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PubMed1990

Activation of a Bean Chitinase Promoter in Transgenic Tobacco Plants by Phytopathogenic Fungi.

The temporal and spatial expression of a bean chitinase promoter has been investigated in response to fungal attack. Analysis of transgenic tobacco plants containing a chimeric gene composed of a 1.7-kilobase fragment carrying the chitinase 5B gene promoter fused to the coding region of the gus A gene indicated that the chitinase promoter is activated during attack by the fungal pathogens Botrytis cinerea, Rhizoctonia solani, and Sclerotium rolfsii. Although induction of [beta]-glucuronidase activity was observed in tissues that had not been exposed to these phytopathogens, the greatest induction occurred in and around the site of fungal infection. The increase in [beta]-glucuronidase activity closely paralleled the increase in endogenous tobacco chitinase activity produced in response to fungal infection. Thus, the chitinase 5B-gus A fusion gene may be used to analyze the cellular and molecular details of the activation of the host defense system during pathogen attack.

Journal Article↗
PubMed1997

Salicylic Acid Interferes with Tobacco Mosaic Virus Replication via a Novel Salicylhydroxamic Acid-Sensitive Mechanism.

Salicylic acid (SA) induces resistance to all plant pathogens, including bacteria, fungi, and viruses, but the mechanism by which SA engenders resistance to viruses is not known. Pretreatment of tobacco mosaic virus (TMV)-susceptible (nn genotype) tobacco tissue with SA reduced the levels of viral RNAs and viral coat protein accumulating after inoculation with TMV. Viral RNAs were not affected equally, suggesting that SA treatment interferes with TMV replication. Salicylhydroxamic acid (SHAM), an inhibitor of the mitochondrial alternative oxidase, antagonized both SA-induced resistance to TMV in nn genotype plants and SA-induced acquired resistance in resistant (NN genotype) tobacco. SHAM did not inhibit induction of the PR-1 pathogenesis-related protein or induction of resistance to Erwinia carotovora or Botrytis cinerea by SA. This indicates that SA induces resistance to TMV via a novel SHAM-sensitive signal transduction pathway (potentially involving alternative oxidase), which is distinct from that leading to resistance to bacteria and fungi.

Journal Article↗
PubMed2004

Properties of a polygalacturonase-inhibiting protein isolated from 'Oroblanco' grapefruit.

Polygalacturonase inhibiting protein (PGIP) was extracted from 'Oroblanco' grapefruit type (triploid pummelo-grapefruit) albedo tissue, purified and partially characterized. Extraction was carried out at 4 degrees C with a high ionic strength extraction buffer. After dialysis and concentration by ultrafiltration the extract was chromatographed on concanavalin A-Sepharose. The PGIP activity was bound by the lectin and then eluted using 250 mM alpha-methyl mannopyranoside, resulting in a 17-fold purification of the PGIP and demonstrating its glycoprotein nature. The anion-exchange and size-exclusion chromatography steps that followed gave a PGIP that was 857-fold purified relative to the initial tissue extract, and having a 44 kDa molecular weight, as estimated by SDS-PAGE electrophoresis. PGIP inhibition activity was tested with endo-polygalacturonase (EC 3.2.1.15) produced by Penicillium italicum and Botrytis cinerea. The radial diffusion and reducing sugar assays showed that P. italicum and B. cinerea endo-PGs were affected by PGIP, whereas no endo-PG activity was detected in the culture filtrate of P. digitatum. In vitro tests revealed that PGIP inhibited P. italicum and B. cinerea growth. By contrast, the influence of PGIP on P. digitatum, growth was negligible, perhaps because this fungus does not produce endo-PG. Following heating for 10 min at 65 degrees C the inhibitory activity of PGIP was reduced by 43%. PGIP activity decreased further as heating temperature increased, and was completely suppressed after heating at 100 degrees C for 10 min.

Journal Article↗
PubMed1994

HM17, a new polyene antifungal antibiotic produced by a new strain of Spirillospora.

