Mycophagous soil amoeba: Interactions with three plant pathogenic fungi

An amoeba of the order Leptomyxida was isolated from wheat take-all decline soil and was found to attack and lyse hyphae and spores of Gaeumannomyces graminis var. tritici and Phytophthora cinnamomi. The amoeba enveloped portions of hyphae of both fungi and penetrated the cell walls by means of fine holes. One-week old chlamydospores and hyphal swellings of P. cinnamomi were also attacked in this way, protoplast lysis being completed within 1 h. Hyphal fragments which could be ingested by the amoeba were lysed leaving amorphous cell debris. Three-week old chlamydospores of P. cinnamomi were enclosed within large food vacuoles and completely digested in about 20 h. Pigmented conidia of Cochliobolus sativus were transported across the substratum for up to several hours but were not perforated or lysed.     

Amoebae from a take-all suppressive soil which feed on Gaeumannomyces graminis tritici and other soil fungi

Amoebae were isolated from soil of the Waite Institute permanent pasture plot which is suppressive to take-all of wheat. Nine species of amoebae belonging to eight genera were tested for their mycophagy against Gaeumannomyces graminis var. tritici, Cochliobolus sativus and Phytophthora cinnamomi. Members of the genera, Gephyramoeba, Mayorella, Saccamoeba, Thecamoeba and an unidentified species of the order Leptomyxida, were mycophagous. Feeding of mycophagous amoebae and their ability to perforate and lyse melanized propagules of fungi are discussed.     

Fluorescence microscopy to study colonization of conidia and hyphae of Cochliobolus Sativus by soil microorganisms

Epi-fluorescence microscopy facilitated observations of the colonization of hyphae and conidia of C. sativus and other plant pathogenic fungi in soil. Of six fluorochromes examined, acridine orange and europium chelate effectively differentiated cells of bacteria and actinomycetes from the melanized fungal structures. Under u.v. radiation, the colonizing organisms fluoresced intensively while the pigmented conidia appeared dark. Periodic observations of hyphal fragments and conidia of C. sativus incubated in nonsterile soil revealed rapid colonization of their surface primarily by bacteria. Whereas up to 80% of the hyphal fragments of C. sativus were lysed after 32 days' incubation in soil, the conidia remained intact. However, disorganization of protoplasts and disappearance of conidial septa were evident after the fourth week. After 2, 3 and 5 weeks incubation of the plant pathogens in soil, no significant difference was noticed between microbial colonization of conidia on 8.0 μm pore size “Nucle-pore” membranes and those placed directly on the soil surface.     

Fine structure of a new mycophagous amoeba and its feeding on Cochliobolus sativus

An unidentified mycophagous soil amoeba is described. The pigmented soil-borne fungus Cochliobolus sativus and four other fungal species, both pigmented and hyaline, were utilized as food. Spores were ingested and lysed within digestive vacuoles by general wall erosion. This contrasts with the wall perforation mechanism described for other mycophagous amoebae. Ultrastructural studies of trophozoites showed that large quantities of electron dense granules were released into the digestive vacuoles during fungal cell lysis. These were incorporated into the amoebal protoplast. Bacteria were commonly present in the amoebal protoplasts and within digestive vacuoles. Their possible role as endosymbionts is discussed.     

Mycophagous amoebae in Australian forest soils

Twenty-seven soils, mostly from forested locations in southeastern Australia and Western Australia, were surveyed for the presence of mycophagous vampyrellid amoebae. Characteristic perforations and lysis of conidia of Cochliobolus sativus incubated in 20 of these soils indicated the presence of these amoebae. Isolations of Arachnula impatiens and an amoeba which may be a species of Theratromyxa were obtained from 15 soils. A. impatiens perforated and lysed hyphae and chlamydospores of Phytophthora cinnamomi in culture.     

Soils suppressive to Gaeumannomyces graminis var. tritici: Induction by other fungi

Rhizosphere soils were obtained from wheat plants growing in fumigated soil inoculated with one of the pathogens, Rhizoctonia solani, Gibberella zeae, Fusarium culmorum, Cochliobolus sativus and Pythium irregulare or one of the non-pathogenic fungi, Gaeumannomyces graminis var. graminis (Ggg) or a Phialophora-like fungus (Plf). Using a pot bioassay, these soils were tested for suppression of Gaeumanomyces graminis var. tritici (Ggt) and the fungus involved in the initial induction. G. zeae was the only fungus that induced suppression to Ggt and to itself. Ggg but not Plf induced suppression to Ggt although both fungi induced suppression to themselves.Fungi capable of inducing suppression of take-all have two characteristics in common, they induce suppression of themselves and their saprophytic survival is restricted to organic matter.     

