In vivo-Untersuchungen zur Entstehung und Funktion der Chlamydosporen von Botrytis cinerea Pers. am Wirt-Parasit-System Fuchsia hybrida – B. cinerea

In vivo-investigations on the formation and function of chlamydospores of Botrytis cinerea Pers. in the host-parasite-system Fuchsia hybrida–B. cinerea On naturally with Botrytis cinerea Pers. infected and artificially inoculated outdoor- and greenhouse-plants of Fuchsia hybrida the extra- and intramatrical formation of the B. cinerea- chlamydospores was investigated. The chlamydospores served 1. as structures of survival, which were tested with regard to their tolerance of drought, nutrient- and oxygen-deficiency, attack by bacteria and pH-requirements. 2. The chlamydospores represented dispersal units, which were capable of germination. 3. The chlamydospores could function as structures of infection, because after chlamydospore germination the outgrowing mycelium – either directly or after production of macroconidia – could serve as secondary inoculum and start new infections.     

Über Entstehung und Keimung der Chlamydosporen von Botrytis cinerea Pers.

On the genesis and germination of the chlamydospores of Botrytis cinerea Pers. The chlamydospores of Botrytis cinerea are hyaline single cells of extremely variable form and size. They are formed under conditions unfavourable for growth as terminal or intercalary cells by transformation of vegetative mycelium parts and are liberated by hyphal disintegration. The chlamydospore genesis in vitro in aging malt agar cultures began about after one month. But the chlamydospore formation could also be initiated earlier by different conditions of culture. The chlamydospores germinated either with hypha or by microconidia — a herewith first described mode of germination. Intermediates of these both modes of chlamydospore germination could be regulated very differentiatedly by transferring the chlamydospores into malt solution (2%)and/or destilled water and by changing the duration of stay in the individual media. Under adverse external conditions no germination occurred. The three Botrytis cinerea-isolates did not show any differences in habitus, genesis and germination of their chlamydospores. Also in vivo on outdoor- and greenhouse-tomatoplants the occurrence of chlamydospores was no rarity. Since the chlamydospores are produced under very different adverse conditions of growth but cannot survive a period of drought lasting longer than three months without damage, they do not represent long-termed resistant perennating structures, but temporary stages of the fungus for intervening periods.     

Phytophthora cinnamomi: the Dynamics of Chlamydospore Formation and Survival

Chlamydospore activity was investigated by inoculating 10 day old seedlings of Lupinus angustifolius with Phytophthora cinnamomi. Infected roots were excised and buried in 4 non-sterile media: – glass beads, gravel, sand or soil at different water potentials and incubated at 22°C. Roots were examined and chlamydospores counted initially and after 10, 20 and 30 days. Large numbers of spores formed in the roots buried in the various media. Spore formation was rapid in glass beads and gravel, and slower in soil, but more chlamydospores survived the 30 day period when roots were buried in soil. Nylon mesh squares 1 cm²were inoculated with 38 chlamydospores and buried in the same 4 media. 70–100% of chlamydospores germinated and colonised 90–100% of the mesh, producing mycelia and an increased number of chlamydospores. In some instances there was a ten-fold increase in spore numbers and numbers were still increasing at 28 days. Maximum numbers and maximum survival occurred on mesh buried in soil. The role of the chlamydospores is considered, as that of a dynamic unit with a saprobic phase in the life cycle of the fungus which is independent of host, and has the capacity to increase both population density and distribution of the pathogen.     

Microcycle speculation in theClaviceps purpurea 244

The mutant strainClaviceps purpurea 244 forming hyphae composed mainly from sclerotiumlike cells was found to sporulate both in liquid and solid media, particularly in the form of terminal chlamydospores (4.0 × 6.5 μm). Chlamydospores produced during submerged cultivation germinated, new chlamydospores being formed directly from germ tubes, or, occasionally, conidia (the so-called microcycle) or new vegetative mycelium were formed. The ultrastructures of the chlamydospores and vegetative cells was identical. The cytoplasm was filled with ribosomes and contained lipid inclusions and vacuoles with membrane invaginations. Strain 244 cultivated under submerged conditions produced 150 μg/ml clavins, with elymoclavin predominating (82 %). The parent strainClaviceps purpurea 129 only produced chlamydospores on the vegetative mycelium, whereas no microcycle was detected; under submerged conditions it produced mainly agroclavin (85 %) at a concentration of 4 mg/ml.     