An antifungal antibiotic (HM17) was obtained from a new isolate classified to the genus Spirillospora on the basis of its chemical and morphological properties. On solid media this antibiotic strongly inhibited the growth of strains of Fusarium oxysporum formae speciales albedinis, Botrytis cinerea, Gaeumaniomyces graminis and several other fungi known to be plant and human pathogens. Antifungal activity in culture collection strains of Spirillospora has not so far been reported. The u.v. absorption spectrum and physico-chemical characteristics place HM17 in the methylpentaene sub-group of polyene macrolides. HM17 is different from other known methylpentaenes. This is the first report of polyene production by a Spirillospora.

Actinomycetales↗
PubMed2004

Fungicide activity through activation of a fungal signalling pathway.

Fungicides generally inhibit enzymatic reactions involved in fungal cellular biosynthesis. Here we report, for the first time, an example of fungicidal effects through hyperactivation of a fungal signal transduction pathway. The OSC1 gene, encoding a MAP kinase (MAPK) related to yeast Hog1, was isolated from the fungal pathogen Colletotrichum lagenarium that causes cucumber anthracnose. The osc1 knockout mutants were sensitive to high osmotic stress and showed increased resistance to the fungicide fludioxonil, indicating that Osc1 is involved in responses to hyperosmotic stress and sensitivity to fludioxonil. The Osc1 MAPK is phosphorylated under high osmotic conditions, indicating activation of Osc1 by high osmotic stress. Importantly, fludioxonil treatment also activates phosphorylation of Osc1, suggesting that improper activation of Osc1 by fludioxonil has negative effects on fungal growth. In the presence of fludioxonil, the wild-type fungus was not able to infect the host plant because of a failure of appressorium-mediated penetration, whereas osc1 mutants successfully infected plants. Analysis using a OSC1-GFP fusion gene indicated that Osc1 is rapidly translocated to the nucleus in appressorial cells after the addition of fludioxonil, suggesting that fludioxonil impairs the function of infection structures by activation of Osc1. Furthermore, fludioxonil activates Hog1-type MAPKs in the plant pathogenic fungi Cochliobolus heterostrophus and Botrytis cinerea. These results strongly suggest that fludioxonil acts as a fungicide, in part, through activation of the MAPK cascade in fungal pathogens.

Amino Acid Sequence↗
PubMed2005

Quantification of allele-specific expression of a gene encoding strawberry polygalacturonase-inhibiting protein (PGIP) using Pyrosequencing.

Recent studies indicate that allele-specific differences in gene expression are a common phenomenon. The extent to which differential allelic expression exists might be underestimated, due to the limited accuracy of the methods used so far. To demonstrate allele-specific expression, we investigated the transcript abundance of six individual, highly homologous alleles of a polygalacturonase-inhibiting protein gene (FaPGIP) from octoploid strawberry (Fragaria x ananassa). We applied the highly quantitative Pyrosequencing method which, for the gene under study, detected allele frequency differences as small as 4.0 +/- 2.8%. Pyrosequencing of RT-PCR products showed that one FaPGIP allele was preferentially expressed in leaf tissue, while two other alleles were expressed in a fruit-specific way. For fruits that were inoculated with Botrytis cinerea a strong increase in overall FaPGIP gene expression was observed. This upregulation was accompanied by a significant change in FaPGIP allele frequencies when compared with non-treated fruits. Remarkably, in the five cultivars tested, the allele frequency in cDNA from the inoculated fruits was similar to that in genomic DNA, suggesting uniform upregulation of all FaPGIP alleles present as a result of pathogenesis-related stress. The results demonstrate that when Pyrosequencing of RT-PCR products is performed, novel allele-specific gene regulation can be detected and quantified.

Alleles↗
PubMed2006

Salicylic acid-mediated innate immunity in Arabidopsis is regulated by SIZ1 SUMO E3 ligase.

