Mycotoxin Profile and Phylogeny of Pathogenic Alternaria Species Isolated from Symptomatic Tomato Plants in Lebanon.

The tomato is one of the most consumed agri-food products in Lebanon. Several fungal pathogens, including Alternaria species, can infect tomato plants during the whole growing cycle. Alternaria infections cause severe production and economic losses in field and during storage. In addition, Alternaria species represent a serious toxicological risk since they are able to produce a wide range of mycotoxins, associated with different toxic activities on human and animal health. Several Alternaria species were detected on tomatoes, among which the most important are A. solani, A. alternata, and A. arborescens. A set of 49 Alternaria strains isolated from leaves and stems of diseased tomato plants were characterised by using a polyphasic approach. All strains were included in the recently defined phylogenetic Alternaria section and grouped in three well-separated sub-clades, namely A. alternata (24 out of 49), A. arborescens (12 out of 49), and A. mali morpho-species (12 out of 49). One strain showed high genetic similarity with an A.limoniasperae reference strain. Chemical analyses showed that most of the Alternaria strains, cultured on rice, were able to produce alternariol (AOH), alternariol methyl ether (AME), altenuene (ALT) and tenuazonic acid (TA), with values up to 5634, 16,006, 5156, and 4507 mg kg-1, respectively. In addition, 66% of the strains were able to co-produce simultaneously the four mycotoxins investigated. The pathogenicity test carried out on 10 Alternaria strains, representative of phylogenetic sub-clades, revealed that they were all pathogenic on tomato fruits. No significant difference among strains was observed, although A. alternata and A. arborescens strains were slightly more aggressive than A. mali morpho-species strains. This paper reports new insights on mycotoxin profiles, genetic variability, and pathogenicity of Alternaria species on tomatoes.

Antifungal Biphenyl Derivatives from Sorbus pohuashanensis Leaves Infected by Alternaria tenuissi and Their Effect against Crop Pathogens.

Eight natural biphenyl-type phytoalexins exhibiting antifungal effect were isolated from the leaves of Sorbus pohuashanensis, which invaded by Alternaria tenuissi, and their growth inhibition rate towards A. tenuissi were 50.3 %, 54.0 %, 66.4 %, 58.8 %, 48.5 %, 51.0 %, 33.3 %, and 37.0 %, respectively. In vivo activity assay verified the protective effect of these natural biphenyls on tobacco leaves. The observation of mycelial morphology revealed that these compounds possessed adverse effects on mycelial growth of A. tenuissi. Subsequently, the most potent active compounds, 3',4',5'-trimethoxy[1,1'-biphenyl]-4-ol (3) and 3,4,4',5-tetramethoxy-1,1'-biphenyl (4), were conducted to the further antifungal evaluation and showed significant activity against the other four crop pathogens, Fusarium graminearum, Helminthosporium maydis, Sclerotinia sclerotiorum, and Exserohilum turcicum. Further, the structure-activity relationships and biosynthesis of these compounds were speculated in this work.

Antimicrobial activity of the volatile compound 3,5-dichloro-4-methoxybenzaldehyde, produced by the mushroom Porostereum spadiceum, against plant-pathogenic bacteria and fungi.

AIMS: In this study, volatile compounds released from mycelia of some aromatic mushrooms were investigated for their inhibitory activity against plant-pathogenic bacteria and fungi. METHODS AND RESULTS: A screening revealed that volatile compounds from mycelia of Porostereum spadiceum remarkably inhibited the colony formation of plant-pathogenic bacteria, including Clavibacter michiganensis subsp. michiganensis and Ralstonia solanacearum while also inhibiting the conidial germination of plant-pathogenic fungi including Alternaria brassicicola and Colletotrichum orbiculare. The volatile compounds were isolated from the culture filtrate of P. spadiceum, and 3,4-dichloro-4-methoxybenzaldehyde (DCMB) was identified as a major compound. DCMB significantly inhibited bacterial colonization at 10 µg ml-1 and fungal conidial germination at 0·1-1 µg ml-1 as a vapour. CONCLUSIONS: This is the first report on the production of the volatile compound DCMB by P. spadiceum and on the antimicrobial activity of DCMB against plant-pathogenic bacteria and fungi at low concentrations. It may be possible to use the compound as an agent for protecting crops from bacterial and fungal diseases during cultivation and storage. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides an understanding of antimicrobial activity of the mushroom volatile compound that may be useful as a novel biological control agent for protecting various plant diseases.

