Effective seed sterilization methods require optimization across maize genotypes

Studies of plant-microbe interactions using synthetic microbial communities (SynComs) often require the removal of seed-associated microbes by seed sterilization before inoculation to provide gnotobiotic growth conditions. A diversity of seed sterilization protocols have been developed in the past and have been used on different plant species with various amounts of validation. From these studies, it has become clear that each plant species requires its own optimized sterilization protocol. It has, however, so far not been tested if the same protocol works equally well for different varieties and seed sources of one plant species. We evaluated six seed sterilization protocols on two different varieties (Sugar Bun & B73) of maize. All unsterilized maize seeds showed fungal growth upon germination on filter paper, highlighting the need for a sterilization protocol. A short sterilization protocol with hypochlorite and ethanol was sufficient to prevent fungal growth on Sugar Bun germinants, however, a longer protocol with heat treatment and germination in fungicide was needed to obtain clean B73 germinants. This difference may have arisen from the effect of either genotype or seed source. We then tested the protocol that performed best for B73 on three additional maize genotypes from four sources. Seed germination rates and fungal contamination levels varied widely by genotype and geographic source of seeds. Our study shows that consideration of both variety and seed source is important when optimizing sterilization protocols and highlights the importance of including seed source information in plant-microbe interaction studies that use sterilized seeds.

利用合成微生物群落（SynComs）开展植物-微生物互作研究时，通常需在接种前通过种子灭菌去除种子携带的微生物，以构建悉生生长条件。过往已开发出多种种子灭菌方案，并已在不同植物物种中应用，且验证程度各不相同。现有研究已明确，每一植物物种均需适配专属的优化灭菌方案。但截至目前，尚无研究验证同一灭菌方案是否可同等适用于同一植物物种的不同品种与种子来源。我们针对玉米的两个不同品种（Sugar Bun与B73）评估了六种种子灭菌方案。所有未灭菌的玉米种子在滤纸上萌发时均出现真菌生长，这凸显了灭菌方案的必要性。一种含次氯酸盐与乙醇的短时长灭菌方案即可有效阻止Sugar Bun萌发样本的真菌生长，但获取无菌B73萌发样本则需采用包含热处理与杀菌剂萌发步骤的更长时长方案。这种差异可能源于基因型或种子来源的影响。随后，我们将适配B73的最优方案应用于来自四个来源的另外三种玉米基因型。种子萌发率与真菌污染水平因基因型与种子地理来源差异呈现显著波动。本研究表明，在优化灭菌方案时，需同时考虑品种与种子来源因素，并强调了在使用灭菌种子开展植物-微生物互作研究时，纳入种子来源信息的重要性。