Common Soil Fungi Research Articles

Linking ex planta fungi with their endophytic stages: Perisporiopsis, a common leaf litter and soil fungus, is a frequent endophyte of Hevea spp. and other plants

Abstract It has been hypothesized that endophytic fungi originate from local environmental sources, and endophyte identity and composition can be predicted based on surrounding ex planta fungi. As part of a pilot study to characterize endophytes and ex planta fungi (e.g. saprotrophs) of wild Hevea trees, endophytes were isolated with almost identical ITS sequences, but these were unidentifiable using GenBank database. Along with the endophytes, ex planta fungi on Hevea leaves were also collected at the sampling sites. The objective of this study was to determine if selected unknown Hevea endophytes could be linked with the ex planta fungi using DNA sequences. Methods included BLAST searches, phylogenetic analysis, and morphological characterizations of the endophytes and saprotrophs. Results show that the BLAST-unidentifiable Hevea endophytes belong in the fungal genus Perisporiopsis (Parodiopsidaceae, Dothideomycetes, Ascomycota). In addition, this Hevea endophyte is at least 98 % identical to >20 other sequences from GenBank, demonstrating that Perisporiopsis is a common endophyte from various plants, and a leaf litter and soil fungus. Connecting a DNA sequence with a name that has biological meaning enables microbial (i.e. fungal) biologists to make more accurate inferences about the functional ecology of a particular species in a community or ecosystem.

Soils of a Mediterranean hot spot of biodiversity and endemism (Sardinia, Tyrrhenian Islands) are inhabited by pan‐European, invasive species of Hypocrea/Trichoderma

We have used a Mediterranean hot spot of biodiversity (the Island of Sardinia) to investigate the impact of abiotic factors on the distribution of species of the common soil fungus Trichoderma. To this end, we isolated 482 strains of Hypocrea/Trichoderma from 15 soils comprising undisturbed and disturbed environments (forest, shrub lands and undisturbed or extensively grazed grass steppes respectively). Isolates were identified at the species level by the oligonucleotide BarCode for Hypocrea/Trichoderma (TrichOKEY), sequence similarity analysis (Trichoblast) and phylogenetic inferences. The majority of the isolates were positively identified as pan-European and/or pan-global Hypocrea/Trichoderma species from sections Trichoderma and Pachybasium, comprising H. lixii/T. harzianum, T. gamsii, T. spirale, T. velutinum, T. hamatum, H. koningii/T. koningii, H. virens/T. virens, T. tomentosum, H. semiorbis, H. viridescens/T. viridescens, H. atroviridis/T. atroviride, T. asperellum, H. koningiopsis/T. koningiopsis and Trichoderma sp. Vd2. Only one isolate represented a new, undescribed species belonging to the Harzianum-Catoptron Clade. Internal transcribed spacer sequence analysis revealed only one potentially endemic internal transcribed spacer 1 allele of T. hamatum. All other species exhibited genotypes that were already found in Eurasia or in other continents. Only few cases of correlation of species occurrence with abiotic factors were recorded. The data suggest a strong reduction of native Hypocrea/Trichoderma diversity, which was replaced by extensive invasion of species from Eurasia, Africa and the Pacific Basin.

Alternative reproductive strategies of Hypocrea orientalis and genetically close but clonal Trichoderma longibrachiatum, both capable of causing invasive mycoses of humans

