Evaluation of the biological control by the yeast <Emphasis Type="Italic">Torulaspora globosa</Emphasis> against <Emphasis Type="Italic">Colletotrichum sublineolum</Emphasis> in sorghum

The yeasts are microorganisms with great potential for biotechnological applications in diverse areas. The biological control of phytopathogens by yeasts has showed satisfactory results under laboratory conditions, and it has already produced commercial formulations. With this as focus, this work aims to perform in vitro and in vivo evaluations of the action of a Torulaspora globosa yeast strain (1S112), isolated from sugarcane rhizosphere, against the phytopathogenic mold Colletotrichum sublineolum, the causative agent of anthracnose in sorghum. In vitro experiments included the antagonism test in Petri dishes with morphological hyphal evaluation; yeast killer activity; siderophore, volatile compound and hydrolytic enzyme production. In vivo experiments were conducted in greenhouse conditions with a sorghum variety susceptible to C. sublineolum by evaluating the anthracnose disease for 6 weeks. The results indicated that the yeast strain significantly controlled the fungal growth, either in vitro or in vivo. The strain of T. globosa exhibited killer activity against two sensitive strains, which is a novel capacity for this species. The yeast did not produce siderophores, volatile compounds or hydrolytic enzymes, although it has reduced the mycelial growth, resulting in hyphal deformities but not cell death. The yeast controlled the anthracnose disease in sorghum, either inoculated before or after the fungal spores, suggesting that the competition for space and nutrients to dominate the mold and killer toxin production, altering the hyphal morphology, are mechanisms utilized by the yeast in the biocontrol.     

Development of <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> on Tolerant and Susceptible <Emphasis Type="Italic">Olea europaea</Emphasis> L. cultivars: A Microscopic Analysis

Colletotrichum acutatum is a cosmopolitan and damaging plant pathogen of temperate, subtropical, and tropical fruits and causes anthracnose on olive (Olea europaea L.). Three olive cultivars showing a variable response to infection by C. acutatum were selected to a preliminary study of pathogen development. Fruit samples, from susceptible and tolerant cultivars, were taken at 0, 24, 48, 72, and 192 h after inoculation for a microscopic and histological study of the infection and colonization process. The aim of this study was to compare the infection process: conidial germination, germ tube and appressorium formation, hyphal growth, and mesocarp colonization in susceptible and tolerant olive cultivars as a condition for further exploration of disease development, which is required to develop cultivars with improved resistance to anthracnose. The rate of mesocarp colonization differed between the susceptible and tolerant cultivars, and both intracellular hemibiotrophy and subcuticular intramural necrotrophy were observed. Hemibiotrophic infection predominated in the moderately tolerant cultivar.     

Agrobacterium-Mediated Transformation and Insertional Mutagenesis in <Emphasis Type="Italic">Colletotrichum acutatum</Emphasis> for Investigating Varied Pathogenicity Lifestyles

Colletotrichum acutatum is a cosmopolitan pathogen causing economically important diseases known as anthracnose on a wide range of hosts. This fungus exhibits varied pathogenicity lifestyles and the tools essential to understand the molecular mechanisms are still being developed. The transformation methods currently available for this species for gene discovery and functional analysis involve protoplast transformation and are laborious and inefficient. We have developed a protocol for efficient Agrobacterium tumefaciens-mediated transformation (ATMT) of C. acutatum. Using this protocol we were able to transform C. acutatum isolates belonging to different genetic groups and originating from different hosts. The transformation efficiency was up to 156 transformants per 10⁴ conidia, with >70% transformants showing single location/single copy integration of T-DNA. Binary vector pBHt2-GFP was constructed, enabling green fluorescence protein tagging of C. acutatum strains, which will be a useful tool for epidemiology and histopathology studies. The ATMT protocol developed was used to identify putative pathogenicity mutants, suggesting the applicability of this technique for rapid generation of a large panel of insertional mutants of C. acutatum leading to the identification of the genes associated with the varied lifestyles.     

Pseudomonads: major antagonistic endophytic bacteria to suppress bacterial wilt pathogen, <Emphasis Type="Italic">Ralstonia solanacearum</Emphasis> in the eggplant (<Emphasis Type="Italic">Solanum melongena</Emphasis> L.)

