Fungus Alternaria Brassicicola Research Articles

New cytotoxic tricycloalternarenes from fungus Alternaria brassicicola

Seven previously undescribed metabolites, designated as tricycloalternarenes Q–W (1–7), were isolated and identified from the fermented rice substrate of fungus Alternaria brassicicola. The planar and absolute structures of all new compounds were determined on the basis of extensive NMR spectroscopic data, a modified Mosher’s method, X-ray crystallographic analysis, and electronic circular dichroism (ECD) spectral analyses. All the isolates were evaluated for in vitro cytotoxicity against five human tumor MM231, MM468, HeLa, SW1990, and SW480 cell lines, and compounds 1, 2, 5, and 7 showed selective cytotoxicity against certain human tumor cell lines with IC50 values ranging from 12.83 to 32.87 μM, with no obvious cytotoxicity to the normal LO2 cell.

Responses to Hydric Stress in the Seed-Borne Necrotrophic Fungus Alternaria brassicicola.

Alternaria brassicicola is a necrotrophic fungus causing black spot disease and is an economically important seed-borne pathogen of cultivated brassicas. Seed transmission is a crucial component of its parasitic cycle as it promotes long-term survival and dispersal. Recent studies, conducted with the Arabidopsis thaliana/A. brassicicola pathosystem, showed that the level of susceptibility of the fungus to water stress strongly influenced its seed transmission ability. In this study, we gained further insights into the mechanisms involved in the seed infection process by analyzing the transcriptomic and metabolomic responses of germinated spores of A. brassicicola exposed to water stress. Then, the repertoire of putative hydrophilins, a group of proteins that are assumed to be involved in cellular dehydration tolerance, was established in A. brassicicola based on the expression data and additional structural and biochemical criteria. Phenotyping of single deletion mutants deficient for fungal hydrophilin-like proteins showed that they were affected in their transmission to A. thaliana seeds, although their aggressiveness on host vegetative tissues remained intact.

Beneficial and Pathogenic Arabidopsis Root-Interacting Fungi Differently Affect Auxin Levels and Responsive Genes During Early Infection.

Auxin (indole-3-acetic acid, IAA) is an important phytohormone involved in root growth and development. Root-interacting beneficial and pathogenic fungi utilize auxin and its target genes to manipulate the performance of their hosts for their own needs. In order to follow and visualize auxin effects in fungi-colonized Arabidopsis roots, we used the dual auxin reporter construct DR5::EGFP-DR5v2::tdTomato and fluorescence microscopy as well as LC-MS-based phytohormone analyses. We demonstrate that the beneficial endophytic fungi Piriformospora indica and Mortierella hyalina produce and accumulate IAA in their mycelia, in contrast to the phytopathogenic biotrophic fungus Verticillium dahliae and the necrotrophic fungus Alternaria brassicicola. Within 3 h after exposure of Arabidopsis roots to the pathogens, the signals of the auxin-responsive reporter genes disappeared. When exposed to P. indica, significantly higher auxin levels and stimulated expression of auxin-responsive reporter genes were detected both in lateral root primordia and the root elongation zone within 1 day. Elevated auxin levels were also present in the M. hyalina/Arabidopsis root interaction, but no downstream effects on auxin-responsive reporter genes were observed. However, the jasmonate level was strongly increased in the colonized roots. We propose that the lack of stimulated root growth upon infection with M. hyalina is not caused by the absence of auxin, but an inhibitory effect mediated by high jasmonate content.

Contribution of cell wall peroxidase‐ and NADPH oxidase‐derived reactive oxygen species to Alternaria brassicicola‐induced oxidative burst in Arabidopsis

SummaryCell wall peroxidases and plasma membrane‐localized NADPH oxidases are considered to be the main sources of the apoplastic oxidative burst in plants attacked by microbial pathogens. In spite of this established doctrine, approaches attempting a comparative, side‐by‐side analysis of the functions of extracellular reactive oxygen species (ROS) generated by the two enzymatic sources are scarce. Previously, we have reported the role of Arabidopsis NADPH oxidase RBOHD (respiratory burst oxidase homologue D) in plants challenged with the necrotrophic fungus Alternaria brassicicola. Here, we present results on the activity of apoplastic class III peroxidases PRX33 (At3g49110) and PRX34 (At3g49120) investigated in the same Arabidopsis–Alternaria pathosystem. ROS generated by Arabidopsis peroxidases PRX33 and PRX34 increase the necrotic symptoms and colonization success of A. brassicicola. In addition, the knockdown of PRX33 and PRX34 transcript levels leads to a reduced number of host cells showing an extracellular burst of ROS after inoculation with A. brassicicola. Our results also reveal an age‐dependent transcript distribution of ROS‐producing peroxidase and NADPH oxidase enzymes, and some potential new components of the RBOHD, PRX33 and PRX34 signalling networks.

