紫芝孢子收集条件及其萌发技术优化

doi:10.3969/j.issn.1004-1524.20240985

浙江农业学报 ›› 2025, Vol. 37 ›› Issue (8): 1634-1647.DOI: 10.3969/j.issn.1004-1524.20240985

紫芝孢子收集条件及其萌发技术优化

韦娇君¹(), 王灿琴¹^,^*(), 阎勇¹, 韦仕岩¹, 罗阳兰¹, 王晓国¹, 周茂荣²

1.广西壮族自治区农业科学院微生物研究所,广西南宁 530007
2.田林县经济作物站,广西田林 533300

收稿日期:2024-11-14 出版日期:2025-08-25 发布日期:2025-09-03
作者简介:韦娇君(1986—),女,壮族,广西南宁人,硕士,助理研究员,研究方向为食用菌育种与栽培。E-mail:weixinjun1986@126.com
通讯作者: *王灿琴,E-mail:wcanqin@163.com
基金资助:
广西重点研发计划(桂科AB22035030);广西农业科学院基本科研业务专项(桂农科2024YP079);创新联盟专项——科技先锋队“强农富民”“六个一”专项行动(桂农科盟202408);广西田林县特色作物试验站资助项目(桂TS2022017)

Optimization of spore collection conditions and germination technology of Ganoderma sinense

WEI Jiaojun¹(), WANG Canqin¹^,^*(), YAN Yong¹, WEI Shiyan¹, LUO Yanglan¹, WANG Xiaoguo¹, ZHOU Maorong²

1. Institute of Microbiology, Guangxi Academy of Agricultural Sciences, Nanning 530007, China
2. Cash Crop Station of Tianlin, Tianlin 533300, Guangxi, China

Received:2024-11-14 Online:2025-08-25 Published:2025-09-03
Contact: WANG Canqin

摘要/Abstract

摘要： 为给紫芝遗传育种提供理论基础,以野生大型真菌DZ020菌株为研究对象,通过形态学鉴定、ITS序列分析,鉴定其分类地位;研究DZ020孢子收集方法,利用单因素试验考察培养基、温度、热刺激、pH值、碳源、保存时间和木屑浸出液对孢子萌发的影响,并采用正交试验和响应曲面法(Box-Behnken设计)优化孢子萌发条件。结果表明,采集的野生大型真菌DZ020依据子实体形态和ITS序列分析鉴定为紫芝。纸箱法收集的孢子萌发率显著高于套袋法和钟罩法,且其污染率显著低于套袋法和钟罩法。单因素试验获得最佳固体培养基配方:6%琼脂+2%牛肉膏+2%蛋白胨,最适温度为30 ℃,最适pH值为4.5,最佳保存时间为1 d,且孢子经过水浴刺激、在培养基中添加麦芽糖和木屑浸出液可提高萌发率。正交试验结果表明,水浴时间、麦芽糖质量分数对孢子萌发率的影响显著,红锥木浸出液质量分数对萌发率的影响不显著。Box-Behnken响应面法优化结果表明,红锥木浸出液质量分数与麦芽糖质量分数的交互作用不显著(p>0.05),红锥木浸出液质量分数与水浴时间的交互作用极显著(p<0.01),麦芽糖质量分数与水浴时间的交互作用显著(p<0.05);优化后的红锥木浸出液质量分数为19.0%,麦芽糖质量分数为1.6%,水浴时间为41.0 min,在此优化条件下萌发率达3.876%。本研究为紫芝遗传育种中单孢菌株的获得提供了有效方法和参考依据。

关键词: 紫芝, 孢子收集, 萌发, 单因素试验, 正交试验, Box-Behnken设计(BBD)法

Abstract:

