The evolution of ectomycorrhizal symbiosis and host-plant switches are the main drivers for diversification of Amanitaceae (Agaricales, Basidiomycota).

BACKGROUND: Evolutionary radiation is widely recognized as a mode of species diversification, but the drivers of the rapid diversification of fungi remain largely unknown. Here, we used Amanitaceae, one of the most diverse families of macro-fungi, to investigate the mechanism underlying its diversification. RESULTS: The ancestral state of the nutritional modes was assessed based on phylogenies obtained from fragments of 36 single-copy genes and stable isotope analyses of carbon and nitrogen. Moreover, a number of time-, trait-, and paleotemperature-dependent models were employed to investigate if the acquisition of ectomycorrhizal (ECM) symbiosis and climate changes promoted the diversification of Amanitaceae. The results indicate that the evolution of ECM symbiosis has a single evolutionary origin in Amanitaceae. The earliest increase in diversification coincided with the acquisition of the ECM symbiosis with angiosperms in the middle Cretaceous. The recent explosive diversification was primarily triggered by the host-plant switches from angiosperms to the mixed forests dominated by Fagaceae, Salicaceae, and Pinaceae or to Pinaceae. CONCLUSIONS: Our study provides a good example of integrating phylogeny, nutritional mode evolution, and ecological analyses for deciphering the mechanisms underlying fungal evolutionary diversification. This study also provides new insights into how the transition to ECM symbiosis has driven the diversification of fungi.

High rate of gene family evolution in proximity to the origin of ectomycorrhizal symbiosis in Inocybaceae.

The genomes of ectomycorrhizal (ECM) fungi have a reduced number of genes encoding Carbohydrate-Active EnZymes (CAZymes), expansions in transposable elements (TEs) and small secreted proteins (SSPs) compared with saprotrophs. Fewer genes for specific peptidases and lipases in ECM fungi are also reported. It is unclear whether these changes occur at the shift to the ECM habit or are more gradual throughout the evolution of ECM lineages. We generated a genomic dataset of 20 species in the ECM lineage Inocybaceae and compared them with six saprotrophic species. Inocybaceae genomes have fewer CAZymes, peptidases, lipases, secondary metabolite clusters and SSPs and higher TE content than their saprotrophic relatives. There was an increase in the rate of gene family evolution along the branch with the transition to the ECM lifestyle. This branch had very high rate of evolution in CAZymes and had the largest number of contractions. Other significant changes along this branch included expansions in transporters, transposons-related genes and communication genes such as fungal kinases. There is a high concentration of changes in proximity to the transition to the ECM lifestyle, which correspond to the identified key changes for the gain of this lifestyle.

Parvodontia relampaga sp. nov.: A Cystostereaceae fungal pathogen that is the causal agent of relampago blight of woody plants in Florida, USA.

Starting in the fall of 2019, mortality, blight symptoms, and signs of white fungal mycelia were observed on external host tissues of non-native landscape trees as well as numerous native trees, understory shrubs, and vines throughout northern and central Florida, USA. We determined that the fungus is an undescribed species of Basidiomycota based on morphological characteristics and DNA sequence analysis. Phylogenetic analyses of the internal transcribed spacer (ITS), large subunit (LSU), and translation elongation factor 1-alpha (tef1) regions revealed that this novel plant pathogen is an undescribed taxon of the genus Parvodontia (Cystostereaceae, Agaricales). We propose the name Parvodontia relampaga sp. nov. which describes its unique morphological features and phylogenetic placement. We confirmed the pathogenicity of P. relampaga in greenhouse inoculations on host plants from which strains of this novel pathogen were isolated, including the non-native gymnosperm Afrocarpus falcatus, the non-native and commercially important Ligustrum japonicum, and the native tree Quercus hemisphaerica. P. relampaga was also detected on a total of 27 different species of woody host plants, including such economically and ecologically important hosts as Fraxinus, Ilex, Magnolia, Persea, Prunus, Salix, Vitis, and Vaccinium. For this new plant disease, we propose the name "relampago blight," which refers to the lightning-like rhizomorph growth (relámpago means 'lightning' in Spanish). This study presents a newly discovered fungal taxon with a wide host range on both angiosperms and gymnosperms that may be an emerging pathogen of concern in Florida and the Gulf Coast region.

