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Cross compatibility and authentication of hybrid genotypes among species of Pleurotus were studied using lignocellulotic enzymes production and Internal transcribed spacer (ITS) of rDNA. Schematic procedure of hyphal anastomosis hybridization between the species of Pleurotus was employed in this study. Formation of clamp connexion, changes in morphological hyphal arrangement and general increased in the enzymes production by the hybrid strains over their wild types is an evidence for the compatibility of crossed genotypes. Highest produced enzyme was manganese peroxidase by hybrid LN 97 with 3.25 Uml-1, followed by laccase with 3.06 Uml-1 obtained in LN 91 (hybrid). Lignin peroxidase was the least enzyme produced, but with better performance in hybrid LN 97 (2.75 Uml-1). Dendrogram of relationship among the genotypes revealed two major clusters. Cluster II contained singleton (LN 95), hybrid strain which totally separated from both parents. Most strains clustered away from their parents, authenticating the hybrids produced. Current study shows the Pleurotus employed for this investigation are cross compatible with high expression of heterosis in hybrid strains relative to the parents on the basis of enzymes produced. This technique could serve as a good tool in breeding programs for Pleurotus strain improvement.
ISSN 1068-3674, Russian Agricultural Sciences, 2016, Vol. 42, No. 6, pp. 423430. Allerton Press, Inc., 2016.
PLANT GROWING
Compatibility Study using Hybridization Procedure among Pleurotus Genotypes and Authentication by Enzyme Expression and ITSR of rDNA1
E. A. Adebayo*, J. K. J Oloke, M. A. Azeez, A. A. Ayandele, and O. N. Majolagbe
Department of Pure and Applied Biology, Ladoke Akintola University of Technology, P.M.B. 4000, Ogbomoso, Nigeria *e-mail: [email protected], [email protected]
Received May 5, 2016
AbstractCross compatibility and authentication of hybrid genotypes among species of Pleurotus were studied using lignocellulotic enzymes production and Internal transcribed spacer (ITS) of rDNA. Schematic procedure of hyphal anastomosis hybridization between the species of Pleurotus was employed in this study. Formation of clamp connexion, changes in morphological hyphal arrangement and general increased in the enzymes production by the hybrid strains over their wild types is an evidence for the compatibility of crossed genotypes. Highest produced enzyme was manganese peroxidase by hybrid LN 97 with 3.25 Uml1, followed by laccase with 3.06 Uml1 obtained in LN 91 (hybrid). Lignin peroxidase was the least enzyme produced, but with better performance in hybrid LN 97 (2.75 Uml1). Dendrogram of relationship among the genotypes revealed two major clusters. Cluster II contained singleton (LN 95), hybrid strain which totally separated from both parents. Most strains clustered away from their parents, authenticating the hybrids produced. Current study shows the Pleurotus employed for this investigation are cross compatible with high expression of heterosis in hybrid strains relative to the parents on the basis of enzymes produced. This technique could serve as a good tool in breeding programs for Pleurotus strain improvement.
Keywords: anastomosis, laccase, lignin peroxidase, manganase peroxidase, oyster mushroom DOI: 10.3103/S106836741606001X
INTRODUCTIONMushroom is a macrofungus with a distinctive fruiting body, which can be either epigeous or hypogeous and large enough to be seen with the naked eye and to be picked by hand [1]. Pleurotus genus has 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 [2]. 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 [3], extra-cellular enzymes [4], flavoring agents [5], -glucosidases [6], antimicrobials [7] and vitamins [8]. Biomass, and intra and extracellular polysaccharides (EPS) are also the aim of several studies [7].
Besides its importance for food production, Pleurotus species are important in applications such as paper pulp bleaching, cosmetics, and other potential industrial uses [9]. Pleurotus species also plays a role in increasing macrophage and lymphocyte activities[10], reducing cholesterol levels [11], enhancing the
anti-complementary properties of polysaccharides[12], and increasing antihepatoma and antisarcoma activities [13]. Basidiomycetes fungi especially Pleurotus species are the most efficient lignin-degrading organisms that produce mainly laccases, lignin peroxidases and manganese peroxidases. These enzymes present a non-specific biocatalyst mechanism and have been used for bioremediation process due to their ability to degrade azo, heterocyclic, reactive and polymeric dyes [14]. Prospection of fungi is the ability to secret high levels of lignin-degrading enzymes with desirable properties for biotechnological applications. These applications have stimulated research on compatibility potential and specific aspects of the molecular biology of these organisms. Cultivars of the oyster mushrooms are readily affected by environmental conditions, making them difficult to differentiate. Disputes between farmers and spawn suppliers related to cultivated strains are becoming more frequent. Pleurotus species is the most commonly cultivated edible mushroom, and its consumption is continuously increasing, many attempts have been made to standardize the distribution of various Pleurotus cultivars for mushroom farming [9]. Identical strains with different commercial names or different strains with the same name often occur in the cultivation and
