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PubMed2002

Novel aspects of tomato root colonization and infection by Fusarium oxysporum f. sp. radicis-lycopersici revealed by confocal laser scanning microscopic analysis using the green fluorescent protein as a marker.

The fungus Fusarium oxysporum f. sp. radicis-lycopersici is the causal agent of tomato foot and root rot disease. The green fluorescent protein (GFP) was used to mark this fungus in order to visualize and analyze the colonization and infection processes in vivo. Transformation of F oxysporum f. sp. radicis-lycopersici was very efficient and gfp expression was stable for at least nine subcultures. Microscopic analysis of the transformants revealed homogeneity of the fluorescent signal, which was clearly visible in the hyphae as well as in the chlamydospores and conidia. To our knowledge, this is the first report in which this is shown. The transformation did not affect the pathogenicity. Using confocal laser scanning microscopy, colonization, infection, and disease development on tomato roots were visualized in detail and several new aspects of these processes were observed, such as (i) the complete colonization pattern of the tomato root system; (ii) the very first steps of contact between the fungus and the host, which takes place at the root hair zone by mingling and by the attachment of hyphae to the root hairs; (iii) the preferential colonization sites on the root surface, which are the grooves along the junctions of the epidermal cells; and (iv) the absence of specific infection sites, such as sites of emergence of secondary roots, root tips, or wounded tissue, and the absence of specific infection structures, such as appressoria. The results of this work prove that the use of GFP as a marker for F. oxysporum f. sp. radicis-lycopersici is a convenient, fast, and effective approach for studying plant-fungus interactions.

Fusarium↗
PubMed2003

Interactions in the tomato rhizosphere of two Pseudomonas biocontrol strains with the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici.

The fungus Fusarium oxysporum f. sp. radicis-lycopersici causes foot and root rot of tomato plants, which can be controlled by the bacteria Pseudomonas fluorescens WCS365 and P. chlororaphis PCL1391. Induced systemic resistance is thought to be involved in biocontrol by P. fluorescens WCS365. The antifungal metabolite phenazine-1-carboxamide (PCN), as well as efficient root colonization, are essential in the mechanism of biocontrol by P. chlororaphis PCL1391. To understand the effects of bacterial strains WCS365 and PCL1391 on the fungus in the tomato rhizosphere, microscopic analyses were performed using different autofluorescent proteins as markers. Tomato seedlings were inoculated with biocontrol bacteria and planted in an F. oxysporum f. sp. radicis-lycopersici-infested gnotobiotic sand system. Confocal laser scanning microscope analyses of the interactions in the tomato rhizosphere revealed that i) the microbes effectively compete for the same niche, and presumably also for root exudate nutrients; ii) the presence of either of the two bacteria negatively affects infection of the tomato root by the fungus; iii) both biocontrol bacteria colonize the hyphae extensively, which may represent a new mechanism in biocontrol by these pseudomonads; and iv) the production of PCN by P. chlororaphis PCL1391 negatively affects hyphal growth and branching, which presumably affects the colonization and infecting ability of the fungus.

Fusarium↗
PubMed2004

Role of chemotaxis toward fusaric acid in colonization of hyphae of Fusarium oxysporum f. sp. radicis-lycopersici by Pseudomonas fluorescens WCS365.

Pseudomonas fluorescens WCS365 is an excellent competitive colonizer of tomato root tips after bacterization of seed or seedlings. The strain controls tomato foot and root rot (TFRR) caused by the phytopathogenic fungus Fusarium oxysporum f. sp. radicis-lycopersici. Under biocontrol conditions, fungal hyphae were shown to be colonized by WCS365 bacteria. Because chemotaxis is required for root colonization by WCS365 cells, we studied whether chemotaxis also is required for hyphae colonization. To that end, an in vitro assay was developed to study hyphae colonization by bacteria. The results indicated that cells of the cheA mutant FAJ2060 colonize hyphae less efficiently than cells of wild-type strain WCS365, when single strains were analyzed as well as when both strains were applied together. Cells of WCS365 show a chemotactic response toward the spent growth medium of F. oxysporum f. sp. radicis-lycopersici, but those of its cheA mutant, FAJ2060, did not. Fusaric acid, a secondary metabolite secreted by Fusarium strains, appeared to be an excellent chemo-attractant. Supernatant fluids of a number of Fusarium strains secreting different levels of fusaric acid were tested as chemo-attractants. A positive correlation was found between chemo-attractant activity and fusaric acid level. No chemotactic response was observed toward the low fusaric acid-producer FO242. Nevertheless, the hyphae of FO242 still were colonized by WCS365, suggesting that other metabolites also play a role in this process. The possible function of hyphae colonization for the bacterium is discussed.

