HL3/5 did not overcome the mlo5 resistance by being generally more vigorous. In the resistant barley line, papillae were larger than in the susceptible line; however, both isolates were associated with papillae of the same diameter at the time of penetration. We suggest that the mlo5 resistant barley line confers two different forms of resistance: isolate-specific and isolate-nonspecific.     

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.     

Production and characterisation of monoclonal antibodies to cell wall components of the flax rust fungus

Monoclonal antibodies have been raised against an haustorium-enriched sample prepared from flax leaves infected with the biotrophic flax rust pathogen Melampsora lini. The monoclonal antibodies were produced following conventional and co-immunisation procedures and the range of antibody specificities was compared. The preparation used as immunogen for the conventional protocol was a crude isolate of haustoria consisting of approx. 65% fungal haustoria, the other components being mainly mesophyll cells or cell wall and chloroplast fragments. Following hybridoma production, 40% of positive cell lines produced antibodies that reacted with haustoria and other fungal cells, but 60% bound to plant cells in the infected leaves. For the co-immunisation protocol, the preparation used for immunisation consisted of the crude isolate of haustoria mixed with serum raised against an haustorium-depleted leaf homogenate. In two fusions, 92-94% of the antibodies reacted with fungal cells, including 3 cell lines that localised specifically to the cell wall of haustoria. Only 6-8% of the antibodies produced via co-immunisation reacted with plant cells. The antigens targeted by the three haustorium-specific monoclonal antibodies are incorporated into the wall at early stages of haustorium development, remain in the wall throughout haustorium maturation, and are present in both compatible and incompatible interactions. The epitopes recognised by the monoclonal antibodies are oligosaccharide in nature and the antigens are highly resistant to extraction from the wall. These results highlight the value of the co-immunisation protocol for the production of monoclonal antibodies to specific components in an impure preparation and provide direct evidence for molecular differentiation within the wall of the haustorium of M. lini.     

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.     

Histological responses of host and non-host plants to <Emphasis Type="Italic">Hyaloperonospora parasitica</Emphasis>

Differences in Hyaloperonospora parasitica development and plant tissue responses were compared for 10 cruciferous hosts (including both resistant and susceptible genotypes), 3 leguminous and 1 graminaceous non-host species. Cotyledons, or true leaves in the case of Triticum aestivum and Pisum sativum, were studied at 2, 8, 24 h and 3, 5, 7 days post inoculation (dpi). The high levels of zoosporangial germination observed on all species tested, as well as on glass slides, suggested that inhibition of germination did not play a significant role in distinguishing host versus non-host resistance. During the early stages of infection, at spore germination and host penetration, there was no evidence of a clear-cut difference between Brassica host species which displayed a hypersensitive, partially resistant or susceptible reaction compared with non-host species. Haustoria formation was the key infection phase for the establishment of biotrophy. Across all tested species, haustoria were initiated inside the epidermal cells. However, there were significant differences in the frequency and timing of haustorial formation and the final size of haustoria among the tested species at early infection stage. Fully developed haustoria were never observed in Raphanus raphanistrum, Triticum aestivum, Lupinus angustifolius nor Trifolium subterraneum. Instead, the haustorium development appears to abort in the penetrated epidermal cells of these species. Although haustoria were formed in the epidermal and mesophyll cells of Sinapsis alba and Pisum sativum, subsequent hyphal growth and/or continued haustoria formation were rare or few, respectively. Hypersensitive reaction was the key resistance response observed among the host and non-host resistant species tested. It is noteworthy that, in the initial stages of pathogenesis, there was no differentiating point that separated the non-host species from those that were hosts.     

Infection of resistant and susceptible cultivars of wheat by Pyrenophora tritici-repentis

Conidial germination, appressorial formation. penetration of epidermal walls, formation of intracellular vesicles and growth of intracellular hyphae in epidermal cells occurred within 12 h of inoculation. Hyphae then grew slowly between mesophyll cells for the next 12 h. Some papillae formed beneath appressoria and most infected epidermal cells retained stain by 24 h after inoculation, indicating major changes in cellular physiology. Slight differences between cultivars in some of these events were not related to resistance.
On the second day. intercellular hyphae emerged more extensively from the infection sites into the mesophyll of the susceptible cultivar Banks, and formed significantly larger mycelia than in the resistant cultivar BH1146 by 3-5 days from inoculation. Rapid intercellular growth then continued in the susceptible cultivar but not in the resistant cultivar. Necrotic lesions expanded faster in the susceptible cultivar from day 3. By day 10. most lesions in this cultivar were large and light brown with a conspicuous chlorotic margin but those in the resistant cultivar were small and dark brown with inconspicuous chlorosis.     

