Infection process of Colletotrichum dematium on mulberry leaves: An unusual method of sporulation

Abstract

The pre-penetration and infection process of Colletotrichum dematium on mulberry leaf was investigated by scanning electron microscope. Conidia produced on germination appressoria directly or at the end of short germ tubes. Appressoria were formed mostly over cuticle, but sometimes over stomata also. At 72 h post-inoculation, an extensive network of sub-cuticular runner hyphae (RH) was produced. The RH were traceable by the cuticular bulgings on leaf surface. The RH emerged to leaf surface through ruptured cuticle to form secondary infection hyphae (SIH). The SIH re-entered the leaf tissue by sending penetration branches through stomata. Conidia were formed singly on short conidiophores from the RH and SIH, at short intervals. The conidia developed on RH were exposed to leaf surface through ruptured cuticle. Some times conidia were released through stomata also. The RH and SIH had thick knots from which hyphal branches and conidia were developed. Definite acervuli were not developed.     

Scanning Electron Microscopy of the Infection Process of Cercospora henningsii on Cassava Leaves

Germination, penetration and sporulation of Cercospora henningsii (Allesch.) on cassava leaves were studied by scanning electron microscopy. Conidia started to germinate 9 h postinoculation producing one to two germ tubes. The germ tubes entered the leaf tissue through the abaxial surface by direct penetration of the epidermis without forming appressoria. The cassava leaf is characterized by its papillose epidermis on the abaxial surface. The penetrations occurred at smooth areas of the leaf epidermis between the papillae. The germ tubes did not enter stomata even when they passed over stomatal openings. Leaf spots started to appear 9 days after the inoculation (dpi), and the emergence of conidia occurred 14 dpi. The symptoms appeared first on the abaxial leaf surface, followed 2 days later on the adaxial. Conidia emerged in clusters through ruptured epidermis on both sides of the leaves. Conidia emerged also through the epidermal papillae and the leaf veins. Even though small groups of conidia emerged through stomata also, emergence through stomata appeared to be random rather than a preferred route. Each conidium was born on a short conidiophore with a swollen base.     

Mode of Infection of Phyllosticta maculata on Banana as Revealed by Scanning Electron Microscopy

The initial infection stages of Phyllosticta maculata on banana were studied using scanning electron microscopy. Conidial germination on the banana leaf surface commenced within 3 h postinoculation to produce a long and slender germ tube. The hyphae developed secondary branches and mostly grew randomly across the leaf surface. Appressoria were formed at the apex of the germ tubes within 18 h postinoculation and were variable in shape. A layer of an extracellular matrix surrounded the appressoria at the pathogen–host interface. On the fruit surface, conidia germinated to produce predominantly swollen germ tubes which functioned as lateral appressoria together with some slender ones. These germ tubes were formed within 3 h postinoculation. There was no stomatal penetration apparent on the leaf; instead, direct penetration through the cuticle with and without the formation of appressoria was observed. Cuticular degradation on the leaf surface was evident with a circular, darkened area around the point of penetration by hyphae or appressoria. The significant role of pycnidia and conidia in the epidemiology of the disease was further demonstrated in naturally infected leaf samples.     

Microscopic Observation on the Development of Puccinia striiformis in the Spike and Stem of Wheat

The majority of germ tubes of the pathotype CYR32 of Puccinia striiformis f.sp. tritici formed on the surface of spike organs of the susceptible wheat cv. Suwon 11 penetrated through the stomatal pore, only a few germ tubes formed small appressoria over the stomata. In the lemma, palea and glume, the stripe rust fungus spread between the parenchyma cells close to the inner epidermal layer, but the fungus did not develop between the thick‐walled cells near the outer epidermal layer of these organs. In the awn and stem, spread of the stripe rust was confined to the intercellular spaces of the chlorophyll parenchyma, beneath the invaded stomatal pore of the epidermis and the urediniospores to be released disrupted the epidermis. In the caryopsis, the spread of hyphae was restricted to the intercellular spaces of the pericarp cells.     