Afectación intestinal por un hongo dematiáceo / Intestinal disease by a dematiaceous fungi

Resumen Introducción: Los hongos dematiáceos se caracterizan por la presencia de abundante melanina en su pared celular. Presentan una distribución mundial, siendo más comunes en climas tropicales y subtropicales. Producen infecciones cutáneas y subcutáneas, además de enfermedades alérgicas, neumonías, abscesos cerebrales o infecciones diseminadas. Caso Clínico: Presentamos el caso de un paciente con adenocarcinoma de recto intervenido quirúrgicamente con hallazgo incidental de divertículo de Meckel y en el cual en el estudio anatomopatológico reveló la presencia de un hongo dematiáceo

Introduction: Dematiaceous fungi are characterized by the presence of brown melanine or melanine like pigments in their cell wall. They are generally distributed worldwide, being more common in tropical and subtropical climates. The clinical syndromes are often cutaneous and subcutaneous infections, but can be also responsible of allergic diseases, pneumonias, cerebral abscesses or disseminated infections. Clinical Case: We present the case of a patient with a diagnosis of rectal adenocarcinoma intervening surgically and with an incidental finding of Meckel's Diverticulum. The anatomopathological study revealed the presence of a dematiaceous fungi.

Development and immunopathological characteristics of an Alternaria-induced chronic rhinosinusitis mouse model.

Airborne fungi are associated with upper and lower airway inflammatory diseases. Alternaria is commonly found in nasal secretions and induces the production of chemical mediators from sinonasal mucosa. This study aimed to establish an Alternaria-induced chronic rhinosinusitis (CRS) mouse model and determine the influence of host allergic background on the immunopathological characteristics of CRS. BALB/c mice were used for establishing the CRS model. Alternaria was intranasally instilled for 8 or 16 weeks with or without ovalbumin (OVA) presensitization. Total serum IgE and Alternaria-specific IgE levels were measured by enzyme-linked immunosorbent assay (ELISA). Interleukin (IL)-4, IL-10, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α levels in nasal lavage fluid (NLF) and splenocytes were measured by ELISA and their mRNAs and levels of associated transcription factors in sinonasal mucosa were determined with quantitative reverse-transcriptase polymerase chain reaction (RT-PCR). Hematoxylin-eosin staining and periodic acid-Schiff staining were performed to evaluate histological changes. Total serum IgE was increased in both allergic and non-allergic CRS. IL-4 was strongly expressed in NLF in both allergic and non-allergic CRS at 16 weeks and not only eosinophils but also neutrophils were increased in NLF of non-allergic CRS mice. The levels of Th1, Th2, and Treg cytokines and transcription factor mRNAs were significantly increased in sinonasal mucosa of non-allergic CRS mice. Both inflammatory cell infiltration and goblet cell hyperplasia were increased in CRS mice. Repeated intranasal instillation of Alternaria results in sinonasal inflammation with inflammatory cell infiltration. The sinonasal mucosal immune responses against Alternaria were shown to differ depending on the host allergic background.

Exogenous polyamines enhance resistance to Alternaria alternata by modulating redox homeostasis in apricot fruit.

The effects of exogenous polyamines treatment on reactive oxygen species (ROS) metabolism in apricot fruits were systematically analyzed through the investigation of their curative and preventive effects on black spot disease. Results showed that 1.5 mM spermine (Spm), 1.5 mM spermidine (Spd) and 10 mM putrescine (Put) treatment significantly inhibited black spot development, additionally, the efficacy of this control was dependent upon the type of polyamines used and concentration level applied. Further studies have shown that exogenous polyamines treatments significantly improved production of O2- and H2O2, and increased the activities and gene expression levels of NADPH oxidase (NOX), super oxide dismutase (SOD), catalase (CAT) ascorbate peroxidase (AXP) and glutathione reductase (GR) in apricot fruit. Increased ascorbic acid (AsA) and reduced glutathione (GSH) content were also observed after exogenous polyamines treatment. These results have revealed that postharvest polyamines treatment effectively enhanced disease resistance through the maintenance of homeostasis in apricot fruits.

