Studies on the seed-borne phases of dark leaf spot Alternaria brassicicola and grey leaf spot Alternaria brassicaeof brassicas

Tests in Britain on samples of basic and commercial Brassica oleracea seed between 1976 and 1978 showed that many lots were infected with Alternaria brassicicola. A. brassicae was uncommon in basic seed in these years and in commercial seed harvested in 1976 and 1977 but was frequent in seed harvested in 1978. Most affected seeds were contaminated by surface-borne spores and mycelium of A. brassicicola but many were internally infected by the fungus situated within the seed-coat and in some seeds in the embryo tissues. Superficial contamination by the fungus declined rapidly after 2 yr in cabbage seeds stored at 10 °C, 50% r.h. but internal infection persisted for up to 12 yr. In some samples, internal infection was commonly associated with small shrivelled seeds. Surface contaminated and internally infected seeds transmitted the disease but seedling infection was more closely correlated with the latter.     

Isolation of 12 polymorphic microsatellite loci in the phytopathogenic fungus Alternaria brassicicola

Twelve polymorphic microsatellite markers were isolated from the phytopathogenic fungus Alternaria brassicicola, the causal agent of black spot of crucifers. An enrichment protocol was used to isolate microsatellite loci, which were then analysed in a collection of 46 isolates sampled from seven different countries. The number of alleles detected in 12 loci ranged from two to 10 (mean 3.5). Investigation of cross‐species amplifications showed that the designed primers were specific to A. brassicicola.     

In silico analysis of RNA interference components and miRNAs-like RNAs in the seed-borne necrotrophic fungus Alternaria brassicicola

RNA interference is a mechanism of suppressing gene expression in plants, animals and fungi. This regulation mechanism involves three main enzymes, Dicers (Dcr), Argonautes (Ago) and RNA Dependent RNA Polymerases (Rdrp) allowing to produce smallRNAs. RNA interference and smallRNAs have a role in the plant–microorganisms interaction, either in a pathogenic or in a symbiotic relationships. Alternaria brassicicola is a pathogenic fungus of the Brassicaceae plants. During plant infection, it is able to transmit itself vertically and horizontally, giving advantages for new infection and dissemination. To investigate RNA interference and the presence of smallRNAs in A. brassicicola, an in silico analysis was achieved. Two DCR, 4 AGO and 3 RDRP genes were identified comforting the presence of smallRNAs in A. brassicicola. SmallRNA sequencing from wild-type strain and DCR deleted mutants allowed the identifcation of 17 miRNAs in A. brassicicola. The synthesis of these miRNAs is only weakly influenced by the inactivation of DCR genes suggesting the possible existence of an alternative Dicer-independent miRNA synthesis pathway. Target's prediction of A. brassicicola miRNAs identified genes in the fungus and in the plant model Arabidopsis thaliana. Some miRNAs were predicted to target A. thaliana genes involved in the methylation of histone and in the disease resistance.     

Alternaria on cruciferous plants. 4.Alternaria species on seed of some cruciferous crops and their pathogenicity

The incidence ofAlternaria spp. on seed samples of cruciferous vegetable crops was surveyed between 1990 and 1992. Some commercial seed lots of crucifers which are commonly grown in Japan were infested withAlternaria species. ThreeAlternaria species were encountered on the seed samples ofBrassica campestris, B. orelacea, andRaphanus sativus. The most frequently detected species wereA. japonica andA. alternata onB. campestris, A. brassicicola onB. oleracea, andA. japonica andA. alternata onR. sativus, respectively.Alternaria brassicae was not detected in this study.Alternaria brassicicola isolates from these crops produced necrotic lesions on all of the crucifer seedlings inoculated, whileA. japonica induced different reactions in different plants or plant parts depending on isolates used in inoculation tests. In contrast, most isolates ofA. alternata could not produce necrotic lesions on foliage leaves of crucifers inoculated, although some of them produced clear lesions only on cotyledons.Alternaria alternata associated with these cruciferous crop seeds was considered to be an oppotunistic parasite of these crops.     

