The role of primary germ tubes in the life cycle of Blumeria graminis: The primary germ tube is responsible for the suppression of resistance induction of a host plant cell

When the conidia of Blumeria graminis f. sp. hordei (Bgh) are inoculated on barley coleoptile cells they produce short germ tubes called primary germ tubes (PGTs) about 2 h after inoculation. We evaluated the positive role of a PGT in inducing accessibility of the host cell under the germ tube. When an appressorium (APP) penetrated the same cell on which a PGT was present, the ratio of haustorium formation (penetration efficiency) was significantly higher than when an APP penetrated the cell adjacent to the one on which a PGT was present. When an APP penetrated the cell laterally adjacent to the one on which a PGT was present we killed the cell under the PGT by puncturing it with a microneedle and then investigated the penetration efficiency of the cell adjacent to the dead cell. As a control we killed the cell longitudinally adjacent to the one on which a PGT was present and investigated the penetration efficiency of the laterally adjacent cell. The results showed that the penetration efficiency of the former was significantly lower than that of the latter. This suggests that some accessibility factor might transfer from a cell on which a PGT is present to a laterally adjacent cell. The existence of a conidium body but not a PGT was not effective for induced accessibility of the host cell. Moreover, when a Bgh germling was removed 6 h after inoculation and another germling was transferred to the same cell, the penetration efficiency was significantly higher than that of control. As a control, a Bgh germling was transferred to a cell on which no germling was present. These results suggest that the existence of PGT is effective for induced accessibility of a host cell when penetrated by Bgh. However, it is unclear whether or not a PGT secretes some substance(s) which suppresses the resistance induction of a host cell.     

The role of primary germ tubes (PGT) in the life cycle of Blumeria graminis: The stopping of PGT elongation is necessary for the triggering of appressorial germ tube (AGT) emergence

The conidia of Blumeria graminis f. sp. hordei (Bgh), following contact with a host surface, first form a short germ tube, called the primary germ tube (PGT), and then an elongating germ tube emerges. It differentiates into an appressorial germ tube (AGT), and then the AGT elongates and swells. It forms a hooked, appressorial lobe that penetrates the epidermal cell wall of the host. In a series of infections, the positive role of PGT in the morphogenesis of the fungus is unclear except for the possibility reported by Carver and Ingerson that the growth of a long germ tube, with the potential to differentiate into an appressorium, seems to be dependent on the perception of a suitable host surface through contact with the PGT. Therefore, the aim of the present study is to further clarify the role of PGT in the morphogenesis of the fungus. When the conidia of Bgh were inoculated onto the coleoptile surface whose cuticle was removed with cellulose acetate, the emergence of the AGT was delayed. This delay was related to the length of the PGT. That is, on the cuticleless coleoptile surface the PGT tended to continue elongating without stopping. If there were gaps on the coleoptile surface such as a cell border on the more hydrophilic substratum like cuticleless coleoptile surface, the PGT stopped elongating there and after that the AGT emerged. Moreover, the length of PGT in the beginning of AGT emergence was same as that of the PGT after appressorium formation. This means that PGT elongation had stopped when AGT began to emerge. Therefore, it is necessary to stop the PGT elongation for the triggering of AGT emergence.     

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     

The rapid and accurate determination of germ tube emergence site by Blumeria graminis conidia

