First Report of Umbel Browning and Stem Necrosis Caused by Diaporthe angelicae on Carrot in France.

In 2011, carrot (Daucus carota L.) seed production occurred on 2,900 ha, which accounts for approximately 25% of the area devoted to the production of vegetable fine seeds. Since 2007, symptoms of umbel browning have been regularly observed in carrot production areas located in the central region. Initially, triangular necrotic lesions appeared on carrot umbels that later spread to the entire umbels and often progressed to the stems. Diseased umbels became dried prematurely, compromising seed development. The loss in seed production was estimated at approximately 8% of the harvested carrot umbels during the cropping seasons of spring and summer 2007 and 2008 in France. In collaboration with seed companies, diseased carrot stems were collected from seven fields of seed production (eight plants per field) and a fungus was isolated from the tissue. The cultures were grown on malt (2%) agar (1.5%) medium and incubated for 2 weeks at 22°C in darkness. Young fungal colonies were white and a brownish green pigmentation developed when the colonies became older. The same color was observed from the top and on the reverse of the colonies. To induce sporulation, isolates were grown on water agar (1.5%) medium in the presence of carrot stem fragments for 1 week at 22°C in darkness, followed by 1 week at 22°C in white light under a 16-h photoperiod. Pycnidia were produced on stem fragments and contained alpha and beta conidia typical of the genus Diaporthe (2). Alternatively, pycnidia were also obtained on malt agar medium after 2 weeks of culture at 25°C in white light under a 12-h photoperiod. The size of alpha and beta conidia was 6.3 ± 0.5 × 2.3 ± 0.4 µm and 23.3 ± 1.8 × 0.9 ± 0.2 µm, respectively (n = 170). In order to confirm the identification at the genus level and determine the species, DNA was extracted from the mycelium of three representative isolates and the ITS regions of the ribosomal DNA were amplified using universal primers (1). The sequences of the amplified products (GenBank Accession Nos. KF240772 to KF240774) were 100% identical with the ITS sequence of a Diaporthe angelicae isolate deposited in the NCBI database (CBS 111592 isolate, KC343027). To confirm pathogenicity, the three isolates of D. angelicae were inoculated on carrot umbels in the greenhouse. A total of nine plants were inoculated (three plants per isolate). Using a micropipette, 10 µl of a conidial suspension containing alpha and beta conidia (105 conidia mL-1) were deposited at the base of the primary umbel and two secondary umbels, which were wounded before inoculation using a scalpel blade. Seven inoculated plants developed triangular, necrotic lesions that were typical umbel browning. D. angelicae was re-isolated on malt agar medium from the inoculated diseased carrot umbels. To our knowledge, this is the first report of D. angelicae in carrot cultivated for seed production in France. The disease resembles the lesions described in the Netherlands in 1951 on carrot inflorescence caused by Phomopsis dauci (3). In future experiments, it would be crucial to precisely determine if D. angelicae could be transmitted to the seeds. References: (1) M. A. Innis et al. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA, 1990. (2) J. M. Santos and A. J. L. Philips. Fungal Divers. 34:111, 2009. (3) J. A. von Arx. Eur. J. Plant Pathol. 57:44, 1951.

Impact of the unfolded protein response on the pathogenicity of the necrotrophic fungus Alternaria brassicicola.

The unfolded protein response (UPR) is an important stress signalling pathway involved in the cellular development and environmental adaptation of fungi. We investigated the importance of the UPR pathway in the pathogenicity of the plant necrotrophic fungus Alternaria brassicicola, which causes black spot disease on a wide range of Brassicaceae. We identified the AbHacA gene encoding the major UPR transcription regulator in A. brassicicola. Deletion of AbHacA prevented induction of the UPR in response to endoplasmic reticulum stress. Loss of UPR in mutants resulted in a complete loss of virulence and was also associated with a cell wall defect and a reduced capacity for secretion. In addition, our results showed that the UPR was triggered by treatment of mycelia with camalexin, i.e. the major Arabidopsis thaliana phytoalexin, and that strains lacking functional AbHacA exhibited increased in vitro susceptibility to antimicrobial plant metabolites. We hypothesize that the UPR plays a major role in fungal virulence by altering cell protection against host metabolites and by reducing the ability of the fungus to assimilate nutrients required for growth in the host environment. This study suggests that targeting the UPR pathway would be an effective plant disease control strategy.