Area under disease progress curve and apparent infection rate of Alternaria blight disease of Indian mustard (Brassica juncea) at different plant age

Plant age has a major influence on the incidence of Alternaria blight disease in Indian mustard crops. Disease progression was monitored twice a week on the two chosen Indian mustard cultivars viz., Varuna and Rohini throughout the season. Severity of blight caused by Alternaria brassicae and Alternaria brassicicola decreased with delay in sowing. Calculation for A-value (Area under disease progress curve – AUDPC) and r-value (apparent infection rate) in crops sown on different dates could identify the speed of progress in the disease on leaves and pods, as the crop does not posses resistance to the pathogen till date. Thus, the probable dates of sowing enabling slow disease progress or the weather conditions coinciding with the different crop phenological stages demarcated the advantageous dates of sowing from the disadvantageous ones. However, cultivar Varuna is more susceptible as compared to the other cultivar Rohini, as apparent infection rate both on leaves and pods was higher in former. Highest per cent disease severity (PDS) for season highly correlated with date of sowing, i.e. delayed date of sowing increased PDS.     

Survival and perpetuation of Alternaria brassicae causing Alternaria blight of oilseed crucifers

Modes of survival and perpetuation of Alternaria brassicae attacking rapeseed and mustard were examined. The pathogen remained viable in diseased plant debris and seeds of infected plants which served as primary sources of inoculum. The pathogen was internally seed-borne and infected seeds resulted in damping-off of seedlings. Air-borne conidia produced on aerial parts of the infected plants were responsible for secondary spread of the disease.     

Detection of Genetic Variation in Alternaria brassicae by RAPD Fingerprints

Genetic variation in fourteen isolates of Alternaria brassicae collected from different geographical regions of the world was determined by RAPD (random amplified polymorphic DNA) analysis. Twenty random primers were tried to amplify genomic DNA of A. brassicae. Based on the PCR (polymerase chain reaction) amplification of genomic DNA of A. brassicae with four oligonucleotide random primers, fingerprints were generated for each isolate and the amplifed products were compared. Using this technique, intra- and intercontinental genetic variation among isolates of A. brassicae could be distinguished.     

TmpL,a Transmembrane Protein Required for Intracellular Redox Homeostasis and Virulence in a Plant and an Animal Fungal Pathogen

The regulation of intracellular levels of reactive oxygen species (ROS) is critical for developmental differentiation and virulence of many pathogenic fungi. In this report we demonstrate that a novel transmembrane protein, TmpL, is necessary for regulation of intracellular ROS levels and tolerance to external ROS, and is required for infection of plants by the necrotroph Alternaria brassicicola and for infection of mammals by the human pathogen Aspergillus fumigatus. In both fungi, tmpL encodes a predicted hybrid membrane protein containing an AMP-binding domain, six putative transmembrane domains, and an experimentally-validated FAD/NAD(P)-binding domain. Localization and gene expression analyses in A. brassicicola indicated that TmpL is associated with the Woronin body, a specialized peroxisome, and strongly expressed during conidiation and initial invasive growth in planta. A. brassicicola and A. fumigatus ΔtmpL strains exhibited abnormal conidiogenesis, accelerated aging, enhanced oxidative burst during conidiation, and hypersensitivity to oxidative stress when compared to wild-type or reconstituted strains. Moreover, A. brassicicola ΔtmpL strains, although capable of initial penetration, exhibited dramatically reduced invasive growth on Brassicas and Arabidopsis. Similarly, an A. fumigatus ΔtmpL mutant was dramatically less virulent than the wild-type and reconstituted strains in a murine model of invasive aspergillosis. Constitutive expression of the A. brassicicola yap1 ortholog in an A. brassicicola ΔtmpL strain resulted in high expression levels of genes associated with oxidative stress tolerance. Overexpression of yap1 in the ΔtmpL background complemented the majority of observed developmental phenotypic changes and partially restored virulence on plants. Yap1-GFP fusion strains utilizing the native yap1 promoter exhibited constitutive nuclear localization in the A. brassicicola ΔtmpL background. Collectively, we have discovered a novel protein involved in the virulence of both plant and animal fungal pathogens. Our results strongly suggest that dysregulation of oxidative stress homeostasis in the absence of TmpL is the underpinning cause of the developmental and virulence defects observed in these studies.     

Expression of an endochitinase gene from <Emphasis Type="Italic">Trichoderma virens</Emphasis> confers enhanced tolerance to <Emphasis Type="Italic">Alternaria</Emphasis> blight in transgenic <Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) czern and coss lines

An endochitinase gene ‘ech42’ from the biocontrol fungus ‘Trichoderma virens’ was introduced to Brassica juncea (L). Czern and Coss via Agrobaterium tumefaciens mediated genetic transformation method. Integration and expression of the ‘ech42’ gene in transgenic lines were confirmed by PCR, RT-PCR and Southern hybridization. Transgenic lines (T₁) showed expected 3:1 Mendelian segregation ratio when segregation analysis for inheritance of transgene ‘hpt’ was carried out. Fluorimetric analysis of transgenic lines (T₀ and T₁) showed 7 fold higher endochitinase activity than the non-transformed plant. Fluorimetric zymogram showed presence of endochitinase (42 kDa) in crude protein extract of transgenic lines. In detached leaf bioassay with fungi Alternaria brassicae and Alternaria brassicicola, transgenic lines (T₀ and T₁) showed delayed onset of lesions as well as 30–73 % reduction in infected leaf area compared to non-transformed plant.     