Perforation and lysis of fungal spores in natural soils

Conidia of Cochliobolus sativus and five other pigmented fungi lysed when incubated in natural soil. Lysis followed perforation of the spore wall by holes of varying dia. Three possible causes of perforation were investigated, namely autolysis, mechanical puncture by soil animals and enzymatic erosion by soil micro-organisms. Results indicated that soil micro-organisms were the likely causal agents although no micro-organism able to perforate conidia has yet been isolated. Colonization of conidia by the soil microflora was studied by electron microscopy. On the basis of these direct observations, possible perforation mechanisms are suggested. Reports of perforation of fungal, plant and bacterial cell walls are briefly summarized and the perforation phenomenon discussed in relation to the biodegradation of pigmented fungal propagules in soils.     

Sorption of benzoic acid onto soil colloids and its implications for allelopathy studies

The fate of allelochemicals in the soil environment largely determines the expression of allelopathy in the natural environment. In allelopathy research, the sorption of allelochemicals onto soil particles has been less well studied than their degradation. A study was carried out to evaluate the growth of cucumber (Cucumis sativus var Marketmore 76) and radish (Raphanus sativus var Crimson giant) in soil amended with 1, 5, 10 and 20 mg l^–1 benzoic acid as model allelopathic substance. Growth of both cucumber and radish was not inhibited in soil amended with benzoic acid. A labeled study indicates that sorption of benzoic acid onto soil particles increases with concentration. Benzoic acid isotherms of both soils were non-linear, with an N value of 0.875 for a garden soil and 0.891 for a garden soil + sand, and they may explain the reason for the limited allelopathic effect of benzoic acid at concentrations often recorded in natural soil.     

Studies on soil fumigation—IV: Effects on fungi

The effect of soil fumigation with a chloropicrin-methyl bromide mixture(1:1) at 440 kg·ha^?1 on the fungal flora of a wheat-field has been investigated. Recolonization of fumigated soil and the occurrence of fungi on roots of wheat growing in fumigated and untreated soil were also followed. Very few fungi survived in fumigated soil that had been covered with polythene sheeting, but in uncovered fumigated soil some fungi survived especially at or near the soil surface.Study of recolonization of covered fumigated soil showed that some fungi, notably species of Chaetomium and Mortierella, appeared to survive fumigation and then increase in number, but many of the fungi recolonizing surface soil (2.5 cm), Alternaria, Stemphylium, Mucor, Cladosporium, Epicoccum), appeared to have come from the air. Recolonization of surface soil was not uniform; high counts were often due to the spores of one or a few fungi and samples collected a few cm apart might show different fungi in high number. In subsurface soil (5–22.5 cm) recolonization was much slower and even 117 days after fumigation the number of colonies and species of fungi was low compared with untreated soil.The common fungi on roots of plants grown in uncovered fumigated or untreated soil were very similar though initially there were fewer fungi on roots from fumigated soil. The main differences recorded were that Chaetomium species were more frequent on roots from fumigated soil and that. in general, Cylindrocarpon destruetans, Embellisia chlamydospora, species of Pythium and Rhizoctonia, and Gaeumannomyces graminis were more common on roots in untreated soil.     

Electron microscopical studies of the colonization of conidia of Cochliobolus sativus by soil microorganisms

Conidia of Cochlioholus sativus were placed onto natural soil, incubated for periods up to 100 days, recovered and examined by scanning and transmission electron microscopy. Lysis of conidia and their colonization by soil microorganisms were studied.     

Seasonal dynamics of amoebae in the root canopy of Zygophyllum dumosum in the Negev Desert, Israel

Water availability and nutrient limitation are the most important driving forces in hot desert ecosystems, determining vegetation cover and biological activity. In this study the amoeba population was determined in the upper soil layer (0-10 and 10-20 cm) under and between 4 individual Zygophyllum dumosum shrubs. The soil samples were taken from the different layer beneath the shrub and between the shrubs (control) along four seasons. Total number of amoeba was significantly higher in the Z. dumosum root canopy (2935 individual/g soil) in comparison to control samples (625 individual/g soil). Amoebal density was highest at 10-20 cm during the winter, spring and summer. In autumn, during heavy dewfall amoebal density was greatest in the top 10 cm of soil.     

