Extraneous DNA interferes with PCR studies of endophytic fungi. A procedure was developed with which to evaluate the removal of extraneous DNA. Wheat (Triticum aestivum) leaves were sprayed with Saccharomyces cerevisiae and then subjected to physical and chemical surface treatments. The fungal ITS1 products were amplified from whole tissue DNA extractions. ANOVA was performed on the DNA bands representing S. cerevisiae on the agarose gel. Band profile comparisons using permutational multivariate ANOVA (PERMANOVA) and non-metric multidimensional scaling (NMDS) were performed on DGGE gel data, and band numbers were compared between treatments. Leaf surfaces were viewed under variable pressure scanning electron microscopy (VPSEM). Yeast band analysis of the agarose gel showed that there was no significant difference in the mean band DNA quantity after physical and chemical treatments, but they both differed significantly (p < 0.05) from the untreated control. PERMANOVA revealed a significant difference between all treatments (p < 0.05). The mean similarity matrix showed that the physical treatment results were more reproducible than those from the chemical treatment results. The NMDS showed that the physical treatment was the most consistent. VPSEM indicated that the physical treatment was the most effective treatment to remove surface microbes and debris. The use of molecular and microscopy methods for the post-treatment detection of yeast inoculated onto wheat leaf surfaces demonstrated the effectiveness of the surface treatment employed, and this can assist researchers in optimizing their surface sterilization techniques in DNA-based fungal endophyte studies.

Effective chemical hybridizing agents (CHAs) or male gametocides enhance cross pollination in plant breeding and genetic analysis of traits. The present study examined the efficacy and optimum concentration of four CHAs, namely: 2-chloroethyl phosphonic acid (Ethrel), ethyl 4'-fluorooxanilate (E4FO), 2, 4-dichlorphenoxy acetic acid (2, 4-D) and Promalin ® (1.8% GA 47 -gibberellins A 4 +A 7 and 1.8% 6-BA-benzyladenine) on pollen sterility and seeding of a tef line (DZ-01-3186). Seed-derived and individually grown tef plants were treated with foliar applications of the four CHAs (at four levels each) sprayed once at the early booting stage, and bagged to control cross pollination. Female fertility was assessed by recording seed set following controlled pollinations. Although all the CHAs caused some pollen sterility, their efficacy levels varied from 9.77 to 99.50% in treated plants compared to the control (6.68 +/-1.04%). Pollen sterility increased with increasing CHA concentration. Near-complete pollen sterility (99.50 ± 0.50%) was achieved by the application of E4FO at rates of 1500 to 3000 ppm, and Ethrel at 5000 ppm. All the CHAs significantly reduced seed yield, with E4FO, Ethrel and Promalin ® at 5000 ppm causing the highest reduction, essentially yielding no seed. In the period following the application of the CHAs, plants treated with E4FO (1000 -1500 ppm) exhibited stigmas that remained fertile. Hence, it is recommended that E4FO (at 1000 -1500 ppm) can be used as a chemical emasculation agent for tef, with the least phytotoxicity and the highest female fertility.

Fungal endophytes have been documented in almost all terrestrial plant groups. Although the endophyte infection syndrome in agronomic cultivars is well studied, relatively little work addresses questions of spatial ecology and fire effects on epichloae endophyte infection in native grasses, and none, to our knowledge, in sub-Saharan Africa. We sampled seven populations of the native Festuca costata Nees along the spline of the Drakensberg range in South Africa at several spatial scales, including both recently burned and unburned stands. We tested epichloae presence and prevalence with immunoblot assays, PCR and genetic sequencing. We found epichloae endophytes were present and prevalent (38-98% infection rates depending on location). Variation in infection rates occurred primarily among locations, but also among bunches. There was little evidence that endophyte infection rates varied with fire. Novel evidence of epichloae infection of a native Festuca in South Africa opens the door to several new research questions, from the phylogenetic relationship between epichloae of sub-Saharan Africa and other continents to the ecological advantages or disadvantages that endophytes confer upon their hosts, especially in a fire-prone ecosystem vulnerable to global environmental change.

Non-symbiotic nitrogen-fixing bacterial diazotrophs closely associated with the roots of grasses probably contribute most of the new nitrogen acquired to sustain productive natural grasslands, yet their ecology is poorly understood, especially in southern Africa. We looked for genetic evidence, using qPCR and gel electrophoresis, for the presence of the bacterial nifH gene associated with the roots of four grass species (20 plants each) in a mesic grassland in South Arica, which would indicate the potential for N fixation by diazotrophs. Grasses most tolerant of low N (Aristidajunciformis) were predicted to harbour the most diazotrophs, especially compared to those most responsive to fertiliser N (Eragrostiscurvula). However, the nifH gene was found in all 80 root samples and did not differ in copy number between species. Sequencing of a representative sample confirmed the identity of the nifH gene. The recently burned half of the grassland had 60% more nifH genes than the area burned 15 months previously, suggesting that grass growth stimulated by fire could recruit diazotrophs. Given their ubiquity and importance in the N economy of grasslands, research is required to characterise root-associated diazotroph communities, quantify their N fixation rates, and understand their environmental controls.