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PubMed1996

Application of differential display RT-PCR to the analysis of gene expression in a plant-fungus interaction.

Establishment of a plant-pathogen interaction involves differential gene expression in both organisms. In order to isolate Botrytis cinerea genes whose expression is induced during its interaction with tomato, a comparative analysis of the expression pattern of the fungus in planta with its expression pattern during in vitro culture was performed by differential display of mRNA (DDRT-PCR). Discrimination of fungal genes induced in planta from plant defense genes induced in response to the pathogen was attempted by including in this comparative analysis the expression patterns of healthy tomato leaves and of tomato leaves infected with two different pathogens, either Rhytophthora infestans or tobacco necrosis virus (TNV). Using a limited set of primer combinations, three B. cinerea cDNA fragments, ddB-2, ddB-5 and ddB-47, were isolated representing fungal genes whose expression is enhanced in planta. Northern blot analysis showed that the transcripts detected with the cDNA clones ddB-2 and ddB-5 accumulated at detectable levels only at late time points during the interaction. The cDNA clone ddB-47 detected two different sizes of transcripts displaying distinct, transient expression patterns during the interaction. Sequence analysis and database searches revealed no significant homology to any known sequence. These results show that the differential display procedure possesses enough sensitivity to be applied to the detection of fungal genes induced during a plant-pathogen interaction. Additionally, four cDNA fragments were isolated representing tomato genes induced in response to the infection caused by B. cinerea, but not by P. infestans.

Amino Acid Sequence↗
PubMed1994

Effect of chitinase antisense RNA expression on disease susceptibility of Arabidopsis plants.

Chitinases accumulate in higher plants upon pathogen attack are capable of hydrolyzing chitin-containing fungal cell walls and are thus implicated as part of the plant defense response to fungal pathogens. To evaluate the relative role of the predominate chitinase (class I, basic enzyme) of Arabidopsis thaliana in disease resistance, transgenic Arabidopsis plants were generated that expressed antisense RNA to the class I chitinase. Young plants or young leaves of some plants expressing antisense RNA had < 10% of the chitinase levels of control plants. In the oldest leaves of these antisense plants, chitinase levels rose to 37-90% of the chitinase levels relative to vector control plants, most likely because of accumulation and storage of the enzyme in vacuoles. The rate of infection by the fungal pathogen Botrytis cinerea was measured in detached leaves containing 7-15% of the chitinase levels of control plants prior to inoculation. Antisense RNA was not effective in suppressing induced chitinase expression upon infection as chitinase levels increased in antisense leaves to 47% of levels in control leaves within 24 hours after inoculation. Leaves from antisense plants became diseased at a slightly faster rate than leaves from control plants, but differences were not significant due to high variability. Although the tendency to increased susceptibility in antisense plants suggests that chitinases may slow the growth of invading fungal pathogens, the overall contribution of chitinase to the inducible defense responses in Arabidopsis remains unclear.

Arabidopsis↗
PubMed1994

Structure and expression of an inhibitor of fungal polygalacturonases from tomato.

A polygalacturonase inhibitor protein (PGIP) was characterized from tomato fruit. Differential glycosylation of a single polypeptide accounted for heterogeneity in concanavalin A binding and in molecular mass. Tomato PGIP had a native molecular mass of 35 to 41 kDa, a native isoelectric point of 9.0, and a chemically deglycosylated molecular mass of 34 kDa, suggesting shared structural similarities with pear fruit PGIP. When purified PGIPs from pear and tomato were compared, tomato PGIP was approximately twenty-fold less effective an inhibitor of polygalacturonase activity isolated from cultures of Botrytis cinerea. Based on partial amino acid sequence, polymerase chain reaction products and genomic clones were isolated and used to demonstrate the presence of PGIP mRNA in both immature and ripening fruit as well as cell suspension cultures. Nucleotide sequence analysis indicates that the gene, uninterrupted by introns, encodes a predicted 36.5 kDa polypeptide containing amino acid sequences determined from the purified protein and sharing 68% and 50% amino acid sequence identity with pear and bean PGIPs, respectively. Analysis of the PGIP sequences also revealed that they belong to a class of proteins which contain leucine-rich tandem repeats. Because these sequence domains have been associated with protein-protein interactions, it is possible that they contribute to the interaction between PGIP and fungal polygalacturonases.

