[Identification and biological characterization of pathogen causing black spot of Pseudostellaria heterophylla in Fujian province].

In Zherong county, Fujian province, the black spot of Pseudostellaria heterophylla often breaks out in the rainy season from April to June every year. As one of the main leaf diseases of P. heterophylla, black spot seriously affects the yield and quality of the medicinal material. To identify and characterize the pathogens causing black spot, we isolated the pathogens, identified them as a species of Alternaria according to Koch's postulates, and then tested their pathogenicity and biological characteristics. The results showed that the pathogens causing P. heterophylla black spot were A. gaisen, as evidenced by the similar colony morphology, spore characteristics, sporulation phenotype, and the same clade with A. gaisen on the phylogenetic tree(the maximum likelihood support rate of 100% and the Bayesian posterior probability of 1.00) built based on the tandem sequences of ITS, tef1, gapdh, endoPG, Alta1, OPA10-2, and KOG1077. The optimum conditions for mycelial growth of the pathogen were 25 ℃, pH 5-8, and 24 h dark culture. The lethal conditions for mycelia and spores were both treatment at 50 ℃ for 10 min. We reported for the first time the A. gaisen-caused black spot of P. heterophylla. The results could provide a theoretical basis for the diagnosis and control of P. heterophylla leaf spot diseases.

Histone H3 gene is not a suitable marker to distinguish Alternaria tenuissima from A. alternata affecting potato.

Potato Alternaria leaf blight is one of the economically most important disease in potato production worldwide. A recent study reported a quick method to distinguish main Alternaria pathogens A. tenuissima, A. alternata, and A. solani using partial histone H3 gene sequences. Using this method, our collection of 79 isolates from 8 provinces in China were presumably separated into A. tenussima and A. alternata. But in depth morphological and genetic analysis casted doubt on this identification. Culture morphologies of six presumed A. alternata isolates (PresA_alt) and six presumed A. tenuissima isolates (PresA_ten) were not significantly different. PresA_ten isolates also produced conidia in branched chains which supposed to be A. aternata. Phylogenetic analyses were conducted using internal transcribed spacer region (ITS) and five genes commonly used for species identification including glyceraldehyde-3-phosphate dehydrogenase (GPDH), translation elongation factor 1-alpha (TEF1), ß-tubulin, plasma membrane ATPase (ATPase), and calmodulin genes. The results showed that GPDH and TEF1 sequences of PresA_alt and PresA_ten isolates were identical. The 12 isolates did not cluster by presumed species neither by individual or concatenated sequence comparisons. The phylogeny-trait association analysis confirmed that the two group isolates were undistinguishable by those molecular markers. Analysis of histone H3 gene sequences revealed variable intron sequences between PresA_ten and PresA_alt isolates, but the amino acid sequences were identical. Our results indicate that the previously published method to distinguish Alternaria species based on histone H3 gene sequence variation is inaccurate and that the prevalence of A. tenuissima isolates in China was likely overestimated.

Computational identification and antifungal bioassay reveals phytosterols as potential inhibitor of Alternaria arborescens.

Alternaria arborescens is a major pathogen for crops like tomato, tangerine and so on and its control is mostly dependent on the application of chemical agents. Plants as the sources of natural products are very attractive option for developing eco-friendly and natural antifungal agents. In this study, we modeled three-dimensional structure of chorismate synthase (CS) enzyme from A. arborescens. Docking studies of phytosterols, namely, γ-sitosterol and ß-sitosterol, with CS showed them to be potential inhibitor of CS. To explore the stability and conformational flexibility of all the AaCS complex systems, molecular dynamics simulations were performed. None of the putative inhibitors as well as ß- and γ-sitosterol showed interaction with the FMNH2 binding pocket of the tomato CS (major host of A. arborescens) indicating their suitability as antifungal compounds inhibiting the shikimate pathway without causing any harm to the host. An in vivo antifungal bioassay showed a significant reduction in fungal growth in the presence of ß-sitosterol (500 ppm) which resulted in ∼23% and ∼17% reduction in fungal fresh and dry weight, respectively, at 8 days after inoculation. This study provides experimental evidence establishing natural sterols like ß-sitosterol can be useful in curbing A. arborescens damage in an eco-friendly manner.Communicated by Ramaswamy H. Sarma.

Effects of ultraviolet-c treatment on growth and mycotoxin production by Alternaria strains isolated from tomato fruits.