The fungal Clitocybe nebularis lectin binds distinct cell surface glycoprotein receptors to induce cell death selectively in Jurkat cells.

Clitocybe nebularis lectin (CNL) is a GalNAcß1-4GlcNAc-binding lectin that exhibits an antiproliferative effect exclusively on the Jurkat leukemic T cell line by provoking homotypic aggregation and dose-dependent cell death. Cell death of Jurkat cells exhibited typical features of early apoptosis, but lacked the activation of initiating and executing caspases. None of the features of CNL-induced cell death were effectively blocked with the pan-caspase inhibitor or different cysteine peptidase inhibitors. Furthermore, CNL binding induced Jurkat cells to release the endogenous damage-associated molecular pattern molecule high-mobility group box 1 (HMGB1). A plant lectin with similar glycan-binding specificity, Wisteria floribunda agglutinin (WFA) showed less selective toxicity and induced cell death in Jurkat, Tall-104, and Hut-87 cell lines. HMGB1 release was also detected when Jurkat cells were treated with WFA. We identified the CD45 and CD43 cell surface glycoproteins on Jurkat cells as the main targets for CNL binding. However, the blockade of CD45 phosphatase activity failed to block either CNL-induced homotypic agglutination or cell death. Overall, our results indicate that CNL triggers atypical cell death selectively on Jurkat cells, suggesting the potential applicability of CNL in novel strategies for treating and/or detecting acute T cell leukemia.

Biochemical and transcriptomic analyses of the symbiotic interaction between Cremastra appendiculata and the mycorrhizal fungus Coprinellus disseminatus.

BACKGROUND: Cremastra appendiculata is a rare terrestrial orchid with a high market value as an ornamental and medicinal plant. However, the species depends entirely on fungi for seed germination under natural conditions. In a previous study, we have successfully isolated and identified the mycorrhizal fungus Coprinellus disseminatus which was able to induce the germination of C. appendiculata seeds. We then speculated that C. disseminatus may do so by breaking the testa imposed dormancy of the seeds. In this study, biochemical and transcriptomic analyses were used to characterize the germination of C. appendiculata seeds, collected at different stages of germination, as affected by C. disseminatus. RESULTS: The lignocellulose in the seeds coat of C. appendiculata was degraded by the mycorrhizal fungus resulting in facilitated absorption of water. The rate of decline in lignin content was 67 and 73% at 6 and 12 days after sowing, respectively. The water content increased from 13 to 90% during symbiosis. A total of 15,382 genes showing significantly different levels of expression (log2 FPKM≥2.0, Qvalue≤0.05) were successfully identified among all libraries, where the highest number of DEGs was shared between 6 days versus 0 day after symbiotic germination. Gene annotation results suggested that 15 key genes related water-status, such as DHN gene family and Xero 1 were down-regulated. The genes zeaxanthin epoxidase ZEP, 9-cis-epoxycarotenoid dioxygenase NCED3 and ß-carotene hydroxylase involved in the biosynthesis of abscisic acid (ABA) were significantly down-regulated in 6 days as compared to 0 day after symbiotic germination. CONCLUSIONS: This work demonstrates that mycorrhizal fungus C. disseminatus can stimulate C. appendiculata seeds germination through a mechanism of breaking the testa imposed dormancy and inducing water absorption of the embryo.

The cold - resistance mechanism of a mutagenic Volvariella volvacea strain VH3 with outstanding traits revealed by transcriptome profiling.

