Isolation and structure elucidation of a new antifungal and antibacterial antibiotic produced by Streptomyces sp. 201

An antibacterial and antifungal antibiotic was isolated from the culture filtrate of Streptomyces sp. 201, and its structure was determined as 2-methyl-heptyl isonicotinate by extensive use of NMR spectroscopy. The compound exhibited marked antimicrobial activity against Bacillus subtilis, Shigella sp., Klebsiella sp., E. coli, Proteus mirabilis, and the pathogenic fungi, Fusarium moniliforme, F. semitectum, F. oxysporum, F. solani and Rhizoctonia solani.     

Morphological and Molecular Characterization of Fusarium solani Isolates

Fusarium solani is a species complex (FSSC) containing isolates that cause diseases in important crops such as root and fruit rot of Cucurbita spp., root and stem rot of pea, sudden death syndrome of soybean, foot rot of bean and dry rot of potato tubers during storage. Based on host range tests, F. solani were subdivided into different formae specialis (f. sp.) and varieties, while DNA sequences of 28S rDNA, internally transcribed spacers (ITS) rDNA and elongation factor (EF-1α) distinguished the ' F. solani complex' in 50 subspecific lineages. In this study we characterized, by cultural, morphological and molecular criteria, 34 isolates of F. solani obtained from potato, other crops and soil. The 34 isolates in the FSSC showed wide variability for their cultural, morphological and molecular traits. The wide variability observed with amplified fragment-length polymorphism (AFLP) and mini-microsatellite analyses is in agreement with the polymorphism observed, in a previous study, within FSSC. Nine of 34 isolates in the FSSC, classified as F. solani var. coeruleum , were morphologically distinguishable from the other F. solani isolates but they were distributed in different clusters; moreover, the nine isolates showed instability of the coeruleum pigmentation of the colonies, supporting the ambiguity of the taxa of this variety of F. solani. Using sequence data from ITS plus 5.8S rDNA region, the isolates were classified into different clades. In particular eight isolates were classified into a well-supported clade including F. solani f. sp . pisi , nine into a clade including only isolates of F. solani f. sp . radicicola and four into a clade including F. solani f. sp . cucurbitae , but this classification could not be used if is not in agreement with host specificity. Two of the nine F. solani var. coeruleum isolates were phylogenetically distinct from all the other FSSC strains.     

Sudden-death syndrome of soybean is caused by two morphologically and phylogenetically distinct species within the Fusarium solani species complex--F. virguliforme in North America and F. tucumaniae in South America

Soybean sudden-death syndrome has become a serious constraint to commercial production of this crop in North and South America during the past decade. To assess whether the primary etiological agent is panmictic in both hemispheres, morphological and molecular phylogenetic analyses were conducted on strains selected to represent the known pathogenic and genetic diversity of this pathogen. Maximum-parsimony analysis of DNA sequences from the nuclear ribosomal intergenic spacer region and the single copy nuclear gene translation elongation factor 1-α, together with detailed morphological comparisons of conidial features, indicate that SDS of soybean in North and South America is caused by two phylogenetically and morphologically distinct species. Fusarium virguliforme sp. nov., formally known as F. solani f. sp. glycines, is described and illustrated for the SDS pathogen in North America, and F. tucumaniae sp. nov. is proposed for the South American pathogen. The molecular phylogenetic results challenge the forma specialis naming system because pathogenicity to soybean might have evolved convergently in F. tucumaniae and F. virguliforme. Phylogenetic evidence indicates the two SDS pathogens do not share a most recent common ancestor, since F. tucumaniae was resolved as a sister to a pathogen of Phaseolus vulgaris, F. phaseoli comb. nov. All three pathogens appear to have evolutionary origins in the southern hemisphere since they are deeply nested within a South American clade of the F. solani species complex.     