Large amounts of tomato fruits and derived products are produced in China and may be contaminated by Alternaria mycotoxins, which may have the potential risks for human health. There is thus an increasing interest in reducing the mycotoxins. In the present study, 26 Alternaria strains isolated from tomato black rots were identified according to morphological and molecular grounds, and their mycotoxigenic abilities for alternariol (AOH), alternariol monomethyl-ether (AME) and tenuazonic acid (TeA) were also investigated. The results showed that A. alternate was the predominant species with incidence values of 65.4% (17/26), followed by A. brassicae (7/26) and A. tenuissima (2/26). A. alternate isolates showed the highest capacity for AOH, AME and TeA production among the studied isolates either in vitro or in vivo, suggested that A. alternata may be the most important mycotoxin-producing species in tomato fruits. Thus, UV-C irradiation was used to reduce the mycotoxin produced by A. alternata in our study. The results showed that low dose of UV-C irradiation (0.25 kJ/m2) could effectively inhibit mycotoxins production and penetration in tomatoes. Upon treatment with UV-C, there was 79.6, 76.4 and 51.4% of reduction in AOH, AME and TeA penetration when compared to untreated fruits. This may be associated with the enhanced phenolics by UV-C irradiation. In fact, the induced phenolics were including p-coumaric, ferulic and pyrocatechuic acids, of which p-coumaric acid (1.0 mM) displayed the highest reduction of TeA with 60.2%, whereas ferulic acid (1.0 mM) showed strong inhibitory effects on the AOH and AME production by 59.4 and 79.1%, respectively. Therefore, the application of UV-C irradiation seems to be a promising method for reducing the potential risk of Alternaria mycotoxins in fruits and also for enhancing phenolics of processing products.

Isolation of taxol producing endophytic fungus Alternaria brassicicola from non-Taxus medicinal plant Terminalia arjuna.

In the present study, an endophytic fungal strain was isolated from its non-Taxus host plant Terminalia arjuna and identified as Alternaria brassicicola based on its morphological characteristics and internal transcribed spacer sequence analysis. This fungus was grown in potato dextrose broth and analyzed for the presence of taxol by using chromatographic and spectrometric techniques. The ethyl acetate extract of A.brassicicola was subjected to column chromatography. Among the different fractions, the fraction 7 showed positive to taxol, which was further confirmed by UV absorption, HPLC, FTIR spectra and LC-ESI-MS by comparing with the authentic taxol (Paclitaxel). The peaks of fraction 7 obtained by UV spectroscopy, FTIR and HPLC analysis were quite similar to that of standard taxol confirming the presence of taxol. A parent ion peak of m/z 854.95 was observed in the LC-ESI-MS spectrum which was similar to paclitaxel with reported m/z of 854 [M+H]+ ion. A. brassicicola produced about 140.8 µg/l taxol as quantified through HPLC. Present study results suggest that the endophytic fungus A.brassicicola serves as a potential source for the production of taxol isolated from non-Taxus plant.

Changes of volatile organic compounds and bioactivity of Alternaria brassicae GL07 in different ages.

Plant endophytes are rich in secondary metabolites and are widely used in medicine, chemical, food, agriculture, and other fields. Here, an endophytic fungus is isolated from Ginkgo biloba L. leaves and identified as Alternaria brassicae GL07 through genotypic characterizations. It can produce fruity scented volatiles. The analysis of volatile organic compounds (VOCs) was done by gas chromatography-mass spectrometry. A total of 32 components were identified; and at different culture times, the composition of VOCs was different. It had more components in the first two weeks, but a fewer components on the 21st day. More olefins, ketone, aldehyde, and alcohol were found in the growth period and more amines and esters were found in the decline period. Also, 2,5-dihydroxyacetophenone, ß-ionone, and nonanal were found. They were the same ingredients in Ginkgo essential oils and some of them were isolated for the first time in the volatile constituents of endophytes. The antioxidant activity and whitening activities of all extracts were also evaluated. When cultured for 10 days, it had the strongest 2,2-diphenyl-2-picrylhydrazyl radical (IC50 , 0.56 g/L), hydroxyl radical scavenging ability (IC50 , 0.47 g/L), reducing ability, and tyrosinase inhibition ability (IC50 , 5.18 g/L), which may be due to a large amount of ketones and alcohols produced during the log phase. This demonstrates the potential of A. brassicae GL07 to produce bioactive compounds and to be used for perfume and cosmetic industries.

Secondary metabolite profiles of small-spored Alternaria support the new phylogenetic organization of the genus.

The group of the small-spored Alternaria species is particularly relevant in foods due to its high frequency and wide distribution in different crops. These species are responsible for the accumulation of mycotoxins and bioactive secondary metabolites in food. The taxonomy of the genus has been recently revised with particular attention on them; several morphospecies within this group cannot be segregated by phylogenetic methods, and the most recent classifications proposed to elevate several phylogenetic species-groups to the taxonomic status of section. The purpose of the present study was to compare the new taxonomic revisions in Alternaria with secondary metabolite profiles with special focus on sections Alternaria and Infectoriae and food safety. A total of 360 small-spored Alternaria isolates from Argentinean food crops (tomato fruit, pepper fruit, blueberry, apple, wheat grain, walnut, pear, and plum) was morphologically identified to species-group according to Simmons (2007), and their secondary metabolite profile was determined. The isolates belonged to A. infectoria sp.-grp. (19), A. tenuissima sp.-grp. (262), A. arborescens sp.-grp. (40), and A. alternata sp.-grp. (7); 32 isolates, presenting characteristics overlapping between the last three groups, were classified as Alternaria sp. A high chemical diversity was observed; 78 different metabolites were detected, 31 of them of known chemical structure. The isolates from A. infectoria sp.-grp. (=Alternaria section Infectoriae) presented a specific secondary metabolite profile, different from the other species-groups. Infectopyrones, novae-zelandins and phomapyrones were the most frequent metabolites produced by section Infectoriae. Altertoxin-I and alterperylenol were the only compounds that these isolates produced in common with members of section Alternaria. None of the well-known Alternaria toxins, considered relevant in foods, namely alternariol (AOH), alternariol monomethyl ether (AME), tenuazonic acid (TeA), tentoxin (TEN) or altenuene (ALT), were produced by isolates of this section. On the other hand, strains from section Alternaria (A. tenuissima, A. arborescens, and A. alternata sp.-grps.) shared a common metabolite profile, indistinguishable from each other. AOH, AME, ALT, TEN, and TeA were the most frequently mycotoxins produced, together with pyrenochaetic acid A and altechromone A. Alternaria section Alternaria represents a substantial risk in food, since their members in all types of crops are able to produce the toxic metabolites.