Ultrastructural Characterization of Infection and Colonization of Maize Leaves by Colletotrichum graminicola, and by a C. graminicola Pathogenicity Mutant

ABSTRACT Observations were made of the ultrastructure of infection and colonization of leaves of a susceptible maize inbred by Colletotrichum graminicola and by a C. graminicola pathogenicity mutant. The mutant causes no symptoms on either maize leaves or stalks. Prior evidence suggested that it is deficient in production of signal peptidase, responsible for cleavage of signal peptides from proteins destined for transport through the endoplasmic reticulum. There was no significant difference in the process of infection or colonization by the mutant and wild-type strains up to 48 h after inoculation. Both the mutant and the wild type produced globose, melanized appressoria within 24 h after inoculation on the host surface. By 36 h, both strains had penetrated the host epidermal cells directly. The host cells frequently formed papillae in response to appressoria, but these were not usually successful in preventing fungal ingress in either case. Penetration was followed by formation of irregularly shaped, swollen infection hyphae. Infection hyphae of both strains grew biotrophically for a relatively short time (less than 12 h). One or more hyphal branches was produced from each infection hypha, and these invaded adjacent mesophyll cells. Both strains of the fungus grew cell-to-cell, setting up new biotrophic interactions in each cell, between 36 and 48 h after inoculation. Papillae were frequently formed by the mesophyll cells, but these were not successful in preventing fungal ingress. The first noticeable difference between the mutant and the wild type was related to their interaction with mesophyll cells. Cells invaded by the wild type died relatively quickly, whereas those infected by the mutant appeared to survive longer. The most dramatic difference between the mutant and wild type occurred when the mutant completely failed to make a transition to necrotrophic growth, while the wild type made that switch at 48 to 72 h after inoculation. The mutant may be unable to secrete sufficient quantities of one or more proteins that are necessary to support the switch between biotrophy and necrotrophy.     

Uromyces Viciae-fabae Haustorium Formation in Susceptible and Resistant Faba Bean Lines

Haustorium formation by the faba bean rust (Uromyces viciae-fabae) was studied on susceptible and resistant faba bean lines. The resistant lines showed incomplete resistance, based on late acting hypersensitivity or on non-hypersensitive resistance acting before haustorium formation. Histological observations on infected leaves showed that both the number of haustoria per infection unit and their developmental stage was reduced in both resistant lines. Isolation of haustoria confirmed that both the number and the size of haustoria were reduced in resistant lines, irrespective of whether the resistance was associated with hypersensitivity. Plant age had no detectable effect on both parameters.     

Host responses to fungal penetration in Evysiphe graminis f.sp. hordei infections in barley

Resistance mechanisms restricting penetration and establishment were investigated in an incompatible interaction using an avirulent race CC/3 (BMV 1 + 4) of Erysiphe graminis f.sp hordei on barley cv. Athos (BMR 2 + 5) and a compatible interaction using the universal susceptible cv. Golden Promise (BMR 0). In both interactions: (i) auto-fluorescence of the host cell wall occurred adjacent to the primary germ tube tip after 4 h, and by 10 h near to the appressorial germ tube; (ii) strongly fluorescing papillae developed after 12 h, being frequently associated with failed penetrations; and (iii) only 30% of the attempted penetrations from appressorial lobes resulted in an incipient haustorium at 16 h. Hypersensitive reactions occurred in cells with an incipient haustorium in 30% of appressorial penetrations on Athos, but only in 4% of those on Golden Promise. Twenty per cent of hypersensitively reacting cells in Athos appeared dead by 14 h using trypan blue (membrane exclusion test), compared with 5% for neutral red, suggesting that plasma membrane damage is an early event in the hypersensitive response. Haustorial death was associated with host cell death, but did not precede it; appressorial death occurred 2-4 h after that of the host cell.     

