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High production and good quality are always the principal goals for agriculturally important crops, without the exception of mushrooms. P. pulmonarius is one of the commercially important edible mushrooms throughout the world. The yield performance improvement was carried out by cross bred P. pulmonarius with P. sapidus and P. ostreatus. The highest rate of 0.587 mm/days for spawn ramification and 53.33 % for percentage spawn productivity were obtained in hybrids LN LL910. The least day (11 and 12th) of the primodia mushroom sporophore were recorded in LL910 and LN 97 respectively, while longest day of 19th was recorded in wild type (NE 07). The highest biological efficiency (109.30 %) and production rate (3.77 %) obtained by LL910, while the least of 33.0 and 0.79 % were obtained by NE 07 for biological efficiency and production rate respectively. The morphological and molecular characterization of the hybrid strains established their true variation from their wild type. LL 910 (JF68088) is located at seventh subclusters from the root with boostrap value of 32 %, while only one parent (LAU 09: JF736658) out of the two has the close boostrap value of 43 % at the first subcluster to the root, with the other parent LAU 10 (JF736659) shows distance relationship after Blast. LN 97 (JF680992) is located at outgroup, while the parent strains NE 07 (boostrap value: 11 %) and LAU 09 (boostrap value: 44 %) located at tenth and second subclusters respectively. The results obtained from this study have shown the improved performance of the hybrids strain over wild type strains.[PUBLICATION ABSTRACT]
World J Microbiol Biotechnol (2013) 29:10291037 DOI 10.1007/s11274-013-1266-8
ORIGINAL PAPER
Improving yield performance of Pleurotus pulmonarius through hyphal anastomosis fusion of dikaryons
Elijah Adegoke Adebayo Julius K. Oloke
Achana Yadav Madumita Barooah
Tarun Chandral Bora
Received: 28 September 2012 / Accepted: 15 January 2013 / Published online: 26 January 2013 Springer Science+Business Media Dordrecht 2013
Abstract High production and good quality are always the principal goals for agriculturally important crops, without the exception of mushrooms. P. pulmonarius is one of the commercially important edible mushrooms throughout the world. The yield performance improvement was carried out by cross bred P. pulmonarius with P. sapidus and P. ostreatus. The highest rate of 0.587 mm/days for spawn ramication and 53.33 % for percentage spawn productivity were obtained in hybrids LN LL910. The least day (11 and 12th) of the primodia mushroom sporophore were recorded in LL910 and LN 97 respectively, while longest day of 19th was recorded in wild type (NE 07). The highest biological efciency (109.30 %) and production rate (3.77 %) obtained by LL910, while the least of 33.0 and 0.79 % were obtained by NE 07 for biological efciency and production rate respectively. The morphological and molecular characterization of the hybrid strains established their true variation from their wild type. LL 910 (JF68088) is located at seventh subclusters from the root with boostrap value of 32 %, while only one parent (LAU 09: JF736658) out of the two has the close boostrap value of 43 % at the rst subcluster to the root, with the other parent LAU 10 (JF736659) shows distance relationship
after Blast. LN 97 (JF680992) is located at outgroup, while the parent strains NE 07 (boostrap value: 11 %) and LAU 09 (boostrap value: 44 %) located at tenth and second subclusters respectively. The results obtained from this study have shown the improved performance of the hybrids strain over wild type strains.
Keywords Pleurotus Yield Hybridization
Performance Mushroom
Introduction
The development and improvement of edible mushroom strains should be an active topic for continuous research within the eld of mushroom cultivation owing to their enormous importance to humans, especially Pleurotus species. Pleurotus are characterized by a white spore print, attached to gills, often with an eccentric stip, or no stip at all. They are highly nutritive as they contain good quality proteins, vitamins minerals. They are low calorie food with very little fat and are highly suitable for obese persons, with no starch and very low sugars, they are the delight of the diabetics (Miles and Chang 1997). Fungi of the Pleurotus genus have an important place among the commercially employed basidiomycetes because they have gastronomic, nutritional and medicinal properties and can be easily cultivated on a large range of substrates. Besides the studies in solid culture aiming for the production of fruit bodies, the submerged culture of the genus Pleurotus has also been studied by several authors with the most varied objectives including the production of liquid inoculum, extra-cellular enzymes (Garzillo et al. 1994), avoring agents (Martin 1992), b-glucosidases (Marois et al.
