The cysteine rich necrotrophic effector SnTox1 produced by Stagonospora nodorum triggers susceptibility of wheat lines harboring Snn1

The wheat pathogen Stagonospora nodorum produces multiple necrotrophic effectors (also called host-selective toxins) that promote disease by interacting with corresponding host sensitivity gene products. SnTox1 was the first necrotrophic effector identified in S. nodorum, and was shown to induce necrosis on wheat lines carrying Snn1. Here, we report the molecular cloning and validation of SnTox1 as well as the preliminary characterization of the mechanism underlying the SnTox1-Snn1 interaction which leads to susceptibility. SnTox1 was identified using bioinformatics tools and verified by heterologous expression in Pichia pastoris. SnTox1 encodes a 117 amino acid protein with the first 17 amino acids predicted as a signal peptide, and strikingly, the mature protein contains 16 cysteine residues, a common feature for some avirulence effectors. The transformation of SnTox1 into an avirulent S. nodorum isolate was sufficient to make the strain pathogenic. Additionally, the deletion of SnTox1 in virulent isolates rendered the SnTox1 mutated strains avirulent on the Snn1 differential wheat line. SnTox1 was present in 85% of a global collection of S. nodorum isolates. We identified a total of 11 protein isoforms and found evidence for strong diversifying selection operating on SnTox1. The SnTox1-Snn1 interaction results in an oxidative burst, DNA laddering, and pathogenesis related (PR) gene expression, all hallmarks of a defense response. In the absence of light, the development of SnTox1-induced necrosis and disease symptoms were completely blocked. By comparing the infection processes of a GFP-tagged avirulent isolate and the same isolate transformed with SnTox1, we conclude that SnTox1 may play a critical role during fungal penetration. This research further demonstrates that necrotrophic fungal pathogens utilize small effector proteins to exploit plant resistance pathways for their colonization, which provides important insights into the molecular basis of the wheat-S. nodorum interaction, an emerging model for necrotrophic pathosystems.     

Detection and identification of bacterial endosymbionts in arbuscular mycorrhizal fungi belonging to the family Gigasporaceae

Intracellular bacteria have been found previously in one isolate of the arbuscular mycorrhizal (AM) fungus Gigaspora margarita BEG 34. In this study, we extended our investigation to 11 fungal isolates obtained from different geographic areas and belonging to six different species of the family Gigasporaceae. With the exception of Gigaspora rosea, isolates of all of the AM species harbored bacteria, and their DNA could be PCR amplified with universal bacterial primers. Primers specific for the endosymbiotic bacteria of BEG 34 could also amplify spore DNA from four species. These specific primers were successfully used as probes for in situ hybridization of endobacteria in G. margarita spores. Neighbor-joining analysis of the 16S ribosomal DNA sequences obtained from isolates of Scutellospora persica, Scutellospora castanea, and G. margarita revealed a single, strongly supported branch nested in the genus Burkholderia.     

Antagonism of Bacillus spp. Isolated from Marine Biofilms Against Terrestrial Phytopathogenic Fungi

We aimed at determining the antagonistic behavior of bacteria derived from marine biofilms against terrestrial phytopathogenic fungi. Some bacteria closely related to Bacillus mojavensis (three isolates) and Bacillus firmus (one isolate) displayed antagonistic activity against Colletotrichum gloeosporioides ATCC 42374, selected as first screen organism. The four isolates were further quantitatively tested against C. gloeosporioides, Colletotrichum fragariae, and Fusarium oxysporum on two culture media, potato dextrose agar (PDA) and a marine medium-based agar [yeast extract agar (YEA)] at different times of growth of the antagonists (early, co-inoculation with the pathogen and late). Overall antagonistic assays showed differential susceptibility among the pathogens as a function of the type of culture media and time of colonization (P < 0.05). In general, higher suppressive activities were recorded for assays performed on YEA than on PDA; and also when the antagonists were allowed to grow 24 h earlier than the pathogen. F. oxysporum was the most resistant fungus while the most sensitive was C. gloeosporioides ATCC 42374. Significant differences in antagonistic activity (P < 0.05) were found between the different isolates. In general, Bacillus sp. MC3B-22 displayed a greater antagonistic effect than the commercial biocontrol strain Bacillus subtilis G03 (Kodiak). Further incubation studies and scanning electronic microscopy revealed that Bacillus sp. MC3B-22 was able to colonize, multiply, and inhibit C. gloeosporioides ATCC 42374 when tested in a mango leaf assay, showing its potential for fungal biocontrol. Additional studies are required to definitively identify the active isolates and to determine their mode of antifungal action, safety, and biocompatibility.     

