Cytological karyotyping and characterization of a 410 kb minichromosome in Nectria haematococca MPI

Karyotypes of the cucurbit pathogen Nectria haematococca MPI (anamorph Fusarium solani f. sp. cucurbitae race 1) was studied using the two standard strains ATCC18098 and ATCC18099. Complete separation of all chromosomes was difficult with pulsed field gel electrophoresis due to both the large size and co-migration of chromosomes. In contrast, cytological karyotyping was done successfully with fluorescence microscopy combined with the germ tube burst method for sample preparation to visualize mitotic metaphase chromosomes. For each strain the basic chromosome number (CN) was nine, which revises previous chromosome estimates of n = 4. Chromosomes were morphologically characterized by their sizes, intensely fluorescing segments, and protrusion of rDNA. In addition to the basic chromosome complement, ATCC18098 had a mini-chromosome of ~410 kb present as a single copy in somatic nuclei. Chromosome fluorescence in situ hybridization indicated that this mini-chromosome is not a derivative from the other chromosomes in the genome. In addition, crossing experiments suggested that it was transmitted in a Mendelian manner to the ascospore progeny.     

Diversity of cutinases from plant pathogenic fungi: differential and sequential expression of cutinolytic esterases by Alternaria brassicicola

Plant cuticles provide a protective layer that has to be penetrated by fungal pathogens. Evidence is provided for a differential and sequential induction of two classes of cutinolytic esterases by Alternaria brassicicola. Serine esterases with cutinolytic activities were expressed by conidia germinating on host surfaces. The enzymes were not induced by surface wax or cutin monomers. They were only expressed during initial (24 h) contact of conidia with cutin on host surfaces freed from wax, and with cutin in aqueous suspensions. In contrast, contact with cutin had no immediate effect on the expression of CUTAB1, a gene encoding two cutinase isozymes with crucial functions in the saprophytic utilization of cutin. Presence of a cutin monomer or prolonged exposure to cutin was required for the induction of CUTAB1 expression. The differential induction of cutinolytic esterases indicates a sequential recognition of cutin as a barrier to be penetrated and to be utilized as a carbon source in saprophytic stages.     

Fusarium polycaprolactone depolymerase is cutinase.

Polycaprolactone (PCL), a synthetic polyester, is degraded by a variety of microorganisms, including some phytopathogens. Many phytopathogens secrete cutinase, a serine hydrolase that degrades cutin, the structural polymer of the plant cuticle. We compared wild-type strains and a cutinase-negative gene replacement mutant strain of Fusarium solani f. sp. pisi (D. J. Stahl and W. Schäfer, Plant Cell 4:621-629, 1992) and a wild-type strain of Fusarium moniliforme to show that Fusarium cutinase is a PCL depolymerase. The wild-type strains, but not the mutant strain, (i) degraded PCL and used it as a source of carbon and energy, (ii) showed induction of secreted PCL depolymerase and an esterase activity of cutinase when grown in the presence of cutin, and (iii) showed induction of PCL depolymerase and an esterase activity of cutinase when grown in the presence of a hydrolysate of PCL, which contains PCL oligomers that are structurally similar to the natural inducers of cutinase. These results together with other details of regulation and conditions for optimal enzyme activity indicate that the Fusarium PCL depolymerase, required for degradation and utilization of PCL, is cutinase.     

Fusarium solani species complex isolates conspecific with Fusarium solani f. sp. cucurbitae race 2 from naturally infected human and plant tissue and environmental sources are equally virulent on plants, grow at 37 degrees C and are interfertile

In a previous taxonomic study based on multilocus sequencing of Fusarium from clinical specimens and hospital environments, the most common lineage was Fusarium solani species complex group 1 (FSSC 1) which is conspecific with F. solani f. sp. cucurbitae race 2, a pathogen of cucurbit fruits. The aims of our study were to determine if clinical and environmental isolates of FSSC 1 are plant pathogens and members of the same biological species as cucurbit isolates, and to determine if all isolates can germinate, grow and sporulate at 37 degrees C. Isolates from the different sources did not differ in virulence on zucchini fruits. All FSSC 1 isolates were pathogenic and produced more rot than FSSC isolates from plant hosts other than cucurbits. Both mating types were found among isolates from each of the sources, and all isolates were sexually compatible with cucurbit isolates. All isolates germinated, grew and sporulated at 37 degrees C. This is the first report in which plant pathogenicity has been verified for a collection of human clinical isolates. Our data are consistent with the hypothesis that all FSSC 1 isolates, regardless of source, are a single biological species, equally virulent plant pathogens and tolerant of the human body temperature.     