Amino Acid Sequence↗
PubMed1996

Antimicrobial peptides from Mirabilis jalapa and Amaranthus caudatus: expression, processing, localization and biological activity in transgenic tobacco.

The cDNAs encoding the seed antimicrobial peptides (AMPs) from Mirabilis jalapa (Mj-AMP2) and Amaranthus caudatus (Ac-AMP2) have previously been characterized and it was found that Mj-AMP2 and Ac-AMP2 are processed from a precursor preprotein and preproprotein, respectively [De Bolle et al., Plant Mol Biol 28:713-721 (1995) and 22:1187-1190 (1993), respectively]. In order to study the processing, sorting and biological activity of these antimicrobial peptides in transgenic tobacco, four different gene constructs were made: a Mj-AMP2 wild-type gene construct, a Mj-AMP2 mutant gene construct which was extended by a sequence encoding the barley lectin carboxyl-terminal propeptide, a known vacuolar targeting signal [Bednarek and Raikhel, Plant Cell 3: 1195-1206 (1991)]; an Ac-AMP2 wild-type gene construct; and finally, an Ac-AMP2 mutant gene construct which was truncated in order to delete the sequence encoding the genuine carboxyl-terminal propeptide. Processing and localization analysis indicated that an isoform of Ac-AMP2 with a cleaved-off carboxyl-terminal arginine was localized in the intercellular fluid fraction of plants expressing either wild-type or mutant gene constructs. Mj-AMP2 was recovered extracellularly in plants transformed with Mj-AMP2 wild-type gene construct, whereas an Mj-AMP2 isoform with a cleaved-off carboxyl-terminal arginine accumulated intracellularly in plants expressing the mutant precursor protein with the barley lectin propeptide. The in vitro antifungal activity of the AMPs purified from transgenic tobacco expressing any of the four different precursor proteins was similar to that of the authentic proteins. However, none of the transgenic plants showed enhanced resistance against infection with either Botrytis cinerea or Alternaria longipes.

Amino Acid Sequence↗
PubMed1989

Fungal air-borne spores as health risk factors among workers in alimentary industries.

A survey to evaluate the occurrence of air-borne fungal spores in two different food industries, dairies and bakeries, was conducted. Our data revealed considerable fungal pollution in the environments of both industries, as well as some differences in the distribution of the genera of fungi recovered. Noteworthy was the frequent finding of numerous fungi frequently responsible for allergic rhinitis, asthma and other diseases, or well-known for their production of mycotoxins in foods or characterized by their degradative activity against various substances. Aspergillus, Candida, Fusarium, Geotrichum, Mucor and Penicillium were the most common genera identified in dairies while Alternaria, Aspergillus, Botrytis, Candida, Cladosporium, Penicillium and Saccharomyces occurred more frequently in bakeries. The survey showed that fungi can play a significant role in allergic and non-allergic diseases in modern working environments.

Air Microbiology↗
PubMed1995

A group-I intron in the mitochondrial small subunit ribosomal RNA gene of Sclerotinia sclerotiorum.

A 1,380-bp intervening sequence within the mitochondrial small subunit ribosomal RNA (mt SSU rRNA) gene of the fungus Sclerotinia sclerotiorum has been sequenced and identified as a group-I intron. This is the first report of an intron in the mt SSU rRNA gene. The intron shows close similarity in secondary structure to the subgroup-IC2 introns from Podospora (ND3i1, ND5i2, and COIi5) and Neurospora (ND5i1). The intron has an open reading frame (ORF) that encodes a putative protein of 420 amino acids which contains two copies of the LAGLI-DADG motif. The ORF belongs to a family of ORFs identified in Podospora (ND3i1, ND4Li1, ND4Li2, ND5i2, and COIi5) and Neurospora (ND5i1). The putative 420-aa polypeptide is also similar to a site-specific endonuclease in the chloroplast large subunit ribosomal RNA (LSU rRNA) gene of the green alga Chlamydomonas eugametos. In each clone of S. sclerotiorum examined, including several clones which were sampled over a 3-year period from geographically separated sites, all isolates either had the intron or lacked the intron within the mt SSU rRNA gene. Screening by means of Southern hybridization and PCR amplification detected the intron in the mt SSU rRNA genes of S. minor, S. trifoliorum and Sclerotium cepivorum, but not in other members of the Sclerotiniaceae, such as Botrytis anamorphs of Botryotinia spp., or in other ascomycetous and basidiomycetous fungi.