BACKGROUND: The straw mushroom (Volvariella volvacea) is one of the important vegetables that is popular for its delicious taste. However, the straw mushroom is sensitive to low temperature, resulting in economic loss during transportation and storage. We obtained a novel straw mushroom strain, named VH3, via ultraviolet mutagenesis. RESULTS: Our study revealed that VH3 exhibited high cold resistance compared to an ordinary straw mushroom cultivar, V23. We found that the electrolyte leakages of VH3 were always significantly lower than that of V23 treated with 4 °C for 0 h, 2 h,4 h, 8 h, 16 h, and 24 h. Before cold treatment (0 h), there were no difference of MDA contents, SOD activities, and CAT activities between VH3 and V23. At the late stage (8 h, 26 h, and 24 h) of cold treatment, the MDA contents of VH3 were lower while both the SOD and CAT activities were higher than those of V23. To investigate the potential mechanisms of VH3 cold resistance, we performed transcriptome sequencing to detect the transcriptome profiling of VH3 and V23 after 0 h and 4 h cold treatment. Transcriptome sequencing revealed that 111 differentially expressed genes (DEG) between V23 (0 h) and VH3 (0 h) (V23-0_vs_VH3-0), consisting 50 up-regulated and 61 down-regulated DEGs. A total of 117 DEGs were obtained between V23 (4 h) and VH3(4 h) (V23-4_vs_VH3-4), containing 94 up-regulated and 23 down-regulated DEGs. Among these DEGs, VVO_00021 and VVO_00017 were up-regulated while VVO_00003, VVO_00004, VVO_00010, and VVO_00030 were down-regulated in V23-0_vs_VH3-0 and VH3-4_vs_V23-4. KEGG and GO analysis revealed that the 6 DEGs were annotated to pathways related to cold stress. Besides, the GA3 content was also decreased in VH3. CONCLUSIONS: Collectively, our study first revealed that the increased cold resistance of VH3 might be caused by the expression change of VVO_00003, VVO_00004, VVO_00017, VVO_00021, and VVO_00030, and decreased GA3.

Burkholderia perseverans sp. nov., a bacterium isolated from the Restinga ecosystem, is a producer of volatile and diffusible compounds that inhibit plant pathogens.

Gram-negative, aerobic, rod-shaped, non-spore-forming, motile bacteria, designated CBAS 719 T, CBAS 732 and CBAS 720 were isolated from leaf litter samples, collected in Espírito Santo State, Brazil, in 2008. Sequences of the 16S rRNA, gyrB, lepA and recA genes showed that these strains grouped with Burkholderia plantarii LMG 9035 T, Burkholderia gladioli LMG 2216 T and Burkholderia glumae LMG 2196 T in a clade of phytopathogenic Burkholderia species. Digital DNA-DNA hybridization experiments and ANI analyses demonstrated that strain CBAS 719 T represents a novel species in this lineage that is very closely related with B. plantarii. The genome sequence of the type strain is 7.57 Mbp and its G + C content is 69.01 mol%. The absence of growth on TSA medium supplemented with 3% (w/v) NaCl, citrate assimilation, ß-galactosidase (PNPG) activity, and of lipase C14 activity differentiated strain CBAS 719 T from B. plantarii LMG 9035 T, its nearest phylogenetic neighbor. Its predominant fatty acid components were C16:0, C18:1 ω7c, cyclo-C17:0 and summed feature 3 (C16:1 ω7c and/or C15:0 iso 2-OH). Based on these genotypic and phenotypic characteristics, the strains CBAS 719 T, CBAS 732 and CBAS 720 are classified in a novel Burkholderia species, for which the name Burkholderia perseverans sp. nov. is proposed. The type strain is CBAS 719 T (= LMG 31557 T = INN12T).

In search of the spectral composition of an effective light trap for the mushroom pest Lycoriella ingenua (Diptera: Sciaridae).

