Evidence of mixotrophic carbon-capture by n-butanol-producer Clostridium beijerinckii.

Recent efforts to combat increasing greenhouse gas emissions include their capture into advanced biofuels, such as butanol. Traditionally, biobutanol research has been centered solely on its generation from sugars. Our results show partial re-assimilation of CO2 and H2 by n-butanol-producer C. beijerinckii. This was detected as synchronous CO2/H2 oscillations by direct (real-time) monitoring of their fermentation gasses. Additional functional analysis demonstrated increased total carbon recovery above heterotrophic values associated to mixotrophic assimilation of synthesis gas (H2, CO2 and CO). This was further confirmed using 13C-Tracer experiments feeding 13CO2 and measuring the resulting labeled products. Genome- and transcriptome-wide analysis revealed transcription of key C-1 capture and additional energy conservation genes, including partial Wood-Ljungdahl and complete reversed pyruvate ferredoxin oxidoreductase / pyruvate-formate-lyase-dependent (rPFOR/Pfl) pathways. Therefore, this report provides direct genetic and physiological evidences of mixotrophic inorganic carbon-capture by C. beijerinckii.

First Report of Anthracnose Stalk Rot of Maize Caused by Colletotrichum graminicola in Switzerland.

Maize stem samples exhibiting symptoms of anthracnose were collected from a field near Zurich, Switzerland, in September of 2012 and were sent to the fungal genetics laboratory, Centro Hispano-Luso de Investigaciones Agrarias (CIALE) at the University of Salamanca, Spain, for further analysis. The stem samples exhibited glossy, black, and irregularly shaped lesions. Tissue samples, approximately 5 mm2, were dissected from below the epidermis. The tissue samples were surface disinfested for 1 min in 20% sodium hypochlorite and cultured on one half strength acidified PDA supplemented with ampicillin (2). Monoconidial isolates from three different stems were grown on potato dextrose agar (PDA) and had dark gray aerial mycelium with orange spore masses. Conidia were falcate, slightly curved, tapered toward the tips with an average length of 31.77 µm and an average width of 4.76 µm and produced in acervuli with setae, consistent with descriptions of C. graminicola Ces. Wils. Conidial suspensions were prepared for each isolate, and were inoculated onto the leaves of 2-week-old maize plants by laying the plants horizontally in a tray (in pots with their root systems intact) and placing 7.5-µl droplets of a 106 conidial suspension on the leaf surface. The trays were covered and plants were incubated overnight at 23°C. The plants were then returned to their upright position and grown in a growth chamber at 25°C with a 12-h light cycle (3). After 6 days, the inoculated plant leaves exhibited lesions that were elongated and irregularly shaped with necrotic centers and chlorotic margins. The water-inoculated controls did not show symptoms. Microscopic examination revealed the production of conidia on the surface of the leaves, identical to the original isolates. Genomic DNA was extracted using the protocol of Baek and Kenerley (1). A region of the ribosomal DNA repeat was amplified and sequenced using the universal primers ITS4 and ITS5. The resulting sequences were 100% identical to each other and 100% identical to C. graminicola sequences in GenBank. One representative sequence was deposited in GenBank under accession no. KF597538. The 100 most similar sequences in GenBank were used to construct a phylogenetic tree using the neighbor-joining method. The phylogenetic analysis revealed that the isolates clustered within the C. graminicola clade, consistent with their identification as C. graminicola. To our knowledge, this is the first report of anthracnose on maize caused by C. graminicola in Switzerland. Previous reports have demonstrated that the pathogen exists in neighboring countries Germany and France. References: (1) J.-M. Baek and C. M. Kenerley. Fungal Genet. Biol. 23:34, 1998. (2) S. A. Sukno et al. Appl. Environ. Microbiol. 74:823, 2008. (3) W. A. Vargas et al. Plant Physiol. 158:1342, 2012.

First Report of Apple Bitter Rot Caused by Colletotrichum godetiae in the United Kingdom.

