twenty - two pathogenic fungi were isolated from the fruiting bodies and compost of a. aegerita . the isolates were collected from gimje , iksan , gunsan of chonbuk , and chilgok of gyeongbuk province from january to april 2009 . pure cultures of the collected isolates were grown in pre - sterilized plates on potato dextrose agar ( pda ) using standard laboratory techniques . the cultures were incubated at 25 in a biological oxygen demand incubator for 1 wk . morphological characters such as colony color , size of hypha , and color and shape of conidia were recorded . conidia and conidiophores were observed under a microscope and by scanning electron microscopy ( jsm-5410lv ; jeol , tokyo , japan ) . these pure mycelia were used for sequence analysis of the rdna its region ( its-1 region , 5.8s gene , and its-2 region ) . the genomic dna was extracted using a dneasy plant mini kit ( qiagen , valencia , ca , usa ) following the manufacturer 's instructions . amplification and sequencing of the isolates were performed using a pair of universal primers : its1 ( 5'-tccgtaggtgaacctgcgg-3 ' ) and its4 ( 5'-tcctccgcttattgatatgc-3 ' )   for the region containing its1 , its2 , and the 5.8s rdna . the amplification was conducted in a 20 l reaction mixture containing 50 nm genomic dna , 10 mm tris - hcl , 1.5 mm mgcl2 , 50 mm kcl , 0.01% gelatin , 0.2 mm dntp , 200 ng of each primer , and 1 unit taq dna polymerase ( promega , madison , wi , usa ) . the reaction mixtures were denatured at 94 for 10 min and subjected to 35 cycles of 1 min at 94 , annealing at 55 for 1 min , extension at 72 for 1 min , and a final extension step of 7 min at 72. the amplified pcr product was separated on a 1.5% agarose gel , followed by purification with a dna purification kit ( core - one ; core - bio , seoul , korea ) , according to the manufacturer 's instructions . both amplicon strands were sequenced using the same primers , reactions were monitored with bigdye terminator cycle sequencing kits ( applied biosystems , foster city , ca , usa ) , and run on an abiprism 3130 automated dna sequencer ( applied biosystems ) , as described previously . the rdna its sequence data were analyzed using the dnastar program ( dnastar inc , madison , wi , usa ) and aligned by the clustal w method . twenty - two pathogenic fungi were isolated from the fruiting bodies and compost of a. aegerita . the isolates were collected from gimje , iksan , gunsan of chonbuk , and chilgok of gyeongbuk province from january to april 2009 . pure cultures of the collected isolates were grown in pre - sterilized plates on potato dextrose agar ( pda ) using standard laboratory techniques . the cultures were incubated at 25 in a biological oxygen demand incubator for 1 wk . morphological characters such as colony color , size of hypha , and color and shape of conidia were recorded . conidia and conidiophores were observed under a microscope and by scanning electron microscopy ( jsm-5410lv ; jeol , tokyo , japan ) . for dna extraction , mycelia cultures were raised individually on pda at 25 for 7 days . these pure mycelia were used for sequence analysis of the rdna its region ( its-1 region , 5.8s gene , and its-2 region ) . the genomic dna was extracted using a dneasy plant mini kit ( qiagen , valencia , ca , usa ) following the manufacturer 's instructions . amplification and sequencing of the isolates were performed using a pair of universal primers : its1 ( 5'-tccgtaggtgaacctgcgg-3 ' ) and its4 ( 5'-tcctccgcttattgatatgc-3 ' )   for the region containing its1 , its2 , and the 5.8s rdna . the amplification was conducted in a 20 l reaction mixture containing 50 nm genomic dna , 10 mm tris - hcl , 1.5 mm mgcl2 , 50 mm kcl , 0.01% gelatin , 0.2 mm dntp , 200 ng of each primer , and 1 unit taq dna polymerase ( promega , madison , wi , usa ) . the reaction mixtures were denatured at 94 for 10 min and subjected to 35 cycles of 1 min at 94 , annealing at 55 for 1 min , extension at 72 for 1 min , and a final extension step of 7 min at 72. the amplified pcr product was separated on a 1.5% agarose gel , followed by purification with a dna purification kit ( core - one ; core - bio , seoul , korea ) , according to the manufacturer 's instructions . both amplicon strands were sequenced using the same primers , reactions were monitored with bigdye terminator cycle sequencing kits ( applied biosystems , foster city , ca , usa ) , and run on an abiprism 3130 automated dna sequencer ( applied biosystems ) , as described previously . the rdna its sequence data were analyzed using the dnastar program ( dnastar inc , madison , wi , usa ) and aligned by the clustal w method . and one isolate each was mucor spp . , penicillium spp . , and aspergillus spp . ( table 1 ) . contamination in mushrooms that spreads mainly due to contaminated air or by a worker can be easily identified from the outside , as the contamination is green to dark green , black , yellowish green , or red in color due to the spores ( fig . caused damage in a similar manner by competing for space resulting in growth inhibition and hampered fruiting body development . the pathogens grew quicker than the mushrooms and occupied all of the surface area , which prevented growth of the mushrooms . penicillium spp . appeared as a green - gray colored powder and turned black as timed passed . the spores of trichoderma spp . and penicillium spp . were round and globular in shape . the mucor spp . and aspergillus spp . had shapes similar to an onion flower stake , and their spores had spikes on the outer surface ( fig . similar morphological characters were reported in penicillium spp . by jo et al . , and in mucor spp . , and aspergillus spp . . trichoderma spp . initially produced a dense pure white mycelium , which was difficult to distinguish from the mushroom mycelium . however , the mycelial mat gradually turned green in color due to heavy sporulation , which is a characteristic symptom of green mold disease . this produced brownish lesions and spots on the developing fruiting bodies , which later joined and completely covered the mushroom fruiting bodies . the emerging fruiting bodies in the affected portion of the substrate were badly spotted , brownish in color , and showed reduced growth and yield . have a green , green - yellow , or white color on the mushroom bottles and cause parasitic damage , compete with other mushrooms for nutrients , and produce a mycotoxin . differences in the morphological characteristics of the four species are described in table 2 and fig . they developed hyaline mycelia and produced conidiophores on exposed fertile branches after 1 wk . the conidia of trichoderma are generally oblong , rarely globose , and mostly green or hyaline but rarely yellow . the sizes of the t. longibrachiatum , t. harzianum , t. pleuroticola , and t. atroviride conidia were 3.8~5.4  2.7~3.2 m , 2.7~3.7  2.4~3.4 m , 2.6~3.7  2.1~3.0 m , and 2.7~4.0  2.4~3.4 m , respectively . the conidia size and length / weight ratio of t. longibrachiatum was the largest followed by t. atroviride , whereas the size of the t. pleuroticola hyphae was longest followed by t. longibrachiatum , and the smallest was t. atroviride . the conidia of all four species were ovate and green in color , while the t. pleuroticola conidia were yellowish green . growth of t. atroviride was slower than that of the other species , and conidia were restricted to concentric rings . t. longibrachiatum tended to form fasciculate conidiation initially , which turned coalescent , often forming greenish yellow conidial crusts with dense conidiation . t. longibrachiatum was distinguished by aggregated conidiophores with weakly developed pustules . as a representative isolate , t. longibrachiatum was usually cultivated until dark green spore bands were 3 cm from the colony center , as the pathogen was mixed together with white mushroom mycelia and light - green pathogen spores . t. harzianum was mass - produced at the end of mycelium with black spores , unlike t. longibrachiatum and t. pleuroticola . differences in the anamorphs , growth characteristics , and dna sequences were observed among the species . for the closely related species , significant differences were more likely visible in the trichoderma anamorph morphology than in the anatomy or morphology of the telemorph . seaby   reported that distinguishing trichoderma spp . using classic microscopic features alone was difficult , as their characteristics differ widely on different media , and spore sizes vary significantly with incubation temperature . virens was characterized by predominantly effuse , penicillate type conidiation , which was sparingly branched and fertile to the apex . the most critical and vulnerable stage of mushroom cultivation is the culture stage , so the utmost care has to taken at this time . if mushrooms become infected at the culture stage , none of them can be harvested . the rdna its sequence data of 38 isolates were compared and analyzed phylogenetically , including 29 isolates from jeonbuk and gyeongbuk provinces , six isolates of trichoderma spp . , and one isolate each of mucor , penicillium , and aspergillus spp . with the publicly available gene sequences by blast against the genbank database ( http://www.ncbi.nlm.nih.gov/ ) . the size of the entire rdna its region was 500~600 bp , yielding 539 bp for trichoderma spp . , 515 bp for penicillium spp . and aspergillus spp . , and 600 bp for mucor spp . the nucleotide sequences obtained from the genbank blast search in the rdna its region of the isolates was compared with that of other pathogens ( table 1 ) . the phylogenetic analysis of the nucleotide sequence revealed that 26 isolates of trichoderma were divided into four taxa , namely t. harzianum , t. pleuroticola , t. longibrachiatum , and t. atroviride . however , jb20 , jb21 , and jb22 were classified as mucor 