Endophytic bacteria of eggplant, cucumber and groundnut were isolated from different locations of Goa, India. Based on in vitro screening, 28 bacterial isolates which effectively inhibited Ralstonia solanacearum, a bacterial wilt pathogen of the eggplant were characterized and identified. More than 50% of these isolates were Pseudomonas fluorescens in which a vast degree of variability was found to exist when biochemical characteristics were compared. In greenhouse experiments, the plants treated with Pseudomonas isolates (EB9, EB67), Enterobacter isolates (EB44, EB89) and Bacillus isolates (EC4, EC13) reduced the wilt incidence by more than 70%. All the selected isolates reduced damping off by more than 50% and improved the growth of seedlings in the nursery stage. Most of the selected antagonists produced an antibiotic, DAPG, which inhibited R. solanacearum under in vitro conditions and might have been responsible for reduced wilt incidence under in vivo conditions. Also production of siderophores and IAA in the culture medium by the antagonists was recorded, which could be involved in biocontrol and growth promotion in crop plants. From our study we conclude that Pseudomonas is the major antagonistic endophytic bacteria from eggplants which have the potential to be used as a biocontrol agent as well as plant growth-promoting rhizobacteria. Large scale field evaluation and detailed knowledge on antagonistic mechanism could provide an effective biocontrol solution for bacterial wilt of solanaceous crops.     

Hydroxamate siderophores of the ectomycorrhizal fungi <Emphasis Type="Italic">Suillus granulatus</Emphasis> and <Emphasis Type="Italic">S. luteus</Emphasis>

Despite indications that S. granulatus and S. luteus release iron-chelating compounds, the exact spectrum of ferric hydroxamates synthesized by these two Suillus species remained unclear. Hence the aim of this study was to identify all of the main siderophores produced by these two ectomycorrhizal fungal species under pure culture conditions. By means of HPLC and LC–MS analyses we show that S. granulatus releases cyclic and linear fusigen, ferrichrome, coprogen and triacetylfusarinine C into the nutrient medium, while S. luteus culture filtrates contain cyclic and linear fusigen, ferricrocin and coprogen. All of the different siderophores were identified on basis of reference compounds and their specific MS spectra which were recorded on a high resolution MS in positive electrospray ionisation mode. Initial HPLC separations were performed on a C-18 stationary phase, using an acidic eluent (0.1% formic acid in water and acetonitrile) in gradient mode. The potential of these two ectomycorrhizal fungal species to produce siderophores representing three different groups of hydroxamates is discussed in relation to its ecological significance.     

<Emphasis Type="Italic">STAYGREEN</Emphasis> (<Emphasis Type="Italic">CsSGR</Emphasis>) is a candidate for the anthracnose (<Emphasis Type="Italic">Colletotrichum orbiculare</Emphasis>) resistance locus <Emphasis Type="Italic">cla</Emphasis> in Gy14 cucumber

Key message

Map-based cloning identified a candidate gene for resistance to the anthracnose fungal pathogen Colletotrichum orbiculare in cucumber, which reveals a novel function for the highly conserved STAYGREEN family genes for host disease resistance in plants.

Abstract

Colletotrichum orbiculare is a hemibiotrophic fungal pathogen that causes anthracnose disease in cucumber and other cucurbit crops. No host resistance genes against the anthracnose pathogens have been cloned in crop plants. Here, we reported fine mapping and cloning of a resistance gene to the race 1 anthracnose pathogen in cucumber inbred lines Gy14 and WI 2757. Phenotypic and QTL analysis in multiple populations revealed that a single recessive gene, cla, was underlying anthracnose resistance in both lines, but WI2757 carried an additional minor-effect QTL. Fine mapping using 150 Gy14?×?9930 recombinant inbred lines and 1043 F₂ individuals delimited the cla locus into a 32 kb region in cucumber Chromosome 5 with three predicted genes. Multiple lines of evidence suggested that the cucumber STAYGREEN (CsSGR) gene is a candidate for the anthracnose resistance locus. A single nucleotide mutation in the third exon of CsSGR resulted in the substitution of Glutamine in 9930 to Arginine in Gy14 in CsSGR protein which seems responsible for the differential anthracnose inoculation responses between Gy14 and 9930. Quantitative real-time PCR analysis indicated that CsSGR was significantly upregulated upon anthracnose pathogen inoculation in the susceptible 9930, while its expression was much lower in the resistant Gy14. Investigation of allelic diversities in natural cucumber populations revealed that the resistance allele in almost all improved cultivars or breeding lines of the U.S. origin was derived from PI 197087. This work reveals an unknown function for the highly conserved STAYGREEN (SGR) family genes for host disease resistance in plants.