Abnormal germling development by brown rust and powdery mildew on cer barley mutants

The barley leaf rust fungus forms appressoria over host leaf stomata and penetrates via the stomatal pore. High levels of avoidance to leaf rust fungi have been described in some wild accessions of Hordeum species where a prominent wax layer on the stomata inhibits triggering of fungal appressorium differentiation. Leaf rust avoidance has not yet been found in H. vulgare. Since cuticular leaf waxes are implicated in the avoidance trait, we screened 27 eceriferum (cer) mutant lines of H. vulgare for avoidance to barley leaf rust. These mutations affect leaf waxes. Reduction in numbers of germ tubes forming appressoria over stomata was found in some lines, but the greatest reduction (ca 30%) was less than previously found in wild barley spp. or in an accession of H. chilense used here as a check. In one line (cer-zh654), avoidance was due to a combination of factors. Firstly, fewer germ tubes oriented towards stomata and so failed to contact them. Secondly, some germ tubes that encountered stomata did not form appressoria but over-grew them. In this line, therefore, the fungus tended to fail both to locate and to respond to stomata. The appressoria of barley powdery mildew form on leaf epidermal cells that they penetrate directly. On certain cer lines, a proportion of germlings of the barley powdery mildew fungus developed abnormally, suggesting that germlings failed to recognise and/or respond to the leaf surface waxes on these mutants.     

落叶松-杨栅锈菌在感病杨树寄主上发育过程的组织学研究

采用荧光染色技术、光学显微镜和电子显微镜技术,系统研究了落叶松-杨栅锈菌在感病杨树叶片上的发育过程。结果表明,在侵染前期（接种12h以内）,锈菌夏孢子在杨树叶片上萌发,利用芽管或附着胞穿透叶表气孔后形成气孔下囊,进而在胞间产生侵染菌丝。进入活体营养生长阶段（接种后24-96h）,锈菌不断产生大量吸器来满足营养需求的同时,侵染菌丝在叶肉细胞间隙蔓延分枝生长至形成菌落结构。最终在产孢阶段（接种120h之后）产孢菌丝分化形成的夏孢子在表皮下聚集成堆,待成熟后突破表皮显露出来。     

Conidia of the tomato powdery mildew <Emphasis Type="Italic">Oidium neolycopersici</Emphasis> initiate germ tubes at a predetermined site

The emergence of germ tubes from the conidia of powdery mildew fungi is the first morphological event of the infection process, preceding appressoria formation, peg penetration and primary haustoria formation. Germination patterns of the conidia are specific in powdery mildew fungi and therefore considered useful for identification. In the present study, we examined conidial germination of the tomato powdery mildew Oidium neolycopersici KTP-01 in order to clarify whether germ tube emergence site in KTP-01 conidia is determined by the first contact of the conidia to leaves (as found for the conidia of barley powdery mildew), or alternatively is predetermined and is unrelated to contact stimulus. Highly germinative conidia of KTP-01 were collected from conidial pseudochains on conidiophores in colonies on tomato leaves using two methods involving an electrostatic spore attractor and a blower. In the electrostatic spore attraction method, the conidia were attracted to the electrified insulator probe of the spore collector—this being the first contact stimulus for the conidia. In addition, the blowing method was used as a model of natural infection; pseudochain conidia were transferred to detached leaves by air (1 m/s) from a blower. Thus, landing on the leaves was the first contact for the conidia. Furthermore, conidia were also blown onto an artificial membrane (Parafilm-coated glass slides forming a hydrophobic surface) or solidified agar plates in Petri dishes (hydrophilic surface). Eventually, almost all conidia on the probe and on tomato leaves or artificial hydrophobic and hydrophilic surfaces synchronously germinated within 6 h of incubation, indicating that the first contact of the conidia with any of the aforementioned substrata was an effective germination induction signal. Germ tube emergence sites were exclusively subterminal on the conidia. Moreover, the germ tubes emerged without any relation to the sites touched first on the conidia. Thus, the present study strongly indicates that conidia of O. neolycopersici produce germ tubes at a predetermined site.     

