Ultrastructure of the Infection of Sorghum bicolor by Colletotrichum sublineolum

ABSTRACT Ultrastructural studies of the infection of susceptible and resistant cultivars of Sorghum bicolor by Colletotrichum sublineolum were conducted. Initial penetration events were the same on both susceptible and resistant cultivars. Germ tubes originating from germinated conidia formed globose, melanized appressoria, that penetrated host epidermal cells directly. Appressoria did not produce appressorial cones, but each penetration pore was surrounded by an annular wall thickening. Inward deformation of the cuticle and localized changes in staining properties of the host cell wall around the infection peg suggests that penetration involves both mechanical force and enzymic dissolution. In compatible interactions, penetration was followed by formation of biotrophic globular infection vesicles in epidermal cells. Filamentous primary hyphae developed from the vesicles and went on to colonize many other host cells as an intracellular mycelium. Host cells initially survived penetration. The host plasma membrane invaginated around infection vesicles and primary hyphae and was appressed tightly to the fungal cell wall, with no detectable matrix layer at the interface. Necrotrophic secondary hyphae appeared after 66 h and ramified through host tissue both intercellularly and intracellularly, forming hypostromatic acervuli by 114 h. Production of secondary hyphae was accompanied by the appearance of electron-opaque material within infected cells. This was thought to represent the host phytoalexin response. In incompatible interactions, infection vesicles and primary hyphae were formed in epidermal cells by 42 h. However, they were encrusted with electron-opaque material and appeared dead. These observations are discussed in relation to the infection processes of other Colletotrichum spp. and the host phytoalexin response.     

Histology of infection by Erysiphe graminis f.sp. hordei in spring barley lines with various levels of partial resistance

The histology of resistance to infection by conidia of Erysiphe graminis f.sp. hordei has been studied in barley lines with various levels of partial resistance. Resistance reduced the proportion of conidia which formed haustoria and was expressed more strongly in fifth than in first formed leaves. Where attempted penetration from the first appressorial lobe failed, a second and sometimes a third lobe could be formed. Most penetrations were from first lobes, a few from second lobes and a negligible number from third lobes. Adult plant resistance decreased penetration success from both first and second lobes. An unidentified component of resistance appeared to impede the development of less vigorous conidia before they stimulated a host cell response. However, in all hosts the majority of failed penetration attempts stimulated a localized host response implicating this response in resistance. Some attempts caused hypersensitive death of epidermal cells in both resistant and susceptible hosts; the proportion of dead cells was higher in fifth than first leaves. Stomatal subsidiary cells were more susceptible to penetration than interstomatal cells and in resistant leaves the majority of successful penetrations were into subsidiary cells. The pathogen was also able to avoid resistance to penetration by establishing endophytic infection following entry of germ tubes through stomates.     

小麦的种和品种对白粉病抗性的初步研究

 在对白粉病具不同抗性的11个小麦的种和品种上进行的按种试验表明,在接种后8小时内,分生孢子达到最高萌发率(约为60%),并形成附着孢。在抗病的和感病的小麦叶片上,分生孢子的萌发率和萌发速度没有差异。接种后12小时,在感病的材料上,发现真菌侵入寄主,但在抗病的材料上,发现最早侵入时间,至少要比在感病材料上约晚4小时。接种后36小时,在高感的材料上,萌发的分生孢子有83%已侵入寄主,其中约有70~80%已形成吸胞。在感病、抗病和高抗材料上的侵入率,分别为70,45,27%。形成或开始形成吸胞的分别为55~61,14~27,4~8%。在抗病材料的表皮细胞内,真菌形成的吸胞较小,并有点畸形。在抗病材料的叶面上,真菌的菌丛稀薄,产孢也很少。     

Prepenetration stages in infection of clematis by Phoma clematidina

A detailed study of conidial germination, germ-tube growth and the formation of infection structures in Phoma clematidina , the causal agent of clematis wilt, is described for two clematis varieties differing in disease resistance. On both the resistant and susceptible varieties, the fungus entered leaves and stems by direct penetration of the cuticle, often, but not always, following the formation of infection structures. More germ tubes per conidium were formed on the susceptible host, but these germ tubes were on average shorter than on the resistant host. Although germ tubes regularly entered the plant via trichomes, stomata were not found to be sites of entry. Following penetration of the cuticle of resistant plants, germ-tube growth was sometimes restricted to the subcuticular region, and halo formation occurred at the sites where penetration was attempted. Subcuticular growth and halo formation were not observed on susceptible plants. These observations may partly explain the resistance of small-flowered clematis varieties to P. clematidina .     