Certain fungus gnats, like Lycoriella ingenua are notorious pests in agriculture, especially in mushroom production. While larvae cause mainly direct crop damage, adults are vectors of several dangerous fungal pathogens. To promote the development of pesticide-free management methods, such as light trapping, we measured the spectral sensitivity of L. ingenua compound eyes with electroretinography and performed two different behavioural experiments to reveal the wavelength dependence of phototaxis in this species. The spectral sensitivity of the compound eyes is bimodal with peaks at 370 nm (UV) and 526 nm (green). Behavioural experiments showed that attraction to light as a function of wavelength depends on light intensity. In our first experiment, where the minimal photon flux (105-109 photons/cm2/s) needed for eliciting a phototactic response was determined wavelength by wavelength, phototaxis was strongest in the green spectral range (~526 nm). In the other behavioural experiment, where wavelength preference was tested under a higher but constant light intensity (~1013 photons/cm2/s), the highest attraction was elicited by UV wavelengths (398 nm). Our results suggest that both UV and green are important spectral regions for L. ingenua thus we recommend to use both UV (~370-398 nm) and green (~526 nm) for trapping these insects.

Facultative symbiosis with a saprotrophic soil fungus promotes potassium uptake in American sweetgum trees.

Several species of soil free-living saprotrophs can sometimes establish biotrophic symbiosis with plants, but the basic biology of this association remains largely unknown. Here, we investigate the symbiotic interaction between a common soil saprotroph, Clitopilus hobsonii (Agaricomycetes), and the American sweetgum (Liquidambar styraciflua). The colonized root cortical cells were found to contain numerous microsclerotia-like structures. Fungal colonization led to increased plant growth and facilitated potassium uptake, particularly under potassium limitation (0.05 mM K+ ). The expression of plant genes related to potassium uptake was not altered by the symbiosis, but colonized roots contained the transcripts of three fungal genes with homology to K+ transporters (ACU and HAK) and channel (SKC). Heterologously expressed ChACU and ChSKC restored the growth of a yeast K+ -uptake-defective mutant. Upregulation of ChACU transcript under low K+ conditions (0 and 0.05 mM K+ ) compared to control (5 mM K+ ) was demonstrated in planta and in vitro. Colonized plants displayed a larger accumulation of soluble sugars under 0.05 mM K+ than non-colonized plants. The present study suggests reciprocal benefits of this novel tree-fungus symbiosis under potassium limitation mainly through an exchange of additional carbon and potassium between both partners.

Chemistry and Toxicology of Major Bioactive Substances in Inocybe Mushrooms.

Mushroom poisoning has always been a threat to human health. There are a large number of reports about ingestion of poisonous mushrooms every year around the world. It attracts the attention of researchers, especially in the aspects of toxin composition, toxic mechanism and toxin application in poisonous mushroom. Inocybe is a large genus of mushrooms and contains toxic substances including muscarine, psilocybin, psilocin, aeruginascin, lectins and baeocystin. In order to prevent and remedy mushroom poisoning, it is significant to clarify the toxic effects and mechanisms of these bioactive substances. In this review article, we summarize the chemistry, most known toxic effects and mechanisms of major toxic substances in Inocybe mushrooms, especially muscarine, psilocybin and psilocin. Their available toxicity data (different species, different administration routes) published formerly are also summarized. In addition, the treatment and medical application of these toxic substances in Inocybe mushrooms are also discussed. We hope that this review will help understanding of the chemistry and toxicology of Inocybe mushrooms as well as the potential clinical application of its bioactive substances to benefit human beings.

Pyrosequencing and Taxonomic Composition of the Fungal Community from Soil of Tricholoma matsutake in Gyeongju.