The development of appressoria by germinating Blumeria graminis conidia depends on its germ tubes making contact with the host surface. Low angle, low temperature scanning electron microscopy showed that 80% or more of first-formed germ tubes of f. spp. hordei, tritici and avenae conidia emerged from close to the host leaf surface, and so made contact with it allowing them to become functional primary germ tubes. Light microscopy of f. sp.hordei confirmed this result and, in addition, showed that germ tubes frequently emerged close to, and contacted, various hydrophobic and hydrophilic artificial substrata. Geometric models of conidium-substratum interfaces were developed and a “surface point method” was derived to predict the frequency with which contact would result if germ tube emergence was a random phenomenon. However, observed contact frequencies were far higher (c. three to eight times) than predicted. Thus, the germ tube emergence site was determined as a response to substratum contact. In part, this appeared to be a non-specific response. Nevertheless, germ tube contact frequencies were greater on the curved surface of leaf epidermal cells than on planar surface, suggesting that specific recognition of leaf surface characteristics may promote directional emergence. The area of contact required to stimulate directional germ tube emergence was very small: contact with a microneedle tip or with a spiders’ suspension thread was sufficient for many conidia. Similarly, on leaves, the only contact is between the tips of a limited number of conidial wall projections and the edges of epicuticular leaf wax plates. Micromanipulation to roll conidia so that their original site of contact with a leaf was rotated away from it, led to the majority of first-formed germ tubes growing away from the leaf, i.e. emerging close to the site of original contact. The experiments indicated that the site of germ tube emergence is determined within 1 min of deposition. This implicated the release of conidial extracellular materials in recognition of the conidium-leaf surface contact site.     

青杨叶锈病菌(Melampsora larici-populina Kleb.)侵染过程的超微结构研究

 采用电子显微镜技术对青杨叶锈病菌(Melampsora larici-populina Kleb.)的侵染过程进行了研究。发现该菌夏孢子萌发产生1~3个芽管,且具较多的树杈状分枝。芽管由气孔侵入,侵入前不形成明显的附着胞或仅个别芽管形成附着胞。芽管侵入气孔后在气孔腔内形成气孔下囊,再分化出圆形的膨大体而产生1~2支初生菌丝。初生菌丝在寄主细胞间扩展,与叶肉细胞壁接触后分化出吸器母细胞,吸器母细胞中的细胞器与胞间菌丝相同,双核。吸器母细胞产生侵入钉侵入叶肉细胞内部形成吸器,成熟吸器由细长具颈环的管状颈部和膨大的吸器体组成,此时胞间菌丝在吸器母细胞处分化出次生菌丝,在叶肉细胞间扩展形成次生菌落,产生孢子堆。病菌在寄主细胞间隙或沿寄主细胞壁延伸时,寄主细胞仍保持正常状态。     

Biology of the Acacia gall rust, Uromycladium tepperianum

Germinating teliospores of Uromycladium tepperianum formed penetration pegs, which directly penetrated host epidermal cells, on the tip cell of three-celled germ tubes. Diploid nuclei within teliospores appeared to divide soon after germination commenced. Only one of the daughter nuclei moved out of the teliospores with the developing germ tubes. Reactions of seedlings inoculated with teliospores from different host species and differences in teliospore dimensions indicated that distinct genotypes of U. tepperianum adapted to particular host species occur in Australia. Limited hyphal growth occurred in two of 20 African Acacia spp. inoculated with teliospores from Acacia saligna. Fungal development was, however, restricted by necrosis of surrounding host cells. U. tepperianum appears to be suitably host-specific for use as a biological control agent against Ac. saligna , a weed in South Africa.     

Early vascular sporulation: a possible role in the virulence of Verticillium albo-atrum in wilt of alfalfa

Serial subculturing of an alfalfa field isolate of Verticillium albo-atrum produced two variants which were significantly less virulent than the original. When conidia of the original isolate, stored at − 70 °C, were infused into alfalfa cuttings, the pathogen grew from spore trapping sites into contiguous vessels and began to sporulate within 24 h. The frequency of vessels in which sporulation occurred at 24 h was similar in alfalfa genotypes known to differ in disease ratings and in host resistance to lateral penetration of vessels. With conidia of the two variants as inoculum, hyphae were found to penetrate much more extensively from trapping ites into adjacent vessels by 24 h. However, the more virulent original isolate sporulated two to three times more frequently. An isolate of V. albo-atrum from potato, which was non-pathogenic to alfalfa, had limited lateral penetrating ability in alfalfa but never sporulated by 24 h in laterally penetrated vessels. Of parameters studied, including sporulation and radial growth, only early vascular sporulation in laterally penetrated vessels correlated with the greater virulence of the original alfalfa isolate.     