2002), antimicrobials (Wisbeck et al. 2002) and vitamins
E. A. Adebayo (&) J. K. Oloke
Department of Pure and Applied Biology, Ladoke Akintola University of Technology, P.M.B. 4000, Ogbomoso, Nigeriae-mail: [email protected]
A. Yadav T. C. Bora
Biotechnology Division, North East Institute of Science and Technology, CSIR, Jorhat 785006, Assam, India
M. BarooahDepartment of Agricultural Biotechnology, Assam Agricultural University, Jorhat 13, India
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(Solomko and Eliseeva 1998). Biomass, intra and extra-cellular polysaccharides (EPS) are also the aim of several studies.
In recent years, there has been a trend toward discovering ways of treating mushrooms so as to give them added value. For example, Wermer and Beelman (2002) have reported on growing mushrooms enriched in selenium. Owing to the above mentioned importance of mushroom (Pleurotus species) to human, the development of edible mushroom strains improvement that will increase the yields production in term of fruit body and other biochemical substances should be a continuous research in mushroom cultivation, especially Pleurotus species. Till date, strain improvement of P. pulmonarius among the Pleurotus species has been a great task, due to its inter-sterility behavior (Bao et al. 2004) and ecologolical spec-icity (Kuo 2009) factors responsible for low performance of P. pulmonarius in several regions, especially in cold weather condition (Kuo 2009).
The hybridization through protoplast fusion of dikaryotic and monokaryotic of Pleurotus species has been reported (Toyomasu and Mori 1978). Since the rst cross breeding, which happened in 1983, generating two Agaricus bisporus hybrids Horst U1 and Horst U3 (Fritsche 1983), a lot of hybrids have been created, including Lentinula edodes (Zhang and Molina 1995) and Pleurotus ostreatus (Ma et al. 2004). However, no report is available on the strains improvement of P. pulmonarius using interstrain crosses by hyphal anastomosis fusion.
The present investigation was undertaken to improve the yield performance of the P. pulmonarius strain through Cross breeding by hyphal anastomosis fusion.
Materials and methods
The dikaryotic mycelium of P. pulmonarius LAU 09,P. sapidus- NE 07 and P.ostreatus- NE 08 were isolated from fruit body, characterized to the species level and registered at GenBank database. The strain was maintained on the potato dextrose agar (PDA) slant at 4 C, until further uses.
Strains hybridization
The hybridization was carried between two different dikaryons strains of Pleurotus species. The mating compatibility of P. pulmonarius LAU 09 with eight other strains of Pleurotus mentioned above was determined through interstrains pairing among dikaryons isolates and strains compatibility was determined by scoring the presence of clamp formations (Eger et al. 1976). Pairings were performed in 90 mm Petri dishes containing 20 ml of PDA, inoculated with 6 mm agar plug from actively growing
(7 days culture) dikaryons strains. In each plate, the agar plugs of two monocultures to be tested were place 20 mm apart and incubated at 25 C until a well developed contact zone at juncture was established (12 days).
Temperature tolerance of the wild and hybrid strains
The temperature optima for mycelium linear growth were assessed on potatoes dextrose agar (PDA) for wild, mutant and hybrid strains with 5 % of yeast extract agar (YEA) over the range of 1535 C with a 5 C increment.
Experiments were conducted on Petri dishes (Zervakis et al. 2001), incubated for 7 days and linear growth rates were determined by Y = KrX ? C (where Y is the distance, X is time), with diameter of mycelia growth.
The optima pH for mycelia growth
The pH optima for mycelia growth were carried out on potato dextrose broth (PDB) with 5 % of yeast extract powder (YE) over the range of 49, adjusted with (0.1 M) of HCl and NaOH. Experiments were carried out in 250 ml asks containing 100 ml substrate. The asks were inoculated with a plug (6 mm) and incubated at 25 C for 7 days at 150 rpm. The mycelia mats were harvested and quantied (Oloke et al. 2009).
Morphological examination
The actively growing culture (5 days old) of wild, mutant and hybrid strains were smeared on slides with cover slip and stained with Lactophenol blue and examined under the light microscope (OLYMPUS CX41).
Spawn production
Paddy grains (100 g/bottle) were washed four times, boiled for 45 min and dried. The dried grains were mixed with 1 %w/w of Calcium carbonate (CaCO3), dispensed in bottles and sterilized at 121 C for 30 min. The sterile grains were inoculated with 6 plugs (6 mm) of actively growing culture, incubated at 23 2 C and mycelia running were recorded 3 days interval. The following parameters were determined; rate of mycelia ramication, ramication days, weight of ramied mycelia and spawn productivity.