Tricholoma matsutake is an ectomycorrhizal fungus that has a symbiotic relationship with the root of Pinus densiflora. Soil microbial communities greatly affect the growth of T. matsutake, however, few studies have examined the characteristics of these communities. In the present study, we analyzed soil fungal communities from Gyeongju and Yeongdeok using metagenomic pyrosequencing to investigate differences in fungal species diversity, richness, and taxonomic composition between the soil under T. matsutake fruiting bodies (Sample 2) and soil where the fairy ring of T. matsutake was no longer present (Sample 1). The same spot was investigated three times at intervals of four months to observe changes in the community. In the samples from Yeongdeok, the number of valid reads was lower than that at Gyeongju. The operational taxonomic units of most Sample 2 groups were less than those of Sample 1 groups, indicating that fungal diversity was low in the T. matsutakedominant soil. The soil under the T. matsutake fruiting bodies was dominated by more than 51% T. matsutake. From fall to the following spring, the ratio of T. matsutake decreased. Basidiomycota was the dominant phylum in most samples. G-F1-2, G-F2-2, and Y-F1-2 had the genera Tricholoma, Umbelopsis, Oidiodendron, Sagenomella, Cladophialophora, and Phialocephala in common. G-F1-1, G-F2-1, and Y-F1-1 had 10 genera including Umbelopsis and Sagenomella in common. From fall to the following spring, the amount of phyla Basidiomycota and Mucoromycota gradually decreased but that of phylum Ascomycota increased. We suggest that the genus Umbelopsis is positively related to T. matsutake.

A precise relationship among Buller's drop, ballistospore, and gill morphologies enables maximum packing of spores within gilled mushrooms.

Basidiomycete fungi eject basidiospores using a surface tension catapult. A fluid drop forms at the base of each spore and, after reaching a critical size, coalesces with the spore and launches it from the gill surface. It has long been hypothesized that basidiomycete fungi pack the maximum number of spores into a minimal investment of biomass. Building on a nascent understanding of the physics underpinning the surface tension catapult, we modeled a spore's trajectory away from a basidium and demonstrated that to achieve maximum packing the size of the fluid drop, the size of the spore, and the distance between gills must be finely coordinated. To compare the model with data, we measured spore and gill morphologies from wild mushrooms and compared measurements with the model. The empirical data suggest that in order to pack the maximum number of spores into the least amount of biomass, the size of Buller's drop should be smaller but comparable to the spore size. Previously published data of Buller's drop and spore sizes support our hypothesis and also suggest a linear scaling between spore radius and Buller's drop radius. Morphological features of the surface tension catapult appear tightly regulated to enable maximum packing of spores. If mushrooms are maximally packed and Buller's drop radii scale linearly with spore radii, we predict that intergill distance should be proportional to spore radius to the power 3/2.

Endophytic bacteria promote the quality of Lyophyllum decastes by improving non-volatile taste components of mycelia.

Endophytic bacteria are always related to the host different traits, including the secondary metabolites production. However, the effect and mechanism of endophytic bacteria in the mushrooms fruit body on mycelia are still not clear. In this study, we investigated the effect of endophytic bacterial metabolites on the quality of Lyophyllum decastes mycelia. Soluble sugars, starch, protein, free amino acids, 5'-Nucleotides, EUC, and organic acids contents of mycelia were analyzed. We found that endophytic bacterial metabolites significantly increased the contents of soluble sugars, starch, protein, free amino acids, organic acids, and EUC. The present study thus suggests that endophytic bacteria could promote the quality of Lyophyllum decastes by improving non-volatile taste components of mycelia.

Exogenous l-ascorbic acid regulates the antioxidant system to increase the regeneration of damaged mycelia and induce the development of fruiting bodies in Hypsizygus marmoreus.

