芳香杯伞Clitocybe fragrans属于真菌界、担子菌门、伞菌纲、伞菌目、白蘑科。主要分布在我国的高原地区,在黑龙江、吉林、河北、山西、云南等多地均有分布(魏铁铮和姚一建2015)。我国对野生蕈菌驯化已有近千年的历史,是世界上最早对食用菌进行栽培研究的国家之一,曾经首先对香菇、木耳、银耳等野生蕈菌驯化栽培成功(王建瑞和图力古尔2006;王刚等2006)。近几年,冷杉侧耳Pleurotus abieticola R.H. Petersen & K.W. Hughes、絮缘蘑菇 Agaricus subfloccosus (J.E. Lange) Hlaváček、脆木耳 Auricularia fibrillifera Kobayasi等先后被驯化成功(张晓宇等2019;梁倩倩等2020;罗智檜等2020)。芳香杯伞属于白蘑科,但是目前对于白蘑科的驯化栽培报道较少(香永田等1987;李晓华等2004),白蘑科里菌种的驯化栽培一直是业界难点。杯伞属仅有大杯伞Clitocybe maxima和漏斗形杯伞Clitocybe infundibuliformis有驯化成功报告(曾金凤1985,1991;颜振兰2001),而芳香杯伞作为白蘑科的一个野生菌种,至今未见驯化栽培的研究报道。
本研究以野生芳香杯伞子实体组织分离获得的菌种为材料,对其进行了形态学和ITS鉴定(刘红霞2014),随后,对其菌丝生长的生物学特性和驯化栽培进行了系统研究,筛选出适宜的母种、原种、栽培种培养基,明确了其生长条件及驯化栽培技术。研究结果对河北省野生蘑菇资源的保护利用、丰富我国食用菌栽培品种具有重要意义。
1 材料与方法
1.1 供试菌株
供试菌株采自于河北省平泉市,组织分离培养后获得菌种,经形态学及ITS鉴定为芳香杯伞Clitocybe fragrans,现保存于河北师范大学应用真菌学实验室。
1.2 菌丝体营养特性研究
1.2.1 不同碳源对菌丝体生长的影响
固定基础培养基中的碳素含量,碳源更换为与葡萄糖等量碳素的10种不同碳源。基础培养基配方为：葡萄糖20 g,蛋白胨3 g,磷酸二氢钾3 g,无水硫酸镁1.5 g,琼脂20 g,加水定容至1 L。以无碳培养基作为对照,在平板上进行培养,用直径6 mm的打孔器打孔,取菌丝接种至平板,每种碳源进行5个重复处理,25 ℃培养箱恒温暗培养。十字划线法在相同时间段内测量菌丝体直径,当有一个菌丝即将满板时,结束培养。
菌丝生长速度(mm/d)=菌落半径(mm)/生长天数(d)
1.2.2 不同氮源对菌丝体生长的影响
固定基础培养基中的氮素含量,氮源更换为与蛋白胨等量氮素的10种氮源,以无氮培养基作为对照。其他步骤同碳源影响实验。
1.2.3 不同C/N对菌丝体生长的影响
固定基础培养基中葡萄糖的含量,改变蛋白胨的含量,配制成C/N比不同的9种培养基。其他步骤同碳源影响实验。
1.2.4 不同无机盐对菌丝体生长的影响
以基础培养基作为对照,将无机盐硫酸镁更换为等质量浓度的9种不同的无机盐。其他步骤同碳源影响实验。
1.2.5 正交试验
以筛选出的菌丝体碳源、氮源、C/N、无机盐为4个单因素,分别挑选出最佳的3个水平,进行L9(34)正交试验,共9组。
1.3 菌丝体生长环境条件研究
1.3.1 不同温度对菌丝体生长的影响
根据最适正交配方制作固体培养基用于温度筛选。在19-43 ℃间以3 ℃为一个梯度设定不同的温度梯度。
1.3.2 初始pH值对菌丝体生长的影响
在pH 4.5-8.5间以0.5为一个梯度设定不同初始pH梯度。
1.3.3 不同光照条件对菌丝体生长的影响
分别设定24 h连续黑暗、12 h光照+12 h黑暗、24 h连续光照,3个不同光照处理方案。
1.4 芳香杯伞菌种培养基研究
1.4.1 母种培养基
根据相关参考文献及菌丝体营养特性,设计以下11种母种培养基配方(初始pH自然)：A：PDA+木屑煮汁培养基：CPDA培养基+木屑100 g (取煮汁);B：大豆饼粉培养基：CPDA培养基中马铃薯替换为大豆饼粉20 g;C：马铃薯蔗糖培养基：CPDA培养基中葡萄糖替换为蔗糖;D：CPDA培养基：马铃薯200 g (去皮切片煮汁),葡萄糖20 g,磷酸二氢钾3 g,蛋白胨3 g,无水硫酸镁1.5 g,琼脂20 g,水1 L;E：玉米粉培养基：CPDA培养基中马铃薯替换为玉米粉20 g;F：牛粪煮汁培养基：CPDA培养基中葡萄糖替换为牛粪100 g (取煮汁);G：麦粒煮汁培养基：CPDA培养基中马铃薯和葡萄糖替换为小麦粒200 g (取煮汁);H：粪草培养料培养基：CPDA培养基中葡萄糖替换为发酵后粪草料100 g (取煮汁);I：PDA+麸皮煮汁培养基：CPDA培养基+麸皮100 g (取煮汁);J：PDA培养基：马铃薯200 g (去皮切片煮汁),葡萄糖20 g,琼脂20 g,水1 L;K：正交配方(见2.1)。
1.4.2 原种培养基
