Identification of Ganoderma Isolates from Egypt Based on Morphological Characters and ITS1-rDNA Genetic Marker

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Since then, over 290 taxonomic names in the genus of Ganoderma have been published, indicating that this genus is morphologically complex (Roberts, 2004).
The Taxonomic classification of Ganoderma species is constructed essentially with respect to morphological features of the basidiocarps, geographical origin, physiological and developmental characters, and chemical components such as secondary metabolites and host specificity (Zheng et al. 2009).However, the morphological concept for Ganoderma identification is still controversial particularly for the tropical species and can be misguiding due to distinct factors, such as crossbreeding and hybridization (Olson & Stenlid, 2002), ambiguous speciation and convergent evolution (Zhou, et al., 2015).As a result, the concept of species in this genus is not well established nor universally accepted.Over the last few decades, it has been demonstrated that the morphology and culture characteristics of species from the same genus can be enormously influenced by growth conditions.Identification of Ganoderma species depend on morphology data may occur many synonyms because the number of species the form of the basidiocarp has been affected by the environment.The basidiospores by latitude and altitude and in some species, in southern latitudes, the context color more dark than Northern latitudes on the European continent.A remarkable effect has been shown on the color, size, and brightness of the basidiocarp, and the existence, absence or longitude of the stipe by the age and environment (Moncalvo, 2000).
Ganoderma diversity seems not yet totally discovered, at least in tropical Africa where it and the polypore mycobiota have also been insufficiently probed.Through literature scanning and Index Fungorummyco bank (http://www.indexfungorum.org/Names/Names.asp.), around 473420 unique names have been utilized in Ganoderma, compared to less than one-third which are valid names (Ahmed-Reda., 2007;Douanla-Meliand Langer, 2009).Molecular procedures could be used to characterize and utilized to describe and distinguish the species.With the improvement of PCR-based techniques, the use of molecular data for taxonomic purposes have been utilized broadly to determine clashing information from morphological attributes (Rolimetal, 2011 andZakaria et al., 2005).The Internal Transcribed Spacer (ITS) regions are probably the most important regions in fungi for molecular systematics within a genus for being generally conserved DNA regions within one species because the DNA region has a considerably higher sequence variation between different species.The highly conserved ribosomal genes, which flank the ITS regions, are ideal for universal primer targeting and as a result the ITS regions can be amplified by PCR, then the sequences analyzed, compared and evolutionary trees produced.The ITS regions, a gene marker for fungi are highly variable and for this reason are useful in separating related species and strains of Ganoderma was sequenced for the intent of a molecular analysis.(Wang et al., 2009).The region of the ribosomal internal transcribed spacer (ITS) are likely the most important regions in fungi for molecular systematics within a genus due to the ITS regions generally conserved DNA regions within one species but, in contrast, the DNA region has a considerably higher sequence variation between different species.ITS is also used to measure the genetic distances between fungal different groups (Del-Prado R et al., 2010).The highly conserved ribosomal genes, which flank the ITS regions, are ideal for universal primer targeting and therefore the ITS regions can be amplified by polymerase chain reaction (PCR), the sequences analyzed and compared, and evolutionary trees produced.
In phylogenetic studies based upon the sequences of (ITS) regions of ribosomal DNA (rDNA), it was demonstrated that extensive assemblage of morphological characters has taken place during the development of Ganoderma (Hong and Jung, 2004) after in advance research (Zakaria et al., 2009).In addition, molecular mycology was performed easily by the accessibility of fungi DNA sequences in GenBank.Furthermore, the ability to be fit to identify some certain fungi with the aid of using DNA sequences only has confirmed the effectiveness of molecular mycology in issues where conventional taxonomic techniques did not produce conclusive stable classification groups (Ekandjo and Chimwamurombe, 2012).
The aim of the present study was to collect samples of Ganodermabasidiocarps from different hosts (Navel orange, Oil palm, Fan palm, Casuarina and Morus) in Giza and Qalyubia governorates in Egypt; making pure cultures of Ganoderma from its basidiocarps and then, identifying themto species level according to morphological characters as well as ITS1-rDNA genetic marker.Small pieces of basidiocarps were soaked for 1 min.in hydrogen peroxide (5%) solution for surface sterilization, rinsed with sterile water and air dried.Internal tissues of fruiting bodies were removed and cultured on Potato Dextrose Agar (PDA) medium.Standard aseptic laboratory procedures were used, and all plates were incubated at 25°C.Emerging colonies of Ganoderma fungi were sub-cultured onto PDA medium till pure cultures were got.The purified isolates were preserved on the same medium at 4-5ºC until used (Kandan et al., 2010).Morphological identification: Macro-morphological features: Morphological characters such as, type of basidiocarp (stipitate/sessile/dimidiate, imbricate, concave, number of concentric zones, etc.), laccate and non-laccate, in addition, margin shape (lobed, fertile/sterile, rounded/ acute) and color (brown, white, reddish, etc.), pores color, tube size and color, context were recorded.Micro-morphological features: For internal morphology, free-hand sections were taken from the cutis, context and from the tube layer of each sample (3) respectively.a block of tube layer was used to isolate basidiospores.Then, the sectioned material was handle with KOH (10%), washed with water to loosen the hyphae.Lactoglycerine (50%) was used as mounting media.Spores were scraped from the pore surface into the mounting solution for observation.Styles of the hyphal system i.e. number, color and diameter were examined.the diameter of Hyphae (20) was measured for each with caution averting collapse hyphae.The diameter and shape of basidiospores were also measured as described before.Caution was considered to avoid very young and immature spores.The slides were examined using light 10X eyepiece of microscope and 10X, 40X and oil immersion (i.e.100X), objectives.Photographs were taken using Motic p 410 attached with photomicrography unit.(Foroutan and Vaidya, 2007;Gottlieb and Wright, 1999).