Soil Vampyrellid amoebae that cause small perforations in conidia of Cochliobolus sativus

The parasitic habits of two mycophagous amoebae, members of the Vampyrellidae isolated from soil, were studied under laboratory conditions. The amoeboid organisms resembled Theratromyxa weberi and Vampyrella vorax. Both organisms lysed conidia of Cochliobolus sativus and chlamydospores o Thielaviopsis hasicola within digestive cysts. Perforations 1 μm dia or less were observed in walls of the lysed fungus spores. The mycophagous Theratromyxa sp. and V. vorax were differentiated in laboratory culture chambers on the basis of morphology, encystment, excystment and diameter of perforations produced in walls of conidia of C. sativus. Both organisms differed from a previously-described species of Vampyrella which causes large perforations and annular depressions in spore walls.     

Effects of vermicompost amendment as a basal fertilizer on soil properties and cucumber yield and quality under continuous cropping conditions in a greenhouse

Purpose

We examined the effects of vermicompost application as a basal fertilizer on the properties of a sandy loam soil used for growing cucumbers under continuous cropping conditions when compared to inorganic or organic fertilizers.

Materials and methods

A commercial cucumber (Cucumis sativus L.) variety was grown on sandy loam soil under four soil amendment conditions: inorganic compound fertilizer (750 kg/ha,), replacement of 150 kg/ha of inorganic compound fertilizer with 3000 kg/ha of organic fertilizer or vermicompost, and untreated control. Experiments were conducted in a greenhouse for 4 years, and continuous planting resulted in seven cucumber crops. The yield and quality of cucumber fruits, basic physical and chemical properties of soil, soil nutrient characteristics, and the soil fungal community structure were measured and evaluated.

Results and discussion

Continuous cucumber cropping decreased soil pH and increased electrical conductivity. However, application of vermicompost significantly improved several soil characteristics and induced a significant change in the rhizosphere soil fungal community compared to the other treatments. Notably, the vermicompost amendments resulted in an increase in the relative abundance of Ascomycota, Chytridiomycota, Sordariomycetes, Eurotiomycetes, and Saccharomycetes, and a decrease in Glomeromycota, Zygomycota, Dothideomycetes, Agaricomycetes, and Incertae sedis. Compared to the organic fertilizer treatment, vermicompost amendment increased the relative abundance of beneficial fungi and decreased those of pathogenic fungi. Cucumber fruit yield decreased yearly under continuous cropping conditions, but both inorganic and organic fertilizer amendments increased yields. Vermicompost amendment maintained higher fruit yield and quality under continuous cropping conditions.

Conclusions

Continuous cropping decreased cucumber yield in a greenhouse, but basic fertilizer amendment reduced this decline. Moreover, basal fertilizer amendment decreased beneficial and pathogenic fungi, and the use of vermicompost amendment in the basic fertilizer had a positive effect on the health of the soil fungal community.

     

Interaction between earthworms and arbuscular mycorrhizal fungi on the degradation of oxytetracycline in soils

The interactive impact of earthworms (Eisenia fetida) and arbuscular mycorrhizal fungi (Rhizophagus intraradices, AM fungi) on the degradation of oxytetracycline (OTC) in soils was studied under greenhouse conditions. Treatments included maize plants inoculated vs. not inoculated with AM fungi and treated with or without earthworms at low (1 mg kg⁻¹ soil DM) or high (100 mg kg⁻¹ soil DM) OTC rates. The root colonization rate, the hyphal density of mycorrhizae, the residual OTC concentration in soils, catalase, dehydrogenase, urease, soil microbial biomass C, Shannon–Wiener index (H) for microbial communities from T-RFLP profiles were measured at harvest. The results indicated that earthworms and AM fungi would individually or interactively enhance OTC decomposition and significantly decreased the residual OTC concentration at both high and low OTC rates. Both earthworms and AM fungi could promote the degradation of OTC by increasing soil microbial biomass C at both high and low OTC rates. The effect of soil enzyme activity and soil microbial diversity on OTC decomposition was different between high and low OTC rates. Hyphomicrobium and Bacillus cereus were dominant bacteria, and Thielavia and Chaetomium were dominant phyla of fungi at all occasions. Earthworm activity stimulated the growth of Hyphomicrobium and Thielavia, while AM fungi may stimulate B. cereus, Thielavia and Chaetomium, resulting in greater OTC decomposition. The interaction between earthworms and AM fungi in affecting the degradation of OTC may be attributed to different mechanisms, depending on soil microbial biomass, function (enzyme activity) and communities (the abundance of Hyphomicrobium, B. cereus, Thielavia and Chaetomium) in the soil.     

