Taxonomic and functional diversity of cultured seed associated microbes of the cucurbit family

Eman M Khalaf, Manish N Raizada

Endophytes are microbes that colonize plant internal tissues without causing disease. In particular, seed-associated endophytes may be vectors for founder microbes that establish the plant microbiome, which may subsequently contribute beneficial functions to their host plants including nutrient acquisition and promotion of plant growth. The Cucurbitaceae family of gourds (e.g., cucumbers, melons, pumpkin, squash), including its fruits and seeds, is widely consumed by humans. However, there is limited data concerning the taxonomy and functions of seed-associated endophytes across the Cucurbitaceae family. Here, bacteria from surface-sterilized seeds of 21 curcurbit varieties belonging to seven economically important species were cultured, classified using 16S rRNA gene sequencing, and subjected to eight in vitro functional tests. In total, 169 unique seed-associated bacterial strains were cultured from selected cucurbit seeds. Interestingly, nearly all strains belonged to only two phyla (Firmicutes, Proteobacteria) and only one class within each phyla (Bacilli, γ-proteobacteria, respectively). Bacillus constituted 50 % of all strains and spanned all tested cucurbit species. Paenibacillus was the next most common genus, while strains of Enterobacteriaceae and lactic acid bacteria were also cultured. Phylogenetic trees showed limited taxonomic clustering of strains by host species. Surprisingly, 33 % of strains produced the plant hormone, indole-3-acetic acid (auxin), known to stimulate the growth of fruits/gourds and nutrient-acquiring roots. The next most common nutrient acquisition traits in vitro were (in rank order): nitrogen fixation/N-scavenging, phosphate solubilisation, siderophore secretion, and production of ACC deaminase. Secretion of extracellular enzymes required for nutrient acquisition, endophyte colonization and/or community establishment were observed. Bacillus strains had the potential to contribute all tested functional traits to their hosts. The seeds of economically important cucurbits tested in this study have a culturable core microbiota consisting of Bacillus species with potential to contribute diverse nutrient acquisition and growth promotion activities to their hosts. These microbes may lead to novel seed inoculants to assist sustainable food production. Given that cucurbit seeds are consumed by traditional societies as a source of tryptophan, the precursor for auxin, we discuss the possibility that human selection inadvertently facilitated auxin-mediated increases in gourd size.