An ERF2-like transcription factor regulates production of the defense sesquiterpene capsidiol upon Alternaria alternata infection.

Capsidiol is a sesquiterpenoid phytoalexin produced in Nicotiana and Capsicum species in response to pathogen attack. Whether capsidiol plays a defensive role and how its biosynthesis is regulated in the wild tobacco Nicotiana attenuata when the plant is attacked by Alternaria alternata (tobacco pathotype), a notorious necrotrophic fungus causing brown spot disease, are unknown. Transcriptome analysis indicated that a metabolic switch to sesquiterpene biosynthesis occurred in young leaves of N. attenuata after A. alternata inoculation: many genes leading to sesquiterpene production were strongly up-regulated, including the capsidiol biosynthetic genes 5-epi-aristolochene synthase (EAS) and 5-epi-aristolochene hydroxylase (EAH). Consistently, the level of capsidiol was increased dramatically in young leaves after fungal inoculation, from not detectable in mock control to 50.68±3.10 µg g-1 fresh leaf at 3 d post-inoculation. Capsidiol-reduced or capsidiol-depleted plants, which were generated by silencing EAHs or EASs by virus-induced gene silencing, were more susceptible to the fungus. In addition, this sesquiterpene when purified from infected plants exhibited strong anti-fungal activities against A. alternata in vitro. Furthermore, an ERF2-like transcription factor was found to positively regulate capsidiol production and plant resistance through the direct transactivation of a capsidiol biosynthetic gene, EAS12. Taken together, our results demonstrate that capsidiol, a phytoalexin highly accumulated in N. attenuata plants in response to A. alternata infection, plays an important role in pathogen resistance independent of jasmonate and ethylene signaling pathways, and its biosynthesis is transcriptionally regulated by an ERF2-like transcription factor.

Antifungal Effect of Magnolol and Honokiol from Magnolia officinalis on Alternaria alternata Causing Tobacco Brown Spot.

In this study, two phenol compounds, magnolol and honokiol, were extracted from Magnolia officinalis and identified by LC-MS, 1H- and 13C-NMR. The magnolol and honokiol were shown to be effective against seven pathogenic fungi, including Alternaria alternata (Fr.) Keissl, Penicillium expansum (Link) Thom, Alternaria dauci f.sp. solani, Fusarium moniliforme J. Sheld, Fusarium oxysporum Schltdl., Valsa mali Miyabe & G. Yamada, and Rhizoctonia solani J.G. Kühn, with growth inhibition of more than 57%. We also investigated the mechanisms underlying the potential antifungal activity of magnolol and honokiol. The results showed that they inhibited the growth of A. alternata in a dose-dependent manner. Moreover, magnolol and honokiol treatment resulted in distorted mycelia and increased the cell membrane permeability of A. alternata, as determined by conductivity measurements. These results suggest that magnolol and honokiol are potential antifungal agents for application against plant fungal diseases.

The methionine biosynthesis regulator AaMetR contributes to oxidative stress tolerance and virulence in Alternaria alternata.