Sporophore/fruit body production (rice straw as substrate)
Paddy straw was shredded, soaked for 24 h (1 straw: 3 water). The straw was boiled for 3 h (pasteurization), dried in sterile condition and packed into sterile bags (1.4 kg/bag),
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inoculated with spawn (510 %w/w) and carefully shaken down to allow even spawn distribution throughout the bag. The bags were incubated in the dark at 25 3 C, until fully colonized. The colonized bags were transferred into mushroom house and watering was carried out periodically for sporophore production.
Amino acids sequence and composition analysis
The nucleotides sequence of wild and mutant strains were translated into different amino acid compositions to show the variation among strains using http://www.geneplot.plasmodb.org
Web End =http://www.geneplot.plas http://www.geneplot.plasmodb.org
Web End =modb.org .
DNA extraction and ITS amplication
Mycelia were grown on potato dextrose agar, harvested using a scalpel, transferred into Epperdorf tubes, small amount of autoclaved rened sand (Sigma) was added and ground to ne paste with pestle-like stick (High Media), 400 ll of DNA Extraction buffer pH 8 (1 M TrisCl pH8.0; 1 M NaCl; 200 mM EDTA pH 8.0; 10 % SDS; 0.1 % b-Mercaptoethanol) was added and centrifuged at 4 C (12,000g) for 10 min. To the collected supernatant, 300 ll Phenol and 300 ll Chloroform: Isoamylalcohol (24:1) were added and mixed gently. This was centrifuged (12,000g, 4 C for 10 min), and aqueous phase was collected and 500 ll chilled Isopropanol was added and incubated at -20 C overnight. After the incubation, it was centrifuged (12,000g, 4 C for 10 min), and the pellet was washed with chilled 70 % ethanol centrifuged for 5 min. The dried pellet was resuspended in 50 ll of Tris EDTA(10 Mm Tris and 1 mM EDTA, pH 8.0) buffer. Amplication of the ITS region of the rRNA gene was carried out with a modied method of Gardes and Bruns (1993), using primers ITS1-F and ITS4-B. The nal concentration of 25 ll PCR reaction volume consists of 200 lM each of dATP, dCTP, dGTP and dTTP, 2.5 mM MgCl2, 10X Taq. DNA Polymerase and 20Pico mole of each of the two primers (Banglore Genei). The PCR proles have initial denaturation step of 94 C for 85 s followed by 25 amplication cycles of denaturation, annealing and extension. The temperature and times for these steps were 95 C for 35 s, 55 C and 55 s and 72 C for 2 min with further incubation at 72 C for 10 min. The amplied PCR products were resolved on a 1.2 % agarose gel, and stained with Ethidium bromide. A 1 kb ladder DNA marker (GeneRulerTM) was used as a size standard.
Sequencing and phylogenetic analysis
The PCR products were puried using Exonuclease I and Shrimp Alkaline phosphatasa in buffer (EXOSAP Kits).
Both strands of the amplied region were sequenced using uorescent dye terminator chemistry and were run on ABI 3130 (4 capillary) or 3730XI (96 capillary) Automated Sequencer (Perkin Elmer Applied Biosystems, Foster City, CA), following the manufacturers protocols. Sequencing primers were ITS1-F, 5.8S, 5.8SR and ITS4-B. Oligonucleotide sequences for primers 5.8S and 5.8SR were given in Vilgalys and Hester (1990). Sequence contigs were assembled and edited using Sequencer 3.0 software (Gene codes Corporation, Ann Arbor, MI).
Phylogenetic trees were constructed by using all cloned sequences together with all nonredundant large subunit (nLSU) sequences of named Pleurotus species obtained from GenBank. The multiple alignments of all the sequences were performed using CLUSTAL W (http://www.ebi.ac.uk/Tools/msa/clustalw2/
Web End =http://www.ebi.ac.uk/ http://www.ebi.ac.uk/Tools/msa/clustalw2/
Web End =Tools/msa/clustalw2/ ), followed by manual adjustments. The phylogenetic analyses was carried out using sequence data of ITS 5.8 s and 28 s ribosomal RNA gene from LAU 09 (wild) and LAU 60 (mutant) of P. pulmonarius and corresponding GenBank data of related species. The identical sequences were merged into one input sequence when running the computer programs to generate the phylogenetic trees constructed by UPGMA, Neighbor-joining (NJ) and parsimony methods. The boostrap test for estimating the reliability of phylogenetic tree topology was performed using 100 replications by the SEQBOOT program (Felsenstein 1989). The consensus tree was obtained by running the consense program (Felsenstein 1989).