Amino Acid Sequence↗
PubMed1994

Visualization of mitotic chromosomes in filamentous fungi by fluorescence staining and fluorescence in situ hybridization.

Mitotic chromosomes of the plant pathogenic filamentous fungi Botrytis cinerea and Alternaria alternata were observed. Chromosomes prepared by the germ tube burst method were stained with the fluorescent dye 4',6-diamidino-2-phenylindole (DAPI) to yield figures with good resolution. Using this method, component chromosomes were clearly distinguished and the chromosome number could be determined. Fluorescence in situ hybridization (FISH) was also successfully applied to the specimens, revealing one ribosomal RNA gene cluster, or nucleolus organizer region (NOR) in the genome of each fungus. A long attenuated chromatid thread expanding from a condensed metaphase chromosome, which had been called a thread-like structure in B. cinerea, was proved to be an NOR. This is the first report of the successful application of FISH to the chromosomes of filamentous fungi.

Alternaria↗
PubMed1980

The distribution of the NADPH regenerating mannitol cycle among fungal species.

The mannitol cycle is an important NADPH regenerating system in Alternaria alternata. The cycle is built up to the following enzymes: mannitol 1-phosphate dehydrogenase, mannitol 1-phosphatase, mannitol dehydrogenase and hexokinase. The net reaction of one cycle turn is: NADH + NADP+ + ATP leads to NAD+ + NADPH + ADP + Pi. The enzymes needed for an operating cycle were found in Aspergillus, Botrytis, Penicillium, Pyricularia, Trichothecium, Cladosporium and Thermomyces all genera belonging to Fungi Imperfecti. The only genus of this class lacking the cycle was Candida. No genera from the classes Basidiomycetes and Phycomycetes showed any mannitol 1-phosphate dehydrogenase or mannitol 1-phosphatase activities. The genera investigated, belonging to Ascomycetes, Gibberella, Ceratocystis and Neurospora all lacked mannitol 1-phosphate dehydrogenase. It was concluded that the mannitol cycle is an important and widespread pathway for NADH oxidation and NADP+ reduction in the organisms belonging to the class Fungi Imperfecti.

Fungi↗
PubMed1982

Cell wall degradation in the autolysis of filamentous fungi.

A systematic study on autolysis of the cell walls of fungi has been made on Neurospora crassa, Botrytis cinerea, Polystictus versicolor, Aspergillus nidulans, Schizophyllum commune, Aspergillus niger, and Mucor mucedo. During autolysis each fungus produces the necessary lytic enzymes for its autodegradation. From autolyzed cultures of each fungus enzymatic precipitates were obtained. The degree of lysis of the cell walls, obtained from non-autolyzed mycelia, was studied by incubating these cell walls with and without a supply of their own lytic enzymes. The degree of lysis increased with the incubation time and generally was higher with a supply of lytic enzymes. Cell walls from mycelia of different ages were obtained. A higher degree of lysis was always found, in young cell walls than in older cell walls, when exogenous lytic enzymes were present. In all the fungi studied, there is lysis of the cell walls during autolysis. This is confirmed by the change of the cell wall structure as well as by the degree of lysis reached by the cell wall and the release of substances, principally glucose and N-acetylglucosamine in the medium.

Aspergillus nidulans↗
PubMed1975

Role of glycosidases in the membranlytic, antifungal action of Saponins.

In studies on the membranlytic action of various saponins on mycelium of Botrytis cinerea and Rhizoctonia solani digitonin, alpha-hederin and tomatin caused considerable leakage of free amino acids, while aescin and theasaponin were less effective. Cyclamin significantly damaged cell membranes of R. solani, but did not change the selective permeability of B. cinerea. Cell membrane disruption was accompanied by an enzymatic conversion of saponins into their corresponding aglycones in cell membrane vicinity, an effect which was significantly inhibited by aldonolactones, known inhibitors of beta-glycosidases. These results lead to the conclusion that the hardly water soluble aglycones are the active part of the saponin molecules, the saponins themselves being only water soluble transport forms. It follows, that the presence of appropriate glycosidases in cell membranes, capable of converting saponins into their aglycones, is a prerequisite for the membranlytic action of saponins. The similarity of the membranlytic effects of saponins towards fungi and erythrocytes is descussed.

