防风根际真菌的分离鉴定及其生防潜力评价

doi:10.13560/j.cnki.biotech.bull.1985.2022-0501

摘要/Abstract

摘要：

作为世界性分布的镰刀属常见病原真菌，尖孢镰刀菌（Fusarium oxysporum）和木贼镰刀菌（F. equiseti）对包括经济作物及药用植物等的生长均有较大危害。利用源自于植物根际土壤的有益微生物防控尖孢镰刀菌和木贼镰刀菌引起的真菌性病害，是目前较为理想的植物病害管理策略。为获得可有效抑制尖孢镰刀菌和木贼镰刀菌的生防菌源，于防风根际土壤中分离纯化真菌104株，基于平板对峙法筛选获得1株对尖孢镰刀菌和木贼镰刀菌具有显著抑制效果的菌株MR-43，结合形态特征、ITS序列分析，将其确定为Sirastachys castanedae（GenBank登录号：OK287148.1），隶属于Sirastachys组进化分支，发现其可宿生于植物根际土壤。基于盆栽试验法，探究MR-43在防风栽培土壤中的定殖能力，评价MR-43对由尖孢镰刀菌引起的防风枯萎病和由木贼镰刀菌引起的防风根腐病的防病能力，及其对防风植株的促生能力。结果显示，Sirastachys castanedae MR-43对尖孢镰刀菌、木贼镰刀菌的抑菌率为57%以上，且经多次验证抑菌效果稳定；菌株MR-43可稳定定殖于防风栽培土壤并实现有效扩繁；菌株MR-43的孢子悬液可有效控制防风枯萎病和防风根腐病，平均防效达68.52%，与多菌灵、代森锰锌的防病效果无显著性差异（P>0.05）；此外，MR-43对防风植株生长具有明显促进作用。因此，Sirastachys castanedae MR-43在防风枯萎病、根腐病等真菌性病害管理方面具有较好的开发价值及应用潜力。

关键词: 尖孢镰刀菌, 木贼镰刀菌, 根际微生物, 拮抗真菌, 葡萄穗霉科, 生物防控, 菌种鉴定, 植物促生

Abstract:

Fusarium oxysporum and F. equiseti worldwide are the common fungal pathogens that cause a lot of damages on the growths of economic crops or medicinal plants. One ideal strategy for controlling the fungal diseases from F. oxysporum and F. equiseti is to utilize rhizospheric microorganisms. In order to obtain the biocontrol microorganisms against F. oxysporum and F. equiseti, 104 fungi from rhizospheric soil of Saposhnikovia divaricata plants were screened and isolated. Subsequently, strain MR-43 was selected as a potential stock for biocontrol due to the significantly antibiotic activity against F. oxysporum and F. equiseti. Based on the analysis of morphological traits and rDNA internal transcribed spacers（ITS）, strain MR-43 was identified as Sirastachys castanedae（GenBank No. OK287148.1）, belonging to a species in section Sirastachys of Stachybotryaceae, as a new record of S. castanedae from rhizospheric soil of S. divaricata. Based on the pot experiment under natural outdoor conditions, colonization rule in the soil of S. divaricate growing was studied, the antifungal efficacy of MR-43 against Fusarium wilt caused by F. oxysporum and root rot disease caused by F. equiseti of S. divaricata were assessed, and its growth promotion abilities of S. divaricata was also determined. The results indicated that MR-43 showed the strongest inhibition rate against F. oxysporum and F. equiseti, which showed up to 57% inhibition. The multiple test confirmed that the antifungal effect was relatively stable. The S. castanedae MR-43 successfully colonized and formed a stable population in soil where S. divaricata was growing. Inoculation of the MR-43 spore suspension delayed the occurrence of S. divaricata Fusarium wilt and root rot disease, suppressed disease lesions with a strong control efficacy of 68.52%, the control effect was not significantly different compared with fungicide treatment of 70% Mancozeb WP and 50% Carbendazim WP. Moreover, MR-43 presented a consistently positive effect on the growth promoting of S. divaricata plants. In nutshell, S. castanedae MR-43 could be considered for the developing potential biocontrol agent for the management of S. divaricata Fusarium wilt and root rot disease.

Key words: Fusarium oxysporum, Fusarium equiseti, rhizospheric microorganism, antagonistic fungi, Stachybotryaceae, biocontrol, strain identification, growth promotion

孙卓, 王妍, 韩忠明, 王云贺, 赵淑杰, 杨利民. 防风根际真菌的分离鉴定及其生防潜力评价[J]. 生物技术通报, 2023, 39(1): 264-273.

SUN Zhuo, WANG Yan, HAN Zhong-ming, WANG Yun-he, ZHAO Shu-jie, YANG Li-min. Isolation, Identification and Biocontrol Potential of Rhizospheric Fungus of Saposhnikovia divaricata[J]. Biotechnology Bulletin, 2023, 39(1): 264-273.