First Report of an Alternaria Leaf Spot Caused by Alternaria brassicae on Crambe abyssinicia in Australia.

Crambe abyssinicia Hochst. is grown sporadically worldwide for its value as a source of high erucic acid industrial oils and secondary commercial products. While there is increasing interest in cropping C. abyssinicia in Australia, for these potentials and also as a source of oil for biodiesel production, currently, there have been no commercial crops of this species. In September 2004, inspection of a small experimental field crop in Beverley, Western Australia indicated the presence of significant leaf spotting just prior to commencement of flowering. The symptoms of this disease included as many as 10 to 15 spot lesions per leaf that were generally rounded and varied between 0.5 to 11 mm in diameter. Clusters of these lesions were often associated with chlorosis of the region of leaves where they occurred. More than 95% of plants inspected showed these symptoms. When affected leaves were incubated in moist chambers, typical conidia of Alternaria brassicae (Berk.) Sacc. were observed. The description of these conidia matched that of the Commonwealth Mycological Institute for this pathogen (1) showing obclavate conidia 105 to 210 µm long and 20 to 30 µm thick, with 11 to 15 transverse septa and 0 to 3 longitudinal or oblique septa, predominantly with a pronounced beak 5 to 8 µm thick extending 0.3 to 0.5 µm of the length of the conidium. Single-spore isolations were made onto potato dextrose agar. Subcultures of these isolates were identified using a polymerase chain reaction (PCR)- based assay (2). This assay involved the use of two sets of A. brassicae-specific primers selected for conventional and real-time PCR. The colonies were confirmed to belong to A. brassicae. In a pathogenicity test to confirm Koch's postulates, single-spore isolates were inoculated onto cotyledons and leaves of 10-day-old C. abyssinicia seedlings. Symptoms on inoculated plants appeared within a period of 14 days of inoculation, matching those found on the affected plants in the field, and A brassicae was reisolated. A. brassicae causes an important worldwide disease of crucifers, for example, it can be a devastating disease of rapeseed and the other cruciferous crops in the United States and Canada. Since A. brassicae has already been reported on other species of crucifers Australia-wide, it may pose a threat to any potential Crambe spp. industry in this country. References: (1) M. B. Ellis No. 162 in: Descriptions of Pathogenic Fungi and Bacteria. CMI, Kew, England, 1966. (2) T. Guillemette et al. Plant Dis. 88:490, 2004.

Samsonia erythrinae gen. nov., sp. nov., isolated from bark necrotic lesions of Erythrina sp., and discrimination of plant-pathogenic Enterobacteriaceae by phenotypic features.

Bacterial strains isolated from diseased erythrina (Erythrina sp.) trees in Martinique (French West Indies) were studied using phenotypic tests, 16S rDNA sequence analysis and DNA-DNA hybridization. Numerical analysis of phenotypic characteristics showed that these strains formed an homogeneous phenon among plant-pathogenic Enterobacteriaceae, and gave useful and updated information for the identification of these bacteria. Results of DNA-DNA hybridization indicated that strains from erythrina belonged to a discrete genomospecies (89-100% hybridization) and had low levels of DNA relatedness (2-33% hybridization) with reference strains of phytopathogenic Erwinia, Brenneria, Pectobacterium, Pantoea and Enterobacter species. 16S rDNA sequence analysis using three different methods revealed that the position of strain CFBP 5236T isolated from erythrina was variable in the different trees, so that strains from erythrina could not be assigned to any recognized genus. It is proposed that these strains are included in a new genus, Samsonia. The name Samsonia erythrinae is proposed for the new species. The G+C content of the DNA of the type strain, CFBP 5236T (= ICMP 13937T), is 57.0 mol%.

Enumeration and characterization of cellulolytic bacteria from refuse of a landfill.

