Multiwell plates for complementation tests of Fusarium.

Vegetative compatibility group (VCG) is a phenotype used to subdivide and categorize populations of Fusarium species (Correll et al. 1987 Phytopathology 77:1640-1646)

Molecular Identification of Fusarium Species in Gibberella fujikuroi Species Complex from Rice, Sugarcane and Maize from Peninsular Malaysia

The objective of this study was to identify Fusarium species in the Gibberella fujikuroi species complex from rice, sugarcane and maize as most of the Fusarium species in the species complex are found on the three crops. Isolates used were collected from the field and obtained from culture collection. The Fusarium isolates were initially sorted based on morphology and identifications confirmed based on the DNA sequence of the translation elongation factor 1-α (TEF-1α) gene. Based on the closest match of BLAST analysis, five species were recovered, namely, F. sacchari, F. fujikuroi, F. proliferatum, F. andiyazi and F. verticillioides. This is the first report regarding F. andiyazi from rice in Malaysia and Southeast Asia. The phylogenetic tree generated by using the neighbor joining method showed that isolates from the same species were grouped in the same clade. The present study indicated that Fusarium species in the G. fujikuroi species complex are widespread in rice, sugarcane and maize in Peninsular Malaysia. The findings also suggest that the use of morphological characters for identification of Fusarium species in the G. fujikuroi species complex from the three crops will lead to incorrect species designation

ChLae1 and ChVel1 Regulate T-toxin Production, Virulence, Oxidative Stress Response, and Development of the Maize Pathogen Cochliobolus heterostrophus

LaeA and VeA coordinate secondary metabolism and differentiation in response to light signals in Aspergillus spp. Their orthologs, ChLae1 and ChVel1, were identified in the maize pathogen Cochliobolus heterostrophus, known to produce a wealth of secondary metabolites, including the host selective toxin, T-toxin. Produced by race T, T-toxin promotes high virulence to maize carrying Texas male sterile cytoplasm (T-cms). T-toxin production is significantly increased in the dark in wild type (WT), whereas Chvel1 and Chlae1 mutant toxin levels are much reduced in the dark compared to WT. Correspondingly, expression of T-toxin biosynthetic genes (Tox1) is up-regulated in the dark in WT, while dark-induced expression is much reduced/minimal in Chvel1 and Chlae1 mutants. Toxin production and Tox1 gene expression are increased in ChVEL1 overexpression (OE) strains grown in the dark and in ChLAE1 strains grown in either light or dark, compared to WT. These observations establish ChLae1 and ChVel1 as the first factors known to regulate host selective toxin production. Virulence of Chlae1 and Chvel1 mutants and OE strains is altered on both T-cms and normal cytoplasm maize, indicating that both T-toxin mediated super virulence and basic pathogenic ability are affected. Deletion of ChLAE1 or ChVEL1 reduces tolerance to H2O2. Expression of CAT3, one of the three catalase genes, is reduced in the Chvel1 mutant. Chlae1 and Chvel1 mutants also show decreased aerial hyphal growth, increased asexual sporulation and female sterility. ChLAE1 OE strains are female sterile, while ChVEL1 OE strains are more fertile than WT. ChLae1 and ChVel1 repress expression of 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis genes, and, accordingly, melanization is enhanced in Chlae1 and Chvel1 mutants, and reduced in OE strains. Thus, ChLae1 and ChVel1 positively regulate T-toxin biosynthesis, pathogenicity and super virulence, oxidative stress responses, sexual development, and aerial hyphal growth, and negatively control melanin biosynthesis and asexual differentiation

Global gene expression and knockout analysis reveals genes associated with fungal fruiting body development in Neurospora crassa