Cutinase is not required for fungal pathogenicity on pea.

Cutinase, a fungal extracellular esterase, has been proposed to be crucial in the early events of plant infection by many pathogenic fungi. To test the long-standing hypothesis that cutinase of Nectria haematococca (Fusarium solani f sp pisi) is essential to pathogenicity, we constructed cutinase-deficient mutants by transformation-mediated gene disruption of the single cutinase gene of a highly virulent N. haematococca strain. Four independent mutants were obtained lacking a functional cutinase gene, as confirmed by gel blot analyses and enzyme assays. Bioassays of the cutinase-deficient strains showed no difference in pathogenicity and virulence on pea compared to the wild type and a control transformant. We conclude that the cutinase of N. haematococca is not essential for the infection of pea.     

Two linear plasmids in mitochondria of Fusarium solani f. sp. cucurbitae

Two linear plasmid-like DNAs designated pFSC1 (9.2 kbp) and pFSC2 (8.3 kbp) were found in an isolate of the plant pathogenic fungus Fusarium solani f. sp. cucurbitae race 1. The plasmids were maternally inherited and copurified with mitochondrial DNA obtained from a mitochondria-enriched cell fraction suggesting that they are located in mitochondria. The plasmids did not share extensive sequence similarity. No homology was detected between either plasmid and the nuclear or mitochondrial genome when cloned plasmids were used as probes in Southern hybridization analyses. The fungus was cured of plasmids by ethidium bromide treatment. Compared to the plasmid-containing isolate, plasmid-cured derivatives had reduced pathogenicity on a susceptible plant host, Cucurbita maxima "Pink Banana."     

Production of cutinolytic esterase by filamentous bacteria

Thirty-eight strains of filamentous bacteria, many of which are thermophilic or thermotolerant and commonly found in composts and mouldy fodders, were examined for their ability to produce cutinolytic esterase (cutinase) in culture media supplemented with cutin, suberin or cutin-containing agricultural by-products. Initially, the ability of culture supernatants to hydrolyse the artificial substrate p-nitrophenyl butyrate was determined by spectrophotometric assays. Only one bacterium, Thermoactinomyces vulgaris NRRL B-16117, exhibited cutinolytic esterase production. The enzyme was highly inducible, was repressed by the presence of glucose in the medium and hydrolysed both apple and tomato cutins. Inducers included apple cutin, apple pomace, tomato peel, potato suberin and commercial cork. Unlike similar fungal enzymes, the T. vulgaris cutinolytic esterase was not inducible by cutin hydrolysate. The cutinolytic esterase exhibited a half-life of over 60 min at 70 degrees C and a pH optimum of >/= 11.0. This study indicates that thermophylic filamentous bacteria may be excellent commercial sources of heat-stable cutin-degrading enzymes that can be produced by fermentation of low cost feedstocks.     

Sewage and community shower drains are environmental reservoirs of Fusarium solani species complex group 1, a human and plant pathogen

In two recent studies, clinical isolates in the Fusarium solani species complex (FSSC) were sequenced; one of the most common lineages was FSSC Group 1 (FSSC 1), a phylogenetic species that is synonymous with F. solani f. sp. cucurbitae race 2, a pathogen of cucurbit fruits. FSSC 1 was also identified in sink and shower drains in two hospitals. The environmental sources of FSSC 1 are important for understanding the epidemiology of both human and plant diseases caused by this organism. FSSC 1 was detected in sewage influent at all six tested urban wastewater treatment plants (WWTPs) in California with a concentration ranging from 75 to 413 colony-forming units (cfu) l⁻¹, a mean of 246 ± 52 cfu l⁻¹ and a median of 254 cfu l⁻¹. During the treatment process, the concentration of FSSC 1 in the solid and liquid fractions diminished. FSSC 1 was detected in five and six of 14 community shower drains by culturing and polymerase chain reaction, respectively, whereas FSSC DNA was detected in all drains. FSSC accounted for 17 ± 6% ( n  = 14) of the total fungal DNA in the drains. FSSC 1 was rarely isolated from post-harvest cucurbit fruits and was not found in cucurbit fields in California.     

