Polysaccharide-rich fraction of Agaricus brasiliensis enhances the candidacidal activity of murine macrophages

A polysaccharide-rich fraction (ATF) of medicinal mushroom Agaricus brasiliensis was evaluated on the candidacidal activity, H2O2 and nitric oxide (NO) production, and expression of mannose receptors by murine peritoneal macrophages. Mice received three intraperitoneal (i.p.) injections of ATF and after 48 h their peritoneal resident macrophages were assayed against Candida albicans yeast forms. The treatment increased fungicidal activity and it was associated with higher levels of H2O2, whereas NO production was not affected. We also found that the treatment enhances mannose receptor expression by peritoneal macrophages, which are involved in the attachment and phagocytosis of non-opsonized microorganisms. Treatment of animals with ATF was able to enhance the clearance of C. albicans during the first 6 h after the experimental i.p. infection. Our results suggest that this extract can increase host resistance against some infectious agents through the stimulation of microbicidal activity of macrophages.

Modelització de la producció de melanina a partir de polifenol oxidasa d'Agaricus bisporus

Les melanines són un grup heterogeni de polímers producte de reaccions enzimàtiques en els teixits vegetals que contenen compostos fenòlics o polifenòlics. Estudis recents han descobert algunes propietats benèfiques de les melanines sobre la salut, tals com antioxidants, antiinflamatòries, immunològiques i propietats anti-tumorals. Així, no només la seva eliminació ha de ser examinada, sinó que també podria considerar-se la seva addició a aliments funcionals de nova creació. D’aquesta manera, es requereix conèixer el mecanisme cinètic de la lanogènesi abans de la seva possible utilització industrial. S’ha desenvolupat un model cinètic per explicar la formació de melanina a partir de L-tirosina utilitzant polifenol oxidasa d’Agaricus bisporus i monitoritzant l'absorbància de la solució. Aquesta expressió permet descriure la formació de melanina en funció del temps de reacció i obtenir alguns paràmetres importants que defineixen el producte, com el coeficient d'extinció. L’absorbància comença a créixer després d'un període de latència en què es produeixen productes intermedis incolors. El coeficient d'extinció dels productes resultants no és un valor constant, perquè depèn de les condicions de cada experiment. La tirosinasa tingué un menor efecte catalitzador sobre la L-tirosina (primera reacció que catalitza), que sobre L-DOPA (segona reacció).

Desenvolvimento in vitro de Agaricus brasiliensis em meios suplementados com diferentes farelos

O cogumelo Agaricus brasiliensis normalmente é cultivado em meios à base de batata ou composto orgânico. O objetivo deste trabalho foi avaliar o desenvolvimento in vitro da linhagem ABL 97/11 de A. brasiliensis, cultivada em meio à base de composto suplementado com diferentes concentrações de farelos de milho, trigo, arroz e soja, à temperatura constante de 28ºC. Avaliou-se, diariamente, o diâmetro da colônia e obteve-se, aos seis dias de cultivo, a massa miceliana. A adição de farelos de soja ou arroz não favorece o desenvolvimento in vitro de A. brasiliensis. O meio de cultura suplementado com 20% de farelo de trigo apresenta as maiores massa miceliana e velocidade de crescimento, comparado aos meios suplementados com outros farelos, na mesma concentração. Na concentração de 10% de farelo, o milho promove a maior velocidade de crescimento do cogumelo.

Caracterización de Cryphonectria hypovirus (CHV) mediante patrones RFLP en cepas hipovirulentas de Cryphonectria parasitica (Murrill) M.E. Barr de Cataluña