 racemosus f. recemosus , penicillium crustosum , and aspergillus tubigenesis , respectively . among the trichoderma spp . , 16 isolates ( 55.2% ) were identified as t. harzianum , six as t. pleuroticola ( 20.7% ) , two as t. longibrachiatum , and the remaining two as t. atroviride . isolates were different in their cultural and morphological characteristics , as described earlier ( table 2 , fig . the introduction of dna sequencing and cladistic analysis in the early 1990s opened a new era for fungal systematics by providing a new set of independently derived data that could be analyzed in tandem with more classically derived data . , isolated from p. ostreatus and p. eryngii beds , were grouped into three species . virens ( 70.8% ) , t. longibrachiatum ( 16.7% ) , and t. harzianum ( 12.5% ) . in addition to showing phylogenetic relationships among trichoderma spp . , studies in which phenotype and dna analyses were combined have tended to confirm species monophyly , as distinguished by bissett [ 27 , 28 ] ( fig . 4 ) . many trichoderma spp . have demonstrated antifungal or plant - growth - stimulating activities , which has led to their exploitation as biological control agents , and some isolates are used in commercially available applications . caused damage in a similar manner by competing for space resulting in growth inhibition and hampered fruiting body development . the pathogens grew quicker than the mushrooms and occupied all of the surface area , which prevented growth of the mushrooms . . appeared as a green - gray colored powder and turned black as timed passed . the spores of trichoderma spp . and penicillium spp . were round and globular in shape . the mucor spp . and aspergillus spp . had shapes similar to an onion flower stake , and their spores had spikes on the outer surface ( fig . similar morphological characters were reported in penicillium spp . by jo et al . , and in mucor spp . , and aspergillus spp . . trichoderma spp . initially produced a dense pure white mycelium , which was difficult to distinguish from the mushroom mycelium . however , the mycelial mat gradually turned green in color due to heavy sporulation , which is a characteristic symptom of green mold disease . this produced brownish lesions and spots on the developing fruiting bodies , which later joined and completely covered the mushroom fruiting bodies . the emerging fruiting bodies in the affected portion of the substrate were badly spotted , brownish in color , and showed reduced growth and yield . have a green , green - yellow , or white color on the mushroom bottles and cause parasitic damage , compete with other mushrooms for nutrients , and produce a mycotoxin . differences in the morphological characteristics of the four species are described in table 2 and fig . they developed hyaline mycelia and produced conidiophores on exposed fertile branches after 1 wk . the conidia of trichoderma are generally oblong , rarely globose , and mostly green or hyaline but rarely yellow . the sizes of the t. longibrachiatum , t. harzianum , t. pleuroticola , and t. atroviride conidia were 3.8~5.4  2.7~3.2 m , 2.7~3.7  2.4~3.4 m , 2.6~3.7  2.1~3.0 m , and 2.7~4.0  2.4~3.4 m , respectively . the conidia size and length / weight ratio of t. longibrachiatum was the largest followed by t. atroviride , whereas the size of the t. pleuroticola hyphae was longest followed by t. longibrachiatum , and the smallest was t. atroviride . the conidia of all four species were ovate and green in color , while the t. pleuroticola conidia were yellowish green . growth of t. atroviride was slower than that of the other species , and conidia were restricted to concentric rings . t. longibrachiatum tended to form fasciculate conidiation initially , which turned coalescent , often forming greenish yellow conidial crusts with dense conidiation . t. longibrachiatum was distinguished by aggregated conidiophores with weakly developed