Microconidia Of Fusarium Oxysporum Research Articles

Development of quantitative proteomics using iTRAQ based on the immunological response of Galleria mellonella larvae challenged with Fusarium oxysporum microconidia.

Galleria mellonella has emerged as a potential invertebrate model for scrutinizing innate immunity. Larvae are easy to handle in host-pathogen assays. We undertook proteomics research in order to understand immune response in a heterologous host when challenged with microconidia of Fusarium oxysporum. The aim of this study was to investigate hemolymph proteins that were differentially expressed between control and immunized larvae sets, tested with F. oxysporum at two temperatures. The iTRAQ approach allowed us to observe the effects of immune challenges in a lucid and robust manner, identifying more than 50 proteins, 17 of them probably involved in the immune response. Changes in protein expression were statistically significant, especially when temperature was increased because this was notoriously affected by F. oxysporum 104 or 106 microconidia/mL. Some proteins were up-regulated upon immune fungal microconidia challenge when temperature changed from 25 to 37°C. After analysis of identified proteins by bioinformatics and meta-analysis, results revealed that they were involved in transport, immune response, storage, oxide-reduction and catabolism: 20 from G. mellonella, 20 from the Lepidoptera species and 19 spread across bacteria, protista, fungi and animal species. Among these, 13 proteins and 2 peptides were examined for their immune expression, and the hypothetical 3D structures of 2 well-known proteins, unannotated for G. mellonella, i.e., actin and CREBP, were resolved using peptides matched with Bombyx mori and Danaus plexippus, respectively. The main conclusion in this study was that iTRAQ tool constitutes a consistent method to detect proteins associated with the innate immune system of G. mellonella in response to infection caused by F. oxysporum. In addition, iTRAQ was a reliable quantitative proteomic approach to detect and quantify the expression levels of immune system proteins and peptides, in particular, it was found that 104 microconidia/mL at 37°C over expressed many more proteins than other treatments.

Activity of Chlorine Dioxide in a Solution of Ions and pH Against Thielaviopsis basicola and Fusarium oxysporum.

Chlorine dioxide (ClO2) is a disinfestant used to control pathogens in water. To determine if interactions between inorganic ions and pH levels effect ClO2 activity in vitro, concentrations of ClO2 (0, 1, 3, 5, 7, 9, 22, 24, 46, 58, and 70 mg/liter) were mixed for 10 min in solutions containing a nitrogen and hard water solution with equal concentrations of ammonium, nitrate, and synthetic hard water (0 and 100 mg/liter) and a divalent metal ion solution with equal concentrations of copper, iron, manganese, and zinc (0, 1, 3, and 5 mg/liter) at pH 5 and 8. Macro- and microconidia of Fusarium oxysporum f. sp. narcissi or conidia and aleuriospores of Thielaviopsis basicola were injected into each suspension for 30 s, captured on filter paper disks that were flushed with water, and plated on 50% potato dextrose agar. Spore germination was quantified after 1 day. ClO2 activity had a similar effect on both fungal species and all types of propagules with interactions among the divalent metal ion solution, nitrogen and hard water solution, and pH treatments. A higher concentration of ClO2 was required at pH 8 than at pH 5 to achieve a lethal dose resulting in 50% mortality of spores (LD50). The addition of the divalent metal ion solution required an increase in ClO2 concentration to maintain a LD50. When combined with the nitrogen and hard water solution, the divalent metal ion solution placed a higher demand on ClO2 at pH 5 and a lower demand on ClO2 at pH 8, thus requiring an increase and decrease in a ClO2 concentration, respectively, to achieve a LD50. Chlorine dioxide doses resulting in 50% mortality ranged from 0.5 to 7.0 mg/liter for conidia of F. oxysporum, 0.5 to 11.9 mg/liter for conidia of T. basicola, and 15.0 to 45.5 mg/liter for aleuriospores of T. basicola.

Fusarium wilt of field-grown muskmelon in British Columbia

A wilt disease of field-grown muskmelon (Cucumis melo L. var. cantalupensis) was observed during the 1999 and 2000 growing seasons on three farms in the semi-arid interior region near the Okanagan Valley of British Columbia. Initial symptoms were yellowing and wilting of leaves, which sometimes occurred unilaterally; in some cases, this was followed by plant death prior to flowering. The crown area of diseased plants had dark brown lesions that progressed into lateral branches for distances of 45—75 cm. Some lesions also extended into the developing fruit, which frequently became infected and rotted. Stem lesions were accompanied by dark brown exudate droplets and necrosis extended into the cortical and vascular regions. Plants with fruit collapsed when temperatures exceeded 25-s30°C in mid-July and early August. White mycelium developed on the infected crown area and on fruits collected from mature plants in the field, after incubation under high humidity conditions, and macroconidia and microconidia of Fusarium oxysporum Schlechtend.:Fr. were produced. A number of muskmelon cultivars were affected, and the most susceptible were Galia and Castella, while Athena appeared unaffected in the field. Pathogenicity tests demonstrated that muskmelon was the only susceptible host and no disease symptoms developed on cucumber (Cucumis sativus L.), watermelon (Citrullus vulgaris Eckl. & Zeyh), or squash (Cucurbita pepo L.). Based on symptomology and host range, the pathogen was identified as F. oxysporum Schlechtend. f. sp. melonis (Leach & Currence) W.C. Snyder & H.N. Hans. Inoculation of a set of differential muskmelon cultivars revealed that the pathogen was F. oxysporum f. sp. melonis race 1. The pathogen was recovered from diseased transplants in a propagation greenhouse and from seed recovered from naturally infected fruits on mature plants in the field. This is the first report of fusarium wilt of muskmelon in British Columbia, and the first report of race 1 of F. oxysporum f. sp. melonis in Canada.