In order to provide a theoretical basis for the genetic breeding of Ganoderma sinense, the wild macrofungi DZ020 strain was used as the research object, and its taxonomic status was identified through morphological identification and ITS sequence analysis. The spore collection method of DZ020 was explored. The effects of medium, temperature, thermal stimulation, pH value, carbon source, storage time and sawdust extract on spore germination were investigated by single factor test. The spore germination conditions were optimized by orthogonal test and response surface method (Box-Behnken design). The results showed that the collected wild macrofungi DZ020 was identified as G. sinense based on fruiting body morphology and ITS sequence analysis. The germination rate of spores collected by the carton method was significantly higher than that by the bagging method and the clock cover method, and the pollution rate was significantly lower than the bagging method and the clock cover method. Single factor test results showed that the optimum solid medium was 6% agar + 2% beef extract+ 2% peptone, the optimal temperature, pH value and storage time for the spore germination were 30 ℃, 4.5, and 1 d, respectively, the germination rate of spores could be increased by water bath stimulation, adding maltose and sawdust extract of Castanopsis hystrix to the medium. The results of orthogonal test showed that the water bath time and maltose mass fraction had significant effects on the germination rate, and the mass fraction of sawdust extract of C. hystrix had no significant effect on the germination rate. The results of Box-Behnken response surface method optimization showed that there was no significant(p>0.05) interaction between the mass fractions of sawdust extract of C. hystrix and maltose, the interaction between the mass fraction of sawdust extract of C. hystrix and water bath time was significant (p<0.01), and the interaction between maltose mass fraction and water bath time was significant (p<0.05). The optimized mass fractions of sawdust extract of C. hystrix and maltose were 19.0% and 1.6%, respectively, the water bath time was 41.0 min. Under the optimized conditions, the germination rate was 3.876%. This study provided an effective method and reference basis for the acquisition of single spore strains in the genetic breeding of G. sinense.

Key words: Ganoderma sinense, spore collection, germination, single factor experiment, orthogonal experiment, Box-Behnken design (BBD) method

中图分类号:

S567.31

韦娇君, 王灿琴, 阎勇, 韦仕岩, 罗阳兰, 王晓国, 周茂荣. 紫芝孢子收集条件及其萌发技术优化[J]. 浙江农业学报, 2025, 37(8): 1634-1647.

WEI Jiaojun, WANG Canqin, YAN Yong, WEI Shiyan, LUO Yanglan, WANG Xiaoguo, ZHOU Maorong. Optimization of spore collection conditions and germination technology of Ganoderma sinense[J]. Acta Agriculturae Zhejiangensis, 2025, 37(8): 1634-1647.

图/表 16

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培养基 Culture medium	pH值 pH value	培养基成分含量 Composition content of medium
ZM1	自然Natural	2%琼脂、2%葡萄糖2% agar powder, 2% glucose
ZM2	自然Natural	2%琼脂、98%无菌水2% agar powder, 98% sterile water
ZM3	自然Natural	2%琼脂、2%可溶性淀粉、20%马铃薯2% agar powder, 2% starch, 20% potato
ZM4(PDA)	自然Natural	2%琼脂、2%葡萄糖、20%马铃薯2% agar powder, 2% glucose, 20% potato
ZM5	自然Natural	2%琼脂、20%灵芝浸出液2% agar powder, 20% ganoderma lucidum extract
ZM6	自然Natural	2%琼脂、10%灵芝浸出液、20%马铃薯2% agar powder, 10% ganoderma lucidum extract, 20% potato
ZM7	自然Natural	3%琼脂、10%麦芽汁、20%马铃薯2% agar powder, 10% wort, 20% potato
ZM8	自然Natural	2%琼脂、2%牛肉膏、2%蛋白胨2% agar powder, 2% beef extract, 2% peptone
ZM9	自然Natural	2%琼脂、2%牛肉膏、2%蛋白胨、20%马铃薯2% agar powder, 2% beef extract, 2% peptone, 20% potato