     

Growth characteristics and growth modeling of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and <Emphasis Type="Italic">Planktothrix agardhii</Emphasis> under iron limitation

Although iron is a key nutrient for algal growth just as are nitrogen and phosphorus in aquatic systems, the effects of iron on algal growth are not well understood. The growth characteristics of two species of cyanobacteria, Microcystis aeruginosa and Planktothrix agardhii, in iron-limited continuous cultures were investigated. The relationships between dissolved iron concentration, cell quota of iron, and population growth rate were determined applying two equations, Monod’s and Droop’s equations. Both species produced hydroxamate-type siderophores, but neither species produced catechol-type siderophores. The cell quota of nitrogen for both M. aeruginosa and P. agardhii decreased with decreasing cell quota of iron. The cell quota of phosphorus for M. aeruginosa decreased with decreasing cell quota of iron, whereas those for P. agardhii did not decrease. Iron uptake rate was measured in ironlimited batch cultures under different degrees of iron starvation. The results of the iron uptake experiments suggest that iron uptake rates are independent of the cell quota of iron for M. aeruginosa and highly dependent on the cell quota for P. agardhii. A kinetic model under iron limitation was developed based on the growth characteristics determined in our study, and this model predicted accurately the algal population growth and iron consumption. The model simulation suggested that M. aeruginosa is a superior competitor under iron limitation. The differences in growth characteristics between the species would be important determinants of the dominance of these algal species.     

Susceptibility of <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> strains different in oxalate production to infection by the mycoparasite <Emphasis Type="Italic">Coniothyrium minitans</Emphasis>

Three strains of Sclerotinia sclerotiorum, namely Ep-1PB (PB), Ep-1PK (PK) and Ep-1PNA5 (A5), were compared for the production of oxalic acid (OA) on potato dextrose agar (PDA) and Maxwell agar medium (MAM) and for mycelial susceptibility to infection by the mycoparasite Coniothyrium minitans on PDA. Results showed that strain PB produced negligible oxalate, whereas strain PK was detected to produce oxalate, but much less than that produced by strain A5. The three investigated strains differed slightly in mycelial growth rates and mycelial biomass on PDA. However, colonies of strains PB and PK formed on PDA were more susceptible to invasion by C. minitans than colonies of strain A5. Meanwhile, amendment of synthetic oxalate in PDA at 0.25–2.00 mg g⁻¹ medium suppressed aggressiveness of C. minitans in invasion of colonies of S. sclerotiorum strain PB developed on this medium. These results suggest that infection of hyphae of S. sclerotiorum is negatively affected by the presence of oxalate. The importance of oxalate degradation by C. minitans in its mycoparasitism on hyphae of S. sclerotiorum provides a clue for improvement of the biocontrol efficacy of C. minitans in the future.     

Gene transfer into <Emphasis Type="Italic">Solanum tuberosum</Emphasis> via <Emphasis Type="Italic">Rhizobium</Emphasis> spp.

Agrobacterium tumefaciens-mediated transformation (ATMT) is the preferred technique for gene transfer into crops. A major disadvantage of the technology remains the complexity of the patent landscape that surrounds ATMT which restricts its use for commercial applications. An alternative system has been described (Broothaerts et al. in Nature 433:629-633, 2005) detailing the propensity of three rhizobia to transform the model crop Arabidopsis thaliana, the non-food crop Nicotiana tabacum and, at a very low frequency, the monocotyledonous crop Oryza sativa. In this report we describe for the first time the genetic transformation of Solanum tuberosum using the non-Agrobacterium species Sinorhizobium meliloti, Rhizobium sp. NGR234 and Mesorhizobium loti. This was achieved by combining an optimal bacterium and host co-cultivation period with a low antibiotic regime during the callus and shoot induction stages. Using this optimized protocol the transformation frequency (calculated as % of shoots equipped with root systems with the ability to grow in rooting media supplemented with 25 μg/ml hygromycin) of the rhizobia strains was calculated at 4.72, 5.85 and 1.86% for S. meliloti, R. sp. NGR234 and M. loti respectively, compared to 47.6% for the A. tumefaciens control. Stable transgene integration and expression was confirmed via southern hybridisation, quantitative PCR analysis and histochemical screening of both leaf and/or tuber tissue. In light of the rapid advances in potato genomics, combined with the sequencing of the potato genome, the ability of alternative bacteria species to genetically transform this major food crop will provide a novel resource to the Solanaceae community as it continues to develop potato as both a food and non-food crop.     

