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.     

Conidia of <Emphasis Type="Italic">Erysiphe trifoliorum</Emphasis> attempt penetration twice during a two-step germination process on non-host barley leaves and an artificial hydrophobic surface

In the present study, using a high-fidelity digital microscope, we observed the sequence of appressorial development on the germ tubes of a powdery mildew fungus isolated from red clover leaves. Based on its morphological characteristics and rDNA internal transcribed spacer (ITS) sequences, the fungus was identified as Erysiphe trifoliorum, and one of its isolates, designated as KRCP-4N, was used in this work. The conidial germination of isolate KRCP-4N was studied on host (red clover) and non-host (barley) leaves, as well as on an artificial hydrophobic membrane (Parafilm). More than 90% of conidia germinated synchronously and developed dichotomous appressoria (symmetrical double-headed appressoria) on all substrata used. On host leaves, all appressorium-forming conidia developed hyphae (colony-forming hyphae) from conidial bodies without extending germ tubes from the tips of the appressoria. On non-host leaves and on Parafilm-covered glass slides, however, all conidia extended germ tubes from one side of dichotomous appressoria (two-step germination). In addition to the dichotomous appressoria, we detected a few conidia that produced hooked appressoria and extended germ tubes from the tip of the appressorium. Penetration attempts by KRCP-4N conidia on barley leaves were impeded by papillae formed at penetration sites beneath these two types of appressorium. From these results, we conclude that the “two-step germination” of E. trifoliorum KRCP-4N conidia is the result of an unsuccessful penetration attempt, causing diversity in appressorial shape.     

A Method for Staining Infection Hyphae in Pine Leaves

Fungus-inoculated Pinus radiata leaves were fixed and then stained with periodic acid-Schiff reagent. Pieces of leaf with fungal material on the surface were removed. These pieces were stained in lactophenol cotton blue for a few minutes and then mounted in dilute lactophenol cotton blue. Microscopic examination of fungal material inside and outside the mounted leaf pieces revealed the following: conidia and germ tubes on the leaf surface were red, appressoria remained unstained, and infection hyphae within the leaf were stained blue. This differential staining method was particularly useful for distinguishing germ tubes from infection hyphae arising from appressoria.     

绿僵菌侵染小菜蛾体表过程的显微观察

采用扫描电镜研究了小菜蛾Plutella xylostella体表结构对绿僵菌入侵行为的影响及绿僵菌的侵染过程。结果表明: 绿僵菌孢子在小菜蛾体表萌发后可形成附着胞,寄主体表结构影响形成附着胞的快慢、多少及穿透体壁时芽管长度, 在平缓结构区和刺状结构区比嵴状结构区更易形成附着胞,且芽管较短。在所有结构区,LF68菌株穿透芽管均短于LD65菌株的芽管。接种后7 h,分生孢子在小菜蛾体表开始萌发,LF68与LD65菌株分别于接种后10 h和13 h出现侵染构造穿透体壁。     

Targeted destruction of fungal structures of Erysiphe trifoliorum on flat leaf surfaces of Marchantia polymorpha

In this study, we observed the germination behaviour of airborne conidia from powdery mildews that settle on thalloid surfaces. We inoculated thalli (flat, sheet‐like leaf tissues) and gemmae (small, flat, sheet‐like leaf tissues that propagate asexually via bud‐like structures) of the common liverwort (Marchantia polymorpha) with conidia from tomato powdery mildew (Oidium neolycopersici; KTP‐02) and red clover powdery mildew (Erysiphe trifoliorum; KRCP‐4N) and examined their germination and subsequent appressorium formation under a high‐fidelity digital microscope. Conidial bodies and germ tubes of the inoculated KRCP‐4N conidia were destroyed on both the thalli and gemmae. The destruction of these fungal structures was observed only for KRCP‐4N conidia inoculated onto M. polymorpha on both leaf surfaces. No differences in destruction of the KRCP‐4N fungal structures between thalli and gemmae were observed. At 4 h post‐inoculation, destruction of the germ tube tip was observed when it reached the gemmae leaf surface. At 6 h post‐inoculation, the conidial bodies and germ tubes were destroyed. In contrast, KTP‐02 conidia were not destroyed and formed normal, well‐lobed appressoria on the surface of M. polymorpha gemmae.     

