Optimisation of the growth conditions and genetic characterisation of Pleurotus species
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A comprehensive genetic and optimisation of the growth conditions study of fifteen Pleurotus species and strains from Belgian Coordinated Collections of Microrganisms/ Mycotheque de l’ Université Catholique de Louvain (Agro) Industrial Fungi and Yeasts Collection was carried out. The optimal environmental conditions for the Pleurotus strains were determined by establishing variability in growth response and primordium development on agar media over a range of growth parameters: osmotic potential ( 0.5 MPa to 5.0 MPa), temperature (5 to 40 °C) and pH (2 to 12). The maximum growth rates and the optimum conditions for growth varied for the different strains. A Pleurotus ostreatus strain able to grow and fruit at elevated temperatures (25 °C) was identified that has potential for cultivation in the tropics. Nine strains were identified with high growth rates. Primordia were produced at low pH (4 to 6), at suboptimal growth temperatures ( 25 °C), and under moderate water stress (0.5 to 3.5 MPa). An investigation into the effect of micro-solute concentration changes on the growth response of the Pleurotus species was carried out on agar media using micro-concentrations of polyethylene glycol (PEG) 6000, potassium chloride (KCl) and sodium chloride (NaCl). The solutes at micro-concentrations ranging from 0.35 g l-1 to 1.00 g l-1 for PEG 6000, 1.50 g l-1 to 3.85 g l-1 for KCl and 0.60 g l-1 to 1.54 g l-1 for NaCl produced a two to three fold increase in the growth rate of the Pleurotus strains, which has potential application in oyster mushrooms (Pleurotus species) spawn production and cultivation. The optimum micro concentrations of combined KCl and NaCl solutes of 2.70 g l-1 KCl: 1.08 g l-1 NaCl or 3.85 g l-1 KCl: 1.54 g l-1 NaCl at 20 °C; and 1.50 g l-1 KCl: 0.60 g l-1 NaCl or 2.70 g l-1 KCl: 1.08 g l-1 NaCl for both 25 °C and 30 °C; of PEG 6000 of 0.90 g l-1 (20 °C) and 0.65 g l-1 (25 and 30 °C) were identified. Development of alternative spawn substrate protocols was done by varying the boiling, soaking periods for the substrates (maize [Zea mays] grain, maize cobs, pumpkin seeds [Cucurbita sp.] and bean seeds [Phaseolus vulgaris]). Maize grain, pumpkin seeds and maize cobs with a water content of 35 to 39 %, 41 to 47 % and 64 % respectively have potential use as alternative substrates to wheat (Triticum aestivum) grain in Pleurotus species spawn production. The feasibility of creating novel media formulations for culturing Pleurotus strains was investigated by developing various whole wheat grain and crushed wheat grain mixtures. The novel media formulations of 1:3 (w/v) to 1:5 (whole wheat grain: distilled water) and 1:3 to 1:15 (crushed wheat grain: distilled water) were determined to be optimal for use as low-cost alternative media for culturing Pleurotus species for spawn production and cultivation purposes. An investigation in the use of Tyndallisation as a means of sterilising the growth media for spawn production was carried out by modifying primarily the boiling times and number of days of incubation. Tyndallisation of 30 minutes boiling daily for three days (wide-mouthed bottles) and 5 minutes boiling for four days (narrow-mouthed bottles) were optimal for production of Pleurotus species spawn. Alternative single substrates and substrate mixtures (1:1) were determined by growth and basidiome production on thatch grass (Agropyron sp.), banana fronds (Musa sapientum), wheat straw, sage (Coleochloa setifera); banana + sage, banana + grass, banana + wheat straw and sage + grass. Grass and sage were the best substrates singly and in combination with banana. Biological efficiencies greater than 140 % were obtained. Positive and negative synergistic effects on primordium development dependant on the strain and substrate mixture were noted. Genetic variability of Pleurotus species and strains was investigated by sequencing the internal transcribed spacer (ITS) region of ribosomal DNA. The 15 Pleurotus strains separated into four clusters. The internal transcribed spacer sequences analysis suggested that strain 31674 was misidentified as P. sajor-caju instead of P. ostreatus. The value of noting the history of the strain in phylogenetic analysis was proposed. This study recommends BCCM/MUCL strain 31017 (P. sajor-caju) for spawn production and cultivation processes due to having the characteristics of high growth rate, high biological efficiency, prolific primordial formation and moderate xerotolerance which are highly desirable for spawn production and cultivation processes.