lentinula edodes was cultured in 500 ml of sterile potato dextrose broth at 25 for 28 days with standing . as shown in fig .
1 , the culture filtrate exhibited antibacterial activity , forming a clear zone of 22 mm on the paper disk method   against ralstonia solanacearum , a causal agent of tomato bacterial wilt disease . to purify the antibacterial compound ,
the culture filtrate was subjected to octadecyl - silica column chromatography and eluted with different concentrations from 5% to 100% methanol .
none of the 50-l aliquots of methanol eluate showed an antimicrobial effect on the bacteria , but the water fraction that passed through the column maintained antibacterial activity .
the ph of the passed fraction was 3.9 , suggesting the presence of an organic acid .
therefore , qualitative analyses of organic acids from the culture filtrate of l. edodes were carried out by high - performance liquid chromatography ( hplc ) .
hplc was performed on a hitachi chromaster apparatus ( tokyo , japan ) , which consisted of a pump , autosampler , column oven , uv - vis detector , and hpx-87 column ( i.d .
4.6  300 mm , particle size of 5 m , aminex therapeutics , kenmore , wa , usa ) ; temperature was maintained at 25. the flow rate was 0.6 ml / min .
the mobile phase was 4 mm h2so4 in water for a total running time of 25 min .
qualitative analysis of organic acids in the culture filtrate of l. edodes was performed by hplc and the results were compared with those of authentic organic acids .
oxalic acid was a dominant component in the culture filtrate of l. edodes , accounting for 50% of the hplc analysis data .
in addition to oxalic acid , phytic acid , malonic acid , and fumaric acid were major components .
quantitative analysis of oxalic acid in the culture filtrate of l. edodes was carried out under the same hplc conditions as were used in qualitative analyses . for quantitative analysis , a standard curve of oxalic acid
was prepared in the concentration range of 200~2,000 g / ml using four different concentrations .
the coefficient of the correlation value for the standard curve of oxalic acid was 0.9982 .
thus , the content of oxalic acid in the culture filtrate of l. edodes was 0.07% .
louis , mo , usa ) was tested for its antibacterial activity against r. solanacearum using the paper disk method .
5,000mg / l ) were added to the filter paper disks ( 5 mm diameter ) , placed on nutrient agar , and overlaid with soft agar ( 0.8% ) mixed with bacterial cells ( 5  10 ) .
clear zones that had formed around the paper disks were evaluated as growth inhibition of the bacterial cells .
the antibacterial activities of nine different organic acids present in the culture filtrate of l. edodes shown in fig . 2 were examined against r. solanacearum .
each clear zone of 34 , 27 , and 12 mm was formed on concentrations of 5,000 , 100 , and 500 mg / l , but not on other concentrations of oxalic acid .
antibacterial activity on 500mg / l oxalic acid was compared to the positive control , tetracycline ( 30mg / l ) .
furthermore , the antibacterial effect of oxalic acid was evaluated against eight different phytopathogenic bacteria : xanthomonas campestris pv .
vesicatoria , escherichia coli , and a gram - positive bacteria strain , bacillus subtilis .
different concentrations of oxalic acid from 300 to 100mg / l were added to nutrient broth containing different bacterial cells and cultured at 28 for 24 hr .
as shown in table 1 , oxalic acid exhibited antibacterial activities against the bacterial strains tested at the minimum inhibitory concentration ( mic ) of 250mg / l , except x. campestris pv .
vesicatoria were the most sensitive , and cell growth was inhibited at an mic of 200mg / l .
interestingly , oxalic acid , like antibiotics such as tetracycline , had a bactericidal effect and the growth of bacterial cells could not be recovered on nutrient media . in preliminary experiments ,
the ph of media containing oxalic acid was ph 3.5~4.5 ; we further evaluated whether the antibacterial activity of oxalic acid was a result of low ph .
the ph 3.5~4.5 media were adjusted with hcl and the r. solanacearum cells were cultured in the media .
the bacterium was grown in the media , although the growth rate was slightly different corresponding to ph ( data not shown ) , indicating that low ph is not main factor affecting antibacterial activation .
taken together , the results suggest that oxalic acid is critical factor responsible for antibacterial activity .
oxalic acid , a saturated short chain dicarboxylic acid , occurs naturally in many plants   and fungi   and forms insoluble calcium or magnesium oxalate crystals , soluble sodium , or potassium oxalate . in previous study ,
different organic acids were identified in japanese apricot fruit by hplc analysis and their antibacterial activities were determined against e. coli , b. subtilis , staphylococcus aureus , and streptococcus suis . however , oxalic acid showed relatively low antibacterial activities towards these bacterial species with an mic of 1,300mg / l compared to the mic of 500 mg / l found in this study .
oxalic acid produced by plant pathogenic fungi provide a low ph , which results in maximal activities of fungal enzymes that degrade the cell wall of the host plant . in artificial cultivation of l. edodes mushroom
, l. edodes may degrade wood sawdust by producing lignocellulytic enzymes ( laccase , cellulase , and xylanase ) and the resulting product , glucose , is used in energy metabolism .
the sms after 4 cycles of harvest was obtained from a mushroom farm , mixed with water ( 1 : 3 ratio w / v ) , and incubated with shaking for 2 hr .
the mixture was then filtered through two layers of miracloth ( calbiochem , la jolla , ca , usa ) , centrifuged for 10 min at 5,000 g and the supernatant was used as the water extract of sms .
water extract from the natural mushroom substrate that had not been inoculated with l. edodes was used as a control . to quantitatively analyze oxalic acid ,
the one peak at 6.63 min indicating oxalic acid was detected at 1000 mau in the water extract of a sms sample , but not in the water extract from natural mushroom substrate .
thus , the extract of sms of l. edodes may be an eco - friendly control agent for bacterial plant diseases .
 the culture filtrate of lentinula edodes shows potent antimicrobial activity against the plant pathogenic bacteria ralstonia solanacearum . 
 bioassay - guided fractionation was conducted using diaion hp-20 column chromatography , and the insoluble active compound was not adsorbed on the resin . 
 further fractionation by high - performance liquid chromatography ( hplc ) suggested that the active compounds were organic acids . 
 nine organic acids were detected in the culture filtrate of l. edodes ; oxalic acid was the major component and exhibited antibacterial activity against nine different phytopathogenic bacteria . 
 quantitative analysis by hplc revealed that the content of oxalic acid was higher in the water extract from spent mushroom substrate than in liquid culture . 
 this suggests that the water extract of spent l. edodes substrate is an eco - friendly control agent for plant diseases . 
Identification of antibacterial substances
Quantitative analysis of oxalic acid
Antibacterial activity of oxalic acid