1 The article is published in the original.
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spawn market. Incorrectly designated strains can result in huge economic losses for farmers. Therefore, precise identification and classification of commercial lines of edible Pleurotus species strains are of major importance. The establishment of the compatibility nature of the Pleurotus species would play a major in improvement of yield production and quality of mushroom products, which is major target in mushroom cultivation.
The PCR investigations based on nucleotide sequences of the internal transcribed spacer (ITS) located between the nuclear rDNA 18S and 28S subunit genes, have made it possible to determine the relationships between fungal species from the genus Ganoderma [15]. Thanks to their nucleotide conservation, the sequences of which constitute useful species-specific markers for taxonomy and phylogeny of Basidiomycota. By comparison, in nuclear ITS sequences, which are to date the most investigated sequences in phylogenetic studies, numerous intra-species nucleotide variations were found. For example, 10 nucleotide variations have been described in the ITS1 and ITS4 sequences from the ITS2 sequences by two isolates (D383 and D1136) of P. cornucopiae [16]. So, when using nuclear ITS sequences to determine phylogenetic relatedness it is necessary to study numerous isolates from the same species. Hence, this study was designed to investigate the compatibility and authenticate hybrid strains among selected species of Pleurotus crossbred with P. Pulmonarius using enzymes activities and internal transcribed spacer region of rDNA.
MATERIALS AND METHODSStrains CollectionThe dikaryotic mycelium of P. pulmonarius LAU 09, P. ostreatus LAU 10, P. citrinopileatus NE 01,P. cornucopiae NE 02, Lentinus sajor-caju NE 05, andP. sapidus NE 07 were isolated and characterized at the Department of Pure and Applied Biology, Ladoke Akintola University of Technology, Ogbomoso, Nigeria. Strains were maintained on potato dextrose agar (PDA) slant at 4C for further uses.
Hybridization of P. pulmonarius with other Species of Pleurotus
Hybridization was carried out between two different dikaryon strains of Pleurotus species. Mating compatibility of P. pulmonarius LAU 09 with five other species of Pleurotus mentioned above were determined through interstrain pairing among dikaryon isolates and strains compatibility was assessed by scoring the presence of clamp formations [17]. Pairing was initiated in 90 mm Petri dishes containing 20 mL of PDA, inoculated with 6 mm agar plug from actively growing (7 d culture) dikaryon strains. In each plate, the agar plugs of two monocultures to be crossed were placed 20 mm apart and incubated at 25C until a well devel-
oped contact zone at juncture was established 12 day after incubation.
Enzymes AssayStrains were grown in PDB with 5% of yeast extract (YE). Experiments were carried out in 250 mL Erlenmeyer flask with 50 mL of substrate, inoculated with a plug (6 mm), incubated at 25C for 5 day with shaking at 150 rpm. Cultures were sieved (Whatman paper II), centrifuged and the filtrates were used as crude enzymes. The assayed enzymes were laccase, manganese peroxidase and lignin peroxidase.
LaccaseLaccase activity was determined via the oxidation of 2,2-azino-bis (3-ethylbenzthiazoline)-6-sulfonate (ABTS) (Sigma). Reaction mixture containing 0.1 mL of 0.3 mM ABTS in 100 mM of Sodium acetate (pH 3.5) and 0.0.1 mL of crude enzyme solution was incubated at 40C for 1 min. ABTS oxidation was monitored by the increased in absorbance at 420 nm ( = 36000 M1 cm1). One unit was defined as lumol of ABTS oxidized per minute and activity was expressed in Uper mL per min [18].
Manganese Peroxidase (MnP)
Manganese peroxidase (MnP) was assayed in a mixture of 0.9 mL of 50 mM Sodium lactate buffer (pH 4.0) containing 0.3 mM of Manganous ions (Mn2+) and 0.1 mL of crude enzyme solution, incubated at 40C for 1 min. The reaction was started by the addition of 200 L of 40 mM H2O2 and absorbance was measured at 270 nm ( = 8100 M1 cm1) [19]. One unit was defined as 1 mol complex Mn3+ lactate formed per mL per min.
