Genetic Diversity and Association Characters of Bacteria Isolated from Arbuscular Mycorrhizal Fungal Spore Walls

Association between arbuscular mycorrhizal fungi (AMF) and bacteria has long been studied. However, the factors influencing their association in the natural environment is still unknown. This study aimed to isolate bacteria associated with spore walls of AMF and identify their potential characters for association. Spores collected from coastal reclamation land were differentiated based on their morphology and identified by 18S rDNA sequencing as Funneliformis caledonium, Racocetra alborosea and Funneliformis mosseae. Bacteria associated with AMF spore walls were isolated after treating them with disinfection solution at different time intervals. After 0, 10 and 20 min of spore disinfection, 86, 24 and 10 spore associated bacteria (SAB) were isolated, respectively. BOX-PCR fingerprinting analysis showed that diverse bacterial communities were associated to AMF spores. Bacteria belonging to the same genera could associate with different AMF spores. Gram positive bacteria were more closely associated with AMF spores. Isolated SAB were characterized and tested for spore association characters such as chitinase, protease, cellulase enzymes and exopolysaccharide production (EPS). Among the 120 SAB, 113 SAB were able to show one or more characters for association and seven SAB did not show any association characters. The 16S rDNA sequence of SAB revealed that bacteria belonging to the phyla Firmicutes, Proteobacteria, Actinobacteria and Bactereiodes were associated with AMF spore walls.

丛枝菌根真菌（arbuscular mycorrhizal fungi, AMF）与细菌的共生关联长期以来备受学界关注。然而，自然环境中影响二者关联的关键因素仍未明确。本研究旨在分离AMF孢子壁附着的细菌，并鉴定其潜在的关联特性。研究从沿海围垦土地采集AMF孢子，通过形态学特征对其进行区分，并经18S核糖体DNA测序鉴定为苏格兰管柄囊霉（Funneliformis caledonium）、白柄球囊霉（Racocetra alborosea）以及摩西管柄囊霉（Funneliformis mosseae）。采用不同时长的消毒溶液处理AMF孢子后，分离其孢子壁附着的细菌（spore associated bacteria, SAB）。在孢子消毒0、10和20分钟后，分别分离得到86株、24株和10株孢子附着细菌。BOX-PCR指纹图谱分析结果显示，AMF孢子附着的细菌群落具有丰富的多样性；同一菌属的细菌可与不同种的AMF孢子形成关联。革兰氏阳性菌与AMF孢子的关联更为紧密。对分离得到的孢子附着细菌进行表征，并检测其与孢子关联相关的特性，包括几丁质酶、蛋白酶、纤维素酶活性及胞外多糖（exopolysaccharide, EPS）合成能力。在120株孢子附着细菌中，113株可表现出至少一种关联相关特性，剩余7株未表现出任何关联特性。对孢子附着细菌的16S核糖体DNA序列分析结果显示，厚壁菌门（Firmicutes）、变形菌门（Proteobacteria）、放线菌门（Actinobacteria）及拟杆菌门（Bacteroidetes）为AMF孢子壁附着的细菌类群。