Morphological and Molecular Characterization of Fusarium solani Isolates

Fusarium solani is a species complex (FSSC) containing isolates that cause diseases in important crops such as root and fruit rot of Cucurbita spp., root and stem rot of pea, sudden death syndrome of soybean, foot rot of bean and dry rot of potato tubers during storage. Based on host range tests, F. solani were subdivided into different formae specialis (f. sp.) and varieties, while DNA sequences of 28S rDNA, internally transcribed spacers (ITS) rDNA and elongation factor (EF-1α) distinguished the ' F. solani complex' in 50 subspecific lineages. In this study we characterized, by cultural, morphological and molecular criteria, 34 isolates of F. solani obtained from potato, other crops and soil. The 34 isolates in the FSSC showed wide variability for their cultural, morphological and molecular traits. The wide variability observed with amplified fragment-length polymorphism (AFLP) and mini-microsatellite analyses is in agreement with the polymorphism observed, in a previous study, within FSSC. Nine of 34 isolates in the FSSC, classified as F. solani var. coeruleum , were morphologically distinguishable from the other F. solani isolates but they were distributed in different clusters; moreover, the nine isolates showed instability of the coeruleum pigmentation of the colonies, supporting the ambiguity of the taxa of this variety of F. solani. Using sequence data from ITS plus 5.8S rDNA region, the isolates were classified into different clades. In particular eight isolates were classified into a well-supported clade including F. solani f. sp . pisi , nine into a clade including only isolates of F. solani f. sp . radicicola and four into a clade including F. solani f. sp . cucurbitae , but this classification could not be used if is not in agreement with host specificity. Two of the nine F. solani var. coeruleum isolates were phylogenetically distinct from all the other FSSC strains.     

Purification and characterization of a 40.8-kDa cutinase in ungerminated conidia of Botrytis cinerea Pers.: Fr

Cytoplasmic soluble proteins from ungerminated conidia of Botrytis cinerea exhibited cutinase activity. A 40.8-kDa cutinase was purified to homogeneity from this crude conidial protein extract. This cutinase does not correspond either to constitutive or to induced lytic cutin enzymes already described by other authors. The possible role of this constitutive cutinase in the induction of other cutinolytic proteins in the early stages of infection of plants by B. cinerea is discussed.     

Induction of a Biopolyester Hydrolase (Cutinase) by Low Levels of Cutin Monomers in Fusarium solani f. sp. pisi

Cutin hydrolysate induced the production of an extracellular cutinase by glucose-grown Fusarium solani f. sp. pisi. The rate of production depended on the amount of cutin hydrolysate added up to 80 mug/ml, and saturation was attained at this level. Glucose was found to be a repressor of cutinase production. A radial immunodiffusion assay for cutinase was developed, and the induction of cutinase by cutin hydrolysate was confirmed by this direct assay. When cutinase was induced by cutin hydrolysate, exogenous labeled phenylalanine was incorporated into cutinase, which was shown to be the major (>70%) protein in the extracellular fluid. Induction of cutinase by cutin hydrolysate was not inhibited by actinomycin D and was stimulated ( approximately 100%) by cordycepin. Addition of cycloheximide with the inducer, or up to 12 h after the addition of the inducer, resulted in a nearly immediate cessation of cutinase production. Deoxyglucose, an inhibitor of proten glycosylation, inhibited the induction of cutinase by cutin hydrolysate. omega-Hydroxy fatty acids were more effective in inducing cutinase than any of the other more polar acids of cutin. Experiments with derivatives and analogues of omega-hydroxy C(16) acid indicated that a free hydroxyl group at the omega-position was the most important factor determining the cutinase-inducing activity. n-Aliphatic primary alcohols with 14 or more carbon atoms induced cutinase, and n-C(16) was the most effective inducer. These results strongly suggest that the monomers function as the chemical signal which induces the extracellular hydrolase.     