Lignin Peroxidase (LiP)
Lignin peroxidase (LiP) activity was determined by monitoring the oxidation of veratryl alcohol (Sigma) at 310 nm for 1 minute at 40C [20]. Reaction mixture contained 0.1 mL of enzyme solution, 0.8 mL of 2 mM veratryl alcohol in 10 mM Sodium acetate with pH3.5. Reaction was started by adding 200 L of 0.1 mM H2O2. One unit was defined as 1 mol of veratralde-hyde released per min using extinction coefficient of 3900 M1 cm1.
Genomic DNA Extraction, Amplification Reaction and Sequencing for rDNA ITS
Genomic DNA for the isolates of Pleurotus species was extracted from the fungal hyphae using the DNA extraction protocol of White et al. [21]. Spectral analysis of genomic DNA for quantification and determi-
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Table 1. Strains identification with Genbank accession numbers for isolates of Pleurotus species
Strains Name Nature Genbank accession
LAU 09 P. pulmonarius Wild isolate JF736658 LAU 10 P. ostreatus Wild isolate JF736659 NE 01 P. citrinopileatus Wild isolate JF736661 NE 02 P. cornucopiae Wild isolate JF736662 NE 05 L. sajor-caju Wild isolate JF736663 NE 07 P. sapidus Wild isolate JF736664 LAU09 LAU10 (LL910) Pleurotus species Hybrid isolate JF680988 LAU09 NE01 (LN91) Pleurotus species Hybrid isolate JF680989 LAU09 NE02 (LN92) Pleurotus species Hybrid isolate JF680990 LAU09 NE05 (LN95) Pleurotus species Hybrid isolate JF680991 LAU09 NE07 (LN97) Pleurotus species Hybrid isolate JF680992
nation of its purity was done using spectrophotometric method. Absorbance of genomic DNA was measured at 260 nm to determine the concentration of DNA in solution while the absorbance at 280 nm was also taken to determine the extent of protein contamination in the extracted DNA. A preparation of good quality DNA should exhibit the following spectral properties; A260/A280 1.8 and A280/A260 0.55. Readings
were taken in Nanodrop (Thermo SCIENTIFIC). PCR was carried out using internal transcribed spacer (ITS) DNA assay with the following base nucleotide primers ITS1-F (CTTGGTCATTTAGAGGAAGTAA) and ITS4-B (CAGGAGACTTGT ACACGGTCCAG) by Gerdes and Bruns [22] method. The amplification program consisted of one cycle of initial denaturation at 94C for 1 minute 25 s, followed by 30 cycles of 95C for 35 s, 55C for 55 s and 72C for 2 min with final extension of 72C for 10 min. DNA amplification was performed in a thermal cycler system (Gene Amp 2700 Applied Biosystems). PCR products were purified using Exonuclease I and Shrimp Alkaline phosphatase in buffer (EXOSAP Kits). Both strands of the amplified region were sequenced using fluorescent dye terminator chemistry and were run on ABI 3130 (4 capillaries) or 3730X1 (96 capillaries) Automated Sequencer (Perkin Elmer Applied Biosystems, Foster City, A), 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 [23]. Sequence contigs were assembled and edited using Sequencer 3.0 software (Gene codes Corporation, Ann Arbor, MI). The sequences have been deposited in GenBank.
Phylogenetic Analysis
Phylogenetic trees were constructed by using all cloned sequences together with all non-redundant large subunit (nLSU) sequences of named Pleurotus
species obtained from GenBank. Multiple alignments of all the sequences were performed using CLUSTAL W (http://www.ebi.ac.uk/Tools/msa/clustalw2/), followed by manual adjustments. Phylogenetic analysis was performed with PAUP* v4.0 b10. Neighbor-joining method, using the Tamura-nei nucleotide substitution model was adopted. Boostrap test for estimating the reliability of phylogenetic tree topology was performed using 1000 replications by the SEQBOOT program. The consensus tree was obtained by running the consense program [24]. Tree view program [25] was used to view the phylogenetic tree.
