维普资讯 http://www.cqvip.com 食用菌学报@2006.13(4):33 ̄35 文章编号:1005—9873(2006)04—0033—03 榆黄蘑“宁黄16号"松杉木屑分离、驯化和栽培研究 阮瑞国,丁李春,潘祥华,陈华,罗仰奋 (福建省宁德市农业科学研究所,福建福安355003) 摘 曩:从榆黄蘑1号分离驯化得到的“宁黄16号”是适应松杉木屑栽培的菌株。“宁黄16号”菌丝生长适应 温度范围广(15 ̄30℃),发菌时间短(24 ̄28 d),在松杉木屑培养料上室内栽培的生物学效率达63.6 A0。 关键词:栽培I组织分离I驯化}松杉木屑I榆黄蘑 中图分类号:S646.140.22 文献标识码:A 榆黄蘑(Pleurotus f £r{ D户 f 以f“ )又名黄金 含水量65 9,5,pH自然。将培养料I和II号分别 菇、金顶侧耳,隶属于担子菌亚门(Basidio— 装入30 cm×12 cm×0.05 cm的聚丙稀塑料袋, mycotina),层菌纲(Hymenomycetes),伞菌目 每袋装干料0.25 kg,l28℃、1.5 Mpa灭菌2 h (Agaricales),侧耳科(Pleurotaceae),侧耳属 后备用。 (Pleurotus),是一种珍稀的药用菌。目前,榆黄 1.2菌种分离和驯化 蘑的研究主要集中在生物学特性和栽培技术方 1.2.1组织分离 面,传统的栽培料多采用阔叶树木屑,由于阔叶 取八成熟的新鲜、朵型圆整、健壮的榆黄蘑1 树木、林资源逐年减少,而松杉木屑栽培榆黄蘑 号子实体,按文献[1~4]的方法挑取组织块 的研究国内外尚未见报道,为此我们进行了松杉 (O.5 cm×0.5 cm),分别接入1号和2号组织分 木屑分离、驯化和栽培榆黄蘑“宁黄16号”的研 离培养基,每种培养基2O支试管,27±1℃培养。 究,现将试验结果报道如下。 1.2.2纯培养 1材料与方法 挑取2号培养基上正常生长的菌落尖端菌 丝进行纯培养_1 ]。 1.1材料 1.2.3子实体形成试验 1.1.1供试菌株 在2号培养基上驯化后的菌丝接入培养料II, 榆黄蘑1号引自福建省农业厅食用菌总站。 置于27±1℃下培养1O~12 d。取i00袋长满菌 1.1.2供试培养基 丝的菌袋,出现原基的菌袋直接置于温度18~ 组织分离培养基。1号培养基(PDA培养 28℃,湿度85 ~9O%的栽培房,促使子实体形 基):马铃薯200 g,葡萄糖2O g,琼脂2O g,pH自 成。观察子实体色泽、形态等栽培特点。经6次栽 然;2号培养基:马铃薯200 g,琼脂2O g,葡萄糖 培试验,最后确定适应松杉木屑栽培的优良新菌 2O g,松杉木屑煮液1000 mL(松杉木屑200 g加 株,命名为“宁黄16号”。 水1000 mL煮沸30 min,取其滤液加水至 1.3“宁黄16号”生物学特性 1000 mL),pH自然。分离培养基制成试管斜面 1.3.1茵丝适宜生长温度、pH值试验 (18 mm×180 mm)备用。 设5℃、1O℃、15℃、18℃、2O℃、25℃、 培养料工:阔叶树木屑77 ,麦麸2O 9,5,石膏 28℃、3O℃、32℃、33℃、35℃和38℃12个温 粉2 9,6,蔗糖1 ,含水量65 9/6,pH自然;培养料 度梯度,每个处理3次重复,采用直径9 cm的培 Ⅱ:松杉木屑77 (新鲜松、杉木屑各一半,未经 养皿,在2号培养基上接入直径0.5 cm的“宁黄 任何预处理),麦麸2O ,石膏粉2 9/5,蔗糖l 9/6, 16号”菌种块,培养7 d后,测量菌落直径。 收稿日期:2005—08—30原稿}2006—04—29修改稿 基金项目:福建省宁德市科技局重点科技项目(2004Z08) 作者简介:阮瑞国(1 958一),男,助理研究员,主要从事食(药)用菌遗传育种和栽培研究工作。 维普资讯 http://www.cqvip.com 食用菌学报 第l3卷 2号培养基,灭菌后用1 mol/L HC1和 l mol/L NaOH溶液调整pH至4.0、5.0、5.5、 菌丝体;而2号培养基的20支试管斜面,因为松 杉木屑汁液含有的苯酚类和萜烯类物质具有芳 勰∞ 香油类,对榆黄蘑菌丝生长有抑制作用,分离培 ∞ 6.0、6.5、7.0、7.5和8.0八个pH梯度,接入直 径为0.5 cm的“宁黄16号”菌种块,置于27土 1℃下培养7 d后,测量菌落直径。 1.3.2栽培试验 养时14支试管菌丝不萌发,4支试管受污染,2 支试管菌丝生长正常。 2.2不同温度对宁黄16号菌丝生长的影响 试验结果表明。在1O~35℃范围内榆黄蘑 用培养料I和Ⅱ打穴接种1O “宁黄16号” 菌种块各300袋,置于18 ̄30℃,相对湿度60 新菌株宁黄16号菌丝均能生长,菌丝生长适宜 ~65%的培养室培养,待菌丝长满袋后,室内排 温度为18~30℃,温度高于35℃或低于10℃ 场栽培,常规管理[ 。榆黄蘑1号接种在培养料 菌丝生长受到抑制(表1)。 I上作对照。 2.3不同pH对宁黄16号菌丝生长的影响 2结果与分析 pH为4.0时。菌丝生长慢。长势弱,但在接 种10 d后,菌丝生长较健壮;pH为5.0~7.0时, 2.1组织分离 菌丝生长旺盛,长速快;pH为5.5~6.5时,菌丝 组织分离后。菌丝20 h后萌发,不同培养基 生长速度最快;pH超过7.5,菌丝生长慢,长势也 1 l l l之间菌丝萌发复活有差异。试验结果表明,从1 差。可见菌丝生长适宜的pH为5.0~7.0,最适 2 5 8 0 1 Z l , 号培养基的2O支试管斜面成功地分离到榆黄蘑pH为5.5~6.5。 加 加∞ 船 表i 不同温度对宁黄l6号菌丝生长的影响 Table 1 Effect of temperature on the growth of Ninghuang No.16 mycellum l1.OO 3.96 2.70 1.76 ”“/”茵丝生长受抑制,“一 茵丝较白,但稀疏,“+”菌丝长势较弱,“++”茵丝长势一般,“+++”茵丝长势较旺盛,“+ +++”茵丝长势最旺盛 “/”No detectable myeelial growth,“”white,comparatively sparse mycelium,“+’’weak mycelium,“++”, intermediate mycelial growth,“+++”vigorous mycelium,“++++”luxuriant myeelium 衰2不同pH对宁黄16号菌丝生长的影响 Table 2 Effect of pH on the growth of Nlnghuang No.16 mycellum 测试项目pH Parameters tested 4.0 5.0 5.5 6.0 6.5 7.0 7.5 8.0 ”“+”茵丝长势弱,“+++”茼丝长势旺盛,“++++”茵丝长势最旺盛 “+”weak myeelium,“+++”,vigorous mycelium,“++++”luxuriant myeelium ¨¨ 维普资讯 http://www.cqvip.com 第4期 阮瑞国等t榆黄蘑“宁黄l6号”松杉木屑分离、驯化和栽培研究 35 ”榆黄蘑1号在II培养料上茵丝生长受抑制 ”No growth of P.citrinopileatus No.1 mycelium occurred on substrate II 2.4栽培试验 食用菌菌种的提纯、复壮。