Screening of Nonfilamentous Bacteria for Production of Cutin-Degrading Enzymes

Two hundred thirty-two nonfilamentous bacterial strains, including saprophytes, plant pathogens, and opportunistic plant and human pathogens, were screened for the ability to produce cutinases (cutin-degrading esterases). Initially, esterase activity of culture filtrates of strains grown in nutrient broth-yeast extract medium supplemented with 0.4% apple or tomato cutin was determined by a spectrophotometric assay utilizing the model substrate p-nitrophenyl butyrate. The culture filtrates of the 10 Pseudomonas aeruginosa strains tested exhibited the highest esterase activity, with values of >500 nmol/min/ml. Of these 10 strains, 3 (K799, 1499A, and DAR41352) demonstrated significant induction (10-fold or above) of esterase activity by addition of cutin to nutrient broth-yeast extract medium. The ability of culture filtrates of the three strains to cause release of apple cutin monomers was confirmed by a novel high-performance liquid chromatography technique. Monomer identification was confirmed by gas chromatography-mass spectroscopy analyses. Addition of the nonionic detergent n-octylglucoside stimulated cutinase activity of culture filtrates from strains K799 and DAR41352, but not that of filtrates from strain 1499A. Time course studies in nutrient broth-yeast extract medium supplemented with apple cutin indicated maximal levels of cutinase in the culture fluids after cultures entered stationary phase. Incubation temperatures below the optimal temperature for growth (37°C) led to maximal production of cutinase.     

Cutinase in Cryphonectria parasitica, the chestnut blight fungus: suppression of cutinase gene expression in isogenic hypovirulent strains containing double-stranded RNAs.

Plant-pathogenic fungi produce cutinase, an enzyme required to degrade plant cuticles and facilitate penetration into the host. The absence of cutinase or a decrease in its production has been associated with a decrease in pathogenicity of the fungus. A set of isogenic strains of Cryphonectria parasitica, the chestnut blight fungus, was tested for the presence and amounts of cutinase activity. The virulent strain of C. parasitica produced and secreted significantly higher amounts of cutinase than the hypovirulent strains. Use of both nucleic acid and polyclonal antibody probes for cutinase from Fusarium solani f. sp. pisi showed that cutinase in C. parasitica is 25 kDa in size and is coded by a 1.1-kb mRNA. Both mRNA and protein were inducible by cutin hydrolysate, while hypovirulence agents suppressed the level of mRNA and the enzyme. Since all the strains had the cutinase gene, the suppression of expression was due to the hypovirulence agents. The data presented are the first report indicating that hypovirulence agents in C. parasitica regulate a gene associated with pathogenicity in other plant-pathogenic fungi.     

Cross-Reactive Antigens Between Pea and Some Fungal Plant Pathogens

Antiserum to pea was used to analyse cross-reactive antigens (CRA) between pea and some fungal plant pathogens with different levels of specificity towards this host by using both double diffusion and immunoblotting techniques. Non pathogens of pea were also included in the study. Nectria haematococca MPVI, the three formae speciales dianthi, lycopersici and pisi of Fusarium oxysporum and Ascochyta pisi produced strong reactions with both techniques. In N. haematococca MPI, F. solani f. sp. phaseoli, V. dahliae and Phoma medicaginis var. pinodella instead, reactions were not detected by double diffusion but only the more sensitive immunoblotting technique. No CRA were observedin, the non-specific pathogens Rhizoctonia solani, Sclerotium rolfsii and Sclerotinia sclerotiorum, as well as in the non-pathogen Phytophthora capsici. The immunoblotting patterns of the most reactive fungi showed common bands with molecular weights of 84, 75 and 62 kDa. Some bands were present only in the specific pathogens N. haematococca MPVI and F. oxysporum f.sp. pisi. The possible involvement in host-parasite interactions of cross-reactive antigens which are, present in the analyzed fungi is discussed.     