Induced accessibility and enhanced inaccessibility at the cellular level in barley coleoptiles. XIV. Evidence for elicitor(s) and suppressor(s) of host inaccessibility from Erysiphe graminis

The release of elicitors and suppressors by Erysiphe graminis, the powdery mildew pathogen of barley, was investigated by microscopy in combination with micromanipulation. The elicitors enhance inaccessibility whereas the suppressors prevent the action of the elicitors. Conidia were deposited onto barley coleoptiles and incubated for intervals that varied from 1-8 h. These conidia were termed the inducer conidia since they determined whether their presence would induce the cells of the host to become inaccessible to subsequent inoculations with the fungus. At specified intervals after inoculation the conidial germlings were removed from cells with a micromanipulator. The coleoptiles were then incubated for an additional 8 h after which, a new germling of the fungus was transferred to the same cell from which the inducer germling had been removed. This new germling was called the challenge germling since it was used to determine if it could challenge the host cell to express either induced accessibility or enhanced inaccessibility. The ability of these challenge germlings to penetrate the barley host cell was then assessed after they had been incubated on the cell for an additional 19 h. This allowed the determination of whether inaccessibility had been enhanced in the host. Inaccessibility was enhanced in the host only when the inducer conidia were incubated on host cells for more than 7 h. Scanning electron microscopy revealed that these challenge germlings did not penetrate into the host cell. Thus, enhanced inaccessibility had occurred. The results indicate that the E. graminis germling released a material that enhanced the inaccessibility of the barley host cells. We refer to this material as an elicitor. The transfer of a challenge germling to a coleoptile was made at various times after the removal of the inducer germling from the tissue. This allowed us to determine that more than 2 h is required for the enhancement of inaccessibility after the removal of the inducer germling from the tissue. If a germling, either the same or a different germling, was left on the host cell continuously, then enhanced inaccessibility did not occur. Rather, susceptibility occurred. These results suggest that the E. graminis germling releases a material that suppresses inaccessibility. We refer to this material as a suppressor. Thus, the results indicate that E. graminis conidia release an elicitor that enhances inaccessibility of barley cells and that they also release a suppressor that prevents enhanced inaccessibility in the barley cell.     

Microscopical studies of the infection of gerbara flowers byBotrytis cinerea

The formation of lesions on ray florets of gerbera flowers caused by single conidia ofBotrytis cinerea was studied in two cultivars infected by two isolates of the pathogen. No differences in reaction after inoculation with conidia of either isolate were seen on either cultivar. The conidia produced usually one germ tube not longer than 10 m, but conidia with five germ tubes were also seen. Direct penetration of germ tubes through the upper cuticle of ray florets was observed. No appressoria or other specialised structures were observed before penetration, and degradation of the cuticle did not occur. Germination of conidia and subsequent flower infection was dependent on the availability of free water, but not on the addition of external nutrients.Between 18 to 25°C, fungal development usually stopped after cuticle penetration, two to four cells around the site of penetration becoming necrotic. This number did not increase when inoculated flowers were subsequently placed at 4°C, conditions conductive for the formation of spreading lesions. When flowers were incubated constantly at 4°C, lesions became visible 3 days after inoculation as a group of 10 to 14 cells. Initially from a vesicle-like structure, mycelium spread subcuticularly or in the lumen of epidermal cells resulting in the death of 40 to 50 cells at 18 days after inoculation. Ungerminated conidia and conidial germlings which has not yet penetrated the cuticle did not cause any visible symptoms in underlying epidermal cells.     

Light and Scanning Electron Microscopy Studies on the Infection of Oriental Lily Leaves By Botrytis Elliptica

Light, scanning electron and fluorescent microscopy were used to observe the infection process of Botrytis elliptica on leaves of oriental lily (cv. Star Gazer). At 20 °C and 100% relative humidity, conidia germinated on both adaxial and abaxial foliar surfaces, but germ tubes failed to invade epidermal cells on the adaxial surface. On abaxial surfaces, short (< 20 m) swollen germ tube appressoria penetrated through stomatal openings (19%), through the epidermis near guard cells (52%), or directly through epidermal cells (29%). Esterase activity was detected on germ tubes and conidia after 6 h of incubation, and deformation of the cuticle on abaxial surfaces of lily was observed surrounding infection sites. By 3 h after inoculation, almost 70% of the conidia had germinated, but no penetration was observed. At 6 h after inoculation, almost one-third of germinated conidia had penetrated epidermal cells, and water-soaked lesions were associated with 20% of the penetrations. By 9 h after inoculation, approximately 60% of the germinated conidia had penetrated plant tissues, and water-soaked lesions were associated with 60% of the infections. Fluorescent microscopy with a specific fungal stain allowed assessment of successful infection and visualization of sub-epidermal hyphae. We conclude that penetration of abaxial foliar surfaces of oriental lilies by B. elliptica occurs via short swollen germ tube appressoria mostly near stomata.     

