Bacteria are happy together to promote plant growth

In this study, we designed a dual-species bacterial consortium that can promote plant growth synergistically. Combined analyses of transcriptome, metabolome, mutant construction, and metabolic modeling revealed that they cooperate with each other through cross-feeding.

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Bacteria survive badly in solitary

When I was a bachelor student taking the Plant Nutrition course in 2015, professor Qirong Shen introduced bio-fertilizers. I was immediately shocked by the comparative photos.

A star bacterium, Bacillus velezensis SQR9, which carried his name’s acronym, could promote plant growth and protect plants against pathogens. Through his passionate course, I became interested in plant beneficial bacteria and decided to do my bachelor thesis with him in 2016. Shortly after, I met my direct supervisor Dr. Zhihui Xu who investigated the biocontrol mechanisms of B. velezensis SQR9 during his PhD. He found an intriguing phenomenon that despite the high inoculation of B. velezensis into the soil, the relative abundance of Bacillus spp. surrounding the plant root was very low. Other researchers also found that Bacillus spp. were only able to colonize the plant roots in the initial 48h and became undetectable in the following month [1]. It seems Bacillus spp. could hardly survive in the natural rhizosphere environment. So how to explain the plant-growth-promoting and biocontrol functions if they were not surviving properly? What did B. velezensis SQR9 do in the limited 48h before they vanish? We hypothesized that B. velezensis SQR9 serves as a pioneer and subsequently induces the assemblage of other indigenous beneficial bacteria that synergistically promote the growth of the host plant.

Find a friend

To test this hypothesis, we compared the rhizosphere bacterial composition of plant that was inoculated by B. velezensis SQR9 with plant without bacterial inoculation. The result showed that several genera were induced by strain SQR9. These genera were predicted to be “friends” of strain SQR9, among which Pseudomonas spp. were the most prominent. I defended my bachelor’s with this brief result leaving many mysteries unsolved.

To explore the biological meaning, I continued this study as a five-year combined post-graduate and PhD student under the supervision of professors Ruifu Zhang, Qirong Shen, and associate professor Zhihui Xu at Nanjing Agricultural University (NJAU). I started isolating bacteria from the rhizosphere, a long process of repeated isolation experiment numerous times for half a year, a tiring and boring process. However, I did manage to obtain my desired bacterial genera, including Pseudomonas spp.. Since bacteria principally live attached to surface as highly concentrated adherent communities, also called biofilms, we chose biofilm as the investigation model. Our favorite Bacillus spp. like to reside on the air-liquid surface as floating biofilm. We screened hundreds of isolates and found Pseudomonas stutzeri XL272, this time carrying my initials as strain designation, could form biofilm synergistically with B. velezensis SQR9!

How do they interact?

Our scientific goal now was to study how do B. velezensis SQR9 “becomes friend” with P. stutzeri XL272. In addition, we were also curious about whether their friendship is stable if the environment changes? As we had narrow experience in studying microbial interaction at that time, we invited 10 scientists from all over the world to Nanjing and hosted an international workshop on the topic of “Plant-Microbe Interactions in the Rhizosphere” in 2018. We received plenty of useful ideas that guided our follow-up research. Moreover, I got lots of support and encouragement from the experts which meant a lot to me as a young scientist. During the workshop, we made a connection with professor Ákos T. Kovács from Technical University of Denmark (DTU). I joined his group as a guest PhD student in December of 2019 supported by a Chinese Scholar Council fellowship.

After years of experiments, data analysis, and discussion with collaborators and colleagues, we are slowly beginning to understand what happens between the two species. Under certain conditions, such as static rich culture conditions or natural soil environment, they could share food (amino acids, organic acid, sugar) and clothes (biofilm extracellular matrix) with each other. However, when there were limited nutrients or when they are forced to live without distance (shaking culture), their friendship collapse, P. stutzeri XL272 would inhibit the growth of B. velezensis SQR9. So, nutrients and spatial segregation matter. Just like humans, we are willing to share with friends when we have extra food but not when we are starving. Also, proper fences make good neighbors. We need private time instead of partying continuously.

What about the host plant?

After making friends, are these microbes beneficial for a host plant? The answer is yes! The dual-species consortium could help plants grow better than working alone.  You could learn more by reading our paper "Bacillus velezensis stimulates resident rhizosphere Pseudomonas stutzeri for plant health through metabolic interactions".  https://www.nature.com/articles/s41396-021-01125-3

 We are only at the beginning of investigating these inter-species interactions and their practical use. In the future, we aim to create bio-fertilizers using these two beneficial bacteria, studying their co-evolution, their interactions with other bacteria, and so on. Follow us and explore more interesting science stories!

This story wouldn’t have been possible without Jiyu Xie and Taimeng Tan (PhD students from NJAU) and their experimental support. Additionally, Viktor Hesselberg-Thomsen (PhD student from DTU) and Mikael Lenz Strube (associate professor from DTU) aided our transcriptome data analysis and bioinformatics area. Just in time at the revision steps of the manuscript, MSc student Daoyue Zheng (MSc student from NJAU) contributed by constructing a metabolic model that nicely fitted the experimental data. Last but not least, Anna Dragoš (assistant professor from University of Ljubljana, Slovenia), Qirong Shen, Ruifu Zhang, Ákos T. Kovács, and my direct supervisor Zhihui Xu all contributed to designing the project and revising the paper. Without your help and support, I wouldn’t be able to finish this challenging project.

Cheers virtually!

Follow us on Twitter:

Xinli Sun @XinliSun
Zhihui Xu @ZhihuiXu2
Ákos T. Kovács @EvolvedBiofilm
Anna Dragoš @Anna_Dragos
Mikael L. Strube @Mikael_lenz

Reference

[1] Gamez RM, Ramirez S, Montes M, Cardinale M. Complementary dynamics of banana root colonization by the plant growth-promoting rhizobacteria Bacillus amyloliquefaciens Bs006 and Pseudomonas palleroniana Ps006 at spatial and temporal Scales. Microbial Ecology 2020; 80: 656–668.

Xinli Sun

PhD, Nanjing Agricultural University