La detecció de soques hipovirulentes a Catalunya ha fet necessari l'estudi genètic de Cryphonectria hypovirus. Mitjançant mostrejos, es van obtenir les soques hipovirulentes i es va comprovar el seu caràcter hipovirulent mitjançant l'extracció del dsRNA. Mitjançant RT-PCR i RFLP es va estudiar la diversitat genètica de les poblacions de Cryphonectria hypovirus. En aquest estudi s'ha observat que la hipovirulència a Catalunya és encara un fenomen localitzat. D'altra banda, s'ha obervat certa diversitat genètica del hipovirus, fet que suggereix que C. hypovirus va ser introduït a Catalunya reiterades vegades. Aquestes reiterades introduccions de C. hypovirus també s'han observat a nivell parcel•lari. La majoria de les mostres pertanyen al subtipus CHV1-I, la qual cosa significa que és d'esperar un increment de la presència del hipovirus a les masses, en ser aquest subtipus el més adequat per dispersar en el medi. S'han detectat altres soques amb patrons RFLP encara no descrits, que podrien pertànyer a altres subtipus. Per tant, s'hauria de fer un estudi genètic més detallat d'aquestes mostres. D'altra banda, hi ha certa diversitat de GCVs associada a les soques amb l'hipovirus. Aquest fet podria suggerir que els GCVs presents a Catalunya tenen una bona capacitat per transmetre el virus o bé seria una altra evidència que C. hypovirus ha estat introduït reiteradament a Catalunya. Finalment s'ha detectat una zona especialment interessant amb diversitat de hipovirus i amb hipovirus encara no descrits, així com soques que podrien pertànyer a CHV1-E. Això suggereix que a la zona podria haver encara més diversitat del hipovirus i per tant podria ser interessant realitzar-hi nous mostreigs.

Some species of Perenniporia Murrill (Poriales, Basidiomycotina) from Southern Brazil

Perenniporia contraria, P. martius, P. medulla-panis, P. ohiensis, P. piperis, P. stipitata and P. tephropora are described in this paper. A key to these species is presented. Five species studied in culture are included, through the respective species code. P. tephropora is a new record from Southern Brazil. A check-list of names Perenniporia species known throughout the world is given.

Characterization of the chitinolytic system during the mycoparasitic interaction between Trichoderma aggressivum f. aggressivum and different host strains of Agaricus bisporus /

Green mould is a serious disease of commercially grown mushrooms, the causal agent being attributed to the filamentous soil fungus Triclzodenna aggressivum f. aggressivu11l and T. aggressivum f. ellropaellm. Found worldwide, and capable of devastating crops, this disease has caused millions of dollars in lost revenue within the mushroom industry. One mechanism used by TricllOdenlla spp. in the antagonism of other fungi, is the secretion of lytic enzymes such as chitinases, which actively degrade a host's cell wall. Therefore, the intent of this study was to examine the production of chitinase enzymes during the host-parasite interaction of Agaricus bisporus (commercial mushroom) and Triclzodemza aggressivum, focusing specifically on chitinase involvement in the differential resistance of white, off-white, and brown commercial mushroom strains. Chitinases isolated from cultures of A. bisporus and T. aggressivu11l grown together and separately, were identified following native PAGE, and analysis of fluorescence based on specific enzymatic cleavage of 4-methylumbelliferyl glucoside substrates. Results indicate that the interaction between T. aggressivulll and A. bisporus involves a complex enzyme battle. It was determined that T. aggressivum produces a number of chitinases that appear to correlate to those isolated in previous studies using biocontrol strains of T. Izarziallilm. A 122 kDa N-acetylglucosaminidase of T. aggressivu11l revealed the highest and most variable activity, and is therefore believed to be an important predictor of antifungal activity. Furthermore, results indicate that brown strain resistance of mushrooms may be related to high levels of a 96 kDa N-acetylglucosaminidase, which showed elevated activity in both solitary and dual cultures with T. aggressivum. Overall, each host-parasite combination produced unique enzyme profiles, with the majority of the differences seen between day 0 and day 6 for the extracellular chitinases. Therefore, it was concluded that the antagonistic behaviour of T. aggressivli1ll does not involve a typical response, always producing the same types and levels of enzymes, but that mycoparasitism, specifically in the form of chitinase production, may be induced and regulated based on the host presented.