Cell Membrane↗
PubMed1981

[Molds in foods of the Czechoslovak Socialistic Republik (author's transl)].

The mycoflora of 13.527 g of food powders which were normal upon sensory evaluation as well of 22.017 pieces of surface sterilized grains and nuts was examined in the Czechoslovak Socialist Republic. 147.027 colonies of 44 genera of molds were isolated and identified from samples. Colonies of Penicllium sp., Aspergillus sp., and Cladosporium sp. occured most often, less often or quite infrequently Rhizopus sp., Mucor sp., Absidia sp., Alternaria sp., Circinella sp., Trichoderma sp., Botrytis sp., Paecilomyces sp., Geotrichum sp., Syncephalastrum sp., Scopulariopsis sp., Phoma sp., Auerobasidium sp., Fusarium sp., Thamnidium sp., Cunninghamella sp., Stemphylium sp., Torula sp., Trichothecium sp., Verticillium sp., Cephalosporium sp., Mortierella sp., Neurospora sp., Ostracoderma sp., Arthrinium sp., Monodyctis sp., Papularia sp., Acremonium sp., Chaetomium sp., Chrysosporium sp., Beauveria sp., Cephaliophora sp., Daktylosporium sp., Drechslera sp., Gliomastix sp., Helminthosporium sp., Humicola sp., Monilia sp., Sepedonium sp., Ulocladium sp., and Wallemia sp.

Food Microbiology↗
PubMed1990

Mycoflora and nutritional value of shelled melon seeds (Citrulus vulgaris Schrad.) in Nigeria.

Thirteen fungi were isolated from mouldy shelled melon seeds. The fungi more frequently isolated included species of Mucor (11.25%), Rhizopus (13.75%), Aspergillus (36.25%), Macrophoma (2.50%), Penicillium (8.75%), Alternaria (5.00%), Fusarium (8.75%), Botrytis (6.25%), Torula (3.75%) and Geotrichum (3.75%). Healthy, shelled melon seeds inoculated with Fusarium solani for 7 or 14 days caused increases in the free fatty acid (f.f.a.) content of the healthy seeds from an initial value of 1.06% to 2.19% after 7 days and 4.23% after 14 days. Similar results were obtained when other fungal isolates were used as inocula but Fusarium solani effected the greatest increase in the f.f.a. content followed by Aspergillus niger and Penicillium notatum. The fungi also caused decreases in the crude protein from 35.51% to 25.16%, crude fibre from 4.30% to 1.35% and total carbohydrate content from 4.28% to 3.01% of the seeds after 14 days infection. Aflatoxin was detected (0.20 micron/g) in the infected seeds being sold in the markets and also from seeds inoculated with spores (0.40 micron/g) of A/ flavus and 0.50 micron/g) from a mixture of spores of various Aspergillus strains after incubation for 14 days.

Aflatoxins↗
PubMed1991

[Isolation and characterization of colloidal soluble polysaccharides in raspberry juice].

Depending on the preparation method, the colloid content of raspberry juices treated with pectolytic enzymes, can be up to 2.5 g/L. Analysis of the polysaccharides shows that there are in particular, arabinans, arabinogalactans type II and rhamnogalacturonans. While the content of arabinans could be reduced by the action of arabinases, the arabinogalactans and rhamnogalacturonans were not degraded by conventional pectic enzymes. Besides these cell-wall polysaccharides, it was possible to isolate a high molecular-mass beta-glucan, which is thought to originate from Botrytis cinerea. Technical problems in juice clarification or filtration may be caused by this beta-glucan or the high content of residual pectic substances.

Beverages↗
PubMed1996

Structural and antifungal properties of a pathogenesis-related protein from wheat kernel.