ungi can serve as highly tractable models for understanding genetic basis of sexual development in multicellular organisms. Applying a reverse-genetic approach to advance such a model, we used random and multitargeted primers to assay gene expression across perithecial development in Neurospora crassa. We found that functionally unclassified proteins accounted for most upregulated genes, whereas downregulated genes were enriched for diverse functions. Moreover, genes associated with developmental traits exhibited stage-specific peaks of expression. Expression increased significantly across sexual development for mating type gene mat a-1 and for mat A-1 specific pheromone precursor ccg-4. In addition, expression of a gene encoding a protein similar to zinc finger, stc1, was highly upregulated early in perithecial development, and a strain with a knockout of this gene exhibited arrest at the same developmental stage. A similar expression pattern was observed for genes in RNA silencing and signaling pathways, and strains with knockouts of these genes were also arrested at stages of perithecial development that paralleled their peak in expression. The observed stage specificity allowed us to correlate expression upregulation and developmental progression and to identify regulators of sexual development. Bayesian networks inferred from our expression data revealed previously known and new putative interactions between RNA silencing genes and pathways. Overall, our analysis provides a fine-scale transcriptomic landscape and novel inferences regarding the control of the multistage development process of sexual crossing and fruiting body development in N. crassa

Differences in fitness of strains of Cochliobolus heterostrophus near-isogenic for toxin production

Cochliobolus heterostrophus strains near-isogenic except for a gene for toxin production were examined for differences in fitness on normal cytoplasm maize in the field and greenhouse. A mixture of two strains (one race O, non-toxin-producing, and one race T, toxin-producing) was used to inoculate a field plot of Cornell 281 in Ames, IA in 1983 and 1984. Different pairs of strains were used each year. The frequency of the race T strain decreased significantly (47% to 20% over 9 weeks in 1983 and 24% to 15% over 8 weeks in 1984) compared with the race O strain in both field seasons, showing that the race T strains were less fit than the race O strains. Differences in fitness of near-isogenic strains were also expressed as differences in lesion length. Lesions produced by race T lines were significantly shorter (an average of 28% in the greenhouse and 25% in the field) than those produced by race O lines. This was true for strains from four backcross generations of the fungus and on three genotypes of maize. No significant differences, however, were observed when infection efficiency and spore germinability of race T and race O strains were compared. Additional studies showed that the laboratory strains were less fit than naturally-occurring field strains. I concluded that the toxin gene or a gene closely linked to it reduces pathogenic fitness. Debilitation associated with the toxin locus could explain the rapid decrease in frequency of race T after susceptible Texas male sterile cytoplasm maize was replaced with normal cytoplasm maize following the 1970 southern leaf blight epidemic.</p

Influence of Substrate Modulus on Gecko Adhesion

<div>Sheet 1: Whole Animal Gecko Trials</div><div>All trials were done in an environmental chamber, set to 25 C and 35 %$RH. The specimens had thirty minutes to acclimate to the conditions, then were tested vertically on  two sets of substrates (cellulose acetate and PDMS) using a force-sensing apparatus, similar to the one used by Niewiarowski et al., 2008. This force-sensing apparatus was measured the force that the specimens were capable of withstanding (for safety of the gecko, a maximum of 20 N was set). </div><div><br></div><div>Before starting an individual experiment, the gecko had a small strip of medical tape carefully placed around the mouth, avoiding the nostrils, and was fitted with two harnesses around their pelvis, which were then attached to a force sensor positioned vertically on a motorized track. The gecko was placed on the substrate and encouraged to take a few steps, ensuring natural adhesion to the substrate.  After each trial the substrate was cleaned.</div><div><br></div><div>Within each substrate set, gecko trials were performed randomly on the different modulus substrates, with a total of three trials per substrate. Geckos were tested a maximum of three times before having at least a 24 hour rest period. The trials with the highest force for each gecko on each substrates are presented here.</div><div><br></div><div>Sheet 2: Isolated Lamella Adhesion</div><div>Skin sheds from 7 naturally molting Tokay geckos were collected and stored in a -20 C freezer. Single lamella were isolated from gecko sheds and adhered to small glass plates with cyanoacrylate glue such that the skin was anchored to the glass, but the setal strip was free to flex. Shear adhesion trials were done with ethanol/water cleaned glass and solvent extracted 1000 micron PDMS substrates.</div><div><br></div><div>The substrate was brought in contact with the lamella in the normal force direction by motor and loaded to a force of around 5 mN. The lamella was then loaded in the shear direction with a velocity of 5 microns/s such that the substrate would move towards the direction of the tip of the toe, with respect to the lamella. The trial was ended manually when the shear force dramatically dropped, reached a plateau for at least a minute, or the lamella reached the end of the substrate, and the maximum force was taken to be the shear adhesion force. </div><div><br></div