Statistical analysis
ANOVA for the mycelia yield production under different conditions of growth factors was performed using SPSS software (Version 16). The data used were in triplicates. The accession means for the mycelia yield measured were to compare the performance of the strains. Accession differences were determined by Duncan Multiple Range Test (DMRT).
Results
The compatibility test with inter-isolate crosses of dikaryons between P. pulmouarius-LAU 09 and P. ostreatus-LAU10 and P. sapidus- NE 07 which gave a positive result of hybridization by forming clamp connections at the line of juncture that are shown in Figs. 1 and 2 for LL910 and LN 97 respectively.
The Linear growth rates of wild and hybrid strains of P. pulmonarius at different temperature ranges were showed in Table 1, with satisfactory growth obtained at 20 C and optima growth temperature at 2530 C for all the strains, while LN 97 (hybrid) produced high yield at all evaluated
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Fig. 1 The clamp connection between LAU 09 and LAU 10 (LL 910 hybrid)
Fig. 2 The clamp connection between LAU 09 and NE 07 (LN97 hybrid)
Fig. 3 The hyphae arrangement of LAU 09
Fig. 4 The hyphae arrangement of LAU 10
temperature values. The means of mycelia yield obtained by LN 97 (hybrid) was signicantly different (p \ 0.05)
from all other strains in all evaluated temperature values except at 25 C. while LL910 (hybrid) was also signicantly different (p \ 0.05) from all other strains in all evaluated temperature values except at 1520 C The highest mean value of linear growth rates (1.764 mm/days) was obtained by LL910 at 25 C, while the lowest mean value of linear growth rates (0.213 mm/days) was obtained by NE 07 at 15 C.
The highest means of dried mycelia weight (0.44 g) was obtained by LN 97 (hybrid) at pH 7, and lowest mycelia yield (0.20 g) was obtained at pH 9 by LAU 10 (wild) (Table 2). The means of mycelia yield by LN 97 were signicantly different (p \ 0.05) at pH 5, 6, 7, 8 and 9.
The highest means of spawn ramication rates(0.587 cm/days), highest means of weight of mycelia ramication (6.400 g) and highest means of percentage spawn productivity (53.33 %) with shortest days of ramication (12 days) were obtained by LL910 (hybrid). The lowest means of spawn ramication rates (0.412 cm/days) was obtained by LAU 09, and lowest means of percentage spawn productivity (23.12 %), lowest values of weight of mycelia ramication (3.700 g) and longest days of ramication (16 days) obtained by LAU 10 (wild) (Table 3). The means values of RT, WMR and PDT obtained by LL910 (hybrid) was signicantly different (p \ 0.05) from the obtained values of RT, WMR and PDT by all other strains.