Scaling up tests on virulence of the cassava green mite fungal pathogen <Emphasis Type="Italic">Neozygites tanajoae</Emphasis> (Entomophthorales: Neozygitaceae) under controlled conditions: first observations at the population level

Virulence of entomopathogens is often measured at the individual level using a single host individual or a group of host individuals. To what extent these virulence assessments reflect the impact of an entomopathogen on their host in the field remains largely untested, however. A methodology was developed to induce epizootics of the cassava green mite fungal pathogen Neozygites tanajoae under controlled conditions to evaluate population-level virulence of two (one Beninese and one Brazilian) isolates of the entomopathogen—which had shown similar individual-level virulence but different field impacts. In unrepeated separate experiments we inoculated mite-infested potted cassava plants with either 50 or 25 live mites (high and low inoculum) previously exposed to spores of N. tanajoae and monitored the development of fungal infections for each isolate under the same conditions. Both isolates caused mite infections and an associated decline in host mite populations relative to the control (without fungus) in all experiments, but prevalence of the fungus varied with isolate and increased with inoculum density. Peak infection levels were 90% for the Beninese isolate and 36% for the Brazilian isolate at high inoculum density, and respectively 17% and 25% at low inoculum density. We also measured dispersal from inoculated plants and found that spore dispersal increased with host infection levels, independent of host densities, whereas mite dispersal varied between isolates. These results demonstrate that epizootiology of N. tanajoae can be studied under controlled conditions and suggest that virulence tests at the population level may help to better predict performance of fungal isolates than individual-level tests.     

Isolation and phylogenetic analysis of bacteria with antimicrobial activities from the Mediterranean sponges Aplysina aerophoba and Aplysina cavernicola

The aim of this study was to isolate bacteria with antimicrobial activities from the marine sponges Aplysina aerophoba and Aplysina cavernicola. The obtained 27 isolates could be subdivided into eight phylogenetically different clusters based on comparative sequence analysis of their 16S rDNA genes. The sponge isolates were affiliated with the low (Bacillus) and high G+C Gram-positive bacteria (Arthobacter, Micrococcus), as well as the alpha-Proteobacteria (unknown isolate) and gamma-Proteobacteria (Vibrio, Pseudoalteromonas). One novel Bacillus species was identified and two species were closely related to previously uncharacterized strains. Isolates with antimicrobial activity were numerically most abundant in the genera Pseudoalteromonas and the alpha-Proteobacteria. The sponge isolates show antimicrobial activities against Gram-positive and Gram-negative reference strains but not against the fungus Candida albicans. A general pattern was observed in that Gram-positive bacteria inhibited Gram-positive strains while Gram-negative bacteria inhibited Gram-negative isolates. Antimicrobial activities were also found against clinical isolates, i.e. multi-resistant Staphylococcus aureus and Staphylococcus epidermidis strains isolated from hospital patients. The high recovery of strains with antimicrobial activity suggests that marine sponges represent an ecological niche which harbors a hitherto largely uncharacterized microbial diversity and, concomitantly, a yet untapped metabolic potential.     