Nonhost versus host resistance to the grapevine downy mildew, Plasmopara viticola, studied at the tissue level

Following inoculation of host and nonhost plants with Plasmopara viticola, the grapevine downy mildew, a histological survey was undertaken to identify the stage where its development is contained in nonhosts and in resistant host plants. Three herbaceous nonhost species, Beta vulgaris, Lactuca sativa, and Capsicum annuum, and three grapevine species displaying different level of resistance (Vitis vinifera [susceptible], Vitis riparia [partially resistant] and Muscadinia rotundifolia [totally resistant]) where inoculated by P. viticola using a controlled leaf disk inoculation bioassay. During the early steps of infection, defined as encystment of zoospores on stomata, penetration of the germ tube, and production of the vesicle with the primary hypha, there was no evidence of a clear-cut preference to grapevine tissues that could attest to host specificity. The main difference between host grapevine species and nonhosts was observed during the haustorium formation stage. In nonhost tissues, the infection was stopped by cell wall-associated defense responses before any mature haustorium could appear. Defense responses in resistant grapevines were triggered when haustoria were fully visible and corresponded to hypersensitive responses. These observations illustrate that, for P. viticola, haustorium formation is not only a key stage for the establishment of biotrophy but also for the host specificity and the recognition by grapevine resistance factors.     

Microtubule dynamics in compatible and incompatible interactions of soybean hypocotyl cells with Phytophthora sojae

The arrangement of microtubules in soybean ( Glycine max ) cells was examined during compatible and incompatible interactions of hypocotyls of soybean cv. Harosoy (susceptible) and cv. Haro 1272 (resistant) with race 1 of the soybean-specific pathogen Phytophthora sojae . Both reaction types were similar during the first 3 h after zoospore inoculation in terms of the number of cells penetrated, and depth penetrated into the cortex. By 3 h postinoculation, clear differences had developed between the two interaction types: incompatible interactions were characterized by a hypersensitive response that was confined to single penetrated cells; while compatibly responding cells appeared unchanged. Both types of response were characterized by autofluorescence of cell walls or cytoplasm and, at 6 h after inoculation, complete disorganization of cell cytoplasm. Reorientation and loss of microtubules was seen in the early stages of the incompatible interaction in association with cellular hypersensitivity, but not in compatible responses. In cells adjacent to those that reacted hypersensitively, there was little evidence of change in microtubule orientation. Treatment of hypocotyls with the microtubule depolymerizer oryzalin prior to inoculation did not alter the compatible response, but led to breakdown of the incompatible response. Changes in microtubule orientation and state are thus among the first structural changes that are visible within cells during incompatibility in this system.     

小麦与条锈病菌不亲和互作的超微结构

 采用条锈菌同一小种的野生型菌系和弱毒突变菌系,分别接种同一小麦品种的方法,研究了不亲和互作的超微结构特征。在不亲和互作中,条锈菌的胞间菌丝、吸器母细胞和吸器明显受抑。吸器可以在发育早期受抑坏死,也可迟滞至吸器体形成之后坏死。吸器外质膜严重皱褶,并出现孔洞,吸器外间质加宽,沉积大量电子致密物质。侵染位点的小麦叶肉细胞表现与过敏性坏死反应相关联的一系列变化。细胞壁内侧还出现乳突状或颗粒状沉积物等防御结构或物质。     

Variation in the early development of Bremia lactucae on lettuce cultivars with different levels of field resistance

The early stages of development of Bremia lactucae (lettuce downy mildew) were examined on lettuce cultivars possessing high (Iceberg and Regina di Maggio) and low (Great Lakes and Plenos) levels of field resistance. Germ tubes, appressoria, penetration, primary and secondary vesicles, intercellular hyphae and haustoria were observed 3. 6 and 24 h after inoculation of cotyledons and of leaf discs from adult plants. Differences were observed between cv. Iceberg and susceptible genotypes in the percentage of spores germinating and the incidence and speed of development of infection structures. Secondary vesicles were first observed 24 h and 6 h after inoculation in Iceberg and susceptible genotypes, respectively. The lowest incidence of secondary vesicle formation 24 h after inoculation (48 and/or 43%) was recorded in Iceberg and Regina di Maggio, and the highest incidence (68%) occurred in Plenos. The formation of intercellular hyphae and haustoria was not observed in cv. Iceberg some 24 h after inoculation. There were significant differences in the lengths of germ tubes formed on different cultivars. Those on cv. Iceberg were longer than those formed on susceptible cultivars. The results indicate that the field resistance of B. lactucae may result from mechanisms which are effective in the early stages of infection.     