Culture age impacts Plectosporium alismatis propagule yields and subsequent desiccation and UV-radiation tolerance

The effect of culture age on yields, desiccation tolerance and resistance to ultraviolet radiation of Plectosporium alismatis, a potential mycoherbistat of aquatic weeds in Australian rice fields, was studied. P. alismatis was grown in a liquid basal medium supplemented with malt extract and sodium nitrate and harvested after 7, 14 or 21 days incubation. Although chlamydospore yields harvested from 14-day-old liquid cultures were significantly higher (29.2×10⁵ chlamydospores mL^?1) than chlamydospore yields harvested from 7-day-old liquid cultures (1.07×10⁵ chlamydospores mL^?1) or from 21-day-old liquid cultures, the germination of freshly-harvested chlamydospores from 7-day-old cultures (72.7%) was significantly reduced when propagules were grown for 14 days (55.3%). When exposed to UV-radiation, conidia and chlamydospores harvested from 14-day-old cultures germinated at a lower rate (<20%) than conidia and chlamydospores harvested from 7-day-old cultures (>40%). When conidia and chlamydospores were dried and subsequently exposed to UV, less than 30% of propagules harvested from 7-day-old cultures germinated, whereas less than 10% of propagules harvested from 14-day-old cultures germinated. A three-way analysis of variance including culture age, UV exposure and type of propagules confirmed that the culture age had more impact on the germination of fresh or dry propagules (P=0.00001 and P=0.0004, respectively) than the type of propagules considered (P=0.5). These results demonstrate that the culture age impacts significantly propagule yields and germination of P. alismatis conidia and chlamydospores, particularly after stress caused by dehydration and/or exposure to UV-B radiation.     

Influence of carbon and nitrogen sources on the transition from yeast-like cells to chlamydospores inAureobasidium pullulans

The transition from yeast-like cells to chlamydospores ofAureobasidium pullulans can be induced by growing the microorganism on a glucose medium with a limiting nitrogen source and a low buffer capacity. When glucose is used as the carbon source, a concentration higher than 3% (w/v) is required to induce the transition. On the other hand, growth limiting concentrations of the N source (ammonium sulphate) are not required, and higher concentrations actually stimulate the appearance of chlamydospores. Other N sources, such as glutamate or ammonium phosphate, do not induce the transition from yeast-like cells to chlamydospores.     

Characterization of aggregates produced by the potential mycoherbistat Plectosporium alismatis in submerged culture: germination,UV-radiation tolerance and infectivity

Plectosporium alismatis, a potential mycoherbistat of Alismatacae spp., has been previously shown to produce aggregates which contain chlamydospores in liquid culture. Weevaluated the impact of medium composition on the formation and composition of aggregates. In shake flasks cultures using 5.74 gL^?1 sodium nitrate, 8.8 gL^?1 malt extract or glucose and 0.1% Tween 80, P. alismatis formed small, uniform (diameter of 75% aggregates <720 µm), dense, melanised aggregates containing 10⁴ conidia and 10³ chlamydospores, these numbers remained unchanged during growth. All 7-day-old aggregates exposed to desiccation or/ and UV-radiation germinated. In bioassays using leaf discs of Alisma plantago-aquatica, P. alismatis aggregates caused necrosis, regardless of whether aggregates had been exposed to desiccation and/or UV-radiation prior to application on leaf discs, whereas other propagules (10³ propagules disc^?1) exposed to drying and UV-radiation stress were unable to cause necrosis. This preliminary research shows the potential of aggregates to be used as part of a formulation of biocontrol agents, provided adequate conditions for optimal aggregate yields are found.     