Surface Ultrastructural Studies on the Germination,Penetration and Conidial Development of Aspergillus Flavus Link : Fries Infecting Silkworm,Bombyx Mori Linn

Aspergillosis is a common disease of the silkworm Bombyx mori Linn., caused by an insect mycopathogen Aspergillus flavus Link:Fries. The present study reveals the germination, penetration and conidial development of A. flavus on the larval integument of B. mori under SEM. Four different strains (NB18, KA, NB4D2 and NB7) of B. mori was surface inoculated with ca. of 1 x 10(6) conidia/ml. Each conidium germinated on the cuticle approximately 6 h after inoculation, forming a humpy or suctorial appressoria within 24 h. The hyphae which entered into haemocoel 2 day post-inoculation, grew and multiplied extensively, forming a mycelial complex, causing death of the host larva in about 4-5 days. This occurred with minimal breakdown of the internal tissues. Death of the host was followed by ramification of the fungus through the mesodermal and epidermal tissues, leading to larval mummification about 5-6 days after inoculation. Extensive fungal growths on the entire larval body followed, consisting of aerial hyphae, which developed branched conidiophores. The aerial hyphae with abundant conidiophores formed a confluent yellowish green fungal mat over the entire larval body in 6-7 days of post-inoculation. The tip of each emerging conidiophores gradually dilated and developed to become a bulbous head known as the vesicle. A large number of conidiogenous cells were produced over the entire surface of vesicle, which later developed into finger-like projections termed as sterigmata or phialides. The phialides matured within 2 days after the aerial hyphae emerged as evidenced by chains of conidia at their tips. The conidia were globose with externally roughened walls. The life cycle of the fungus on B. mori was completed in six to seven days.     

The morphology of the imperfect states of powdery mildews (Erysiphaceae)

In contrast to popular belief, a rich variety of morphological characteristics exists in the imperfect states of powdery mildews. Because it has been generally assumed that species cannot be distinguished by their appressoria, haustoria, conidiophores, conidia, fibrosin bodies, and conidial germ tubes, their morphology has received little attention and several older publications have even been forgotten. As with the perfect states, few species can be recognized by one characteristic of the imperfect state alone but many species can be identified when a combination of several characteristics is used. Important characteristics are the location of the mycelium, the production of conidia singly or in chains, the presence or absence of conspicuous fibrosin bodies, the appressoria, the size and shape of the conidia, and the position and type of their germ tubes. Many species are associated with particular families or genera of plants and therefore these are included in a key to identify 131 species of powdery mildew. This key shows how much and especially how little is known about many species. It is hoped that this review will stimulate study of the morphology of the imperfect states of numerous species. Consideration of both the perfect and the imperfect state should result in a more natural classification of several genera, for exampleUncinula andErysiphe which at present include both species which produce conidia in long chains and those which produce conidia singly. It appears that there are two lines of development of the imperfect states. One is characterized by lobed appressoria and conidiophores which produce conidia singly. The other is characterized by more or less rounded, unlobed appressoria and conidiophores which produce conidia in chains. A better knowledge of all the different imperfect states may provide more information regarding the evolution of powdery mildews.     

Fine Structure of Germinating Botrytis fabae Sardina Conidia

The fine structure of germinating Botrytis fabae conidia wasstudied using both chemically stained sections and freeze-etchedreplicas. Germinating conidia have fewer organelles than restingconidia, glycogen is absent, and prevacuoles have disappeared.Endoplasmic reticulum which occurs as small strands close tothe cell wall of resting conidia becomes, on germination, multiplesheets surrounding the nuclei. A cross wall is formed at thebase of the germ tube soon after germination commences. Thenew wall material which appears to be continuous with this septalwall is produced, at least partly, from a new wall layer laiddown in the centre of the old conidial wall. An apical corpuscleis present at the apex of young germ tubes. Freeze-etched preparationsshow the formation of lomasomes by the passage of vesicles throughthe plasmalemma of conidia and germ tubes. In young hyphae lomasomescontain a complex arrangement of branching tubules. Some ofthe particles on the outer plasmalemma of young hyphae are arrangedin a geometrical pattern.     

