Taiwan Provice of China; CN, China; CDN, Canada; NL, Netherlands; GB, United kingdom; USA,. United States of America; I, Italy; D, Germany; ARM, Armenia; ...
Morphological and Genetic Characteristics of Different Collections of Ganoderma P. Karst. Species Susanna Badalyan 1 , Narine Gharibyan 1 , Mirco lotti 2 and Alessandra Zambonelli 1
,
2
Laboratory of Fungal Biology and Biotechnology, Faculty of Biology, Yerevan State University,
Manoogian St. , Yerevan 0025 , Armenia;
2
1
Aleg
Dipartimento di Protezione e Valorizzione Agroalimentare,
University of Bologna, via Fanin 46, Bologna 40127, Italy Email: badalyan_ s@ yahoo. com; ngarib60@ yahoo. com; miele99@ agrsci. unibo. it; zambonel@ agrsci . unibo. it Abstract Extensive research on biology of Basidiomycetes mushrooms has markedly increased due to their potential use for the production of new biotech-products ( pharmaceuticals, nutriceuticals, etc. ) . In spite of their commercial importance there are many gaps to be filled in the current knowledge on their taxonomy and biology. It is reported that white-rot species of genus Ganoderma P. Karst. possess different medicinal effects and have been used as traditional medicines in many countries. More than 250 Ganoderma species are described , however variable morphological characteristics makes species identification difficult.
Ganoderma collections of 22 strains were isolated in Armenia, France, Iran , Italy and China. Collections were genetically identified using nuclear rDNA-ITS sequences data. Cultural ( morphological, ecological, physiological )
characteristics and growth parameters of mycelial colonies and pellets,
presence of
chlamydospores were thoroughly described in submerged and static culture&. using different nutrient media. Two collections of G. lucidum from Armenia genetically were identified as G. lucidum, two from three French collections as G. lucidum, one as Trametes gibbosa , four Italian and one Chinese collections also belongs to G.
lucidum species. Armenian, Italian and French collections of G. lucidum are placed in the European G. lucidum clade. Seven collections of G. applanatum from Armenia and one from Iran are identified as G. adspersum species which is originally reported for Armenian mycobiota. One collection of G. applanatum from Armenia was confirmed by molecular analysis. One Ganoderma sp. strain from France and two strains from Iran were identified as belonging to G. resinaceum. The French G. resinaceum is closely related with G.
lucidum sequences from USA. The sequence analyses revealed that G.
lucidum from Armenia were
monophyletic with respect to the Italian G. lucidum genotype. The genetic and morpho-physiological data presented here could assist further molecular taxonomic studies of Ganoderma species. Keywords. Ganoderma , Systematics, Genetic and Morphological Characteristics
Introduction Extensive research on biology of Basidiomycetes mushrooms has markedly increased due to their potential use for the production of different biotech-products ( enzymes, pharmaceuticals, nutriceuticals, etc. ) ( Wasser, 2002 ; Saltarelli et al. , 2009) . It is reported that several species of the polypore genus Ganoderma . 247.
Proceedings of the 18th Congress of the International Society for Mushroom Science
P. Karst. ( Ganodermataceae , Basidiomycota) , particularly Garwderma lucidum complex , possess medicinal effects and have been used as traditional medicines in many Asian countries ( Stamets, 2001). Bioactive compounds, such as polysaccharides, triterpens, etc. with immune-modulating, hypoglycemic, antimicrobial, antioxidant and other therapeutic effects were isolated from these mushrooms (Lin et al. , 1995; Park et al. , 1997; Lee et al., 1998; Wasser and Weis, 1999). In nowadays biotech-products, dietary supplements and functional food additives with different formulations
( tablets,
powders,
teas,
etc. )
obtained from
biotechnologically produced fruiting bodies and mycelium of Garwderma species are broadly available in the world market (Wasser et al. , 2000) . In spite of the commercial importance of Garwderma species, there are many gaps to be filled in the current knowledge on their taxonomy and biology ( Rivard en, 1985 ; Rivarden , 1991; Moncalvo, 1995; Moncalvo et al. , 1995a; Moncalvo et al. , 1995b; Moncalvo and Rivarden, 1997; Hong and Jung, 2004) . The fruiting bodies of Garwderma species are laccate or non-laccate and vary in colour from orange-red to brown. They are white-rot mushrooms occurring on conifers and hardwoods throughout the world. Several species also have industrial applications with the ability to degrade lignin and cellulose ( Adaskaveg and Gilbertson, 1994; Elissetche et al. , 2001). Root and stem rot caused by these mushrooms result in worldwide losses of crop and trees ( Martinez et al. , 1994 ; Miller et al. , 1994 ) . The genus Garwderma is taxonomically the most complex in the family Ganodermataceae. The macro-and micromorphological characters of both fruiting bodies and mycelia of Garwderma species are variable which created several synonymous names and makes this genus most difficult among the polypores to classify ( Rivarden, 1985; Rivarden, 1991; Moncalvo et al. , 1995a; Moncalvo et al. , 1995b; Moncalvo and Rivarden, 1997).