Life Cycle of Botryodiplodia theobromae, a Soft Rot Pathogen of Yam

Botryodiplodia theobromae Pat. forms chlamydospores and specialised hyphae in diseased yam tissues in which condition it tides over unfavourable periods. With favourable conditions the chlamydospores germinate and the specialised hyphae formed serve as sources of infection to new hosts and infect through wounds. Germination tubes infect by means of terminal appressoria, and nutrition within the host is effected by production of haustoria.     

Ganoderma carocalcareus sp. nov., with crumbly-friable context parasite to saprobe on Anthocleista nobilis and its phylogenetic relationship in G. resinaceum group

A new species Ganoderma carocalcareus (Basidiomycota, Ganodermataceae) was collected on living trunk and dead stumps of Anthocleista nobilis (Gentianaceae) in waterlogged swamps in the Mbalmayo Forest Reserve, Cameroon, and identified on the basis of morphology and phylogenetic analyses inferred from mitochondrial small subunit (mtSSU) and internal transcribed spacer (ITS1-5.8S-ITS2) rDNA sequences. Distinct phenotypic characteristics of the new species are dimorphism of basidiomata and variability in context structure and texture over developmental stages. The young basidiomata is ungulate to punk-shaped with context composed of vegetative hyphae attended by scattered, orbicular, smooth, thick-walled chlamydospores, and the mature basidiomata is cushion- to bracket-like with context entirely consisting of chlamydospores masses. This ontogeny intimates the origin of chlamydospores, for which the biogenesis correlates the vanishing of vegetative hyphae throughout the basidiomata maturation. Morphological comparison included Tomophagus colossus (=G. colossus), G. subamboinense and G. weberianum, the known Ganodermataceae species producing chlamydospores and or gasterospores in basidiomata tissues, and G. resinaceum, the closest species with regard to morphology. It followed that G. carocalcareus could not be assigned to these or any other known Ganoderma species. Analyses of mtSSU and ITS rDNA sequence data resolved G. carocalcareus in the G. resinaceum group as a distinct species, but being a close relative of both G. subamboinense and G. weberianum.     

Impact of nutritional conditions on yields,germination rate and shelf-life of Plectosporium alismatis conidia and chlamydospores as potential candidates for the development of a mycoherbicide of weeds in rice crops

The effect of nutritional conditions on spore qualities was investigated in order to select which propagules, conidia or chlamydospores, would be most suitable for mycoherbicide development. Plectosporium alismatis was grown in a liquid basal medium supplemented with glucose and a mineral nitrogen source (sodium nitrate) or an organic nitrogen source (casamino acids). Conidial and chlamydospore yields, germination rate and shelf-life were compared. Two growth models were developed: on one hand, sodium nitrate added as the sole nitrogen source was partially utilised (8%), resulting in poor growth (1.77±0.02 mg mL^?1; 6±1.7×10⁵ conidia mL^?1). Under these conditions, P. alismatis produced dense, melanised-like aggregates that contained chlamydospores (12.4±0.7×10⁴ chlamydospores mL^?1). Germination rates of chlamydospores and conidia produced under these conditions was high (80%). Twenty percent of chlamydospores were able to germinate after 4 months storage at 25°C, while survival of conidia declined rapidly (<2%). When casamino acids were added to the liquid medium as the sole nitrogen source, P. alismatis produced sparser pellets resulting in high dry weights (5.37±0.09 mg mL^?1 and high conidia numbers (9.6±1.5×10⁶ conidia mL^?1), while no chlamydospore were observed. The germination rate of conidia produced in casamino acids was low (33±13%) after 8 h incubation and microcycle conidiation occurred. Five percent of these conidia germinated after 4 months storage. These data indicate that chlamydospores may be suitable for mycoherbicide development, provided further optimisation of yields is achieved.     