在生产上,将组织分 宁黄16号在培养料II上菌丝发满菌袋时间 离物用于生产性栽培,一般先进行出菇试验验证 为24~28 d,18~28℃下子实体均能生长。全生 后,才进行大面积生产L4]。试验采用组织分离技 育期91 d,可采菇4潮,总产量47.7 kg,平均袋 术获得了理想的适应松杉木屑栽培的榆黄蘑菌 产量0.159 kg,生物学效率63.6 ;而对照榆黄 株“宁黄16”号,其菌丝生长适温15~3O℃,子实 蘑1号在培养料II上菌丝生长受抑,在培养料I 体生长适温18 ̄28℃;成活率高,在松杉木屑培 上菌丝生长期为28 d,全生育期105 d,共采收4 养料上,生物学转化率达63.6 9,6;子实体中等大, 潮菇,总产量7o.2 kg,平均袋产量0.234 kg,生 菌盖金黄色。这为榆黄蘑栽培开辟了新的培养 物学效率达93.6 料来源,也为我国松杉木屑资源的科学利用找到 2.5子实体形态特征 了一条新的途径。 出发菌株榆黄蘑1号子实体形态特征:菌盖 “宁黄16号”菌株适应性较强,在福建省闽 漏斗形,直径3~12 am,草黄色至金黄色,光滑, 东地区自然条件下,用常规方法进行室内栽培便 肉质l菌褶白色,稍密,不分叉,狭窄至稍宽,与菌 可收到良好效果,经济效益显著,可为城乡菇民 柄延生;菌肉白色,有菌香味}菌柄长2~12 am, 增加一条致富的途径,是值得推广的一个优良榆 粗0.5~1.5 am,偏生,白色至淡黄色,基部常相 黄蘑品种。 连。“宁黄l6号”子实体形态特征:子实体中等 大,近叠状丛生,菌盖2~5 am,光滑,金黄色,初 参考文献 期扁半球形,内卷,有细条纹,后期近漏斗状;菌 [1]黄年来.中国食用菌百科[M].北京t农业出版社, 盖薄,菌肉白色,菌褶片状淡白色,较密,向下延 1993.177. 生,长短菌褶相间,越向盖缘,短菌褶越多;菌柄 [2]贾身茂.食用菌制种技术[M].北京:中国林业出版 近中央生变为偏心生至侧生,内实,白色,长2~ 社,1990.135~138. 12 cm,菌柄粗0.2~O.5 am,基部相连形成一丛。 [3]杨庆尧.食用菌生物学基础[M].上海:上海科学技 术出版社。1981.242. 3讨论 [4]陈明杰。余智晟,凌霞芬,等.草菇组织分离的的遗 组织分离是一个无性繁殖过程,广泛应用于 传变异研究(Ⅱ)[J]。食用菌学报,2000,7(2):1~4. 维普资讯 http://www.cqvip.com ACTA EDULIS FUNGI@2006.13(4):36~38 Domestication and CulCUltivatitlVatlon of Plon o l-'leurotteurot tsel‘‘ trt noptl。’ tus ea StrainNinghuang No.16 on a Substrate Containing Pine and Fir Sawdust RUAN Rui-guo ,DING Li-chun,PAN Xiang-hua,CHEN Hua,LUO Yang-fen (Agricultural Research Institute of Ningde City t Fu’all,Fujian 355003,China) Abstract:A new strain of Pleurotus citrinopileatus,Ninghuang No.16,suitable for cultivation on substrates containing pine and fir sawdust,has been isolated and domesticated from P.ci trinopilea tus strain No.1. Mycelial growth of the new strain was recorded over the temperature range 1 5~30℃,and colonization of bags containing 0.25 kg of pine/fir sawdust,based substrate was complete after 24~28 d.Fruit body formation proceeded normally at 18--28℃and four mushroom flushes were completed after 91 d producing a total of 47.7 kg of fruit bodies from 300 bags(average 0.159 kg per bag)at a biological efficiency of 63.6%. Key words:Mushroom cultivation;Fruit body tissue;Mycelial propagation;Pine and fir sawdust; Pleurotus cff ,Io口ff 口m Pleurotus cjf,f凡D口 fe口f“ ,a rare medicinal fungus,is assigned to the Basidiomycotina, Tissue isolation medium No. 1 (PH unadjusted)consisted of 200 g potato,20 g glucose,20 g agar,1000 mL water.Tissue Hymenomycetes,Agaricales,Pleurotaceae and Pleurotus,and the biological characteristics and cultivation of this mushroom have been the isolation medium No. 2(PH unadjusted) consisted of 200 g potato,20 g glucose,20 g agar and 1000 mL extract of pine and fir sawdust.Sawdust extract was prepared by suspending 100 g each of pine and fir sawdust in subject of considerable research.Sawdust from broad.1eaved trees is the cOnventional cultivation medium for尸. citrinopileatus. However。