Development of a SCAR marker for molecular detection and diagnosis of Tilletia controversa Kühn,the causal fungus of wheat dwarf bunt

Tilletia controversa Kühn (TCK) is an important quarantine pathogen that causes wheat dwarf bunt and results in devastating damage to wheat production. The fungus is difficult to be distinguished from T. caries and T. laevis, which cause wheat common bunt, based on morphological, physiological and symptomatological characteristics of the pathogens. The traditional detection of the fungus can be a long and tedious process with poor accuracy. The inter-simple sequence repeat (ISSR) technique has been used for identifying molecular markers for detection of TCK. Of 28 ISSR primers screened, ISSR-859 amplified a specific 678 bp DNA fragment from all TCK isolates but not from any isolates of the common bunt fungi or other pathogenic fungi tested. Based on the fragment sequence, a pair of sequence characterized amplified region (SCAR) primers was designed, which amplified a 372 bp DNA fragment specifically in TCK. The SCAR marker was detected using as low as 1 ng template DNA of TCK, and was also detected using broken teliospores and DNA from asymptomatic wheat samples. We developed the SYBR Green I and TaqMan Green I and TaqMan real-time polymorphism chain reaction methods to detect TCK with the detection limit of 0.1 fg with asymptomatic wheat samples. Further work is needed to develop a rapid test kit for this pathogenic fungus using the designed specific primers.     

Dieter Seidel‐65 Jahre

Fruit extract of Solanum xanthocarpum was evaluated for its toxicity against Alternaria brassicae, the causal agent of Alternaria blight of Indian mustard [Brassica juncea (L.) Czern. &; Coss]. The mass obtained after vacuum drying of the crude methanolic extract was utilised for further sequential fractionation using n-hexane, ethyl acetate, n-butanol and methanol. Among the crude and different fractions tested, methanolic fraction was most effective with a minimum inhibitory concentration (MIC) of 62.5 μg/ml. The methanolic fraction was further fractionated using open column liquid chromatography into five subfractions (I–V) to identify the antifungal bioactive compounds. Among the five subfractions (SFs) tested SF IV was most effective at inhibiting A. brassicae conidial germination and thereby inhibited lesion development of Alternaria blight at a concentration of 15.625 μg/ml or higher. Furthermore, bioautography of SF IV with Alternaria alternata and diagnosis with Dragendorff reagent indicated that SF IV contains a mixture of bioactive alkaloids, namely a₁ (R_f = 0.12) and a₂ (R_f = 0.22). The potential of using S. xanthocarpum as a resource for the development of biofungicides is discussed.     

Climatic factors influencing spore production in Alternaria brassicae and Alternaria brassicicola

Sporulation in A. brassicae and A. brassicicola on naturally-infected leaf discs of oilseed rape and cabbage required humidities equal to or higher than 91.5% and 87% r.h. respectively. The optimum temperatures for sporulation were 18–24°C for A. brassicae and 20–30°C for A. brassicicola at which temperatures both fungi produced spores in 12–14 h. Above 24°C sporulation in A. brassicae was inhibited. At sub-optimal temperatures sporulation times for A. brassicicola were significantly longer than for A. brassicae with the differences increasing with decrease in temperature. Interrupting a 16-h wet period at 20°C with a period of 2 h at 70% or 80% r.h. did not affect sporulation in either fungus but a dry interruption of 3–4 h inhibited sporulation in both. Exposure of both fungi to alternating wet (18 h at 100% r.h., 20°C) and dry periods (6 or 30 h at 5565% r.h., 20°C) did not affect the concentration of spores produced in each wet period. Sporulation times were not affected by either the host type of the age of the host tissue. White light (136 W/m²) inhibited sporulation in A. brassicae with the degree of inhibition increasing with increasing light intensity. The effect of light on sporulation in A. brassicicola was not tested.     