Occurrence of itraconazole-tolerant micromycetes in the soil and food products

Unexpected pathogens from the environment represent considerable risk for humans with impaired health. We examined the occurrence of itraconazole tolerant micromycetes in soil and in maize products. Five concentrations of itraconazole (2.5–12.5 μg/mL) selected according to known treatment schedules for human patients were incorporated into Sabouraud agar with chloramphenicol and Rose Bengal and diluted samples were inoculated onto the agar surface. After 7-d growth at 22°C colonies ofAlternaria sp.,Aspergillus clavatus. A. glaucus group,A. flavus. A, fumigatus, A. niger group,A. ochraceus group,A. ochraceus, Chœtomium sp.,Cladosporium cladosporioides. Cylindrocarpon sp.Doratomyces sp.,Fusarium sp.,F. moniliforme. F. oxysporum. F. solani, F. subglutinans. Marianaea elegans, Mortierella sp.,Mucor sp.,Myrothecium sp.,Penicillium sp.,Rhizopus sp.,Scopulariopsis brevicaulis. Sepedonium sp.,Stachybotrys chartarum. Stemphylium sp.,Torula humicola andTrichoderma viride were isolated.     

Comparison of the hydrolysis of polyethylene terephthalate fibers by a hydrolase from Fusarium oxysporum LCH I and Fusarium solani f. sp. pisi

The hydrolysis of polyethylene terephthalate (PET) fibers by two fungal hydrolases was investigated. The hydrolase from a newly isolated Fusarium oxysporum strain (LCH 1) was more efficient in releasing terephthalic acid from PET fibers compared to the enzyme from F. solani f. sp. pisi DSM 62420 when equal amounts of p-nitrophenyl butyrate-hydrolyzing activity were employed. PET fabrics treated under the same conditions with the enzyme from F. oxysporum LCH 1 also showed a considerably higher increase in hydrophilicity compared to fabrics treated with the enzyme from F. solani f. sp. pisi DSM 62420.     

Diversity in soil fungi from undisturbed and disturbed Celtis tala and Scutia buxifolia forests in the eastern Buenos Aires province (Argentina)

The rhizospheric soil microfungi from a native forest (undisturbed and disturbed) were studied using soil dilution plate and soil washing methods. Fungi were isolated using slightly acid and alkaline culture media. 54 taxa were isolated: 49 from undisturbed forest soil and 37 from disturbed forest soil. Acremonium sp., Aspergillus ustus, Coemansia pectinata, Doratomyces stemonitis, Fusarium solani, F. oxysporum, Gliocladium roseum, Humicola fusco-atra, Mortierella sp., Penicillium lilacinum, Trichoderma harzianum, and T koningii, showed the highest frequency, in both, undisturbed and disturbed forests. In undisturbed soil forest the biodiversity index was 3.97 whereas in disturbed ones was 3.89.     

sti35, a stress-responsive gene in Fusarium spp.

A stress-induced mRNA was identified in the phytopathogenic fungus Fusarium oxysporum f. sp. cucumerinum. Treatment of the fungus with ethanol resulted in the induction of a major mRNA species encoding a protein of approximate Mr 37,000. A full-length cDNA clone of the induced message was obtained. RNA blot analysis indicated that the mRNA was induced by various other stresses, including treatment with copper(II) chloride and heat (37 degrees C). However, it was not greatly induced by treatment with phaseollinisoflavan, an antifungal isoflavonoid produced by Phaseolus vulgaris (French bean). In contrast, phaseollinisoflavan induced the homologous mRNA in the related bean pathogen Fusarium solani f. sp. phaseoli. A genomic clone of the F. solani f. sp. phaseoli gene was obtained, and both this and the cDNA clone from F. oxysporum f. sp. cucumerinum were sequenced. The latter indicated an open reading frame of 320 codons encoding a 34,556-dalton polypeptide. The corresponding reading frame in F. solani f. sp. phaseoli was 324 codons, 89% identical to the F. oxysporum f. sp. cucumerium sequence, and was interrupted by a short intron. The gene was designated sti35 (stress-inducible mRNA). Although computer homology searches were negative, the cloned gene was observed to cross-hybridize to DNAs of other filamentous fungi, Saccharomyces cerevisiae, and soybean. Thus, sti35 appears to be a common gene among a variety of eucaryotes.     