Mycosporine-alanine: A self-inhibitor of germination from the conidial mucilage ofColletotrichum graminicola

On soft substrata conidia ofColletotrichum graminicola germinated by the formation of germ tubes, whereas on hard, hydrophobic substrata they germinated by the formation of appressoria. Regardless of the substratum, conidia did not germinate at high conidium concentrations, suggesting the presence of an endogenous self-inhibitor. Bioassays to test for the inhibition of germination demonstrated the presence of a low-molecular-weight self-inhibitor in the mucilage which surrounds conidia as they are produced in acervuli. The inhibitory material exhibited an absorbance maximum of 310 nm, indicating that it is a member of the mycosporine family of compounds. The inhibitor was purified to homogeneity and shown by mass spectrometric analysis to have a molecular mass consistent with the structure corresponding to mycosporine-alanine.     

Infection of Subterranean Clover (Trifolium subterraneum) by Kabatiella caulivora

Kabatiella caulivora is a serious pathogen of clover ( Trifolium ) spp. Subterranean clover ( T. subterraneum ) cv. Woogenellup was inoculated with K. caulivora , to study the attachment and germination of conidia, germ-tube penetration of the plant surface, and histochemistry and ultrastructure of changes in the host associated with lesion development. The foliar architecture caused the conidia to concentrate at the base of leaflets and on the petiolules (between the leaflets and petioles). Epidermal cells immediately beneath conidia and, occasionally, also adjacent cells developed a yellow-brown discoloration 1 day post-inoculation. Penetration appeared to be directly through the cuticle, characterized by constricted hyphae at the point of entry. No appressoria were observed. In leaves, invasion was restricted to the area proximal to the petiolule and leaf mid-rib. In petioles and petiolules, the hyphae initially remained between the epidermal cells and first layer of mesophyll cells before moving intercellularly through the mesophyll tissue towards phloem tissues. The cuticle was occasionally degraded in petiole and petiolule infections, the loss of epidermal and mesophyll cell wall components was detected, and chloroplasts and starch grains were disrupted. Plants developed macroscopic symptoms 10–11 days post-inoculation with necrotic lesions occurring on leaves, petioles and petiolules. Sporulation occurred approximately 15–18 days post-inoculation when affected plants collapsed. This information may be useful for breeding programmes aimed at selecting varieties with improved resistance to the clover scorch disease.     

Role of topography sensing for infection-structure differentiation in cereal rust fungi

Over 90% of the germ tubes of Puccinia graminis tritici (wheat stem rust) and Puccinia hordei (barley brown rust) differentiate appressoria on encountering stomata.There has been controversy as to the role of host topographical signals in the highly precise and efficient induction of these infection structures over stomata by cereal rusts. In the present study, polystyrene replicas of microfabricated silicon wafers, bearing precise microtopographies of defined dimensions, were used to investigate the influence of ridge spacing and height on infection-structure induction by P. graminis tritici and P. hordei. It was found that artificial topographical signals alone can induce a reproducibly high percentage (83–86%) of germ tubes to differentiate infection structures. Multiple, closely spaced (1.5 μm) ridges which were 2.0 μm high provided the most inductive topography. Differentiation on flat surfaces and over single ridges was < 4%. Appressorium induction commonly initiated a cascade of differentiation events involving the formation of infection pegs, vesicles, infection hyphae, and occasionally haustorial mother cells. It is suggested that the close spacing of cell junctions associated with the dumbbell-shaped guard cells of cereal stomatal complexes provide inductive signals for infection-structure formation by cereal rusts in vivo. Received: 17 October 1996 / Accepted: 5 December 1996     

Stomatal lock-open, a consequence of epidermal cell death, follows transient suppression of stomatal opening in barley attacked by Blumeria graminis