Simple sequence repeat markers distinguish among morphotypes of Sphaeropsis sapinea

Sphaeropsis sapinea is a fungal endophyte of Pinus spp. that can cause disease following predisposition of trees by biotic or abiotic stresses. Four morphotypes of S. sapinea have been described from within the natural range of the fungus, while only one morphotype has been identified on exotic pines in the Southern Hemisphere. The aim of this study was to develop robust polymorphic markers that could be used in both taxonomic and population studies. Inter-short-sequence-repeat primers containing microsatellite sequences and degenerate anchors at the 5' end were used to target microsatellite-rich areas in an S. sapinea isolate. PCR amplification using an annealing temperature of 49 degrees C resulted in profiles containing 5 to 10 bands. These bands were cloned and sequenced, and new short-sequence-repeat (SSR) primer pairs were designed that flanked microsatellite-rich regions. Eleven polymorphic SSR markers were tested on 40 isolates of S. sapinea representing different morphotypes as well as on 2 isolates of the closely related species Botryosphaeria obtusa. The putative I morphotype was found to be identical to B. obtusa. Otherwise, the markers clearly distinguished the remaining three morphotypes and, furthermore, showed that the C morphotype was more closely related to the A than the B morphotype. The B morphotype was the most genetically diverse, and the isolates could be further divided based on their geographic origins. Sequencing of different alleles from each locus showed that the most polymorphic markers had mutations within a microsatellite sequence.     

Temperature constraints on the growth and functioning of root organ cultures with arbuscular mycorrhizal fungi

In this study we investigated the effects of temperature on fungal growth and tested whether the differences in fungal growth were related to the effects of temperature on carbon movement to, or within, the fungus. Growth curves and C uptake-transfer-translocation measurements were obtained for three arbuscular mycorrhizal fungi (AMF) isolates cultured within a 6-30 degrees C temperature range. A series of experiments with a model fungal isolate, Glomus intraradices, was used to examine the effects of temperature on lipid body and 33P movement, and to investigate the role of acclimation and incubation time. Temperature effects on AMF growth were both direct and indirect because, despite clear independent root and AMF growth responses in some cases, the uptake and translocation of 13C was also affected within the temperature range tested. Root C uptake and, to a lesser extent, C translocation in the fungus, were reduced by low temperatures (< 18 degrees C). Uptake and translocation of 33P by fungal hyphae were, by contrast, similar between 10 and 25 degrees C. We conclude that temperature, between 6 and 18 degrees C, reduces AMF growth, and that C movement to the fungus is involved in this response.     

Detection and Identification of Bacterial Endosymbionts in Arbuscular Mycorrhizal Fungi Belonging to the Family Gigasporaceae

Intracellular bacteria have been found previously in one isolate of the arbuscular mycorrhizal (AM) fungus Gigaspora margarita BEG 34. In this study, we extended our investigation to 11 fungal isolates obtained from different geographic areas and belonging to six different species of the family Gigasporaceae. With the exception of Gigaspora rosea, isolates of all of the AM species harbored bacteria, and their DNA could be PCR amplified with universal bacterial primers. Primers specific for the endosymbiotic bacteria of BEG 34 could also amplify spore DNA from four species. These specific primers were successfully used as probes for in situ hybridization of endobacteria in G. margarita spores. Neighbor-joining analysis of the 16S ribosomal DNA sequences obtained from isolates of Scutellospora persica, Scutellospora castanea, and G. margarita revealed a single, strongly supported branch nested in the genus Burkholderia.     

Rhizonin, the first mycotoxin isolated from the zygomycota, is not a fungal metabolite but is produced by bacterial endosymbionts

Rhizonin is a hepatotoxic cyclopeptide isolated from cultures of a fungal Rhizopus microsporus strain that grew on moldy ground nuts in Mozambique. Reinvestigation of this fungal strain by a series of experiments unequivocally revealed that this "first mycotoxin from lower fungi" is actually not produced by the fungus. PCR experiments and phylogenetic studies based on 16S rRNA gene sequences revealed that the fungus is associated with bacteria belonging to the genus Burkholderia. By transmission electron microscopy, the bacteria were localized within the fungal cytosol. Toxin production and the presence of the endosymbionts were correlated by curing the fungus with an antibiotic, yielding a nonproducing, symbiont-free phenotype. The final evidence for a bacterial biogenesis of the toxin was obtained by the successful fermentation of the endosymbiotic bacteria in pure culture and isolation of rhizonin A from the broth. This finding is of particular interest since Rhizopus microsporus and related Rhizopus species are frequently used in food preparations such as tempeh and sufu.     

