We report the draft genome sequences of 59 Gram-positive bacterial strains that were isolated from Vietnamese crop plants. The strains were assigned to nine different Bacillus or Brevibacillus species. Ten strains classified as being Bacillus spp. (3 strains), Brevibacillus spp. (6 strains), or Lysinibacillus sp. (1 strain) could not be identified to the species level.
ABSTRACT
We report the draft genome sequences of 59 Gram-positive bacterial strains that were isolated from Vietnamese crop plants. The strains were assigned to nine different Bacillus and Brevibacillus species. Ten strains classified as being a Bacillus sp. (3 strains), Brevibacillus sp. (6 strains), or Lysinibacillus sp. (1 strain) could not be identified to the species level.
ANNOUNCEMENT
Endophytic and plant-associated Gram-positive bacteria were isolated from Vietnamese crop plants such as coffee, black pepper, maize, orange trees, dragon trees, tomato, and cabbage (for details, see Table 1). Samples were obtained from the soil adjacent to plant roots, the surface, and the inner tissue of different plant parts such as root, stem, and leaf (Table 1). Samples from inner tissues were propagated after surface sterilization using 70% ethanol and 1% sodium hypochlorite (1). Brevibacillus spp. were found to be enriched when soil samples adherent to plant roots were incubated with shaking for a further 2 weeks. Diluted samples were incubated on either half-strength tryptic soy broth or tryptone-yeast extract-glucose agar (2) for 3 to 5 days at 30°C. In order to enrich endospore-forming bacteria, single colonies were picked from agar plates, diluted in 0.5 ml 0.9% NaCl, and heat treated for 20 min at 80°C. Only strains that were able to suppress fungal plant pathogens, such as Fusarium oxysporum, Phytophthora palmivora, or Neoscytalidium dimidiatum, under in vitro conditions were used in further experiments. As a first step for characterizing these strains more completely, the isolates underwent genome sequencing, and their taxonomy based on their draft genome sequences was evaluated.
TABLE 1.
List of the 59 endospore-forming Gram-positive bacteria isolated from Vietnamese crop plants
| GenBank accession no. | SRA accession no. | Sample name | Estimated genome size (bp) | G+C content (%) | Total no. of raw reads | Genome coverage (×) | No. of contigs | Contig N50 (bp) | Total no. of genes | Isolation source and date (day-mo-yr) | Taxonomy according to dDDH and ANIb results | dDDH (%)a | ANIb (%)b | Similar type strain |
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| JABMIW000000000 | SRR12133238 | BT2.2 | 3,708,421 | 44.0 | 7,888,450 | 28.0 | 22 | 556,510 | 3,798 | Dragon tree stem, 5-5-2019 | B. altitudinis | 86.20 | 98.26 | B. altitudinis DSM 21631 |
| VDDR00000000 | SRR12104977 | A8 | 5,071,716 | 35.4 | 5,427,083 | 52.0 | 52 | 307,045 | 5,877 | Coffee root, 1-9-2018 | B. cereus | 90.50 | 98.74 | B. cereus ATCC 14579 |
| VEPO00000000 | SRR12104976 | A22 | 5,812,849 | 35.2 | 5,059,544 | 56.0 | 57 | 315,527 | 6,032 | Coffee root, 1-9-2018 | B. cereus | 74.20 | 96.80 | B. cereus ATCC 14579 |