We have purified and characterized a protein from the water-soluble fraction of wheat kernel (Triticum aestivum cv. S. Pastore) consisting of a single polypeptide chain blocked at its N-terminus by a pyroglutamate residue; the complete amino acid sequence has been determined by automated sequence analysis performed on peptide fragments obtained by enzymatic hydrolyses of the protein. Homology studies have shown that this protein is very similar (97% sequence identity) to the previously characterized wheatwin1 as well as to other members of the pathogenesis-related (PR) proteins of class 4; in analogy with wheatwin1, we have termed this protein wheatwin2. Both wheatwin1 and wheatwin2 have specific antifugal activity toward the wide-host-range pathogen Botrytis cinerea and the wheat-specific pathogenic fungi of wheat Fusarium culmorum and Fusarium graminearum of groups 1 and 2. On the basis of their structural and functional properties, wheatwin1 and wheatwin2 can be classified as members of the PR4 protein family; this represents the first report concerning the presence of this kind of protein in wheat.

Amino Acid Sequence↗
PubMed1994

Growth and conidiation of Trichoderma viride are affected by non-steroidal antiinflammatory agents.

Nonsteroid antiinflammatory agents (NSAIA's) (inhibitors of cyclooxygenase and lipoxygenase) of several structural series inhibited growth of Trichoderma viride. The most potent growth inhibitors were indomethacin and its derivative repanidal which inhibited in the range of 0.1 mmol/L. The weakest inhibitors were acetylsalicylic acid and lysine salicylate which exerted only a weak effect at concentrations above 1 mmol/L. The inhibition of growth was accompanied by a stimulation of conidiation in the dark. A light pulse increased the efficiency of these drugs to stimulate the conidiation. Saccharomyces cerevisiae was as sensitive to NSAIA's as T. viride while Botrytis cinerea was less sensitive. The results indicate that arachidonate metabolism may play a role in the growth of fungi and may participate also in the process of conidiation.

Anti-Inflammatory Agents, Non-Steroidal↗
PubMed2002

Copper complexes with bioactive ligands. Part II--Antifungal activity.

Antifungal activity of new copper(II) complexes of 2-methylthionicotinate (2-MeSNic) of the composition Cu(2-MeSNic)2(MeNia)(2).4H2O (where MeNia is N-methylnicotinamide), and Cu(2-MeSNic)2(Nia)(2).2H2O (where Nia is nicotinamide) and Cu(2-MeSNic)2L2 (where L is isonicotinamide, iNia, or ethyl nicotinate, EtNic) were tested on various strains of filamentous fungi by the macrodilution method. Most sensitive against copper(II) adducts with bioactive ligands were Rhizopus oryzae and Microsporum gypseum (IC50 1.5-2.3 mmol/L). The adducts with Nia, MeNia and EtNic at 5 mmol/L induced morphological changes in growing hyphae of Botrytis cinerea, mainly their intensive branching attached to release of cytoplasm with partial growth inhibition. Inhibition of sporulation (> 90%) of Alternaria alternata by Cu(2-MeSNic)2.H2O was observed as a change in the color of the colonies. The highest resistance was marked by B. cinerea and Fusarium moniliforme (average IC50 values 4.25 and 3.13 mmol/L, respectively). The presence of all bioactive ligands in copper(II) complexes caused an increase in the inhibition effect against model fungi (except significant inhibition activity of EtNic on R. oryzae).

Alternaria↗
PubMed1975

Morphogenic effects of ramihyphin A in filamentous fungi.

Ramihyphin A at subfungistatic concentrations stimulates ramification of hyphae of filamentous fungi. Stimulation of terminal ramification of hyphae that can be observed particularly in phytopathogenic fungi is most frequent. Hyphae of Microsporon canis, Trichophyton mentagrophytes, Blastomyces dermatitidis, Coccidioides immitis and Histoplasma capsulatum ramify intensively laterally. Stimulation of the lateral ramification was observed in Monilia fructigena, Penicillium marneffei and Penicillium chrysogenum. The antibiotic induces also formation of vesicular structures in phytopathogens. Due to the substantial ramification of hyphae, both terminal and lateral, the growth of colonies is interrupted. The addition of the antibiotic to a growing colony of Botrytis cinerea induces dichotomic ramification of terminal hyphae after 3 h of growth. Lateral hyphae begin to grow later and further ramify dichotomically. Dense bundles of ramified hyphae are formed after 24 h due to the unbalanced ramification and the colony no longer increases its size.

Antifungal Agents↗