Selection and Characterization of Microorganisms Utilizing Thaxtomin A, a Phytotoxin Produced by Streptomyces scabies

Thaxtomin A is the main phytotoxin produced by Streptomyces scabies, a causal agent of potato scab. Thaxtomin A is a yellow compound composed of 4-nitroindol-3-yl-containing 2,5-dioxopiperazine. A collection of nonpathogenic streptomycetes isolated from potato tubers and microorganisms recovered from a thaxtomin A solution were examined for the ability to grow in the presence of thaxtomin A as a sole carbon or nitrogen source. Three bacterial isolates and two fungal isolates grew in thaxtomin A-containing media. Growth of these organisms resulted in decreases in the optical densities at 400 nm of culture supernatants and in 10% reductions in the thaxtomin A concentration. The fungal isolates were identified as a Penicillium sp. isolate and a Trichoderma sp. isolate. One bacterial isolate was associated with the species Ralstonia pickettii, and the two other bacterial isolates were identified as Streptomyces sp. strains. The sequences of the 16S rRNA genes were determined in order to compare thaxtomin A-utilizing actinomycetes to the pathogenic organism S. scabies and other Streptomyces species. The nucleotide sequences of the γ variable regions of the 16S ribosomal DNA of both thaxtomin A-utilizing actinomycetes were identical to the sequence of Streptomyces mirabilis ATCC 27447. When inoculated onto potato tubers, the three thaxtomin A-utilizing bacteria protected growing plants against common scab, but the fungal isolates did not have any protective effect.     

Simple Sequence Repeat Markers Distinguish among Morphotypes of Sphaeropsis sapinea

Sphaeropsis sapinea is a fungal endophyte of Pinus spp. that can cause disease following predisposition of trees by biotic or abiotic stresses. Four morphotypes of S. sapinea have been described from within the natural range of the fungus, while only one morphotype has been identified on exotic pines in the Southern Hemisphere. The aim of this study was to develop robust polymorphic markers that could be used in both taxonomic and population studies. Inter-short-sequence-repeat primers containing microsatellite sequences and degenerate anchors at the 5′ end were used to target microsatellite-rich areas in an S. sapinea isolate. PCR amplification using an annealing temperature of 49°C resulted in profiles containing 5 to 10 bands. These bands were cloned and sequenced, and new short-sequence-repeat (SSR) primer pairs were designed that flanked microsatellite-rich regions. Eleven polymorphic SSR markers were tested on 40 isolates of S. sapinea representing different morphotypes as well as on 2 isolates of the closely related species Botryosphaeria obtusa. The putative I morphotype was found to be identical to B. obtusa. Otherwise, the markers clearly distinguished the remaining three morphotypes and, furthermore, showed that the C morphotype was more closely related to the A than the B morphotype. The B morphotype was the most genetically diverse, and the isolates could be further divided based on their geographic origins. Sequencing of different alleles from each locus showed that the most polymorphic markers had mutations within a microsatellite sequence.     

Rhizonin, the First Mycotoxin Isolated from the Zygomycota, Is Not a Fungal Metabolite but Is Produced by Bacterial Endosymbionts

Rhizonin is a hepatotoxic cyclopeptide isolated from cultures of a fungal Rhizopus microsporus strain that grew on moldy ground nuts in Mozambique. Reinvestigation of this fungal strain by a series of experiments unequivocally revealed that this “first mycotoxin from lower fungi” is actually not produced by the fungus. PCR experiments and phylogenetic studies based on 16S rRNA gene sequences revealed that the fungus is associated with bacteria belonging to the genus Burkholderia. By transmission electron microscopy, the bacteria were localized within the fungal cytosol. Toxin production and the presence of the endosymbionts were correlated by curing the fungus with an antibiotic, yielding a nonproducing, symbiont-free phenotype. The final evidence for a bacterial biogenesis of the toxin was obtained by the successful fermentation of the endosymbiotic bacteria in pure culture and isolation of rhizonin A from the broth. This finding is of particular interest since Rhizopus microsporus and related Rhizopus species are frequently used in food preparations such as tempeh and sufu.     