The invasion of pea roots, pisum sativum l., by soil microorganisms, Acanthamoeba palestinensis (reich) and pseudomonas SP

Pea roots (Pisum sativum L. cultivar Tall Sugar White) were inoculated with Pseudomonas sp., isolated from the roots of Timothy grass (Phleum pratensis L.). Microscopic examination showed that the bacterium had invaded many epidermal and outer cortical cells of the root. The presence of the soil amoeba, Acanthamoeba palestinensis (Reich), which readily ingests Pseudomonas sp., did not alter the root damage. The amoebae were also found in the epidermis and outer cortex of pea roots. No amoebae were found inside pea roots when Pseudomonas sp. was absent. Bacterial invasion also occurred in pea roots grown in garden soil previously sterilized by γ-irradiation and inoculated with Pseudomonas sp., but not in the same soil inoculated and unsterilized. Timothy grass roots were similarly uninfected in inoculated unsterilized garden soil.     

Suppression of other soil microorganisms by mycelium of arbuscular mycorrhizal fungi in root-free soil

The influence of mycelium of two arbuscular mycorrhizal (AM) fungi, Glomus intraradices and Glomus mosseae, on other soil microorganisms, was examined in root-free soil with and without organic substrate amendment in terms of cellulose. The AM fungi were grown in symbiosis with cucumber in a compartmented growth system, which allowed AM fungal external mycelium to grow into root-free compartments. The fungicide Benomyl was applied to the root-free compartments to create an alternative non-mycorrhizal control treatment. Whole cell biomarker fatty acids were employed to quantify different groups of soil microorganisms including the two AM fungi. Abundance of most microbial groups were reduced by external mycelium of both AM fungi, though differential effects on the microbial community composition were observed between the two AM fungi as revealed from principal component analysis. Inhibition of other soil microorganisms was more pronounced in root-free soil with mycelium of G. mosseae than with mycelium of G. intraradices. In general, cellulose increased the amount of biomarker fatty acids of most groups of soil microorganisms, but cellulose did not affect the influence of AM fungi on other soil microorganisms. Benomyl suppressed growth of the external mycelium of the two AM fungi and had limited non-target effects on other microbial groups. In conclusion, our results show differential effects of external mycelium of AM fungi on other soil microbial communities, though both AM fungi included in the study overall inhibited most microbial groups as examined using whole cell biomarker fatty acids.     

Origin and properties of β-glucosidase activity of tomato-field soil

The distribution of β-glucosidase activity in a tomato-field soil was examined. Of the total activity found, > 50% was in the < 2mm soil fraction, 20% in the organic debris and a significant proportion in roots. In an attempt to determine the origin of β-glucosidase in this soil, the properties of β-glucosidase of various fungal and bacterial isolates from the soil, and of plant materials, were studied.Selective inhibition of bacterial or fungal growth in re-moistened, over-dried, inoculated soil indicated that fungi were a more important source of β-glucosidase in this soil. Monierella, Actinomucor, Coniochaeta and Penicillium were the principal fungi isolated from the soil by the dilution-plate technique, comprising over 60% of the total isolates. Remoistened oven-dried soil, inoculated with Mortierella and Actinomucor spp exhibited higher β-glucosidase activity after incubation than did soil inoculated with other strains.The β-glucosidase activity of extracts from cultured fungal strains had similar pH optima and Q₁₀ values to those of soil extracts. The β-glucosidase of extracts from isolates of bacteria and actinomycetes had similar Q₁₀ values, but higher pH optima, than did that of soil extracts.These results indicate that fungi, mainly some of the mucoraceous fungi, may be the primary source of β-glucosidase in tomato-field soil.     