Blumeria graminis f.sp. hordei (Bgh) attack disrupted stomatal behaviour, and hence leaf water conductance (g(l)), in barley genotypes Pallas and Ris?-S (susceptible), P01 (with Mla1 conditioning a hypersensitive response; HR), and P22 and Ris?-R (with mlo5 conditioning papilla-based penetration resistance). Inoculation caused some stomatal closure well before the fungus attempted infection. Coinciding with epidermal cell penetration, stomatal opening in light was also impeded, although stomata of susceptible and mlo5 lines remained largely able to close in darkness. Following infection, in susceptible lines stomata closed in darkness but opening in light was persistently impeded. In Ris?-R, stomata recovered nearly complete function by approximately 30 h after inoculation, i.e. after penetration resistance was accomplished. In P01, stomata became locked open and unable to close in darkness shortly after epidermal cells died due to HR. In the P22 background, mlo5 penetration resistance was often followed by consequential death of attacked cells, and here too stomata became locked open, but not until approximately 24 h after pathogen attack had ceased. The influence of epidermal cell death was localized, and only affected stomata within one or two cells distance. These stomata were unable to close not only in darkness but also after application of abscisic acid and in wilted leaves suffering drought. Thus, resistance to Bgh based on HR or associated with cell death may have previously unsuspected negative consequences for the physiological health of apparently 'disease-free' plants. The results are discussed in relation to the control of stomatal aperture in barley by epidermal cells.     

Histological and cytological studies of plant infection by <Emphasis Type="Italic">Erysiphe euonymi</Emphasis>-<Emphasis Type="Italic">japonici</Emphasis>

Powdery mildew caused by Erysiphe euonymi-japonici (Eej) is an increasingly serious fungal disease on Euonymus japonicus that is an important ornamental plant. However, little is currently known about infection and pathogenesis of Eej on E. japonicus. Here, we report plant infection by Eej at the histological and cytological levels. Eej caused severe disease symptoms with white and snow-like colonies on leaf surfaces of E. japonicus. Microscopic observations were conducted continuously to define infection process of Eej on E. japonicus. Eej conidia germinated to produce appressorial germ tubes on leaf surfaces and formed irregular haustoria in plant epidermal cells at 6 h post-inoculation (hpi) and 12 hpi, respectively. After uptaking nutrients from host cells by haustoria, Eej formed numerous hyphae and extensive colonization on leaf surfaces at 96 hpi and finally produced abundant conidiophores and new conidia on leaf surfaces at 168 hpi. In addition, there was consistently a single nucleus in different Eej infection structures and haustorial development could be divided into three major stages, including formation of penetration peg, formation of haustorial neck and initial haustorium, and maturation of haustorium. These results provide useful information for further determination of Eej pathogenesis and finally controlling the disease.     

Symptoms and Prepenetration Events Associated with the Infection of Common Bean by the Anamorph and Teleomorph of Glomerella cingulata f.sp. phaseoli

Glomerella cingulata f.sp. phaseoli and Colletotrichum lindemuthianum are the teleomorph and anamorph, respectively, of the pathogen causing anthracnose in common bean. The mechanisms relating to the sexual reproduction of this plant pathogen are still unclear, as are the infection structures involved and the symptoms produced. In the present study, bean plants were inoculated with ascospores and conidia, and the events taking place within the following 120 h were investigated using light microscopy and scanning electron microscopy. The symptoms exhibited by plants inoculated with the ascospores were milder than in those inoculated with conidia. Microscopy revealed that most of ascospores produced germ tubes and appressoria at an early stage (24 h after inoculation). From 48 h onwards, the formation of hyphae and the production of germ tubes and appressoria were great. In contrast, infections originating from conidia developed more slowly, and at 24 and 48 h, many non‐germinated conidia were present, whereas only few conidia developed germ tubes and appressoria. Ascospore germination and appressorium formation were similar on both resistant and susceptible cultivars. Hence, the symptoms and the temporal sequence of events associated with the infection of bean plants by the two fungal forms differed, although the structures produced were similar. This is the fist report comparing symptoms and prepenetration events between anamorph and teleomorph of G. cingulata f.sp. phaseoli in common bean.     

