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6. Le Xuan Tham 33(3): 29-39 T¹p chÝ Sinh häc 9-2011 Nghiªn cøu sù ph©n hãa sinh ®Þa häc cña nÊm h−¬ng (Lentinula edodes ) vµ loµi míi - B¹ch kim h−¬ng (Lentinula platinedodes sp. nov.) ph¸t hiÖn ë C¸t Tiªn, Nam ViÖt Nam Lª Xu©n Th¸m, NguyÔn Nh¦ CH¦¥NG Së Khoa häc vµ C«ng nghÖ L©m §ång Ph¹m Ngäc D−¬ng V−ên Quèc gia C¸t Tiªn Bïi Hoµng Thiªm Së Khoa häc vµ C«ng nghÖ §ång Nai Trong c¸c ®ît kh¶o s¸t vÒ ®a d¹ng nÊm h×nh th¸i c¸ thÓ vµ ph©n hãa sinh ®Þa häc cña h−¬ng (Shiitake) thuéc chi Lentinula Earle, Lentinula edodes vµ kh¶ n¨ng cïng nguån gèc chóng t«i ® ph¸t hiÖn nhiÒu chñng ®Þa lý ph©n cña chi Lentinula . hãa ®Æc s¾c tõ vïng nói cao Sa Pa, Cao B»ng, B¾c ViÖt Nam, tõ vïng nói cao Langbiang, §µ i. PH¦¥NG PH¸P nghiªn cøu L¹t, L©m §ång vµ ®Õn vïng chuyÓn tiÕp tõ cao nguyªn xuèng ®ång b»ng - V−ên quèc gia 1. Chñng nÊm h−¬ng (VQG) C¸t Tiªn, §ång Nai, Nam ViÖt Nam, M−êi chñng nÊm h−¬ng thuéc chi Lentinula : ® ph©n tÝch so s¸nh víi c¸c chñng ë NhËt B¶n, (1) . Chñng nÊm h−¬ng Sa Pa (SP) nguån gèc Trung Quèc vÒ h×nh th¸i vµ cÊu tróc DNA [20]. hoang d¹i, ®−îc thu thËp t¹i huyÖn Sa Pa, Lµo Trong ®ã míi giíi thiÖu s¬ bé h×nh th¸i vÒ 1 loµi Cai khi mäc ré vµo nh÷ng th¸ng mïa ®«ng cã t¸n nÊm tr−ëng thµnh mµu b¹ch kim, dù kiÕn (3/2008, 2011) gi¸ l¹nh ( ≤ 7 °C), do ng−êi d©n lµ míi: Lentinula platinedodes , sp. nov., rÊt gÇn b¶n ®Þa thu h¸i trong rõng cung cÊp; (2) . Chñng víi loµi chuÈn Lentinula boryana - chØ ph©n bè nÊm h−¬ng Lentinula edodes nguån gèc hoang ë vïng Trung, Nam Mü. d¹i ë Cao B»ng, tõ Së Khoa häc & C«ng nghÖ Nghiªn cøu cña TrÞnh Tam KiÖt (1981) [8] Cao B»ng; (3, 4) . Chñng nÊm h−¬ng D2 vµ L170 cho thÊy chØ ghi nhËn mét loµi ë ViÖt Nam: ®−îc nu«i trång nhiÒu n¨m t¹i §µ L¹t, tõ ViÖn Lentinula edodes (Berk.) Pegl. víi nÊm h−¬ng NÊm Tottori, NhËt B¶n; (5) . Chñng nÊm h−¬ng c¸c vïng nói cao B¾c ViÖt Nam gi¸p giíi víi V©n Nam, Trung Quèc (YuC) do Lª Xu©n Trung Quèc, nhÊt lµ vïng Cao B»ng, song ® chØ Th¸m s−u tËp; (6) . Chñng nÊm h−¬ng ë thµnh ra mét sè nÐt ph©n hãa ®Æc biÖt: bao chung d¹ng phè Tr−êng Sa, Trung Quèc (ChsC); (7) . Chñng bét dÔ tan biÕn, hÖ sîi