boletus griseipurpureus ( boletaceae ) is a wild edible ectomycorrhizal mushroom , rich in protein and low in fat ( 1 ) .
an analysis of the internal transcribed spacer ( its ) region for b. griseipurpureus from the south of thailand revealed a monophylogenetic clade ( 1 ) .
however , only its sequences can not indicate the differences among its populations at the subspecies level .
the second largest subunit of rna polymerase ii ( rpb2 ) is more variable and more informative than the its region ( 3 ) and has been used to constitute phylogenetic trees of genera , such as cortinarius and to resolve the species concepts in strobilomyces ( 4 ) .
therefore , this marker has the potential to discriminate the subspecies ( 3 ) and thus it was chosen in this study .
mushroom extracts are generally used in dietary supplements and in combination with herbs ( 5 ) for their potential health benefits .
ethanolic extracts of pleurotus and russula inhibited both the gram - positive and the gram - negative bacteria ( 7 ) . for this reason ,
this study aimed to identify the genetic variation of b. griseipurpureus and also to evaluate any antibacterial activities .
basidiomes were collected under acacia magium in surat thani and songkhla provinces and under eucalypts in roi et and ubon ratchathani provinces , in thailand .
basidiomes were assigned to the b. griseipurpureus corner taxon on the basis of the morphological features ( 2 ) .
similar to another study ( 1 ) , amplification was performed in a 50 l reaction mixture , frpb2 - 5f ( fungal rpb2 - 5forward ) and brpb2 7.1r ( basidiomycete rpb2 7.1 reward ) ( biodesign , thailand ) used as primers .
sequences were then edited using bioedit and mega ( molecular evolutionary genetics analysis ) ( version 5.05 ) was applied to analyze the aligned sequences carried out by the clustal w analysis .
bootstrapping was performed with 1000 replicates of the neighbor - joining ( nj ) analyses using 1000 replicates .
basidiomes were dried at 40c for 16 hours before 70 g was blended and soaked in 500 ml of either methanol or ethyl acetate at room temperature for 1 week .
the residue was re - extracted twice with 500 ml of the solvent , reduced to dryness , dissolved in the respective solvent to a concentration of 200 mg / ml and stored at 4c .
the antibacterial activity against escherichia coli atcc 25922 , staphylococcus aureus atcc 29523 and methicillin - resistant staphylococcus aureus ( mrsa 189 ) inoculated as compared with mcfarland standard no .
0.5 , was evaluated using the agar well diffusion method on mueller hinton agar ( becton dickinson and company , france ) . the extracts ( 80 l )
were placed in 6 mm wells ; the plates in triplicate were incubated at 37 + 2c for 24 hours and the diameter of inhibition zones was determined .
the best extract was determined by the minimal inhibition concentration ( mic ) ( 9 ) .
similar to another study ( 1 ) , amplification was performed in a 50 l reaction mixture , frpb2 - 5f ( fungal rpb2 - 5forward ) and brpb2 7.1r ( basidiomycete rpb2 7.1 reward ) ( biodesign , thailand ) used as primers .
sequences were then edited using bioedit and mega ( molecular evolutionary genetics analysis ) ( version 5.05 ) was applied to analyze the aligned sequences carried out by the clustal w analysis .
bootstrapping was performed with 1000 replicates of the neighbor - joining ( nj ) analyses using 1000 replicates .
basidiomes were dried at 40c for 16 hours before 70 g was blended and soaked in 500 ml of either methanol or ethyl acetate at room temperature for 1 week .
the residue was re - extracted twice with 500 ml of the solvent , reduced to dryness , dissolved in the respective solvent to a concentration of 200 mg / ml and stored at 4c .
the antibacterial activity against escherichia coli atcc 25922 , staphylococcus aureus atcc 29523 and methicillin - resistant staphylococcus aureus ( mrsa 189 ) inoculated as compared with mcfarland standard no . 0.5 , was evaluated using the agar well diffusion method on mueller hinton agar ( becton dickinson and company , france ) .
the extracts ( 80 l ) were placed in 6 mm wells ; the plates in triplicate were incubated at 37 + 2c for 24 hours and the diameter of inhibition zones was determined .
the best extract was determined by the minimal inhibition concentration ( mic ) ( 9 ) .
the polymerase chain reaction ( pcr ) products from the amplified rdna were 1,100 bp in size on 1.5% agarose gels .
the rdna rpb2 regions were cloned and the nucleotide sequences were used to constitute a phylogenetic tree ( figure 1 ) together with 13 genbank sequences .
b. griseipurpureus from ubon ratchathani and roi et had a greater sequence identity than that from the southern provinces of thailand ( surat thani and songkhla ) .
