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.
ABSTRACT
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.
ANNOUNCEMENT
Banana (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 (2–7). 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 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 coasts—plantation SB, Chiapas (14°54′25″N, 92°26′26″W; average temperature, 26.3°C; average annual rainfall, 2,158 mm), and plantations SO and RE, Tabasco (17°37′31″N, 92°57′05″W; 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. Then, the roots were washed in 200 ml sterile phosphate-buffered saline (PBS)-Silwet Maxx (Arysta LifeScience, Mexico) (0.02% [vol/vol]) on sterile bottles and shaken at 250 rpm, 4°C for 20 min. Afterward, washed roots were taken off and submerged again in PBS-Silwet Maxx under sterile conditions, shaken at 250 rpm at 4°C for 20 min, and sonicated (VCX-130PB Ultrasonic Processor; Fisher Scientific) at 70% frequency for 5 min. The rhizosphere fraction was recovered by centrifugation (3,857 × g, 4°C, 20 min) and kept at −80°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.
TABLE 1.
Characteristics and SRA accession numbers of 16S rRNA sequences obtained in this study
| Location | Soil type | Sample type | Sampling date (mo/yr) | Sample name | SRA accession no. fora: |
||
|---|---|---|---|---|---|---|---|
| Rep 1 | Rep 2 | Rep 3 | |||||
| Chiapas SB | Loamy | Bulk soil | 12/2017 | 16S bulk soil Chiapas Rep 1, 2, 3 |
SRR12963519 | SRR12963518 | SRR12963562 |
| Rhizosphere-control | 12/2017 | 16S rhizosphere Chiapas Rep 2, 3 |
SRR12963551 | SRR12963540 | |||
| Rhizosphere-biostimulant | 12/2017 | 16S rhizosphere Chiapas Biostimulant, Rep 1, 2, 3 |
SRR13237956 | SRR13237955 | SRR13237944 | ||
| Tabasco SO | Sandy loam | Bulk soil | 2/2018 | 16S bulk soil tab sandy T1 Rep 1, 2, 3 |
SRR12963529 | SRR12963523 | SRR12963522 |
| Rhizosphere-control | 2/2018 | 16S rhizosphere tab sandy T1 Rep 2, 3 |
SRR12963521 | SRR12963520 | |||
| Rhizosphere-biostimulant | 2/2018 | 16S rhizosphere tab sandy T1 Biostimulant, rep 1, 2, 3 |
SRR13237935 | SRR13237934 | SRR13237933 | ||
| Bulk soil | 6/2018 | 16S bulk soil tab sandy T2 Rep 1, 2, 3 |
SRR12963559 | SRR12963558 | SRR12963557 | ||
| Rhizosphere-control | 6/2018 | 16S rhizosphere tab sandy T2 Rep 1, 2, 3 |
SRR12963556 | SRR12963555 | SRR12963554 | ||
| Rhizosphere-biostimulant | 6/2018 | 16S rhizosphere tab sandy T2 Biostimulant, Rep 1, 2, 3 |
SRR13237954 | SRR13237953 | SRR13237952 | ||
| Bulk soil | 12/2018 | 16S bulk soil tab sandy T3 Rep 1, 2, 3 |
SRR12963543 | SRR12963542 | SRR12963541 | ||
| Rhizosphere-control | 12/2018 | 16S rhizosphere tab sandy T3 Rep 1, 2, 3 |
SRR12963539 | SRR12963538 | SRR12963537 | ||
| Rhizosphere-biostimulant | 12/2018 | 16S rhizosphere tab sandy T3 Biostimulant, Rep 1, 2, 3 |
SRR13237945 | SRR13237943 | SRR13237942 | ||
| Tabasco SO | Clay loam | Bulk soil | 2/2018 | 16S bulk soil tab clay T1 Rep 1, 2, 3 |
SRR12963517 | SRR12963516 | SRR12963570 |
| Rhizosphere-control | 2/2018 | 16S rhizosphere tab clay T1 Rep 1, 2, 3 |
SRR12963569 | SRR12963568 | SRR12963567 | ||
| Rhizosphere-biostimulant | 2/2018 | 16S rhizosphere tab clay T1-biostimulant, rep 1, 2 | SRR13237932 | SRR13237931 | |||