The common soil fungus Trichoderma (teleomorph Hypocrea, Ascomycota) shows increasing medical importance as an opportunistic human pathogen, particularly in immunocompromised and immunosuppressed patients. Regardless of the disease type and the therapy used, the prognosis for Trichoderma infection is usually poor. Trichoderma longibrachiatum has been identified as the causal agent in the majority of reported Trichoderma mycoses. As T. longibrachiatum is very common in environmental samples from all over the world, the relationship between its clinical and wild strains remains unclear. Here we performed a multilocus (ITS1 and 2, tef1, cal1 and chit18-5) phylogenetic analysis of all available clinical isolates (15) and 36 wild-type strains of the fungus including several cultures of its putative teleomorph Hypocrea orientalis. The concordance of gene genealogies recognized T. longibrachiatum and H. orientalis to be different phylogenetic species, which are reproductively isolated from each other. The majority of clinical strains (12) were attributed to T. longibrachiatum but three isolates belonged to H. orientalis, which broadens the phylogenetic span of human opportunists in the genus. Despite their genetic isolation, T. longibrachiatum and H. orientalis were shown to be cosmopolitan sympatric species with no bias towards certain geographical locations. The analysis of haplotype association, incongruence of tree topologies and the split decomposition method supported the conclusion that H. orientalis is sexually recombining whereas strict clonality prevails in T. longibrachiatum. This is a rare case of occurrence of sexual reproduction in opportunistic pathogenic fungi. The discovery of the different reproduction strategies in these two closely related species is medically relevant because it is likely that they would also differ in virulence and/or drug resistance. Genetic identity of environmental and clinical isolates of T. longibrachiatum and H. orientalis suggests the danger of nosocomial infections by Hypocrea/Trichoderma and highlights the need for ecological studies of spore dispersal as source of invasive human mycoses.

Aspergillus flavus: the major producer of aflatoxin

SUMMARY Aspergillus flavus is an opportunistic pathogen of crops. It is important because it produces aflatoxin as a secondary metabolite in the seeds of a number of crops both before and after harvest. Aflatoxin is a potent carcinogen that is highly regulated in most countries. In the field, aflatoxin is associated with drought-stressed oilseed crops including maize, peanut, cottonseed and tree nuts. Under the right conditions, the fungus will grow and produce aflatoxin in almost any stored crop seed. In storage, aflatoxin can be controlled by maintaining available moisture at levels below that which will support growth of A. flavus. A number of field control measures are being utilized or explored, including: modification of cultural practices; development of resistant crops through molecular and proteomic techniques; competitive exclusion using strains that do not produce aflatoxin; and development of field treatments that would block aflatoxin production. Aspergillus flavus Link (teleomorph unknown) kingdom Fungi, phyllum Ascomycota, order Eurotiales, class Eurotiomycetes, family Trichocomaceae, genus Aspergillus, species flavus. Aspergillus flavus has a broad host range as an opportunistic pathogen/saprobe. It is an extremely common soil fungus. The major concern with this fungus in agriculture is that it produces highly carcinogenic toxins called aflatoxins which are a health hazard to animals. In the field, A. flavus is predominantly a problem in the oilseed crops maize, peanuts, cottonseed and tree nuts. Under improper storage conditions, A. flavus is capable of growing and forming aflatoxin in almost any crop seed. It also is a pathogen of animals and insects. In humans it is predominantly an opportunistic pathogen of immunosuppressed patients. http://www.aspergillusflavus.org, http://www.aflatoxin.info/health.asp, plantpathology.tamu.edu/aflatoxin, http://www.aspergillus.org.uk.

Dose- and time - dependent abundance of some soil-microbes at chlorsulfuron herbicide application

Introduction Intensification of crop production practices, has enhanced the pesticide use in the agriculture. Among the xenobiotics the herbicides are the most frequently applied agrochemicals (KECSKES, 1976), for improve the yield-production and quality. Apart from the desired toxic effects of some herbicides on weeds, however, the treatments may also have adverse effects on non-target organisms in the soil, in plants (SZENTPETERY et al. 2005, JOLANKAI et al. 2006) and the rhizosphere, like to other results on the GMO-s (VILLANYI et al. 2006). Either the structure or the function of microbial communities may be influenced by pesticide applications. In case of some herbicides the fast degradation, metabolisation is also known by several soil microbes (KATAI et al. 2002). Mechanisms of such toxic or metabolic effects are highly depending on the types and rates of pesticides, the chemical formulations and also the microbial species, exposed. Herbicides are generally most toxic to phototrophic microorganisms through the disruption of photosynthesis (DELORENZO et al. 2000). Beside the reduced applyed rates, the “new generation” herbicides are offering less harmful effects to the beneficial soil-living organisms, by keeping its weed-control capacity throughout the whole vegetation periods (NAGY, 1989). In the agriculture in comparison with conventional herbicides, the Glean (with a 75 % of chlorsulfuron content) can be still 100 times more efficient against the weeds. Regarding the soil biological consequences, an active role in the detoxification of chlorsulfuron by moulds and Actinomycetes was reported in an earlier study (JOSHI et al., 1985). The biodegradation pathway of chlorsulfuron, operated by Aspergillus niger, a common soil fungus was studied under laboratory conditions by BOSCHIN et al. (2003). The fate of chlorsulfuron herbicid could be potentially managed by some soil-microbes in the soils. Several microbial groups from the soil was studied, therefore in their sensitivity to the various doses of the chlorsulfuron (Glean 75) herbicide. There was a timedependent effect considered, when the 3-weeks and 3-months abundance was also assessed in a soil-incubation model-experiment. A simplified, rapid and economic plating method was developed to estimate the abundance of specific microbes from the herbicide treated soil at different application rates.