根据相关参考文献及菌丝体营养特性,设计以下5种原种培养基配方,配方如下：高粱粒培养基：高粱98%,石膏1%,糖1%;玉米粒培养基：玉米粒98%,石膏1%,糖1%;荞麦培养基：荞麦98%,石膏1%,糖1%;麦粒培养基：小麦98%,石膏1%,糖1%;小米培养基：小米98%,石膏1%,糖1%。
将高粱粒用冷水浸泡12 h后,加热蒸煮至八分熟时,按比例加入石膏粉及葡萄糖,搅拌均匀后装入单头大试管(3 cm×20 cm),121 ℃高压灭菌1 h。待试管温度降至室温,接入直径为12 mm的菌丝块。22 ℃培养箱培养,当菌丝长满试管时可作为原种。
栽培料配方为粪草培养料98%,石膏1%、糖1%。使用前需对其进行人工二次发酵处理,处理方法为,将粪草培养料进行预湿,含水量保持在60%-65%,按比例加入石膏和糖,搅拌均匀后于55-60 ℃烘箱中进行二次发酵3-5 d,期间每天早晚进行翻料处理,待发酵臭味消失后,即发酵完成。将长好的原种接至发酵好的栽培料菌袋中(菌袋规格140 mm×280 mm×0.045 mm),于22 ℃、湿度60%的发菌培养室,在黑暗条件下进行发菌处理。将发菌完成的菌袋进行覆土出菇,出菇温度为20-25 ℃,湿度为80%-90%,并给予一定的散射光;昼夜温差10 ℃左右刺激原基发生,保持1 d两次通风;当原基形成后,加大通风,直至子实体发育成熟。
2 结果与分析
2.1 芳香杯伞菌丝体生物学特性
表1 不同营养物质对芳香杯伞菌丝生长的影响
Table 1
| 培养条件 Conditions | 不同因素 Different nutritional factors | 菌丝长势 Mycelial growth vigor | 生长速度 Mycelial growth rate (mm/d) | 显著性差异 Significance levels | |
|---|---|---|---|---|---|
| 0.05 | 0.01 | ||||
| 碳源 Carbon source | 乳糖Lactose | + | 0.39±0.02 | g | G |
| 果糖Fructose | + | 0.79±0.02 | f | E | |
| 葡萄糖Glucose | ++ | 1.32±0.03 | e | D | |
| 木糖Xylose | + | 0.77±0.03 | F | E | |
| 半乳糖Galactose | + | 0.20±0.02 | h | G | |
| 山梨糖醇Sorbitol | ++ | 2.09±0.03 | a | A | |
| 蔗糖Sucrose | +++ | 1.86±0.03 | b | B | |
| 甘露醇Mannitol | +++ | 1.80±0.05 | b | B | |
| 麦芽糖Maltose | ++ | 1.50±0.03 | d | C | |
| 可溶性淀粉Starch | ++ | 1.62±0.05 | c | C | |
| 空白CK | +++ | 1.70±0.09 | c | C | |
| 氮源 Nitrogen source | 钼酸铵Ammonium molybdate | ++ | 1.33±0.06 | d | DEF |
| 氯化铵Ammonium chloride | ++ | 1.39±0.09 | d | D | |
| 硝酸铵Ammonium nitrate | +++ | 1.86±0.07 | a | AB | |
| 尿素Urea | + | 1.08±0.12 | e | F | |
| 亮氨酸Leucine | +++ | 1.56±0.03 | bc | CD | |
| 蛋白胨Peptone | +++ | 1.42±0.12 | cd | CDE | |
| 牛肉膏Beef extract | +++ | 1.64±0.03 | b | BC | |
| 硫酸铵Ammonium sulfate | +++ | 1.64±0.07 | b | BC | |
| 空白CK | + | 1.28±0.09 | d | EF | |
| 酵母浸粉Yeast extract powder | +++ | 2.03±0.07 | a | A | |
| 碳氮比 | 酵母膏Yeast extract paste | + | 0.72±0.0 | f | G |
| C/N | 10:1 | + | 1.03±0.03 | d | C |
| 15:1 | + | 1.17±0.06 | bc | BC | |
| 20:1 | ++ | 1.17±0.06 | bc | BC | |
| 25:1 | ++ | 1.19±0.03 | b | BC | |
| 30:1 | ++ | 1.25±0.06 | b | AB | |
| 35:1 | ++ | 1.42±0.06 | a | A | |
| 40:1 | ++ | 1.14±0.07 | bcd | BC | |
| 45:1 | ++ | 1.06±0.03 | cd | C | |
| 50:1 | ++ | 1.03±0.03 | d | C | |
