不同基质对指状节丛孢孢子捕食器官形态建成的影响*

利用牛粪、土壤等基质诱导捕食线虫真菌指状节丛孢产生孢子捕食器官。孢子捕食器官形成过程与正常情况下捕食器官的形成基本相似;不同基质诱导孢子捕食器官的能力不同, 同种内不同菌株间形成孢子捕食器官的能力也有异别。预培养实验,温度处理实验的结果表明基质中诱导孢子捕食器官的主要诱导因子可能是某些化合物,这些物质在100℃下基本上丧失诱导活性;在诱导孢子捕食器官的过程中,基质中的微生物所起的作用不大。     

Spread and change in stress resistance of Shiga toxin-producing Escherichia coli?O157 on fungal colonies

To elucidate the effect of fungal hyphae on the behaviour of Shiga toxin-producing Escherichia coli (STEC) O157, the spread and change in stress resistance of the bacterium were evaluated after coculture with 11 species of food-related fungi including fermentation starters. Spread distances of STEC O157 varied depending on the co-cultured fungal species, and the motile bacterial strain spread for longer distances than the non-motile strain. The population of STEC O157 increased when co-cultured on colonies of nine fungal species but decreased on colonies of Emericella nidulans and Aspergillus ochraceus. Confocal scanning microscopy visualization of green fluorescent protein-tagged STEC O157 on fungal hyphae revealed that the bacterium colonized in the water film that existed on and between hyphae. To investigate the physiological changes in STEC O157 caused by co-culturing with fungi, the bacterium was harvested after 7 days of co-culturing and tested for acid resistance. After co-culture with eight fungal species, STEC O157 showed greater acid resistance compared to those cultured without fungi. Our results indicate that fungal hyphae can spread the contamination of STEC O157 and can also enhance the stress resistance of the bacteria.     

Morphogenesis and significance of hyphal coiling by nematode-trapping fungi in mycoparasitic relationships

Abstract The mycoparasitic behaviour of some nematode-trapping fungi was investigated. These organisms interacted with other soil fungi by hyphal coiling around the host hyphae. A detailed study with Arthrobotrys oligospora revealed that different fungi, representing all taxonomic groups, were attacked.
In dual cultures, the interaction between A. oligospora and Rhizoctonia solani occurred soon after hyphal contact, irrespective of the nutrient level of the medium. Coiling was also observed when the two organisms were grown in sterilized soil. The coils possessed a high metabolic activity compared to the surrounding hyphae, as was indicated by fluorescence microscopy of fluorescein diacetate (FDA)-stained preparations. On the ultrastructural level, developing coils showed an abundance of membranous vesicles which developed from tubular-shaped endoplasmic reticulum. At the site of coiling, a strong cell wall proliferation was observed in the Rhizoctonia cells. The cytoplasm of these cells subsequently disintegrated. The death of the cells was confirmed in vital staining experiments. Penetration of intact Rhizoctonia cells was not observed. The interaction between A. oligospora and R. solani is interpreted in terms of competition for nutrients.     

Development and fate of electron-dense microbodies in trap cells of the nematophagous fungusArthrobotrys oligospora

The development of electron-dense microbodies in cells of capture organs of the nematophagous fungus Arthrobotrys oligospora was studied with different ultrastructural techniques. Kinetic experiments revealed that the synthesis of these microbodies started in a very early stage of trap formation; the organelles originated from special regions of endoplasmic reticulum by budding. Mature organelles were surrounded by a single membrane of approximately 9 nm (KMnO4-fixation) and lacked crystalline inclusions. The presence of the electron-dense microbodies was independent of the conditions during which the traps had developed. The organelles remained intact during aging of the trap cells. They were also observed in the trophic hyphae after capture and penetration of nematodes. However, the distribution patterns of these organelles in the trophic hyphae, which were identical to those observed after germination of isolated traps on different cultivation media, suggested that their presence must be explained by dilution of organelles in newly formed cells.     

Circadian Changes in Anxiolysis-Related Behavior of Syrian Hamsters. Correlation with Hypothalamic GABA Release

The recently developed method of studying fungi inside a soil by fluorescence microscopy was used to examine the periodism of trap formation by predacious fungi. Therefore, this method was supplemented by a technique, which also permitted serial sections of the soil and the counting of capture organs of Arthrobotrys oligospora in the soil. The combination of these methods also allowed spotting of the traps at their exact location, thus giving evidence about their spatial, three dimensional pattern and demonstrating the relative predatory success in soil. It was shown that, in spite of a virtually homogeneous induction by eelworms, the distribution is distinctly periodical. A period of 29.65 ± 4.14 h was calculated. Additional EDX-measurements of the mycelia showed maxima of mineral content, the distance of which agreed with the period length observed. These mineral accumulations obviously marked the sites of potential trap formation.     