Herbivory-responsive calmodulin-like protein CML9 does not guide jasmonate-mediated defenses in Arabidopsis thaliana.

Calcium is an important second messenger in plants that is released into the cytosol early after recognition of various environmental stimuli. Decoding of such calcium signals by calcium sensors is the key for the plant to react appropriately to each stimulus. Several members of Calmodulin-like proteins (CMLs) act as calcium sensors and some are known to mediate both abiotic and biotic stress responses. Here, we study the role of the Arabidopsis thaliana CML9 in different stress responses. CML9 was reported earlier as defense regulator against Pseudomonas syringae. In contrast to salicylic acid-mediated defense against biotrophic pathogens such as P. syringae, defenses against herbivores and necrotrophic fungi are mediated by jasmonates. We demonstrate that CML9 is induced upon wounding and feeding of the insect herbivore Spodoptera littoralis. However, neither different CML9 loss-of-function mutant lines nor overexpression lines were impaired upon insect feeding. No difference in herbivore-induced phytohormone elevation was detected in cml9 lines. The defense against the spider mite Tetranychus urticae was also unaffected. In addition, cml9 mutant lines showed a wild type-like reaction to the necrotrophic fungus Alternaria brassicicola. Thus, our data suggest that CML9 might be a regulator involved only in the defense against biotrophic pathogens, independent of jasmonates. In addition, our data challenge the involvement of CML9 in plant drought stress response. Taken together, we suggest that CML9 is a specialized rather than a general regulator of stress responses in Arabidopsis.

Anti-inflammatory fusicoccane-type diterpenoids from the phytopathogenic fungus Alternaria brassicicola.

Altering the cultivation conditions, such as temperature, media compositions, illumination, aeration, the shape of the culturing flask, etc., is regarded as a useful strategy for the exploitation of structurally novel and bioactive secondary metabolites, which inspired us to explore the chemical and pharmacological diversities from the genetically powerful fungus Alternaria brassicicola. As a result, twelve fusicoccane-type diterpenoids, including eight new ones, termed brassicicenes Q-X (1-8), were isolated from a modified rice medium. Biosynthetically, all these compounds were derived from the mevalonic acid (MVA) pathway, and their structures incorporating absolute configurations were assigned by the interpretation of spectroscopic (HRESIMS and 1D and 2D NMR) analyses, chemical conversion, a modified Mosher's method, 13C NMR calculation, and single-crystal X-ray diffraction (Cu Kα). Structurally, compound 1 was an unusual 16-nor-dicyclopenta[a,d]cyclooctane diterpenoid bearing a fused cyclopent-2-en-1-one moiety. In addition, compound 3 was found to show significant anti-inflammatory activity against the production of NO, TNF-α, and IL-1β at 10 μM. Further western blot and immunofluorescence experiments found the mechanism of action to be that 3 inhibited the NF-κB-activated pathway, highlighting it as a promising starting point for the development of new anti-inflammatory agents.

A mitovirus isolated from the phytopathogenic fungus Alternaria brassicicola.

Alternaria brassicicola is one of the causal agents of alternaria blackspot in rapeseed. In this study, a dsRNA segment was isolated and sequenced from the fungus. The complete nucleotide sequence of the dsRNA was 2506 bp in length and, using the fungal mitochondrial genetic code, was predicted to contain a single large open reading frame (ORF) in the positive strand. This ORF was predicted to encode a protein with 719 amino acids that contains characteristic conserved motifs of the RNA-dependent RNA polymerase (RdRp). BLAST analysis revealed that this protein had significant sequence similarity to the RdRp from viruses of the genus Mitovirus. These results indicated that the dsRNA segment represents the replicative form of a mitovirus, which is tentatively designated "Alternaria brassicicola mitovirus 1" (AbMV1) and is a new member of the genus Mitovirus in the family Narnaviridae.