| 无机盐 Mineral salt | 氯化钾Potassium chloride | ++ | 0.98±0.02 | b | BC |
| 氯化镁Magnesium chloride | ++ | 0.65±0.02 | d | D | |
| 硫酸镁Magnesium sulfate | ++ | 1.15±0.10 | a | AB | |
| 碳酸钠Sodium carbonate | + | 0.39±0.03 | e | E | |
| 硫酸钠Sodium sulphate | +++ | 1.27±0.11 | a | A | |
| 硫酸钾Potassium sulphate | ++ | 0.80±0.06 | cd | CD | |
| 空白CK | ++ | 0.93±0.06 | bc | BC | |
| 硫酸锰Manganese sulphate | - | - | |||
| 硫酸铜Cupric sulfate | - | - | |||
| 硫酸锌Zinc sulphate | - | - | |||
| 硝酸钠Sodium nitrate | - | - | |||
注：“+++”表示菌丝生长势较强,“++”表示菌丝生长势一般,“+”表示菌丝生长势弱,下同
Note: “+++” indicates vigorous growth, “++” indicates moderate growth, “+” indicates weak growth. The same below.
图1
图1
不同培养条件对芳香杯伞菌丝生长的影响
Fig. 1
Effects of different culture condition on mycelial growth of Clitocybe fragrans.
以筛选的适宜芳香杯伞菌丝生长的碳源、氮源、C/N、无机盐为单因素,进行4因素3水平L9(34)正交试验(表2和表3)。不同正交配方对芳香杯伞菌丝菌落形态以及生长速度、菌丝干重影响差异较大,从极差R与r看出,菌丝生长速度各因素的影响顺序为D<C<A<B,菌丝生物量各因素的影响顺序为C<D<B<A (表3)。综合分析,适宜菌丝生长的最佳组合为A3B1C3D2,即7号培养基,在此配方下,芳香杯伞菌丝生长速度与菌丝体干重均极显著(P<0.01)高于其他组。据此分析适宜芳香杯伞的碳源为蔗糖、氮源为酵母浸粉、C/N为40:1、无机盐为硫酸镁。正交配方为：蔗糖17.3 g,酵母浸粉1.8 g,磷酸二氢钾3 g,无水硫酸镁1.5 g,琼脂20 g,水1 L。
表2 芳香杯伞培养基配方优化正交试验因素水平表
Table 2
| 水平 Level | 因素 Factor | |||
|---|---|---|---|---|
| 碳源 Carbon source | 氮源 Nitrogen source | C/N | 无机盐 Mineral salt | |
| 1 | 山梨糖醇Sorbitol | 酵母浸粉Yeast extract powder | 35:1 | 硫酸钠Sodium sulphate |
| 2 | 甘露醇Mannitol | 牛肉膏Beef extract | 30:1 | 硫酸镁Magnesium sulfate |
| 3 | 蔗糖Sucrose | 蛋白胨Peptone | 40:1 | 氯化钾Potassium chloride |
表3 芳香杯伞培养基配方优化正交试验结果
Table 3
| 试验号 Text number | A：碳源 Carbon source | B：氮源 Nitrogen source | C：C/N | D：无机盐 Mineral salt | 菌丝生长速度 Mycelial growth rate (mm/d) | 菌丝干重 Mycelial dry weight (g/d) |
|---|---|---|---|---|---|---|
| 1 | 1 | 1 | 1 | 1 | 0.96±0.03cd | 17.10±1.39c |
| 2 | 1 | 2 | 2 | 2 | 0.86±0.08cd | 12.93±0.39de |
| 3 | 1 | 3 | 3 | 3 | 0.55±0.02d | 9.50±0.21f |
| 4 | 2 | 1 | 2 | 3 | 0.9±0.04cd | 16.57±2.16c |
| 5 | 2 | 2 | 3 | 1 | 1.12±0.07bc | 11.50±0.35ef |
| 6 | 2 | 3 | 1 | 2 | 0.95±0.06cd | 10.90±0.32ef |
| 7 | 3 | 1 | 3 | 2 | 1.56±0.04a | 28.90±1.85a |
| 8 | 3 | 2 | 1 | 3 | 1.24±0.04b | 25.00±0.42b |
| 9 | 3 | 3 | 2 | 1 | 0.73±0.26d | 14.93±0.66cd |