Hypsizygus marmoreus is an important commercial edible fungus, but the lack of basic studies on this fungus has hindered further development of its commercial value. In this study, we found that the treatment of damaged vegetative mycelia with 1 mM l-ascorbic acid (ASA) significantly increased the antioxidant enzyme activities (GPX, GR, CAT and SOD) and antioxidant contents (GSH and ASA) and reduced the ROS levels (H2O2 and O2-) in mechanically damaged mycelia. Additionally, this treatment increased mycelial biomass. At the reproductive stage, our results demonstrated that the treatment of damaged H. marmoreus mycelia with 2.24 mM ASA significantly increased the antioxidant enzyme activities (GPX, GR, GST, TRXR and CAT), endogenous ASA contents and GSH/GSSG ratios in different developmental stages and significantly decreased the MDA and H2O2 contents. Furthermore, this study showed that the expression levels of the antioxidant enzyme genes were consistent with the enzyme activities. Damaged mycelia treated with ASA regenerated 2-3 d earlier than the control group and showed significantly enhanced fruiting body production. These results suggested that exogenous ASA regulated mycelia intracellular ASA content to increase mycelial antioxidant abilities, induce the regeneration of damaged mycelia and regulate the development of fruiting bodies in H. marmoreus.

Rainstorm effects on the biocontrol efficacy of the decay fungus Chondrostereum purpureum against birch sprouting in boreal forests.

In forest regeneration areas, alongside roads and railways, under electric power lines and above gas pipe lines, there is a need for regular sprout control. A biocontrol method against broadleaved sprouting with formulations including the decay fungus Chondrostereum purpureum (Pers. Ex Fr.) Pouzar has been shown to be effective. Yet, heavy rain during spreading of this fungal inoculum on freshly cut stumps may affect the efficacy of the treatment, i.e., stump mortality during the following years. Thus, we performed an experiment where freshly cut birch stump surfaces (Betula pendula Roth and Betula pubescens Ehrh.) were treated with fungal inoculum under heavy irrigation and without it. Furthermore, two different adjuvants which aimed to fix the fungal inoculum to freshly cut stumps during irrigation and to protect against solar radiation were tested. Our results revealed that the artificial rainstorm treatment caused a delay in the efficacy of C. purpureum, but after three growing seasons, there was no significant difference in the mortality of birch stumps treated under irrigation or without it (stump mortalities 74 and 86%, respectively). Adjuvants did not improve the efficacy in stumps treated under irrigation nor in those treated without irrigation. KEY POINTS: • Heavy rain delayed the sprout control efficacy of a fungus Chondrostereum purpureum. • Final efficacy of formulations was the same in wet and dry conditions. • No additional adjuvants are needed to improve formulations.

Dillapiole in Piper holtonii as an Inhibitor of the Symbiotic Fungus Leucoagaricus gongylophorus of Leaf-Cutting Ants.

Plants of the Piperaceae family are studied for their diverse secondary metabolism with a vast array of compounds that act as chemical defense agents against herbivores. Of all the agricultural pests, the management of insects is a highly significant challenge in the Neotropics, and ants of the Attini tribe pose a major problem. Due to their symbiotic association with the fungus Leucoagaricus gongylophorus (Möller) Singer (Agaricaceae), the species of Atta and Acromyrmex have exhaustive foraging activity which has intensified as deforestation and monoculture farming have increased. The control of leaf-cutting ants is still carried out with synthetic products with negative consequences to the environment and human health. In search for natural and sustainable alternatives to synthetic pesticides, Piper holtonii C. DC. was selected among other plant species after field observations of the foraging activity of Atta cephalotes, which revealed that P. holtonii was never chosen by ants. In vitro evaluation of an ethanol extract of the leaves of P. holtonii resulted in promising inhibitory activity (IC50 102 ppm) against L. gongylophorus. Subsequently, bioassay-guided fractionation led to the isolation of the phenylpropanoid dillapiole, which was also detected in the essential oil. This compound demonstrated inhibition of the fungus with an IC50 of 38 ppm. Considering the symbiotic relationship between the Attini ants and L. gongylophorus, the negative effect on the survival of one of the organisms will affect the survival of the other, so dillapiole or standardized essential oil extracts of P. holtonii containing this active principle could be a unique and useful source as a control agent for leaf cutting-ants.