recent years have seen a major reduction in broad 1eaved forest resources and alternative substrates need to be identified. Therefore,since to our knowledge there are no reports describing the use of pine and fir sawdust to cultivate P。citrinopileatus,we have examined the possibility of using these materials to isolate,domesticate and cultivate 1000 mL water and boiling for 30 min.After filtering to remove the solid residue,the filtrate was re.adjusted to 1000 mL with water. Cultivation substrates:substrate I(pH unadjusted)consisted of 77%broad.1eaved tree sawdust,20%wheat bran,2%gypsum and 1% sucrose,and had a water content of 65%: substrate II(PH unadjusted)consisted of 77% pine and fir sawdust(in equal amounts)。20% wheat bran,2%gypsum and 1%sucrose,and had a water content of 65%. Substrate 尸.citrinopileatus・strain Ninghuang No.16. 1 Materials and Methods 1.1 Materiais 1.1.1 Fungal strain (O.25 kg)was transferred to polypropylene bags(30 cm×12 cm x 0.05 cm)and sterilized No. 1 was 尸leurotus cff,l凡D口ifP口f“ at 128℃。1+5 Mpa for 2 h. 1.2 Isolation and domestication of strain Ninghuang No.1 6 obtained from the Edible Fungi Station, Agricultural Office of Fujian Province. 1.1.2 Cultivation media Received:Aug.30,2005;Accepted:April 29,2006 1.2.1 Propagation of fungal mycelia from fruit Sponsored by the Foundation of Ningde Science and Technology Bureau(2004Z08) C0rresp0nding author.Tel:+86。593。6263165 E-mail:ndnks@163.com 维普资讯 http://www.cqvip.com No.4 RUAN Rui-guo。DING Li-chun,PAN Xiang-hua,et al 37 body tissue Fresh,rounded and vigorous Pleurotus citrinopileatus fruit bodies(80%mature)were used for isolating fungal mycelium.Blocks of tissue(0.5 cm×0.5 cm)were transferred to slants of No.1 and No.2 media(20 of each) and incubated at 27±1℃[1~ . 1.2.2 Isolation of pure cultures Pure cultures were obtained by sub-culture of normal growing mycelia from the slant cultures described in section 1.2.2[卜 . 1.2.3 Fruiting Domesticated mycelium from media No.2 was sub・cultured on to substrate II(100 bags) and incubated at 27±1℃for 10~12 d.After colonization,the bags were transferred to a mushroom house(temperature 18~28℃, relative humidity 85%~9O%)for fruiting,and fruit bodies of good color and shape were selected for further selection experiments. After repeating this procedure five times,a new strain of good quality and suitable for cultivation on pine and fir sawdust substrate was selected and designated“Ninghuang No. 16”strain. 