Reversible modifications of target proteins by small ubiquitin-like modifier (SUMO) proteins are involved in many cellular processes in yeast and animals. Yet little is known about the function of sumoylation in plants. Here, we show that the SIZ1 gene, which encodes an Arabidopsis SUMO E3 ligase, regulates innate immunity. Mutant siz1 plants exhibit constitutive systemic-acquired resistance (SAR) characterized by elevated accumulation of salicylic acid (SA), increased expression of pathogenesis-related (PR) genes, and increased resistance to the bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Transfer of the NahG gene to siz1 plants results in reversal of these phenotypes back to wild-type. Analyses of the double mutants, npr1 siz1, pad4 siz1 and ndr1 siz1 revealed that SIZ1 controls SA signalling. SIZ1 interacts epistatically with PAD4 to regulate PR expression and disease resistance. Consistent with these observations, siz1 plants exhibited enhanced resistance to Pst DC3000 expressing avrRps4, a bacterial avirulence determinant that responds to the EDS1/PAD4-dependent TIR-NBS-type R gene. In contrast, siz1 plants were not resistant to Pst DC3000 expressing avrRpm1, a bacterial avirulence determinant that responds to the NDR1-dependent CC-NBS-type R gene. Jasmonic acid (JA)-induced PDF1.2 expression and susceptibility to Botrytis cinerea were unaltered in siz1 plants. Taken together, these results demonstrate that SIZ1 is required for SA and PAD4-mediated R gene signalling, which in turn confers innate immunity in Arabidopsis.

Arabidopsis↗
PubMed1985

Volumetric aerobiological survey of conidial fungi in the North-East Netherlands. I. Seasonal patterns and the influence of metereological variables.

In order to obtain actual data about the qualitative and quantitative occurrence of airborne fungi in The Netherlands, a viable-volumetric sample survey with the Andersen sampler was undertaken. From April 1981 to the end of June 1983, one day a week samples were taken on the unobstructed rooftop of a hospital in the N-E Netherlands. An average of 268 Colony Forming Units (CFU), per sample, per cubic metre of air were found. Almost three-quarters of the total catch consisted of seven genera, Cladosporium predominating. Botrytis showed in surprisingly high numbers when compared with other surveys, while Alternaria occurred in very low concentrations. Most atmospheric airborne spora were found between May and September. Aspergillus and Penicillium prevailed in the autumn and winter months, although were present the year-round. Momentary weather conditions seemed less important for the overall spore picture than the average meteorological characteristics for a whole season. The obtained aero-mycological information may be useful in determining clinical strategies for skin testing and serological investigations in patients with suspected mould allergies.

Air Microbiology↗
PubMed1991

Significance of sampling height of airborne particles for aerobiological information.

Pollen and spore counts from Burkard traps for routine pollen and spore sampling placed at 15 m above ground and at ground level were compared. Daily counts of most pollen types were higher on the ground than at roof level, but the counts were significantly correlated. The ratios of pollen frequencies at high and low levels varied between 1.0 and 11.5. The most prominent differences were recorded for herbaceous pollen (e.g. Artemisia counts 11.5 and Poaccae counts 4.4 times higher at ground level) and in Botrytis and Ustilaginales spores. Tree pollen grains and basidiomycetous spores were more equally distributed. Wind speed did not affect the variation of pollen frequencies at either height. Large spores are not so unevenly distributed as previously supposed. Artemisia and grass pollen was detected 1 to 2 weeks earlier at ground level than on the roof. It is therefore concluded that especially the beginning of flowering should be monitored at a low level.

Air Pollutants↗
PubMed1998

Aeroallergens and viable microbes in sandstorm dust. Potential triggers of allergic and nonallergic respiratory ailments.