kh«ng thuÇn nhÊt. Sau nÊm h−¬ng nguyªn gèc nói Langbiang, §µ L¹t nµy, chóng t«i còng nhÊn m¹nh thêm vµ chñng thuéc Lentinula edodes do Tr−¬ng B×nh Nguyªn Sa Pa cã nhiÒu ®Æc ®iÓm ph©n hãa gÇn víi loµi s−u tËp; (8, 9) . Hai chñng thuéc loµi Lentinula Lentinula lateritia h¬n lµ L. edodes vµ ®Ò nghÞ lateritia (L.l, L.l’) hoang d¹i ®−îc thu thËp t¹i lµ mét thø ®Þa lý míi [20]. vïng B¾c C¸t Tiªn (C¸t Léc, Bï Sa) vµ nói Trong c«ng tr×nh nµy chóng t«i ph©n tÝch Langbiang, L©m §ång ë ®é cao gÇn 2000 m; c¸c chñng nÊm h−¬ng Shiitake ë B¾c vµ Nam (10) . Trong ®ît kh¶o s¸t gi÷a mïa m−a (5- ViÖt Nam s©u vÒ h×nh th¸i vµ ph©n tö, so s¸nh 10/2008) vµ ®Çu n¨m (4/2009) t¹i tuyÕn Th¸c víi c¸c chñng loµi míi ph¸t hiÖn ë Nam C¸t Trêi, Nam C¸t Tiªn thu ®−îc mÉu cña mét loµi Tiªn, trong ®ã x¸c lËp c¬ së ®Çy ®ñ cho loµi míi rÊt gièng víi loµi chuÈn Lentinula boryana , cã Lentinula platinedodes sp. nov., tiÕn mét b−íc thÓ lµ míi Lentinula sp., bao gåm 2 d¹ng vÒ quan hÖ chñng lo¹i ph¸t sinh, sù ph¸t sinh Lentinula sp.2 vµ Lentinula sp.3. C¸c chñng loµi 29 nµy hiÖn ®ang ®−îc l−u gi÷ t¹i B¶o tµng NÊm, Ph©n lËp gièng thuÇn khiÕt trªn m«i tr−êng PGA VQG C¸t Tiªn vµ Së Khoa häc vµ C«ng nghÖ, c¶i tiÕn; Kh¶o s¸t tèc ®é lan t¬ trªn m«i tr−êng L©m §ång. Toµn bé vËt liÖu gièng nÊm gèc h¹t; Kh¶o s¸t tèc ®é lan t¬ trªn m«i tr−êng mïn ®−îc b¶o tån t¹i B¶o tµng gièng chuÈn - VTCC c−a; Kh¶o s¸t qu¸ tr×nh ra qu¶ thÓ. thuéc ViÖn Vi sinh vËt vµ C«ng nghÖ sinh häc, 5. Qui tr×nh ph©n tÝch rDNA §¹i häc quèc gia Hµ Néi vµ Phßng C«ng nghÖ Vi sinh øng dông, Së Khoa häc vµ C«ng nghÖ C¸c b−íc ph©n tÝch rDNA ®−îc tiÕn hµnh L©m §ång. theo Lª Xu©n Th¸m vµ nnk. (2010) [20]. 2. Ph©n tÝch h×nh th¸i C¸c s¶n phÈm PCR tinh s¹ch ®−îc khuÕch C¸c mÉu vËt sau khi ph©n tÝch, chôp ¶nh ®¹i víi bé kÝt ABI PRISM Cycle sequencing vµ ®−îc ®Þnh lo¹i trªn c¬ së c¸c t− liÖu cña: Pegler ®Öm 5x sequence (Perkin-Elmer Applied (1975, 1983) [14, 15], Singer (1986) [19], Biosystem) víi hçn hîp ph¶n øng. Hibbett vµ nnk. (1998) [5], Hibbett (2001) [6], Tr×nh tù cña ITS vµ D1/D2 26S rDNA cña TrÞnh Tam KiÖt (1981) [8], Mata & Petersen c¸c chñng nÊm ®−îc ®äc trùc tiÕp trªn m¸y ®äc (2000, 2001) [9, 10], kÕt hîp tra cøu vµ so s¸nh tr×nh tù tù ®éng 3100 Avant. Sau ®ã, kÕt qu¶ trªn internet. tr×nh tù ®−îc so s¸nh víi c¸c tr×nh tù cña c¸c loµi ® ®−îc x¸c ®Þnh trong GenBank. 