Enhancement of nodulation by some arid climate strains of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> biovar <Emphasis Type="Italic">trifolii</Emphasis> using protoplast fusion

Fourteen randomly clover indigenous nodulated Rhizobium strains were isolated from different locations in Saudi Arabia. They were identified as different strains of the genus Rhizobium leguminosarum biovar trifolii and characterized for their intrinsic antibiotic resistance against a range of antibiotics, nodulation capability and plasmid profiles. Results revealed the presence of high molecular weight plasmids (megaplasmids) in all the selected strains. Based on the ability for nodulation production, two weak strains (RtI1 and RtI2) and one efficient strain (RtA1) were selected for protoplast fusion and the numbers of nodules produced by the intra-specific protoplast fusion strains were investigated. Results clearly confirmed the effective role of the protoplast fusion in enhancing both nodulation production capacity of Rhizobium species and their range of antibiotic resistance. Protoplast fusion of the local Rhizobium species resulted in 1.93- to 5.67-fold increase in nodulation number compared to their parental strains, which was considered an excellent result concerning agricultural practices, especially the formation of nitrogen-fixing root nodules on legume crop plants. Protoplast fusion also produced fusants with a wide range of antibiotic resistance, another advantage added to the new strains against environmental stresses. In conclusion, protoplast fusion proved its efficiency as a tool for constructing a second generation of Rhizobia with much better characteristics for efficient applications in arid land.     

Genetic variation of <Emphasis Type="Italic">Colletotrichum magnum</Emphasis> isolated from <Emphasis Type="Italic">Carica papaya</Emphasis> as revealed by DNA fingerprinting

Mexico is one of the five largest producers of papaya worldwide, but losses caused by pathogens, mainly fungus, at the pre- and post-harvest stages are often more than 50% of the crop. Papaya anthracnose, caused by three different species of the Colletotrichum genus in Mexico, occupies a preponderant place in this problem. Although two of these species, C. gloeosporiodes and C. truncatum, have been characterized morphologically and genotypically, this has not occurred with C. magnum, the third species involved, about which there is very little information. Because of this, it is vital to know its genetic characterization, much more so considering that the studies carried out on the other two species reveal a wide genetic diversity, differences in pathogenicity and in the response to fungicides of the different strains characterized. In this work, Colletotrichum spp. isolates were collected at different papaya orchards in the south-southeast of Mexico. C. magnum isolates identified by species-specific primers were characterized by morphological and molecular approaches. Differences in colony characteristics resulted in five morphological groups. AP-PCR, DAMD and ISSR markers were found to be very efficient for revealing the interspecific variability of this species. The high genetic variability found in the accessions of C. magnum was linked to the geographical area where they were collected. Isolates from Chiapas State were the most variable, showing point mutations in the ITS1-ITS2 region. These results will enable a better phytosanitary management of anthracnose in papaya in this region of Mexico.     

Association Between the <Emphasis Type="Italic">cfx</Emphasis>A Gene and Transposon Tn<Emphasis Type="Italic">4555</Emphasis> in <Emphasis Type="Italic">Bacteroides distasonis</Emphasis> Strains and Other <Emphasis Type="Italic">Bacteroides</Emphasis> Species