Molecular Detection of Plasmodiophora brassicae, Causal Agent of Clubroot of Crucifers, in Plant and Soil.

Clubroot of crucifers, caused by Plasmodiophora brassicae, recently has been identified in canola (Brassica napus) fields in Alberta, Canada. An effective strategy for managing the disease is to avoid planting cruciferous crops in P. brassicae-infested soil, because the pathogen produces resting spores that can remain infectious for many years. A simple, one-step polymerase chain reaction (PCR) protocol was developed to detect the pathogen in plant and soil samples. The primers TC1F and TC1R, based on a P. brassicae partial 18S ribosomal RNA (rRNA) gene sequence from GenBank, yielded a 548-bp product in the optimized PCR. A second pair of primers, TC2F and TC2R, which amplified a fragment of the 18S and internal transcribed spacer (ITS) 1 regions of the rDNA repeat, also was tested and produced a 519-bp product. Neither set of primers amplified any DNA fragment from noninfected plant hosts, noninfested soil, or common soil fungi and bacteria tested in this study. Quantities of 100 fg or less of total P. brassicae DNA, or 1 × 103 resting spores per gram of soil, could be detected consistently using these primers and PCR protocol, corresponding to an index of disease of 11% or lower when the soil was bioassayed. The protocol also enabled detection of P. brassicae in symptomless root tissue 3 days after inoculation with the pathogen. Therefore, the PCR assay described in this study could provide a reliable diagnosis for routine detection of P. brassicae in plant and soil materials in a specific and rapid manner.

Bioaccumulation and secondary gold nugget formation

Experimental evidence suggests that microorganisms are important in the cycling of Au (1). Recent microcosm studies with auriferous soils from Australian mine sites have shown that the resident microbiota are capable of solubilising up to 80 wt.% of the Au contained in these soils (1). A number of common soil bacteria, actinomycetes, archaea, and fungi have been shown to precipitate Au(I/III)-complexes under a wide range of experimental conditions (2). Morphological evidence for the occurrence of micrometre sized bacterioform structures on secondary Au grains has been reported in earlier studies (3). However, neither the biological origin of these structures nor associated organisms or processes have been identified. Gold grains for this study were obtained from the auriferous soils overlying mineralisation at the Tomakin Parkand the Hit or Miss Gold Mine in moderate New South Wales and tropical Queensland, respectively. The grains were 200 to 2200 μm in diameter and generally showed little sign of physical transport. Analyses of gold grain digests that amounted to an average fineness of 99.2% established their secondary origin. Scanning electron microscopy (SEM) revealed bacterioform on untreated grains from both sites. The presence of active bacterial biofilms on the surface of Au grains was confirmed using confocal stereo laser microscopy (CSLM) combined with nucleic acid staining. Isolation, amplification and separation of 16S ribosomal DNA from these biofilms and identification of the organisms present using DNA sequence analysis showed that unique, sitespecific bacterial communities are associated with these Au grains that differ from those dominating the surrounding soil. 16S ribosomal DNA clones bearing 99 % similarity to a known metallophilic bacterium are present on all 16S rDNA positive grains from both locations. The ability of this metallophilic bacterium to accumulate AuCl4 complexes was tested under in-vitro conditions, and biologically active cells typically accumulated twice as much Au as lysed cell preparations. These results indicate that bacteria that are associated with Au grains may be able to actively reduce AuCl4 and accumulate metallic Au, and thus contribute to the authigenic formation of secondary Au nuggets in the regolith.

