Construction of a plant growth-promoting synthetic bacterial community from the sugarcane microbiome

Author(s)

Jaderson Silveira Leite Armanhi

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Instituto de Biologia
• Defense date: 2018-08-20

Examining board members

Paulo Arruda; Alessandra Alves de Souza; Siu Mui Tsai; Elíbio Leopoldo Rech Filho; Daniel Guariz Pinheiro

• Advisor: Paulo Arruda

Abstract

Plant-associated microbial communities can promote plant growth and development by several mechanisms, such as the production of phytohormones, repression of pathogens, stresses resistance and nutrient uptake. Culturing these beneficial microbes is an essential step towards understanding these mechanisms. However, traditional methods of microbial cultivation and isolation are commonly based on screening for known plant growth-promoting microbial activities or specific phylogenetic groups. Therefore, although these approaches provide a better understanding of well-studied microbial traits, they tend to limit the discovery of novel essential functions in plant microbe interaction. Besides, as these methods are exclusively based on axenic cultures, they are time-consuming, expensive and may result in losses of relevant biological information due to strict mutual dependences between microbes. In sugarcane, in the past decades, considerable efforts have been made to study diazotrophic bacterial communities. More recently, our research group has shown that there are several other highly abundant microbial groups inhabiting sugarcane that have never been investigated regarding its functional role in plants. Here we described innovative approaches to screen, target and validate plant beneficial microorganisms based on the ecological context, such as relative abundance and colonization dynamics, regardless of their taxonomic affiliation or preselected functions. First, we developed a strategy called "community-based culture collection" (CBC) based on isolating microbes by selecting colonies that may contain single or multiple microorganisms. The rationale is to bypass all given impasses by traditional techniques, reduce the laborious work of pure cultures isolation and allow naturally occurring communities to be stored. We developed a multiplexing 16S rRNA gene amplicon sequencing method that allowed identifying the bacterial composition of each isolated community in a high throughput manner. Then, we described a bioinformatics pipeline to cross reference the stored bacteria and all bacterial diversity associated with sugarcane, with the purpose of constructing a synthetic community composed exclusively of highly abundant bacteria in the sugarcane plant. Then, the synthetic bacterial community was validated for its plant growth promoting ability in inoculation experiments in a plant model. We have applied these procedures to explore beneficial bacteria in association to sugarcane as most of them are still understudied. The sugarcane CBC was created by isolating 5,137 microbial colonies from root and stalks. After sequencing and cross-referencing, we found that we successfully recovered bacterial groups accounting for 15.9% and 61.6¿65.3% of the rhizosphere and the endophytic microbiomes of stalks, respectively. The synthetic community was composed of several highly abundant bacteria in sugarcane and inoculated in maize plants. Members of the synthetic bacterial community efficiently colonized plant organs, displaced the natural microbiota and dominated 53.9% of the rhizosphere bacterial abundance. Also, inoculated plants dramatically increased biomass by 3.4-fold. Taken together, the results presented in this work comprise innovative methods applicable to large scale microbial isolation and identification, and the concept of designing synthetic microbial communities based on plant microbiome profile for screening plant beneficial microorganisms (AU)

• FAPESP's process: 16/04322-0 - Cross-talk between microorganism-microorganism and plant-microorganism from a representative bacterial collection of the sugarcane microbiome
• Grantee: Jaderson Silveira Leite Armanhi
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)