Table 4 shows the distribution of the total fresh mushroom harvested and primodia formation days for wild and hybrid strains, with the shortest primodia formation days (11 days) and highest total mushroom weight (1,093 g) obtained by LL910 (hybrid), while the longest primodia formation days (19 days) and lowest total mushroom weight obtained by NE07 (wild). The biological efciency (BE) and production rate (PR) of 109.30 21.58 and
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Table 1 Linear growth rates (mm/day) of wild and hybrid strains of P. pulmonarius at different temperature ranges
Temp (C) strains 15 20 25 30 35
LAU 09 0.455 0.05d 1.061 0.11b 1.500 0.31b 1.092 0.48b 0.857 0.08b
LAU 10 0.509 0.39c 0.962 0.22c 1.031 0.27d 1.022 0.26c 0.905 0.08b
NE 07 0.213 0.01e 1.056 0.04b 1.026 0.01d 1.030 0.01c 0.546 0.04c
LL910 0.638 0.04b 1.089 0.07b 1.764 0.18a 1.575 0.08a 1.262 0.09a
LN 97 0.768 0.06a 1.218 0.05a 1.490 0.07c 1.566 0.06a 1.322 0.05a
The mean values on the same column followed by different letters are signicantly different at 0.05 probability level according to Duncan Multiple Range Test
Table 2 Weight of dried mycelia yield of wild, mutant and hybrid strains of P. pulmonarius at different pH values
pH strains 4 5 6 7 8 9
LAU 09 0.39 0.03a 0.38 0.01a 0.39 0.02a 0.37 0.02b 0.40 0.04a 0.30 0.02b
LAU 10 0.26 0.01c 0.32 0.01b 0.28 0.02b 0.26 0.02d 0.24 0.02c 0.20 0.02c
NE 07 0.34 0.04b 0.30 0.03b 0.30 0.02b 0.34 0.04c 0.33 0.03b 0.30 0.06b LL910 0.36 0.03b 0.37 0.01a 0.38 0.01a 0.32 0.02c 0.32 0.04b 0.30 0.0b
LN 97 0.36 0.02b 0.38 0.02a 0.41 0.03a 0.44 0.02a 0.42 0.00a 0.42 0.03a
The mean values on the same column followed by different letters are signicantly different at 0.05 probability level according to Duncan Multiple test
Table 3 Spawn ramication rates (cm/day) of wild, mutant and hybrid strains of P. pulmonarius
Strains RT (cm/day) RD (day)
WMR (g) PDT (%)
LAU 09 0.412 0.19b 16 4.900 0.11c 30.62 0.72c
LAU 10 0.425 0.10b 16 3.700 0.17d 23.12 1.08e
NE 07 0.437 0.02b 16 4.200 0.15c 26.25 0.95d LL910 0.578 0.05a 12 6.400 0.20a 53.33 1.73a
LN 97 0.587 0.02a 12 6.100 0.05b 50.83 0.48ab
The mean values on the same column followed by different letters are signicantly different at 0.05 probability level according to Duncan Multiple Range Test
RT ramication rate, RD ramication days, WMR weight of mycelia ramication, PDT spawn productivity
3.77 1.05 respectively were obtained by LL910 (hybrid), while lowest BE (33.00 7.93) and PR(0.79 0.04) were produced by NE07 (wild) (Table 5). The obtained mushroom yield in the hybrid strains in this study, especially in LL910 is better than the wild type strains.
Table 6 shows differences in amino acid compositions between wild and hybrid strains of P. pulmonarius. The type of amino acids produced does not vary among the strains, but there is variation in number of amino acids produced. The highest number (63) of amino acid was obtained by LL910 and LN 97 (hybrids), while the least number of amino acids (47) produced by NE 07 (wild). The
initial amino acid (IA) is aspartic acid (D) for both wild and hybrid strains. The terminal amino acids obtained for all strains are the same (Asparagine- N) except LN 97 (hybrid) which has E (glutamic acid).
The inter-crosses of the hyphal arrangement with a gel-like substance inside the lament of the hybrid LL910 (Fig. 5) showed a positive test of compatibility test between LAU 09 (wild) and LAU 10 (wild) Figs. 3 and 4 respectively. The hyphal arrangement of the hybrid (LL910) strains showed a diverged hyphal structure quite different from both parents. This might be responsible for its better adaptability to physiological parameters and high yield production rates. The hyphal arrangement of the hybrid strain (LN97Fig. 8) shows a signicant different from its wild types (LAU09Fig. 6 and NE07Fig. 7), in that the hyphal was coiled together in parallel form with segmented appendages, which indicate that an hybrid has been produced which is different from its wild types.