Detection of Two Fungal Biocontrol Agents against Root-knot Nematodes by RAPD Markers

The strain ZK7 of Pochonia chlamydosporia var. chlamydosporia and IPC of Paecilomyces lilacinus are highly effective in the biological control against root-knot nematodes infecting tobacco. When applied, they require a specific monitoring method to evaluate the colonization and dispersal in soil. In this work, the randomly amplified polymorphic DNA (RAPD) technique was used to differentiate between the two individual strains and 95 other isolates, including isolates of the same species and common soil fungi. This approach allowed the selection of specific fragments of 1.2 kb (Vc1200) and 2.0 kb (Vc2000) specific for ZK7, 1.4 kb (P1400) and 0.85 kb (P850) specific for IPC, using the random Primers OPL-02, OPD-05, OPD-05 and OPC-11, respectively. These fragments were cloned, sequenced, and used to design sequence-characterized amplification region (SCAR) primers specific for the two strains. In classical polymerase chain reaction (PCR), with serial dilution of ZK7 and IPC pure culture DNAs template, the detection limits of these oligonucleotide SCAR-PCR primers were found to be 10, 1000, 500, 100 pg, respectively. In the dot blotting, digoxigenin (DIG)-labeled amplicons from these four primers specifically recognized the corresponding fragments in the DNAs template of these two strains. The detection limit of these amplicons were 0.2, 0.2, 0.5, 0.5 mug, respectively.

Biotransformation of 3-Methyl-4-nitrophenol, a Main Product of the Insecticide Fenitrothion, by Aspergillus niger

Biotransformation of the environmental pollutant 3-methyl-4-nitrophenol (MNP), a newly characterized estrogenic chemical, and the primary breakdown product of the heavily used insecticide fenitrothion was investigated using a common soil fungus. In 96 h, daily culture sacrifice, extraction, and analysis showed that the filamentous fungus, Aspergillus niger VKM F-1119, removed more than 85% of the MNP present in solution (original concentration = 25 mg/L), mostly through biodegradation. Additionally, in 16-day time-course studies, A. niger was capable of biotransformation of MNP at concentrations as high as 70 mg/L. Gas chromatography mass spectroscopy (MS) analyses of culture fluid extracts indicated the formation of four metabolites: 2-methyl-1,4-benzenediol, 4-amino-3-methylphenol, and two singly hydroxylated derivatives of MNP. Culture scale up and metabolite analysis by liquid chromatography MS resulted in the confirmation of the original metabolites plus the detection of an azo derivative metabolite that has not been previously reported before during MNP biodegradation by any micro-organisms.

Taking the Fungal Highway: Mobilization of Pollutant-Degrading Bacteria by Fungi

The capacity of fungi to serve as vectors for the dispersion of pollutant-degrading bacteria was analyzed in laboratory model systems mimicking water-saturated (agar surfaces) and unsaturated soil environments (glass-bead-filled columns). Two common soil fungi (Fusarium oxysporum and Rhexocercosporidium sp.) forming hydrophilic and hydrophobic mycelia, respectively, and three polycyclic aromatic hydrocarbon degrading bacteria (Achromobacter sp. SK1, Mycobacterium frederiksbergense LB501TG, and Sphingomonas sp. L138) were selected based on the absence of mutual antagonistic effects. It was shown that fungal hyphae act as vectors for bacterial transport with mobilization strongly depending on the specific microorganisms chosen: The motile strain Achromobacter sp. SK1 was most efficiently spread along hyphae of hydrophilic F. oxysporum in both model systems with transport velocities of up to 1 cm d(-1), whereas no dispersion of the two nonmotile strains was observed in the presence of F. oxysporum. By contrast, none of the bacteria was mobilized along the hydrophobic mycelia of Rhexocercosporidium sp. growing on agar surfaces. In column experiments however, strain SK1 was mobilized by Rhexocercosporidium sp. It is hypothesized that bacteria may move by their intrinsic motilitythrough continuous (physiological) liquid films forming around fungal hyphae. The results of this study suggest that the specific stimulation of indigenous fungi may be a strategy to mobilize pollutant-degrading bacteria leading to their homogenization in polluted soil thereby improving bioremediation.