Aeroallergens and antigens in sandstorm dust, extracts of which were skin prick test (SPT) positive in allergic patients, were detected by rocket immunoelectrophoresis and ELISA. Fungi and bacteria isolated by agar settle plates and soil dilution and soil washing methods were enumerated and identified. Cat dander, Acacia, Alternaria, Aspergillus, Chenopodium, Cladosporium, Bermuda grass, Pithecellobium, Prosopis, Rumex, cultivated rye, and Washingtonia palm allergens were detected by both methods. Viable microbes including 1892 +/- 325 colony-forming units (cfu) of bacteria, and 869 +/- 75 cfu of fungi were isolated per gram of dust by the soil dilution method. Randomly selected microbial colonies on streaking and subculture were found to consist of between two and seven mixed colonies. Fungi including Alternaria, Aspergillus, Botrytis, Cladosporium, Mortierella, Mucor, Mycelia sterilia, Penicillium, Pythium, Ulocladium, Verticillium, and some yeasts were isolated. Actinomyces, Bacillus, Pseudomonas, and mostly coagulase-negative Staphylococcus species were identified, but the bulk of unidentified bacterial isolates were mainly mixed colonies of rods, cocci, coccobacilli, and some filamentous types. Six-hour agar settle-plate counts during sandstorms were 100 and 40% higher for bacteria and fungi, respectively, than without sandstorms. The most abundant aeroallergens were those of Acacia, Alternaria, Aspergillus, Bermuda grass, Cladosporium, cultivated rye, Prosopis, and cat dander. Pithecellobium dulce, Rumex crispus, and Washingtonia palm allergens were detectable for the first time in Riyadh. IgE reactivities of the dust in man were demonstrated by ELISA using sera from atopic, exposed, and normal subjects. These results indicate that sandstorm dust is a prolific source of potential triggers of allergic and nonallergic respiratory ailments, and the methods mentioned here should be routinely used for quick sampling of the environment.

Air Pollutants↗
PubMed2006

Brassica oleracea pollen, a new source of occupational allergens.

BACKGROUND: Vegetable pollen is a rare source of occupational allergens. Occupational allergy has only been described in the case of paprika pollen and tomato pollen. We describe a new source of occupational pollen allergy. AIM: To study the incidence and the impact of broccoli and cauliflower pollen allergy in employees involved in classical plant breeding. METHODS: Fifty-four employees of five companies working with cauliflower (Brassica oleracea botrytis) and broccoli (B. oleracea italica/cymosa) pollen were eligible for complete evaluation. Allergy to cauliflower and broccoli pollen was evaluated by questionnaire and determination of sensitization by radioallergosorbent test (RAST) and skin-prick tests (SPT). SPT and RAST were performed with a panel of commercial and homemade extracts from cauliflower and broccoli pollen. RESULTS: Work-related symptoms such as rhinitis, conjunctivitis, asthma and urticaria caused by B. oleracea pollen were reported by 44% of the participants (24/54), of whom all but one had positive SPT for cauliflower- and/or broccoli-pollen/flower extracts and 58% (14/24) had positive RAST results. Symptoms had developed within the first 2 years in 33% of the patients. Six patients had to stop or change work. CONCLUSIONS: Brassica oleracea pollen is a new source of occupational allergen with strong allergenic potential leading to symptoms in almost half of the exposed employees.

Adult↗
PubMed1992

Isolation and characterization of three chitinases from Trichoderma harzianum.

Three proteins which display chitinase activity were purified from the supernatants of Trichoderma harzianum CECT 2413 grown in minimal medium supplemented with chitin as the sole carbon source. Purification was carried out after protein precipitation with ammonium sulphate, adsorption to colloidal chitin and digestion, and, finally, chromatofocusing. By this procedure, two chitinases of 42 kDa (CHIT42) and 37 kDa (CHIT37) were purified to homogeneity, as judged by SDS/PAGE and gel filtration, whereas a third, of 33 kDa (CHIT33), was highly purified. The isoelectric points for CHIT42, CHIT37 and CHIT33 were 6.2, 4.6 and 7.8, respectively. The three enzymes displayed endochitinase activities and showed different kinetic properties. CHIT33 was able to hydrolyze chitin oligomers of a polymerization degree higher than n = 4, its Km for colloidal chitin being 0.3 mg/ml. CHIT42 and CHIT37 were able to hydrolyze chitin oligomers with a minimal polymerization degree of n = 3, their Km values for colloidal chitin being 1.0 mg/ml and 0.5 mg/ml respectively. With regard to their lytic activity with purified cell walls of the phytopathogenic fungus Botrytis cinerea, a hydrolytic action was observed only when CHIT42 was present. Antibodies against CHIT42 and CHIT37 specifically recognized the proteins and did not display cross-reaction, suggesting that each protein is encoded by a different gene.

