Tropical palm endophytes exhibit low competitive structuring when assessed using co-occurrence and antipathogen activity analysis

Leaf-inhabiting fungal and bacterial endophytes are at their most diverse in tropical rainforest plant hosts, with some influencing host plant fitness as either symbionts or pathogens. Endophyte activity and community composition is thought to depend on competition amongst co-occurring species for resources. Here, we reveal the strength of competitive interactions between endophytes by combining e-DNA metabarcoding to characterize the community with culturing assays to ascertain their potential activity. The endophyte community associated with the understory palm Astrocaryum sciophilum was determined by extracting eDNA from 120 leaflets of eight plants located in a lowland rainforest in French Guiana. This revealed 516 fungal and 606 bacterial Operational Taxonomic Units (OTUs). Co-occurrence analysis of the most abundant OTUs revealed that direct fungal-fungal interactions were overall more negative than bacterial-bacterial interactions. This was confirmed by C-score calculations for the whole endophyte community, revealing significantly greater levels of species segregation for fungi but not bacteria when compared with simulated random communities. Following the culturing of 131 fungal, and 66 bacterial Astrocaryum endophytes collected from the same leaves, Minimal Inhibitory Concentrations of ethyl acetate culture extracts were evaluated against the fungus Trichophyton rubrum and the bacterium Staphylococcus aureus. Overall, a minority of extracts demonstrated antipathogen activity, with greater antifungal activity expression for both fungi and bacteria when compared with antibacterial activity. In order to explore whether this activity translated into competitive structuring of the community, a BLAST was conducted to match metabarcoding sequences with Sanger derived culture sequences. This demonstrated that cultures displaying antipathogen activity were significantly more likely to co-occur with fungi if they were bacteria, and bacteria if they were fungi. Nevertheless, overall correlation values averaged around zero. These results indicate that for Astrocaryum palms, fungal endophytes are likely to play a greater role in determining colonization success of subsequent endophytes whether these be fungal or bacterial, but that overall endophyte communities do not display strong patterns of community structuring through competition. Endophyte communities are thus likely to reach relative stasis in mature leaves, where the diverse community of fungi and bacteria amongst other factors prevent the subsequent establishment of pathogens.

栖息于叶片的真菌与细菌内生菌（endophytes）在热带雨林宿主植物中展现出最高的多样性，其中部分类群可作为共生体或病原菌，对宿主植物的适合度产生影响。目前认为，内生菌的活性与群落组成取决于共存物种间对资源的竞争作用。本研究通过结合环境DNA元条形码测序（e-DNA metabarcoding）表征群落结构，与培养实验以确定其潜在活性，揭示了内生菌间竞争互作的强度。与林下棕榈Astrocaryum sciophilum相关联的内生菌群落，通过从法属圭亚那低地雨林中8株植株的120张小叶中提取环境DNA（eDNA）得以解析。该分析共鉴定出516个真菌与606个细菌操作分类单元（Operational Taxonomic Units，OTUs）。对丰度最高的OTUs进行共现分析后发现，整体上真菌-真菌间的直接互作相较于细菌-细菌间的互作，表现出更强的负向作用。通过对整个内生菌群落进行C-score（C评分）计算，该结果得到了验证：相较于模拟的随机群落，真菌群落呈现出显著更高的物种分离水平，而细菌群落则无此特征。在对从同一叶片中分离得到的131株真菌与66株细菌Astrocaryum内生菌进行培养后，本研究评估了乙酸乙酯培养提取物对真菌红色毛癣菌（Trichophyton rubrum）与细菌金黄色葡萄球菌（Staphylococcus aureus）的最低抑菌浓度（Minimal Inhibitory Concentrations，MIC）。整体而言，仅有少数提取物表现出抗病原菌活性；相较于抗菌活性，真菌与细菌来源的提取物均展现出更强的抗真菌活性。为探究该活性是否会转化为群落的竞争性构建，本研究通过BLAST（Basic Local Alignment Search Tool）将元条形码测序序列与桑格测序得到的培养序列进行比对。该分析表明，具有抗病原菌活性的培养物，若为细菌则更易与真菌共现，若为真菌则更易与细菌共现。尽管如此，整体的相关系数平均值约为0。上述结果表明，对于Astrocaryum棕榈而言，真菌内生菌在决定后续内生菌（无论为真菌还是细菌）的定殖成功率方面，可能发挥着更为重要的作用；但整体而言，内生菌群落并未通过竞争展现出显著的群落构建模式。因此，成熟叶片中的内生菌群落很可能会达到相对稳定的状态：其中多样的真菌与细菌群落等多种因素，会阻碍后续病原菌的定殖。