Isolation, morphological and molecular characterization of phytate-hydrolysing fungi by 18S rDNA sequence analysis

Abstract

Phytate is the primary storage form of phosphate in plants. Monogastric animals like poultry, pigs and fishes have very low or no phytase activities in their digestive tracts therefore, are incapable to efficiently utilize phytate phosphorus from the feed. Phytase from microbial sources are supplemented to feedstuff of these to increase the uptake of phytate phosphorus. In the present work efforts were made to isolate and characterize proficient phytase producing fungi from soil. Phytase producing fungi were isolated using phytate specific medium. Fungal isolates were selected according to their higher phytase activities. These isolates were further characterized and identified by morphological and microscopic analysis and confirmed by amplification of 18S rRNA gene, using specific primers. This gene was subsequently sequenced and phylogenetic affiliations were assigned. Fungal isolates were identified as various species of Aspergillus. Phytases from these fungi could be utilized as a feed additive in poultry and swine industries.

PRODUCTION AND CHARACTERIZATION OF PHYTASE FROM INDIGENOUS Aspergillus niger ISOLATES

“…Betancur et al (2012) also selected 26 for phytase producing fungal isolates. Zone of hydrolysis (12 ± 0.3 to 32 ± 0.2 mm) of different fungal isolates reported by Gontia-Mishra et al (2013) were comparable with zone of hydrolysis (25.03±0.1 to 49.00±0.11mm) observed in present study. Zones of hydrolysis were in contrast with reported zone of hydrolysis (8 ± 0.2 to 19 ± 0.1 mm) of Gupta et al (2014).…”

“…Microscopic characteristics were visualized using 400X magnification under bright field microscope. Screening for phytase production: Indigenous A. niger isolates were screened for phytase production following the method of Gontia-Mishra et al (2013). PSM was prepared by mixing: 15g glucose, 2g NH4NO3, 0.5g KCl, 0.5g MgSO4.7H2O, 0.3g MnSO4.7H2O, 0.001g FeSO4.7H2O and 20g agar in 1liter distilled water (pH 5.6).…”

“…Gupta et al (2014) screened isolated fungal isolates on PSM agar with Ca-phytate 0.5% as substrate. In another study PSM agar with 0.5% sodium phytate as substrate was used to screen different fungal isolates (Gontia-Mishra et al, 2013). Gangoliya et al (2015) used 0.1% Ca phytate as substrate to screen the A. fumigatus on PSM agar of pH 5.5.…”

“…Cobalt chloride 0.2%, ammonium molybdate 6.25% and ammonium vanadate 0.42% were used. Gontia-Mishra et al (2013) also screened Aspergillus spp. for phytase production through cobalt chlorie staining.…”

“…Phytase added in monogastric animal feed helps to cope with problem of non-bioavailability and environmental contamination of P (Casey and Walsh, 2004). Different microbes have been screened for their phytase production ability on phytase screening medium (PSM) (Howson and Davis, 1983) supplemented with different substrates: Calcium phytate (Gupta et al, 2014) and sodium phytate (Gontia-Mishra et al, 2013). Molecular weights of phytases have been determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).…”