Enumeration and phenotypic characterization of aerobic cellulolytic bacteria were performed on fresh, 1 year old and 5 years old refuse samples of a French landfill site. Numbers of cellulolytic bacteria ranged from 1.1x10(6) to 2.3x10(8) c.f.u. (g dry wt.)(-1) and were lower in 5 years old refuse samples. A numerical analysis of phenotypic data based on 80 biochemical tests and performed on 321 Gram-positive isolates from refuse, revealed a high phenotypic diversity of cellulolytic bacteria which were distributed into 21 clusters. Based on the phenotypic analysis and the sequencing of 16S rDNA of five representative strains of major clusters, the predominant cellulolytic groups could be assigned to the family of Bacillaceae and to the genera Cellulomonas, Microbacterium and Lactobacillus. Furthermore, chemical parameters such as pH, carbohydrates and volatile solid contents influenced the composition of the cellulolytic bacterial groups which were reduced essentially to the family of Bacillaceae in the oldest refuse samples.

Induced defence responses limit Hartig net formation in ectomycorrhizal birch roots.

Roots of clonal birches (Betula pendula) were inoculated with the ectomycorrhizal fungi Paxillus involutus (isolates P0 and Mi) and Hebeloma cylindrosporum (strains D1 and D105). These fungi showed different rates of mycorrhiza formation in vitro. Mature mycorrhizas were obtained after only 2-4 d with H. cylindrosporum, whereas 6-8 d were necessary with P. involutus isolate P0, and P. involutus isolate Mi was not able to form mature mycorrhiza during the 10 d of the experiment. Temporal changes in PAL activity and the expression of genes encoding intracellular pathogenesis-related proteins were followed after inoculating birch roots with these fungi. Transient increase of PAL activity, and transient induction of expression of the wound-inducible Bet v1-SC1 gene, were observed in roots challenged with both H. cylindrosporum strains and the P. involutus isolate P0. These changes were found to coincide with hyphal penetration between root cells during Hartig net formation, and were never observed in roots inoculated with the poorly aggressive P. involutus isolate Mi. Examination of mycorrhizal root sections under u.v. light indicated the presence of phenolic compounds in the host cell walls at the vicinity of the Hartig net. These results strongly suggest that hyphal penetration between the root cells triggers a transient defence response which, in turn, could limit Hartig net formation to the outer layer of the root cortex.

Molecular cloning and expression analysis of dihydroflavonol 4-reductase gene in flower organs of Forsythia x intermedia.

The expression, during flower development, of the gene encoding the anthocyanin pathway key enzyme dihydroflavonol 4-reductase (DFR) was investigated in floral organs of Forsythia x intermedia cv. 'Spring Glory'. Full-length DFR and partial chalcone synthase (CHS) cDNAs, the gene of interest and a flavonoid pathway control gene respectively, were obtained from petal RNA by reverse transcription PCR. Whereas for CHS northern blot analysis enabled the study of its expression pattern, competitive PCR assays were necessary to quantify DFR mRNA levels in wild-type plants and in petals of 2 transgenic clones containing a CaMV 35S promoter-driven DFR gene of Antirrhinum majus. Results indicated a peak of CHS and DFR transcript levels in petals at the very early stages of anthesis, and different expression patterns in anthers and sepals. In comparison to wild-type plants, transformants showed a more intense anthocyanin pigmentation of some vegetative organs, and a dramatic increase in DFR transcript concentration and enzymatic activity in petals. However, petals of transformed plants did not accumulate any anthocyanins. These results indicate that other genes and/or regulatory factors should be considered responsible for the lack of anthocyanin production in Forsythia petals.

Heterologous transformation of Agrocybe aegerita with a bacterial neomycin-resistance gene fused to a fungal promoter-like DNA sequence.

DNA sequences of the basidiomycete Agrocybe aegerita were cloned in E. coli based on their ability to drive the expression of the bacterial promoterless tetracycline (Tc)-resistance gene. A 0.48% frequency of the cloned sequences promoted antibiotic-resistance. The sequence conferring the highest Tc resistance (40 µg/ml) was selected to drive the expression in E. coli of two other promoterless genes encoding chloramphenicol and neomycin resistance. One of the derivative vectors, pN13-A2, carrying a chimeric neomycin-resistance gene, was used to transform an A. aegerita neomycin-sensitive strain by protoplast electroporation. Transformation frequencies ranged from 1 to 2.8 transformants per µg of DNA per 10(3) viable cells, in a relatively high background of spontaneous-resistant colonies (2% of the surviving protoplasts). Molecular analyses showed that transformation had occurred by the integration of pN13-A2 sequences, either ectopically or at the resident locus carrying the A. aegerita promoter-like sequence, with probable molecular rearrangements. The nucleotide sequence of the promoter-like fragment revealed the presence of a CT motif that is known to be involved in a promoter function in some highly expressed genes of filamentous fungi.