The influence of the primary germ tube on infection of barley by Erysiphe graminis f. sp. hordei

In each of three separate genotype combinations of Erysiphe graminis f. sp. hordei and barley the probability of infection success by an appressorium was reduced by close proximity to a primary germ tube interacting with the same host cell, whether this germ tube was produced by the same or another conidium. Infection success of appressoria was unaffected by events in an adjacent cell, whether associated with other conidia or with the primary germ tube of the same conidium. This interaction between germ tubes and underlying host cells should be considered when mechanisms of resistance to E. graminis f. sp. hordei are investigated.     

Extracellular matrix of <Emphasis Type="Italic">Magnaporthe oryzae</Emphasis> may have a role in host adhesion during fungal penetration and is digested by matrix metalloproteinases

Spores and infection structures such as germ tubes and appressoria of Magnaporthe oryzae, the fungus causing blast disease of wheat, produced an extracellular matrix (ECM) on the surfaces of host leaves during fungal differentiation. The chemical components and function of the ECM were studied to understand the pathological roles using two immunological techniques and ECM-digesting enzymes. The ECM was characterized by fibrous and amorphous materials, located in the spaces between fungal cell walls and plant cuticles. Immunohistochemical and immunoelectron microscopy suggested that ECM includes components positively reacted with antibodies of four animal cell adhesion factors (collagen VI, vitronectin, fibronectin and laminin) and an animal integrin α3. ECM, incubated on a cellulose membrane, was rapidly digested by matrix metalloproteinases (collagenase and gelatinase B), resulting in the detachment of most infection structures from membrane surfaces. Both ultrastructural observation and immunological responses showed that more ECM was located at the appressoria than at the spores and germ tubes. This result suggested that appressoria needed a powerful adhesion force for aggressive action of penetration pegs into plant cuticles. An erratum to this article can be found at     

Histopathology of the initial stages of the interaction between rose flowers and Botrytis cinerea

The early stages of the interaction between flowers of the cut rose cv. Sonia andBotrytis cinerea was investigated by scanning electron microscopy and light microscopy. Infection of petals by conidial germ tubes evoked a susceptible reaction. In contrast to general findings nutrient addition to the inoculum was not a prerequisite for this phenomenon. At the lower side of germ tube tips the cuticle was penetrated by infection pegs. Already at this early stage of the infection process, the infection sites were macroscopically visible as scattered white spots. After penetration, pegs enlarged to form infection hyphae, which invaded the periclinal wall of outer epidermal cells. At those sites, the petals formed outgrowths of variable appearance at their abaxial side. Thee outgrowths consisted of remanants of collapsed epidermal cells and of infection hyphae. Subsequent intra- and intercellular growth of hyphae led to a collapse of epidermal and mesophyll cells. The symptoms described generally developed within 24 h. After subsequent incubation the lesions became necrotic. Eventually, the necrosis would spread leading to the death of whole petals.     

