Poster presentations

Abstract

The key to maximising the nutritional value of lignocellulosic materials is in disrupting the plant cell walls to allow complete access to nutrients and not creating extra anti-nutritional factors (Castro et al., 1994). In particular, steam and pressure treatments to disrupt lignocellulosics in a way which allows improved utilisation of cell wall polysaccharides by rumen microbes (Castro and Machado, 1990). Rumen fungi produce a wide range of polysaccharide degrading enzymes and are as primary colonisers of fibrous plant materials that degrade lignin-containing plant cell walls. With the advancement of molecular enumeration methods, in particular 18S rDNA gene probing methods, researchers were able to monitor fungal species within the rumen (Stahl et al., 1998). Quantitative competitive PCR (QC-PCR) techniques play an important role in nucleic acid quantification because of their significant lower cost of equipment and consumables (Franz et al., 2001).The aim of this study was to use QC-PCR to determine the effect of sugarcane pith treated with low temperature steam plus 0.9% H 2 SO 4 on in vitro growth of rumen anaerobic fungi. Material and methods Rumen fungi were isolated from pre-incubated wheat straw in the rumen of fistulated sheep and then method of Joblin (1981) was used to grown under anaerobic conditions at 39ºC for 3 days. These isolates were used (1:9) as a source of fungi inoculum. Serum bottles (four replicate per sample) containing fungi culture medium, 1g of sugarcane pith as untreated (UTP) or treated with low temperature steam (at 135 ºC for 80 min) and 0.9 % H 2 SO 4 (STP) and 1ml antibiotic solution were used to culture the isolated fungi at 39ºC in an incubator. To preparing fungi pure culture, sub-culturing was done three times. Total genomic DNA was isolated from pure culture samples using Guanidine Thiocyanate-Silica Gel method. A universal PCR primer pair GAF (F): 5'-GAG GAA GTA AAA GTC GTT AAC AAG GTT TG-3' and GAF(R): 5'-GAAATT CAC AAA GGG TAG GAT GAT TT-3' was used to amplify a specific region of 18S rDNA from anaerobic rumen fungi. Standard control DNA was constructed to use in the QC-PCR and was shown to amplify under the same reaction condition and the same amplification efficiency as the target DNA. The PCR was performed in a final volume of 25µl sealed in a capillary tip, and thermocycling was carried out in a model 2000 (Biometra). The PCR amplification condition was as follows: denaturation at 94°C for 4min followed by 35 cycles of 94 °C for 30s; 56°C for 30s; and 72°C for 1min followed by a final extension at 72°C for 5min. The PCR products were separated by electrophoresis on agarose gels, stained with ethidium bromide, and visualized by UV transillumination. The relative intensities of PCR products were used to compare fungal biomass under different samples. The signal intensity was quantified by Image J 1.29x and expressed in arbitrary units. The data was analysed using the GLM procedure of SAS for a completely randomized design.