The tangerine pathotype of A. alternata, which produces a unique host-selective ACT toxin causes brown spots on citrus leaves and fruits. In this study, we report a methionine biosynthesis regulator (MetR), which belong to bZIP transcription factor, is required for methionine metabolism, oxidative stress tolerance and pathogenicity. We generated two ΔAaMetR mutants in the tangerine pathotype of Alternaria alternata and investigated the resulting mutant phenotypes. The ΔAaMetR disruption mutant grew poorly in the absence of methionine and unable to produce conidia. Furthermore, pathogenicity tests have shown that ΔAaMetR mutant on their tangerine host can neither penetrate nor cause disease. These ΔAaMetR mutants exhibit an increased sensitivity to exogenous H2O2 and many ROS generating oxidants. To elucidate the transcription network of AaMetR, we performed RNA-Seq experiments on wild-type and ΔAaMetR mutant and identified genes that were differentially expressed between the two genotypes. Transcriptome data demonstrated that AaMetR contributes in many other biological processes including ROS detoxification, sulfur transfer, and amino acid metabolism. Comparative transcriptome analysis indicated that the ΔAaMetR mutant up-regulated several genes involved in cysteine and methionine metabolism. In conclusion, our results highlight the global regulatory role of AaMetR in cysteine and methionine metabolism and provide new insights into the crucial role of ROS detoxification, sporulation and pathogenicity in the tangerine pathotype of A. alternata.

Pathogenicity and transmission of fungi detected on Passiflora alata seeds / Patogenicidade e transmissão de fungos detectados em sementes de Passiflora alata

Passion fruit is usually propagated by seeds because of the ease and lower cost in seedling production. However, the seed is the most efficient agent for the spread of pathogens. The damages from seed-borne diseases occur mainly during the germination stages or at the formation of seedlings in nurseries. Considering the need for knowledge on the pathology of sweet passion fruit seeds, the objective was to evaluate the transmission and pathogenicity of the fungi Alternaria sp., Botrytis fabae, Cladosporium cladosporioides, Fusarium spp. and Lasiodiplodia theobromae, known as potentially pathogenic to this crop, and isolated from sweet passion fruit seeds. Therefore, tests on seed health, germination and seedling emergence in a sterilized commercial substrate were conducted using seeds from this species, inoculated with those fungal isolates. Leaves, stems and fruit from this plant were also inoculated with the same fungi. Alternaria sp., Fusarium spp. and L. theobromae were identified in seedlings obtained from inoculated seeds, confirming the transmission of these fungi by seeds. L. theobromae was also considered the most harmful fungus to passion fruit crop, as it causes seed rot and other disease symptoms on the leaves, stem and fruit. These findings inferred that healthy seeds of sweet passion fruit are essential for producing seedlings and to prevent the spread of the diseases caused by these fungi to exempt areas.(AU)

O maracujazeiro geralmente é propagado por meio de sementes em virtude da facilidade e do menor custo na produção de mudas. No entanto, a semente é o agente mais eficiente de disseminação de patógenos, sendo que os danos decorrentes das doenças transmitidas por elas ocorrem principalmente durante os estágios de germinação ou na formação de mudas nos viveiros. Considerando a necessidade de informações acerca da patologia de sementes de maracujá-doce nesse contexto, objetivou-se obter informações sobre a transmissão e a patogenicidade dos fungos Alternaria sp., Botrytis fabae, Cladosporium cladosporioides, Fusarium spp. e Lasiodiplodia theobromae, isolados de sementes de maracujá-doce e potencialmente patogênicos à cultura. Para tanto, testes de sanidade, germinação e emergência de plântulas em substrato comercial esterilizado foram conduzidos com sementes dessa espécie, inoculadas com esses isolados. Folhas, colo e frutos dessa planta também foram inoculados com os mesmos fungos. Alternaria sp., Fusarium spp. e L. theobromae foram identificados em plântulas obtidas de sementes inoculadas, confirmando a transmissão por sementes. L. theobromae foi considerado o mais agressivo à cultura do maracujá, por ter causado podridão nas sementes, além de maiores lesões nas folhas, no colo da planta e nos frutos. Dessa forma, infere-se que a obtenção de sementes de maracujá-doce sadias é imprescindível para a produção de mudas, evitando-se assim a disseminação desses patógenos em áreas isentas.(AU)

Seeds of weeds as an alternative host of phytopathogens / Sementes de plantas daninhas como hospedeiras alternativas de fitopatógenos