Development of appressoria on conidial germ tubes of <Emphasis Type="Italic">Erysiphe</Emphasis> species

Modes of branching of appressoria on conidial germ tubes of 36 Erysiphe spp. were studied. Only unlobed appressoria, termed alobatus pattern, were seen in E. lonicerae, E. magnifica and E. symphoricarpi. Viewed from above with light or scanning electron microscopes, other species had ± irregular lobing, but from below in the plane of contact with the substrate successive dichotomous branchings at 120° were seen to produce a five-lobed appressorium within 6 h. Each division produced a temporarily dormant outward-facing lobe and an inward limb that continued growth and division to form the axis of curved, hooked, single- or double-headed symmetrical or asymmetrical structures in a helicoid cyme-like pattern. Outlines of extracellular material after removal of germinated conidia confirmed this manner of branching. After 36 h some lobes re-divided forming botryose or jigsaw patterns even extending with extra appressoria to form candelabra-like structures. Conidia developed only one true germ tube; rarely secondary unswollen tubes emerged from spare shoulders or ends. The same true germ tubes developed initially on host surfaces, where secondary tubes and/or extensions from appressorial lobes grew into colony-forming hyphae. Lobed appressoria of Neoerysphe and Phyllactinia also branched at 120°. Podosphaera xanthii exhibited a simpler branching pattern.     

The Infection Process of Pestalotiopsis Longisetula Leaf Spot on Strawberry Leaves

Pestalotia leaf spot, caused by the fungus Pestalotiopsis longisetula Guba, has become the major disease affecting strawberry production in Brazil. Strawberry seedlings with 4–5 leaves were inoculated with a conidial suspension of P. longisetula (2 × 10⁵ conidia/ml), and leaf samples were collected at 48, 72, 96 and 144 h after inoculation (hai) for observation in the scanning electron microscope. Conidia germinated within 48 hai. At 72 hai, conidia had formed very long germ tubes over the epidermal cells without any evidence of appressorial formation nor direct penetration. At 96 hai, fungal hyphae grew inter‐ and intracellularly in the lacunous parenchyma and also through tracheary elements. Pycnidia were first observed on the leaf surface at 96 hai. At 144 hai, conidia of P. longisetula were first liberated from the pycnidia. This study adds new information to better understand of the infection process of P. longisetula that may help in developing more effective disease control strategies.     

Development of Infection Structures by the Direct-Penetrating Soybean Rust Fungus (Phakopsora pachyrhizi Syd.) on Artificial Membranes

The development of infection structures by the directly infecting soybean rust fungus of different artificial membranes was followed by light and scanning electron microscopy. On water agar uredospores developed germ tubes without appressoria. On dialysis membranes more than 80% of the uredospores formed appressoria. With low frequencies (1–7%) also primary hyphae and/or penetration hyphae were present. When cellulose nitrate membrane filters with pore diameters ≤ 0.2 μm were used, uredospores germinated but showed a strongly reduced appressoria formation. Membranes with pores ≥ 0.1 μm allowed a development of infection structures similar to that on dialysis membranes. In experiments with paraffin oil incorporated into collodion membranes more than 90% of the uredospores formed appressoria, about 50% of the appressoria developed hyphae. Ungerminated spores and germ tubes always contained 2 nuclei. In fully developed appressoria 4 nuclei were present. Compared with stomata entering rust fungi appressoria formation by Phakopsora pachyrhizi occurred more frequently and seemed to be less dependent on specific stimuli. Moreover, in most cases only few of the appressoria formed penetration or primary hyphae. The induction of these structures seemed to be dependent on further unknown stimuli.     