| K1 | 0.790 | 1.140 | 1.050 | 0.937 | ||
| K2 | 0.990 | 1.073 | 0.830 | 1.123 | ||
| K3 | 1.177 | 0.743 | 1.077 | 0.897 | ||
| R | 0.387 | 0.397 | 0.247 | 0.226 | ||
| k1 | 13.177 | 20.857 | 17.667 | 14.510 | ||
| k2 | 12.990 | 16.477 | 14.810 | 17.577 | ||
| k3 | 22.943 | 11.777 | 16.633 | 17.023 | ||
| r | 9.953 | 9.080 | 2.857 | 3.067 |
注：数据为6次的平均值和标准差;Kn和kn分别代表n水平的菌丝平均生长速度和菌丝平均干重;R和r为该因素的极差
Note: The data were average value of six replicates; Kn and kn indicate the average of mycelial growth rate and mycelial dry weight respectively at n level. R and r are ranges.
2.2 芳香杯伞菌丝体生长环境条件
图2
图2
不同温度和初始pH对芳香杯伞菌丝生长的影响
A：温度;B：初始pH
Fig. 2
Effects of different temperature and initial pH on the mycelial growth of Clitocybe fragrans.
A: Temperature; B: Initial pH.
不同光照条件处理下,菌丝体生长速度差异不大。但在黑暗处理下,菌丝生长速度及菌落形态略优于其他处理条件(图1)。
2.3 芳香杯伞菌种培养基
2.3.1 母种培养基
表4 不同培养基对芳香杯伞菌丝生长的影响
Table 4
| 培养基 Medium | 培养基名称 Medium name | 菌丝长势 Mycelial growth vigor | 生长速度 Mycelial growth rate (mm/d) | 显著性差异 Significance levels | |
|---|---|---|---|---|---|
| 0.05 | 0.01 | ||||
| 母种培养基 Stock culture media | A：PDA+木屑煮汁Boiled sawdust juice | +++ | 1.24±0.04 | de | CD |
| B：大豆饼粉培养基Soybean meal medium | +++ | 1.49±0.05 | c | B | |
| C：马铃薯蔗糖培养基Potato sucrose medium | +++ | 1.02±0.01 | f | EF | |
| D：CPDA | +++ | 1.26±0.10 | de | CD | |
| E：玉米粉培养基Corn flour medium | +++ | 1.79±0.07 | b | A | |
| F：牛粪煮汁培养基Boiled cow dung juice medium | ++ | 1.32±0.07 | d | BCD | |
| G：麦粒煮汁培养基Boiled wheat juice medium | ++ | 1.17±0.05 | e | DE | |
| H：粪草培养料培养基Compost substrate medium | ++ | 1.96±0.04 | a | A | |
| I：PDA+麸皮煮汁Boiled bran juice | ++ | 1.78±0.04 | b | A | |
| J：PDA | ++ | 1.38±0.04 | cd | BC | |
| K：正交配方Orthogonal formula | ++ | 0.97±0.05 | f | F | |
| 原种培养基 Mother spawn media | 高粱Sorghum | +++ | 2.44±0.02 | a | A |
| 玉米粒Corn | ++ | 1.34±0.07 | b | B | |
| 荞麦Buckwheat | + | 0.4±0.02 | d | D | |
| 小麦Wheat | ++ | 1.07±0.04 | c | C | |
| 小米Millet | ++ | 1.32±0.04 | b | B | |
图3
图3
不同培养基对芳香杯伞菌丝生长和菌落形态的影响
A-K：不同母种培养基
Fig. 3
Effects of different culture medium on the mycelial growth and colonial morphology of Clitocybe fragrans.