Characterization of culturable bacteria isolated from the cold-water coral Lophelia pertusa

Microorganisms associated with corals are hypothesized to contribute to the function of the host animal by cycling nutrients, breaking down carbon sources, fixing nitrogen, and producing antibiotics. This is the first study to culture and characterize bacteria from Lophelia pertusa, a cold-water coral found in the deep sea, in an effort to understand the roles that the microorganisms play in the coral microbial community. Two sites in the northern Gulf of Mexico were sampled over 2 years. Bacteria were cultured from coral tissue, skeleton, and mucus, identified by 16S rRNA genes, and subjected to biochemical testing. Most isolates were members of the Gammaproteobacteria, although there was one isolate each from the Betaproteobacteria and Actinobacteria. Phylogenetic results showed that both sampling sites shared closely related isolates (e.g. Pseudoalteromonas spp.), indicating possible temporally and geographically stable bacterial-coral associations. The Kirby-Bauer antibiotic susceptibility test was used to separate bacteria to the strain level, with the results showing that isolates that were phylogenetically tightly grouped had varying responses to antibiotics. These results support the conclusion that phylogenetic placement cannot predict strain-level differences and further highlight the need for culture-based experiments to supplement culture-independent studies.     

Biohydrogenation of linoleic acid by rumen fungi compared with rumen bacteria

AIMS: To investigate biohydrogenation of linoleic acid by rumen fungi compared with rumen bacteria, and to identify the fungus with the fastest biohydrogenation rate. METHODS AND RESULTS: Biohydrogenation of linoleic acid by mixed rumen fungi and mixed rumen bacteria were compared in vitro. With mixed rumen bacteria, all biohydrogenation reactions were finished within 100 min of incubation and the end product of biohydrogenation was stearic acid. With mixed rumen fungi, biohydrogenation proceeded more slowly over a 24-h period. Conjugated linoleic acid (CLA; cis-9, trans-11 C18 : 2) was an intermediate product, and vaccenic acid (VA; trans-11 C18 : 1) was the end product of biohydrogenation. Fourteen pure fungal isolates were tested for biohydrogenation rate. DNA sequencing showed that the isolate with the fastest rate belonged to the Orpinomyces genus. CONCLUSIONS: It is concluded that rumen fungi have the ability to biohydrogenate linoleic acid, but biohydrogenation is slower in rumen fungi than in rumen bacteria. The end product of fungal biohydrogenation is VA, as for group A rumen bacteria. Orpinomyces is the most active biohydrogenating fungus. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to demonstrate that rumen fungi can biohydrogenate fatty acids. Fungi could influence CLA content of ruminant products.     

大连地区海泥样品中分离的五株海洋放线菌的研究

对从大连小平岛地区海面下10~20 m处的海泥样品中分离的5株海洋放线菌进行了生理特征和抗菌活性的研究。抗菌活性实验初步表明,菌株S097,S187和S233具有较好的拮抗革兰氏阳性菌、革兰氏阴性菌和真菌测试菌株的活性,尤其是菌株S233对绿脓杆菌和白色假丝酵母的抑制活性很强。16 s rDNA序列分析结果表明,5株放线菌(S097,S187,S233,S239,L180)分别与Streptomyces argenteolusCGMCC 4.1693、S.flavofuscusNRRL B-8036、S.variabilisNRRL B-3984T、S.lit-m ocidiniNRRL B-3635和S.sulphureusNRRL B-1627T显示出最高的序列同源性(99%),这是这些菌种首次报道在大连地区的海泥样品中得到分离。利用I型聚酮合成酶(PKSI)兼并引物从菌株S187中扩增出了PKSI片段,揭示了该菌株生产I型聚酮类化合物的潜在能力。本文的研究结果为进一步开发利用大连地区海泥中的海洋放线菌资源奠定了基础。     

Electrotransformation of the human pathogenic fungus Scedosporium prolificans mediated by repetitive rDNA sequences

The regions encoding the 5.8S rRNA and the flanking internal transcribed spacers (ITSI and ITSII) from two isolates of the human pathogenic fungus Scedosporium prolificans and one isolate of the taxonomically related species Pseudallescheria boydii (S. apiospermum) were sequenced. The sequences of the two S. prolificans isolates were identical. However, there were minor differences between both species. Phylogenetic analysis of known fungal sequences confirmed a close relationship between S. prolificans and P. boydii. An attempt was made to transform S. prolificans by electroporation using a plasmid vector, pMLF2, bearing the Escherichia coli hygromycin B phosphotransferase gene (hph) under the control of Aspergillus nidulans promoter and terminator sequences. To increase transformation efficiency, the sequenced ribosomal cluster of S. prolificans was used to construct a new vector for homologous recombination.     