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.     

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.     

Formation of highly branched hyphae by Colletotrichum acutatum within the fruit cuticles of Capsicum spp.

This study showed that Colletotrichum acutatum penetrates the cuticle layer of Capsicum spp. fruits by forming a previously uncharacterized structure from appressoria. This unusual structure was localized in the cuticle layer. The structure, formed within 24 h post‐inoculation (hpi), was a highly branched, well‐differentiated hypha which penetrated the epidermal cell at 72 hpi. The novel structure, with abnormally thick walls (about 250 nm), often formed multiple branches in the affected chilli pepper. This dendroid structure, probably required for penetration, was formed exclusively in the cuticle layer of chilli pepper fruits and was not found when C. acutatum was inoculated onto pepper petals, mango leaves, or fruits of tomato and aubergine. Colletotrichum acutatum produced similar dendroid structures within resistant chilli pepper fruits, but eventually these structures turned dark brown and no further infection in the epidermal cells occurred, implicating the presence of inhibitors of the formation and development of the dendroid penetration structure in the resistant line.     

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。     

Histopathology of Infection and Colonization of Susceptible and Resistant Port-Orford-Cedar by Phytophthora lateralis

ABSTRACT Port-Orford-cedar (POC) root disease, caused by Phytophthora lateralis, continues to kill POC in landscape plantings and natural forests in western North America. POC trees resistant to P. lateralis have been identified and propagated. Cytological observations of P. lateralis in susceptible and resistant roots and stems were made with light and transmission electron microscopy to identify resistance mechanisms. No differences in infection pathway and initial colonization were observed between susceptible and resistant roots, although there were differences in the rate and extent of development. Germ tubes formed appressoria, and penetration hyphae grew either between or directly through epidermal cell walls; inter- and intracellular hyphae colonized the root cortex. In susceptible roots, hyphae penetrated into the vascular system within 48 h of inoculation. In contrast, hyphae in roots of resistant seedlings grew more slowly in cortical cells and were not observed to penetrate to the vascular tissues. In resistant roots, infection was marked by general thickening of cortical cell walls, wall appositions around penetrating hyphae, collapse of cortical cells, and accumulation of osmophillic granules around hyphae. In susceptible stems, hyphae grew inter- and intracellularly in all cells of the secondary phloem except fiber cells, but were concentrated in sieve and parenchyma cells in the functional phloem. The pattern of penetration and colonization of hyphae was similar in the resistant stems, except that hyphae were found in the fiber cells of the xylem. In resistant stems, there were fewer hyphae in the functional phloem, and cytological changes such as damaged nuclei and disintegrated cytoplasm were evident. Structural changes in resistant stems included collapsed cells, wall thickening, secretory bodies, apposition of electron dense materials, and crystals in cell walls.     

Cuticular penetration of β-cypermethrin in insecticide-susceptible and resistant strains of Bactrocera dorsalis

Semi-thin sectioning and transmission electron microscope techniques were employed to investigate the cuticle thickness, integument structure, and fat body of larvae from susceptible and resistant strains of Bactrocera dorsalis. The results showed that the cuticle of β-cypermethrin-resistant strains (25.96 ± 1.00 μm) was thicker than that of susceptible strains (19.36 ± 0.82 μm). The number of chitin layers in the endocuticle of β-cypermethrin-resistant strains (98.00 ± 3.61 layers) was more than that in susceptible strains (75.67 ± 2.40 layers). Compared with susceptible strains, the laminated structure of the chitin layers in the endocuticle of resistant strains revealed higher density and more distinctive structure, and the interspace of epidermal cells was thicker. Fat body in the resistant insects contained more fat granules than those in susceptible insects. Moreover, HPLC analysis showed that the cuticular penetration of β-cypermethrin into larvae of resistant strains was slower than that of susceptible strains. In addition, the metabolism of β-cypermethrin in resistant strains was faster than that in susceptible strains, indicating that the resistant strains could enhance detoxification metabolism. These results indicated that cuticle thickness, fat body, laminated structure of the chitin layers, and interspace of epidermal cells might be correlated with cuticular penetration between susceptible and resistant strains, suggesting that the resistant strains could decrease the rate of penetration of insecticide into the internal cavity.     

