Researchers map entire population of microorganisms in sugarcane

Complete inventory of more than 35,000 species of bacteria and fungi in sugarcane has been published in Scientific Reports with a new method for studying their functions.

By Diego Freire  |  Agência FAPESP – A single sugarcane sample is home to 23,811 types of bacteria and 11,727 different groups of fungi, according to researchers at the University of Campinas’s Biology Institute (IB-UNICAMP) in São Paulo State, Brazil, who have produced the first ever complete map of the microorganism communities that inhabit sugarcane tissue, from roots to shoots and leaves.

The research project was carried out at the Central Laboratory for High-Performance Technology in Life Sciences (LaCTAD), which is supported by FAPESP under its Multi-User Equipment Program (EMU).

Two articles on the project have been published in the journal Scientific Reports, now part of Springer Nature: the first on June 30, reporting the production of the complete sugarcane microbiome, i.e., all the microorganisms that inhabit the plant’s various organs; the second on July 12, presenting a new method of isolating and growing sugarcane collections for the study of the functions they perform in the plant.

The microbiota of plants, like those of animals, mediate important interactions between the organism and its environment, which in the case of plants include the conversion of atmospheric nitrogen into compounds used in protein synthesis. According to the researchers, however, without a complete map of the microbiome, it is impossible to know everything about this relationship, which needs to be better understood if progress is to be made in developing biotechnology for sustainable agriculture.

“A community of microorganisms inhabits all higher organisms and plays a key role in the immune system. This is true of animals, including humans, as well as plants. They protect the organism against pathogens,” said Paulo Arruda, a professor at IB-UNICAMP’s Department of Genetics and Evolution.

“However, scientists know about little more than half a dozen of the functions performed by bacteria and fungi in plants. Studies of their diversity, structure and impact on economically important crops are rare. These two papers offer a new picture of the sugarcane microbiome and how to assess it to develop technology based on the association between plants and microorganisms.”

The researchers produced a complete inventory of the bacterial and fungal communities associated with sugarcane, mapping their structure and composition in detail. The study identified more than 35,000 species of bacteria and fungi that inhabit the interior and surfaces of the roots, shoots and leaves.

“These many thousands of microorganisms certainly aren’t just contributing to the half-dozen functions of the plant microbiota we know about. We’re looking at a veritable black box of biology, and it’s waiting to be explored,” said Arruda, who also heads the Protein Kinase Chemical Biology Center (SGC-Unicamp), supported by FAPESP under its Partnership for Technological Innovation Program (PITE).

Map of microorganisms

Mass identification of the species of microorganisms that inhabit sugarcane was made possible by a second-generation sequencer, which sequences microbiome DNA markers present in samples of roots, shoots and leaves without the need to isolate and grow each species of bacterium or fungus in a laboratory, as has always been performed hitherto.

“Until very recently, technological constraints limited researchers to growing bacteria in the lab, which takes a long time and results in a small number of samples, especially because many species don’t grow in a culture medium. Thus, we only had superficial knowledge of the communities of microorganisms that inhabit each tissue of the plant,” said Rafael Soares Correa de Souza, a researcher affiliated with UNICAMP’s Center for Molecular Biology and Genetic Engineering (CBMEG).

Furthermore, he went on, “most research in the field focused on analysis of the root microbiome, identifying the bacteria and other microorganisms associated with it and those that were linked to nutrient uptake and other processes”.

The researchers’ second-generation sequencing technology enabled them to identify the entire sugarcane microbiome, including communities that live both inside and outside the roots, stalks and leaves in different stages of development. They now have a complete “map” that describes the most abundant bacteria and fungi, as well as their functions and possible biotechnological applications, and affording access to a world of diversity hitherto unknown to science.

They produced their own sequencing protocol for this purpose, collecting samples of roots, shoots and leaves separately and washing each sample in a special solution. The gray water was centrifuged slowly to remove the soil and other environmental residues. The isolated microorganisms were then centrifuged at high speed to precipitate cells containing the genetic material to be sequenced. The same procedure was performed for the microorganisms that live inside the plant, and the tissues were then shredded in a blender to expose them.

In addition to the map, the researchers have built up collections of samples representing the microorganisms that live in the sugarcane microbiome.

“These collections are stored so that they can be accessed easily, enabling researchers to select microorganisms for inocula that represent different communities and can be studied to determine what beneficial effects they have on plants. Now we have a map and the biological resources needed to move ahead with the research,” said Jaderson Silveira Leite Armanhi, also a researcher affiliated with CBMEG.

In addition to FAPESP, the project was funded by Spanish energy company Repsol and Repsol Sinopec Brasil S.A. Researchers affiliated with the Biotechnology and Plant Genomics Center at the Technical University of Madrid (UPM) in Spain also took part.

The results of the mapping exercise are presented in the article “Unlocking the bacterial and fungal communities in assemblages of the sugarcane microbiome” by Rafael Soares Correa de Souza, Vagner Katsumi Okura, Jaderson Silveira Leite Armanhi, Beatriz Jorrín, Núria Lozano, Márcio José da Silva, Manuel González-Guerrero, Laura Migliorini de Araújo, Natália Cristina Verza, Homayoun Chaichian Bagheri, Juan Imperial and Paulo Arruda, available at www.nature.com/articles/srep28774.

The article “Multiplex amplicon sequencing for microbe identification in community-based culture collections” by Jaderson Silveira Leite Armanhi, Rafael Soares Correa de Souza, Laura Migliorini de Araújo, Vagner Katsumi Okura, Piotr Mieczkowski, Juan Imperial and Paulo Arruda, can be read at www.nature.com/articles/srep29543.