Chemotactic Factors↗
PubMed2005

Influence of fusaric acid on phenazine-1-carboxamide synthesis and gene expression of Pseudomonas chlororaphis strain PCL1391.

Production of the antifungal metabolite phenazine-1-carboxamide (PCN) by Pseudomonas chlororaphis strain PCL1391 is essential for the suppression of tomato foot and root rot caused by the soil-borne fungus F. oxysporum f. sp. radicis-lycopersici. The authors have shown previously that fusaric acid (FA), a phytotoxin produced by Fusarium oxysporum, represses the production of PCN and of the quorum-sensing signal N-hexanoyl-l-homoserine lactone (C(6)-HSL). Here they report that PCN repression by FA is maintained even during PCN-stimulating environmental conditions such as additional phenylalanine, additional ferric iron and a low Mg(2+) concentration. Constitutive expression of phzI or phzR increases the production of C(6)-HSL and abolishes the repression of PCN production by FA. Transcriptome analysis using P. chlororaphis PCL1391 microarrays showed that FA represses expression of the phenazine biosynthetic operon (phzABCDEFGH) and of the quorum-sensing regulatory genes phzI and phzR. FA does not alter expression of the PCN regulators gacS, rpoS and psrA. In conclusion, reduction of PCN levels by FA is due to direct or indirect repression of phzR and phzI. Microarray analyses identified genes of which the expression is strongly influenced by FA. Genes highly upregulated by FA are also upregulated by iron starvation in Pseudomonas aeruginosa. This remarkable overlap in the expression profile suggests an overlapping stress response to FA and iron starvation.

Fusaric Acid↗
PubMed2005

Enrichment for enhanced competitive plant root tip colonizers selects for a new class of biocontrol bacteria.

Our group studies tomato foot and root rot, a plant disease caused by the fungus Forl (Fusarium oxysporum f.sp. radicis-lycopersici ). Several bacteria have been described to be able to control the disease, using different mechanisms. Here we describe a method that enables us to select, after application of a crude rhizobacterial mixture on a sterile seedling, those strains that reach the root tip faster than our best tomato root colonizer tested so far, the Pseudomonas fluorescens biocontrol strain WCS365. Of the five tested new isolates, four appeared to be able to reduce the number of diseased plants. Analysis of one of these strains, P. fluorescens PCL1751, suggests that it controls the disease through the mechanism 'competition for nutrients and niches', a mechanism novel for biocontrol bacteria. Moreover, this is the first report describing a method to enrich for biocontrol strains from a crude mixture of rhizobacteria. Another advantage of the method is that four out of five strains do not produce antifungal metabolites, which is preferential for registration as a commercial product.

Agriculture↗
PubMed2006

Pip, a novel activator of phenazine biosynthesis in Pseudomonas chlororaphis PCL1391.

Secondary metabolites are important factors for interactions between bacteria and other organisms. Pseudomonas chlororaphis PCL1391 produces the antifungal secondary metabolite phenazine-1-carboxamide (PCN) that inhibits growth of Fusarium oxysporum f. sp. radius lycopersici the causative agent of tomato foot and root rot. Our previous work unraveled a cascade of genes regulating the PCN biosynthesis operon, phzABCDEFGH. Via a genetic screen, we identify in this study a novel TetR/AcrR regulator, named Pip (phenazine inducing protein), which is essential for PCN biosynthesis. A combination of a phenotypical characterization of a pip mutant, in trans complementation assays of various mutant strains, and electrophoretic mobility shift assays identified Pip as the fifth DNA-binding protein so far involved in regulation of PCN biosynthesis. In this regulatory pathway, Pip is positioned downstream of PsrA (Pseudomonas sigma factor regulator) and the stationary-phase sigma factor RpoS, while it is upstream of the quorum-sensing system PhzI/PhzR. These findings provide further evidence that the path leading to the expression of secondary metabolism gene clusters in Pseudomonas species is highly complex.