1.3 Biological characteristics of“Nlnghuang No. 16”strain 1.3,1 Optimum temperature and pH for mycelial growth Discs(0.5 cm dia)of medium No.2 containing“Ninghuang No.16”mycelium were inoculated at the center oftplates(9 cm dia)of the same medium and incubated at twelve different temperatures(5℃,10℃,15℃, 18℃,2O℃,25℃,28℃,3O℃,32℃,33℃, 35℃,38℃).Radial growth was determined after seven days. Plates of medium No.2,adjusted to pH 4.O,5.O,5.5,6.O,6.5,7.O,7.5 Or 8.0 with either 1 mol/L HCl or 1 mol/L NaOH,were inoculated as above and incubated at 27±1℃. Radial growth was determined after seven days. 1.3.2 Cultivation experiments Cultivation bags containing either substrate I or II(300 bags of each)were inoculated with 10%substrate II containing mycelium of “Ninghuang No.16”and incubated at 18-- ̄30℃ and 60%~65%relative humidity.After the substrate was fully colonized,the bags were arranged on shelves for fruiting using standard management procedures[ .Substrate i inocu- lated with P.citrinopileatus strain No.1 served as contro1. 2 Results and Analysis 2.1 Propagation of fungal mycelium from fruit body tissue Mycelial growth was evident after 20 h incubation at 27±1℃.AII 20 tubes of media No.1 successfully supported the growth of P. citrinopileatus mycelium.However,only two tubes containing media No.2 produced normal mycelia,14 failed to support mycelial growth, and four were contaminated。 2.2 Effect of temperature on mycelial growth of Ninghuang No.1 6 Growth of Nin ghuang No.1 6 mycelium occurred over the temperature range 1O~35℃ with relatively rapid growth recorded between 18-- ̄30℃.No mycelial growth was detected at temperatures>35℃or<10℃(see Table 1 in the Chinese version). 2.3 Effect of pH on mycelial growth of Nlnghuang No.16 Growth of Ninghuang No.1 6 mycelium occurred over the pH range 5.0~7.0 with the most rapid and vigorous growth recorded at pH values between 5.5 and 6.5.Mycelial growth was much slower and less vigorous at pH 4.0 and at pH 7.5 and above(see Table 2 in the Chinese version). 2.4 Mushroom cultivation Colonization of substrate II by Ninghuang No.16 mycelium was complete after 24~28 d, and fruit body formation proceeded normally at 18~28℃.Four mushroom flushes were completed after 91 d and produced a total of 维普资讯 http://www.cqvip.com 38 ACTA EDULIS FUNGI Vo1.13 47.7 kg of fruit bodies(average 0.159 kg per bag)at a biological efficiency of 63.6%.No mycelial growth of P.citrinopileatus No.1 occurred on substrate II,while bags containing substrate 1 were fully colonized after 28 d. Four mushroom flushes were completed after 105 d and produced a total of 70.2 kg of fruit bodies(average 0.234 kg per bag)at a biological efficiency of 93.6%. 