Carbohydrate Conformation↗
PubMed2006

The major allergen of Alternaria alternata (Alt a 1) is expressed in other members of the Pleosporaceae family.

There is general consensus regarding the scarce cross-reactivity existing between Alternaria alternata and other allergenic moulds such as Aspergillus fumigatus, Penicillium notatum or Cladosporium herbarum. However, A. alternata has been shown to have a very significant level of allergenic cross-reactivity with other fungi belonging to the Pleosporaceae family. To date, no biological identity or homologies with other proteins have been described for Alt a 1, and it remains unclear whether the major allergen Alt a 1 contributes to the cross-reactivity shown for these moulds. Specific quantification of Alt a 1 in culture filtrates of Stemphylium botryosum, Ulocladium botrytis, Curvularia lunata, Alternaria tenuissima, C. herbarum, Penicillium chrysogenum and Asp. fumigatus, and immunoblotting using culture filtrate extracts from the above-mentioned moulds and rabbit serum anti-recombinant Alt a 1 have shown significant amounts of Alt a 1 in culture filtrates as well as antigenic components ranging from 14 to 20 kDa that strongly react with the specific serum for all taxonomically related species (Pleosporaceae family). No reactions were revealed in culture filtrates of Cladosporium, Penicillium and Aspergillus. These results restrict the cross-reactivity phenomenon due to Alt a 1 to the scope of the taxonomical term of family.

Allergens↗
PubMed2006

Skin test reactivity to molds in pre-school children with newly diagnosed asthma.

BACKGROUND: The aim of the present study was to evaluate the prevalence of sensitization to molds and to house dust mites (HDM) in pre-school children with newly-diagnosed asthma. METHODS: From 1996 to 2000, 122 children 1 to 6 years of age with fresh asthma treated in the Kuopio University Hospital, Kuopio, Finland, were recruited in the study; 94% attended. Skin prick tests were performed to common inhalant allergens and to 10 molds. The homes were surveyed for moisture damage. RESULTS: A majority of the houses had signs of dampness and moisture. One-third of the children reacted to inhalant allergens. There were 11 positive reactions to molds in five children who all reacted to at least one animal dander or seasonal pollen. Aspergillus fumigatus, Botrytis cinerea, and Cladosporium herbarum were the most common mold allergens. There were only two children with positive reactions to HDM, and none of them reacted to molds. No associations were found between the presence or degree of moisture problems at home and mold or HDM allergy. CONCLUSION: Sensitization to molds seems to play only a minor role in pre-school children with newly-diagnosed asthma in a northern climate where the incidences of mold and HDM allergies are rather low.

Age Factors↗
PubMed1995

Partial characterization of chitinolytic enzymes from Streptomyces albidoflavus.

Streptomyces albidoflavus NRRL B-16746 secreted three types of chitinolytic enzymes: N-acetyl-glucosaminidase, chitobiosidase and endochitinase. Optimal activity for all three types of enzymes occurred at pH 4-6; however 55-74% of the chitobiosidase and endochitinase activity was detectable at pH 8-10. Chitobiosidase activity originated from two strongly acidic (pI < 3.0) proteins with molecular mass of 27 kDa and 34 kDa, while endochitinase activity originated from five major acidic proteins (pI 5.1, 5.3, 5.75, 5.8-5.9 and 6.4) with molecular mass of 59, 45, 38.5, 27 and 25.5 kDa. Purified chitobiosidases significantly reduced spore germination and germ tube elongation of Botrytis cinerea and Fusarium oxysporum. Chitinolytic enzymes with significant activity at pH 4-10 may be used, transgenically, to reduce the growth and/or development of a broad spectrum of insects and fungi that are major economic pests.

Acetylglucosaminidase↗
PubMed2006

Fungi and ochratoxin A detected in healthy grapes for wine production.