Nematophagous Fungi - Study by Fluorescence Microscopy and EDX-Technique of the Periodicity of Trap Formation in Soil

The recently developed method of studying fungi inside a soil by fluorescence microscopy was used to examine the periodism of trap formation by predacious fungi. Therefore, this method was supplemented by a technique, which also permitted serial sections of the soil and the counting of capture organs of Arthrobotrys oligospora in the soil. The combination of these methods also allowed spotting of the traps at their exact location, thus giving evidence about their spatial, three dimensional pattern and demonstrating the relative predatory success in soil. It was shown that, in spite of a virtually homogeneous induction by eelworms, the distribution is distinctly periodical. A period of 29.65 ± 4.14 h was calculated. Additional EDX-measurements of the mycelia showed maxima of mineral content, the distance of which agreed with the period length observed. These mineral accumulations obviously marked the sites of potential trap formation.     

Assessment of germination and carnivorous activities of a nematode-trapping fungus Arthrobotrys dactyloides in fungistatic and fungicidal soil environment

Carnivorism is the ability of nematode-trapping fungi to trap and digest the nematodes by sophisticated devices called traps. Delivery of nematode-trapping fungi in soil for bio-control of pest nematodes often fails or gives inconsistent results. Possible reasons for failure could be the effect of soil fungistasis on germination of nematode-trapping fungi in soil environment, use of avirulent species and sensitivity of these fungi to fungicidal residues in soil. Exploitation of nematode-trapping fungi for nematode control demands that it be compatible with fungicides applied in soil or crops and proliferate in soil. This investigation represents is one of the first to evaluate the effect of fungicides on the nematode-trapping fungus Arthrobotrys dactyloides. A. dactyloides showed in vitro carnivorous potential against Meloidogyne incognita, Meloidogyne javanica, Meloidogyne graminicola, Helicotylenchus dihystera and Heterodera cajani. Conidia of A. dactyloides exposed to agricultural soils showed poor germination but formed conidial traps, which captured and killed the soil nematodes. Conidial traps, which trapped the nematodes, grew well in all soils after killing and nutrient absorption from nematode body. Soil amended with 20 mg ai kg⁻¹ of carbendazim and thiram, 30 mg ai kg⁻¹ of mancozeb, 50 mg ai kg⁻¹ of captan, and 100 mg ai kg⁻¹ of carboxin completely checked the conidial trap formation and nematode capturing. 30, 50 and 100 mg ai kg⁻¹ of metalaxyl adversely affected the conidial trap formation and nematode capturing in soil. Propiconazole inhibited 15.2% conidial trap formation up to 50 mg ai kg⁻¹ but caused 93.3% inhibition of conidial traps formation and complete inhibition of nematode capturing at 100 mg ai kg⁻¹. Sulphur, triademefon, and tricyclazole showed least toxic effect on conidial trap formation and nematode capturing activities of A. dactyloides in soil up to 100 mg ai kg⁻¹.     

Surface polymers of the nematode-trapping fungus Arthrobotrys oligospora

The nematophagous fungus Arthrobotrys oligospora captures nematodes using adhesive polymers present on special hyphae (traps) which form a three-dimensional network. To understand further the adhesion mechanisms, A. oligospora surface polymers were visualized by transmission electron microscopy and characterized by chemical methods. Both traps and hyphae were surrounded by a fibrillar layer of extracellular polymers which stained with ruthenium red. The polymer layer was resistant to most of the chemicals and enzymes tested. However, part of the layer was removed by sonication in a Tris-buffer or by extraction in a chaotropic salt solution (LiCl), and the structure of the polymers was modified by treatment with Pronase E. Chemical analysis showed that the crude extracts of surface polymers removed by sonication or LiCl solution contained neutral sugars, uronic acids and proteins. Gel chromatography of the extracts revealed that the major carbohydrate-containing polymer(s) had a molecular mass of at least 100 kDa, containing neutral sugars (75% by weight, including glucose, mannose and galactose), uronic acids (6%) and proteins (19%). There was more polymer in mycelium containing trap-bearing cells than in vegetative hyphae. SDS-PAGE of the extracted polymers showed that the trap-forming cells contained at least one protein, with a molecular mass of approx. 32 kDa, not present on vegetative hyphae. Examining the capture of nematodes by traps of A. oligospora in which the layer of surface polymers was modified, or removed by chemical or enzymic treatments, showed that both proteins and carbohydrate surface polymers were involved in the adhesion process.     