Fatty acid composition and change in Collembola fed differing diets: identification of trophic biomarkers

To assess the potential of fatty acid (FA) compositions to act as biomarkers in the soil food web, two species of Collembola, Folsomia candida and Proisotoma minuta, were switched to four possible diets: Cladosporium cladosporioides (a common soil fungus), Panagrellus redivivus (a bacteria feeding nematode), Zea mays (maize) and Alnus glutinosa (alder). The change in FA content of the Collembola was observed over the following 39 days. The four diets produced significant shifts in the FA compositions of the Collembola, with P. redivivus causing the most extreme changes; Collembola fed P. redivivus gained complex FA compositions similar to those of the nematode diet. Changes in the relative abundances of some FAs were found to follow negative exponential curves, as the components either accumulated in, or were removed from, the FA pool in the Collembola; abundance half-lives varied between 0.5 and 22.4 days, indicating that Collembolan FA compositions changed readily with the input of new exogenous components. The results demonstrate that Collembolan FA compositions are influenced by diet, and that the abundances of FAs such as i15:0, i17:0 and 18:1(n-7) may be used as biomarkers of nematode consumption by Collembola. In contrast, the C 20 polyunsaturated FAs cannot be used as biomarkers for nematode predation as Collembola possess the ability to biosynthesise high abundances of these compounds when not provided by the diet.

A DNA-based assay identifies Batrachochytrium dendrobatidis in amphibians.

Chytridiomycosis caused by Batrachochytrium dendrobatidis (Chytridiomycota) has been implicated in declines of amphibian populations on four continents. We have developed a sensitive and specific polymerase chain reaction-based assay to detect this pathogen. We isolated B. dendrobatidis from captive and wild amphibians collected across North America and sequenced the internal transcribed spacer regions of the rDNA cassette of multiple isolates. We identified two primers (Bd1a and Bd2a) that are specific to B. dendrobatidis under amplification conditions described in this study. DNA amplification with Bd1a/Bd2a primers produced a fragment of approximately 300 bp from B. dendrobatidis DNA but not from DNA of other species of chytrids or common soil fungi. The assay detected 10 zoospores or 10 pg of DNA from B. dendrobatidis and detected infections in skin samples from a tiger salamander (Ambystoma tigrinum), boreal toads (Bufo boreas), Wyoming toads (Bufo baxteri), and smooth-sided toads (Bufo guttatus). This assay required only small samples of skin and can be used to process a large number of samples.

Effects of fungal food quality and starvation on the fatty acid composition of Protaphorura fimata (Collembola)

The lipid pattern of animals is influenced by species, life stage, environmental conditions and diet. We investigated the effects of food quality and starvation on the phospholipid (PLFA) and neutral lipid (NLFA) fatty acid pattern of the collembolan Protaphorura fimata. Collembolans were fed with two common soil fungi, Agrocybe gibberosa and Chaetomium globosum, of which the cellular lipid composition was analysed. A. gibberosa was grown on agar with different nitrogen contents, resulting in altered fatty acid patterns and C:N ratios, i.e. fungi of different food quality. Collembolans did not mirror the lipid composition of the fungal diet as the pattern of major NLFAs in P. fimata was vice versa. Presumably, altered food quality of fungi caused compensatory responses by the collembolans, thereby diminishing the fungal signal. In a further experiment P. fimata (previously maintained with C. globosum) was kept without food for up to 4 weeks. Starvation resulted in a decline in the total amount of NLFAs; however, it did not affect the fatty acid pattern, indicating that NLFAs were degraded indiscriminately. Generally, the PLFA profile of the collembolans changed only slightly due to variations in diet quality or starvation.