Cultural characteristics:
Culture studies were done to ensure that our data and findings are compatible with those of earlier studies.All tested isolates (3 replicates) were grown up on malt extract agar (MEA) mediaat 25°C.The actively growing margin of mycelia was used for inoculation.
Formation of chlamydospores was examined using bright-field microscopy (Hong and Jung, 2004).One drop of Melzer's reagent was mixed with a chlamydospores on a clean, grease-free slide and was allowed to stand for 10-20 minutes and reactions were recordedusing40X and oil immersion (i.e.100X) (Leonard,2006).Scanning electron microscopy: Fungal isolates were grown on MEA medium at 25ºC for two weeks (Adaskaveg and Gilbertson, 1989).Examination and Photographing of Chlamydospores were carried out using a Jeol Scanning Electron Microscope (T.330A) in the Central Laboratory, Faculty of Agriculture, Ain Shams University.

Molecular identification:
DNA extraction: The DNA extractions were performed using the modified CTAB method (Wu et al., 2009) as follows: About 0.2 g mycelium samples were homogenized in liquid nitrogen in cooled mortar, transferred to an 2ml ependorafcontain 800 µL extraction buffer (2%CTAB, 100 mM/L Tris-HCl, 20 mM/L EDTA, 1.4 M/LNaCl, 7%ßmercaptoethanol and pH 8.0) and mixed gently.The mixture was incubated for 30 min at 60°C in water bath, the homogenate was extracted with chloroform/ isoamyl alcohol (24:1) and centrifuged at 12,000 rpm for 15 min.The supernatant was transferred to new tube and incubated with 1.5 volume of precipitation buffer (1%CTAB, 0.05M/L Tris-HCl, 0.01M/L EDTA, pH 8.0) for 30 min at room temperature, and centrifuged at 12,000 rpm to obtain DNA pellet.Next, the sediment was dissolved in TE buffer (10 mM/L Tris-HCl (pH 8.0), 0.1 mM/L EDTA (pH 8.0), 1M/L NaCl)) and 1µL of RNase A (2.5 U/ml) was added to the solution at 37°C for 30 min to digest RNA.Then the DNA was precipitated by 2.5 volume absolute ethanol and 1/10 volume 3 mol/L NaAC for 1 h at -20°C.The ethanol precipitation was then washed with 70% ethanol, dried and suspended in 100µL of TE buffer.DNA concentrations were estimated and standardized against the known concentrations of DNA on 1.6 % (w/v) agarose gels.