AIMS: The mycoflora of healthy grapes (i.e. without visible symptoms of rot) for wine production in Portuguese wine-making regions was assessed and its potential for ochratoxin A (OTA) production evaluated. The OTA content of grapes was also determined. METHODS AND RESULTS: A total of 386 fungal strains were isolated by plating methods. The most frequent genera found in grapes were non-ochratoxigenic species: Cladosporium (28%), Penicillium (24%), Botrytis (13%) and Aspergillus (9%). Two OTA-producing strains were isolated, belonging to the species Aspergillus carbonarius and Aspergillus ochraceus. OTA was detected in three of four grape samples, up to 116 ng l(-1). CONCLUSIONS: OTA is being produced in healthy berries by Aspergillus species, namely A. carbonarius, at levels below the maximum recommended limit of 2,000 ng l(-1) in wine. SIGNIFICANCE AND IMPACT OF THE STUDY: The OTA concentration detected in healthy Portuguese grapes does not represent a risk to wine regarding the legal limit established.

Aspergillus ochraceus↗
PubMed2006

Antimicrobial activity of carvacrol related to its chemical structure.

AIMS: To investigate the relation between the chemical structure and the antimicrobial activity of carvacrol, eugenol, menthol and two synthesized carvacrol derivative compounds: carvacrol methyl ether and carvacryl acetate against bacteria, Escherichia coli, Pseudomonas fluorescens, Staphylococcus aureus, Lactobacillus plantarum, Bacillus subtilis, a yeast Saccharomyces cerevisiae and one fungi Botrytis cinerea. METHODS AND RESULTS: The antimicrobial activity was tested in liquid and vapour phases, by both broth liquid and microatmosphere methods, respectively. The same classification of the compound's antimicrobial efficiency was found with both methods. Eugenol and menthol exhibited a weaker antimicrobial activity than carvacrol, the most hydrophobic compound. Carvacryl acetate and carvacrol methyl ether were not efficient, indicating that the presence of a free hydroxyl group is essential for antimicrobial activity. CONCLUSIONS: The different extents of antimicrobial aroma compounds' efficiency showed that hydrophobicity is an important factor and the presence of a free hydroxyl group and a delocalized system allows proton exchange. SIGNIFICANCE AND IMPACT OF THE STUDY: This study has identified the importance of the hydrophobicity and the chemical structure of phenolic aroma compounds for antimicrobial activity and may contribute to a most rational use of these compounds as antimicrobial agent.

Anti-Infective Agents↗
PubMed2000

Humus bacteria of Norway spruce stands: plant growth promoting properties and birch, red fescue and alder colonizing capacity.

We studied the potential of the humus layer of the Norway spruce stands to supply beneficial rhizobacteria to birch (Betula pendula), alder (Alnus incana) and fescue grass (Festuca rubra), representatives of pioneer vegetation after clear-cutting of the coniferous forest. Axenically grown seedlings of these species were inoculated with the acid spruce humus, pH 3.7-5.3. Actinorhizal propagules, capable of nodulating alder, were present in high density (10(3) g(-1)) in humus of long-term limed plots, whereas plots with nitrogen fertilization contained almost none (</=10 g(-1)). The genera most frequently found in the humus were Bacillus, Paenibacillus, Arthrobacter, Nocardia, Rhodococcus and Pseudomonas, independently of prior liming or fertilization of the plots. The taxa found in the seedling roots differed from that in humus by the prevalence of the Gram-negative genera Pseudomonas, Alcaligenes and Comamonas. Enrichment cultures of the roots on nitrogen-free media yielded Paenibacillus and Rhodococcus species. Nitrogen-fixing R. erythropolis and a novel Paenibacillus, closest by full sequence of 16S rDNA to P. durus, represented new classes of nitrogen-fixing rhizosphere bacteria. In addition, nitrogen-fixing R. fascians was found in the humus. The rhizoflora and humus contained high proportions of bacteria antagonistic towards plant pathogenic Rhizoctonia sp., Botrytis cinerea and Fusarium culmorum. The antagonistic isolates also commonly produced siderophores and/or cell wall degrading enzymes.

Journal Article↗