球毛壳菌固态发酵产抗植物病原真菌活性物质的工艺优化

doi:10.3864/j.issn.0578-1752.2023.11.006

摘要/Abstract

摘要：

【目的】优化球毛壳菌（Chaetomium globosum）秸秆固态发酵的培养基组成和发酵条件，提高发酵粗提物的抗真菌活性，为球毛壳菌生物农药的开发及秸秆绿色资源化提供参考。【方法】首先，以粗提物抗9种植物病原真菌菌丝生长的抑制率为评价指标，对培养基中的秸秆（水稻、小麦、玉米、油菜）和氮源（豆粕、麦麸、氯化铵）进行筛选，确定最适发酵培养基组成。随后进行发酵条件的单因素优化，以粗提物对辣椒疫霉的抑制率为评价指标，初步确定各发酵条件的较优范围及对粗提物抗真菌活性影响的程度。基于单因素优化的结果，利用正交设计对发酵条件进行正交优化，各参数的范围如下：发酵时间18—36 d、发酵温度26—32 ℃、培养基初始含水量70%—85%、秸秆与麦麸质量比4﹕1—1﹕1、秸秆粒径20—>100目。最后，获得球毛壳菌秸秆固态发酵产抗真菌活性物质的最优发酵条件并进行验证。【结果】在筛选固态发酵培养基组成时发现，球毛壳菌以小麦秸秆和麦麸组成的培养基进行发酵所获得的粗提物的抗真菌效果总体上优于其他组成的培养基。在发酵条件单因素优化中，菌液接种量对粗提物抗真菌效果影响不显著，故不对其进行后续的正交优化。正交优化中各发酵条件对球毛壳菌发酵粗提物抗真菌活性的影响极为显著（P<0.001），其影响程度依次为小麦秸秆与麦麸质量比>发酵时间>培养基初始含水量>发酵温度>小麦秸秆粒径。正交优化获得的最优发酵条件为发酵时间24 d、发酵温度26 ℃、培养基初始含水量80%、小麦秸秆与麦麸质量比4﹕1、小麦秸秆粒径60—100目。经发酵条件优化后，1 mg·mL^-1的球毛壳菌发酵粗提物对核盘菌、辣椒疫霉、稻瘟病菌、桃褐腐病菌、尖镰孢、黄色镰孢、鞘腐霉、禾谷镰孢、水稻纹枯病菌的抑制率分别为100%、92.86%、85.94%、83.90%、76.12%、73.02%、66.18%、58.96%、52.99%。【结论】经过发酵培养基组成和发酵条件优化后，球毛壳菌的发酵粗提物具有较高的抗真菌活性，可为后续分离、纯化球毛壳菌利用秸秆固态发酵产生的抗真菌活性物质打下基础。

关键词: 球毛壳菌, 秸秆, 植物病原真菌, 固态发酵, 抗真菌活性

Abstract:

【Objective】The objective of this study is to optimize the medium composition and fermentation conditions of straw solid-state fermentation of Chaetomium globosum, improve the antifungal activity of fermentation crude extract, and to provide references for the development of biopesticides of C. globosum and the green resource utilization of straw.【Method】Firstly, using the inhibition rate of crude extracts against the mycelia growth of 9 plant pathogenic fungi as evaluation index, straw (rice, wheat, maize, rape) and nitrogen source (bean pulp, wheat bran, ammonium chloride) in the medium were screened to determine the optimal composition of fermentation medium. Then the single factor optimization of fermentation conditions was carried out, and the inhibition rate of crude extracts against Phytophthora capsici was taken as the evaluation index to determine the optimal range of each fermentation condition and the degree of influence on the antifungal activity of crude extracts. Based on the results of single factor optimization, orthogonal design was used to optimize the fermentation conditions. Parameters were as follows: fermentation time 18-36 d, fermentation temperature 26-32 ℃, initial water content of medium 70%-85%, mass ratio of straw to wheat bran 4﹕1-1﹕1, straw particle size 20->100 mesh. Finally, the optimal fermentation conditions for producing antifungal active substances by solid state fermentation of straw from C. globosum were obtained and verified.【Result】When the composition of solid fermentation medium was screened, it was found that the antifungal effect of crude extracts obtained from the medium composed of wheat straw and wheat bran was generally better than that of other medium. In the single factor optimization of fermentation conditions, the inoculation amount of fungal solution had no significant effect on the antifungal effect of crude extract, so the subsequent orthogonal optimization was not carried out. In the orthogonal optimization, the effects of fermentation conditions on the antifungal activity of the crude extracts were extremely significant (P<0.001), and the effects were as follows: mass ratio of wheat straw to wheat bran>fermentation time>initial water content of medium>fermentation temperature>particle size of wheat straw. The optimal fermentation conditions obtained by orthogonal optimization were as follows: fermentation time 24 d, fermentation temperature 26 ℃, initial water content of medium 80%, mass ratio of wheat straw to wheat bran 4﹕1, particle size of wheat straw 60-100 mesh. After optimization of fermentation conditions, the inhibitory rates of 1 mg·mL^-1 crude extract from the fermentation of C. globosum against Sclerotinia sclerotiorum, P. capsica, Magnaporthe oryzae, Monilinia fructicola, Fusarium oxysporum, Fusarium culmorum, Sarocladium oryzae, Fusarium graminearum and Rhizoctonia solani were 100%, 92.86%, 85.94%, 83.90%, 76.12%, 73.02%, 66.18%, 58.96% and 52.99%, respectively.【Conclusion】After the optimization of fermentation medium composition and fermentation conditions, the crude extracts of C. globosum had high antifungal activity, which could lay a foundation for the subsequent separation and purification of antifungal active substances produced by straw solid fermentation of C. globosum.

Key words: Chaetomium globosum, straw, plant pathogenic fungi, solid state fermentation, antifungal activity

廖宏娟, 江玉梅, 冶霞, 张志斌, 马童雨, 朱笃. 球毛壳菌固态发酵产抗植物病原真菌活性物质的工艺优化[J]. 中国农业科学, 2023, 56(11): 2106-2117.

LIAO HongJuan, JIANG YuMei, YE Xia, ZHANG ZhiBin, MA TongYu, ZHU Du. Optimization of Solid State Fermentation for Production of Active Substances Against Plant Pathogenic Fungi from Chaetomium globosum[J]. Scientia Agricultura Sinica, 2023, 56(11): 2106-2117.

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图/表 9

表1

表2

秸秆与氮源种类对球毛壳菌粗提物抗真菌活性的影响"