Accumulation of New Polypeptides in Ri T-DNA-Transformed Roots of Tomato (Lycopersicon esculentum) during the Development of Vesicular-Arbuscular Mycorrhizae.

Root-inducing transferred-DNA (Ri T-DNA)-transformed roots of tomato (Lycopersicon esculentum) were in vitro inoculated with surface-sterilized vesicular-arbuscular mycorrhizal leek root pieces. About 1 week after inoculation, the infection of the transformed root culture by the fungal endophyte was confirmed by photonic microscopy. Total proteins were extracted from the mycorrhizal roots and analyzed by two-dimensional polyacrylamide gel electrophoresis. Control gels were run with proteins extracted from noninoculated roots mixed with purified intraradical vesicles and extraradical hyphae. Comparison of the resulting patterns revealed the presence of two polypeptides with estimated apparent masses of 24 and 39 kDa that were detected only in infected roots. Polypeptides with similar migration parameters were not detected in roots challenged with spore extracts, suggesting that the accumulation of the polypeptides was directly linked to root colonization by the fungus rather than to induction by fungus-derived elicitors.

Codon reading scheme in Mycoplasma pneumoniae revealed by the analysis of the complete set of tRNA genes.

The 33 genes encoding the complete set of tRNA species in Mycoplasma pneumoniae have been cloned and sequenced. They are organized into 5 clusters in addition to 9 single genes. No redundant gene was found, indicating that 33 tRNAs correspond to 32 different anticodons and decode all 62 codons used in this organism. There is only one single tRNA for each of the Ala, Leu, Pro, and Val family boxes. Therefore, a simplified decoding system resembling that recently described for Mycoplasma capricolum (1) has to also exist in M.pneumoniae. However, analysis of the anticodon set and codon usage revealed features characteristic of the latter: (i) there is no obvious preference toward AT rich synonymous codons, (ii) CGG codons are assigned for arginine and are translated by tRNA Arg(UCG), and (iii) CNN or GNN anticodons are encountered in the Ser, Thr, Arg, and Gly family boxes. We thus propose that this codon-anticodon recognition pattern has emerged in the 'M.pneumoniae cluster' under a genomic economization strategy but without the influence of AT pressure.

Differential detection of Mycoplasma pulmonis and Mycoplasma arthritidis with species-specific DNA probes.

Mycoplasma pulmonis and Mycoplasma arthritidis are both significant causes of infection in colonies of rodents, which are common experimental animals for biomedical research. Since differential diagnosis has proven difficult due to similar homology between these two murine Mycoplasma species, the development of a reliable identification system is of importance. In this study DNA probes specific for M. pulmonis and M. arthritidis were generated after cross-hybridization between two reference strains and cloning of the specific DNA fragments into the pGEM-3 blue vector. Two clones, harboring plasmids pGEM-89 (specific for M. pulmonis) and pGEM-31 (specific for M. arthritidis) were selected based on their specificity upon colony hybridization and were used as species-specific probes. These probes could detect in dot blots 150 pg of M. pulmonis DNA or 300 pg of M. arthritidis DNA with no visible hybridization with up to 100 ng of heterologous DNA. When hybridized with dot blots of culture grown organisms, the pGEM-89 probe produced a positive signal with all 12 isolates of the M. pulmonis strains tested, but none of the four M. arthritidis, and vice versa for the pGEM-31 probe. We thus anticipate that these probes will be useful for routine detection and identification of mycoplasmal infections of rodents.

The gene for a 4.5S RNA homolog from Mycoplasma pneumoniae: genetic selection, sequence, and transcription analysis.

In an effort to make an inventory of the tRNA genes of Mycoplasma pneumoniae, a DNA fragment was found to contain a sequence that can be folded into a hairpin structure very similar to that of the 4.5S RNA of Escherichia coli. Recombinant plasmids carrying this region were able to complement E. coli strains that were deficient in 4.5S RNA. S1 mapping showed that the mature transcript is only 79 nucleotides long.