Infection Process of Plectosporium tabacinum on False Cleavers (Galium spurium)

A native fungus, Plectosporium tabacinum (van Beyma) M. E. Palm, W. Gams et Nirenberg, has potential as a bioherbicide for the control of both herbicide-resistant and herbicide-susceptible false cleavers. Limited information is available on the infection process of P. tabacinum. P. tabacinum spore distribution pattern, germination, penetration, and colonization on false cleavers leaves were examined using confocal, light, and scanning electron microscopy. The results demonstrated that conidia were distributed over the entire surface of leaves and cotyledons. More than 90% of the conidia germinated on the leaf surface 6-8 h after inoculation. Penetration of the leaf epidermis by conidia started 8-10 h after inoculation. Histological observation showed that no appressoria were formed by P. tabacinum, but its hyphae produced appressed club-like structures that penetrated the cuticle and epidermal layers. No stomata or other natural openings were observed on the upper leaf surface of false cleavers seedlings. Penetration occurs directly on epidermal cells with more frequent intercellular penetrations. Hyphal penetration was visualized at a depth of 30 and 40 üm after 8 and 16 h of incubation, respectively. Secondary hyphae colonized mesophyll cells 16 h after inoculation. Even spore distribution, short spore germination time, club-like infection structure formation, direct penetration, quick colonization, and mucous secretion on false cleavers leaves may contribute to the kill of false cleavers by P. tabacinum. Slow spore germination and germ tube growth, low spore germination numbers, and no infection structure formation on Brassica napus leaves may be factors affecting the host selectivity of P. tabacinum.     

A new species ofScopulariopsis from soil

A new species ofScopularoipsis described from soil is characterized by the conidiophores, branches, and sporogenous cells being distinctly swollen. The conidia have a well-marked germ slit running more or less longitudinally down the spore. TheScopulariopsis state ofMicroascus doguetii is re-examined and evidence is presented which indicates that the striations on the walls of the spores are germ slits.     

Production and Dispersal of Conidia of Peronospora pisi on Pea Leaves

The number of crops of conidiophores and conidia of Peronospora pisi produced on a single lesion on leaflets of pea (Pisum sativum L.) was determined both in vitro and in vivo by artificial inoculation. Only two crops were produced in vitro whereas five crops were discerned under in vivo conditions. There was a marked difference in the size of conidiophores and conidia formed under the above two conditions. Temperature had a marked effect on the penetration of pea leaflets by conidial germ tubes of P. pisi. The pathogen penetrated the leaf tissues at 12-14°C in 8.0-8.5 h whereas more time (9.5-16.5 h) was required for penetration at higher temperatures (15-18°C). Use of systemic fungicides has been suggested to control downy mildew of pea because of its capacity to produce more than one crop of propagules on the same lesion. Maximum spore discharge in P. pisi was observed from 9.00 to 15.00 hours. However, presence of light, high temperature, lack of moisture on leaf surface and wind velocity affected spore discharge.     

Development of special hyphal branches of Phyllactinia corylea on host and non-host surfaces

Hyphae of Phyllactinia corylea produce two kinds of special branches on the host surface: adhesion bodies which serve as fungal attachment and stomatopodia which enter the leaf through stomata. Conidial germination on host and non-host surfaces was examined with a scanning electron microscope to explain the stimuli responsible for development of the special branches, and the involvement of host recognition in the process. Conidia germinated within 4 h on host and non-host surfaces, but on non-host surfaces the emergence of the germ tube was not always directed towards the substratum. Adhesion bodies were formed from the tips of germ tubes at the first contact point on host and non-host surfaces. Development of stomatopodia was more specific and they were formed precisely over stomata on the host surface. Stomatopodia-like structures were occasionally formed over finely ridged leaf veins on the host surface and over some fine scratches on synthetic surfaces. The experiments showed that while conidial germination and development of adhesion bodies are in response to contact stimuli, the development of stomatopodia is a response to precise topographical signals, and the directional emergence and attached growth of germ tubes involve host recognition.This revised version was published online in October 2005 with corrections to the Cover Date.