Weeds compete with agricultural crops for water, light, nutrients and space, besides having an extensive seed bank. However, another aspect to be considered relates to few studies pointing out weeds as hosts of phytopathogenic fungi. Many fungi, the main cause of diseases in plants, are known to use seeds as an efficient means of survival and dispersal. The objective of this work was to evaluate the health of weed seeds and the pathogenicity of fungi associated to plants of agricultural importance. The seeds were collected manually in Cerrado areas located in the municipality of Gurupi, Tocatins, Brazil. The blotter test method was used to evaluate seed health. The incidence of fungi was evaluated through an individual analysis of seeds using a stereoscopic and an optical microscope. The pathogenicity of fungi from weed seeds was evaluated by inoculation in plants of agronomic interest and, when pathogenic, we inoculated them in the host plant of the fungus. Weed seeds have been identified in fungi of the genus Alternaria, Aspergillus, Bipolaris, Cladosporium, Curvularia, Fusarium, Nigrospora, Papularia, Rhizopus and Pythium. The seeds of Acanthospermum australe, Bidens pilosa, Cenchrus echinatus, Digitaria horizontalis, Echinochloa crus-pavonis, Eleusine indica, Ipomoea sp., Pennisetum setosum, Sida rhombifolia, Spermacoce latifolia, Tridax procumbens and Vernonia polyanthes carry and disseminate fungi that, once inoculated, cause infection in plants of agricultural importance, such as Oryza sativa, Phaseolus vulgaris, Vigna unguiculata, Zea mays and Glycine max.(AU)

As plantas daninhas competem com culturas agrícolas por água, luz, nutrientes e espaço, além de possuírem um extenso banco de sementes. Entretanto, outra vertente a ser considerada é quanto aos poucos estudos relacionando plantas daninhas como hospedeiras de fungos fitopatogênicos. É sabido que muitos fungos, principais causadores de doenças em plantas, utilizam as sementes como meio eficiente de sobrevivência e de dispersão. Dessa forma, o trabalho objetivou avaliar a sanidade de sementes de plantas daninhas e a patogenicidade dos fungos associados às plantas de importância agrícola. As sementes foram coletadas manualmente em áreas de cerrado localizadas no município de Gurupi, Tocantins, utilizando o método blotter test para avaliação da sanidade. A incidência dos fungos foi avaliada com auxílio de microscópio estereoscópico e ótico. A patogenicidade dos fungos oriundos das sementes de plantas daninhas foi avaliada por meio da inoculação em plantas de interesse agronômico e, quando patogênico, a inoculação foi na própria planta daninha hospedeira do fungo. Foram identificados os fungos dos gêneros Alternaria, Aspergillus, Bipolaris, Cladosporium, Curvularia, Fusarium, Nigrospora, Papularia, Rhizopus e Pythium. As sementes de Acanthospermum australe, Bidens pilosa, Cenchrus echinatus, Digitaria horizontalis, Echinochloa crus-pavonis, Eleusine indica, Ipomoea sp., Pennisetum setosum, Sida rhombifolia, Spermacoce latifolia, Tridax procumbens e Vernonia polyanthes transportam e disseminam fungos que, uma vez inoculados, causam infecção em plantas de importância agrícola, como Oryza sativa, Phaseolus vulgaris, Vigna unguiculata, Zea mays e Glycine max.(AU)

Transcriptomic profiling of Alternaria longipes invasion in tobacco reveals pathogenesis regulated by AlHK1, a group III histidine kinase.

Tobacco brown spot, caused by Alternaria species, is a devastating tobacco disease. To explore the role of a group III histidine kinase (AlHK1) on A. longipes pathogenesis, the invasion progress of A. longipes was monitored. We found that the wild-type strain C-00 invaded faster than the AlHK1-disrupted strain HK∆4 in the early and middle infection stages and the reverse trend occurred in the late infection stage. Then, eight invasion transcriptomes were performed using RNA-Seq and 205 shared, 505 C-00 and 222 HK∆4 specific differentially expressed genes (DEGs) were identified. The annotation results showed seven antioxidant activity genes were specifically identified in the HKΔ4 DEGs. A subsequent experiment confirmed that HKΔ4 was more resistant to low concentrations oxidative stress than C-00. In addition, the results from 1) statistics for the number of DEGs, GO enriched terms, DEGs in clusters with rising trends, and 2) analyses of the expression patterns of some DEGs relevant for osmoadaptation and virulence showed that changes in C-00 infection existed mainly in the early and middle stages, while HKΔ4 infection arose mainly in the late stage. Our results reveal firstly the pathogenesis of A. longipes regulated by AlHK1 and provide useful insights into the fungal-plant interactions.