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.     

MoCDC14 is important for septation during conidiation and appressorium formation in Magnaporthe oryzae

As a typical foliar pathogen, appressorium formation and penetration are critical steps in the infection cycle of Magnaporthe oryzae. Because appressorium formation and penetration are closely co‐regulated with the cell cycle, and Cdc14 phosphatases have an antagonistic relationship with cyclin‐dependent kinases (CDKs) on proteins related to mitotic exit and cytokinesis, in this study, we functionally characterized the MoCDC14 gene in M. oryzae. The Mocdc14 deletion mutant showed significantly reduced growth rate and conidiation. It was also defective in septum formation and nuclear distribution. Septation was irregular in Mocdc14 hyphae and hyphal compartments became multi‐nucleate. Mutant conidia often showed incomplete septa or lacked any septum. During appressorium formation, the septum delimiting appressoria from the rest of the germ tubes was often formed far away from the neck of the appressoria or not formed at all. Unlike the wild‐type, some mutant appressoria had more than one nucleus at 24 h. In addition to appressoria, melanization occurred on parts of the germ tubes and conidia, depending on the irregular position of the appressorium‐delimiting septum. The Mocdc14 mutant was also defective in glycogen degradation during appressorium formation and appressorial penetration of intact plant cells. Similar defects in septum formation, melanization and penetration were observed with appressorium‐like structures formed at hyphal tips in the Mocdc14 mutant. Often a long fragment of mutant hyphae was melanized, together with the apical appressorium‐like structures. These results indicate that MoCDC14 plays a critical role in septation, nuclear distribution and pathogenesis in M. oryzae, and correct septum formation during conidiogenesis and appressorium formation requires the MoCdc14 phosphatase.     

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.     

Morphological Alterations of Metarhizium anisopliae During Penetration of Boophilus microplus Ticks

Chronological histological alterations of Metarhizium anisopliae during interaction with the cattle tick Boophilus microplus were investigated by light and scanning electron microscopy. M. anisopliae invades B. microplus by a process which involves adhesion of conidia to the cuticle, conidia germination, formation of appressoria and penetration through the cuticle. Twenty-four hours post-infection conidia are adhered and germination starts on the surface of the tick. At this time, the conidia differentiate to form appressoria exerting mechanical pressure and trigger hydrolytic enzyme secretion leading to penetration. Massive penetration is observed 72 h post-inoculation, and after 96 h, the hyphae start to emerge from the cuticle surface to form conidia. The intense invasion of adjacent tissues by hyphae was observed by light microscopy, confirming the ability of M. anisopliae to produce significant morphological alterations in the cuticle, and its infective effectiveness in B. microplus.     

A method for staining infection hyphae in pine leaves.

Fungus-inoculated Pinus radiata leaves were fixed and then stained with periodic acid-Schiff reagent. Pieces of leaf with fungal material on the surface were removed. These pieces were stained in lactophenol cotton blue for a few minutes and then mounted in dilute lactophenol cotton blue. Microscopic examination of fungal material inside and outside the mounted leaf pieces revealed the following: condidia and germ tubes on the leaf surface were red, appressoria remained unstained, and infection hyphae within the leaf were stained blue. This differential staining method was particularly useful for distinguishing germ tubes from infection hyphae arising from appressoria.     

The infection process, sporulation and survival of Alternaria helianthi on sunflower

Electron microscopy was used to study the infection of sunflower leaves by Alternaria helianthi. Conidia germinated by producing one to many germ tubes which grew across the leaf surface before forming appressoria. The fungus directly penetrated its host through the cuticle and epidermis. Entry into the host through wounds and stomates was also observed. Extracellular sheaths were found to be associated with germ tubes and intercellular hyphae of A. helianthi. Conidiophores developed through collapsed stomates, from leaf veins, trichomes and also from mycelium growing across the host leaf surface. Microcylic conidia were produced directly from parent conidia under certain conditions. Studies using a volumetric spore trap showed that the airborne spore concentration followed a distinct periodicity with peaks occurring between 0900 and 1100 h each day. Laboratory studies showed that safflower, noogoora burr and bathurst burr could serve as alternative hosts for A. helianthi. The pathogen was readily isolated from sunflower crop debris from a diseased crop that had been harvested 1 yr earlier.     