秸秆与氮源 Straw and nitrogen source	粗提物对不同植物病原真菌的抑制率Inhibition rate of crude extracts against different plant pathogenic fungi (%)
秸秆与氮源 Straw and nitrogen source	A	B	C	D	E	F	G	H	I
小麦秸秆+麦麸 Wheat straw + Bran	97.97±1.35a	72.68±1.05a	77.05±1.33a	71.30±2.14a	63.24±1.56a	62.16±2.14a	61.29±2.53a	49.15±1.18a	42.86±0.94b
小麦秸秆+NH₄Cl Wheat straw + NH₄Cl	80.75±2.32b	11.70±2.27e	34.53±1.19f	11.36±1.05f	28.19±0.77d	16.51±1.95e	0.00±0.48f	14.60±0.67e	49.22±0.91a
小麦秸秆+豆粕 Wheat straw + Bean pulp	63.29±1.65c	17.32±0.31d	49.59±2.17d	2.56±0.86g	17.91±0.36f	4.14±0.46g	-3.73±0.56g	22.22±0.50d	2.63±0.28e
水稻秸秆+麦麸 Rice straw + Bran	94.59±0.89a	77.38±3.74a	71.31±0.74b	64.67±0.85b	59.31±2.75a	55.76±1.56b	48.57±2.35b	40.26±2.64b	40.18±2.59b
水稻秸秆+NH₄Cl Rice straw + NH₄Cl	19.88±1.98e	17.74±0.85d	49.64±1.91d	0.00±0.75g	16.05±0.95f	4.59±0.24g	1.95±0.68e	13.18±0.19e	48.88±2.65a
水稻秸秆+豆粕 Rice straw + Bean pulp	36.71±1.65d	24.41±1.82c	64.22±1.58c	12.81±0.59f	21.27±0.35e	19.84±0.54e	2.24±0.76e	24.44±2.51d	14.91±1.99d
玉米秸秆+麦麸 Maize straw + Bran	98.31±3.37a	74.38±1.84a	70.49±1.88b	63.89±1.55b	47.35±1.50b	46.85±0.89c	47.14±0.96b	46.66±1.90a	43.64±1.77b
玉米秸秆+NH₄Cl Maize straw + NH₄Cl	77.78±3.25b	7.67±1.12f	22.30±1.54g	25.00±1.16d	28.57±0.45d	20.85±2.56e	6.14±0.09d	38.76±2.05b	42.97±1.08b
玉米秸秆+豆粕 Maize straw + Bean pulp	18.09±1.56e	9.45±1.37ef	42.15±0.79e	12.82±1.02f	16.42±1.25f	16.67±1.45e	14.94±0.70c	15.52±1.36e	-12.03±1.5f
油菜秸秆+麦麸 Rape straw + Bran	82.28±2.62b	22.05±1.26c	63.42±1.07c	34.18±1.39c	35.46±0.65c	28.57±1.95d	45.28±3.14b	31.85±1.70c	27.19±2.04c
油菜秸秆+NH₄Cl Rape straw + NH₄Cl	78.26±1.95b	37.36±1.43b	31.22±1.37f	18.94±1.25e	23.02±1.61e	2.75±0.67h	15.79±1.64c	32.11±1.49c	28.28±0.65c
油菜秸秆+豆粕 Rape straw + Bean pulp	21.79±1.17e	22.05±0.59c	43.90±1.46e	11.11±0.65f	15.66±1.63f	12.70±0.40f	5.97±0.11d	23.70±1.86d	-11.40±1.1f

表2

图1

图2

图3

表3

表4

图4

图5

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试验号 Test No.	因素Factor					抑制率 Inhibition ratio (%)
试验号 Test No.	A	B	C	D	E	抑制率 Inhibition ratio (%)
1	1	1	1	1	1	73.94
2	1	2	2	2	2	64.06
3	1	3	3	3	3	83.82
4	1	4	4	4	4	33.24
5	2	1	2	3	4	76.53
6	2	2	1	4	3	76.53
7	2	3	4	1	2	91.35
8	2	4	3	2	1	79.82
9	3	1	3	4	2	81.48
10	3	2	4	3	1	77.35
11	3	3	1	2	4	80.39
12	3	4	2	1	3	83.00
13	4	1	4	2	3	76.70
14	4	2	3	1	4	77.35
15	4	3	2	4	1	36.04
16	4	4	1	3	2	55.17
K₁	63.77	77.16	71.51	81.41	66.79
K₂	81.06	73.82	64.91	75.24	73.02
K₃	80.56	72.90	80.62	73.22	80.01
K₄	61.32	62.81	69.66	56.82	66.88
R	19.74	14.35	15.71	24.59	13.22
主次顺序Order	D>A>C>B>E
优水平Excellent level	A₂	B₁	C₃	D₁	E₃
优组合Excellent combination	A₂B₁C₃D₁E₃

方差来源 Source of variance	偏差平方和 Sum of squares of deviations	自由度 df	F值 F-value	显著性水平 Significance level
A	0.406	3	79.821	P<0.001
B	0.136	3	26.750	P<0.001
C	0.160	3	31.453	P<0.001
D	0.394	3	77.443	P<0.001
E	0.139	3	27.288	P<0.001
误差Error	0.054	32