RESULTSData such as code, name, nature and GenBank accession numbers of Pleurotus isolates used in this study are shown in Table 1. Isolates consist of six wild types (LAU 09, LAU 10, NE 01, NE 02, NE 05 and NE 07) and five hybrid strains (LL 910, LN 91, LN 92, LN 95 and LN 97) of Pleurotus. Compatibility test with inter-isolate crosses of dikaryons between the five isolates of Pleurotus species with P. pulmonarius gave positive result by forming clamp connexions (Table 2). Hybrids LL910, LN 91 and LN 97 showed very high compatibility (+++), followed by LN 95 (++) and low compatibility was observed in LN 92.
Schematic procedure of hybridization between the species of Pleurotus was shown in Fig. 1A,E. Two species with different morphological hyphal arrangement inter-crossed to form clamp connexion of hybrid strain with an interwoven morphological hyphal arrangement.
Table 3 showed the enzymes activity with highest activity obtained in the hybrid strains. Highest enzyme produced is manganese peroxidase with value of 3.25 Uml1 (LN 97), followed by 3.21 Uml1 (LN 92) and least value (0.52 Uml1) obtained from the wild type (NE 01). Laccase is another better produced enzyme with highest value of 3.06 Uml1 in
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Table 2. Inter-isolate crosses of dikaryons of Pleurotus species
+ = Clamp connexion observed (cross compatible) + low compatibility, ++ high compatibility, +++ very high compatibility.
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Strains LAU 09 Resulted
hybrids Compatibility
LAU 10 +++ LL910 Positive NE 01 +++ LN 91 Positive NE 02 +++ LN 92 Positive NE 05 ++ LN 95 Positive NE 07 +++ LN 97 Positive
LN 91, followed by LN 97 (3.00 Uml1) and least value of 0.35 Uml1 from NE 05 (wild type). The least produced enzyme is lignin peroxidase, with appreciable values obtained in hybrid strains as 2.75 Uml1
(LN 97), 1.67 Uml1 (LN 91) and the lowest value of0.30 Uml1 (LAU 10). Manganese peroxidase was produced maximally, followed by Laccase and Lignin peroxidase by all hybrid strains, and lowest enzyme yield were obtained in wild strains. Quantity and quality of Genomic DNA assessed using Nanodrop without RNase treatment is presented in Table 4, which manifested a very good yield of DNAs. Concentration of DNAs varied from 2071.70 ng/L (LAU 30) to 4894.20 ng/L (LN 91). The A260 to A280 DNA concentration was between 1.49 (least) in strain LN 91 and1.99 (highest) in isolate NE 01. Dendrogram of relationship shows two major clusters (Fig. 2). In the Cluster I were virtually all isolates and their hybrids except for LAU 09-NE 05, which was a hybrid between LAU 09 (Pleurotus pulmonarius) and NE 05 (Lentinus sajor-caju) that distinctly separated into cluster II (a singleton). Cluster I was further divided into four sub-clusters, with sub-cluster la containing four isolates (NE 02, NE 01, NE 05 and NE 07) and one hybrid (LAU 09-NE 02). Sub-cluster lb contained one isolate (LAU 09) and its hybrid (LAU09-NE07), sub-cluster Ic included one isolate (LAU 10) and another hybrid (LAU 09-NE 01), while sub-cluster Id was a singleton containing LAU 09-LAU 10 hybrid.
DISCUSSION
Isolates used in this study have been characterized to the species level and registered at the National Centre for Biotechnology information (NCBI) database. The obtained data on the genotypes used in this study has never been reported before in the literature. Formation of clamp connexion and interwoven morphological hyphal arrangement in the hybrid strains implies that the two parents crossed were compatible. The very high compatibility obtained through clamp connexion and pronounced interwoven morphological arrangement in the hybrids may be as a result of close ancestral among the species. Schematic hybridization procedure clearly revealed a simple and novel hybridization method for fungi species. Current study agreed with previous work reported by Lattera et al.[26], that anastomosis induction, followed by formation of clamp connexions, indicated compatibility among two strains and the formation of the corresponding dikaryon. Omoanghe et al. [27] reported a high compatibility between P. tuber-regium and other species of Pleurotus.
A bifactorial sexual compatibility system or tetrapolar heterothallism is characteristic of the genus Pleurotus and is controlled by two unlinked loci with multiple alleles. Mating systems of other Pleurotus species have been reported to elicit tetrapolar in nature [16,
Table 3. Enzymes Activity of Wild and Hybrid Strains of Pleurotus species
LACC: Laccase enzyme; LiP: lignin peroxidase; MnP: Manganese peroxidase.