COMPOUNDS FIGHT ARTERY NARROWING

CIRCULAR COMPOUNDS FROM a common soil fungus show promise in treating Researchers in Japan discovered that beauveriolides I and III, 13-membered cyclic depsipeptides made of three amino acids and a fatty acid, inhibit the buildup of macrophage lipids in a mouse with atherosclerotic arteries { Proc. Natl. Acad. Sci. USA , 101 , 737 (2004)}. Lipid buildup is one of the essential steps of plaque formation, which leads to artery narrowing. The scientists further elucidated the fungal compounds' specific target: the enzyme acyl-CoA:cholesterol acyltransferase (ACAT). Coauthor Hiroshi Tomoda, a professor of lipid biochemistry at Kitasato University, notes that plays multiple roles in our body: cholesterol absorption from the intestine, lipoprotein production in the liver, and atherosclerogenesis in the artery. Therefore, researchers believe that ACAT is an ideal target for treatment of atherosclerosis. Other ACAT inhibitors have already been identified. The major significance of the new ...

Biodegradation of Chlorsulfuron and Metsulfuron‐Methyl by Aspergillus niger in Laboratory Conditions

Two sulfonylurea herbicides, chlorsulfuron and metsulfuron‐methyl, were studied under laboratory conditions, in order to elucidate the biodegradation pathway operated by Aspergillus niger, a common soil fungus, which is often involved in the degradation of xenobiotics. HPLC‐UV was used to study the kinetic of degradation, whereas LC‐MS was used to identify the metabolites structure. In order to avoid the chemical degradation induced by a decrease in pH, due to the production of citric acid by the fungus, the experiments were performed in a buffered neutral medium. No significant degradation for both compounds was observed in mineral medium with 0.2% sodium acetate. On the contrary, in a rich medium, after 28 days the degradations, chemical degradation excluded, were about 30% for chlorsulfuron and 33% for metsulfuron‐methyl. The main microbial metabolites were obtained via cleavage of the sulfonylurea bridge. In addition the fungus seems to be able to hydroxylate the aromatic ring of chlorsulfuron. In the case of metsulfuron‐methyl the only detected metabolite was the triazine derivative, while the aromatic portion was completely degraded. Finally, the demethylation of the methoxy group on the triazine ring, previously observed with a Pseudomonas fluorescens strain, was not observed with A. niger.

Finally, proof of weapons of mass destruction.

Allelopathy (one species' use of chemicals to harm other species) may be a key ingredient in successful invasions of alien plants into established communities. Bais et al. show that in response to elicitation by common soil fungi, spotted knapweed (Centaurea maculosa) launches an ineffective defense against the fungi that results in extensive collateral damage to neighboring plants. Specifically, the flavonoid (-)-catechin, released from the roots of knapweed, produces a massive reactive oxygen species (ROS)-mediated oxidative burst, Ca2+ signaling, and rapid cell death in those unadapted native species whose habitats the plant regularly invades. The roots release both (+) and (-) enantiomers, but only the (-) enantiomer functions as a weapon of mass destruction; the (+) enantiomer inhibits the growth of numerous common soil-borne bacterial pathogens. Eliciting apoptotic response for a competitive advantage is an example of signal cross-talk between the genomes of interacting organisms and highlights how the internal signaling of one organism can be used by others to adjust their phenotypes in an adaptive manner. The study provides strong circumstantial evidence for an allelopathic interaction, but the genetic manipulation of (-)-catechin release would allow researchers to determine if these responses occur in nature. Precise genetic control over the release of secondary metabolites from plants would benefit ecological research.

