Response to Edaphoclimatic Conditions and Crop Management of the Bacterial Microbiome of Musa acuminata Rhizosphere Profiled by 16S rRNA Gene Amplicon Sequencing

Bacterial rhizospheric microbiomes of Musa acuminata cultivated in farms close to the west and east Mexican coasts and with different climate, soils, and crop management practices, were characterized by 16S rRNA gene amplicon sequencing. Results showed that rhizospheric microbiome composition changed along with seasonal weather but were mostly indifferent to soil type.

B anana (Musa spp.) is one of the most produced crops in the world (1). The rhizosphere and endosphere microbiomes of Musa spp. have been intensely investigated to address the major threat to banana production, Fusarium wilt (2)(3)(4)(5)(6)(7). Nevertheless, the dynamic behavior of such microbiomes throughout seasons in different climates, soil types, and crop management practices have not been analyzed, despite the fact that they may provide valuable information to prevent diseases or increase productivity. Here, we report the 16S rRNA gene profiling of the Musa acuminata rhizosphere cultivated in two climate regimes, different soil types, and different types of crop management, which include the addition of biostimulants.
Bulk soil and roots of banana plants conventionally cultivated with or without microbial biostimulants (BioFit RTU and Mycoroot, Innovak, Mexico; 1 kg BioFit RTU 1 1 kg Mycoroot/200 liters water/ha, three 4-month-spaced applications during the cropping year; 2 kg Mycoroot/200 liters water/ha, 2 weeks after each dual application) were sampled (random blocks) in three farms from southern Mexico close to the east and west coastsplantation SB, Chiapas (14°549250N, 92°269260W; average temperature, 26.3°C; average annual rainfall, 2,158 mm), and plantations SO and RE, Tabasco (17°379310N, 92°579050W; average temperature, 26.9°C; average annual rainfall, 3,862 mm) (8,9). The Chiapas samples were collected only during December 2017, while samples from Tabasco were collected during February, June, and December 2018. In addition, the Tabasco samples were collected from banana cultivated in three soil types, sandy loam, clay loam, and silty loam. All samples were triplicated, each one composed of seven plant roots (collected during the inflorescence emission of the mother plant) and seven 20-cm-deep soil columns for rhizosphere and bulk soil samples.
For rhizospheric DNA extraction, excess soil on the roots was mechanically removed.  130PB Ultrasonic Processor; Fisher Scientific) at 70% frequency for 5 min. The rhizosphere fraction was recovered by centrifugation (3,857 Â g, 4°C, 20 min) and kept at 280°C until metagenomic DNA extraction. Bulk soil and rhizosphere (250 mg) metagenomic DNA were extracted with a DNeasy PowerSoil kit (Qiagen, Germany) following the manufacturer's instructions, and after extraction, its integrity and concentration were assessed by agarose gel electrophoresis and UV spectroscopy. 16S V3-V4 rRNA was amplified with 337F/805R primers (25 PCR cycles) and indexed with a Nextera XT index kit v2 (Illumina) (8 PCR cycles) using Phusion (Thermo Fisher) DNA polymerase (10). 16S rRNA amplicon libraries were paired-end sequenced on an Illumina MiSeq platform. A total of 25,675,772 raw reads were obtained for 16S libraries (Table 1). Sequencing reads were analyzed with CLC Genomics Workbench 9.0 and CLC Microbial Genomics module 1.3 (Qiagen, Denmark). Raw reads were overlapped into single longer reads and fixed-length trimmed; chimeras and reads showing ,100 abundance were removed. To identify operational taxonomic units (OTUs), filtered reads were clustered against the SILVA 16S database 138.1 (11) using 97% identity as clustering criteria. A total of 79,182 OTUs were predicted for 16S libraries.
Rhizosphere microbiomes were different from those of their surrounding bulk soil but derived from them. Bulk soil and rhizosphere microbiome composition changed along with seasonal weather and, in some cases, also after biostimulant application; however, soil type did not show any influence.
Data availability. The sequences obtained in this study were made public in the Sequence Read Archive (SRA) (accession numbers are listed in Table 1) via the National Center for Biotechnology Information (NCBI) under the accession number PRJNA673638.