Infection of Mayorella penardi (Gymnamoebia) in oak-hornbeam forest soil by the ectoparasitic fungus Amoebophilus simplex (Zygomycota)

During research on naked amoebae in terrestrial habitats in the oak-hornbeam forests of Malé Karpaty Mts. (Western Slovakia), a mass fungal infection in Mayorella penardi Page, 1972 (Euamoebida: Paramoebidae) was observed. In one of the 243 soil and moss samples examined a remarkably high percentage (80%) of amoebae were infected by the ectoparasitic soil fungus Amoebophilus simplex Barron, 1983 (Zygomycota: Zoopagales), as revealed by detailed light microscopical observations of infected specimens. The parasitic fungus displayed characteristic cylindrical conidia (length 10 μm, breadth 3 μm) projecting out from the posterior part of the amoeba cell. Infecting conidium frequently produced a chain of two to three conidia and, rarely, secondary chains from the distal end. Two to four infections in a single amoeba were frequently observed. From the present study, and from earlier records, it can be deduced that Amoebophilus simplex is specialized in parasitizing mayorellid amoebae, maybe even Mayorella penardi exclusively. Two other species (Korotnevella diskophora and Korotnevella stella) from the family Paramoebidae were recorded in the same sample, but neither of them showed signs of infection.     

Co-remediation of DDT-contaminated soil using white rot fungi and laccase extract from white rot fungi

Purpose

2,2-Bis(p-chlorophenyl)-1,1,1-trichloroethane (DDT), one of the most widely used organochlorine pesticides in soil, was banned in the 1970s for agricultural use because of its detrimental impacts on wildlife and harmful effects on human health via the food chain. However, high levels of DDT are frequently detected in agricultural soils in China. Considering this situation, this study investigated the use of white rot fungi and laccase derived from white rot fungi to co-remediate DDT-contaminated soil.

Materials and methods

A culture of white rot fungi was used to inoculate soil samples and also to extract laccase from. Soil was contaminated with four components of DDT (p,p′-DDE, o,p′-DDT, p,p′-DDD, and p,p′-DDT). Individual DDT components and the sum of the DDT components (p,p′-DDE, o,p′-DDT, p,p′-DDD, and p,p′-DDT—collectively referred to as DDTs) were both analyzed by GC at various stages during the incubation period. The efficacy of co-remediating DDT-contaminated soil using white rot fungi and laccase was tested by investigating how degradation varied with varying amounts of white rot fungi, sterilizing soil, temperature, soil pH, concentrations of DDT, and concentration of the heavy metal ion Cd²⁺.

Results and discussion

“”It was concluded that the reduction of DDTs in soil using white rot fungi and laccase was higher than reduction using only white rot fungi or laccase by nearly 14 and 16 %, respectively. Five milliliters fungi per 15 g soil and 6 U laccase per gram soil were the optimal application rates for remediation, as shown by a reduction in DDTs of 66.82 %. The difference in the reduction of individual DDT components and DDTs between natural and sterilized soils was insignificant. The optimal temperature and pH in the study were 28 °C and 4.5, respectively. In addition, reduction of individual DDT components and DDTs increased with increasing concentrations of DDT and decreased with increasing concentrations of Cd²⁺.

Conclusions

Compared with the remediation of DDT using only white rot fungi or laccase, the co-remediation of DDT using white rot fungi and laccase degraded DDT in soil more rapidly and efficiently; the highest reduction of DDTs was 66.82 %.     

Fraxinus-Glomus-Pisolithus symbiosis: Plant growth and soil aggregation effects

It has been established that arbuscular mycorrhizal (AM) fungi are involved in the conservation of soil structure. However, the effect of ectomycorrhizal (EM) fungi alone or in interaction with AM fungi in soil structure has been much less studied. This experiment evaluated EM and AM fungi effects on soil aggregation and plant growth. Ash plants (Fraxinus uhdei) were grown in pots, and were inoculated with Glomus intraradices and Pisolithus tinctorius separately but also in combination. Our results showed that F. uhdei established a symbiotic association with EM and AM fungi, and that these organisms, when interacting, showed synergistic and additive effects on plant growth compared to singly inoculated treatments. EM and AM fungi prompted changes in root morphology and increased water-stable aggregates. AM fungi affect mainly small-sized macroaggregates, while EM and EM-AM fungi interaction mainly affected aggregates bigger than 0.5 mm diameter. These results suggest that ectomyccorrhizal as well as arbuscular mycorrhizal fungi should be considered in restoration programs with Fraxinus plants.