The phylogenetic analyses was carried out using sequence data of ITS 5.8 and 28 s ribosomal RNA gene from wild and hybrid strains of P. pulmonarius and corresponding GenBank data of related species. The identical sequences were merged into one input sequence when running the computer programs to generate the phylogenetic tree constructed by UPGMA, Neighbor-joining (NJ) and parsimony methods. The outgroup for the hybrid strain of P. pulmonarius LL 910 (accession number: JF680988) for phylogenetic evaluation is an hybrid strain of Pleurotus species (JF680992) from GenBank database. The rooted UPGMA tree (Fig. 9) shows two phylogenetic lineages
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Table 4 Distribution of the total fresh mushroom production (g) over three harvest by wilds and hybrids strain of P. pulmonarius on Saw dust
Strains IP PP 1st Harvest (g) (%) 2nd Harvest (g) (%) 3rd Harvest (g) (%) Total weight (g)
LAU 09 14 32 210 (35.53) 198 (33.50) 183 (30.96) 591
LAU 10 17 37 121 (32.97) 140 (38.15) 106 (28.88) 367
NE 07 19 42 108 (32.97) 102 (30.91) 120 (36.36) 330
LL910 11 29 495 (45.29) 312 (28.55) 286 (26.17) 1,093
LN 97 12 29 199 (31.79) 231 (36.90) 196 (31.31) 626
IP incubation period (required days for formation of primordia), PP Production period, starting with the formation of primordia till third harvest, Weight of mushrooms obtained in 5 replicates, percent production for the strains during each harvest
Table 5 Biological efciency (%) and Production rate (%) of the wilds and hybrids strain of P. pulmonarius
Strains BE (%) PR(%)
LAU 09 59.10 12.4c 1.85 0.65c
LAU 10 36.70 11.25d 0.97 0.76d
NE 07 33.00 7.93d 0.79 0.04d
LL910 109.30 21.58a 3.77 1.05a
LN 97 62.60 14.60b 2.16 0.91b
The mean values on the same column followed by different letters are signicantly different at 0.05 probability level according to Duncan Multiple Range Test
BE Biological Efciency (Productivity) of the each strain, PR Production Rate of the each strain
Table 6 The differences in amino acid compositions between wild and hybrid strains of P. pulmonarius
Strains TAP NA IA TA
LAU 09 5 56 D N
LAU 10 5 56 D N
NE 07 5 45 D N LL910 5 63 D N
LN 97 5 63 D E
TAP type of amino acids produced, NA numbers of amino acids prod, IA initial amino acid, TA terminal amino acid, D aspartic acid, N Asparagine, E glutamic acid
Fig. 5 The hyphal arrangement of LL910 (hybrid) showing a pronounced intercrosses of the hyphae
Fig. 6 The hyphae arrangement of LAU 09
Fig. 7 The hyphae arrangement of NE 07
with thirteen subclusters, arising from the root. One leads to a terminal node containing a strain of P. pulmonarius LAU 09 from GenBank database (JF736658). LL 910 (LAU 09- LAU 10; JF68088) is located at seventh subclusters from the root with boostrap value of 32 %, while only one parent (LAU 09: JF736658) out of the two has the close boostrap value of 43 % at the rst subcluster to the root, with the other parent LAU 10 (JF736659) shows a distance relationship after Blast.
The hybrid strain of P. pulmonarius LN 97 (accession number: JF680992) stand as an outgroup in phylogenetic evaluation of the same hybrid strain (JF680992) from
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GenBank database. The rooted UPGMA tree (Fig. 10) shows two phylogenetic lineages with twelve subclusters, arising from the root. One leads to a terminal node containing a strain of P. ostreatus (EU420068) from GenBank database. LN 97 (LAU 09- NE 07; JF680992) is located at outgroup, while the parent strains NE 07 (boostrap value: 11 %) and LAU 09 (boostrap value: 44 %) located at tenth and second subclusters respectively.
Discussion
The clamp connections formation is proof of true production of hybrid strains form in this study. The protoplast
fusion crosses involving mating type compatible strains of Coprinus macrorhzus (Kiguchi and Yanagi 1985) gave rise to heterokaryons which fruited normally, and heterokaryons were also obtained at a similar frequency when protoplasts incompatible strains were fused, but not surprisingly these failed to develop fruit bodies. Omoanghe et al. (2009) reported the high compatibility betweenP. tuberregium and other species of Pleurotus. A bifactorial sexual compatibility system or tetrapolar heterothallism is characteristic of genus Pleurotus and is controlled by two unlinked loci with multiple alleles. Mating systems of other Pleurotus species that have been tested have also been tetrapolar (Vilgalys and Sun 1994; Vilgalys et al. 1996; Zervakis and Balis 1996). In tetrapolar basidiomycetes, 25 % of random intrastock crosses are expected to be compatible (Carlile and Watkinson 1994).
The results obtained at different evaluated temperature ranges are consistent with an earlier report, that the optimum temperature for mycelia growth for Pleurotus species was established at 2530 C, with no growth observed at 35 C (Zervakis et al. 2001; Kibar and Peksen 2008). The considerable high yield of mycelia obtained at 35 C is a proof that the hybrids strain has the ability to tolerate high range of temperature. The better performance of hybrid strains at pH 5, 6, 7, 8 and 9 were obtained in this study. The broad ranges of pH 3.5 to pH 6 were previously reported as optima pH for fungal growth and products yield (Membre et al. 1999; Restaino et al. 1983). The ability of the hybrid strains to satisfactorily produced mycelia mat at higher pH values above previously reported pH values is an indication of strains improvement.