Strains Enzyme Activity (Uml1)
LACC LiP MnP
LAU 09 1.80 0.06 0.40 0.01 0.58 0.05 LAU 10 2.40 0.12 0.30 0.00 0.52 0.07 NE 01 2.39 0.17 0.50 0.10 2.23 0.12 NE 02 0.55 0.02 0.56 0.08 2.40 0.11 NE 05 0.35 0.08 0.58 0.04 2.43 0.03 NE 07 1.60 0.07 0.41 0.01 2.45 0.14 LN 91 3.06 0.13 1.67 0.14 2.00 0.05 LN 92 2.91 0.16 1.15 0.05 3.21 0.16 LN 95 2.51 0.11 1.21 0.07 3.15 0.11 LN 97 3.00 0.21 2.75 0.11 3.25 0.15 LL910 2.14 0.09 1.34 0.05 3.05 0.04
Table 4. Quantification and purity of extracted Genomic DNA
A260 = Absorbance at 260, A280 = Absorbance at 280.
Strains A260 A280 A260/A280 DNA Con.
(ng/L)
LAU 09 97.536 62.303 1.57 4876.8 LAU 10 51.416 28.181 1.82 2570.8 NE 01 56.373 28.360 1.99 2818.6 NE 02 68.700 35.643 1.93 3435.0 NE 05 90.811 51.455 1.76 4540.5 NE 07 78.004 42.222 1.85 3900.2 LN 91 97.884 65.576 1.49 4894.2 LN 92 78.694 42.990 1.83 3934.7 LN 95 93.563 57.968 1.61 4678.2 LN 97 79.933 43.289 1.85 3996.6 LL910 62.015 33.105 1.87 3100.7
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(a)
(b)
(c)
LAU09
LAU10
LAU09 LAU09
LAU09 LAU09
LAU09
LL910
LL910
PINT
INT
LAU10
Mycelium of each species
Hyphal arrangement of each species
Clamp connexions of hybrid strain
Hyphal arrangement of the hybrid (LL910) showing clamp connexions; PINT: Pronounced interwoven
LN91
NE01 NE01
NE02
LN91
Mycelium of each species
Hyphal arrangement of each species
Clamp connexions of hybrid strain
Hyphal arrangement of the hybrid (LN91) showing clamp connexions; INT: interwoven
INT
LN92
NE02
Mycelium of each species
Hyphal arrangement of each species
Clamp connexions of hybrid strain
Hyphal arrangement of the hybrid (LN92) showing clamp connexions; INT: interwoven
Fig. 1. Schematic procedure of hyphal anastomosis crosses (hybridization) among genotypes of Pleurotus species. (a) Schematic procedure of hybridization between LAU 09 and LAU 10. (b) Schematic procedure of hybridization between LAU 09 and NE 01. () Schematic procedure of hybridization between LAU 09 and NE 02. (d) Schematic procedure of hybridization between LAU 09 and NE 05. (e) Schematic procedure of hybridization between LAU 09 and NE 07.
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(d)
(e)
LAU09 LAU09
LN95
INT
LN95
NE05 NE05
NE07
Mycelium of each species
Hyphal arrangement of each species
Clamp connexions of hybrid strain
Hyphal arrangement of the hybrid (LN95) showing clamp connexions; INT: interwoven
LAU09
LN97
LAU09
NE07
PINC
Mycelium of each species
Hyphal arrangement of each species
Clamp connexions of hybrid strain
Hyphal arrangement of the hybrid (LN97) showing clamp connexions; PINC: pronounced interwoven connexions
Fig. 1. Contd.
28]. In tetrapolar basidiomycetes, 25% of random intrastock crosses are expected to be compatible [29].