3. Ph©n lËp gièng nÊm C©y ph¸t sinh chñng lo¹i ®−îc x©y dùng sö PGA (Potato Glucose Agar) c¶i tiÕn lµ m«i dông phÇn mÒm ClustalX 1.83. KÕt hîp víi tr−êng t¸ch gièng thuÇn khiÕt vµ kh¶o s¸t hÖ sîi ph©n lo¹i truyÒn thèng, c¸c mèi quan hÖ ph¸t trªn m«i tr−êng th¹ch cã thµnh phÇn cho mét lÝt sinh tiÕn hãa cña c¸c taxon ®−îc thÈm ®Þnh vµ m«i tr−êng: 200 g khoai t©y, 100 g cµ rèt, 1 g ®¸nh gi¸. peptone, 100 g gi¸ ®ç, 0,5 g KH 2PO 4, 0,5g MgSO 4, 15 g glucose, 15 g agar. Khoai t©y, cµ ii. KÕT QU¶ Vµ TH¶O LUËN rèt ®−îc gät vá vµ gi¸ ®ç ®−îc röa s¹ch ®un s«i kho¶ng 15-20 phót, läc lÊy n−íc chiÕt, bá x¸c 1. Ph¸t hiÖn nÊm b¹ch kim h−¬ng Lentinula b. M«i tr−êng PGA ®−îc hÊp khö trïng ë platinedodes sp. nov. C¸t Tiªn 121 °C, 1 atm trong 30 phót. M«i tr−êng cÊy chuyÒn gièng cÊp hai vµ kh¶o s¸t hÖ sîi trªn Dù kiÕn ®©y còng lµ loµi míi ghi nhËn cho m«i tr−êng h¹t: 1000 g g¹o løc, 600-700 ml khu hÖ Lentinula ë ViÖt Nam, ®−a tæng sè hiÖn n−íc, 0,5 g CaCO 3. G¹o løc ®−îc nÊu chÝn råi biÕt lªn 2-3 loµi. MÉu thu ®−îc ë Nam C¸t Tiªn trén víi CaCO 3 cho vµo èng nghiÖm vµ hÊp khö bao gåm 2 d¹ng h×nh hÕt søc ®Æc s¾c. LÇn ®Çu trïng ë 120 °C, 1 atm trong 60 phót. tiªn ë vïng Th¸c Trêi nãng Èm ®Çu mïa m−a nhiÖt ®íi (~30 oC), nÊm h−¬ng mäc ré, thÓ hiÖn 4. Nu«i trång ra qu¶ thÓ nh÷ng nÐt gièng víi L. lateritia , song kh¸ phï Mïn c−a lµ nguyªn liÖu chÝnh ®−îc bæ sung hîp víi m« t¶ loµi chuÈn L. boryana ë Trung 10% c¸m g¹o, 10% c¸m b¾p, 5% bét b¸nh dÇu Mü. Nhµ nÊm häc Earle x¸c ®Þnh loµi chuÈn , t×m thÊy ë Cu Ba, Trung Mü, khi ®ã, vµ 1% CaCO 3 vµ n−íc, trén ®Òu ®Ó ®é Èm ®¹t L. cubensis 60-70%. Sau ®ã 1 kg c¬ chÊt ®−îc cho vµo bÞch ®©y lµ loµi duy nhÊt cho chi míi Lentinula t¸ch vµ hÊp khö trïng 120 phót ë 121 °C, 1 atm vµ ra tõ Lentinus Fr., vµo n¨m 1909, dùa vµo mÉu tiÕp tôc hÊp khö trïng lÇn 2 sau 24 h. Gièng vËt do Berkeley vµ Curtis x¸c lËp: Lentinus ®−îc cÊy tõ m«i tr−êng h¹t sang m«i tr−êng cubensis Berk.
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