Anaerobic growth ability and alcohol fermentation activity of microscopic fungi

The method proposed in this study was used to isolate fungi grown under anaerobic conditions and to reveal distinctions in their abundance and species composition in different habitats. The ability of micromycetes of different taxa to grow under anaerobic conditions and ensure alcohol fermentation was determined for a representative sample (344 strains belonging to more than 60 species). The group of fungi growing under anaerobic conditions included species with high, moderate, and low fermentation activity. The ability for anaerobic growth and fermentation depended on the taxonomic affiliation of fungi. In some cases, the expression of these characteristics depended on the habitat from which the strain was isolated. The maximum level of ethanol accumulation in culture liquid (1.2–4.7%) was detected for Absidia spinosa, Aspergillus sp. of group flavus, Aspergillus terreus, Acremonium sp., Mucor circinelloides, Mucor sp., Fusarium oxysporum, F. solani, F. sambucinum, Rhizopus arrhizus var. arrhizus, Trichoderma atroviride, and Trichoderma sp.     

Nicotiana plumbaginifolia plants silenced for the ATP-binding cassette transporter gene NpPDR1 show increased susceptibility to a group of fungal and oomycete pathogens

The behaviour of Nicotiana plumbaginifolia plants silenced for the ATP-binding cassette transporter gene NpPDR1 was investigated in response to fungal and oomycete infections. The importance of NpPDR1 in plant defence was demonstrated for two organs in which NpPDR1 is constitutively expressed: the roots and the petal epidermis. The roots of the plantlets of two lines silenced for NpPDR1 expression were clearly more sensitive than those of controls to the fungal pathogens Botrytis cinerea , Fusarium oxysporum sp., F. oxysporum f. sp. nicotianae , F. oxysporum f. sp. melonis and Rhizoctonia solani , as well as to the oomycete pathogen Phytophthora nicotianae race 0. The Ph gene-linked resistance of N. plumbaginifolia to P. nicotianae race 0 was totally ineffective in NpPDR1 -silenced lines. In addition, the petals of the NpPDR1 -silenced lines were spotted 15%–20% more rapidly by B. cinerea than were the controls. The rapid induction (after 2–4 days) of NpPDR1 expression in N. plumbaginifolia and N. tabacum mature leaves in response to pathogen presence was demonstrated for the first time with fungi and one oomycete: R. solani , F. oxysporum and P. nicotianae . With B. cinerea , such rapid expression was not observed in healthy mature leaves. NpPDR1 expression was not observed during latent infections of B. cinerea in N. plumbaginifolia and N. tabacum , but was induced when conditions facilitated B. cinerea development in leaves, such as leaf ageing or an initial root infection. This work demonstrates the increased sensitivity of NpPDR1 -silenced N. plumbaginifolia plants to all of the fungal and oomycete pathogens investigated.     

Induced Resistance in Cucumbers against Fusarium oxysporum f. sp. cucumerinum and Rhizoctonia solani AG2–2 by Infection of the Cotyledons

Localized infection in cucumber cotyledons with Colletotrichum lagenarium induced resistance against infection after challenge inoculation with Rhizoctonia solani AG2–2 and Fusarium oxysporum f. sp. cucumerinum in the roots. The plants were unprotected in soil that was infested heavily with R. solani or in contact with the mycelium, and induced resistance was not observed. Wounding of the root also negated the effect of induced resistance to F. oxysporum .     