Heavy trap formation by Arthrobotrys oligospora in liquid culture

Abstract The nematode-trapping fungus Arthrobotrys oligospora was grown in liquid culture in modified separatory funnels with vigorous air bubbling. The growth medium was a dilute soya peptone broth supplemented with a trap-inducing substance. The dipeptide l -phenylalanyl- l -valine or its constituents phenylalanine and valine were used as inducers of trap formation. Trap formation started within 2 days after inoculation and the traps increased in number and size during a 7-day period. The traps formed in liquid culture were fully functional in trapping nematodes. At the ultrastructural level they were characterized by the presence of many electron-dense microbodies similar to those in trap cells grown on solid media. Biomass increase, amounting to 6–7 mg dry weight · ml⁻¹, and trap formation were highly synchronized. The mycelial mass was homogeneously dispersed in the growth vessel during the entire growth period. Heavy trap formation in liquid culture by this fungus has not been reported previously.     

An ultrastructural study of cell-cell interactions in capture organs of the nematophagous fungus Arthrobotrys oligospora

Abstract A detailed ultrastructural analysis was made of interactions between individual cells within the same adhesive network (trap) of the nematophagous fungus Arthrobotrys oligospora . These interactions were confined to traps which had captured nematodes, and occurred concurrently with the fungus-nematode interactions. The process was initiated by the anchoring of 2 cells of different loops constituting the trap network, by means of the adhesive coating of these cells. Subsequently, penetration tubes were formed. As in nematode-fungus interactions, the walls of these tubes arose from underneath the original trap cell walls. Two response were observed: either only one of the anchored cells formed a penetration tube, which penetrated the opposite cell and subsequently digested its contents; or both cells formed penetration tubes simultaneously, which were directed against each other. In the latter case, no penetration of other cells was observed, and elongated tubes were occasionally formed. The above mechanism differed from 2 other modes of interaction also observed, namely fusion of intact cells (anastomosis) and development of new hyphae inside dead hyphal cells. In the latter case the newly formed cells developed from the cross-wall of the neighbouring intact cell.     

Thioredoxin1 regulates conidia formation,hyphal growth,and trap formation in the nematode-trapping fungus Arthrobotrys oligospora

Arthrobotrys oligospora, a model nematophagous fungus that produces specific adhesive networks to capture nematodes, has been proposed as a potentially effective biological agent to control harmful plant-parasitic nematodes. Although thioredoxin has been characterized as playing important roles in many cellular processes in other species, its function in nematophagous fungi has not been studied. Here, the function of a thioredoxin homolog, Aotrx1, was investigated in A. oligospora. The encoding gene of Aotrx1 in the nematophagous fungus A. oligospora was knocked out by homologous recombination; strain growth was assessed. The ΔAotrx1 strain of A. oligospora showed a significant decrease in growth rate on different media (PDA, CMY, and TG), a 70% decrease of conidia production, and a lower germination rate compared with the wild type. The mutant strain was unable to form traps to capture nematodes and was more sensitive to SDS and H2O2. Thioredoxin is involved in conidia development, trap formation, normal mycelial growth, and resistance to environmental stresses in the nematode-trapping fungus A. oligospora.     

Nonspecies-specific effects of mouse oviducts on the development of bovine IVM/IVF embryos by a serum free co-culture

In Experiment 1, development of bovine embryos derived from in vitro-matured (IVM) and in vitro-fertilized (IVF) oocytes was examined under 4 culture conditions: 1) co-culture with mouse ampullae continuously for 8 d, 2) co-culture with mouse ampullae that were replaced with fresh ampullae at 48-h intervals, 3) co-culture with bovine granulosa cell monolayers, and 4) culture in medium alone. Culture medium consisted of tissue culture medium 199 (TCM-199) supplemented with 1% fetal calf serum (FCS). Inseminated oocytes were transferred to each of the culture treatment 24 h after insemination and were cultured for 8 d. The number of blastocysts per number of cleaved ova obtained after co-culture with mouse ampullae (42.9%) was significantly (P<0.05) higher than that obtained after co-culture with granulosa cell monolayers (28.3%) or culture without cells (4.2%). In Experiment 2, the developmental ability of bovine IVM/IVF embryos co-cultured with mouse ampullae supplemented with or without serum was examined. When serum was excluded from the culture medium, 26.4% (33 125 ) of the total number of embryos cultured were able to develop to the blastocysts stage using this co-culture system. This value was comparable to that obtained in a serum-supplemented co-culture system (30.7%; 39 125 ). In addition, the developmental ability of embryos that reached to the 4-cell stage or beyond at 46 to 48 h after insemination was not significantly different when the embryos were co-cultured with mouse ampullae with (38.5 vs 44.6%) or without (37.0 vs 33.8%) serum.     