Highest enzymes yield reported in this study by hybrid strains has established increased in yield performance of the hybrid strains of Pleurotus species over their wild types, which is an evidence of strain improvement [17]. Lattera et al. [26] reported an increase in the production of laccase enzyme in hybrid strains from the hybridization of P. ostreatus andP. florida. Strains improvements always affect the yield production of an organism, Wang et al. [30] opined that biological efficiency of P. ostreatus increased with an increase in the ration of wheat bran, while on other hand, Stajic et al. [31] demonstrated that laccase production depends on the Plerotus species type of carbon
and nitrogen sources and their concentration in the medium. Highest production of MnP, laccase and LiP enzymes could be attributed to efficient lignin-degrading ability of Pleurotus species. Basidiomycetes are the most efficient lignin-degrading organisms that produce mainly laccases, manganese peroxidase, and lignin peroxidase [32]. These enzymes, as a non-specific biocatalyst mechanism have been extensively used for bioremediation process due to their ability to degrade azo, heterocyclic, reactive and polymeric dyes[14]. The A260 to A280 DNA concentration obtained between 1.49 (least) in strain LN 91 and 1.99 (highest) in isolate NE 01 indicate low amount of contamination by proteins and RNA [33]. The A260/A280 ratio
obtained for most isolates fell within the acceptable
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52
59 30 31
33
54
gi|329763836|gb|JF736662.1| Pleurotus cornucopiae strain NE02 gi|329763829|gb|JF680990.1| Pleurotus sp. LAU09-NE02 gi|329763835|gb|JF736661.1| Pleurotus citrinopileatus strain NE01 gi|329763837|gb|JF736663.1| Lentinus sajor-caju strain NE05 gi|329763838|gb|JF736664.1| Pleurotus sapidus strain NE07 gi|329763832|gb|JF736658.1| Pleurotus pulmonarius strain LAU09 gi|329763831|gb|JF680992.1| Pleurotus sp. LAU09-NE07 gi|329763833|gb|JF736659.1| Pleurotus ostreatus strain LAU10 gi|329763828|gb|JF680989.1| Pleurotus sp. LAU09-NE01 gi|329763827|gb|JF680988.1| Pleurotus sp. LAU09-LAU10 gi|329763830|gb|JF680991.1| Pleurotus sp. LAU09-LAU05
Ia
Ib
Ic
Id
I
II
9 25
0.02
Fig. 2. Dendrogram of relationship among the strains of Pleurotus, Lentinus and their hybrids using Neighbour joining method.
optimum range (1.80). Quality of extracted DNA was very high with the highest concentration of 4894.20 ng/L (LN 91) and least concentration of 2071.70 ng/L (LAU 30). High DNA concentration obtained might be adduced to very efficient isolation protocol adopted [33]. Amount and quality of the DNA obtained in this study were suitable for PCR amplification and other molecular assays [34]. Dendrogram of relationship revealed that most hybrids and either of their parents are closely related genetically. For instance, LAU 09-NE 02 showed very close resemblance to NE 02 (P. cornucopiae) in sub-cluster Ia than LAU 09 (P. pulmonarius) in sub-cluster Ib, while LAU 09-NE 07 resembles LAU 09 than NE 07 (P. sapidus) and are together in sub-cluster Ib. Both hybrids LAU 09-NE 01 and LAU 09-LAU 10 are distinctly separated from their parents and found in distinct sub-cluster from their parents, while hybrid LAU 09-NE 05 is completely distinct from either of its parents to form a singleton. This is not unexpected as this hybrid is formed from combination of two distinct genomes (P. pulmonarius Lentinus. sajor-caju), thereby giving rise to a new genome expressing high level of heterosis. A similar result was reported in cotton [35] and pointed to the possibility that the cross combinations suggested could also be adopted in breeding programs to increase genetic diversity that was low in the population as observed here. Conical crosses would broaden the genetic window and should aid breeding for high yield and disease resistance by creating better segregating populations [36].
CONCLUSIONS
Results obtained from the current study shows a clear compatibility among the genotypes with formation of clamp connexions. The changes in morphological hyphal arrangement, increment in enzymes production obtained in hybrid strain can be adopted in strain improvement technique. A novel hybridization
procedure or method for fungi species, especially mycelia fungi have been developed in this study. Divergence study that depicted closeness and diversity among the genotypes could serves as a good tool in breeding programs to increase genetic diversity.
REFERENCES1. Chang, S.T. and Miles, P.G., Mushrooms: Cultivation, Nutritional Value, Medicinal Effect, and Environmental Impact, Sulzycki, J., Ed., Boca Raton, FL: CRC Press, 2004, 2nd ed.
2. Pawlik, A., Grzegorrz, J., Joanna, K., Wanda, M., and Jerzy, R., Genetic diversity of the edible mushroom Pleurotus sp. by amplified fragment length polymorphism, Curr. Microbiol., 2012, no. 65, pp. 438445.