Diversity of facultatively anaerobic microscopic mycelial fungi in soils

The numbers of microscopic fungi isolated from soil samples after anaerobic incubation varied from tens to several hundreds of CFU per one gram of soil; a total of 30 species was found. This group is composed primarily of mitotic fungi of the ascomycete affinity belonging to the orders Hypocreales (Fusarium solani, F. oxysporum, Fusarium sp., Clonostachys grammicospora, C. rosea. Acremonium sp., Gliocladium penicilloides, Trichoderma aureoviride, T. harzianum, T. polysporum, T. viride. T. koningii, Lecanicillum lecanii, and Tolypocladium inflatum) and Eurotiales (Aspergillus terreus, A. niger, and Paecilomyces lilacimus), as well as to the phylum Zygomycota, to the order Mucorales (Actinomucor elegans, Absidia glauca, Mucor circinelloides, M. hiemalis, M. racemosus, Mucor sp., Rhizopus oryzae, Zygorrhynchus moelleri, Z. heterogamus, and Umbelopsis isabellina) and the order Mortierellales (Mortierella sp.). As much as 10-30% of the total amount of fungal mycelium remains viable for a long time (one month) under anaerobic conditions.     

Induction of cutinolytic esterase activity during saprophytic growth of cucurbit pathogens, Fusarium solani f. sp. cucurbitae races one and two (Nectria haematococca MPI and MPV, respectively)

Cutins from fruit of Cucurbita maxima and Cucurbita moschata cultivars, apple and a C(16) alcohol (hexadecanol) were used to induce cutinolytic esterase activity during saprophytic growth of strains of the two cucurbit pathogens, Fusarium solani f. sp. cucurbitae, race 1 (Nectria haematococca mating population (MPI) and F. solani f. sp. cucurbitae, race 2 (MPV). Four strains of MPV and 11 strains of MPI were were included in the study. Although we were primarily interested in the two cucurbit pathogens (MPI and MPV), six strains of the pea pathogen F. solani f. sp. pisi (MPVI) were included to provide a comparison since most of the knowledge on cutinase activity in N. haematococca has come from a study of that group. Cutinolytic esterase was induced in all strains from both MPV and MPVI but was not detected in any of the 11 strains from MPI regardless of the induction conditions. The amount of cutinolytic esterase activity induced in the MPV strains differed according to the strain and both the source and the amount of cutin used in the induction medium. Information on the influence of cutin source and pH on the induction of cutinolytic esterase activity during saprophytic growth of strains from MPV demonstrates that the gene is regulated differently from that in MPVI.     

Inhibitory effects of essential oils of medicinal plants from growth of plant pathogenic fungi

Plant cells produce a vast amount of secondary metabolites. Production of some compounds is restricted to a single species. Some compounds are nearly always found only in certain specific plant organs and during a specific developmental period of the plant. Some secondary metabolites of plants serve as defensive compounds against invading microorganisms. Nowadays, it is attempted to substitute the biological and natural agents with chemically synthesized fungicides. In the present research, the antifungal activities of essential oils of seven medicinal plants on mycelial growth of three soilborne plant pathogenic fungi were investigated. The plants consisted of Zataria multiflora, Thymus carmanicus, Mentha pieperata, Satureja hortensis, Lavandual officinolis, Cuminum cyminum and Azadirachta indica. The first five plants are from the family Labiatae. Examined fungi, Fusarium oxysporum f.sp. lycopersici, Fusarium solani and Rhizoctonia solani are the causal agents of tomato root rot. Essential oils of Z. multiflora, T. carmanicus, M. pieperata, S. hortensis and C. cyminum were extracted by hydro-distillation method. Essential oils of L. officinalis and A. indica were extracted by vapor-distillation method. A completely randomized design with five replicates was used to examine the inhibitory impact of each concentration (300, 600 and 900 ppm) of each essential oil. Poisoned food assay using potato dextrose agar (PDA) medium was employed. Results showed that essential oils of A. indica, Z. multiflora, T. carmanicus and S. hortensis in 900 ppm at 12 days post-inoculation, when the control fungi completely covered the plates, prevented about 90% from mycelial growth of each of the fungi. While, the essential oils of M. pieperata, C. cyminum and L. officinalis in the same concentration and time prevented 54.86, 52.77 and 48.84%, respectively, from F. solani growth. These substances did not prevent from F. oxysporum f.sp. lycopersici and R. solani growth. Minimum inhibitory concentration (MIC) of essential oils of T. carmanicus, Z. multiflora and A. indica from R. solani and F. solani growth was 900 and 600 ppm, respectively. In addition, the MIC of essential oils of these plants and essential oil of S. hortensis from F. oxysporum f.sp. lycopersici growth was 900 ppm. The MIC of essential oils of M. pieperata, C. cyminum and L. officinalis from F. solani growth was 900 ppm.     

