Characterization of Seed Mycobiota Using Culture-Dependent and Culture-Independent Approaches.

Seed fungi are potentially important for their roles in seedling microbiome assembly and seedling health, but surveys of full seed fungal communities remain limited. While culture-dependent methods have been used to characterize some members of the seed mycobiota, recent culture-independent studies have improved the ease in identifying and characterizing full seed fungal communities. In this chapter, we describe how to survey seed fungi using both traditional culture-based methods and culture-free metabarcoding. We first describe protocols for the isolation and long-term preservation of fungal strains from individual seeds and for the extraction and amplification of DNA from such fungal isolates for identification with Sanger sequencing. We also detail how to extract, amplify, and sequence fungal DNA directly from individual seeds. Finally, we provide suggestions for troubleshooting media choices, PCR inhibition by isolates and plant tissue, and PCR limitation by low fungal DNA.

A metacommunity ecology approach to understanding microbial community assembly in developing plant seeds.

Microorganisms have the potential to affect plant seed germination and seedling fitness, ultimately impacting plant health and community dynamics. Because seed-associated microbiota are highly variable across individual plants, plant species, and environments, it is challenging to identify the dominant processes that underlie the assembly, composition, and influence of these communities. We propose here that metacommunity ecology provides a conceptually useful framework for studying the microbiota of developing seeds, by the application of metacommunity principles of filtering, species interactions, and dispersal at multiple scales. Many studies in seed microbial ecology already describe individual assembly processes in a pattern-based manner, such as correlating seed microbiome composition with genotype or tracking diversity metrics across treatments in dispersal limitation experiments. But we see a lot of opportunities to examine understudied aspects of seed microbiology, including trait-based research on mechanisms of filtering and dispersal at the micro-scale, the use of pollination exclusion experiments in macro-scale seed studies, and an in-depth evaluation of how these processes interact via priority effect experiments and joint species distribution modeling.

The core seed mycobiome of Pseudotsuga menziesii var. menziesii across provenances of the Pacific Northwest, USA.

Fungal symbionts occur in all plant tissues, and many aid their host plants with critical functions, including nutrient acquisition, defense against pathogens, and tolerance of abiotic stress. "Core" taxa in the plant mycobiome, defined as fungi present across individuals, populations, or time, may be particularly crucial to plant survival during the challenging seedling stage. However, studies on core seed fungi are limited to individual sampling sites, raising the question of whether core taxa exist across large geographic scales. We addressed this question using both culture-based and culture-free techniques to identify the fungi found in individual seeds collected from nine provenances across the range of coastal Douglas-fir (Pseudotsuga menziesii var. menziesii), a foundation tree species in the Pacific Northwest and a globally important timber crop that is propagated commercially by seed. Two key findings emerged: (i) Seed mycobiome composition differed among seed provenances. (ii) Despite variation in the seed mycobiome, we detected four core members, none of which is a known pathogen of Douglas-fir: Trichoderma spp., Hormonema macrosporum, Mucor plumbeus, and Talaromyces rugulosus. Our results support the concept of a core seed microbiome, yet additional work is needed to determine the functional consequences of core taxa for seedling germination, growth, survival, and competition.

Culturable Seed Microbiota of Populus trichocarpa.

Plants harbor a diverse community of microbes, whose interactions with their host and each other can influence plant health and fitness. While microbiota in plant vegetative tissues has been extensively studied, less is known about members of the seed microbiota. We used culture-based surveys to identify bacteria and fungi found in the seeds of the model tree, Populus trichocarpa, collected from different sites. We found that individual P. trichocarpa seeds typically contained zero or one microbe, with common taxa including species of Cladosporium, Aureobasidium, Diaporthe, Alternaria, and Pseudomonas, a bacterium. Pseudomonas isolates were associated with seed mortality and were negatively associated with the occurrence of fungal isolates within Epicoccum, Alternaria, and Aureobasidium from the same seed. Next, we conducted an inoculation experiment with one of the isolated seed microbes, Pseudomonas syringae pv. syringae, and found that it reduced seed germination and increased seedling mortality for P. trichocarpa. Our findings highlight common fungi and bacteria in the seeds of P. trichocarpa, prompting further study of their functional consequences. Moreover, our study confirms that P. syringae pv. syringae is a seed pathogen of P. trichocarpa and is the first report that P. syringae pv. syringae is a lethal seedling pathogen of P. trichocarpa, allowing for future work on the pathogenicity of this bacterium in seedlings and potential antagonism with other seed microbes.