Amplification of ITS region by PCR:
Two specific primers of ITS-1 18-mers were designed and synthesized based on the conserved sequence of the ribosomal Internal Transcriber Spacer (ITS) region 1 of rDNA of Ganoderma boninense (EMBL accession number X78749).´5TTGACT GGG TTG TAG CTG 3´ (forward primer)´5GCG TTA CAT CGC AAT ACA3´ (reverse primer).PCR amplification reaction was performed using PCR thermocycler programmed as, 5 min preheating at 95˚C followed by 35 cycles (94 ˚C for 40 s, 52 ˚C for 40 s, 72 ˚C for 45 s) followed by final 12 min extension at 72˚C.The expected DNA fragment product size was 167 bp.The PCR products were analyzed electrophored on a 1.6% agarose gel, followed by visualized under UV light, photographed and analyzed by documentation using Syngiene Ingenius 3; USA, (Karthikeyan et al., 2007).

Sequenceand Phylogenetic analysis
All the PCR products were sequenced, The Internal Trans-cribed Spacer (ITS) fragment was extracted and purified from agarose gels and prepared to be sequenced using the Gene JET™ PCR Purifi-cation Kit (Fermentas).Finally sequencing of PCR product was done via GATC German Company by ABI 3730xl DNA sequences.The sequence data analysis of the ITS gene was received and analyzed.The Sequence assembly was carried out with the Sequencer v. 4.8 program.Sequences obtained from this study were trimmed with Bio Edit software and later BLASTn searched for closest matches in NCBI GenBank database.Phylogenetic tree was constructed to illustrate the relationships among the homologous fungiby using the Maximum Likelihood method based on the Kimura 2-parameter model (Kimura, 1980).The best DNA sequence similarities with our ITS region were obtained from NCBI GenBank and aligned using CLUSTAL W. The treewas drawn to scale, with brlengths measured in the number of substitutions per site.The analysis involved 15 nucleotide sequences.Phylogenetic tree was conducted using MEGA5 (Tamura. et al., 2011).

Morphological features:
An artificial key was prepared to differentiate the collected species and for the segregation and assignment of correct taxonomy of the studied samples (Foroutan and Vaidya, 2007).

Polymerase chain reaction (amplification)
Sequence and Phylogenetic analysis Amplification of the ITS1 region yielded PCR products of approximately 167 bp (Figure 6).BLASns searches revealed a high similarity (91% to100%) between the ITS sequence of isolates in this investigation with those for various Ganoderma species in GenBank.The highest similarity was with G. resinaceum (98 % to100%), (Figure 7).A phylogeny generated from sequence data separated the studied isolates into two distinct clades.These two clades are labeled A, B (Figure 8).
Clade A is consisted of five isolated Ganoderma from Egypt, with six G. resinaceum strains four of which from India (FJ491954, FJ665694, GU451246 and GU451247), and two strains from Korea, and France (JQ 520204 and FJ 805250) respectively.This clade recorded 94% support in the bootstrap analysis.
Clade B represents G. lucidum from Russia (GU207321, GU207323 and GU181349), which was robustly supported in the bootstrap analysis (100%).G. cupreum was presented as output group.