培养基 Culture medium	pH值 pH value	培养基成分含量 Composition content of medium
ZM1	自然Natural	2%琼脂、2%葡萄糖2% agar powder, 2% glucose
ZM2	自然Natural	2%琼脂、98%无菌水2% agar powder, 98% sterile water
ZM3	自然Natural	2%琼脂、2%可溶性淀粉、20%马铃薯2% agar powder, 2% starch, 20% potato
ZM4(PDA)	自然Natural	2%琼脂、2%葡萄糖、20%马铃薯2% agar powder, 2% glucose, 20% potato
ZM5	自然Natural	2%琼脂、20%灵芝浸出液2% agar powder, 20% ganoderma lucidum extract
ZM6	自然Natural	2%琼脂、10%灵芝浸出液、20%马铃薯2% agar powder, 10% ganoderma lucidum extract, 20% potato
ZM7	自然Natural	3%琼脂、10%麦芽汁、20%马铃薯2% agar powder, 10% wort, 20% potato
ZM8	自然Natural	2%琼脂、2%牛肉膏、2%蛋白胨2% agar powder, 2% beef extract, 2% peptone
ZM9	自然Natural	2%琼脂、2%牛肉膏、2%蛋白胨、20%马铃薯2% agar powder, 2% beef extract, 2% peptone, 20% potato

收集方法 Collecting method	污染率 Pollution rate/%	萌发率 Germination rate/%	培养时间 Incubation time/d
纸箱法Carton	5	0.237±0.064 aA	7
钟罩法Clock cover	60	0.150±0.031 bB	7
套袋法Bagging	75	0.114±0.032 bB	7

收集方法 Collecting method	污染率 Pollution rate/%	萌发率 Germination rate/%	培养时间 Incubation time/d
纸箱法Carton	5	0.237±0.064 aA	7
钟罩法Clock cover	60	0.150±0.031 bB	7
套袋法Bagging	75	0.114±0.032 bB	7

培养条件 Condition	处理 Treatment	萌发率 Germination rate/%	培养时间 Incubation time/d
培养基Culture medium	ZM8	0.458±0.020 aA	7
	ZM7	0.272±0.030 bB	7
	ZM9	0.270±0.010 bB	7
	ZM5	0.048±0.010 cC	7
	ZM6	0.042±0.010 cCD	7
	ZM1	0 dD	10
	ZM2	0 dD	10
	ZM3	0 dD	10
	ZM4	0 dD	10
温度Temperature/℃	30	0.553±0.010 aA	7
	25	0.317±0.020 bB	9
	20	0.168±0.020 cC	15
	15	0.008±0.010 dD	18
	35	0 dD	10
热刺激Thermal stimulation	40 ℃水浴Water bath at 40 ℃	1.425±0.050 aA	7
	CK	0.435±0.020 bB	7
pH值pH value	4.5	1.703±0.070 aA	7
	5.0	0.928±0.040 bB	7
	6.0	0.148±0.030 dD	7
	CK	0.448±0.050 cC	7
	7.0	0 eE	10
	8.0	0 eE	10
	9.0	0 eE	10
碳源Carbon source	麦芽糖Maltose	0.592±0.030 aA	7
	乳糖Lactose	0.550±0.040 abAB	7
	果糖Fructose	0.517±0.010 abAB	7
	海藻糖Trehalose	0.442±0.060 bcABC	7
	CK	0.453±0.060 bcABC	7
	葡萄糖Glucose	0.428±0.010 bcBC	7
	蔗糖Sucrose	0.327±0.070 cC	7
保存时间Storage time/d	1	0.492±0.060 aA	7
	5	0.425±0.050 abAB	7
	10	0.417±0.070 abAB	7
	15	0.390±0.040 abcAB	7
	30	0.333±0.050 bcABC	7
	45	0.258±0.050 cdBC	7
	60	0.183±0.030 dC	7
木屑浸出液Sawdust extract	红锥木Castanopsis hystrix	0.633±0.030 aA	7
	松树Pinus	0.588±0.060 abAB	7
	梨树Pyrus sorotina	0.507±0.010 bcAB	7
	枫树Acer spp	0.490±0.060 bcAB	7
	CK	0.433±0.070 cB	7
	青冈树Quercus glauca Thunb.	0 dC	10
	板栗树Castanea mollissima Bl.	0 dC	10

