Protein extract from Cereus jamacaru (DC.) inhibits Colletotrichum gloeosporioides growth by stimulating ROS generation and promoting severe cell membrane damage.

Mandacaru (Cereus jamacaru DC.), is a cactaceous symbol of caatinga vegetation at Brazilian Northeast region, however, there are no much studies about biochemical properties of this species. Here, the pioneering study brings very relevant data to highlight the importance of research with endemic plants of the caatinga. Afterward, the presence of enzymes such as peroxidase, protease, chitinase, ß-1,3-glucanase, and serine (trypsin) and cysteine (papain) protease inhibitors were evaluated. The peroxidase activity was higher in roots than other tissues. The ß-1,3-glucanase and proteolytic activity were prominent in stem and roots. The chitinase activity and protease inhibitor for both classes analyzed were detected in the stem and fruit peel. Antifungal activity against Colletotrichum gloeosporioides showed the root extract has a promising inhibitory activity on this economical important phytopathogenic fungus. After the contact of the hyphae with root extract increase in membrane permeability, based on Propidium Iodide (PI) uptake, and production of reactive oxygen species (ROS) were detected, compared to negative control. In addition, Scanning Electron Microscopy (SEM) analysis showed morphological damage on hyphae structure indicating that the treatment debilitates either cell membrane or cell wall leading to the cell death C. gloeosporioides.

The histidine kinase slnCl1 of Colletotrichum lindemuthianum as a pathogenicity factor against Phaseolus vulgaris L.

Colletotrichum lindemuthianum, the causal agent of anthracnose, is responsible for significant damage in the common bean (Phaseolus vulgaris L.). Unraveling the genetic mechanisms involved in the plant/pathogen interaction is a powerful approach for devising efficient methods to control this disease. In the present study, we employed the Restriction Enzyme-Mediated Integration (REMI) methodology to identify the gene slnCl1, encoding a histidine kinase protein, as involved in pathogenicity. The mutant strain, MutCl1, generated by REMI, showed an insertion in the slnCl1 gene, deficiency of the production and melanization of appressoria, as well as the absence of pathogenicity on bean leaves when compared with the wild-type strain. The slnCl1 gene encodes a histidine kinase class IV called SlnCl1 showing identity of 97% and 83% with histidine kinases from Colletotrichum orbiculare and Colletotrichum gloesporioides, respectively. RNA interference was used for silencing the histidine kinase gene and confirm slnCl1 as a pathogenicity factor. Furthermore, we identified four major genes involved in the RNA interference-mediated gene silencing in Colletotrichum spp. and demonstrated the functionality of this process in C. lindemuthianum. Silencing of the EGFP reporter gene and slnCl1 were demonstrated using qPCR. This work reports for the first time the isolation and characterization of a HK in C. lindemuthianum and the occurrence of gene silencing mediated by RNA interference in this organism, demonstrating its potential use in the functional characterization of pathogenicity genes.

Activity of chitin deacetylase from Colletotrichum gloeosporioides on chitinous substrates.

Production of chitin deacetylases from the phytopathogenic fungus Colletotrichum gloeosporioides was successfully achieved by submerged fermentation. The highest specific activity of 0.018 U mg(-1) of protein was obtained after 96 h of cultivation at pH 6 and 28°C. Two bands with molecular weights of 35 kDa and 170 kDa determined with SDS-PAGE displayed deacetylase activities as detected in the zymograms. Reacetylated commercial chitosan (52% acetylation degree) was used as substrate for the extracellular crude extract in order to estimate the kinetic parameters of acetate production as undirected deacetylation measurement. The highest acetate production of 12.8 µmol mL(-1) was obtained using 7.5 mg mL(-1) of substrate. The produced enzyme from C. gloeosporioides achieved up to 25% deacetylation of a chitin substrate (hydrolyzed biological chitin) having 80% degree of acetylation, MW of 102×10(3) g mol(-1) and a crystallinity index of ca. 60%.

Pectinolytic enzyme production by Colletotrichum truncatum, causal agent of soybean anthracnose.

BACKGROUND: Colletotrichum truncatum is the most common pathogenic fungus associated with soybean anthracnose, a prevalent disease in Argentina. Pectinolytic enzymes are involved in the pathogenicity of a wide range of plant pathogenic fungi. OBJECTIVES: To explore pectinolytic enzyme production in Argentinian Colletotrichum strains isolated from diseased soybean plants from different geographic locations, as a preliminary step to establish the biological role of the pectinolytic enzymes in the Colletotrichum spp.-soybean system, yet unknown. METHODS: Ten strains were screened for in vitro pectinolytic enzyme production on a defined medium based on pectin as carbon source. RESULTS: All isolates were able to grow in this medium and polymethylgalacturonase (PMG), polygalacturonase (PG) and pectin lyase (PL) activities were detected. On the whole, the peak of polygalacturonases activities preceded the day of maximum growth, while PL activity reached its highest level afterwards. Strain BAFC 3097 (from Santa Fe province) yielded high titles of the three enzymes (1.08U/ml PG, 1.05U/ml PMG, 156U/ml PL), after a short incubation period (7-10 days). Low synthesis of polygalacturonases in cultures containing glucose as unique carbon source suggests that these enzymes are constitutive in contrast with PL, which was not detected. CONCLUSIONS: The disparity observed in enzyme production among strains cannot be related to fungal growth, since no major differences in mycelial yield were found; it was not connected with their geographic origin, but might be associated with differences in virulence among strains not yet evaluated.

