一株植物根际促生真菌浅囊状栓菌SICAU-T41的筛选及全基因组分析

Screening and whole-genome analysis of a plant growth-promoting fungus, Trametes gibbosa SICAU-T41

摘要:

【背景】 多孔菌科(Polyporaceae)的种类较多，形态特征差异较大，至今尚未形成十分完善的分类系统，其中栓菌属是多孔菌科内分类最为混乱的一个类群。【目的】 扩充植物根际促生真菌的资源库，从四川省凉山彝族自治州会理市烤烟根际土壤中分离出1株具有植物促生效果的多孔真菌浅囊状栓菌(Trametes gibbosa) SICAU-T41。旨在通过对菌株SICAU-T41进行全基因组测序，解析其全基因组的结构、功能及其在种属进化关系中的位置，深入挖掘其次级代谢产物基因资源。【方法】 采用传统定向筛选培养基培养、摇瓶培养的方法筛选植物促生真菌，测定其促生特性，并通过盆栽试验和qPCR试验验证其促生能力和定殖能力。利用Illumina和PacBio平台相结合的测序技术对菌株SICAU-T41进行全基因组测序，并进行基因预测、功能注释、次级代谢产物合成基因簇预测、比较基因组学分析。【结果】 从烤烟根际土壤中共分离纯化得到78株不同菌落形态的真菌菌株，并测定其溶解无机磷的能力、产IAA能力、纤维素降解能力、产铁载体能力和拮抗病原菌的能力。筛选得到1株促生能力最强的菌株SICAU-T41，其溶磷量为30.40 mg/L，IAA产量为48.10 mg/L，纤维素降解圈直径为2.03 cm，铁载体晕圈直径(halation diameter, HD)及菌落直径(colony diameter, CD)比值HD/CD为2.00。盆栽试验结果表明，菌株SICAU-T41能显著促进烟苗的生长，烟苗的株高与CK相比增加了74.7%，根长增加了14.4%，叶片数增加了16.7%，叶面积增加了48.4%，根数增加了52.1%，地上部鲜重增加了36.7%，地下部鲜重增加了82.6%。相较于CK，添加菌株SICAU-T41能显著提高其在烤烟根际土壤中的定殖量，定殖量为29.93×10³ copies/μL，较CK提高了123.36%。对菌株SICAU-T41进行全基因组测序，其基因组大小为33.93 Mb，预测的总基因数为10 142个，蛋白质编码基因数为9 843个，G+C含量为56.89%；在eggNOG、KEGG、GO、CAZy数据库中分别注释到7 875、3 444、5 793、448个基因；转运蛋白分类数据库分析(transporter classification database analysis, TCDB)预测到1 129个与多种分泌系统相关的转运蛋白；同时预测到菌株SICAU-T41中存在22个次级代谢产物合成基因簇，涉及萜类、非核糖体肽类、β-内酰胺类、核糖体合成并经翻译后修饰的肽类天然产物(ribosomally synthesized and post-translationally modified peptide secondary metabolites, RiPPs)等多种天然产物的合成。基于比较基因组学分析，菌株SICAU-T41与浅囊状栓菌(Trametes gibbose)的平均核苷酸一致性(average nucleotide identity, ANI)值达98.5%，说明菌株SICAU-T41是一株浅囊状栓菌。【结论】 从烤烟根际土壤中分离到的植物促生真菌SICAU-T41具有多种植物促生特性，能显著促进烟苗的生长。通过对菌株SICAU-T41全基因组序列的测定与分析，在基因层面揭示了该菌的植物促生机制，同时丰富了浅囊状栓菌的基因组数据库。

Abstract:

[Background] The Polyporaceae encompasses numerous species that exhibit highly diverse morphological traits; consequently, no fully satisfactory classification system has yet been established. Among its constituent genera, Trametes is the most taxonomically chaotic group within the family. [Objective] To expand the resource library of plant growth-promoting fungi, we isolated Trametes gibbosa SICAU-T41, a strain of poroid fungus, with plant growth-promoting effects from the rhizosphere soil of flue-cured tobacco in Huili County, Liangshan Prefecture, Sichuan Province. This study aimed to conduct whole-genome sequencing of strain SICAU-T41 to analyze its genomic structure, function, and phylogenetic position and mine its secondary metabolite gene resources. [Methods] Directed screening media and shake-flask culture were employed to isolate plant growth-promoting fungi, the plant growth-promoting characteristics of which were determined. Pot experiments and qPCR were then employed to evaluate the plant growth-promoting effects and colonization of the isolates. Whole-genome sequencing of strain SICAU-T41 was performed by Illumina and PacBio platforms. Gene prediction, functional annotation, prediction of secondary metabolite biosynthetic gene clusters (BGCs), and comparative genomic analysis were then conducted. [Results] A total of 78 fungal strains with distinct colony morphology were isolated and purified from the rhizosphere soil of flue-cured tobacco. Their abilities to solubilize inorganic phosphorus, produce indole-3-acetic acid (IAA), degrade cellulose, produce siderophores, and antagonize pathogens were measured. Strain SICAU-T41, exhibiting the strongest plant growth-promoting potential, was selected. This strain demonstrated the phosphorus-solubilizing capacity of 30.40 mg/L, IAA production of 48.10 mg/L, cellulose degradation zone diameter of 2.03 cm, and siderophore production (HD/CD ratio) of 2.00. Pot experiment results demonstrated that strain SICAU-T41 significantly promoted the growth of tobacco seedlings. Compared with the control (CK), inoculation of this strain increased the seedling height by 74.7%, root length by 14.4%, leaf number by 16.7%, leaf area by 48.4%, root number by 52.1%, aboveground fresh weight by 36.7%, and underground fresh weight by 82.6%. Compared with CK, the addition of SICAU-T41 significantly increased its colonization density in the rhizosphere soil of flue-cured tobacco, which reached 29.93×10³ copies/μL, a 123.36% increase over CK. Whole-genome sequencing revealed that the genome of strain SICAU-T41 had a length of 33.93 Mb, containing 10 142 predicted genes, including 9 843 protein-coding genes, with the G+C content of 56.89%. A total of 7 875, 3 444, 5 793, and 448 genes were annotated in the eggNOG, KEGG, GO, and CAZy databases, respectively. TCDB analysis predicted 1 129 transporters associated with various secretion systems. Furthermore, 22 secondary metabolite BGCs were predicted in the genome of strain SICAU-T41, involved in the synthesis of various natural products such as terpenoids, non-ribosomal peptides (NRPs), β-lactams, and ribosomally synthesized and post-translationally modified peptides (RiPPs). Comparative genomic analysis showed that the average nucleotide identity between strain SICAU-T41 and T. gibbosa reached 98.5%, confirming that SICAU-T41 was a strain of T. gibbosa. [Conclusion] The plant growth-promoting fungus T. gibbosa SICAU-T41, isolated from the rhizosphere soil of flue-cured tobacco, possesses multiple plant growth-promoting properties and significantly enhances tobacco seedling growth. Whole-genome sequencing and analysis of strain SICAU-T41 revealed the genetic basis underlying its plant growth-promoting mechanisms at the genomic level, while also enriching the genomic database of T. gibbosa.