Optimization of spore collection conditions and germination technology of Ganoderma sinense

doi:10.3969/j.issn.1004-1524.20240985

Acta Agriculturae Zhejiangensis ›› 2025, Vol. 37 ›› Issue (8): 1634-1647.DOI: 10.3969/j.issn.1004-1524.20240985

• Crop Science • Previous Articles Next Articles

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

Abstract

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

CLC Number:

S567.31

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.

Figures/Tables 16

References 32

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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

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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