Plant Seeds Commonly Host Bacillus spp., Potential Antagonists of Phytopathogens.

In agriculture, horticulture and plantation forestry, Bacillus species are the most commonly applied antagonists and biopesticides, targeting plant pathogens and insect pests, respectively. Bacillus isolates are also used as bacterial plant biostimulants, or BPBs. Such useful isolates of Bacillus are typically sourced from soil. Here, we show that Bacillus - and other antagonistic microbes - can be sourced from a broad range of plant seeds. We found that culturable Bacillus isolates are common in the seeds of 98 plant species representing 39 families (i.e., 87% of the commonly cultured bacteria belonged to Bacillales). We also found that 83% of the commonly cultured fungi from the seeds of the 98 plant species belonged to just three orders of fungi-Pleosporales, Hypocreales and Eurotiales-that are also associated with antagonism. Furthermore, we confirmed antagonism potential in agaro with seed isolates of Bacillus from Pinus monticola as a representative case. Eight isolates each of seed Bacillus, seed fungi, and foliar fungi, all from P. monticola, were paired in a total of 384 possible pair-wise interactions (with seed and foliar fungi as the targets). Seed Bacillus spp. were the strongest antagonists of the seed and foliar fungi, with a mean interaction strength 2.8 times greater than seed fungi (all either Eurotiales or Hypocreales) and 3.2 times greater than needle fungi. Overall, our study demonstrates that seeds host a taxonomically narrow group of culturable, antagonistic bacteria and fungi.

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.

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.