Behaviour of Phytophthora cinnamomi Zoospores on Roots of Four Avocado Cultivars

Scanning electron microscopy was used to study the behaviour of Phytophthora cinnamomi zoospores on the roots of three tolerant avocado cultivars. Duke 7, G6 and Martin Grande, and a susceptible Edranol cultivar. Zoospores were attracted to the region of cell elongation and encysted on the roots of all cultivars studied. Adhesion of the zoospores appeared to be aided by root slime. Cysts usually produced one germ tube which penetrated the root directly, or formed an appressorium-like swelling before penetration occurred. Extensive growth of germ tubes occurred where zoospores germinated some distance behind the region of elongation. Cysts germinating behind this region often formed branched germ tubes and more than one appressorium-like swelling. There were no clear differences in the type of pre-penetration structures, formed by zoospore cysts, on the roots of the different avocado cultivars.     

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.     

Red rot resistance in interspecific protoplast fusion product progeny of Porphyra yezoensis and P. tenuipedalis (Bangiales, Rhodophyta)

Nine primary regenerants were recovered by interspecific protoplast fusion of Porphyra yezoensis Ueda T‐14 (Py) (cultivated Porphyra) and Porphyra tenuipedalis Miura (Pt). This combination is difficult to achieve with conventional sexual hybridization, yet is important in that non‐cultivated P. tenuipedalis is partially resistant (PR) to red rot disease, caused by the microbial pathogen, Pythium porphyrae Takahashi et Sasaki. Out of the nine primary regenerants, two strains (Py‐Pt‐4 and Py‐Pt‐7) were like the parent, P. tenuipedalis, while the rest were like the other cultivated parent P. yezoensis T‐14 in their life cycle. Red rot resistance was assessed in parents and interspecific fusion product progeny (FPP) by exposing the foliose thalli to equivalent infection and measuring two parameters of the host‐pathogen interactions: supported fungal biomass and amount of disease produced. Intermediate resistance between P. yezoensis T‐14 (1.00) and P. tenuipedalis (0.13) was observed in two of the Py‐type FPP, Py‐Pt‐2F₂ (0.25) and Py‐Pt‐5F₂ (0.23). Stable inheritance of resistance was observed through two subsequent generations. The morphologic and reproductive characteristics of the regenerated foliose thalli, and nature of host‐pathogen interactions were used to further verify the hybrid origin of the FPP. Host‐pathogen interactions were followed using epi‐fluorescence and scanning electron microscopy (SEM). The zoospores encysted at higher rates on the susceptible cultivated parent (P. yezoensis T‐14) germinated immediately and the short germ tubes formed appres‐soria and penetrated the algal cells near the site of encystment. While on the PR parental (P. tenuipedalis) and partially resistant FPP (PRFPP) progeny (Py‐Pt‐2F₂ and Py‐Pt‐5F₂) the low rate of zoospore encystment was followed by cyst germination, but only a few of the germ tubes formed appressoria and penetrated the thallus surface. Long germ tubes (with no appressoria) were seen growing on the thallus surface without host penetration. The minimal rate of encystment concomitant with low rate of appressorium formation on the PR parent and PRFPP was observed as the major factor responsible for the partial resistance in these thalli.     

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.     

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.     

Histopathology of Wheat Seedling Roots Infected with Pythium arrhenomanes

Two-day-old wheat seedlings were placed on the edge of a P. arrhenomanes culture for 3 h at 25°C, and transferred into test tubes (18 mm dia.) containing glass beads and 1 ml of sterile water. Roots were sampled every 6 or 12 h for 84 h, and observed with the scanning electron microscope, or serial sectioned for light microscopy. Roots were colonized extensively in the region of root hair formation near the tip within 30 h after inoculation. Extensive penetration occurred 0.1—2 mm behind the root tip, with hyphae breaching the endodermis and gaining entry into the stele. Behind this area, hyphae remained limited to the outer cortical cells, or did not penetrate at all. Hyphae grew intracellularly, and became irregularly inflated inside cells. In most cases, hyphae penetrated the roots directly through the epidermis with appressoria being formed, or through breaks on the surface. However, histological evidence suggested chemical action taking place both outside and inside the cells.     