A flavoprotein supports cell wall properties in the necrotrophic fungus Alternaria brassicicola

BackgroundFlavin-dependent monooxygenases are involved in key biological processes as they catalyze a wide variety of chemo-, regio- and enantioselective oxygenation reactions. Flavoprotein monooxygenases are frequently encountered in micro-organisms, most of which require further functional and biocatalytic assessment. Here we investigated the function of the AbMak1 gene, which encodes a group A flavin monooxygenase in the plant pathogenic fungus Alternaria brassicicola, by generating a deficient mutant and examining its phenotype.ResultsFunctional analysis indicates that the AbMak1 protein is involved in cell wall biogenesis and influences the melanization process. We documented a significant decrease in melanin content in the Δabmak1 strain compared to the wild-type and complemented strains. We investigated the cell wall morphology and physical properties in the wild-type and transformants using electron and atomic force microscopy. These approaches confirmed the aberrant morphology of the conidial wall structure in the Δabmak1 strain which had an impact on hydrophilic adhesion and conidial surface stiffness. However, there was no significant impairment in growth, conidia formation, pathogenicity or susceptibility to various environmental stresses in the Δabmak1 strain.ConclusionThis study sheds new light on the function of a fungal flavin-dependent monooxygenase, which plays an important role in melanization.

Biotransformation of rutabaga phytoalexins by the fungus Alternaria brassicicola: Unveiling the first hybrid metabolite derived from a phytoalexin and a fungal polyketide

The biotransformations of the rutabaga phytoalexins rutalexin, brassicanate A, isalexin and rapalexin A by the plant pathogenic fungus Alternaria brassicicola are reported. While the biotransformations of rutalexin, brassicanate A, and isalexin are fast, rapalexin A is resistant to fungal transformation. Unexpectedly, biotransformation of rutalexin yields a hybrid metabolite named rutapyrone, derived from rutalexin metabolism and phomapyrone G, a fungal metabolite produced by A. brassicicola. These fungal transformations are detoxification reactions likely carried out by different enzymes. The discovery of rapalexin A resistance to detoxification suggests that this phytoalexin in combination with additional phytoalexins could protect crucifers against this pathogen. Phytoalexins resistant to degradation by A. brassicicola are expected to provide the producing plants with higher disease resistance levels.

Ultra high diluted arsenic reduces spore germination of Alternaria brassicicola and dark leaf spot in cauliflower

ABSTRACT A major problem in cauliflower crop is the fungus Alternaria brassicicola, which causes dark leaf spot on Brassicaceae family. The current use of copper salts in agriculture is questioned. In fact, these products present some disadvantages, connected mainly with their deposits in the soil and toxicity on plants. This work investigated the effects of arsenic treatments, in ultra high diluted form (UHD), prepared by a process of repeated dilution and succussion (shaking), through: 1) in vitro germination experiments, where spores of A. brassicicola were suspended in the treatments; 2) in planta experiments and 3) a field trial, where cauliflower plants infected by the fungus were sprayed with treatments. The results showed that ultra high dilutions of arsenic (where no more molecules of this substance are present) were effective in all the experiments, inhibiting spore germination by 60.0%, controlling fungal disease in in planta experiments (relative efficacy of 42.1%), and, in field trial, decreasing the mean infection level in cauliflower heads by 45.7% and 41.6% in artificially inoculated and naturally infected plants, respectively. This is the first study to demonstrate that ultra high dilutions effectively reduce in vitro spore germination and infection of A. brassicicola in cauliflower plants, both under controlled conditions and in the field. Our research is still very experimental, however, in light of the significant results obtained with ultra-diluted arsenic, and given that its extreme high dilution level precludes any toxicity or accumulation in the environment, the use of UHDs could be considered a potential and reliable approach for sustainable agriculture.

ChemInform Abstract: Structural Revisions of a Class of Natural Products: Scaffolds of Aglycon Analogues of Fusicoccins and Cotylenins Isolated from Fungi.

AbstractNovel brassicicenes L (IV), M (If), and N (V) are isolated from the fungus Alternaria brassicicola and their structures are elucidated.

Structural Revisions of a Class of Natural Products: Scaffolds of Aglycon Analogues of Fusicoccins and Cotylenins Isolated from Fungi.

The reisolation and structural revision of brassicicene D is described, and inspired us to reassign the core skeletons of brassicicenes C-H, J and K, ranging from dicyclopenta[a,d]cyclooctane to tricyclo[9.2.1.0(3,7)]tetradecane using quantum-chemical predictions and experimental validation strategies. Three novel, highly modified fusicoccanes, brassicicenes L-N, were also isolated from the fungus Alternaria brassicicola, and their structures were unequivocally established by spectroscopic data, ECD calculations, and crystallography. The reassigned structures represent the first class of bridgehead double-bond-containing natural products with a bicyclo[6.2.1]undecane carbon skeleton. Furthermore, their stabilities were first predicted with olefin strain energy calculations. Collectively, these findings extend our view of the application of computational predictions and biosynthetic logic-based structure elucidation to address problems related to the structure and stability of natural products.

Simplified Assays for Evaluation of Resistance to Alternaria brassicicola and Turnip Mosaic Virus.