The Bacteroides genus, the most prevalent anaerobic bacteria of the intestinal tract, carries a plethora of the mobile elements, such as plasmids and conjugative and mobilizable transposons, which are probably responsible for the spreading of resistance genes. Production of β-lactamases is the most important resistance mechanism including cephalosporin resistance to β-lactam agents in species of the Bacteroides fragilis group. In our previous study, the cfxA gene was detected in B. distasonis species, which encodes a clinically significant broad-spectrum β-lactamase responsible for widespread resistance to cefoxitin and other β-lactams. Such gene has been associated with the mobilizable transposon Tn4555. Therefore, the aim of this study was to detect the association between the cfxA gene and the presence of transposon Tn4555 in 53 Bacteroides strains isolated in Rio de Janeiro, Brazil, by PCR assay. The cfxA gene was detected in 11 strains and the Tn4555 in 15. The transposon sequence revealed similarities of approximately 96% with the B. vulgatus sequence which has been deposited in GenBank. Hybridization assay was performed in attempt to detect the cfxA gene in the transposon. It was possible to associate the cfxA gene in 11 of 15 strains that harbored Tn4555. Among such strains, 9 presented the cfxA gene as well as Tn4555, but in 2 strains the cfxA gene was not detected by PCR assay. Our results confirm the involvement of Tn4555 in spreading the cfxA gene in Bacteroides species.     

A Species-Specific Polymerase Chain Reaction Assay for Rapid and Sensitive Detection of <Emphasis Type="Italic">Colletotrichum</Emphasis><Emphasis Type="Italic">capsici</Emphasis>

Colletotrichum capsici is an important fungal species that causes anthracnose in many genera of plants causing severe economic losses worldwide. A primer set was designed based on the sequences of the ribosomal internal transcribed spacer (ITS1 and ITS2) regions for use in a conventional PCR assay. The primer set (CcapF/CcapR) amplified a single product of 394 bp with DNA extracted from 20 Mexican isolates of C. capsici. The specificity of primers was confirmed by the absence of amplified product with DNA of four other Colletotrichum species and eleven different fungal genera. This primer set is capable of amplifying only C. capsici from different contaminated tissues or fungal structures, thereby facilitating rapid diagnoses as there is no need to isolate and cultivate the fungus in order to identify it. The sensitivity of detection with this PCR method was 10 pg of genomic DNA from the pathogen. This is the first report of a C. capsici-specific primer set. It allows rapid pathogen detection and provides growers with a powerful tool for a rational selection of fungicides to control anthracnose in different crops and in the post-harvest stage.     

Biocontrol of <Emphasis Type="Italic">Pythium</Emphasis> damping-off in cucumber by arbuscular mycorrhiza-associated bacteria from the genus <Emphasis Type="Italic">Paenibacillus</Emphasis>

The Pythium biocontrol features of 17 Paenibacillus strains, all previously isolated from the rhizosphere, hyphosphere or bulk soil from mycorrhizal and non-mycorrhizal cucumber plants, were examined using a cucumber seedling emergence bioassay. Thirteen strains – four strains of Paenibacillus polymyxa, eight strains of P. macerans and one strain of Paenibacillus sp. – significantly increased the percentage of seedling emergence of seeds inoculated with agar plugs of Pythium aphanidermatum FC42. Overall, the efficacy of Pythium biocontrol did not seem to differ between isolates of Paenibacillus originating from either mycorrhizal or non-mycorrhizal systems. No strains significantly reduced the damping-off incidence caused by the aggressive isolate Pythium sp. B5. Two strains of P. macerans not only reduced the incidence of pre-emergence damping-off by 73%, but they also counteracted the plant growth-depressing effect of P. aphanidermatum FC42, so that 68–82% of the emerged seedlings remained healthy 7 days after sowing. Two strains of P. macerans and one strain of P. polymyxa also significantly increased the percentage of seedling emergence following inoculation with approximately 10⁵ zoospores of P. aphanidermatum FC42. There was no significant difference between the dry weight of three selected bacteria-inoculated and -uninoculated plants in the absence of Pythium; however, the dry weight of bacteria-inoculated plants was significantly higher than that of the uninoculated control plants with bacteria in the presence of P. aphanidermatum FC42.     