A-K: Different stock culture medium.
2.3.2 原种培养基
菌丝体在5种原种培养基上均可以正常生长,但是不同培养基上菌丝长势差异较大,在荞麦培养基上菌丝生长速度较慢,在小米粒培养基上菌丝较纤弱,浓密度较差(表4)。综合分析,选择菌丝洁白浓密,长势较好,且生长速度最快的高粱粒培养基作为芳香杯伞适宜原种培养基。
2.4 驯化栽培
原种接种后22 ℃暗培养,发菌时间为30-35 d。栽培袋接菌后,35-40 d满袋,后移入出菇房,芳香杯伞的原基发生时间较短,为8-10 d,经过7-8 d原基分化成子实体,子实体成熟时菌盖厚度0.25-0.41 cm,菌盖直径1.87-2.93 cm,菌盖向上翻折生长,呈倒伞状;菌盖边缘呈波浪形或圆形;菌盖颜色为白色;菌柄长度3.61-4.08 cm,菌柄中空,扁平;单菇重量范围为1.75-2.51 g,单个子实体重量较轻,菇质较软且易碎,子实体香味浓郁(图4)。
图4
图4
芳香杯伞驯化栽培
A：菌袋出菇;B：单个子实体形态
Fig. 4
The fruit bodies of Clitocybe fragrans obtained by domestic cultivation.
A: Fruiting in sack cultivation; B: Separate fruit body morphology.
3 讨论
芳香杯伞属于白蘑科,白蘑科中的杯伞属、口蘑属、香蘑属等很多野生种类均为优良野生蕈菌,但其人工驯化栽培一直是业界公认难题。而且杯伞属仅有大杯伞Clitocybe maxima和杯蕈Clitocybe infundibuliformis驯化成功,对于芳香杯伞国内外至今未见驯化栽培研究报道。
本研究对野生芳香杯伞菌种分离后,发现其菌丝生长缓慢、纤弱、长势较差,因此对其菌丝生长所需的营养条件进行了研究。芳香杯伞适宜的碳源为蔗糖、山梨糖醇和甘露醇。这与大杯伞的最适碳源是蔗糖相同,与白霜杯伞Clitocybe dealbata的最适碳源是葡萄糖不同。芳香杯伞适宜的氮源为酵母浸粉、牛肉膏和蛋白胨,均为有机氮源,该真菌更偏好有机氮源,可能是有机氮源更有利于菌丝的利用吸收。这与大杯伞最适氮源是尿素不同。不同蘑菇的营养需求不同,如白霜杯伞在无氮的情况下生长最好(冀瑞卿等2005;牛长满等2007)。菌丝体对不同无机盐利用差异较大,培养基中添加硫酸锰、硫酸锌、硫酸铜、硝酸钠菌丝均不生长,不生长的原因是无机盐的浓度不合适还是这4种无机盐的加入抑制了菌丝生长,有待设计试验验证。正交试验结果表明,硫酸镁为菌丝生长最适无机盐,其最适浓度有待进一步筛选确定。
芳香杯伞最适的母种培养基为粪草培养料培养基,可能因为芳香杯伞属于草腐菌,粪草培养料含有的营养物质更有利于菌丝吸收,因此粪草培养料培养基最适宜芳香杯伞菌丝生长;而在正交配方上菌丝生长较弱,可能因为正交配方的营养物质过于单一,而其他配方都添加了天然基质,所含营养丰富。
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