pelB gene in isolates of Colletotrichum gloeosporioides from several hosts.

Colletotrichum gloeosporioides is an important pathogen for a great number of economically important crops. During the necrotrophic phase of infection by Colletotrichum spp, the degradative enzymes of plant cell walls, such as pectate lyase, clearly increase. A gene pelB that expresses a pectate lyase was identified in isolates of C. gloeosporioides in avocado pathogens. Various molecular studies have identified a kind of specialization of C. gloeosporioides isolates with specific hosts; however, there have been no studies of this gene in isolates from hosts other than avocado. The same is true for other species of Colletotrichum. We examined genetic variability in order to design primers that would amplify pelB gene fragments and compared the products of this amplification in C. gloeosporioides isolates from different hosts. Genetic variability was assessed using ISSR primers; the resultant data were grouped based on the UPGMA clustering method. Primers for the pelB gene were designed from selected GenBank sequences using the Primer 3 program at an annealing temperature of 60 degrees C and product amplification of nearly 600 bp. The ISSR primers were efficient in demonstrating the genetic variability of the Colletotrichum isolates and in distinguishing C. gloeosporioides, C. acutatum and C. sublineolum species. The gene pelB was found in C. gloeosporioides, C. acutatum and C. sublineolum. Amplified restriction fragments using MspI did not reveal differences in pelB gene structure in isolates from the three different host species that we investigated.

Host range and genetic relatedness of Colletotrichum acutatum isolates from fruit crops and leatherleaf fern in Florida.

Isolates of Colletotrichum acutatum were collected from anthracnose-affected strawberry, leatherleaf fern, and Key lime; ripe-rot-affected blueberry; and postbloom fruit drop (PFD)-affected sweet orange in Florida. Additional isolates from ripe-rot-affected blueberry were collected from Georgia and North Carolina and from anthracnose-affected leatherleaf fern in Costa Rica. Pathogenicity tests on blueberry and strawberry fruit; foliage of Key lime, leatherleaf fern, and strawberry; and citrus flowers showed that isolates were highly pathogenic to their host of origin. Isolates were not pathogenic on foliage of heterologous hosts; however, several nonhomologous isolates were mildly or moderately pathogenic to citrus flowers and blueberry isolates were pathogenic to strawberry fruit. Based on sequence data from the internal transcribed spacer (ITS)1-5.8S rRNA-ITS2 region of the rDNA repeat, the glutaraldehyde-3-phosphate dehydrogenase intron 2 (G3PD), and the glutamine synthase intron 2 (GS), isolates from the same host were identical or very similar to each other and distinct from those isolated from other hosts. Isolates from leatherleaf fern in Florida were the only exception. Among these isolates, there were two distinct G3PD and GS sequences that occurred in three of four possible combinations. Only one of these combinations occurred in Costa Rica. Although maximum parsimony trees constructed from genomic regions individually displayed little or no homoplasy, there was a lack of concordance among genealogies that was consistent with a history of recombination. This lack of concordance was particularly evident within a clade containing PFD, Key lime, and leatherleaf fern isolates. Overall, the data indicated that it is unlikely that a pathogenic strain from one of the hosts examined would move to another of these hosts and produce an epidemic.

Vegetative compatibility and parasexual segregation in Colletotrichum lindemuthianum, a fungal pathogen of the common bean.

The heterokaryotic and vegetative diploid phases of Colletotrichum lindemuthianum are described using nutritional and biochemical markers. Nitrate non-utilizing mutants (nit), derived from R2047, R89, R73, R65, and R23 isolates, were paired in all possible combinations to obtain heterokaryons. Although pairings R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete vegetative incompatibility, prototrophic heterokaryons were obtained from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89, R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23. Heterokaryons gave rise to spontaneous mitotic segregants which carried markers corresponding to one or the other of the parental strains. Heterokaryons spontaneously produced prototrophic fast-growing sectors too, characterized as diploid segregants. Diploids would be expected to yield auxotrophic segregants following haploidization in basal medium or in the presence of benomyl. Parental haploid segregants were in fact recovered from diploid colonies growing in basal medium and basal medium containing the haploidizing agent. Although barriers to the formation of heterokaryons in some crosses were detected, the results demonstrate the occurrence of parasexuality among vegetative compatible mutants of C. lindemuthianum.