It is evident that traditional taxonomy of the Garwderma complex based on morphological characters of fruiting bodies and mycelia should be reviewed in light of molecular data. Presently, more than 250 Ganoderma species have been described ( Rivarden, 1991 ) .
Recent phylogenetic studies proved that extensive
convergence or parallelism of morphological characters has occurred during the evolution of Garwderma ( Moncalvo et al. , 1995 a; Moncalvo et al. , 1995 b) . Analyses of morphological data proved that host relationships, formation of thick-wall chlamydospores, growth rate and thermophily are important taxonomic criteria to distinguish Garwderma cultures and reveal phylogenetic relationships among species in this complex (Nobles, 1948; Adaskaveg and Gilbertson, 1986; Adaskaveg and Gilbertson, 1989; Hseu, 1990; Wang and Hua, 1991 ; Miller et al. , 1994; Hong and Jung, 2004). Three types of chlamydospores have been described in Garwderma species: abundantly produced ovoid; rarely formed ellipsoid or bullet-shaped ( Moncalvo et al. , 1995 a) . In some Garwderma species chlamydospores can be absent ( Wang and Hua, 1991 ) . It was reported that fast growing cultures of Garwderma species are thermophilic and produce numerous ovoid chlamydospores , while slow-growing cultures are not thermophilic and do not produce chlamydospores ( Moncalvo et al. , 1995a). Studies based on nearly full mt SSU rDNA sequences of 37 strains including sequences from conserved and variable domains show that Garwderma species formed a monophyletic group with a strong bootstrap support even if the relationship of the genus with the other genera of Polyporaceae are still unclear (Hibbett and Thorn, 2001 ; Hong and Jung, 2004). They were grouped into at least six monophyletic groups ( G. colossus group , G. applanatum group , G. tsugae group , Asian G. lucidum group , G. meredithiae group
and G. resinaceum group) distinguishable also by morphological, cultural and mating characters ( Nobles, 1948). High phenotypic plasticity in Garwderma genus was mentioned by several authors ( Steyaert, 1972; Bazzalo and Wright, 1982; Rivarden, 1991; Chen, 1993). It has been suggested that the group is young and strong speciation has not been achieved, yet ( Rivarden, 1991; Moncalvo et al. , 1995b). A molecular survey of taxa labeled as G. lucidum and used in pharmacological studies shows that this name was largely misapplied . 248.
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Genetics and Breeding
al
( Moncalvo, 2005 ) . Recent studies of G.
e
composed of geographically restricted seven species ( G. valesiacum, G. ahamdii , G. tsugae , G. oregonense,
lucidum species complex indicate that it is polyphyletic and
G. resinaceum, G. oerstedii and G. praelongum) ( Moncalvo, 2000) . It was reported that North American G. lucidum and European G. resinaceum are intercompatible in vitro belonging to the same biological species ( Adaskaveg and Gilbertson, 1986) . However it was unacceptable by Moncalvo and coauthors ( Moncalvo et al. , 1995 a) , because demonstration of intercompatibility in vitro does not prove that the two taxa really are interfertile producing viable spores and that genetic exchange is effective in vivo. The species model uses monophyly and the interfertility criteria, i. e. absence of genetic exchange between the two lineages and taxonomic divergence was proposed ( Moncalvo et al. , 1995a). It was shown that there was little correlation between rDNA gene phylogeny and morphology in the G. lucidum complex and it is questionable whether gene phylogenies reflect the phylogeny at the organismallevel ( Moncalvo et al. , 1995 a) . Thus, complex approach in taxonomy of Ganoderma species using molecular, morphological , physiological , ecological and mating data is required. In this paper genetic and morphological characters of Ganoderma samples collected on different hosts from different geographical regions are presented.