Factors responsible for transition of the <Emphasis Type="Italic">Duddingtonia flagrans</Emphasis> carnivorous fungus from the saprotrophic to the zootrophic nutrition type

Quantitative investigation of the factors responsible for trap formation in the nematophagous fungus Duddingtonia flagrans F-882 in submerged liquid culture was carried out. The data obtained suggest a complex program for the regulation of zootrophic nutrition in D. flagrans. Optimal concentrations of such carbon and nitrogen sources as sucrose (0.4%), ammonium ions (0.2%), and tryptone (0.2%) promote trap formation in the case of contact with the nematodes Panagrellus redivivus. Increased concentrations of these compounds, however, inhibit trap formation. The sensitivity of the mycelium to nematode excreta depends on the state of the culture and is increased under limitation by certain nutrient components or in the course of prolonged starvation. A direct correlation was found between the number of caught nematodes and the number of chlamydospores formed on the mycelium. The nutrients obtained from the nematode biomass are used for formation of additional chlamydospores (on average, about 20 chlamydospores per nematode). Environmental and evolutionary aspects of the role of zootrophic nutrition in carnivorous fungi are discussed.     

A new nematode-destroying Harposporium

A mucedinaceous parasite, observed destroying eelworms in a maize-meal-agar plate culture prepared by the addition of some forest duff from western Maryland, is newly described as Harposporium cycloides. Its conidia differ from the crescentic or semicircular conidia of the familiar H. anguillulae in that their curvature extends to an angular magnitude of approximately a full circle. Its chlamydospores, though sometimes found united in pairs, are much more often produced singly than are the chlamydospores of H. anguillulae.     

Influence of pH on the transition from yeast-like cells to chlamydospores in Aureobasidium pullulans

When A. pullulans is grown on a glucose medium with a limiting nitrogen source and low buffer capacity, the yeast-like cells that are originally present undergo a transition to chlamydospores. The initial pH must be around 6 for the transition to take place under optimal conditions. On the above-mentioned medium pH decreases to values below 2 in the first two days; if this decrease is prevented, either by buffering the medium or by repeatedly adjusting the pH to its original value no chlamydospores form.     

Modeling competition for infection sites on roots by nonpathogenic strains of Fusarium oxysporum

By use of plane and solid geometry and probability models, efficiencies of infection and competition for nutrients and infection sites by a nonpathogenic strain of Fusarium oxysporum (C14) with F. oxysporum f. sp. cucumerinum on the rhizoplane of cucumber were calculated. The model is derived from previously published data. Efficiencies for successful infection were 0.04 chlamydospores per infection site for both pathogen and nonpathogen. Observed successful infections by the pathogen in competition with the nonpathogen were close in values to the competition ratio (CR) calculated as the number of chlamydospores on the infection court of the pathogen divided by the total number of both pathogen and nonpathogen at relatively low densities. When total chlamydospores were, on average, closer than 175 μm apart, however, competition for nutrients/mutual inhibition occurred. At such densities there was an overestimation of the effect of competition for infection sites. These relationships were modeled at inoculum densities of pathogen and/or nonpathogen of 5000 chlamydospores per g soil and above, however, in the field, maximum densities of 1000 colony forming units/g (cfu) were observed. Most likely models of competition for infection sites at this density of the pathogen revealed that infection efficiency was only approximately halved, even when 0.98 of the possible 30 infection sites were occupied by the nonpathogen. It is conclude that competition for nutrients and/or infection sites is an insignificant factor in biocontrol of Fusarium wilt diseases by nonpathogenic fusaria.     

Evaluation of the antigen from chlamydospores of Candida albicans in the serodiagnosis of candidiasis

Antigens have been prepared from the chlamydospores and blastospores of Candida albicans and their precipitin patterns were analysed by two-dimensional immunoelectrophoresis using specific antisera.The two antigens were used in routine serological tests of patients suffering from candidiasis. On double-diffusion tests for the detection of circulating antibodies of Candida albicans, the antigen from chlamydospores displays precipitin lines that differ in number and intensity from those obtained with the antigen from blastospores. The results are briefly discussed in the framework of C. albicans antigen standardization.     