Studying the natural defense mechanisms developed by model plants such as Arabidopsis is an important approach towards the improvement of crop species. The availability of mutants as well as the relative easiness to silence any gene in Arabidopsis provides an invaluable source of genotypes that can be used to discover new elements involved in the defense response. Here we describe simple and reliable methods to evaluate susceptibility/resistance to the pathogenic fungus Alternaria brassicicola and the viral pathogen Turnip mosaic virus.

Characterization of a novel single-stranded RNA virus, closely related to fusariviruses, infecting the plant pathogenic fungus Alternaria brassicicola

The alternaria blackspot of rapeseed is one of the most prominent diseases of rapeseed. It is caused by three species of the genus Alternaria: Alternaria brassicicola, Alternaria brassicae, and Alternaria raphanin. Here we report a novel positive-sense RNA virus from an A. brassicicola strain 817-14. The virus has a 6639 nucleotide (nt) long genome, excluding a poly (A)-tail, and was predicted to contain three putative open reading frames (ORF1, ORF2, and ORF3). The large ORF1 encoded a 174-kDa polyprotein (composed of 1522 amino acid residues) containing a conserved RNA-dependent RNA polymerase (RdRp) domain and a helicase domain. The other two smaller ORFs encoded polypeptides with unknown function. Homology search and phylogenetic analysis, based on the RdRp and helicase domains, suggest that this virus is related to and grouped with Sclerotinia sclerotiorum fusarivirus 1 (SsFV1), Rosellinia necatrix fusarivirus 1 (RnFV1), Fusarium graminearum virus-DK21 (FgV1), and Penicillium roqueforti RNA mycovirus 1 (PrRV1), all of which belong to a newly proposed family Fusariviridae. For this study, we designed the virus as “Alternaria brassicicola fusarivirus 1” (AbFV1). Virus elimination revealed that AbFV1 has no conspicuous impact on the biological properties of its host.

Chitin Nanofiber Elucidates the Elicitor Activity of Polymeric Chitin in Plants

Chitin, an N-acetyl-D-glucosamine polymer, is a component of fungal cell walls and a microbe/pathogen-associated molecular pattern that elicits plant defense responses. As polymeric chitin is difficult to handle due to its insolubility in water, many studies on chitin-induced immune responses have used water-soluble low-molecular weight chitin instead. Thus, it is unclear if polymeric chitin can induce resistance. Here, we examined the elicitor activity of chitin nanofiber (CNF) of submicron thickness prepared from polymeric chitin. CNF showed a high dispersing ability in water and induced both reactive oxygen species (ROS) production and chitin-induced defense-related gene expression in Arabidopsis thaliana seedlings. The Arabidopsis chitin elicitor receptor kinase 1 (Atcerk1) mutant, which is impaired in chitin perception, also failed to respond to CNF. CNF exposure triggered ROS generation in suspension-cultured cells from Oryza sativa. Furthermore, pre-treatment of Arabidopsis leaves with CNF effectively reduced pathogen infection by both the fungus Alternaria brassicicola and the bacterium Pseudomonas syringae pv. tomato DC3000. These results demonstrate that CNF has elicitor activity and will help define the role of polymeric chitin in plant immune responses.

EFEITO DE EXTRATOS DE Allamanda blanchetti NO CONTROLE DE Alternaria brassicicola EM MUDAS DE COUVE-MANTEIGA

RESUMO: O objetivo do presente trabalho foi avaliar o efeito do extrato de uma planta nativa da caatinga (Allamanda blanchetti) no controle do fungo Alternaria brassicicola quanto à indução de mecanismos de resistência em mudas de couve-manteiga e ação fungitóxica diretaEm condições de casa de vegetação, as mudas de couve-manteiga foram pulverizadas com os extratos de A. blanchetti extraídos a quente e a frio, nas concentrações 0, 100, 500 e 1000 ppm. Avaliações da severidade da doença foram realizadas a 5, 6, 7, 8, 9 e 10 dias após a inoculação. Peroxidases foram analisadas em cinco tempos, quais sejam, 0, 24, 72, 120 e 168 horas após a pulverização das mudas com os extratos. Além disso, fora avaliado também a capacidade dos extratos a quente e a frio a 0, 100, 500 e 1000 ppm em inibir a germinação dos esporos de A. brassicicola. As concentrações de 1000 ppm dos extratos a quente e a frio foram as mais eficientes no controle da doença. O extrato de A. blanchetti induziu aumento da atividade de peroxidase às 24 horas após a aplicação dos tratamentos e promoveu inibição da germinação dos esporos de A. brassicicola. Estes resultados indicam o potencial de A. blanchetti no controle de A. brassicicola, tanto pela ação direta quanto pela ativação de mecanismos de defesa.