Materials and Methods Ganoderma collections of 20 samples have been investigated (Table 1 ) . The cultures were isolated from immature fruiting bodies found in Armenia ( 8 strains, on Quercus sp. , Fraxinus sp. , Morus alba and Acacia sp. wood) , France ( 4 strains, on Quercus sp. and broad leaves wood) , Iran ( 3 strains, on Populus sp. ,
Fagus sp. and broad leaves wood) , and Italy ( 3 strains, on Castanea sativa and Quercus ilex). Cultures are preserved in the Culture Collection of the Laboratory of Fungal Biology and Biotechnology ( FBBL) , Yerevan State University ( Badalyan et al. , 2005 ) . The fruiting bodies samples were conserved as exsiccata in the Mycological Herbarium of the Mycological Center of the University of Bologna ( CMI-Unibo). All tested samples including 18 mycelial strains and two fruiting bodies samples were genetically identified using nuclear rDNA-ITS sequences data. The primer pair ITS1F-ITS4 ( Gardes and Bruns, 1993) were used to amplify nuclear rDNA-ITS regions of the Ganoderma specimens' by direct PCR technique ( Iotti and Zambonelli, 2006). PCR products were first purified by the NucleoSpin Extract kit ( Machery-Nagel, Dtiren, Germany) and were then sequenced in both directions using primers ITS 1 F and ITS4. Sequences generated in this study were compared with those downloaded from GenBank databases (http: I /www. ncbi . nlm. nih. gov/) obtained from Ganoderma species fruiting bodies of known geographical origin. The rDNA 5. 8S region was excluded by the analysis because it was lacking in many of the sequences deposited in GenBank database. Sequences were aligned by Clustal W (Thompson et al. , 1994) and phylogenetic trees were generated by the neighbour joining ( NJ) and maximum parsimony ( MP) methods using Mega 5. 0 (Tamura et al. , 2007) and Paup 4. OhiO (Swofford, 2000) , respectively. Branch support values ( 1 000 bootstrap replicates) are expressed as percentages. As the topology of the resulting phylogenetic trees was congruent at all supported nodes, only the NJ tree is shown. Cultural studies of Armenian, French and Iranian collections presented in Table 1 were performed on different solid ( 2% malt-extract agar, potato-dextrose agar, glucose-peptone agar) and liquid ( malt-extract) media with a pH 6. 0, at 25"C and 200 rt/min. Colony morphology, texture and pigmentation were observed in 090 mm Petri dishes ( three replicates per strains) and analyzed after an incubation period of seven days in darkness at 25"C using Nobles's and Stalpers's scales (Nobles, 1965; Stalpers, 1978). For micromorphological investigation, sterile microscope cover slips ( 2 em
X2
em) were placed onto MEA . 249.