Disease suppression and crop improvement in moong beans (<Emphasis Type="Italic">Vigna radiata</Emphasis>) through <Emphasis Type="Italic">Pseudomonas</Emphasis> and <Emphasis Type="Italic">Burkholderia</Emphasis> strains isolated from semi arid region of Rajasthan,India

Pseudomonas fluorescens BAM-4, Burkholderia cepacia BAM-6 and B. cepacia BAM-12 isolated from the rhizosphere of moong bean (Vigna radiata L.) showed significant growth-inhibitory activity against a range of phytopathogenic fungi. Light and scanning electron microscopic (SEM) studies showed morphological abnormalities such as fragmentation, swelling, perforation and lysis of hyphae of pathogens by Pseudomonas and Burkholderia. Two of the strains (BAM-4 and BAM-6) produced siderophore in CAS agar plates, whereas all three strains produced chitinase. Bacterization of seeds of moong bean with pseudomonads has been reported as a potential method for enhancing plant growth and yield, and for providing protection against Macrophomina phaseolina. Seed bacterization with these plant growth-promoting rhizobacteria (PGPR) showed a significant increase in seed germination, shoot length, shoot fresh and dry weight, root length, root fresh and dry weight, leaf area and rhizosphere colonization. Yield parameters such as pods, number of seeds, and grain yield per plant also enhanced significantly in comparison to control. The disease suppression and plant growth enhancement along with the positive rhizosphere colonization by these strains indicate their possible use as PGPR/biocontrol agents against charcoal rot.     

Phenol Oxidase Activity of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp245 Mutants with Modified Motility and <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> Sp7 Phase Variants with Different Plasmid Composition

Herein, we reveal the alteration in phenol oxidase enzymes complex production from Azospirillum brasilense Sp245 omegon mutants with polar and lateral flagella dysfunction and from A. brasilense Sp7 phase variants with different plasmid composition. The enzymatic activities for various laccases, tyrosinases, Mnperoxidases, and lignin peroxidases as well as the isomorphic composition of intracellular laccases and tyrosinases were estimated for the studied variants and the parent strains. It was noted that various genetic events correlating with phenotypic heterogeneity in A. brasilense populations affect their phenol oxidase activity level.     

Interaction of <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> and <Emphasis Type="Italic">Glomus intrarradix</Emphasis> in Sugar Cane Roots

Fifteen-day-old variety NA 56-79 sugar cane seedlings were inoculated with Azospirillum brasilense and Glomus intrarradix. This article aims at examining changes in sugar cane root seedlings inoculated with Glomus intrarradix and Azospirillum brasilense, the increase in microbial biomass and the acetylene reduction process as well. The internal root colonization was studied 20 days after inoculation using scanning and a transmission electron microscope. Both microorganisms entered the sugar cane root through the emergent lateral roots. The microorganisms were capable of coexisting both intra and intercellularly, producing changes in the cell wall, thus allowing colonization and interaction between the organisms. These changes increased the number of microorganisms inside the root as well as acetylene nitrogen reduction. Sugar cane plant biomass increased with joint-inoculation. The number of endophytic microorganisms and nitrogen fixing activity increased when they were colonized by Azospirillum and Glomus together.     

<Emphasis Type="Italic">In vitro</Emphasis> biocontrol analysis of <Emphasis Type="Italic">Alternaria alternata</Emphasis> (Fr.) Keissler under different environmental conditions

The species Trichoderma harzianum was analyzed as possible biocontrol agent of Alternaria alternata under different environmental conditions (water activity and temperature). The strains were analyzed macroscopically to obtain the Index of Dominance. The analysis was completed using two microscopic techniques. T. harzianum showed dominance on contact over A. alternata at all testing temperatures and water activities tested except at 0.95 a _w and 15 °C, at which T. harzianum inhibited A. alternata at a distance. Biocontrol was governed by different mechanisms such as competition for space and nutrients, mycoparasitism, and possible antibiosis. Temperature and water activity significantly influenced fungal growth rate.     

Inhibition of matrix metalloproteinase-2 activity by siderophores of<Emphasis Type="Italic"> Pseudomonas</Emphasis> species

To obtain a novel matrix metalloproteinase (MMP) inhibitor produced by bacteria, we have focused on the chelating activity of siderophores. Several siderophore-producing bacteria were isolated from soil using chrome azurol S agar plates and then the effect of siderophores on MMP-2 activity was assayed by gelatin zymography. The results showed that partially purified siderophores from ten isolated strains inhibited MMP-2 activity. Among these strains, two were non-fluorescent and eight were fluorescent Pseudomonas species. From these eight strains, pyoverdine-type siderophores were detected. The Zn²⁺-chelating activity of these siderophores correlated with the inhibition of MMP-2 activity. Therefore, it is considered that siderophores such as pyoverdines inhibit MMP-2 activity by chelating Zn²⁺ on the active site of MMP-2.