Tests of fungicides against Alternaria brassicicola and studies on some factors enhancing attack by the fungus

When various fungicides were sprayed on detached cabbage cotyledons in the laboratory and on young plants in the glasshouse, propineb and mancozeb at 0·2 % a.i. consistently gave good protection against subsequent infection by spores of Alternaria brassicicola. Copper oxychloride, zineb, thiram and maneb showed promise in the laboratory, but were less successful in the glasshouse. Two fentin compounds were active at low concentrations against the fungus but were phytotoxic. Increased pathogenicity of A. brassicicola in the presence of cabbage pollen and of downy mildew infection in the laboratory is reported.     

Production of phytotoxic spore germination liquids by Alternaria brassicae and A. brassicicola and their effect on species of the family Brassicaceae

Experiments assessed the susceptibility of Brassica spp. and non-Brassica spp. in the family Brassicaceae to infection by Alternaria brassicae and A. brassicicola, and determined the sensitivity of the host species to spore germination liquids (SGLs) produced by the pathogens on B. napus leaves. There was a wide range of sensitivity to the pathogens. Brassica spp. were generally more susceptible, and some non-Brassica spp. (Barbarea vulgaris and Capsella bursa-pastoris) were immune to A. brassicicola. Measurable damage was caused by SGLs but with significant variation between host species. Non-hosts and weak hosts also showed necrosis. It was concluded that, in the case of both pathogens, the toxic factors in these SGLs were host-selective. Selectivity in toxin production was also demonstrated in relation to the host surface or growing medium in which spores germinated. A substantial amount of toxin was produced on all Brassicaceae tested but not on unrelated species (Triticum aestivum, Pisum sativum and Lycopersicon esculentum). Neither pathogen produced measurable amounts of toxin when cultured in Czapek (Dox) broth.     

The Effects of Glucosinolates and Their Breakdown Products on Necrotrophic Fungi

Glucosinolates are a diverse class of S- and N-containing secondary metabolites that play a variety of roles in plant defense. In this study, we used Arabidopsis thaliana mutants that contain different amounts of glucosinolates and glucosinolate-breakdown products to study the effects of these phytochemicals on phytopathogenic fungi. We compared the fungus Botrytis cinerea, which infects a variety of hosts, with the Brassicaceae-specific fungus Alternaria brassicicola. B. cinerea isolates showed variable composition-dependent sensitivity to glucosinolates and their hydrolysis products, while A. brassicicola was more strongly affected by aliphatic glucosinolates and isothiocyanates as decomposition products. We also found that B. cinerea stimulates the accumulation of glucosinolates to a greater extent than A. brassicicola. In our work with A. brassicicola, we found that the type of glucosinolate-breakdown product is more important than the type of glucosinolate from which that product was derived, as demonstrated by the sensitivity of the Ler background and the sensitivity gained in Col-0 plants expressing epithiospecifier protein both of which accumulate simple nitrile and epithionitriles, but not isothiocyanates. Furthermore, in vivo, hydrolysis products of indole glucosinolates were found to be involved in defense against B. cinerea, but not in the host response to A. brassicicola. We suggest that the Brassicaceae-specialist A. brassicicola has adapted to the presence of indolic glucosinolates and can cope with their hydrolysis products. In contrast, some isolates of the generalist B. cinerea are more sensitive to these phytochemicals.     

Combined expression of a barley class II chitinase and type I ribosome inactivating protein in transgenic <Emphasis Type="Italic">Brassica juncea</Emphasis> provides protection against <Emphasis Type="Italic">Alternaria brassicae</Emphasis>

Alternaria leaf spot caused by Alternaria brassicae, or A. brassicola, is one of the major fungal diseases of Brassica juncea (Indian mustard). To develop resistance against this fungal disease, the barley antifungal genes class II chitinase (AAA56786) and type I ribosome inactivating protein (RIP; AAA32951) were coexpressed in Indian mustard via Agrobacterium-mediated transformation. The stable integration and expression of transgenes in T₀ plants were confirmed by Southern blot and Western analysis. The transgenic lines showing inheritance in Mendalian fashion (3:1) were further evaluated by in vitro studies and under greenhouse conditions for resistance to the A. brassicae fungal pathogen. The transgenic plants showed up to 44% reduction in A. brassicae hyphal growth in in vitro antifungal assays. In green house screening, the transgenic plants sprayed with A. brassicae spores showed resistance through delayed onset of the disease and restricted number, size, and expansion of lesions as compared to wild type plants. These results indicate that the expression of chitinase and RIP from a heterologous source in B. juncea provide subsequent protection against Alternaria leaf spot disease and can be helpful in increasing the production of Indian mustard.     