细胞乳突的形成和小麦白粉菌成功侵染的关系

 用一套己知抗白粉病单基因的小麦材料,研究了小麦白粉菌诱发寄主表皮细胞产生的乳突与成功侵染的关系。抗病小麦叶片上,诱发产生乳突的分生孢子占测定分生孢子数的58.8%,感病品种为63.8%。这表明抗性不同的小麦材料接种白粉菌之后,叶表皮细胞内形成乳突的百分率相近,然从在抗病材料和感病材料中形成的乳突阻止病原物发育和成功侵染的作用不相同,在抗病叶片上,诱发产生乳突的分生孢于中有88%停留在压力孢阶段,不继续发育,仅有12%能突破乳突形成吸器,使得侵染成功。与此对照,在感病叶片上只有32%诱发乳突的分生孢子不能穿透乳突,而68%则能突破乳突。成功地与寄主建立寄生关系。我们的研究表明,乳突能否成功地阻止白粉菌侵入可能与乳突形成的迟早有关。     

荸荠茎点霉秆枯病菌侵染过程的超微观察

<正>荸荠(Eleocharis dulcis),又称马蹄,为莎草科多年生草本植物,是一种具有食用和药用价值的水生蔬菜。近年来,随着荸荠在我国种植面积的不断扩大,病害发生也呈逐年上升趋势。荸荠茎点霉秆枯病是2009年在湖北省荸荠产区发现的一种新病害,由Phoma bellidis侵染引起,该病在湖北省团风地区发生尤为严重,对荸荠的产量和品质造成严重影响;病害一般在8~12月发生,发病初期在荸荠茎秆上产生圆形或梭形红褐色小斑,随后病斑沿茎     

Resistance of sunflower (Helianthus annuus L.) to terminal bud attack bySclerotinia sclerotiorum (Lib.) de Bary

Resistance of sunflowers to terminal bud attack bySclerotinia sclerotiorum was studied by microscopical observations of infection processes and by genetical analyses of trials showing natural infections. Electron microscope studies showed that there were no differences in leaf morphology between susceptible and resistant genotypes, and that both were contaminated by ascospores. Only on the susceptible genotype was considerable ascospore germination observed, followed by mycelial development and leaf penetration. On the resistant genotype, there was little ascosopore germination and no further development. The genetical studies of percentage natural attack observed on eight inbred lines representing a range of reactions, and the hybrids from a factorial cross of these lines, indicated that inheritance is mainly additive, with few interactions.     

Ultrastructural analysis of responses of host and fungal cells during plant infection

Cellular responses in fungi and in susceptible or resistant hosts during fungus–plant interactions have been studied ultrastructurally to examine their role in pathogenicity. Pathogenicity is determined in some saprophytic fungi by various factors: the production of disease determinants such as the production of host-specific toxins (HSTs) or the extracellular matrix (ECM) by fungal infection structures and H₂O₂ generation from penetration pegs. Three different target sites for HSTs have been identified in host cells in many ultrastructural studies: plasma membranes, chloroplasts, and mitochondria. The mode of action of HSTs is characterized by the partial destruction of the target structures only in susceptible genotypes of host plants, with the result that the fungus can colonize the host. The infection structures of most fungal pathogen secrete ECM on plant surfaces during fungal differentiation, while the penetration pegs of some pathogens produce reactive oxygen species (ROS) in the cell walls and plasma membranes. The pathological roles of ECM and H₂O₂ generation are discussed here in light of ultrastructural evidence. Host and fungal characteristics in the incompatible interactions include the rapid formation of lignin in host epidermal cell walls, failure of penetration pegs to invade lignin-fortified pectin layers, the inhibition of subcuticular hyphal proliferation and the collapse of hyphae that have degraded cell walls within pectin layers of the host. Apoptosis-like host resistant mechanism is also discussed.     

Hemibiotrophic infection of Pisum sativum by Colletotrichum truncatum

The infection of pea ( Pisum sativum ) by Colletotrichum truncatum was studied by light and electron microscopy. These investigations were facilitated by use of an Argenteum pea mutant, which has a readily detachable epidermis. Infection pegs emerging from appressoria penetrated epidermal cells directly. Large intracellular primary hyphae formed a dense stromatic mycelium confined within a single epidermal cell. Primary mycelia gave rise to thinner secondary hyphae which radiated into surrounding cells and caused extensive wall dissolution. Melanized sclerotia developed in the centre of chlorotic water-soaked lesions. Acervuli were not observed. Epidermal cells survived initial penetration by primary hyphae, as shown by their ability to plasmolyse and accumulate Neutral red, but all infected cells were dead when the secondary hyphae had formed. Six cultivars of pea were susceptible, but seven other legumes were resistant. A single isoform of polygalacturonase with a pI of 8·3 and apparent M _r of 40000 was purified from culture filtrates and the TV-terminal amino acid sequence determined. The relevance of the results to the taxonomy of C. truncatum and the relationships between infection process and host range are discussed.     