Effect of soil bacteria on hyphal growth of the arbuscular mycorrhizal fungus Glomus claroideum

The effect of 46 bacterial strains isolated from tilled and non-tilled soils collected at 3 localities on the growth of intraradical hyphae of the arbuscular mycorrhizal (AM) fungusGlomus claroideum was demonstrated. A larger number of stimulatory bacterial isolates was obtained from tilled soils, but the bacteria showing the strongest stimulation of hyphal growth were isolated from a soil that had not been cultivated. Isolates obtained from hyphae of AM fungi showed no substantial stimulatory effects, but produced more uniform effects on hyphal growth than the isolates of bacteria obtained from soil. Bacterial cenoses present in 3 different soils differ significantly in their effects on AM fungi.     

Protein expression by Streptococcus uberis in co-culture with bovine mammary epithelial cells

Three strains of Streptococcus uberis isolated from dairy cows with mastitis were co-cultured with a bovine mammary epithelial cell line (MAC-T) in Dulbecco's modified Eagle's medium without fetal bovine serum. Protein profiles from culture supernatants and bacterial pellets among different treatments were compared by electrophoresis. There were proteins induced or having increased expression in both supernatant and surface-associated samples from S. uberis co-cultured with MAC-T cells. Some of these proteins were recognized by antibodies in serum obtained from a cow infected by S. uberis . In supernatant samples, there were two distinct protein bands at 35 and 36.8 kDa for all three strains of S. uberis co-cultured with MAC-T cells. These two bands were absent when bacterial protein synthesis was inhibited by chloramphenicol. This study clearly indicates that bacterial protein expression was regulated in response to co-culture with mammary epithelial cells.     

Interaction between Laccaria laccata and Trichoderma virens in co-culture and in the rhizosphere of Pinus sylvestris grown in vitro

Interactions between the ectomycorrhizal fungus Laccaria laccata and the soil fungus Trichoderma virens in co-culture and in the rhizosphere of Pinus sylvestris seedlings growing in vitro were investigated by light and scanning electron microscopy. The growth of T. virens was inhibited in co-culture. Shortened, more branched and sometimes deformed or injured hyphae of T. virens were observed in the zone of inhibition. Two-month-old mycorrhizae of P. sylvestris/ L. laccata were inoculated with a conidial suspension of T. virens and examined at intervals of 7-24 h and 2, 3, and 6 days post-inoculation (p.i.). On non-mycorrhizal roots, conidia germination was high and long hyphae formed 3 days p.i. On mycorrhizal roots, short germ tubes were observed only sporadically. At 3 days p.i., the mantle hyphae of L. laccata grew towards the conidia and coiled around them. Extremely dense coils of hyphae were found around clusters of conidia. Deformation of conidia, breaks in conidial walls and their partial degradation were observed 6 days p.i.     

Effects of co-culture and embryo number on the in vitro development of bovine embryos

It is generally accepted that culturing embryos in groups or with somatic cells improves both the yield and quality of the blastocysts obtained. The aims of this study were 1) to compare the yield and quality of the embryos obtained after culture in several number conditions and in several culture systems and 2) to assess the effect of co-culture started at various stages of embryo development. Under cell-free culture conditions (modified synthetic oviduct fluid [mSOF] supplemented with 10% fetal calf serum [FCS] 48 h post insemination, the rate of Day 10 blastocysts was lower when embryos were cultured in small groups (1 to 6 per drop) than in large groups (4 versus 23% ; P < 0.01). There was no group effect when embryos were co-cultured either with Buffalo rat liver (BRL) cells in TCM 199, or in a culture system allowing the progressive development of cumulus cells in mSOF, even if co-culture started at 66 or 114 h post insemination. However, embryos cultured singly had lower cell numbers than embryos cultured in large groups when co-culture started at 114 h post insemination. This suggests that 1) somatic cells improve the development of singly cultured bovine embryos up to the blastocyst stage after the 9-16 cell stage; 2) co-culture affects blastocyst cell number of singly cultured embryos by acting roughly between the 5-8 and the 9-16 cell stage; and 3) cooperation between embryos could replace the effect of co-culture either on the yield of blastocysts or on blastocyst cell number. Blastocysts appeared significantly earlier in co-culture with cumulus cells in mSOF than in co-culture with BRL cells in TCM 199 (detection of the blastocysts: 7.3 +/- 0.1 d post insemination with cumulus cells versus 8.1 +/- 0.1 d with BRL cells; P < 0.001) and had a significant higher number of cells (143 +/- 9 versus 85 +/- 11; P < 0.001). This system thus seems suitable for the culture of small numbers of embryos resulting from in vitro maturation and fertilization of oocytes from individual donor cows.     