Identification and pathogenic characterization of endophytic <Emphasis Type="Italic">Fusarium</Emphasis> species from cowpea seeds

Isolates of Fusarium were obtained and identified from seeds of cowpea, Vigna unguiculata (L.) Walp., by means of blotter tests and slide cultures. Species were differentiated according to the morphology of the macroconidia, microconidia and their arrangement in chains or false heads, the size and type of conidiophore, and the presence or absence of chlamydospores. The species were identified as F. semitectum, F. equiseti, F. oxysporum, F. solani, F. anthophilum, F. sporotrichioides, F. moniliforme, and Fusarium sp. Among the species, F. semitectum was the most frequently detected. None of these species were pathogenic when inoculated in susceptible cowpea cultivar (BR 17- Gurgueia). But, an isolate of F. oxysporum f. sp. tracheiphilum used as a standard of comparison for pathogenicity (control) induced symptoms of yellowing, vascular wilting, and death of a susceptible cowpea cultivar under the same environmental conditions.     

Extracellular xylanase production by Fusarium species in solid state fermentation

Fusarium sp. has been shown to be a promising organism for enhanced production of xylanases. In the present study, xylanase production by 21 Fusarium sp. isolates (8 Fusarium culmorum, 4 Fusarium solani, 6 Fusarium verticillioides and 3 Fusarium equiseti) was evaluated under solid state fermentation (SSF). The fungal isolate Fusarium solani SYRN7 was the best xylanase producer among the tested isolates. The effects of some agriculture wastes (like wheat straw, wheat bran, beet pulp and cotton seed cake) and incubation period on xylanase production by F. solani were optimized. High xylanase production (1465.8 U/g) was observed in wheat bran after 96 h of incubation. Optimum pH and temperature for xylanase activity were found to be 5 and 50 degrees C, respectively.     

Bacillus strains isolated from rhizosphere showed plant growth promoting and antagonistic activity against phytopathogens

Seven bacterial isolates screened from rhizosphere of common bean growing at Uttarakhand Himalaya showed potential plant growth promoting (PGP) and antagonistic activities. Based on 16S rRNA gene sequence the isolate BPR7 was identified as Bacillus sp. BPR7. The strain BPR7 produced IAA, siderophore, phytase, organic acid, ACC deaminase, cyanogens, lytic enzymes, oxalate oxidase, and solubilized various sources of organic and inorganic phosphates as well as potassium and zinc. Strain BPR7 strongly inhibited the growth of several phytopathogens such as Macrophomina phaseolina, Fusarium oxysporum, F. solani, Sclerotinia sclerotiorum, Rhizoctonia solani and Colletotricum sp. in vitro. Cell-free culture filtrate of strain BPR7 also caused colony growth inhibition of all test pathogens. PGP and antifungal activities of Bacillus sp. BPR7 suggest that it may be exploited as a potential bioinoculant agent for P. vulgaris.     

The role of chitosan in protection of soybean from sudden death syndrome caused by Fusarium solani f. sp. glycines

The in vitro antifungal properties of chitosan and its role in protection of soybean from a sudden death syndrome (SDS) were evaluated. Chitosan inhibited the radial and submerged growth of F. solani f. sp. glycines with a marked effect at concentrations up to 1mg/ml indicating antifungal property and at 3mg/ml was able to delay SDS symptoms expression on soybean leaves for over three days after fungal inoculation when applied preventively. Chitosan was able to induce the level of chitinase activity in soybean resulting in the retardation of SDS development in soybean leaves. However, the SDS symptoms gradually appeared and were associated with the reduction of chitinase activity level after five days of infection period. These results suggested the role of chitosan in partially protecting soybeans from F. solani f. sp. glycines infection.     