overall , the b. griseipurpureus sequences were closely related to the b. edulis and b. satanus sequence data in genbank .
the basidiome crude extracts showed a significant antagonistic effect against the growth of the gram - positive and gram - negative bacteria ( p < 0.05 ) and the crude methanol extract exhibited a stronger antibacterial activity than the crude ethyl acetate extract ( p < 0.01 , table 1 ) . the mic of the crude methanol extract was 31
b. 
 griseipurpureus corner from ubon ratchathani = bg ubon ratchathani ( kc887325 ) , b. griseipurpureus from roi et = bg roi et ( kc887326 ) , b. griseipurpureus from surat thani = bg surat thani ( kc887327 ) , b. griseipurpureus from songkhla = bg songkhla ( kc887328 ) .  
data are presented as mean  sd .   values are not different by duncan s multiple range test ( p < 0.05 ) .
the sequences which contained the rpb2 region suggest a monophylogenetic clade among basidiomes of b. griseipurpureus and the phylogenetic tree suggests that b. griseipurpureus has affinity in the boletus group .
these findings are in contrast with the its results that showed b. griseipurpureus was more closely related to tylopilus than the boletus group ( 1 ) . in the future , the rpb2 region should be analyzed in parallel with other molecular markers for the phylogenetic study such as lsu , atp6 , rpb1 , nuc - lsu and mtssu sequences in order to better define the taxa boundaries ( 3 , 4 , 10 , 11 ) .
this will require a more extensive collection of b. griseipurpureus in the region than what was possible in this study .
lastly , the antimicrobial activities of the basidiome extracts suggest that this edible species may have attributes other than food nutrients . as for the extracts of daedalea elegans , the methanol extracts had a greater biological activity ( 5 ) . a crude methanol extract from auricularia polytrica was able to inhibit both s. aureus and e. coli ( 9 ) .
the crude methanol extracts of tylopilus neofelleus basidiomes were active against the gram - positive but were inactive for the gram - negative bacteria ( 12 ) .
however , a crude ethanol extract of russula delica was more effective in inhibiting the growth of the gram - positive than the gram - negative bacteria ( 13 )
. the higher activities of the crude methanol extracts are in concordance with a previous study with strobilomyces ( 14 ) . for this study ,
the crude methanol extract of b. griseipurpureus displayed a stronger antibacterial activity than the crude ethyl acetate extract .
the minimal inhibition concentration of the crude methanolic extract from the basidiocarp of this species ( 31
g / l ) was high , compared to the extracts from tricholoma lobayensis ( 9 ) .
moreover , the basidocarp extracts also available in some plant extracts , such as the crude ethanolic extract of zataria multiflora was able to inhibit 75 strains of mrsa at the concentration 2 - 16 mg / l .
the replacement of antibacterial agents with extracts of medicinal mushrooms and herbs may overcome bacterial activities ( 5 , 9 , 12 - 16 ) .
b. griseipurpureus might be applied in dietary supplements in combination with herbs ( 5 ) and bioactive compounds for potential health benefits .
a further study of this species will help enhance its values and widen its cultivation opportunities .
 background : boletus griseipurpureus corner , an edible mushroom , is a putative ectomycorrhizal fungus . currently , the taxonomic boundary of this mushroom is unclear and its bitter taste makes it interesting for evaluating its antibacterial properties.objectives:the purpose of this study was to identify the genetic variation of this mushroom and also to evaluate any antibacterial activities.materials and methods : basidiocarps were collected from 2 north - eastern provinces , roi et and ubon ratchathani , and from 2 southern provinces , songkhla and surat thani , in thailand . 
 genomic dna was extracted and molecular structure was examined using the rna polymerase ii ( rpb2 ) analysis . 
 antibacterial activities of basidiocarp extracts were conducted with escherichia coli atcc 25922 , staphylococcus aureus atcc 29523 and methicillin - resistant staphylococcus aureus ( mrsa ) 189 using the agar - well diffusion method.results:all the samples collected for this study constituted a monophyletic clade , which was closely related with the boletus group of polypore fungi . for the antibacterial study , it was found that the crude methanol extract of basidiomes inhibited the growth of all bacteria in vitro more than the crude ethyl acetate extract.conclusions:basidomes collected from four locations in thailand had low genetic variation and their extracts inhibited the growth of all tested bacteria . 
 the health benefits of this edible species should be evaluated further . 
1. Background
2. Objectives
3. Materials and Methods
3.1. Molecular Identification
3.2. Basidiocarp Extracts
4. Results
5. Discussion