Amino Acid Sequence↗
PubMed2001

Population dynamics of Pythium aphanidermatum in cucumber grown in closed systems.

Foot and root rot in cucumber, caused by Pythium aphanidermatum (Edson) Fitzp., is an economically important disease in soilless culture systems. Nevertheless, very few data are available on the populations of this pathogen. Therefore, two detection methods, nested PCR (polymerase chain reaction) and plating on a selective medium after concentration of samples, were optimised and evaluated. With both methods very low concentrations of P. aphanidermatum could be detected; i.e. the detection limits were around 0.05 CFU/ml nutrient solution. In addition, real-time quantitative PCR using a Molecular Beacon probe was designed and tested. The potential and limitations of the different detection methods are discussed. With these different detection techniques, the population dynamics of P. aphanidermatum in a cucumber crop was followed. The impact of different disinfection treatments was studied in a greenhouse experiment with a cucumber crop growing on rockwool slabs in 12 independent closed systems. The nutrient solution was recirculated without disinfection (control), after UV-irradiation (250 mJ/cm2), or after slow sand filtration treatment. Part of the crop was inoculated with an isolate of P. aphanidermatum. The non-inoculated part could only become infected through the recirculated nutrient solution. Disease symptoms (stem rot, wilt, and root rot) and the yield loss were recorded in addition to the population dynamics of the pathogen. Very clear differences in the spread of the pathogen and in disease symptoms were measured between the systems with and without disinfection. UV-irradiation and slow sand filtration were both effective in removing the pathogen and protected the crop from disease symptoms. Correlation indices between the final yield and the different measurements during the experiment were calculated.

Colony Count, Microbial↗
PubMed1978

[The importance of antagonistic soil micro-flora for eliminating soil-born fungal pathogens in intensive corn-growing (author's transl)].

In a sandy soil, the antagonistic bacterial and fungal flora was analysed and their effectivity with respect to corn foot and stem rot was investigated. Measures that promoted total micro-flora in soil (organic fertilization) stimulated, in most cases, the antagonists as well. Different organic substances supported various types of antagonists. In soil with inhibited aeration the antagonists were reduced. A full effectivity of the antagonistic biological soil component presupposes the absence of host plants.

Anaerobiosis↗
PubMed2003

Characterization and in vitro expression patterns of an exopolygalacturonase encoding gene from Fusarium oxysporum f.sp. radicis lycopersici.

AIMS: In this work, we report the isolation, characterization and expression pattern in in vitro cultures of an EXOPG encoding gene (pgx2), a novel EXOPG encoding gene of Fusarium oxysporum f.sp. radicis lycopersici, responsible for foot crown and root rot disease in tomato plants. The gene was compared with other fungal polygalacturonases (PGs) previously reported. METHODS AND RESULTS: Partial sequences of the purified EXOPG native protein were used to design primers that amplified a genomic fragment by PCR. The amplified genomic fragment was used as a probe to screen a genomic library. One isolated clone was analysed. The complete genomic, cDNA and the deduced amino acid sequences were compared with other fungal EXOPGs and ENDOPGs. Regulation of pgx2 expression was analysed by Northern blot in in vitro cultures supplemented with different carbon sources. CONCLUSIONS: Pgx2 was present as single copy in the haploid genome of several Fusarium species. PGX2 showed the conserved amino acid motifs typical of PGs and those reported for fungal EXOPGs. Pgx2 was regulated at transcriptional level showing similar expression pattern to other EXOPG encoding gene (pgx1) when the fungus was cultured on different carbon sources suggesting a coordinate expression of both genes. This similarity would be supported by the presence of common putative regulatory motifs in the upstream regions of both genes. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reports the analysis of a novel EXOPG gene of the tomato pathogen F. oxysporum f.sp. radicis lycopersici, a contribution to the understanding of the role of cell-wall-degrading enzymes produced by fungi during pathogenesis.