New model substrates for enzymes hydrolysing polyethyleneterephthalate and polyamide fibres

Recently the potential of enzymes for surface hydrophilisation and/or functionalisation of polyethyleneterephthalate (PET) and polyamide (PA) has been discovered. However, there was no correlation between enzyme class/activity (e.g. esterase, lipase, cutinase) and surface hydrolysis of these polymers and consequently no simple assay to estimate this capability. Enzymes active on the model substrates bis (benzoyloxyethyl) terephthalate and adipic acid bishexyl-amide, were also capable of increasing the hydrophilicity of PET and PA. When dosed at the identical activity on 4-nitrophenyl butyrate, only enzymes from Thermobifida fusca, Aspergillus sp., Beauveria sp. and commercial enzymes (TEXAZYME PES sp5 and Lipase PS) increased the hydrophilicity of PET fibres while other esterases and lipases did not show any effect. Activity on PET correlated with the activity on the model substrate. Hydrophilicity of fibres was greatly improved based on increases in rising height of up to 4.3 cm and the relative decrease of water absorption time between control and sample of the water was up to 76%. Similarly, enzymes increasing the hydrophilicity of PA fibres such as from Nocardia sp., Beauveria sp. and F. solani hydrolysed the model substrate; however, there was no common enzyme activity (e.g. protease, esterase, amidase) which could be attributed to all these enzymes.     

FsFKS1, the 1,3-beta-glucan synthase from the caspofungin-resistant fungus Fusarium solani

The cell wall, a mesh of carbohydrates and proteins, shapes and protects the fungal cell. The enzyme responsible for the synthesis of one of the main components of the fungal wall, 1,3-beta-glucan synthase, is targeted by the antifungal caspofungin acetate (CFA). Clinical isolates of Candida albicans and Aspergillus fumigatus are much more sensitive to CFA than clinical isolates of Fusarium species. To better understand CFA resistance in Fusarium species, we cloned and sequenced FsFKS1, which encodes the Fusarium solani f. sp. pisi beta(1,3)-D-glucan synthase, used RNA interference to reduce its expression and complemented deletion of the essential fks gene of the CFA-sensitive fungus A. fumigatus with FsFKS1. Reduction of the FsFKS1 message in F. solani f. sp. pisi reduced spore viability and caused lysis of spores and hyphae, consistent with cell wall defects. Compensating for the loss of A. fumigatus fks1 with FsFKS1 caused only a modest increase in the tolerance of A. fumigatus for CFA. Our results suggest that FsFKS1 is required for the proper construction of F. solani cell walls and that the resistance of F. solani to CFA is at best only partially due to resistance of the FsFKS1 enzyme to this antifungal agent.     

Fungi associated with Scolytogenes birosimensis (Coleoptera: Curculionidae) infesting Pittosporum tobira

The bark beetle Scolytogenes birosimensis Niijima is suspected to be involved in the decline of Pittosporum tobira (Thunb. ex Murray) Aiton in the coastal areas of Japan. We isolated fungi from adult S. birosimensis in nine different localities in Japan to assess their potential association and predict their contribution to the success of the beetle. Results from morphological identification of associated fungi showed that the beetle was associated with Fusarium solani and Candida spp. Furthermore, molecular analysis showed that F. solani was most closely related to the plant pathogenic fungus F. solani f. sp. mori. Fungal isolation from surface-sterilized, dissected beetles and scanning electron miscroscope (SEM) observation of the body surface suggested that the associated fungi were carried in the pits on the beetles' elytra. These findings contribute to the understanding of the relationships between S. birosimensis and its associated fungi.