Diversity among bacteria causing blotch disease on the commercial mushroom, agaricus bisporus /

A total of 251 bacterial isolates were isolated from blotched mushroom samples obtained from various mushroom farms in Canada. Out of 251 stored isolates, 170 isolates were tested for pathogenicity on Agaricus bisporus through mushroom rapid pitting test with three distinct pathotypes observed: dark brown, brovm and yellow/yellow-brown blotch. Phenotypic analysis of 83 isolates showed two distinct proteinase K resistant peptide profiles. Profile group A isolates exhibited peptides with masses of 45, 18, 16 and 14 kDa and fiirther biochemical tests identified them as Pseudomonasfluorescens III and V. Profile group B isolates lacked the 16-kDa peptide and the blotch causing bacterial isolates of this group was identified as Serratia liquefaciens and Cedecea davisae. Comparative genetic analysis using Amplified Fragment Length Polymorphism (AFLP) on 50 Pseudomonas sp. isolates (Group A) showed that various blotch symptoms were caused by isolates distributed throughout the Pseudomonas sp. clusters with the exception of the Pseudomonas tolaasii group and one non-pathogenic Pseudomonas fluorescens cluster. These results show that seven distinct Pseudomonas sp. genotypes (genetic clusters) have the ability to cause various symptoms of blotch and that AFLP can discriminate blotch causing from non-blotch causing Pseudomonasfluorescens. Therefore, a complex of diverse bacterial organisms causes bacterial blotch disease

An unusual postharvest spotting disease of the comercial mushroom, Agaricus bisporus, caused by a novel pathovar of Pseudomonas tolaasii

An unusual postharvest spotting disease of the commercial mushroom, Agaricus bisporus, which was observed on a commercial mushroom farm in Ontario, was found to be caused by a novel pathovar of Pseudomonas tolaasii. Isolations from the discoloured lesions, on the mushroom pilei, revealed the presence of several different bacterial and fungal genera. The most frequently isolated genus being Pseudomonas bacteria. The most frequently isolated fungal genus was Penicillium. Of the bacteria and fungi assayed for pathogenicity to mushrooms, only Pseudomonas tolaasii was able to reproduce the postharvest spotting symptom. This symptom was typically reproduced 1 to 7 days postharvest, when mushroom pilei were inoculated with 101 to 105 cfu. Of the fungi tested for pathogenicity only a Penicillium sp. and Verticillium fungicola were shown to be pathogenic, however, neither produced the postharvest spotting symptom. The Pseudomonas tolaasii strain isolated from the postharvest lesions differed from a type culture (Pseudomonas tolaasii ATCC 33618) in the symptoms it produced on Agaricus bisporus pilei under the same conditions and at the same inoculum concentration. It was therefore designated a pathovar. This strain also differed from the type culture in its cellular protein profile. Neither the type culture, nor the mushroom pathogen was found to contain plasmid DNA. The presence of plasmid DNA is therefore not responsible for the difference in pathogenicity between the two strains.

Cell wall degrading enzymes and interaction between Trichoderma Aggressivum and Agaricus Bisporus

Agaricus bisporus is the most commonly cultivated mushroom in North America and has a great economic value. Green mould is a serious disease of A. bisporus and causes major reductions in mushroom crop production. The causative agent of green mould disease in North America was identified as Trichoderma aggressivum f. aggressivum. Variations in the disease resistance have been shown in the different commercial mushroom strains. The purpose of this study is to continue investigations of the interactions between T. aggressivum and A. bisporus during the development of green mould disease. The main focus of the research was to study the roles of cell wall degrading enzymes in green mould disease resistance and pathogenesis. First, we tried to isolate and sequence the N-acetylglucosaminidase from A. bisporus to understand the defensive mechanism of mushroom against the disease. However, the lack of genomic and proteomic information of A. bisporus limited our efforts. Next, T. aggressivum cell wall degrading enzymes that are thought to attack Agaricus and mediate the disease development were examined. The three cell wall degrading enzymes genes, encoding endochitinase (ech42), glucanase (fJ-1,3 glucanase) and protease (prb 1), were isolated and sequenced from T. aggressivum f. aggressivum. The sequence data showed significant homology with the corresponding genes from other fungi including Trichoderma species. The transcription levels of the three T. aggressivum cell wall degrading enzymes were studied during the in vitro co-cultivation with A. bisporus using R T -qPCR. The transcription levels of the three genes were significantly upregulated compared to the solitary culture levels but were upregulated to a lesser extent in co-cultivation with a resistant strain of A. bisporus than with a sensitive strain. An Agrobacterium tumefaciens transformation system was developed for T. aggressivum and was used to transform three silencing plasmids to construct three new T. aggressivum phenotypes, each with a silenced cell wall degrading enzyme. The silencing efficiency was determined by RT-qPCR during the individual in vitro cocultivation of each of the new phenotypes with A. bisporus. The results showed that the expression of the three enzymes was significantly decreased during the in vitro cocultivation when compared with the wild type. The phenotypes were co-cultivated with A. bisporus on compost with monitoring the green mould disease progression. The data indicated that prbi and ech42 genes is more important in disease progression than the p- 1,3 glucanase gene. Finally, the present study emphasises the role of the three cell wall degrading enzymes in green mould disease infection and may provide a promising tool for disease management.