Proceedings of the 18th Congress of the International Society for Mushroom Science
Z37096-73 G.lucidum N Z37049-99 G./ucidum F Strain Glu15 I Strain Glu14 I Strain Glu1 I AM906058 G./ucidum I EU498090 G.lucidum I Strain Gl-4 ARM
G. lucidum (type I)
Strain Gl-5 ARM FJ463930 G.lucidum IND X78748-69 G.tsugae CN Strain Gl-1-3 F Z37052-76 G./ucidum USA
100 . - - - -"'
"-Jl_ Z37054-79 G.tsugae CON X78735-56 G.tsugae USA Strain Gl-1-2 F 95
99
Z37051-75 G./ucidum USA AM269772 G.lucidum USA
I
G. lucidum (type II)
Z37062-85 G.resinaceum GB
G. resinaceum X78737-58 G.resinaceum NL
G. applanatum FJ655455 G.applanatum IND FR686556 G.applanatum D
G. lucidum (type III) AF170009-10 G.lucidum RA AF170007-08 G./ucidum RA
I G. lucidum (type IV)
Strain 5036 ARM Strain Ga-1 ARM Strain Ga-2-2 ARM Strain Ga-2-3 ARM Strain Ga-2-1 ARM
' - - - - - - - - - - - - - 1 Strain Ga-2-4 ARM 100
Strain Ga-3 ARM
G. adspersum
Strain Ga-4 IR AM906057 G.adspersum I EF060011 G.adspersum I FJ655452 G.adspersum IND EU162053 G.adspersum B AY456341 G.lucidum USA
G. lucidum (type V) 97
EF188279 G.lucidum CN Z37050-74 G.lucidum CN ' - - - - - - - - - - - - - - - - - - - - - - T.gibbosa strain G~1-1 F
1------1 0.02
Fig. 1
Neighbour-Joining dendrogram based on ITS1-ITS2 rDNA sequences of Ganoderma lucidum. Taxa are labelled by their collection name, GenBank accession number and geographic origin: J, Japan; RC, Taiwan Provice of China; CN, China; CDN, Canada; NL, Netherlands; GB, United kingdom; USA, United States of America; I, Italy; D, Germany; ARM, Armenia; F, France; N, Norway; IND, India; RP, Philippines; RA, Argentina; B, Belgium. The references of the accessions from GeneBank: (Moncalvo et al., 1995a; Moncalvo et al., 1995b; Gottlieb et al., 2000; Edwards et al. , 2004; Guglielmo et al. , 2007; Naumann et al. , 2007; Terho et al. , 2007; Guglielmo et al. , 2008 ; Saltarelli et al. , 2009; Zheng et al. , 2009) .
. 250.
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Genetics and Breeding
plates next to growing mycelia and plates were further incubated at 25 "C ( Badalyan and Sakeyan, 2004) . The cover slips together with mycelia were removed, stained with a 0. 1% methylene-blue aqueous solution and examined under a microscope ( 3 434 Kolleg SHB 45, Germany) using 10 ( 40 ocular/ objective. Photos were taken with a SONY digital camera. Average radial growth rates ( GR) of mycelia were determined according to the formula GR ( mm/d) = r 2 ) I ( tn - t 2 ) where r2 and r n were the radii of the colonies (in mm) on the 2"d( t 2 ) and 7'h ( tJ day of
rn -
growth, respectively ( Buchalo, 1988) . Effect of different temperatures ( 5 , 10, 20, 25 , 30, and 35 "C at pH 6. 0) on mycelial growth of colonies on MEA was tested and average diameters of colonies were determined on days 7'h and 10'h and respective growth rates ( mm/d) were calculated.
Results and Discussion The sequences of Armenian strains Gl-4 and Gl-5 (specimen voucher 5039 and 5039/1, respectively) labelled as G. lucidum were placed in European Ganoderma lucidum clade. Thus G. lucidum from Armenia are monophyletic with respect to the G. lucidum Italian and French isolates analysed in this study and with the Italian G. lucidum genotype ( EU498090) which was recently indicated as a potential candidate for medicinal cope for its biochemical characteristics (Saltarelli et al. , 2009) . One of the four French collections identified as Trametes gibbosa was used as outgroup in the phylogenetic tree (Fig. 1 ) . Seven collections from Armenia and one from Iran labelled as G. applanatum form a separated clade together with sequences of G. adspersum which was originally reported from Armenian mycobiota. These two pecies are phylogenetically well separated but difficult to distinguish based on morphological characters ( Breitenbach and Kranzlin, 1986). Only one Armenian collection of G. applanatum fruiting body (voucher pecimen 5035) was confirmed by molecular analysis. One Ganoderma sp. strain from France and two strains from Iran were identified as G. resinaceum (Table 1 , Fig. 1 ) . The topology of the tree from this study is consistent with the taxonomy of G. lucidum complex proposed by Moncalvo et al. ( Moncalvo et al. , 1995a). Table 1
Tested Ganoderma samples Origination and
Herbarium number
Strain
Substrate
CMI-Unibo
date of culture
Morphological
Molecular
Accession
identification
identification
number
isolation
5037
F-1
Broad leaves wood
France, 2000
Ganoderma sp.