| Bulk soil | 6/2018 | 16S bulk soil tab clay T2 Rep 1, 2, 3 |
SRR12963553 | SRR12963552 | SRR12963550 | ||
| Rhizosphere-control | 6/2018 | 16S rhizosphere tab clay T2 Rep 1, 2, 3 |
SRR12963549 | SRR12963548 | SRR12963547 | ||
| Rhizosphere-biostimulant | 6/2018 | 16S rhizosphere tab clay T2 Biostimulant, rep 1, 2, 3 |
SRR13237951 | SRR13237950 | SRR13237949 | ||
| Bulk soil | 12/2018 | 16S bulk soil tab clay T3 Rep 1, 2, 3 |
SRR12963536 | SRR12963535 | SRR12963534 | ||
| Rhizosphere-control | 12/2018 | 16S rhizosphere tab clay T3 Rep 1, 2, 3 |
SRR12963533 | SRR12963532 | SRR12963531 | ||
| Rhizosphere-biostimulant | 12/2018 | 16S rhizosphere tab clay T3 Biostimulant, rep 1, 2, 3 |
SRR13237941 | SRR13237940 | SRsR13237939 | ||
| Tabasco SO | Silty loam | Bulk soil | 2/2018 | 16S bulk soil tab silty T1 Rep 1, 2, 3 |
SRR12963566 | SRR12963565 | SRR12963564 |
| Rhizosphere-control | 2/2018 | 16S rhizosphere tab silty T1 Rep 1, 2, 3 |
SRR12963563 | SRR12963561 | SRR12963560 | ||
| Rhizosphere-biostimulant | 2/2018 | 16S rhizosphere tab silty T1 Biostimulant, rep 1, 2 |
SRR13237930 | SRR13237929 | |||
| Bulk soil | 6/2018 | 16S bulk soil tab silty T2 Rep 1 |
SRR12963546 | ||||
| Rhizosphere-control | 6/2018 | 16S rhizosphere tab silty T2 Rep 1, 2 |
SRR12963545 | SRR12963544 | |||
| Rhizosphere-biostimulant | 6/2018 | 16S rhizosphere tab silty T2 Biostimulant, rep 1, 2, 3 |
SRR13237948 | SRR13237947 | SRR13237946 | ||
| Bulk soil | 12/2018 | 16S bulk soil tab silty T3 Rep 1, 2, 3 |
SRR12963530 | SRR12963528 | SRR12963527 | ||
| Rhizosphere-control | 12/2018 | 16S rhizosphere tab silty T3 Rep 1, 2, 3 |
SRR12963526 | SRR12963525 | SRR12963524 | ||
| Rhizosphere-biostimulant | 12/2018 | 16S rhizosphere tab silty T3 Biostimulant, rep 1, 2, 3 |
SRR13237938 | SRR13237937 | SRR13237936 | ||
| Tabasco RE | Silty loam | Bulk soil | 2/2018 | 16S bulk soil tab RE T1 Rep 1, 2, 3 |
SRR13234470 | SRR13234469 | SRR13234458 |
| Rhizosphere-control | 2/2018 | 16S rhizosphere tab RE T1 Rep 1, 2, 3 |
SRR13234450 | SRR13234449 | SRR13234448 | ||
| Rhizosphere-biostimulant | 2/2018 | 16S rhizosphere tab RE T1 Biostimulant, rep 1, 2, 3 |
SRR13234447 | SRR13234446 | SRR13234445 | ||
| Bulk soil | 6/2018 | 16S bulk soil tab RE T2 Rep 1, 2, 3 |
SRR13234444 | SRR13234468 | SRR13234467 | ||
| Rhizosphere-control | 6/2018 | 16S rhizosphere tab RE T2 Rep 1, 2, 3 |
SRR13234466 | SRR13234465 | SRR13234464 | ||
| Rhizosphere-biostimulant | 6/2018 | 16S rhizosphere tab RE T2 Biostimulant, rep 1, 2, 3 |
SRR13234463 | SRR13234462 | SRR13234461 | ||
| Bulk soil | 12/2018 | 16S bulk soil tab RE T3 Rep 1, 2, 3 |
SRR13234460 | SRR13234459 | SRR13234457 | ||
| Rhizosphere-control | 12/2018 | 16S rhizosphere tab RE T3 Rep 1, 2, 3 |
SRR13234456 | SRR13234455 | SRR13234454 | ||
| Rhizosphere-biostimulant | 12/2018 | 16S rhizosphere tab RE T3 Biostimulant, rep 1, 2, 3 |
SRR13234453 | SRR13234452 | SRR13234451 | ||
Rep, replicate.
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.
ACKNOWLEDGMENT
This work was supported by Innovak Global, Chihuahua, Mexico.
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Associated Data
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Data Availability Statement
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.