Lipopolysaccharides of plant-growth promoting Pseudomonas sp. strain WCS417r induce resistance in carnation to Fusarium wilt

The numbers of diseased plants could significantly be reduced when microconidia of Fusarium oxysporum f.sp. dianthi were inoculated into the stem and viable-, heat-killed cells or purified LPS of the bacterium Pseudomonas sp. strain WCS417r were applied to the roots. Because the competition between F. o. dianthi and the bacterium could be excluded, the disease suppression seems to be due to an induced resistance. Accumulation of phytoalexins was found in the stem segments. No accumulation of these compounds was found when the plants were bacterized but noninfected. It is concluded that cell surface components present in the lipopolysaccharides of the bacterium are the inducing factors. Increased peroxidase activity could be measured in root washes and root extracts after only bacterial preparations were added. No significant differences in peroxidase activity were found in stem extracts. The possible role of increased peroxidase activity in suppression of Fusarium wilt in carnation is discussed.

In vitro inhibitory activity of xylem exudates from cucurbits towardsFusarium oxysporum microconidia

Experiments were conducted to determine if xylem exudates from three cucurbitaceous plants as well as non-cucurbit species were inhibitory to the growth of microconidia ofFusarium oxysporum f. sp.niveum, causal agent of Fusarium wilt of watermelon. Inhibitory effects of xylem exudates from watermelon(Citrullus lanatus) cultivars differentially susceptible toF.o. f.sp.niveum as well as from watermelon plants previously inoculated with virulent or avirulent races ofF. oxysporum, were compared. Results indicated that xylem fluid contained an inhibitory component that was independent of the resistance status of the cultivar or previous inoculation with virulent or avirulentF.o. f.sp.niveum races. Xylem exudates from cucumber(Cucumis sativis) also inhibited microconidia growth and colony formation while muskmelon(Cucumis melo) and tomato(Lycopersicon esculentum) xylem exudates had little inhibitory activity. Incubation with proteinase K, lysozyme, or boiled xylem fluid suggested that the active component had proteinaceous properties with an oligosaccharide moiety.

Effect of inoculum density, nitrogen source and saprophytic fungi on Fusarium wilt of Mexican lime

Mexican lime seedlings were inoculated with 0, 500, 1000, 2000, 4000 and 8000 microconidia ofFusarium oxysporum f. sp.citri per gram of potting media. The percent infection and mean disease severity rating increased with increasing inoculum density of the pathogen. In potting mix infested withAspergillus ochraceus, Penicillium funiculosum andTrichoderma harzianum at 5000 conidia per gram 2 weeks prior to infestation withF. oxysporum f. sp.citri at 0, 1000, 4000, and 8000 microconidia per gram,A. ochraceus reduced,P. funiculosum increased andT. harzianum had no effect on disease severity or pathogen population. OnlyP. funiculosum showed antagonistic activityin vitro against the pathogen. Disease severity and pathogen propagule densitites were greater and pH was lower in potting media fertilized with NH4−N than in media fertilized with NO3−N.

Surface localization of enzymes in mycelia and microconidia of Fusarium oxysporum.

Localization of enzymes in Fusarium oxysporum was investigated. It was found that invertase and sulfhydryl oxidizing activity were localized in the isolated cell-wall fraction of mycelia and microconidia of this fungus, prepared either by toluene and pronase treatment or mechanical disruption. The distribution of the enzyme activities was quite different from that of intracellular localizing enzymes. The physiological implication of the localization of these enzymes was discussed.

Localization of enzymes in the mycelium and microconidia of Fusarium oxysporum.

Maruyama, Yoshiharu (Cornell University, Ithaca, N.Y.) and Martin Alexander. Localization of enzymes in the mycelium and microconidia of Fusarium oxysporum. J. Bacteriol. 84:307-312. 1962-Extracts prepared from mycelium and microconidia of Fusarium oxysporum f. cubense were fractionated into a soluble and four particulate fractions by differential centrifugation, and the distribution of several enzymes in the isolated cell constituents was examined. Succinic dehydrogenase, cytochrome oxidase, and a large amount of the reduced diphosphopyridine nucleotide (DPNH) cytochrome c reductase and reduced triphosphopyridine nucleotide cytochrome c reductase were associated with one of the particulate fractions prepared from the hyphae; fumarase and DPNH oxidase activities were largely found in the soluble and in a second particulate fraction. The highest recovery and concentration of diphosphopyridine nucleotidase was observed to be bound to a third type of hyphal granule. Aldolase, aconitase, glucose-6-phosphatase, and uricase were recovered entirely with the soluble mycelium constituents. Similar enzyme-distribution patterns were observed in microconidia. Several enzymatic activities of the mycelial extracts were compared with those in the extracts of microconidia.

Distribution of protein and nucleic acids in hyphae and microconidia of Fusarium.

Extracts obtained by manual grinding of the mycelium and microconidia of Fusarium oxysporum were fractionated into four particulate portions—designated P1, P2, P3 and P4—and into a soluble portion by differential centrifugation. Approximately 60% of the protein was recovered in the supernatant fraction whereas the ribonucleic acid content of the P3 and P4 particles was particularly high. The P4 particle fraction was composed of approximately 40% ribonucleic acid and 60% protein and contained a particle with a sedimentation constant of about 85 S. More than 70% of the deoxyribonucleic acid was recovered among the various particle types, but the deoxyribonucleic acid: protein ratio was highest in the P1 granules. Polysaccharides were particularly concentrated in the P3 fraction. On the basis of the distribution of bound hexosamine, the extracts appeared to be free of cell wall fragments. Several methods of disruption of the fungus hyphae were examined.