Hydrosoluble phylloplane components of Theobroma cacao modulate the metabolism of Moniliophthora perniciosa spores during germination.

The surface of plants forms a defense barrier that directly inhibits the first point of contact of microorganisms with the host. To understand this defense mechanism in Moniliophthora perniciosa interaction with Theobroma cacao cv Catongo, the aim of this study was to compare the changes in protein expression in basidiospores of the fungus M. perniciosa in response the leaf water washes (LWW) of two contrasting cocoa varieties for resistance to witches' broom disease. A total of 8.1 × 108 basidiospores were used for each treatment containing washed leaf material. Germinated basidiospores in the absence of LWW were used as control. The proteomic analysis was performed by the 2D-PAGE technique combined with mass spectrometry (MS). Protein extraction was based on the SDS-dense method followed by sonication for cell disruption and phenol extraction. Sixty-four proteins had accumulation of variation when compared to the control (no LWW). Proteins were identified associated with energy (ATP synthase) and protein (BiP) metabolism, whose accumulation was reduced by basidiospores germinated in leaf wash from Catongo cocoa. The reduction in ATP synthase of the basidiospores germinated the Catongo LWW suggests a shift from aerobic to fermentative metabolism of the fungus in response to components of the LWW. Furthermore, proteins involved in virulence were identified along with fungal resistance to polyketide cyclase, glycoside hydrolase, multidrug transporter protein (SFM) and proteins related to oxidative stress and fermentation, such as catalase A and alcohol dehydrogenase (ADH). The data showed an effect of cocoa phylloplane substances on the germination of fungal basidiospores through differences in protein expression patterns in the presence of LWW of the CCN51 and Catongo genotypes. These results may reveal mechanisms of resistance, host susceptibility and pathogen virulence.

The pathogen Moniliophthora perniciosa promotes differential proteomic modulation of cacao genotypes with contrasting resistance to witches´ broom disease.

BACKGROUND: Witches' broom disease (WBD) of cacao (Theobroma cacao L.), caused by Moniliophthora perniciosa, is the most important limiting factor for the cacao production in Brazil. Hence, the development of cacao genotypes with durable resistance is the key challenge for control the disease. Proteomic methods are often used to study the interactions between hosts and pathogens, therefore helping classical plant breeding projects on the development of resistant genotypes. The present study compared the proteomic alterations between two cacao genotypes standard for WBD resistance and susceptibility, in response to M. perniciosa infection at 72 h and 45 days post-inoculation; respectively the very early stages of the biotrophic and necrotrophic stages of the cacao x M. perniciosa interaction. RESULTS: A total of 554 proteins were identified, being 246 in the susceptible Catongo and 308 in the resistant TSH1188 genotypes. The identified proteins were involved mainly in metabolism, energy, defense and oxidative stress. The resistant genotype showed more expressed proteins with more variability associated with stress and defense, while the susceptible genotype exhibited more repressed proteins. Among these proteins, stand out pathogenesis related proteins (PRs), oxidative stress regulation related proteins, and trypsin inhibitors. Interaction networks were predicted, and a complex protein-protein interaction was observed. Some proteins showed a high number of interactions, suggesting that those proteins may function as cross-talkers between these biological functions. CONCLUSIONS: We present the first study reporting the proteomic alterations of resistant and susceptible genotypes in the T. cacao x M. perniciosa pathosystem. The important altered proteins identified in the present study are related to key biologic functions in resistance, such as oxidative stress, especially in the resistant genotype TSH1188, that showed a strong mechanism of detoxification. Also, the positive regulation of defense and stress proteins were more evident in this genotype. Proteins with significant roles against fungal plant pathogens, such as chitinases, trypsin inhibitors and PR 5 were also identified, and they may be good resistance markers. Finally, important biological functions, such as stress and defense, photosynthesis, oxidative stress and carbohydrate metabolism were differentially impacted with M. perniciosa infection in each genotype.