Genome sequence of a novel endornavirus from the phytopathogenic fungus Alternaria brassicicola.

In an effort to discover new mycoviruses from phytopathogenic fungi, a dsRNA molecule of 10,290nt, resembling those associated with the viruses belonging to the family Endornaviridae, was isolated from Alternaria brassicicola, one of the causal agents of rapeseed black spot disease. Genome analysis revealed the presence of a single open reading frame coding for a polyprotein of 3400aa containing conserved viral methyltransferase (MTR), viral RNA helicase 1 (Hel-1), and RNA-dependent RNA polymerase (RdRp) domains. In addition, a cysteine-rich region (CRR) with conserved CXCC motifs, shared among several endornaviruses, was also identified between the MTR and Hel-1 domains. Phylogenetic analysis based on the RdRp sequence strongly suggested that the virus infecting A. brassicicola should be considered a representative of a novel endornavirus species, and this virus was designated as Alternaria brassicicola endornavirus 1 (AbEV1).

Involvement of the caleosin/peroxygenase RD20 in the control of cell death during Arabidopsis responses to pathogens

Caleosins, mostly found in lipid droplets of seeds and leaves, are believed to play physiological roles through their enzymatic capacities to produce oxylipins. We recently identified the caleosin RD20 as a peroxygenase reducing endogenous fatty acid hydroperoxides into their corresponding alcohols. Such oxylipins confer tolerance to oxidative stress by decreasing reactive oxygen species accumulation and by minimizing cell death. RD20 expression being induced by pathogens, we have examined the mode of action of this caleosin in response to biotic stress. Plants overexpressing RD20 exhibited an alteration of their leaf cuticle wax components and an increased resistance to the fungus Alternaria brassicicola. Conversely, silencing RD20 led to an enhanced propagation of the fungus and to reduced severity of the damages caused by the inoculation of the bacteria Pseudomonas syringae pv tomato. We discuss these findings and propose that the major function of RD20 is to generate oxylipins modulating oxidative status and cell death.

Folic acid induces salicylic acid-dependent immunity in Arabidopsis and enhances susceptibility to Alternaria brassicicola.

Folates are essential for one-carbon transfer reactions in all organisms and contribute, for example, to de novo DNA synthesis. Here, we detected the folate precursors 7,8-dihydropteroate (DHP) and 4-amino-4-deoxychorismate (ADC) in extracts from Arabidopsis thaliana plants by Fourier transform ion cyclotron resonance-mass spectrometry. The accumulation of DHP, but not ADC, was induced after infection of plants with Pseudomonas syringae delivering the effector protein AvrRpm1. Application of folic acid or the DHP precursor 7,8-dihydroneopterin (DHN) enhanced resistance in Arabidopsis to P. syringae and elevated the transcript accumulation of the salicylic acid (SA) marker gene pathogenesis-related1 in both the treated and systemic untreated leaves. DHN- and folic acid-induced systemic resistance was dependent on SA biosynthesis and signalling. Similar to SA, folic acid application locally enhanced Arabidopsis susceptibility to the necrotrophic fungus Alternaria brassicicola. Together, the data associate the folic acid pathway with innate immunity in Arabidopsis, simultaneously activating local and systemic SA-dependent resistance to P. syringae and suppressing local resistance to A. brassicicola.

Transcriptional responses of the Bdtf1-deletion mutant to the phytoalexin brassinin in the necrotrophic fungus Alternaria brassicicola.

Brassica species produce the antifungal indolyl compounds brassinin and its derivatives, during microbial infection. The fungal pathogen Alternaria brassicicola detoxifies brassinin and possibly its derivatives. This ability is an important property for the successful infection of brassicaceous plants. Previously, we identified a transcription factor, Bdtf1, essential for the detoxification of brassinin and full virulence. To discover genes that encode putative brassinin-digesting enzymes, we compared gene expression profiles between a mutant strain of the transcription factor and wild-type A. brassicicola under two different experimental conditions. A total of 170 and 388 genes were expressed at higher levels in the mutants than the wild type during the infection of host plants and saprophytic growth in the presence of brassinin, respectively. In contrast, 93 and 560 genes were expressed, respectively, at lower levels in the mutant than the wild type under the two conditions. Fifteen of these genes were expressed at lower levels in the mutant than in the wild type under both conditions. These genes were assumed to be important for the detoxification of brassinin and included Bdtf1 and 10 putative enzymes. This list of genes provides a resource for the discovery of enzyme-coding genes important in the chemical modification of brassinin.