G. resinaceum
JN588588
5038/1
Gl-1-1
Quercus sp.
France, 2002
G. lucidum
Trametes gibbosa
JN58859!
5038
Gl-1-2
Quercus sp.
France, 2002
G. lucidum
G. lucidum
JN588578
5038/2
Gl-1-3
Quercus sp.
France, 2002
G. lucidum
G. lucidum
JN588574
5039
Gl-4
Quercus sp.
Armenia, 2002
G. lucidum
G. lucidum
JN588572
5039/1
Gl-5
Quercus sp.
Armenia, 2002
G. lucidum
G. lucidum
JN588573
5040
Ga-l
Fraxinus sp.
Armenia, 2002
G. applanatum
G. adspersum
JN588580
5041
Ga-2-1
Morus alba
Armenia, 2005
G. applanatum
G. adspersum
JN588583
5041/1
Ga-2-2
Morus alba
Armenia, 2005
G. applanatum
G. adspersum
JN588581
5041/2
Ga-2-3
Morus alba
Armenia, 2005
G. applanatum
G. adspersum
JN588582
5035
Quercus sp.
Armenia, 2002
G. applanatum
G. applanatum
JN588587
5036
Quercus sp.
Armenia, 2003
G. applanatum
G. adspersum
JN588579
Morus alba
Armenia, 2010
G. applanatum
G. adspersum
JN588584
Ga2-4
. 251
Proceedings of the 18th Congress of the International Society for Mushroom Science ( Continued) Herbarium number
Origination and Strain
date of culture
Substrate
CMI-Unibo
isolation
Morphological
Molecular
Accession
identification
identification
number
Ga-3
Acacia sp.
Armenia , 2010
G. applanatu.m
G. adspersum
JN588585
Gr-3
Populus sp.
Iran, 2008
G. resinaceum
G. resinaceum
JN588590
Ga-4
Fagus sp.
Iran , 2008
G. applanatum
G. adspersum
JN588586
Iran, 2008
Garwderma sp.
G. resinaceum
JN588589
Gr-5 5042
Glu1
Castanea sativa
Italy, 2009
G. lucidum
G. lucidum
JN588575
5109
Glul4
Quercus ilex
Italy, 2009
G. lucidum
G. lucidum
JN588576
5110
Glu15
Quercus ilex
Italy' 2009
G. lucidum
G. lucidum
JN588577
All Ganoderma strains showed different species-specific colony morphology on tested media. However, the favorable growing conditions for Ganoderma cultures were MEA medium with pH 6 at the 25 - 30"C . They were fast growing with higher than 7 mm/ d average growth rate at 25 "C : G. resinaceum 10. 5 mm/ d, G.
lucidum 9 . 8 mm/d and G. adspersum 8. 3 mm/d. Different hyphal microstructures (small round-shaped clamps, chlamydospores, apical hyphal enlargements, etc. ) were often described in static and agitated cultures. Numerous, lemon-ovoid and rare ellipsoid chlamydospores in both agar and liquid media were observed in G. resinaceum and G. lucidum cultures respectively, confirming data presented by Moncalvo et al. ( Moncalvo et al. , 1995a). The chlamydospores were relatively rare and round-shaped in G. adspersum. Asexual arthro-and blasto-spores were not detected in any of the Ganoderma strains. Hyphal loops were described only in agar cultures of all species, particularly G. resmaceum.
During submerged growth
Ganoderma strains formed disperse mycelium, pellets and/ or mixture of disperse mycelium with pellets. It is known, that form and shape of myceial pellets depend on culture conditions (pH, temperature and agitation rate) . In our experiment, filamentous pellets were mainly found in G. lucidum strains, whereas smooth and smaller pellets were found in G. adspersum culture.
This study evinced that the sequences of G. lucidum, G. resmaceum, G. adspersum and G. applanatum from specimens coming from European, Tran-Caucasian and Iranian regions are closely related and phylogenetically separated from the East-Asiatic sequences. Further molecular and morphological studies of expanded collections of Ganoderma species will assist phylogenetic and biogeographical studies of this complex group of fungi. The obtained results will also support biotechnological cultivation of medicinal mushroom G.
lucidum and other related Ganoderma species.
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