Denaturation of the high molecular weight protein fraction of mustard (Brassica juncea) and rapeseed (Brassica campestris) by urea or guanidinium hydrochloride

Urea and guanidinium hydrochloride dissociate the 12S protein of mustard and rapeseed to 1.8 S protein and the extent of dissociation depends on the concentration of the denaturant. Mustard (Brassica juncea) protein is more readily dissociated than the rapeseed (Brassica campestris) protein. The reagents denature the protein as evidenced by increase in viscosity, appearance of difference spectra and quenching of fluorescence. Rapeseed protein is denatured more readily than the mustard protein. Analysis of visctosity, spectral and fluoresence data suggests that the first event in the denaturation reaction is the perturbation of the aromatic amino acid residues followed by their exposure to the solvent medium and unfolding of the protein molecule.     

Cytokinin-like substances and accumulation of labelled metabolites at infection sites of Alternaria brassicicola

Green islands were observed on mustard leaves beneath the infection drops containing germinating conidia of Alternaria brassicicola, when the surrounding uninfected tissue had yellowed due to senescence. Green island formation has been correlated with the secretion of cytokinin-like substances by the pathogens. A. brassicicola secreted cytokinin-like substances in a liquid synthetic medium and their application to detached host leaves evoked the formation of green islands in the dark. ¹⁴C studies confirmed that green islands act as metabolic sinks in which photosynthates are retained or accumulated. Cytokinin-like substances appear to be actively involved in infection and pathogenesis of A. brassicicola.     

Loss of EphB6 protein expression in human colorectal cancer correlates with poor prognosis

Erythropoietin-producing hepatocyte (Eph) receptor family constitutes the largest family of tyrosine kinase receptors in the human genome. Loss of EphB6, a kinase-deficient receptor, correlated with a negative outcome in several carcinomas. This study aimed to investigate the expression of EphB6 protein and mRNA levels in colorectal cancers (CRCs) and possible correlations with clinicopathological variables and prognosis. To assess protein expression level, 124 CRCs and 57 colorectal adenomas samples were examined by immunostaining, the mRNA level of 43 paired CRC and the adjacent normal tissues were detected by using SYBR Green real-time PCR method. Decreased expression of EphB6 protein was found in CRC as compared with adenoma and normal tissues (χ² = 10.146, P = 0.001 and χ² = 45.333, P < 0.001, respectively). Low EphB6 mRNA expression was detected in 83.8 % of cancers with negative or low EphB6 protein expression. The loss of EphB6 protein in CRC was positively associated with poorly differentiation (P < 0.001), lymph node metastasis (P = 0.006), Dukes stage (P = 0.002) and depth of invasion (P = 0.016). The patients with lymph node metastasis had a worse prognosis independently of gender, age, tumor site, stage and differentiation (RR = 0.404, CI 0.267–0.213, P < 0.001). Low levels of EphB6 protein expression are associated with a shorter mean duration of survival in colorectal cancer. Our results demonstrated that EphB6 may represent a novel, useful tissue biomarker for the prediction of survival rate in CRC.     

Patterns of microsatellite evolution inferred from the Helianthus annuus (Asteraceae) transcriptome

The distribution of microsatellites in exons, and their association with gene ontology (GO) terms is explored to elucidate patterns of microsatellite evolution in the common sunflower, Helianthus annuus. The relative position, motif, size and level of impurity were estimated for each microsatellite in the unigene database available from the Compositae Genome Project (CGP), and statistical analyses were performed to determine if differences in microsatellite distributions and enrichment within certain GO terms were significant. There are more translated than untranslated microsatellites, implying that many bring about structural changes in proteins. However, the greatest density is observed within the UTRs, particularly 5^′UTRs. Further, UTR microsatellites are purer and longer than coding region microsatellites. This suggests that UTR microsatellites are either younger and under more relaxed constraints, or that purifying selection limits impurities, and directional selection favours their expansion. GOs associated with response to various environmental stimuli including water deprivation and salt stress were significantly enriched with microsatellites. This may suggest that these GOs are more labile in plant genomes, or that selection has favoured the maintenance of microsatellites in these genes over others. This study shows that the distribution of transcribed microsatellites in H. annuus is nonrandom, the coding region microsatellites are under greater constraint compared to the UTR microsatellites, and that these sequences are enriched within genes that regulate plant responses to environmental stress and stimuli.