菜豆锈菌侵染过程的显微和超微观察

 本文报道了通过微分干涉衬显微镜、荧光显微镜及扫描电镜和透射电镜所观察到的菜豆锈菌的侵入和扩展过程。菜豆锈菌夏孢子萌发多产生1个芽管,偶尔也产生双芽管。芽管以气孔侵入为主,也可从表皮直接侵入。侵入前形成或不形成明显的附着胞。气孔侵入的芽管首先在气孔腔内形成气孔下囊,再进一步分化出圆形的膨大体,由膨大体产生1~2支初生菌丝。初生菌丝与叶肉细胞壁接触后分化出吸器母细胞,吸器母细胞进入叶肉细胞内部形成吸器。初生侵染菌丝在产生吸器母细胞的部位的后部产生分枝,形成次生侵染菌丝在叶肉细胞间蔓延。     

Histological and ultrastructural characterization of the leaf infection events of Colletotrichum fructicola on Malus domestica ‘Gala’

Glomerella leaf spot (GLS), characterized by black necrotic spots and severe defoliation, is a destructive foliar disease of apple. Widely grown cultivars such as Gala and Golden Delicious are highly susceptible to GLS. Currently, the infection biology of the causal pathogen, Colletotrichum fructicola, on apple leaves is unclear. In the present study, the penetration and colonization processes of C. fructicola were characterized on apple (cv. Gala) leaves using light and transmission electron microscopy. C. fructicola conidia produced germ tubes 4 hours post-inoculation (hpi) and appressoria at 8 hpi. In melanized appressoria, funnel-shaped appressorial cones formed around the penetration pore. At 12 hpi, C. fructicola produced secondary conidia. After penetration, C. fructicola began to develop infection vesicles at 36 hpi. At 48 hpi, the primary hyphae of C. fructicola were produced from infection vesicles within host epidermal cells; the host epidermal cell plasma membrane remained intact, indicating a biotrophic association. Subsequently, secondary hyphae penetrated epidermal cells and destroyed cell components, initiating necrotrophic colonization. C. fructicola also produced biotrophic subcuticular infection vesicles and hyphae. Together, these results demonstrate that C. fructicola forms special infection structures and colonizes apple leaves in a hemibiotrophic manner, involving intracellular as well as subcuticular colonization strategies. Detailed characterization of the infection process of C. fructicola on apple leaves will assist in the development of disease management strategies and provide a foundation for studies of the molecular mechanism of the C. fructicola–apple leaf interaction.     

Infection of wheat spikes by <Emphasis Type="Italic">Fusarium avenaceum</Emphasis> and alterations of cell wall components in the infected tissue

The infection process of Fusarium avenaceum on wheat spikes and the alteration of cell wall components in the infected host tissue were examined by means of electron microscopy and cytochemical labelling techniques following spray inoculation at growth stage (GS) 65 (mid-flowering). Macroconidia of the pathogen germinated with one to several germ-tubes 6–12 h after inoculation (hai) on host surfaces. The germ-tubes did not penetrate host tissues immediately, but extended and branched on the host surfaces. Hyphal growth on abaxial surfaces of the glume, lemma and palea was scanty 3–4 days after inoculation (dai) and no direct penetration of the outer surfaces of the spikelet was observed. Dense mycelial networks formed on the inner surfaces of the glume, lemma, palea and ovary 36–48 hai. Penetration of the host tissue occurred 36 hai by infection hyphae only on the adaxial surfaces of the glume, lemma, palea and upper part of ovary. The fungus penetrated the cuticle and hyphae extended subcuticularly or between the epidermal wall layers. The subcuticular growth phase was followed by penetration of the epidermal wall, and hyphae spread rapidly inter- and intracellularly in the glume, lemma, palea and ovary. During this necrotrophic colonization phase of the wheat spike, a series of alterations occurred in the host tissues, such as degeneration of cytoplasm and cell organelles, collapse of host cells and disintegration of host cell walls. Immunogold labelling techniques showed that cell walls of spike tissues contained reduced amounts of cellulose, xylan and pectin near intercellular hyphae or infection pegs compared to walls of healthy host tissues. These studies suggest that cell wall degrading enzymes produced by F. avenaceum facilitated rapid colonization of wheat spikes. The different penetration properties of abaxial and adaxial surfaces of the spikelet tissues as well as the two distinct colonization strategies of host tissues by F. avenaceum are discussed. The penetration and colonization behaviour of F. avenaceum in wheat spikelets resembled that of F. culmorum and F. graminearum, although mycotoxins produced by F. avenaceum differed from those of the latter two Fusarium species.     