3. Rosado, F.R., Carbonero, E.R., Kemmelmeier, C., Tischer, C.A., Gorin, P.A.J., and Iaconini, M., A partially 3-0-methylated (1-4)-linked, B-D-salaetan and B-D-mannan from Pleurotus ostreatoroseus, Sing. FEMS Microbiol. Lett., 2002, no. 212, pp. 261265.
4. Garzillo, A.M.V., Di Paolog, S., Kuzzi, M., and Buonocore, V., Hydrolytic properties of extracelluar cellulases from Pleurotus ostreatus, App. Microbiol., 1994, no. 42, pp. 476481.
5. Martin, A.M., Study of the growth and biomass composition of the edible mushroom Pleurotus ostreatus, in Food Science and Human Nutrition, Charalambus, G., Ed., Amsterdam: Elsevier Science Publishers, 1992, pp. 239248.
6. Marois, H., Romas, C., Matos, N., Forgaes, E., Cserhati, T., Almeida, V., Oliveira, J., Darwish, Y., and Illes, Z., Liquid chromatographic and electrophoretic characterization of extracellular -glucosidase of Pleurotus ostreatus grow in organic waste, J. Chroma, 2002, no. B770, pp. 111119.
7. Adebayo, E.A., Oloke, J.K., Majolagbe, O.N., Ajani, R.A., and Bora, T.C., Antimicrobial and anti-inflammatory potential of polysaccharide from Pleurotus pulmonarius LAU 09, Afr. J. Microbiol. Res., 2012, no. 6 (13), pp. 33153323.
8. Solomko, E.F. and Eliseeva, G.S., Biosynthesis of group B vitamins by the fungus Pleurotus ostreatus in
RUSSIAN AGRICULTURAL SCIENCES Vol. 42 No. 6 2016
ADEBAYO et al.
submerged culture, Prikl. Biokhim., 1988, no. 24 (2), pp. 64169.
9. Sigoillot, C., Camerero, S., Vidal, T., Record, E., Asther, M., and Perez-Boada, M., Comparison of different fungal enzymes for bleaching high quality paper pulps, J. Biotechnol., 2005, no. 115, pp. 333343.
10. Kurashige, S., Akusawa, Y., and Endo, F., Effects of Lentinus edodes, Grifola frondosa and Pleurotus ostreatus administration on cancer outbreak and activities of macrophages and lymphocytes in mice treated with a carcinogen N-butyl-N-butanolnitrosamine, Inmunoph. Immunot., 1997, no. 19, pp. 175183.
11. Bobek, P. and Galbavy, S., Hyopocholesferolemic and anti-atherogenic effect of oyster mushroom (Pleurotus ostreatus) in rabbit, Nahrung, 1999, no. 43 (5), pp. 339 342.
12. Kwon, S.I. and Anderson, A.J., Laccase isozymes: Production by an opportunistic pathogen, a Fusarium proliferatum isolate from wheat, Physiol. Molecul. Plant. Pathol., 2001, no. 59, pp. 235242.
13. Wang, H., Gao, J., and Ng, T.B., A new lectin with highly potent antihepatoma and antisarcoma activities from the oyster mushroom Pleurotus ostreatus, Biochem. Biophys. Res. Comm., 2000, no. 275, pp. 810816.
14. Baldrian, P. and Snajdr, J., Production of ligninolytic enzymes by litter-decomposing fungi and their ability to decolorize synthetic dyes, Enz. Microbiol. Technol., 2006, no. 39, pp. 10231029.
15. Moucalvo, J.M., Wang, H.H., and Hseu, R.S., Phylo-genetic relationships in Ganoderma inferred from the internal transcribed spacers and 255 ribosomal DNA sequences, Mycologia, 1995, no. 87 (2), pp. 223238.
16. Vilgalys, R. and Sun, B.L., Ancient and recent patterns of geographic speciation in the oyster mushroom Pleurotus revealed by phylogenetic analysis of ribosomal DNA sequences, Proc. Nat. Acad. Sci., 1994, no. 91, pp. 45994603.
17. Adebayo, E.A., Oloke, J.K., Achana, Y., Barooah, M., and Bora, T.C., Improving yield performance of Pleurotus pulmonarius through hyphal anastomosis fusion of dikaryons, W. J. Microbiol. Biotechnol., 2013, no. 29, pp. 10291037.