Amino Acid Sequence↗
PubMed2001

Biological activity of Pythium oligandrum against Phytophthora species.

Influence of Pythium oligandrum as an a.i. of Polyversum on population dynamic of Phytophthora cryptogea in peat and development of Phytophtora rot on gerbera, Lawson cypress and yew-tree were evaluated. Drenching of peat, artificially infested with P. cryptogea, with Polyversum immediatelly after gerbera planting resulted in significant decrease of colony forming units number within 4 weeks. Concentrations of the biopreparate used had no significant influence on its greater, biological activity. Drenching of plants with Polyversum at conc. 0.05 or 0.1%, after planting into peat infested with P. cryptogea or P. cinnamomi, resulted in the strong suppression of Phytophthora foot or root and stem rot of gerbera, cypress and yew-tree.

Antibiosis↗
PubMed1998

"Toxic sock" syndrome.

For several months, a 17-year-old male athlete noticed that his feet had an itchy, burning sensation at the heels and toes. His foot odor had become so malodorous (somewhat akin to rotting fish) that he would not remove his shoes except immediately before washing his feet. Washing made the odor somewhat milder, but only for a short time. The patient had tried an over-the-counter "athlete's foot" powder without results. He had no other health problems and was not taking any medication. The accompanying figure shows the toes of his right foot.

Adolescent↗
PubMed2003

[A case of anaphylactoid shock occurring immediately after the initiation of second intravenous administration of high-dose immunoglobulin (IVIg) in a patient with Crow-Fukase syndrome].

We report a case of anaphylactoid shock occurring immediately after the initiation of second intravenous administration of high-dose immunoglobulin (IVIg) in a patient with Crow-Fukase syndrome. The patient was a 57-year-old woman, who was admitted to our hospital because of numbness and muscle weakness in the four extremities, difficulty in walking, and foot edema. On admission, her skin was dry and rough, and also showing scattered pigmentation, small hemangiomas, and hypertrichosis in both legs. She had distal dominant muscle weakness, more prominent in her legs, and was not able to walk. Deep tendon reflexes in her four extremities were markedly diminished or absent. She had a glove and stocking type of paresthesia, severe impairment of vibration, and absence of joint position sensation in her four extremities. On laboratory data, serum vascular endothelial growth factor (VEGF) was markedly elevated to 5,184 pg/ml (normal: below 220 pg/ml). Cerebrospinal fluid examination revealed cell counts of 2/microliter and protein level of 114 mg/dl. Abdominal echo showed marked hepatosplenomegaly. On peripheral nerve conduction study, both motor and sensory conduction velocity were undetectable in her legs. We diagnosed her condition as Crow-Fukase syndrome, and started IVIg of polyethyleneglycol-treated gamma-globulin (PEG-glob) at 400 mg/kg/day for 5 consecutive days for polyneuropathy. Since the first IVIg mildly improved muscle weakness, we tried the second IVIg of PEG-glob. However, immediately after the initiation of second IVIg of PEG-glob, she developed hypotention, dyspnea, cold sweating, cyanosis, and became lethargic. We immediately stopped IVIg and started first-aid treatment with epinephrine and corticosteroid for these symptoms. This treatment was successful and the patient fully recovered without any sequelae. Since serum IgE level remained unchanged and lymphocyte stimulation test (LST) was positive against the same rot number of PEG-glob, we diagnosed these symptoms as anaphylactoid shock. Based on the results of LST, we speculated that PEG-glob was the causative agent of anaphylactoid reaction. Anaphylactic or anaphylactoid reaction as adverse effects of IVIg is very rare, and to our knowledge, there are only 4 previous reports of anaphylactic or anaphylactoid reaction caused by IVIg. Therefore, we speculated that the prominent high level of serum VEGF in the present patient might play a significant contributory role in the development of anaphylactoid shock, since the vascular permeability of VEGF is 50,000 times stronger than that of histamine. We consider that it is necessary to carefully monitor IVIg of PEG-glob administration for polyneuropathy in patients with high level of serum VEGF, like Crow-Fukase syndrome.

Anaphylaxis↗