Cutaneous phaeohyphomycosis in a hematopoietic stem cell transplant patient caused by Alternaria rosae: First case report.

Alternaria species have been reported as a rare cause of fungal infection in organ and stem cell transplant recipients, but to date, no reports have been published of infection in humans caused by Alternaria rosae. Here, we report cutaneous A. rosae infection in a 66-year-old farmer with a history of primary myelofibrosis who had undergone allogeneic unrelated donor hematopoietic stem cell transplantation. Forty-nine days post transplant, he presented with a nodule on the thumb with no findings suggestive of disseminated infection. Pathology, culture, and molecular speciation showed the nodule was caused by cutaneous A. rosae. He had been on voriconazole as antifungal prophylaxis, but was found to have a subtherapeutic voriconazole level. He was switched to posaconazole based on published in vitro data showing its superior efficacy in Alternaria treatment. Susceptibility testing showed that the A. rosae isolate was indeed susceptible to posaconazole. His cutaneous lesion remained stable, but he died from respiratory failure secondary to lobar pneumonia. At lung autopsy, A. rosae was not identified in the lungs. We believe this to be the first published report, to our knowledge, of A. rosae infection in humans.

Comparative Genomics of Pathogens Causing Brown Spot Disease of Tobacco: Alternaria longipes and Alternaria alternata.

The genus Alternaria is a group of infectious/contagious pathogenic fungi that not only invade a wide range of crops but also induce severe allergic reactions in a part of the human population. In this study, two strains Alternaria longipes cx1 and Alternaria alternata cx2 were isolated from different brown spot lesions on infected tobacco leaves. Their complete genomes were sequenced, de novo assembled, and comparatively analyzed. Phylogenetic analysis revealed that A. longipes cx1 and A. alternata cx2 diverged 3.3 million years ago, indicating a recent event of speciation. Seventeen non-ribosomal peptide synthetase (NRPS) genes and 13 polyketide synthase (PKS) genes in A. longipes cx1 and 13 NRPS genes and 12 PKS genes in A. alternata cx2 were identified in these two strains. Some of these genes were predicted to participate in the synthesis of non-host specific toxins (non-HSTs), such as tenuazonic acid (TeA), alternariol (AOH) and alternariol monomethyl ether (AME). By comparative genome analysis, we uncovered that A. longipes cx1 had more genes putatively involved in pathogen-plant interaction, more carbohydrate-degrading enzymes and more secreted proteins than A. alternata cx2. In summary, our results demonstrate the genomic distinction between A. longipes cx1 and A. altenata cx2. They will not only improve the understanding of the phylogenetic relationship among genus Alternaria, but more importantly provide valuable genomic resources for the investigation of plant-pathogen interaction.

Alternaria extract activates autophagy that induces IL-18 release from airway epithelial cells.

Alternaria alternata is a major outdoor allergen that causes allergic airway diseases. Alternaria extract (ALT-E) has been shown to induce airway epithelial cells to release IL-18 and thereby initiate Th2-type responses. We investigated the underlying mechanisms involved in IL-18 release from ALT-E-stimulated airway epithelial cells. Normal human bronchial epithelial cells and A549 human lung adenocarcinoma cells were stimulated with ALT-E in the presence of different inhibitors of autophagy or caspases. IL-18 levels in culture supernatants were measured by ELISA. The numbers of autophagosomes, an LC3-I to LC3-II conversion, and p62 degradation were determined by immunofluorescence staining and immunoblotting. 3-methyladenine and bafilomycin, which inhibit the formation of preautophagosomal structures and autolysosomes, respectively, suppressed ALT-E-induced IL-18 release by cells, whereas caspase 1 and 8 inhibitors did not. ALT-E-stimulation increased autophagosome formation, LC-3 conversion, and p62 degradation in airway epithelial cells. LPS-stimulation induced the LC3 conversion in A549 cells, but did not induce IL-18 release or p62 degradation. Unlike LPS, ALT-E induced airway epithelial cells to release IL-18 via an autophagy dependent, caspase 1 and 8 independent pathway. Although autophagy has been shown to negatively regulate canonical inflammasome activity in TLR-stimulated macrophages, our data indicates that this process is an unconventional mechanism of IL-18 secretion by airway epithelial cells.