Investigation of rice blast development in susceptible and resistant rice cultivars using a gfp‐expressing Magnaporthe oryzae isolate

In this study, an isolate of Magnaporthe oryzae expressing the green fluorescent protein gene (gfp) was used to monitor early events in the interaction of M. oryzae with resistant rice cultivars harbouring a blast resistance (R) gene. In the resistant cultivars Saber and TeQing (Pib gene), M. oryzae spores germinated normally on the leaf surface but produced morphologically abnormal germ tubes. Germling growth and development were markedly and adversely affected in leaves of these resistant cultivars. Penetration of host cells was never seen, supporting the idea that disruption of germling development on the leaf surface might be one of the resistance mechanisms associated with Pib function. Thus, this particular R gene appeared to function in the absence of host penetration by the fungal pathogen. Confocal laser scanning microscopy of M. oryzae‐infected susceptible rice cultivars showed the dimorphic growth pattern that is typically observed during the biotrophic and necrotrophic stages of leaf colonization in susceptible cultivars. The suitability of the gfp‐expressing M. oryzae isolate for further research on R‐gene function and identification of resistant genotypes in rice germplasm collections is discussed.     

Uredospore germination and germ tube penetration of Puccinia striiformis in seedling leaves of resistant and susceptible wheat varieties

Differences were observed in spore germination and germ tube penetration of race 60 ofPuccinia striiformis on some wheat varieties. Of the five wheat varieties investigatedTriticum spelta var.album was the most resistant, strongly inhibiting spore germination and retarding germ tube penetration. On the hexaploid wheat varieties the germ tube penetrated through the stomata, whereas on the tetraploid varieties it did so at the junction of two epidermal cells. There was no correlation between density or length of hairs on the leaves and the rate of spore germination and germ tube penetration.Samenvatting Waarnemingen zijn verricht over sporekieming en kiembuispenetratie van fysio 60 vanPuccinia striiformis op vijf tarwevariëteiten. Ten opzichte van de meest vatbare variëteitTriticum dicoccum var.tricoccum gaven de overige variëteiten een duidelijke remming van sporekieming en resistentie tegen kiembuispenetratie te zien (Tabel 1),Triticum spelta var.album toonde zich in beide opzichten het meest resistent. De wijze van penetratie van de kiembuizen verschilde al naar de genetische samenstelling van de onderzochte tarwevariëteiten. Op de drie hexaploïde variëteiten bleek de kiembuis via het huidmondje binnen te dringen (Fig. 2), maar op de twee tetraploïde variëteiten via de wand tussen twee epidermiscellen (Fig. 1).De kiem- en penetratiepercentages bleken niet gecorreleerd te zijn aan dichtheid en lengte van de haren op het blad (Tabel 2).     

Responses of Erysiphe graminis germlings to contact with artificial and host surfaces

Germling development by Erysiphe graminis f. sp. hordei was compared between conidia held in a simulated air-borne state on microthreads constructed from safety-line threads produced by orb--weaving spiders (Araneus diadematus), and conidia inoculated onto glass, agar, or living or dead barley coleoptile epidermes. Suspended conidia germinated but generally produced only multiple short germ tubes. Conidia on living or dead coleoptiles, bathed from beneath with 0.01 Ca(NO₃)₂ solution, generally produced one short germ tube and a second germ tube which elongated and formed a normal appressorium. On glass and agar, multiple short germ tubes were sometimes formed but long germ tubes were formed less frequently than on host epidermis. When conidia with short germ tubes were transferred from microthreads to coleoptiles, they produced a long germ tube which differentiated an appressorium. Conidia with a single short germ tube were also transferred from microthreads so that only the tip of the short germ tube was in contact with a leaf epidermal strip layed on agar, whilst the conidium rested on the agar. Long germ tubes were formed more frequently by such conidia than by controls which had no contact with the leaf epidermis. This suggested that a stimulus causing elongation of the second tube was perceived through the short germ tube in contact with the epidermal strip. Where long germ tubes made contact with the epidermal strip, normal appressoria were formed more frequently than where the long tube made contact with the agar surface alone. The results indicate that germlings develop through distinct stages in response to particular stimuli.