紫芝孢子收集条件及其萌发技术优化

Optimization of spore collection conditions and germination technology of Ganoderma sinense

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水平 Level	因素Factor
水平 Level	(A)红锥木浸出液质量分数 Sawdust extract mass fraction/%	(B)麦芽糖质量分数 Maltose mass fraction/%	(C)水浴时间 Water bath time/min
1	10	1	30
2	20	2	40
3	30	3	50

因素 Factor	水平Level
因素 Factor	-1	0	1
(A)红锥木浸出液质量分数 Sawdust extract mass fraction/%	10	20	30
(B)麦芽糖质量分数 Maltose mass fraction/%	1	2	3
(C)水浴时间Water bath time/min	30	40	50

试验号 Test No.	因素Factor			萌发率 Germination rate/%
试验号 Test No.	A/%	B/%	C/min	萌发率 Germination rate/%
1	10	1	30	3.054±0.220 b
2	30	2	30	3.206±0.213 ab
3	20	3	30	3.222±0.222 ab
4	30	1	40	3.544±0.116 ab
5	20	2	40	3.760±0.195 a
6	10	3	40	3.667±0.250 ab
7	20	1	50	3.437±0.331 ab
8	10	2	50	3.544±0.329 ab
9	30	3	50	3.415±0.253 ab

因素Factor	k₁	k₂	k₃	R
A	3.422	3.473	3.388	0.077
B	3.345	3.503	3.435	0.184
C	3.161	3.657	3.465	0.261

差异源 Source of variance	平方和 Sum of squares	自由度 Degree of freedom	均方 Mean square	F	p
Intercept	105.740	1	105.740	145 048.133	<0.001
A	0.011	2	0.005	7.486	0.188
C	0.376	2	0.188	257.822	0.004
B	0.038	2	0.019	25.943	0.037
Residual	0.001	2	0.001

试验号 Test No.	因素Factor			萌发率 Germination rate/%
试验号 Test No.	A	B	C	萌发率 Germination rate/%
1	-1	0	-1	2.599
2	1	0	1	2.760
3	0	0	0	4.090
4	1	1	0	3.532
5	0	-1	-1	3.010
6	1	-1	0	3.725
7	-1	0	1	3.607
8	0	0	0	3.927
9	-1	-1	0	3.565
10	0	-1	1	3.469
11	0	0	0	3.964
12	0	0	0	3.900
13	0	1	1	3.042
14	-1	1	0	3.494
15	0	0	0	3.700
16	1	0	-1	3.282
17	0	1	-1	3.162

方差来源 Source of variance	平方和 Sum of squares	自由度 Degree of freedom	均方 Mean square	F值 F value	p值 p value
模型Model	2.86	9	0.318 0	22.72	0.000 2
A	0.000 1	1	0.000 1	0.010 3	0.921 9
B	0.040 0	1	0.040 0	2.86	0.134 6
C	0.090 7	1	0.090 7	6.48	0.038 3
AB	0.003 7	1	0.003 7	0.265 9	0.622 0
AC	0.585 2	1	0.585 2	41.81	0.000 3
BC	0.091 8	1	0.091 8	6.56	0.037 5
A²	0.215 7	1	0.215 7	15.41	0.005 7
B²	0.051 7	1	0.051 7	3.70	0.096 0
C²	1.660 0	1	1.660 0	118.59	<0.000 1
残差Residual	0.098 0	7	0.014 0
失拟度Lack of fit	0.018 4	3	0.006 1	0.307 5	0.820 0
绝对误差Pure error	0.079 6	4	0.019 9
总离差Total deviation	2.96	16

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