Involvement of an extracellular fungus laccase in the flavonoid metabolism in Citrus fruits inoculated with Alternaria alternata.

Fungi of the genus Alternaria are responsible for substantial pre-harvest losses in Citrus. In this study a degradative metabolism of flavonoids (flavanones, flavones and polymethoxyflavones) was observed when 'Fortune' mandarin, Citrus limon and Citrus paradisi, fruits were inoculated with Alternaria alternata, a pre-harvest pathogenic fungus. Associated to this flavonic metabolism the de novo synthesis of the phytoalexin scoparone was detected. This metabolism of flavonoids is caused by an extracellular fungus laccase. The kinetic characterisation of this enzyme revealed that the activity was induced by Citrus flavonoids and was dependent on flavonoid concentrations. The enzyme exhibited a Km of 1.9 mM using ABTS as substrate with an optimum pH of 3.5 in citrate buffer 100 mM. The enzyme is active between 15 and 45 °C, the optimum temperature being around 35 °C, although 50% of the initial activity is lost after 45 min at 35 °C. The A. alternata laccase was inhibited by 0.5 mM l-cysteine and by caffeic acid. Study of the substrate specificity of this enzyme revealed that Citrus flavonoids are substrates of A. alternata laccase. These results suggest that the laccase enzyme could be involved in the pathogenesis of A. alternata in Citrus.

[Inhibition of Bacillus pumilus AR03 on Alternaria alternata and Erysiphe cichoracearum on tobacco].

This study aimed to evaluate the efficiency of Bacillus pumilus AR03 against Altenaria alternata and Erysiphe cichoracearum. The antagonistic activities were studied in the way of co-culture on plate, inhibition of pathogen conidia germination on concave slides and LB agar medium. In the investigation, the water suspension of living cells of strain AR03, at 3 x 10(8) cfu . mL-1 had a remarkable inhibition effect on hyphae growth and conidia germination of A. alternata and caused hyphae deformation, shorter and swollen nodes, winding hyphae accumulation, abnormal tubes with tips expanded or deformed. Conidia did not germinate and the tissues of compartment became swollen or formed a round spherical bubble. In addition, the inhibition rate of conidia germination of E. cichoracearum was 91. 3% and 69. 3%, respectively when treated with strain AR03 at 1 x 10(7) cfu . mL-1 and 1.5 x 10(6) cfu . mL-1. Conidia treated by living cells of AR03 became swollen, deformed, the protoplasm of conidia shrinked, disintegrated gradually and separated from the conidia wall. And some conidia were hollow because the protoplasm leaked out from inside. Greenhouse results revealed that the effects of living cells of AR03 with different concentrations were significantly different. Bacterial suspension of AR03 at 3 x 10(8) cfu . mL-1 was strongly antagonistic to E. cichoracearum with the control efficiencies 7 days and 15 days after treatment of two spays being 83.8% and 90.3%, respectively, while the control efficiencies of AR03 at 3 x 10(6) cfu . mL-1 were 70.0% and 73.3%, respectively. Strain AR03 had a long persistence against powdery mildew more than 30 days.

Ultrastructural changes and oxidative stress markers in wild and cultivar Sesamum orientale L. following Alternaria sesami (Kawamura) Mohanty and Behera. inoculation.