Effect of 1-aminocyclopropane-1-carboxylic acid deaminase producing bacteria on the hyphal growth and primordium initiation of Agaricus bisporus

The mechanism of casing soil stimulating the primordium formation of Agaricus bisporus is not well understood so far. Our results showed that 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase (AcdS)-producing bacteria were abundant in the casing soil of A. bisporus and accounted for up to 20 % of total culturable bacteria. A. bisporus produced ACC and ethylene. The supplement of methionine increased the ACC concentrations within the hyphae, and aminooxyacetic acid displayed an opposite effect. Methionine and ACC promoted the ethylene production while CoCl₂ suppressed the production. The AcdS-producing bacterial strain Pseudomonas putida UW4 co-cultured with A. bisporus could attach to hyphae, stimulate the hyphal growth, and reduce the ethylene production of A. bisporus. Added in sterilized casing soil, it induced the primordium formation of A. bisporus. In comparison, its AcdS-deficient mutant UW4-AcdS^? displayed the opposite effects. These results indicated that the inhibitor to the primordium formation of A. bisporus was ethylene; the AcdS-producing bacteria within the casing layer cleaved ACC, lowered the ethylene level in mushroom hyphae, and relieved the inhibition of ethylene. This is a new model of the synergism between bacteria and fungi.     

Ectomycorrhizal hyphae structure components of the soil bacterial community for decreased phosphatase production

Ectomycorrhizal fungi (EMF) provide nutrients to their hosts by means of hyphae that extend beyond nutrient-depleted rhizosphere soil. Soil bacteria may compete with EMF for nutrients or may act synergistically to enhance nutrient supply to hosts. To assess the interactions between hyphae and bacteria, two types of small, sand-filled mesh bags were incubated in a Pseudotsuga menziesii/Betula papyrifera forest. The bags allowed ingrowth by EMF (35-μm mesh) or excluded hyphae (0.5-μm mesh), while allowing migration of soil bacteria. After incubation, bacteria were isolated from bags using a method to enrich for Gram-positive bacteria. Isolates were assayed for phosphatase and N-acetyl glucosaminidase (NAGase) activities to assess the potential to access organic phosphorus and nitrogen. The average phosphatase activities were higher in exclusion than ingrowth bags, while NAGase activities did not differ. Streptomyces isolates, which are expected to be strong competitors and antagonists of EMF, were more prevalent in ingrowth bags and yet had lower phosphatase activities. Furthermore, there were no indications of antagonism between fungi and Streptomyces, as there were no increases in NAGase activities in ingrowth bags. We conclude that fungal hyphae can structure components of the soil bacterial community for decreased extracellular enzyme production.     

A New Fluorescence Staining Assay for Visualizing Living Microorganisms in Soil

5- (and 6-)Sulfofluorescein diacetate (SFDA), which is converted to a fluorescent product by intracellular esterase activity, was used to stain living microorganisms, including bacteria, Saccharomyces cerevisiae, and fungi, in soil. SFDA (1 mM) dissolved in ethyl alcohol was added to an intact soil sample, and the preparation was examined with an epifluorescence microscope. Bright single cells and colonies of live bacteria were observed without interference from the autofluorescence of soil minerals and detritus. Cultured Escherichia coli was killed through heat treatment; thus, SFDA was concluded to stain only living cells. Microbial colonies obtained from natural soils and various cultured strains were tested. It was found that 151 of 154 colonies from natural soils were stained and that hyphae and spores from 1 of 28 cultured microbial strains were not stained. The SFDA method was successfully used to visualize and count bacteria in soil samples from Mount Shiga in Japan.