Finally, Proof of Weapons of Mass Destruction

Allelopathy (one species' use of chemicals to harm other species) may be a key ingredient in successful invasions of alien plants into established communities. Bais et al. show that in response to elicitation by common soil fungi, spotted knapweed (Centaurea maculosa) launches an ineffective defense against the fungi that results in extensive collateral damage to neighboring plants. Specifically, the flavonoid (–)-catechin, released from the roots of knapweed, produces a massive reactive oxygen species (ROS)-mediated oxidative burst, Ca2+ signaling, and rapid cell death in those unadapted native species whose habitats the plant regularly invades. The roots release both (+) and (–) enantiomers, but only the (–) enantiomer functions as a weapon of mass destruction; the (+) enantiomer inhibits the growth of numerous common soil-borne bacterial pathogens. Eliciting apoptotic response for a competitive advantage is an example of signal cross-talk between the genomes of interacting organisms and highlights how the internal signaling of one organism can be used by others to adjust their phenotypes in an adaptive manner. The study provides strong circumstantial evidence for an allelopathic interaction, but the genetic manipulation of (–)-catechin release would allow researchers to determine if these responses occur in nature. Precise genetic control over the release of secondary metabolites from plants would benefit ecological research.

DP003 Molecular identification of Nocardia species: Establishing of reporting criteria for 16S rDNA sequence-based diagnosis

Soil and litter fungi can colonise and decompose many natural materials, including highly resilient proteinaceous compounds of animal origin. The shells of terrestrial gastropods are formed from such a compound (conchiolin) combined with inorganic calcium carbonate. In this study, we investigated fungal communities colonising empty shells of the common terrestrial gastropod Cepaea hortensis. Shells were exposed on the surface of litter from four different forest types (alder alluvial, oak-hornbeam, peat-bog pine and scree forest) and the fungi were surveyed and identified in four 3-month periods. We found 27 fungal species, one fungus forming mycelial cords, seven types of sterile mycelium and streptomycetes colonising the shells. The most frequent fungal species identified were common soil fungi. Multivariate analysis revealed a significant effect of the litter type on the fungal community. Humidity and pH at the locality are likely to be more important for fungal communities than the proteinaceous material of the shell.

Detection of the mycoparasite Stachybotrys elegans, using primers with sequence- characterized amplification regions in conventional and real-time PCR

A specific and sensitive polymerase chain reaction (PCR) assay was developed for the detection of Stachybotrys elegans, a potential biological control agent and mycoparasite of Rhizoctonia solani. A pair of primers (SE-13F and SE-13R) with sequence-characterized amplification regions was designed and assayed in conventional and real-time PCR to detect S. elegans, in pure cultures and in field soil samples. Following conventional PCR amplification, a product of 880 base pairs was amplified from DNA of all isolates belonging to S. elegans. No product was amplified from DNA of isolates belonging to other species of Stachybotrys, common soil fungi, or bacteria, or from DNA of plant tissue, confirming the specificity of the primers for S. elegans. Stachybotrys elegans was also detected and quantified in field soils, 2 days after its inoculation, using real-time PCR conjugated with the fluorescent SYBR® Green I dye. The assay is reliable and has detected as little as 150 ng of DNA per gram of natural soil. The results of the assay were compared with those of the number of colony-forming units of S. elegans per gram of soil recovered from the same soils. The potential of these specific primers in quantitative PCR assays will facilitate the study of the distribution of the fungus in the soil in field studies of biological control of R. solani.

Mycelial colonization by bradyrhizobia and azorhizobia.

This study examines mycelial colonization of common soil fungi by bradyrhizobia and an azorhizobial strain, resulting in the forming of biofilms. The effects of the fungal exudates on a bradyrhizobial strain have also been investigated. Bradyrhizobia gradually colonized the mycelia for about 18 days, after which the biofilm structures collapsed with the release of the rhizobial cell clusters to the culture medium. The azorhizobial strain showed differential colonization of the mycelia. In general, there were no considerable mycotoxin effects of the fungal exudates on the bradyrhizobial strain used, instead the rhizobial strain utilized the exudates as a source of nutrition. This study indicates that the present microbial association with biofilm formation has important implications in the survival of rhizobia under adverse soil conditions devoid of vegetation. Further, it could have developed an as yet unidentified nitrogen fixing system that could have contributed to the nitrogen economy of soils.