Development of a Semi-nested PCR-Based Method for Specific and Rapid Detection of Alternaria solani Causing Potato Early Blight in Soil.

Early blight, caused by Alternaria solani, is one of the most devastating diseases of potato that causes severe yield loss worldwide. The infected potato debris existed in the soil serve as the initial infection sources for the next growing potato. Current identification of A. solani in soil relies primarily on cultural and morphological characteristics, which are time-consuming and inaccurate. In this study, a semi-nested PCR method was developed using primers based on internal transcribed spacer region that is specific to A. solani. 20 isolates including 6 Alternaria species and 10 other species of common potato pathogens were used to examine the specificity of the primers. The primer set ptAsQ-F/ptAs-R was highly specific to A. solani, as a product of 251 bp was amplified only from A. solani isolates and no amplification signal was observed from other tested species. The sensitivity of this method determined using A. solani genomic DNA was 10 fg. This PCR assay was also successfully employed to detect A. solani in soil with the detection sensitivity of one conidia spore in 0.5 g of soil. To the best of our knowledge, this is the first report of molecular detection of A. solani in soil, which provides a useful tool for early and rapid detection of early blight in soil before next growing season.

Identification of A. arborescens, A. grandis, and A. protenta as new members of the European Alternaria population on potato.

Alternaria species, primarily the small-spored Alternaria alternata and the large-spored Alternaria solani, are considered a serious threat to potato cultivation. To develop control strategies, it is important to gain insight into the Alternaria population. Based on the sequence analyses of the internal transcribed spacer region (ITS) and the glyceraldehyde-3-phosphate dehydrogenase gene, the small-spored and large-spored Alternaria isolates could be separated from each other. Sequence analyses of the calmodulin gene and the RNA polymerase second largest subunit showed that besides A. solani also A. grandis and A. protenta were present in the large-spored Alternaria population. Sequence analyses of the Alternaria major allergen gene Alt a 1 and the elongation factor-α revealed that both A. alternata and species belonging to the Alternaria arborescens species complex were present in the small-spored Alternaria population. Furthermore, according to the histone h3 sequence the members of the A. arborescens species complex could be subdivided into two groups. Concerning the fitness, it was concluded that the mycelium growth rate of the large-spored isolates was significantly lower compared to the growth rate of the small-spored isolates. In contrast, the spore-germinating capacity and early growth of the large-spored isolates was greater compared to those of the small-spored isolates. Within the groups of small-spored and large-spored isolates there were no significant differences in fitness between the species.

Biochemical evaluation of resistance responses of potato to different isolates of Alternaria solani.

The resistance phenotypes of nine potato cultivars to five isolates of Alternaria solani, causal agent of early blight, were studied after inoculation and growth under greenhouse conditions. We identified potato cultivars with both susceptible and resistant phenotypes as well as A. solani isolates with varying degrees of aggressiveness. Two potato cultivars and two pathogen isolates were selected for biochemical analysis of phenol production and peroxidase activity after inoculation. Phenol compounds were evaluated 2, 4, 6, and 8 days after inoculation, while peroxidase activities were monitored daily for 10 days. Native polyacrylamide electrophoresis was used to identify one protein with peroxidase activity in extracts taken 6 days after inoculation. Significantly higher peroxidase activity as well as total phenol content in potato was correlated with resistance in the Iranian potato cultivar Diamond. Variability of responses within the same cultivar to different isolates of A. solani suggests genotypic diversity between isolates that results in phenotypic diversity for pathogen aggressiveness.

Alternaria brasiliensis sp. nov., a leaf pathogen on Phaseolus vulgaris.

Alternaria brasiliensis sp. nov., was isolated from leaves of common bean, Phaseolus vulgaris L., showing punctiform non concentric leaf spots of brown color. Besides the symptomatological differences, the new Alternaria species presents distinct type of arrangement of conidia chain, body and beak size. The disease was observed in Montanha county, State of Espirito Santo, Brazil, but not yet reported in the literature. The causal agent of the disease is now described by the first time. The epithet used here is referring to the country where the species has been found.