Defence of Agaricus bisporus against toxic secondary metabolites from Trichoderma aggressivum.

Trichoderma spp are effective competitors against other fungi because they are mycoparasitic and produce hydrolytic enzymes and secondary metabolites that inhibit the growth of their competitors. Inhibitory compounds produced by Trichoderma aggressivum, the causative agent of green mold disease, are more toxic to the hybrid off-white strains of Agaricus bisporus than the commercial brown strains, consistent with the commercial brown strain’s increased resistance to the disease. This project looked at the response of hybrid off-white and commercial brown strains of A. bisporus to the presence of T. aggressivum metabolites with regard to three A. bisporus genes: laccase 1, laccase 2, and manganese peroxidase. The addition of T. aggressivum toxic metabolites had no significant effect on MnP or lcc1 transcript abundance. Alternatively, laccase 2 appears to be involved in resistance to T. aggressivum because the presence of T. aggressivum metabolites results in higher lcc2 transcript abundance and laccase activity, especially in the commercial brown strain. The difference in laccase expression and activity between A. bisporus strains was not a result of regulatory or coding sequence differences. Alteration of laccase transcription by RNAi resulted in transformants with variable levels of laccase transcript abundance. Transformants with a low number of lcc transcripts were very sensitive to T. aggressivum toxins, while those with a high number of lcc transcripts had increased resistance. These results indicated that laccase activity, in particular that encoded by lcc2, serves as a defense response of A. bisporus to T. aggressivum toxins and contributes to green mold disease resistance in commercial brown strains.

Bdellovibrio bacteriovorus HD100 guards against Pseudomonas tolaasii brown-blotch lesions on the surface of post-harvest Agaricus bisporus supermarket mushrooms

BACKGROUND: Pseudomonas tolaasii is a problematic pathogen of cultured mushrooms, forming dark brown 'blotches' on mushroom surfaces and causing spoilage during crop growth and post-harvest . Treating P. tolaasii infection is difficult, as other, commensal bacterial species such as Pseudomonas putida are necessary for mushroom growth, so treatments must be relatively specific. RESULTS: We have found that P. tolaasii is susceptible to predation in vitro by the δ-proteobacterium Bdellovibrio bacteriovorus. This effect also occurred in funga, where B. bacteriovorus was administered to post-harvest mushroom caps before and after administration of the P. tolaasii pathogen. A significant, visible improvement in blotch appearance, after incubation, was observed on administration of Bdellovibrio. A significant reduction in viable P. tolaasii cell numbers, recovered from the mushroom tissue, was detected. This was accompanied by a more marked reduction in blotch severity on Bdellovibrio administration. We found that there was in some cases an accompanying overgrowth of presumed-commensal, non-Pseudomonas bacteria on post-harvest mushroom caps after Bdellovibrio-treatment. These bacteria were identified (by 16SrRNA gene sequencing) as Enterobacter species, which were seemingly resistant to predation. We visualised predatory interactions occuring between B. bacteriovorus and P. tolaasii on the post-harvest mushroom cap surface by Scanning Electron Microscopy, seeing predatory invasion of P. tolaasii by B. bacteriovorus in funga. This anti-P. tolaasii effect worked well in post-harvest supermarket mushrooms, thus Bdellovibrio was not affected by any pre-treatment of mushrooms for commercial/consumer purposes. CONCLUSIONS: The soil-dwelling B. bacteriovorus HD100 preys upon and kills P. tolaasii, on mushroom surfaces, and could therefore be applied to prevent spoilage in post-harvest situations where mushrooms are stored and packaged for sale.

The predatory mite Stratiolaelaps scimitus as a control agent of the fungus gnat Bradysia matogrossensis in commercial production of the mushroom Agaricus bisporus

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)