Ultrastructure of appressorium development by basidiospore germlings of the rust fungusGymnosporangium juniperi-virginianae

Summary Basidiospore germlings ofG. juniperi-virginianae readily formed appressoria (infection structures) on dialysis membranes. These specimens could be effectively freeze-substituted and processed for study with transmission electron microscopy. Appressorium formation on these membranes appeared to be very similar to that occurring on host leaves up to the point of penetration peg formation. A germ tube emerged laterally from each spore, grew until it contacted the membrane, and then differentiated into a swollen appressorium whose end was flattened against the membrane. The fungal wall in contact with the membrane became very thin. A region devoid of most organelles developed in the appressorium tip. Numerous filasomes and microvesicles accumulated in this region. Eventually, a structure known as the appressorial cone formed at the end of the appressorium. This structure was deposited outside the plasma membrane in direct contact with the dialysis membrane. Basidiospores and appressoria appeared to be effectively stuck to the dialysis membrane by a fibrillar, extracellular matrix. This substance appeared as a diffuse network on young germ tubes, but subsequently assumed the appearance of an electron-dense layer or coating on appressoria and basidiospores.     

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。     

Composition and organisation of extracellular matrices around germ tubes and appressoria ofColletotrichum lindemuthianum

Summary The ultrastructure and composition of the extracellular matrices (ECMs) associated with germ tubes and appressoria ofColletotrichum lindemuthianum have been examined. Flexuous fibres (fimbriae), up to 6 m long and 4–30 nm in diameter, protruded from the surface of germ tubes and appressoria. Anionic colloidal gold and lectin cytochemistry showed that ECMs of germ tubes and appressoria contain basic proteins, -D-mannose and -D-galactose residues. A monoclonal antibody, UB26, was raised to infection structures isolated from leaves ofPhaseolus vulgaris infected withC. lindemuthianum. UB26 recognised a protein epitope on two glycoproteins (M_r 133,000 and 146,000). Reductions in the M_r of these proteins after treatment with peptide-N-glycosidase and trifluoromethane sulphonic acid suggest that they carry N- and O-linked side-chains. Immunofluorescence and EM-immunogold labelling showed that glycoproteins recognised by UB26 were restricted to the ECMs around germ tubes and appressoria but fimbriae were not labelled. Unlike appressorial germ tubes formed in vitro, intracellular infection hyphae were not labelled, suggesting that the glycoproteins recognised by UB26 are not present on fungal structures formed within host cells. In liquid culture, these glycoproteins were not released into the medium, suggesting they are physically linked to the cell wall. Also, the glycoproteins were not removed from glass surfaces by ultrasonication. These results suggest that glycoproteins recognised by UB26 may be involved in the adhesion of germ tubes and appressoria to substrata. Our results show that the ECMs of germ tubes and appressoria differ markedly in structure and composition from those of conidia and intracellular hyphae, and that extracellular glycoproteins are associated with specific regions of the fungal cell surface.Abbreviations ECM extracellular matrix - BPA Bauhinia purpurea agglutinin - BSA bovine serum albumin - DIC differential interference contrast - FITC fluorescein isothiocyanate - GNL Galanthus nivalis lectin - GSI-B₄ Griffonia simplicifolia isolectin B₄ - HEPES (N-(2-hydroxyethyl)piperazine-N-(2-ethanesulphonic acid) - IIF indirect immunofluorescence - IPC isopycnic centrifugation - MAb monoclonal antibody - PEG polyethylene glycol - PBS phosphate buffered saline - PNGase peptideN-glycosidase - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - TCS tissue culture supernatant - TEM transmission electron microscopy - TFMS trifluoromethane sulphonic acid     

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.     

Nuclear behavior during the formation of appressoria byAlternaria alternata

Nuclear behavior in the developmental process of appressoria inAlternaria alternata was investigated. In pregerminated conidia, approximately 94% of the conidial cells were uninucleate. The migration of a nucleus into an elongating germ tube from a germinating conidium was confirmed after 2h of incubation at 24±1°C in PDB. Peak frequencies of binucleate and trinucleate germ tubes were detected 1 and 2h after the peak frequency of uninucleate germ tubes, respectively. Four-and five-nucleate germ tubes did not show marked peak frrequencies. A marked peak frequency of the six-nucleate germ tubes occurred about 1 h after the peak frequency of the trinucleate germ tubes, suggesting that the nuclei in the trinucleate germ tubes each divided once within 1 h. The significance of early establishment of multinucleate appressorial cells in the colonization of host plants by pathogenicA. alternata was discussed.     