DISCUSSION
The genus Ganoderma includes several wood-decaying fungi on living trees in addition to dead tree trunks and stumps and has been recorded mostly in tropical and temperate areas.Several studies have been carried out on Ganoderma diseases focusing on economic damage, the severity of the disease and host range in many regions such as America, Asia, the Middle East and Europe (Fernando, 2008).In Egypt, Ganoderma species has been reported as a pathogen on casuarina trees (Ahmed-Reda, 2007;Mahmoud et al., 2007).In this study, identification of Ganoderma species was commonly found to be associated with stem rot of the navel orange, oil palm, fan palm, casuarina, morus which are selected from two governorates (Giza, Qalyubia) in Egypt.
The morphological similarity of Ganoderma species has caused confusion in the identification of these species.Numerous species have been described but many of them were later found to be synonyms or represented species complexes (Muthelo, 2009;Zheng et al., 2009).Identification of Ganoderma isolates was carried out depending on morphological features including macroscopic, microscopic, cultural characteristics and chemical reactions as well as DNA based methods.
Two types of basidiocarps can be produced in Ganoderma on the particular species.These include species with Lacctebasidiocarps having a shiny upper surface (G.lucidumcomplex), or a non-Laccatebasidiocarps with a dull upper surface (G.applanatum complex), (Roberts, 2004).The morphological features of the basidiocarps collected in this study resembled those of the species in the G. lucidum complex, which have laccate pilei.
Micromorphological characters of the basidiocarps such as size and morphology of basidiospores, type of hyphal system, as well as the structure of the pileal crust/cuticale surface have been used in the taxonomy of Ganoderma (Gottlieb and Wright,1999).
The family Ganodermataceaeis characterized by unique double-walled basidiospores.The differences in basidiospore morphology have been reported for different species within this fungal family.Two kinds of basidiocarps produce this type of basidiospore have been distinguished: one with a shiny (laccate), yellowish or reddish brown to black pilear surface, and those with a dull (non-laccate), grey-brown to the black pilear surface (Moncalvo, 2000).All the basidiospores examined in this study shared the same characteristics having a yellowish brown to black pilear surface that is well defined.
The structure of the pileal crust/ cuticle cells (Cutis) is a useful character in the taxonomy of the Ganodermataceae.Fruit bodies of Ganoderma mostly have an hymenioderm or characoderm and anamixoderm.Characteristics of the cuticle cells have also been valuable in distinguishing species in at least Ganoderma lucidum group (Bhosle et al., 2010 ;Steyaert, 1980).
The structure of the cutis of all the isolates examined in this study shared the same cutis structure having a hymenioderm (claviform type).The hyphal system in the Ganodermataceae is usually trimitic, occasionally dimitic, the generative hyphae are hyaline, thin walled, branched, septate or not, and clamped.Clamp connections may often be difficult to observe in dried specimens but are easily observed in the youngest parts of the hymenium and context of fresh specimens.Skeletal hyphae are always pigmented, thick-walled, and arboriform or aciculiform.Binding hyphae are usually colorless with terminal branching (Seo and Kirk, 2000).The hyphal system is trimitic in all the isolates was examined in this study.
Morphology of all the isolates was examined in this study as well as appeared to be similar to a number of species in the G. lucidium complex.However, a positive identification could not be carried out based on morphology alone.Cultures of Ganoderma species produce various hyphal structures, such as generative hyphae with clamp connections, fiber or skeletal hyphae, 'stag-horn' hyphae, cuticular cells and vesicles, and hyphal rosettes as well as chlamdospores.
The most useful characters in distinguishing Ganoderma cultures are chlamydospore production, growth rate and thermophily (Adaskaveg and Gilbertson, 1989).
High phenotypic plasticity at the macroscopic level, uniformity of microscopic characters, and subjective interpretation of various features such as color or consistency, a lack of handy identification keys and absence of type specimens have resulted in the creation of numerous unnecessary names (synonyms),.The absence of a world monograph has also contributed to problems with species circumscriptions and identifications in Ganoderma (Foroutan and Vaidya, 2007).
Ganoderma pfeifferi is highly characteristic because of the cracked and wrinkled resinous layer on the pileus, the sweet scent in winter and the dark brown context which immediately distinguishes it from old specimens of G. lucidum and G. resinaceum (Ryvarden et. al., 1993).
Based on morphology alone the G.lucidum in North America and G. resinaceum in European is considered the same biological species and could not be differentiated from each other (Douanla-

Fig. 7 :
Fig. 7: Aligned sequences of the ITS1 regions of rDNA.Ambiguities and polymorphisms are indicated by question marks.Alignment gaps are indicated by dashes, conserved bases by dots and nucleotide base substitution highlighted in yellow.The DNA sequence from left to right reads from 5´ to 3´

Source of fungal isolates: Samples
this study, all isolate form thickwalled chlamydospores with dextrinoid staining.Ganoderma species based on morphological characteristics were divided into six monophyletic groups (G.olossums group, G. applanatum group, G. tsugae group, Asian G. lucidum group, G. meredithiae group and G. resinaceum group) which included different species.Chlamydospores were observed from the members of the G. resinaceum group and G. oerstedii of the Asian G. lucidum group.Members of the G. resinaceum group had negative chlamydospores(G.subamboinense, G. lucidum ATCC64251 in Taiwan) or dextrinoid staining (G.resinaceum, G. pfeifferi and G. lucidum in North America), but Ganodermaoerstedii had amyloid chlamydospores