Apple is an important crop in United Kingdom, with a total production of 233,750 tonnes in 2011. Symptoms of apple bitter rot were observed on apple fruits (Malus domestica L.) in the Newcastle area, United Kingdom, in October 2008. Lesions were round, 1 to 5 cm in diameter, brown and dry, with acervuli producing yellowish spore masses in concentric bands. Infected material was sent to the W-HRI (University of Warwick) for identification of the causal agent. Fungal isolates with morphological characteristics similar to those of Colletotrichum acutatum sensu lato were isolated from diseased fruits. Monoconidial isolates were grown on PDA at 25°C with a 12-h light period. The cultures were light gray, with cottony aerial mycelium getting darker with age and with color ranging from whitish to dark gray on the reverse side of the colony. The cultures have yellowish spores masses and dark melanized structures similar to acervuli. Colletotrichum spp. are difficult to identify solely on morphology; therefore, representative isolates were used for multi-locus gene sequencing and characterization (1). Genomic DNA was extracted using a modified Chelex100 protocol. Three loci were amplified and sequenced: the ITS region was amplified and sequenced using the universal primers ITS4 and ITS5. Primers TB5 and TB6 were used for the amplification and sequencing of the variable region of the TUB gene. Primers GDF1 and GDR1 were used to amplify a 200-bp intron region of the GAPDH gene. No differences were found among the strains at any of the loci. One sequence for each locus has been deposited in GenBank under accessions KF834206 (ITS), KF834207 (TUB), and KF834208 (GAPDH). In GenBank, ITS sequences matched with 100% identity to C. higginsianum (EU400147) and to C. gloeosporioides (AJ301931 to 972); and with identity between 99.6 and 99.8% with sequences belonging to C. godetiae (part of C. acutatum species complex). The TUB sequences match with 100% identity to more than 25 sequences belonging to C. godetiae. The GAPDH sequences match with 100% identity to JQ948739 and 35 belonging to C. godetiae strains IMI 381927 and CBS 131331. A multilocus phylogenetic tree (ITS, TUB, and GAPDH) was reconstructed using sequences of reference strains belonging to C. higginsianum, C. gloeosporioides, C. godetiae, and related species. The phylogenetic tree confirmed the identity of the strains isolated from apple as C. godetiae. Koch's postulates were tested with representative isolate by artificial inoculation of 12 healthy fruits of the cv. Golden Delicious. Fruit surfaces were sterilized with 70% ethanol, wounded with a sterile needle, and then inoculated with a plug of actively growing mycelium prepared from a 10-day-old culture grown on PDA. Inoculated fruits were incubated in sterile conditions at 25°C with a 12-h photoperiod. In 83% of fruits, symptoms appeared between 7 and 15 days later. The rot begins as light brown, circular lesion getting darker with orange spore masses. Fungal colonies isolated from the lesions and cultured on PDA have identical morphological characteristics of the isolate used for the pathogenicity assay. To the best of our knowledge, this is the first report of apple bitter rot caused by C. godetiae in the United Kingdom. Apple bitter rot is spread worldwide and in moist, temperate regions it is considered one of the most important diseases causing considerable crop losses. Since the losses are more severe under prolonged warm and wet weather conditions, bitter rot caused by C. acutatum species may become an emerging problem in the United Kingdom in the near future, and may require investigation of management practices to control this new disease. References: (1) R. Baroncelli. Colletotrichum acutatum sensu lato: From diversity study to genome analysis. Coventry, United Kingdom, PhD thesis, 2012. (2) U. Damm et al. Stud. Mycol. 73:37, 2012.

CPR1: a gene encoding a putative signal peptidase that functions in pathogenicity of Colletotrichum graminicola to maize.

Colletotrichum graminicola causes anthracnose leaf blight and stalk rot of maize. We used restriction-enzyme mediated insertional (REMI) mutagenesis to identify a gene in this fungus that is required for pathogenicity to both stalks and leaves. The predicted polypeptide encoded by this gene, which we have named CPR1, is similar to a family of proteins that comprise one subunit of the eukaryotic microsomal signal peptidase. The nonpathogenic CPR1 REMI mutant contains a plasmid integration in the 3' untranslated region of the gene, 19 bp downstream from the stop codon. The result is a significant reduction in transcript levels in comparison to the wild type, perhaps as a result of increased transcript instability. We were unable to knock out the CPR1 gene, and it may be essential for viability. Microscopic examination of the REMI mutant on maize leaves revealed that it is fully capable of penetrating and colonizing host cells during the initial, biotrophic phases of the disease interaction but, unlike the wild type, it appears to be unable to switch to a necrotrophic mode of growth. We suggest that the CPR1 REMI mutant may be unable to secrete sufficient quantities of degradative enzymes to support that transition. The CPR1 REMI mutant provides us with a useful tool for future studies of the role of fungal protein transport in this important stalk rot disease of maize.

Restriction enzyme-mediated integration used to produce pathogenicity mutants of Colletotrichum graminicola.

We have developed a restriction enzyme-mediated insertional mutagenesis (REMI) system for the maize pathogen Colletotrichum graminicola. In this report, we demonstrate the utility of a REMI-based mutagenesis approach to identify novel pathogenicity genes. Use of REMI increased transformation efficiency by as much as 27-fold over transformations with linearized plasmid alone. Ninety-nine transformants were examined by Southern analysis, and 51% contained simple integrations consisting of one copy of the vector integrated at a single site in the genome. All appeared to have a plasmid integration at a unique site. Sequencing across the integration sites of six transformants demonstrated that in all cases the plasmid integration occurred at the corresponding restriction enzyme-recognition site. We used an in vitro bioassay to identify two pathogenicity mutants among 660 transformants. Genomic DNA flanking the plasmid integration sites was used to identify corresponding cosmids in a wild-type genomic library. The pathogenicity of one of the mutants was restored when it was transformed with the cosmids.

Evidence for two independent lineages of shiitake of the americas (Lentinula boryana) based on rDNA and beta-tubulin gene sequences.

Portions of ribosomal RNA genes (rDNA) were sequenced from members of the genus Lentinula and were used, along with partial beta-tubulin gene sequences, for phylogenetic reconstructions. The rDNA sequences of L. boryana were separated into two well-defined lineages. Lineage 1 was composed of isolates from Mexico and Costa Rica while lineage 2 encompassed isolates from the United States, Venezuela, and Brazil. The two South American isolates of L. boryana had nearly identical ITS sequences and very closely related beta-tubulin sequences. This high level of similarity may indicate that sexual reproduction occurs among the sampled populations, although this is difficult to reconcile with the large geographic distances (over 4000 km) that separate some of the collecting locations. An alternative explanation may be that the isolates sampled are the product of a rapid population expansion over a large geographic area. Analyses of partial beta-tubulin gene sequences that were rooted using Pleurotus spp. support the hypothesis that L. boryana is monophyletic.