Appressorium formation and nuclear division in Colletotrichum truncatum

Conidia of the soybean anthracnose fungus, Colletotrichum truncatum differentiate to form appressoria required for host invasion when the germ tube touches a hard surface. This thigmotrophic stimulus appears to be translated by the fungus during the second round of nuclear division. Inhibiting the second round of DNA synthesis by fluorodeoxyuridine or hydroxyurea blocked appearance of appressoria but not emergence of the germ tube. DNA synthesis and mitosis resumed upon removal of FUdR but only mycelia formed, and infection structures did not appear. In addition, actinomycin D reversibly blocked development of appressoria and synthesis of polyadenylate, but nuclear division was not affected. The data suggest that anthracnose conidia produce appressoria in response to germ tube contact by altering the messenger program of its germ tube nucleus. This study has also shown that mitochondrial DNA had an unusual bimodal distribution in CsCl at 1.690 and 1.719 g/cm³, respectively.Non-Standard Abbreviations FUdR 5-fluorodeoxyuridine - polyA polyadenylic acid     

Anatomical study on the interaction between the root endophytic fungus <Emphasis Type="Italic">Heteroconium chaetospira</Emphasis> and Chinese cabbage

Chinese cabbage roots colonized by the dematiaceous fungal taxon Heteroconium chaetospira were previously found to become highly resistant to clubroot and Verticillium yellows. The dematiaceous fungus possesses an endophytic nature, but no detailed anatomical studies on endophyte–host plant interactions have so far been provided. Light and electron microscopy revealed that hyphae of H. chaetospira were abundant on and inside the root epidermal cells by 3 weeks following inoculation. The penetration pegs easily breached into epidermal cells, and the infection hyphae penetrated into cortical cells. Some appressorium-like swollen structures formed from intracellular hyphae, but no visible degradation of the host cell walls was evident where the hyphae contacted. No visible signs of host reactions and no invagination of the host plasma membrane around the hyphae were seen in the host cells. By 8 weeks following inoculation, masses of closely packed fungal cells had been formed in some cells of the epidermis and cortical layers, but further hyphal ingress was halted, mostly in the inner cortical cell layer. Thus, root vascular cylinders remained intact.     

Pathogenesis of barley leaves by Helminthosporium teres II: Carbohydrate involvement

Conidia of Helminthosporium teres had negligible difference in germination and germ tube length between the decolorized and non-decolorized host leaves. Appressoria, penetration and colonization were less on decolorized host leaves, but addition of exogenous nutrients stimulated these stages. Leached conidia had reduced germination on decolorized host leaves, while appressoria formation, penetration and colonization were negligible. The addition of nutrients in the external environment, however, enabled some of the leached conidia to penetrate and colonize. Stimulation by the exogenous nutrients in decreasing order were: sucrose > glucose > yeast extract > leaf leachates. Optimum levels for various nutrients tested were 2% (w/v) each of sucrose and glucose, and 0.1% (w/v) yeast extract. Higher concentrations inhibited these stages of infection. Leached conidia and decolorized leaves had smaller amounts of carbohydrates than non-leached conidia and non-decolorized leaves, respectively. Depletion of host carbohydrates reduced appressoria formation, penetration and colonization and loss of carbohydrates from spores reduced germination.     

Rapid induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs during fungus infection of soybean (Glycine max L.) roots or elicitor treatment of soybean cell cultures at the onset of phytoalexin synthesis

The differential regulation of the activities and amounts of mRNAs for two enzymes involved in isoflavonoid phytoalexin biosynthesis in soybean was studied during the early stages after inoculation of primary roots with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea, the causal fungus of root rot disease. In the incompatible interaction, cloned cDNAs were used to demonstrate that the amounts of phenylalanine ammonia-lyase and chalcone synthase mRNAs increased rapidly at the time of penetration of fungal germ tubes into epidermal cell layers (1–2 h after inoculation) concomitant with the onset of phytoalxxin accumulation; highest levels were reached after about 7 h. In the compatible interaction, only a slight early enhancement of mRNA levels was found and no further increase occurred until about 9 h after inoculation. The time course for changes in the activity of chalcone synthase mRNA also showed major differences between the incompatible and compatible interaction. The observed kinetics for the stimulation of mRNA expression related to phytoalexin synthesis in soybean roots lends further support to the hypothesis that phytoalexin production is an early defense response in the incompatible plant-fungus interaction. The kinetics for the enhancement of mRNA expression after treatment of soybean cell suspension cultures with a glucan elicitor derived from P. megasperma cell walls was similar to that measured during the early stages of the resistant response of soybean roots.Abbreviations cDNA copy DNA - CHS chalcone synthase - PAL phenylalanine ammonia-lyase     