18. Machado, K.M.G. and Matheus, D.R., Biodegradation of remazol brilhant blue R by ligninolytic enzymatic complex produced by Pleurotus ostreatus, Brazil.J. Microbiolol., 2006, no. 37, pp. 468473.19. Glenn, J.K., Akileswarean, L., and Gold, M.H., Mn(II) oxidation is the principle function of the extracellular Mn-peroxidase from Phanerochaetes chrysosporium, Arch. Biochem. Biophys., 1986, no. 251, pp. 688 696.
20. Tien, M. and Kirk, K.T., Lignin peroxidase of Phanerochaetes chrysosporium, Meth. Enzymol., 1988, no. 161B, pp. 238249.
21. White, T.J., Bruns, T., Lee, S., and Taylor, J., Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics, PCR Protocols: A Guide to Methods and Applications Academic Press, Innis, M.A., Gelfand, D.H., Sninsk, J.J., and White, T.J., Eds., New York, 1990, pp. 315322.
22. Gardes, M. and Bruns, T.D., ITS primers with enhanced specificity for basidiomycetes- application to
the identification of mycorrhizea and rusts, Mol. Ecol., 1993, no. 2, pp. 113118.
23. Vilgalys, R. and Hester, M., Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species, J. Bacteriol., 1990, no. 172, pp. 42384246.
24. Felsenstein, J., PHYLIP Phylogeny inference package (version 3.2), Cladist., 1989, no. 5, pp. 164166.
25. Page, R., Tree view: An application to display phylogenetic trees on personal computers, Comp. Appl. Biosci., 1996, no. 12, pp. 357358.
26. Lettera, V., Claudia, D.V., Alessandra, P., and Giovanni, S., Low impact strategies to improve ligninolytic enzyme production in filamentous fungi: The case of laccase in Pleurotus ostreatus, C. R. Biol., 2011, no. 334, pp. 781788.
27. Omoanghe, S.I. and Mikiashvill, N.A., Lignocellulolytic enzyme activity, substrate utilization, and mushroom yield by Pleurotus ostreatus cultivated on substrate containing anaerobic digester solids, J. Ind. Microbiol. Biotechnol., 2009, no. 36, pp. 13531362.
28. Zervakis, G. and Balis, C., A pluralistic approach in the study of Pleurotus species with emphasis on compatibility and physiology of the European morphotaxa, Mycol. Res., 1996, no. 100, pp. 717731.
29. Carlile, M.J. and Watkinson, S.C., The Fungi, London, Boston, San Diego, New York, Sydney, Tokyo: Academic Press, 1994.
30. Wang, D., Sakoda, A., and Suzuki, M., Biological efficiency and nutritional value of Pleurotus ostreatus cultivated on spent beer grain, Biores. Technol., 2001, no. 78, pp. 293300.
31. Stajic, M., Persky, L., Friesem, D., Hadar, Y., Wasser, S.P., Nevo, E., and Vukojevic, J., Effect of different carbon and nitrogen sources on laccase and peroxidases production by selected Pleurotus species, Enz. Microb. Technol., 2006, no. 38, pp. 6573.
32. Gomes, E., Aguiar, A.P., Carvlho, C.C., Bonta, R.M., Desilva, R., and Boscolo, M., Ligninases production by Basidiomycetes strains on lignocellulosic agricultural residues and their application in the decolorization of synthetic dyes, Brazil. J. Microbiol., 2009, no. 40, pp. 3139.
33. Tapia-Tussell, R., Lappe, P., Ulloa, M., Ramayo, A.Q., Farfan, M.C., Saavedra, A.L., and Brito, D.P., A rapid and simple method for DNA extraction from yeasts and fungi isolated from Agave fourcroydes, Res. Prot., 2006, no. 33, pp. 6770.
34. Plaza, G.A., Upchurch, R., Brigmon, R.L., and Whitman, W.B., Rapid DNA extractcion for screening soil filamentous fungi using PCR amplification, Pol. J. Environ. Stud., 2004, no. 13 (3), pp. 315318.
35. Ali, M.A., Seyal, M.T., Awan, S.I., Niaz, S., Ali, S., and Abbas, A., Hybrid authentication in upland cotton through RAPD analysis, Austr. J. Crop. Sci., 2008, no. 2 (3), pp. 141149.
36. Rahman, M., Hussain, D., and Zafar, Y., Estimation of divergence among elite cotton cultivars-genotypes by DNA fingerprinting technology, Crop Sci., 2002, no. 42, pp. 21372144.
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