pustules . as a representative isolate , t. longibrachiatum was usually cultivated until dark green spore bands were 3 cm from the colony center , as the pathogen was mixed together with white mushroom mycelia and light - green pathogen spores . t. harzianum was mass - produced at the end of mycelium with black spores , unlike t. longibrachiatum and t. pleuroticola . differences in the anamorphs , growth characteristics , and dna sequences were observed among the species . for the closely related species , significant differences were more likely visible in the trichoderma anamorph morphology than in the anatomy or morphology of the telemorph . seaby   reported that distinguishing trichoderma spp . using classic microscopic features alone was difficult , as their characteristics differ widely on different media , and spore sizes vary significantly with incubation temperature . virens was characterized by predominantly effuse , penicillate type conidiation , which was sparingly branched and fertile to the apex . the most critical and vulnerable stage of mushroom cultivation is the culture stage , so the utmost care has to taken at this time . if mushrooms become infected at the culture stage , none of them can be harvested . the rdna its sequence data of 38 isolates were compared and analyzed phylogenetically , including 29 isolates from jeonbuk and gyeongbuk provinces , six isolates of trichoderma spp . , and one isolate each of mucor , penicillium , and aspergillus spp . with the publicly available gene sequences by blast against the genbank database ( http://www.ncbi.nlm.nih.gov/ ) . the size of the entire rdna its region was 500~600 bp , yielding 539 bp for trichoderma spp . , 515 bp for penicillium spp . and aspergillus spp . , and 600 bp for mucor spp . the nucleotide sequences obtained from the genbank blast search in the rdna its region of the isolates was compared with that of other pathogens ( table 1 ) . the phylogenetic analysis of the nucleotide sequence revealed that 26 isolates of trichoderma were divided into four taxa , namely t. harzianum , t. pleuroticola , t. longibrachiatum , and t. atroviride . however , jb20 , jb21 , and jb22 were classified as mucor 
 racemosus f. recemosus , penicillium crustosum , and aspergillus tubigenesis , respectively . among the trichoderma spp . , 16 isolates ( 55.2% ) were identified as t. harzianum , six as t. pleuroticola ( 20.7% ) , two as t. longibrachiatum , and the remaining two as t. atroviride . isolates were different in their cultural and morphological characteristics , as described earlier ( table 2 , fig . the introduction of dna sequencing and cladistic analysis in the early 1990s opened a new era for fungal systematics by providing a new set of independently derived data that could be analyzed in tandem with more classically derived data . , isolated from p. ostreatus and p. eryngii beds , were grouped into three species . virens ( 70.8% ) , t. longibrachiatum ( 16.7% ) , and t. harzianum ( 12.5% ) . in addition to showing phylogenetic relationships among trichoderma spp . , studies in which phenotype and dna analyses were combined have tended to confirm species monophyly , as distinguished by bissett [ 27 , 28 ] ( fig . 4 ) . many trichoderma spp . have demonstrated antifungal or plant - growth - stimulating activities , which has led to their exploitation as biological control agents , and some isolates are used in commercially available applications .
agrocybe aegerita is an important mushroom cultivated in korea , with good feel and a peculiar fragrance . 
 a. aegerita can be cultivated throughout the year using culture bottles but is more susceptible to contamination than other mushrooms . 
 twenty - two pathogens were isolated from the fruiting bodies and compost of a. aegerita , and seven isolates were isolated from pleurotus ostreatus to compare with the a. aegerita isolates , collected from gimje , iksan , gunsan of chonbuk , and chilgok of gyeongbuk province in 2009 . 
 these isolates were identified based on morphological and molecular characteristics . 
 of the 29 isolates , 26 were identified as trichoderma spp . 
 and the remaining three were aspergillus spp . 
 , mucor spp . , and penicillium spp . 
 a phylogenetic analysis revealed that the 26 isolates of trichoderma were divided into four taxa , namely t. harzianum , t. pleuroticola , t. longibrachiatum , and t. atroviride . among the trichoderma spp . 
 , 16 isolates ( 55.2% ) were identified as t. harzianum , six as t. pleuroticola ( 20.7% ) , two as t. longibrachiatum , and the remaining two were t. atroviride .