Involvement of structurally distinct cupuassu chitinases and osmotin in plant resistance to the fungus Moniliophthora perniciosa.

The cupuassu tree (Theobroma grandiflorum) is a crop of great economic importance to Brazil, mainly for its pulp and seeds, which are used in food industry. However, cupuassu fruit production is threatened by witches' broom disease caused by the fungus Moniliophthora perniciosa. As elements of its defense mechanisms, the plant can produce and accumulate pathogenesis-related (PR) proteins such as chitinases and osmotins. Here, we identified three cupuassu PR proteins (TgPR3, TgPR5 and TgPR8) from cupuassu-M. perniciosa interaction RNA-seq data. TgPR3 and TgPR8 corresponded to chitinases, and TgPR5 to osmotin; they are phylogenetically related to cacao and to Arabidopsis PR sequences involved in biotic and abiotic stress. The TgPR proteins' tridimensional structure was obtained through homology modeling, and molecular docking with chitin and chitosan showed that the TgPR proteins can interact with both cell wall molecules and presented a higher affinity for chitosan. TgPR gene expression was analyzed by RT-qPCR on resistant and susceptible cupuassu genotypes infected by M. perniciosa at 8, 24, 48 and 72 h after infection (hai). The TgPR genes showed higher expression in resistant plants compared to the susceptible ones, mainly for TgPR5 at 8 and 24 hai, while the expression was lower in the susceptible cupuassu plants. To our knowledge, this is the first in silico and in vitro reports of cupuassu PR protein. The data suggested that TgPRs could be involved in recognizing mechanisms of the plant's innate immune system through chitin receptors. Our results also suggest a putative role of chitinase/chitosanase for the TgPR5/osmotin.

The ability of Listeria monocytogenes to form biofilm on surfaces relevant to the mushroom production environment.

Due to its ubiquitous nature, Listeria monocytogenes is a threat to all fresh fruits and vegetables, including mushrooms, which are Ireland's largest horticultural crop. Although fresh cultivated mushrooms (Agaricus bisporus) have not been previously linked with listeriosis outbreaks, the pathogen still poses a threat to the industry, particularly due to its ability to form biofilms. This threat is highlighted by the multiple recalls of mushroom products caused by L. monocytogenes contamination and by previous studies demonstrating that L. monocytogenes is present in the mushroom production environment. In this study, the biofilm formation potential of L. monocytogenes strains isolated from the mushroom production environment was investigated on materials and at temperatures relevant to mushroom production. A preliminary assessment of biofilm formation of 73 mushroom industry isolates was undertaken using a crystal violet assay on polystyrene microtitre plates. The biofilm formation of a subset (n = 7) of these strains was then assessed on twelve different materials, including materials that are representative of the materials commonly found in the mushroom production environments, using the CDC biofilm reactor. Vertical scanning interferometry was used to determine the surface roughness of the chosen materials. All the strains tested using the CDC biofilm reactor were able to form biofilms on the different surfaces tested but material type was found to be a key determining factor on the levels of biofilm formed. Stainless steel, aluminium, rubber, polypropylene and polycarbonate were all able to support biofilm levels in the range of 4-4.9 log10 CFU/cm2, for seven different L. monocytogenes strains. Mushroom industry-specific materials, including growing nets and tarpaulins, were found to support biofilms levels between 4.7 and 6.7 log10 CFU/cm2. Concrete was found to be of concern as it supported 7.7 log10 CFU/cm2 of biofilm for the same strains; however, sealing the concrete resulted in an approximately 2-log reduction in biofilm levels. The surface roughness of the materials varied greatly between the materials (0.7-3.5 log10 Ra) and was found to have a positive correlation with biofilm formation (rs = 0.573) although marginally significant (P = 0.051). The results of this study indicate that L. monocytogenes can readily form biofilms on mushroom industry relevant surfaces, and additionally identifies surfaces of specific concern, where rigorous cleaning and disinfection is required.