The hyphomycete Sorosporetta — Syngliocladium from mole cricket,Scapteriscus vicinus

The hyphomycete Sorosporella sp. was isolated from the mole cricket, Scapteriscus vicinus collected in Alachua Co., Florida. Scanning (SEM) and/or transmission (TEM) electron microscopy were used to study the chlamydospores characteristic of this genus and the conidial production in the alternate state, Syngliocladium. Brick-red chlamydospores, which occur in clusters in insect cadavers, have a fibrous cell wall as revealed by TEM. Pores often occur between walls of adjacent cells. Lipids, including a large central lipid droplet and smaller droplets along the periphery of the cell, are abundant in the cytoplasm. Most organelles were difficult to distinguish due to the density of the cytoplasmic material. Chlamydospores germinate on water or media. On Sabouraud maltose agar, germinating chlamydospores produce a white mycelial mat; synnematous-type growth was sometimes observed. Mycelia may bear conidiophores and ellipsoid conidia.Florida Agricultural Experiment Station Journal Series No. 7789.     

FUNGAL REMAINS IN THE DEVONIAN TRIMEROPHYTE PSILOPHYTON DAWSONII

Fungal reproductive structures were found within aerial axes of the Late Devonian trimerophyte, Psilophyton dawsonii Banks, Leclercq and Hueber from Quebec, Canada. Specimens are spherical to oval, up to 175 μm in maximum diam, and exhibit two distinct wall layers. They are restricted to the inner cortical region of infected axes. The fungal bodies resemble species of Palaeomyces from the well-known Rhynie Chert flora, and are also similar to the chlamydospores commonly found in Pennsylvanian coal ball plants. Among extant organisms they are most similar to oomycetous oogonia and endogonaceous chlamydospores.     

New species ofAcremonium, Cylindrocarpon andVerticillium from soil in the Bonin (Ogasawara) Islands, Japan

Three new soil fungi from the Bonin (Ogasawara) Islands, Japan are described:Acremonium macroclavatum, characterized by large clavate guttulate conidia;Cylindrocarpon boninense, characterized by 3–7-septate clavate macroconidia, terminal or intercalary chlamydospores, rarely produced unicellular clavate microconidia; andVerticillium hahajimaense, characterized by conidial heads bearing cylindrical conidia, and catenulate chlamydospores.     

Necrotrophic Mycoparasitism of Chalara elegans (Thielaviopsis basicola) by a Sterile Basidiomycete

Interactions between Chalara elegans and a sterile hyaline basidiomycete (SHB) were studied by light, scanning and transmission electron microscopy. Young colonies of C. elegans were quickly overgrown by the mycoparasite, with lysis of, hyphae and conidia. Direct penetration of host hyphae and conidia was observed, but simple coiling around these also occurred. Chlamydospore development was inhibited, but 12.5% of mature chlamydospores were able to survive attack for 120 days. Chlamydospore germination was apparently stimulated by the SHB, whereupon lysis followed. Evidence of invasion of intact chlamydospores is also presented.     

Liquid fermentation to produce biomass of mycoherbicidal strains of Fusarium oxysporum

Conditions for optimizing spore production, especially chlamydospores, by host-specific mycoherbicidal strains of Fusarium oxysporum causing vascular wilts in coca (Erythroxylum coca) and poppy (Papaver somniferum) were studied in 2.5-1 fermentors. The fermentor dissolved oxygen and pH had significant effects on the growth characteristics of F. oxysporum strains. The effect of the fungal strain, however was not significant for most of the variables studied except for chlamydospore formation. After 14 days of fermentation, the spore types produced were microconidia and chlamydospores, with very little production of macroconidia. While the total viable counts were significantly higher under high than under low dissolved O₂, the chlamydospore counts were significantly higher under low than under high dissolved O₂. The percentage of chlamydospores obtained, as a proportion of total viable was significantly higher when the fermentor pH was increased, than when it was not. Scaling-up the liquid fermentation to 20 l, yielded log₁₀ c = 6.8 (where c = chlamydospores ml⁻¹) after 14 days' fermentation, with biomass viable counts of log₁₀ v∼8.0 (where v = viable counts g⁻¹ air-dried biomass). A single-step liquid fermentation reported in this study increased chlamydospore yields and reduced the time required for their production with techniques currently available from 5 weeks to less than 2 weeks. Received: 24 April 1997 / Received revision: 6 August 1997 / Accepted: 29 August 1997