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.     

Colonization of wheat seedling leaves by Fusarium species as observed in growth chambers: a role as inoculum for head blight infection?

Fusarium species involved in the Fusarium head blight complex in Western Europe were investigated for their potential to infect and colonize non-damaged wheat leaves and to produce conidia on senescing wheat leaves incubated at high relative humidity. Fusarium avenaceum, Fusarium culmorum, Fusarium graminearum, Fusarium poae and Fusarium tricinctum did not directly penetrate the leaf tissue after conidia germination on the leaf surface. Germ tubes grew on the host surface for 24–36 hr forming a mycelial network. After invading the host, some species formed runner hyphae between cell wall layers or underneath the cuticular layer. Macroscopic symptoms developed on leaves and stems from 7 d post inoculation. Inside leaf tissues, hyphae thickened in diameter and were both inter- and intra-cellular. Fusarium tricinctum formed sporophores which erupted through the leaf surface releasing numerous conidia. Incubation of senescing leaves at 100 % relative humidity for 48 hr resulted in sporulation of all Fusarium spp.     

An unusual method of development and sporulation of Colletotrichum gloeosporioides on castor leaf – an SEM account

Development and sporogenesis of Colletotrichum gloeosporioides on castor leaf differed from that on other known host plants. C. gloeosporioides had three kinds of hyphae on castor leaf: primary infection hyphae (PIH), runner hyphae (RH) and secondary infection hyphae (SIH). The PIH originated from conidia, grew on leaf surface and entered the leaf by direct penetration of the cuticle without forming appressoria. The RH were sub-cuticular hyphae, the track of which was traceable by the bulgings on the leaf surface, and the SIH were the hyphae that emerged to leaf surface from RH through the cuticle or stomata. Conidia were initiated as small protrusions along the lengths of RH and SIH that got differentiated into distinct conidia, each born on a short stumpy conidiophore without forming any congregation. The protrusions from RH emerged to the leaf surface by piercing the cuticle, and they developed into distinct conidia on the leaf surface. The conidia developed from RH and SIH were identical in size and shape. Even though conidia were occasionally found emerged through stomata, that appeared to be random than a preferred route for the discharge of conidia. The penetration and sporogenesis of C. gloeosporioides on castor leaf differed from that reported on mulberry leaf.     

Cytological Study of Wheat Spike Infection by Bipolaris sorokiniana

The infection of wheat spikelets by Bipolaris sorokiniana , the causal agent of black point on grains and grain shrivelling, was examined by light and electron microscopy. Conidia of the pathogen germinated 6–12 h after inoculation on the surfaces of the different spike tissues. Extracellular sheaths were observed on germ tubes and appressoria attached to the surfaces of lemma, palea and seeds, but were only scarcely detected on the surface of conidia. Appressoria, frequently found over grooves, formed penetration hyphae invading the epidermal cell walls. Infection process was similar on the surface of the lemma, palea and glume. Growth of the fungus in the epidermal and parenchyma cells was found predominantly in the cell walls, and hyphae also extended intercellularly and intracellularly. Infection of seeds appeared to occur via two ways: (i) direct infection of the outer layers of the cell walls of the pericarp and (ii) through entering the stigma into the pericarp cells. Secretion of host cell wall hydrolytic enzymes at the apex of the penetrating hyphae may facilitate the spread of the fungus. In addition, toxins secreted by the fungus might explain the rapid death of host cells in contact with or distant to fungal cells. A host response to fungal infection involved the development of appositions between cell wall and plasma membrane in cells adjacent to fungal cells. Fungal hyphae were sometimes also surrounded by electron dense material.