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.     

Cultivar Resistance to Anthracnose Disease of Cowpea (Vigna unguiculata (L.) Walp.) caused by Colletotrichum destructivum O'Gara

The infection process of Colletotrichum destructivum, a hemibiotrophic anthracnose fungus, was studied by light microscopy in two cowpea (Vigna unguiculata) cultivars which differ in disease reaction type. Large, multilobed, intracellular infection vesicles, followed by necrotrophic, radiating, secondary hyphae were produced in tissues of the susceptible cv. IT82E-60. In the resistant cv. TVx3236, both the production of appressoria and their melanisation were impaired, resulting in reduced penetration. Where penetration occurred, the initially-infected epidermal cells underwent a hypersensitive response, restricting the growth of multilobed vesicles and thereby blocking the destructive necrotrophic phase of disease development. The phytoalexins kievitone and phaseollidin accumulated earlier and more rapidly in stem tissues of the resistant cultivar, associated with the appearance of delimited, necrotic spots on inoculated surfaces. In contrast, delayed and slower accumulation of these compounds occurred in the compatible interaction, together with the development of typical spreading, water-soaked, anthracnose lesions.     

Light and scanning electron microscopy studies on the infection process of melon leaves by Colletotrichum lagenarium

Colletotrichum lagenarium is the casual agent of anthracnose disease of melons. Light and scanning electron microscopy were used to observe the infection process of C. lagenarium on the leaves of two melon cultivars differing in susceptibility. On both cultivars conidia began germinating 12 h after inoculation (hai), forming appressoria directly or at the tips of germ-tubes. By 48 hai appressoria had melanised and direct penetration of host tissue had begun. On the susceptible cultivar, infection vesicles formed within 72 hai and developed thick, knotted primary hyphae within epidermal cells. By 96 hai C. lagenarium produced highly branched secondary hyphae that invaded underlying mesophyll cells. After 96 hai, light brown lesions appeared on the leaves, coincident with cell necrosis and invasion by secondary hyphae. While appressoria formed more quickly on the resistant cultivar, fewer germinated to develop biotrophic primary or invasive necrotrophic secondary hyphae than on the susceptible cultivar. These results confirm that C. lagenarium is a hemibiotrophic pathogen, and that resistance in melons restricts colonisation by inhibiting the development of necrotrophic secondary hyphae.     

Histopathological Study of Barley Cultivars Resistant and Susceptible to Rhynchosporium secalis

ABSTRACT Differences in the penetration process by Rhynchosporium secalis were compared in resistant and susceptible barley cultivars at the seedling stage. Percent penetration and percent host cell wall alteration (HCWA) differed significantly among cultivars and isolates as revealed by light microscopy. Based on these two variables, the cultivars were statistically separated into two groups that corresponded to their disease reactions. The resistant cultivars, Johnston and CDC Guardian, showed 81.2 to 99.4% HCWA and 0.1 to 20.1% penetration at encounter sites, whereas the susceptible cultivars, Harrington, Argyle, and Manley, had 30.1 to 78.3% HCWA and 31.8 to 81.8% penetration. In the current study, cv. Leduc, which is susceptible at the seedling stage and resistant at the adult stage, showed the same percent HCWA and penetration as did susceptible cultivars. A significant negative correlation (P < 0.01) was found between percent penetration and percent HCWA for cultivars inoculated with two isolates of the pathogen. Isolate 1 was less virulent than isolate 2 with respect to percent penetration and induced significantly fewer HCWA. Scanning electron microscopy showed various shapes of fungal appressoria but no apparent difference in host reaction between resistant and susceptible cultivars. Transmission electron microscopy revealed interactions between the host and pathogen at various stages of penetration. The resistant cv. Johnston responded by producing appositions, as evidenced by a layer of compact osmiophilic material deposited on the inner side of the cell wall. Infection pegs produced by conidia were unable to penetrate the cuticle where an apposition had formed inside. When penetration occurred in the susceptible cv. Argyle, cytoplasmic aggregates and separation of the plasmalemma were visible from the host cell wall, but the layer of compact osmiophilic material was not always present. Data based on light microscopic observations suggested that HCWA may be one of the mechanisms responsible for resistance that is characterized as penetration prevention rather than as a slow rate of mycelial growth after successful penetration. HCWA occurred in response to attempted cuticle penetration, suggesting that HCWA may produce chemical barriers that help to prevent penetration.