Fig. 8 The hyphal arrangement of LN 97 (hybrid) showing a pronounced interwoven of the hyphae
Fig. 9 The phylogenetic tree of LL910 (hybrid) constructed by UPGMA, with boostrap values from 100 replicates showing maximum similarity with LAU 09 and LAU 10 (wilds) and the homogeneous strains of Pleurotus species
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Fig. 10 The phylogenetic tree of LN97 (hybrid) constructed by UPGMA, with boostrap values from 100 replicates showing maximum similarity with LAU 09 and NE 07 (wilds) and the homogeneous strains of Pleurotus species
The means values of spawn ramication rates, weight of mycelia ramication and percentages spawn productivity showed signicantly differences in LL910 from all other strains. This has established true strains improvement in comparison to wild strain and previous report. Isikhuemhen and LeBauer (2004) reported the average 2030 days for spawn ramication for the Pleurotus species. The highest value of WMR, RT and PDT at shorter RD which obtained by hybrid strains over their wild types are of commercial importance in mushroom production. The shortest primodia formation days (11 days) and highest total mushroom weight (1,093 g) obtained by LL910 (hybrid) obtained in this study is better than that of Vogel and Salmones (2000), who observed pinhead formation after an incubation period of 23 days in commercial culture of P. ostreatus, while Obodai et al. (2003) cited incubation periods of 34 days when using rice straw as substrate. The biological efciency (109.30; 62.60 %) and production rate (3.77;2.16 %) were obtained by LL910 and LN 97 respectively. The obtained values of BE and PR by hybrid strains were higher than the values reported by Salmones et al. (1997), who observed BE values of 16.871.9 % and values of0.481.38 %. The highest BE and PR reported in this study, especially hybrids strain has established increased in yield performance of the hybrid strains of P. pulmonarius LAU 09, which is an evidence of strain improvement.
The differences in amino acids composition obtained in this study might be as result of alteration in nucleotides sequence which has altered the amino acid sequence and eventually will affect the protein synthesis of the organisms. The predicted amino acid sequence consisted of 246 amino acids and showed a high similarity to known fungal pyrG sequences. The deduced amino acid sequence of
pyrG from P. ostreatus showed a 93.09 % similarity to that of pyrG from P. eryngii with 16 amino acid differences (Yin et al. 2005).
The diverged hyphal structure obtained in the hybrid strains different from their parent strains might responsible for their differences in mycelia formation. The interspecic and intergeneric nuclear hybrids colonies were distinguished from both parents because they produced more compact and more vigorously growing mycelium (Yoo et al. 1988). The cladistic position of the hybrid strains in the phylogenetic tree suggested that there was a common ancestor since sub ancestors diverged majorly at early stages of evolution (vilgalys et al. 1996). The previous study on mating compatibility (Zervakis and Balis 1996) reported different phylogenetic lineages between P. cystidious and P. smithii which recently diverged species based on the relatively short branches that separate them. The identication of 12 biological species among 25 Pleurotus morphological species from mating compatibility tests have been reported by Bao et al. (2004). Phylogenetic analyses based on the PCRRFLP data of the partial 26 srDNA has also reported (Bao et al. 2004) which revealed that 9 of the biological species, theP. cornucopiae complex, P. cystidiosus complex, P. salmoneos-tramineus, complex, P. dryinus, P. nebrodensis,P. smithii, and P. ulmarius were congruent with independent phylogenetic lineages.
Conclusion
The result obtained shows the improved performance of hybrid strains over the wild (parent) strains. The hybrid strains gave the highest yield in term of spawn
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productivity, total weight of fresh fruit body, biological efciency and production rate. So also, the morphological and molecular variation with evolutionary divergence of the hybrid strains from the wild type is true evidence of a novel strain of Pleurotus species developed from this study.
Acknowledgments The Director of NEIST, India is gratefully acknowledged for granting facilities available to carry out this research, so also TWAS, Italy; and CSIR for the award of Postgraduate Fellowship given to me and utilized at NEIST, CSIR, Jorhat, India, and also to the authority of LAUTECH, Ogbomoso, Nigeria for granted the study leave to utilized the award.
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