A high throughput profiling method for cutinolytic esterases

A procedure for identifying and profiling cutinolytic esterases was developed by combining traditional plate screen assays with an automated robotic system. In the first phase, the micro-organisms were screened on agar plates with cutin or the model substrate polycaprolactone as the sole carbon sources. In the second phase, p-nitrophenyl esters of fatty acids were used as the substrates in an automated activity assay of liquid culture media. The variables used were pH and the carbon chain length of the fatty acid moiety of the p-nitrophenyl substrate. Finally, ³H-labelled cutin was used as a specific substrate to verify the positive hits and to validate the screening procedure. With pH as the variable in the automatic screen, esterase production of cutinase positive strains typically proceeded in two stages: first an esterase with neutral activity optimum was produced, after which a strong esterolytic response in the alkaline range was detected. With carbon chain length of the fatty acid as the variable best correlation with cutinase production was obtained with strains showing a high ratio of activities towards p-nitrophenyl-butyrate and p-nitrophenyl-palmitate.     

Cutinolytic esterase activity of bacteria isolated from mixed-plant compost and characterization of a cutinase gene from Pseudomonas pseudoalcaligenes

The objective of the current study was to examine cutinolytic esterase (i.e., cutinase) activity by pseudomonads and bacteria isolated from mixed-plant compost. Approximately 400 isolates representing 52 taxa recovered from mixed-plant compost using cuticle baits, along with 117 pseudomonad isolates obtained from a culture collection (i.e., non-compost habitats), were evaluated. The ability of isolates to degrade the synthetic cutin polycaprolactone (PCL) was initially measured. Isolates from 23 taxa recovered from the compost degraded PCL. As well, isolates from 13 taxa of pseudomonads cleared PCL. Secondary screening measured esterase activity induced by the presence of apple cuticle using the chromogenic substrate p-nitrophenyl butyrate. Eighteen isolates representing four taxa (Alcaligenes faecalis , Bacillus licheniformis , Bacillus pumilus , and Pseudomonas pseudoalcaligenes) recovered from compost exhibited substantial esterase activity when grown with cuticle. In contrast, none of the pseudomonad isolates from the culture collection produced appreciable esterase activity. Although degradation of PCL was not correlated with esterase activity, isolates that were unable to degrade PCL failed to produce measureable esterase activities. Zymogram analysis indicated that the esterases produced by bacteria from compost ranged in size from 29 to 47 kDa. A gene from P. pseudoalcaligenes (cutA) was found to code for a cutin-induced esterase consisting of 302 amino acids and a theoretical protein size of 32 kDa. The enzyme was unique and was most closely related to other bacterial lipases (≤48% similarity).     

Hydrolysis of plant cuticle by plant pathogens. Properties of cutinase I, cutinase II, and a nonspecific esterase isolated from Fusarium solani pisi.

The properties of the homogeneous cutinase I, cutinase II, and the nonspecific esterase isolated from the extracellular fluid of cutin-grown Fusarium solani F. pisi (R.E. Purdy and P.E. Kolattukudy (1975), Biochemistry, preceding paper in this issue) were investigated. Using tritiated apple cutin as substrate, the two cutinases showed similar substrate concentration dependence, protein concentration dependence, time course profiles, and pH dependence profiles with optimum near 10.0. Using unlabeled cutin, the rate of dihydroxyhexadecanoic acid release from apple fruit cutin by cutinase I was determined to be 4.4 mumol per min per mg. The cutinases hydrolyzed methyl hexadecanoate, cyclohexyl hexadecanoate, and to a much lesser extent hexadecyl hexadecanoate but not 9-hexadecanoyloxyheptadecane, cholesteryl hexadecanoate, or hexadecyl cinnamate. The extent of hydrolysis of these model substrates by cutinase I was at least three times that by cutinase II. The nonspecific esterase hydrolyzed all of the above esters except hexadecyl cinnamate, and did so to a much greater extent than did the cutinases. None of the enzymes hydrolyzed alpha- or beta-glucosides of p-nitrophenol. p-Nitrophenyl esters of fatty acids from C2 through C18 were used as substrates and V's and Kms were determined...