Alternaria sesami causes leaf spot disease in Sesamum orientale. Conidium germination, inoculation, penetration and colonization of the pathogen on the plant surfaces were studied using scanning electron microscopy. Electron microscopy analysis revealed multiple germ tubes from conidium that spread in all direction across the leaf surfaces. Penetration in the plant surface occured, directly through the epidermis or via stomata with or without the appressoria formation. Hyphal penetration continued through the substomata cavity and some of hyphal branches grew in the intercellular space of mesophyll tissue. Hyphal toxin, caused cell and cell wall damages. Changes in different biochemical parameters in the diseased sesame plants (both in wild and cultivar) were compared to control. Transmission electron microscopy showed structural changes in the chloroplast of diseased plants. Isozyme pattern and assays of different enzymes, namely catalase, acid phosphatase and peroxidase expressed varied level of activities. Meanwhile, esterase, polyphenol oxidase and superoxide dismutase in diseased plants showed remarkable levels compared to control. Due to the infection, chlorophyll content, carbohydrates and total soluble protein decreased whereas free amino acid, proline, phenols and disease-related proteins increased in the host plants. Differential SDS-PAGE band profiling of total soluble proteins were also observed in plants due to the infection.

Potential role of Alternaria and Cladosporium species in canine lymphoplasmacytic rhinitis.

OBJECTIVES: To evaluate the possible role of Alternaria and Cladosporium species in the pathogenesis of canine lymphoplasmacytic rhinitis by comparing the amount of specific fungal DNA in nasal mucosal biopsies between dogs without nasal neoplasia and those with lymphoplasmacytic rhinitis or nasal neoplasia. METHODS: Quantitative real-time polymerase chain reaction (qPCR) assays detecting DNA from Alternaria and Cladosporium fungi were applied to nasal mucosal biopsies collected from dogs with lymphoplasmacytic rhinitis (n = 8), dogs with nasal neoplasia (n = 10) and control animals (n = 10). A copy number for each sample was calculated using a standard curve of known copy number and differences amongst groups were assessed using Kruskal-Wallis tests. RESULTS: No significant difference was found between the groups. Low levels of Alternaria DNA (10-100 copies/PCR) were detected in one sample; very low levels of DNA (<10 copies/qPCR) were detected in 6 samples, and 21 samples were negative. Low levels of Cladosporium DNA were detected in 2 samples; very low levels of DNA in 18; and 8 were negative. CLINICAL SIGNIFICANCE: Results of this study reveal that Alternaria and Cladosporium species are part of the canine nasal flora, and that these fungi are probably not involved in the pathogenesis of lymphoplasmacytic rhinitis.

Leaf proteome profiling of transgenic mint infected with Alternaria alternata.

The genus Mentha has been widely used in food, flavor, culinary, cosmetic and pharmaceutical industries. Substantial damage to this crop happened regularly due to environmental stresses like metal toxicity and pathogen attack. Here, an approach has been taken to raise transgenic mint over-expressing γ-glutamyl-cysteine synthetase (γ-ECS), the rate-limiting enzyme of GSH biosynthesis, resulted enhanced GSH content and its in planta expression confers significant tolerance towards abiotic/biotic stresses viz. metal toxicity - Cd, Zn as well as against infection of Alternaria alternata and Rhizoctonia solani. A differential proteomic analysis through 2-DE and MALDI TOF-TOF MSMS was performed to focus on the altered abundance of functionally important protein species in control and infected transgenic mint. Results showed a significant variation in the protein profile of the infected transgenic plant as compared to the wild/control transgenic counterpart. In addition to protein species related to stress and defense, redox regulation, transcription factors and energy & metabolism, protein species related to signaling and gene regulation as well as cell division also showed differential accumulation in infected transgenic. Hence, proteomics can be used as a tool to decipher the mechanism of action of GSH in providing tolerance against a necrotrophic fungus, A. alternata in transgenic mint. BIOLOGICAL SIGNIFICANCE: The reported work describes a comparative proteomics of non-model unsequenced plants like Mentha. There is a comparative protein profile between transgenic and its wild counterparts under control and infected condition. The work has an impact in crop proteomics and also tries to explain the application of proteomic approach to decipher the mechanism by which a foreign metabolite mediates stress tolerance in plant under control and infected condition. This article is part of a Special Issue entitled: Translational Plant Proteomics.