Colletotrichum sublineolum genetic instability assessed by mutants resistant to chlorate.

The fungus Colletotrichum sublineolum, causal agent of sorghum anthracnose, presents high variability, genetic instability and host specialization. The aims of the present work were to investigate the mechanisms involved in the genetic instability in this species. Mutants resistant to chlorate and unable to use nitrate (Nit mutants), were obtained spontaneously, isolated and characterized for complementation pattern, reversion frequency and RAPD profile. The results showed that chlorate-resistant mutants could be divided into six phenotypic classes that probably represented mutations in the structural nitrate reductase locus (nit1), in the structural nitrite reductase locus (nit6 and niiA of Neurospora and Aspergillus, respectively), in the specific regulator locus (nit3), in the main regulator locus (nit2), in loci that codified the cofactor containing molybdenum necessary for nitrate reductase activity (NitM), and one or more genes responsible for nitrate intake (crn). In addition, the genetic control of this metabolism in C. sublineolum seems to be similar to other fungi species such as Aspergillus, Neurospora and Fusarium. The high reversion frequency (10(-4) to 10(-5)) presented by nit1 mutants suggests that the instability in evaluated strains could be a result of transposable elements activity. The RAPD analysis enabled confirmation that the Nit mutants have a similar genetic background to original strain, and that polymorphism exists among wild-type strains, nit1 mutants and revertants of C. sublineolum. These are important aspects for the later direction of molecular analysis, where these mutants will be used as a tool to isolate the active transposable elements in the C. sublineolum genome.

Vegetative compatibility and parasexual segregation in Colletotrichum lindemuthianum, a fungal pathogen of the common bean

The heterokaryotic and vegetative diploid phases of Colletotrichum lindemuthianum are described using nutritional and biochemical markers. Nitrate non-utilizing mutants (nit), derived from R2047, R89, R73, R65, and R23 isolates, were paired in all possible combinations to obtain heterokaryons. Although pairings R2047/R89, R2047/R73, R65/R73, and R73/R23 showed complete vegetative incompatibility, prototrophic heterokaryons were obtained from pairings R2047/R65, R2047/R23, R65/R89, R65/R23, R73/R89, R89/R23, R2047/R2047, R65/R65, R89/R89, R73/R73, and R23/R23. Heterokaryons gave rise to spontaneous mitotic segregants which carried markers corresponding to one or the other of the parental strains. Heterokaryons spontaneously produced prototrophic fast-growing sectors too, characterized as diploid segregants. Diploids would be expected to yield auxotrophic segregants following haploidization in basal medium or in the presence of benomyl. Parental haploid segregants were in fact recovered from diploid colonies growing in basal medium and basal medium containing the haploidizing agent. Although barriers to the formation of heterokaryons in some crosses were detected, the results demonstrate the occurrence of parasexuality among vegetative compatible mutants of C. lindemuthianum.

Effect of different carbon sources on endochitinase production by Colletotrichum gloeosporioides.

The present work analyzes the production of endochitinase by Colletotrichum gloeosporioides, a phytopathogenic fungus, using six different carbon sources and two pH values. For quantitative assay of endochitinase activity in solution, the synthetic substrate 4-methylumbelliferyl-beta-D-N,N',N"-triacetylchitotrioside was used. The major productions were obtained at pH 7.0 and 9.0, when colloidal chitin and glucose were used, whereas xylose and lactose were not good carbon sources. When testing different concentrations of colloidal chitin, glucose and glucosamine, colloidal chitin 0.5% was the best substrate, giving values of 2.4 U at the fifth day. When using glucose, best production occurred at 0.3% concentration, after 5 days growth, with values of 1.31 U. Endochitinase production was markedly decreased in high levels of glucose and in all glucosamine concentrations tested. SDS-PAGE co-polymerized with glycol-chitin analysis showed three major activity bands of 200, 100, and 95 kDa, when incubated at 50 degrees C.

Purification and characterization of an endochitinase produced by Colletotrichum gloeosporioides.

The phytopathogenic fungus Colletotrichum gloeosporioides was analyzed for chitinase activity, the best production occurring at the fourth day. A 43 kDa endochitinase with specific activity of 413 U microg(-1) protein was purified corresponding to a 75% yield. The optima of temperature and pH for the enzyme were 50 degrees C and pH 7.0, respectively. The enzyme showed a high stability at 50 degrees C and pH 7.0. Values of pH from 5.0 up to 7.0 gave, at least, 50% of maximum activity, suggesting a biotechnological application. Further studies are in progress to determine the possible use of this endochitinase in biological control.