图/表 9

表1 防风枯萎病和根腐病发病程度分级标准

Table 1 Disease grading standard of Fusarium wilt and root rot of S. divaricata

代表值 Representative value	分级标准Grading standard
代表值 Representative value	枯萎病Fusarium wilt	根腐病 Root rot
0级	植株健康，无枯萎症状	健株，无病斑
1 级	植株10%以下叶片发黄	全株10%以下的叶片发病
3 级	11%-25%叶片萎蔫发黄，开始下垂	全株11%-25%的叶片发病
5 级	26%-50%叶片枯萎发黄，开始脱落	全株26%-50%的叶片发病
7 级	植株茎秆瘦弱稀疏，51%-75%叶片发黄，并大量脱落	全株51%-75%的叶片发病
9 级	全株萎蔫发黄，整体脱落，严重枯死	全株76%以上的叶片发病

表2 根际真菌对防风病原真菌的抑制作用

Table 2 Antifungal activities of selected rhizospheric fungi against fungal pathogens of S. divaricata

序号No.	尖孢镰刀菌F. oxysporum/%	木贼镰刀菌F. equiseti/%
MR-43	59.63±2.22 a	57.04±0.19 c
MR-38	56.30±1.28 b	23.89±0.19 d
MR-34	32.16±1.70 c	56.67±0.32 c
MR-24	22.36±0.92 d	61.85±0.31 a
MR-68	14.49±0.78 e	55.19±0.40 c
MR-37	11.55±0.82 f	57.78±0.26 bc
MR-70	9.91±2.31 f	60.00±0.35 ab

图1 菌株MR-43对尖孢镰刀菌、木贼镰刀菌生长的影响 A：尖孢镰刀菌纯培养；B：尖孢镰刀菌与MR-43共培养；C：木贼镰刀菌纯培养；D：木贼镰刀菌与MR-43共培养

Fig. 1 Effects of MR-43 on F. oxysporum and F. equiseti during the dual culture assay A: F. oxysporum. B: F. oxysporum with MR-43 during the dual culture. C: F. equiseti.D: F. equiseti with MR-43 during the dual culture

图2 菌株MR-43菌落形态及显微形态特征 A：菌落正面；B：菌落背面；C：分生孢子；D, E：分生孢子梗；F：产孢瓶体。基准刻度：C-E=10 μm；F=5 μm

Fig. 2 Microscopic morphology characteristics and the colony morphology of strain MR-43 isolate A: Front of colony. B: Back of colony. C: Conidias. D and E: Conidiophores.F: Stipes. Scale bars: C-E =10 μm. F = 5 μm

图3 基于ITS序列构建MR-43近缘关系的系统发育树

Fig. 3 Phylogenetic tree of MR-43 isolate and closely related taxa based on ITS sequences

图4 菌株MRRif-43在防风土壤中的定殖情况

Fig. 4 Colonization of MRRif-43 in the soil of S. divaricata

表3 MR-43孢子悬液对防风真菌性病害的防效

Table 3 Control effect of the MR-43 spore suspension on fungal disease of S. divaricata

不同处理 Treatment	枯萎病Fusarium wilt		根腐病Root rot
不同处理 Treatment	病情指数Disease incidence	防病效果Disease control/%	病情指数Disease incidence	防病效果Disease control/%
A	13.55±2.23 c	73.21±2.36 a	22.66±2.18 c	63.83±2.36 a
B	16.00±3.11 bc	68.37±3.30 ab	23.44±2.11 c	63.44±3.30 a
C	22.18±3.72 b	56.15±5.35 c	32.07±3.66 b	49.99±3.35 b
D	19.59±2.28 bc	61.27±4.51 b	30.85±3.29 b	51.89±2.70 b
CK	50.59±4.29 a	—	64.14±2.18 a	—

图5 MR-43对防风根腐病、枯萎病的防控效果 CK：清水处理；A：哈茨木霉；B：枯草芽孢杆菌；C：50%多菌灵可湿性粉或70%代森锰锌可湿性粉剂；D：MR-43

Fig. 5 Biocontrol effects of MR-43 against the root rot and Fusarium wilt disease of S. divaricata CK: Untreated control（water）. A: T. harzianum. B: B. subtilis. C: Carbendazim 50% WP or Mancozeb 70% WP. D: MR-43

表4 MR-43对防风植株的促生效果

Table 4 Effects of MR-43 on the promoting growth of S. divaricata

不同处理 Treatment	整株长度 Plant length/cm	根长 Root length/cm	整株鲜重 Plant fresh weight/g	根鲜重 Root fresh weight/g	整株干重 Plant dry weight/g	根干重 Root dry weight/g
A	55.73±3.52 a	29.44±3.42 a	8.21±0.40 a	3.88±0.51 a	2.56±0.67 a	1.32±0.30 a
B	51.63±3.58 b	29.34±2.15 a	7.66±1.62 a	3.04±0.47 b	2.49±0.97 a	1.04±0.30 b
C	51.35±4.14 b	28.63±2.84 a	7.89±1.98 a	3.10±0.39 b	2.54±1.01 a	1.06±0.25 b
CK	50.85±3.21 b	28.11±2.03 a	6.44±1.37 b	2.28±0.81 c	1.92±0.60 b	0.85±0.18 c

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