Hypersensitive cell death and post-haustorial defence responses arrest the orange rust (Hemileia vastatrix) growth in resistant coffee leaves

The growth of a coffee orange rust fungus (Hemileia vastatrix Berk and Br.) isolate (race II) and the sequence of responses it induced in leaves of resistant Coffea arabica L. and C. congensis Froehner as well as on a susceptible C. arabica were investigated cytologically and biochemically. The percentages of germinated urediospores and of appressoria formed over stomata as well as the fungal growth inside leaf tissues were similar in resistant and susceptible leaves until the 3rd day after the inoculation. In the susceptible leaves, at the majority of the infection sites (70%) the fungus pursued its growth without apparent inhibition while in the resistant leaves the fungus ceased its growth with higher frequency (34% in C. arabica and 54% in C. congensis) after the formation of at least one haustorium. The first signs of incompatibility, detected 2 days after the inoculation, were cytologically expressed by hypersensitive host cell death (HR), host cell wall autofluorescence and haustoria encasement with callose and β-1,4-glucans. Biochemically, two peaks of phenylalanine ammonia-lyase (PAL) activity were detected by 2 and 5 days after the inoculation. The 1st peak coincided with the early accumulation of phenolic compounds and with the beginning of cell death. The 2nd peak could be related to later accumulation of phenols and the lignification of the host cell walls. About 5–7 days after the inoculation, ultrastructural observations revealed the accumulation of a material partially crystallized in the intercellular spaces around the senescent hyphae, next to dead host cells and in close association with the middle lamella that initially labelled for pectins. It also contained polysaccharides and phenolic-like compounds. Cellulose, hemicellulose, extensins, hydroxyproline-rich glycoproteins and proteins were not detected. The hypertrophy of the host cells in the infection area were also observed around 12 days after the inoculation corresponding macroscopically to the reaction flt.In susceptible plants, cell death was also observed 3 days after the inoculation but only in a reduced percentage of infection sites in which the fungus aborted at an early stage. A late haustorium encasement and stimulation of PAL activity were also observed but these delayed host responses did not prevent fungal growth and sporulation.The intercellular material, only observed in the resistant plants, is here reported for the first time and although its role is unknown it might be the result of plant cell death.     

Spatiotemporal patterns of oxidative burst and micronecrosis in resistance of wheat to brown rust infection

The accumulation of H₂O₂ (oxidative burst) and the progress of pathogen development were studied in compatible and incompatible wheat‐brown rust interactions. The accumulation of H₂O₂ was detected in 98·7% of guard cells with appressoria 8 h post inoculation (hpi). The reaction in both susceptible and resistant plants declined 2–3 days post inoculation (dpi). The second phase of the oxidative burst was observed in the mesophyll and/or epidermis. In susceptible plants it began 4–5 dpi and was detected only in the epidermis. In resistant plants the response was observed in the mesophyll. In moderately resistant plants it was induced 1–3 dpi, and the percentage of infection units reached 80–90% 8 dpi. This corresponded with severe necrotic symptoms. In highly resistant plants, the oxidative burst was short and transient. The percentage of infection units with H₂O₂ accumulation reached its highest level (60–70%) 2 dpi, and decreased thereafter. Four days later, the low percentage and weak DAB staining indicated very low H₂O₂ accumulation. The localization and the time‐course changes of the oxidative burst correlated with the profiles of the micronecrotic response, haustorium mother cell formation and pathogen development termination. An early and localized induction of oxidative burst followed by its rapid quenching correlated with high resistance and almost no disease symptoms. The possible correlation of the oxidative burst and pathogen development patterns with the level and durability of resistance conferred by Lr genes are discussed.