Table_1_Vertical Transmission of Diverse Cultivation-Recalcitrant Endophytic Bacteria Elucidated Using Watermelon Seed Embryos.docx

Seed transmission of endophytic microorganisms is a growing research area in plant biology and microbiology. We employed cultivation versus cultivation-independent approaches on excised embryos from watermelon seeds (6–12 months in storage) and on embryo-derived in vitro seedlings (EIVS) to assess the vertical transmission of endophytic bacteria. Surface-disinfected watermelon seeds bore abundant residual bacteria in the testa and perisperm tissues, predominantly Bacillus spp. propounding the essentiality of excluding all non-embryonic tissues for vertical transmission studies. Tissue homogenates from re-disinfected seed embryos displayed no cultivable bacteria during the 1-week monitoring. Bright-field live microscopy revealed abundant bacteria in tissue homogenates and in embryo sections as intracellular motile particles. Confocal imaging on embryo sections after SYTO-9 staining and eubacterial fluorescent in situ hybridization (FISH) endorsed enormous bacterial colonization. Quantitative Insights Into Microbial Ecology (QIIME)-based 16S rRNA V3–V4 taxonomic profiling excluding the preponderant chloroplast and mitochondrial sequences revealed a high bacterial diversity in watermelon seed embryos mainly Firmicutes barring spore formers followed by Proteobacteria, Bacteroidetes, and Actinobacteria, and other minor phyla. Embryo-base (comprising the radicle plus plumule parts) and embryo-cotyledon parts differed in bacterial profiles with the abundance of Firmicutes in the former and Proteobacteria dominance in the latter. EIVS displayed a higher bacterial diversity over seed embryos indicating the activation from the dormant stage of more organisms in seedlings or their better amenability to DNA techniques. It also indicated embryo-to-seedling bacterial transmission, varying taxonomic abundances for seed embryos and seedlings, and differing phylogenic profiles for root, hypocotyl, and cotyledon/shoot-tip tissues. Investigations on different watermelon cultivars confirmed the embryo transmission of diverse cultivation recalcitrant endophytic bacteria. Firmicutes, Proteobacteria, Actinobacteria, and Bacteroidetes formed the core phyla across different cultivars with 80–90% similarity at genus to phylum levels. Conversely, freshly harvested seeds displayed a dominance of Proteobacteria. The findings revealed that dicot seeds such as in different watermelon cultivars come packaged with abundant and diverse vertical and seedling-transmissible cultivation recalcitrant endophytic bacteria with significant implications for plant biology.

种子内生微生物的传播是植物生物学和微生物学中日益增长的研究领域。本研究采用培养与培养无关的方法，对西瓜种子（储存6-12个月）的剥离胚胎和胚胎来源的体外幼苗（EIVS）进行了研究，以评估内生细菌的垂直传播。表面消毒的西瓜种子在种皮和胚乳组织中残留大量细菌，主要为芽孢杆菌属，这突显了在垂直传播研究中排除所有非胚胎组织的必要性。重新消毒的种子胚胎的组织匀浆在1周的监测期间未培养出细菌。明场活体显微镜揭示了组织匀浆和胚胎切片中的大量细菌作为细胞内可动颗粒。在SYTO-9染色和真细菌原位杂交（FISH）后的胚胎切片的共聚焦成像证实了大量的细菌定殖。基于定量微生物生态学（QIIME）的16S rRNA V3–V4分类学分析排除了主要的叶绿体和线粒体序列，揭示了西瓜种子胚胎中细菌的高多样性，主要属于厚壁菌门，其次是变形菌门、拟杆菌门和放线菌门，以及其他较小的门类。胚基（包括胚根和胚芽部分）和胚子叶部分在细菌组成上存在差异，前者中厚壁菌门丰富，后者中变形菌门占主导。EIVS显示出比种子胚胎更高的细菌多样性，这表明幼苗中更多生物的休眠阶段被激活或其DNA技术更易于应用。这也表明了胚到幼苗的细菌传播，种子胚胎和幼苗中分类学丰度的变化，以及根、下胚轴、子叶/芽尖组织的系统发育特征的不同。对不同西瓜品种的研究证实了多种栽培抗逆内生细菌的胚胎传播。厚壁菌门、变形菌门、放线菌门和拟杆菌门构成了不同品种的核心门类，在属到门水平的相似度达到80-90%。相反，新鲜收获的种子显示出变形菌门的占主导地位。研究结果揭示了双子叶种子，如不同西瓜品种，携带着丰富且多样的垂直和幼苗传播的栽培抗逆内生细菌，这对植物生物学具有重要意义。