2.5 Morphological characteristics of Pleurotus cff, ,l口pffP口 ll fruit body P.citrinopileatus No.1:pileus 3~12 cm in diameter,infundibuliform,grass yellow or golden yellow—coloured,smooth,fleshy;white and slightly dense gills without branching, narrow to slightly broad,decurved to stipe; white flesh with fragrance;stipe 2,、—12 cm in length and 0.5~1.5 in diameter・side growing, white to light yellow-coloured, normally jointed at base. P.citrinopileatus Ninghuang No.16: medium-sized fruit body,grows fascicularly ̄ pileus 2~5 cm in diameter,smooth,golden yellow-coloured,flat and hemispherical—shaped at first,incurved with thin stripe,close to infundibuliform later;thin pileus,white flesh, lamellar and light white gill,slightly dense, decurrent,alternate short and long gills,more short gills near the pileus edge;stipe nearly middle-growing to eccentric,solid inside, white。2~12 cm in length,0.2~0.5 cm in diameter,jointed at base to form a single c】uster. 3 Discussion Mycelial propagation from fruit body tissue is a form of asexual propagation used to purify and rejuvenate strains of edible fungi. When this procedure is employed,fruiting experiments are normally carried out on the newly--derived strain before it is used for large-- scale productionE . As a result of our experiments,we have obtained a new strain, Ninghuang No.16,suitable for cultivation on pine and fir sawdust.Normal mycelial growth was observed on the pine/fir sawdust substrate between 15~3O℃,and fruiting occurred between 18 ̄28℃with矗biological efficiency of 63.6%.The strain has a relatively high survival rate and produces a medium.sized fruit body with a golden yellow pileus.Moreover, the new strain produced higher yields on a broadleaf sawdust based substrate compared to P.citrinopileatus No.1 from which the new strain was developed.Therefore,not only have we identified a new substrate for P. cff厂f Dp2fP以f“ cultivation but have also developed a new science.based method for utilizing pine and fir sawdust in China. P. clf厂f Dpffe以f“ ,strain Ninghuang No. 16,is highly adaptable,and in the eastern areas of Fujian Province,good production levels and significant economic benefit have been achieved using conventional mushroom house.based cultivation methods,thereby contributing to the wealth of farmers in both the city and countryside.P.citrinopileatus, strain Ninghuang No.16,is a mushroom well worth popularizing. References [1]HUANG NL.Edible fungi cyclopedia[M].Beijing: Agricultural Publishing House・1993.177.(in Chinese) [2]JIA MS.Strain producing techniques for edible fungi[M].Beijing,China Forestry Publishing House.1990.135~138.(in Chinese) [3]YANG QR.Biological basis of edible fungi[M]. Shanghai:Shanghai Science and Technology Press, 1981.242.(in Chinese) [43 CHEN MJ,Yu ZS,LIN XF,et a1.Study on the genetic variation in the tissue culture of Volvariella volvacea[J- ̄.Acta Edulis Fungi,2000,7(2):1~4. (in Chinese with English abstract)