Quantification of substratum contact required for initiation of Colletotrichum graminicola appressoria

Colletotrichum graminicola, like many plant pathogenic fungi develop appressoria on germling apices, to facilitate penetration of their host. Induction of these structures occurs after contact with the host surface has been established by the germling. Surface contact and subsequent development of appressoria by germlings of C. graminicola was assessed using interference-reflection microscopy (IRM) and microfabricated pillared silicon substrata. Observations with IRM revealed that under low nutrient conditions, 90% of the germlings developed appressoria once they established 4.5 microm of continuous contact with the substratum. Substrata bearing pillars < or =5 microm in width supported < or =10% appressoria; however, as pillar width was increased the percentage of appressoria formed increased in a sigmoid fashion to a maximum of 80%. The percentage of appressoria produced experimentally on these surfaces was compared to data sets generated from a model designed to calculate the probability of appressorium development on similar pillar arrays at various germ tube contact lengths. These results indicate that germ tubes of C. graminicola require more than 4microm of continuous contact with a hydrophobic substratum for induction of appressoria.     

Conidial germination and germ tube elongation of Phomopsis amaranthicola and Microsphaeropsis amaranthi on leaf surfaces of seven Amaranthus species: Implications for biological control

Microsphaeropsis amaranthi and Phomopsis amaranthicola are potential biological control agents for several Amaranthus species. In an effort to understand the initial infection processes with these pathogens, a study was conducted of the conidial germination and germ tube length (μm) on the weed leaf surfaces at 21 °C and 28 °C. Weeds included Amaranthus rudis, A. palmeri, A. powellii, A. retroflexus, A. spinosus, A. hybridus, and A. albus. For P. amaranthicola, conidial germination and germ tube length varied among the seven weed species at both temperatures, while for M. amaranthi the differences in germ tube lengths were significant among weed species only at 21 °C. While the conidia of M. amaranthi and P. amaranthicola germinated on the leaf surfaces of all seven weed species, temperature appeared to impact the number and length of germ tubes on the leaf surfaces. The percentage of germinated conidia and the length of germ tubes at both temperatures were often greater for M. amaranthi than for P. amaranthicola. In order for the fungal pathogen to successfully infect and kill a weedy host, conidia must germinate and form a germ tube, two processes that vary with host species and temperature for M. amaranthi and P. amaranthicola. The extent to which successive infection processes, e.g., penetration, invasion and colonization, contribute to host specificity warrants study.     

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.     

Appressoria of the nematophagous fungus verticillium suchlasporium

Appressoria of the nematophagous fungus Verticillium suchlasporium infecting eggs of the beetroot cyst nematode Heterodera schachtii were studied by field emission scanning electron microscopy (FESEM). Protoappressoria and mature appressoria were found in both germ tubes and mycelial hyphae, and mucilaginous material was seen where the appressoria contacted the surface of the egg. The role of this material in the penetration of the eggs by the hyphae is discussed.     

Screening the ability of rice roots to overcome the mechanical impedance of wax layers: importance of test conditions and measurement criteria

The development of a wax layer method for screening the ability of rice (Oryza sativa L.) roots to overcome mechanical impedance is described. Wax layers (3 mm thick) made of mixtures of white soft paraffin and paraffin wax were installed 50 mm deep in tubes of sand. The sand was watered with nutrient solution and planted with 3-d old rice seedlings. The numbers of root axes per plant that had penetrated the wax layers 24 d after planting were counted. The ratio of penetrated to total root axes per plant gave a misleading measure of root penetration ability, as rice varieties differed in the ratio of penetrated to total axes in a low impedance (3% wax) control. In non-flooded conditions, a 60% wax layer decreased root penetration (number of roots penetrating the wax layer per plant) to a mean of 74% of the low impedance control, whereas an 80% wax layer decreased mean root penetration to 31% of the control. The best measure of root penetration in non-flooded conditions was the number of axes penetrating an 80% wax layer. Flooding decreased root penetration of a 60% wax layer to a mean of 26% of the low impedance control. The best measure of root penetration in flooded conditions was the number of axes penetrating a 60% wax layer.