Isolation,identification, and in vitro predatory activity of nematophagous fungus Arthrobotrys musiformis and its interaction with nematodes using scanning electron microscopy

We isolated and identified nematophagous fungus Arthrobotrys musiformis from materials derived from cattle and sheep. We also molecularly characterised the native fungal isolates and evaluated the nematophagous activity of the isolates. A total of 19 isolates of A. musiformis were isolated from 1532 samples, and the detection rate of A. musiformis in all samples was 1.24%. These isolates were identified using a light microscope and their 5.8S rDNA, 18S rDNA, 28S rDNA, and the internal transcribed spacers 1 and 2 region. Interaction of the isolate (NPS045) with the nematode targets of the infective larvae (L₃) of Haemonchus contortus and the free-living nematode Caenorhabditis elegans was observed by scanning electron microscopy (SEM). SEM result showed that the two species of nematodes were initially captured at 5?h after being added to the isolates. L₃ was penetrated at 22?h after capture and completely destroyed by the fungus at 68?h. C. elegans was penetrated at 14?h post-capture and was completely digested by the fungus at 24?h. In vitro experimental assay of samples in 24-well plates showed that for the three fungal isolates, the L₃s of Trichostrongylus colubriforms were reduced by 94.80%, 90.17%, and 89.02%.     

Identification and isolation of two ascomycete fungi from spores of the arbuscular mycorrhizal fungus Scutellospora castanea

Two filamentous fungi with different phenotypes were isolated from crushed healthy spores or perforated dead spores of the arbuscular mycorrhizal fungus (AMF) Scutellospora castanea. Based on comparative sequence analysis of 5.8S ribosomal DNA and internal transcribed spacer fragments, one isolate, obtained from perforated dead spores only, was assigned to the genus Nectria, and the second, obtained from both healthy and dead spores, was assigned to Leptosphaeria, a genus that also contains pathogens of plants in the Brassicaceae. PCR and randomly amplified polymorphic DNA-PCR analyses, however, did not indicate similarities between pathogens and the isolate. The presence of the two isolates in both healthy spores and perforated dead spores of S. castanea was finally confirmed by transmission electron microscopy by using distinctive characteristics of the isolates and S. castanea. The role of this fungus in S. castanea spores remains unclear, but the results serve as a strong warning that sequences obtained from apparently healthy AMF spores cannot be presumed to be of glomalean origin and that this could present problems for studies on AMF genes.     

Dense fouling in acid transfer pipelines by an acidophilic rubber degrading fungus

An unique case of dense fouling by an acidophilic, hard rubber (polymerized rubber) degrading fungus in the acid transfer pipelines of a boron enrichment plant located at Kalpakkam, India is reported. In spite of a highly adverse environment for survival (pH 1.5, no dissolved nutrients), the fungus thrived and clogged the pipeline used for transferring 0.1N hydrochloric acid (HCl). Detailed investigations were carried out to isolate and identify the fungus and examine the nutrient source for such profuse growth inside the system. Microscopic observation showed the presence of a thick filamentous fungal biomass. Molecular characterization by 18S rRNA gene sequencing showed 98% similarity of the isolate with the acidophilic fungus Bispora sp. In laboratory studies the fungus showed luxuriant growth (specific growth rate of 13 mg day?1) when scrapings of the hard rubber were used as the sole source of carbon. Scanning electron microscopy revealed extensive incursion of the fungus into the hard rubber matrix. In the laboratory, fungal growth was completely inhibited by the antifungal agent sodium omadine. The study illustrates an interesting example of biofouling under extreme conditions and demonstrates that organisms can physiologically adapt to grow under unfavourable conditions, provided that a nutrient source is available and competition is low. The use of this fungal strain in biodegradation and in development of environmentally compatible processes for disposal of rubber wastes is envisaged.