| VEPP00000000 | SRR12104975 | A24 | 5,689,021 | 35.7 | 2,398,472 | 28.0 | 73 | 233,470 | 5,897 | Black pepper root, 1-9-2018 | B. cereus | 85.50 | 98.13 | B. cereus ATCC 14579 |
| VEPT00000000 | SRR12283339 | A31 | 5,319,678 | 35.3 | 5,328,004 | 40.0 | 102 | 246,002 | 5,556 | Coffee rhizosphere, 1-9-2018 | B. cereus | 90.10 | 98.64 | B. cereus ATCC 14579 |
| VEPQ00000000 | SRR12104956 | A42 | 5,604,011 | 35.6 | 6,770,872 | 80.0 | 42 | 489,606 | 5,708 | Black pepper root, 1-9-2018 | B. cereus | 74.30 | 96.75 | B. cereus ATCC 14579 |
| JABSVB000000000 | SRR12115356 | HB3.1 | 5,796,358 | 34.8 | 5,938,348 | 15.0 | 82 | 478,132 | 5,867 | Orange plant adherent soil, 17-4-2019 | B. cereus | 89.80 | 98.25 | B. cereus ATCC 14579 |
| JABSVE000000000 | SRR12132980 | HD1.4B | 5,660,925 | 34.9 | 5,561,352 | 13.0 | 53 | 831,879 | 5,737 | Tomato root soil, 23-4-2019 | B. cereus | 88.40 | 98.20 | B. cereus ATCC 14579 |
| JABSVC000000000 | SRR12124346 | HD2.4 | 5,660,820 | 34.9 | 8,250,328 | 20.0 | 48 | 378,831 | 5,734 | Tomato root adherent soil, 23-4-2019 | B. cereus | 88.30 | 98.22 | B. cereus ATCC 14579 |
| JABSVF000000000 | SRR12141689 | M2.1B | 5,869,336 | 34.8 | 8,131,108 | 20,0 | 63 | 344,459 | 6,029 | Maize field soil, 6-12-2018 | B. cereus | 73.10 | 96.28 | B. cereus ATCC 14579 |
| VEPR00000000 | SRR12104981 | SN4.3 | 5,594,617 | 35.6 | 3,571,304 | 45.0 | 78 | 146,072 | 5,773 | Ostrinia nubilalis, 1-11-2018 | B. cereus | 73.60 | 96.76 | B. cereus ATCC 14579 |
| VEPS00000000 | SRR12042680 | TK1 | 6,195,299 | 34.7 | 11,965,210 | 30.0 | 363 | 83,852 | 6,483 | Coffee rhizosphere, 1-11-2018 | B. cereus | 73.50 | 95.96 | B. cereus ATCC 14579 |
| VEPU00000000 | SRR12104980 | SN4.1 | 5,443,801 | 35.2 | 4,526,204 | 40.0 | 32 | 868,705 | 5,590 | Ostrinia nubilalis, 1-11-2018 | B. pacificus | 77.20 | 97.10 | B. pacificus EB422 |
| VEPY00000000 | SRR12104982 | CD3-1a | 5,150,560 | 35.2 | 4,168,978 | 56.0 | 40 | 589,852 | 5,431 | Brassica juncea rhizosphere, 1-11-2018 | Bacillus sp. | 62.50 | 94.31 | B. tropicus N24 |
| VEPX00000000 | SRR12104984 | CD3-5 | 5,695,940 | 35.3 | 4,593,634 | 60.0 | 50 | 302,092 | 5,784 | Brassica juncea rhizosphere, 1-11-2018 | Bacillus sp. | 69.60 | 95.45 | B. pacificus EB422 |
| JABSVD000000000 | SRR12132949 | HD1.3 | 5,695,940 | 35.1 | 7,791,502 | 19.0 | 56 | 469,972 | 5,784 | Tomato root soil, 23-4-2019 | Bacillus sp. | 66.30 | 95.04 | B. pacificus EB422 |
| JABMIX000000000 | SRR12141623 | GR2.1 | 4,084,062 | 43.6 | 3,512,065 | 25.0 | 22 | 1,042,180 | 4,240 | Green egg-plant root, 6-12-2018 | B. subtilis | 88.50 | 98.49 | B. subtilis ATCC 6051 |
| JABMIY000000000 | SRR12142185 | DL2.1 | 3,927,651 | 44.0 | 3,260,262 | 24.0 | 46 | 476,949 | 4,159 | Maize field soil, 8-5-2018 | B. tequilensis | 78.50 | 97.25 | B. tequilensis KCTC 13622 |
| VEPW00000000 | SRR12104983 | CD3-2 | 5,958,606 | 35.8 | 2,788,296 | 30.0 | 68 | 236,159 | 6,186 | Brassica juncea rhizosphere, 1-11-2018 | B. tropicus | 72.40 | 96.68 | B. tropicus N24 |
| VEPV00000000 | SRR12104979 | SN1 | 5,335,513 | 35.2 | 5,015,568 | 63.0 | 34 | 456,889 | 5,560 | Ostrinia nubilalis, 1-11-2018 | B. tropicus | 72.50 | 96.70 | B. tropicus N24 |
| VEWT00000000 | SRR12132949 | A25 | 3,833,140 | 46.2 | 5,120,982 | 88.0 | 25 | 981,821 | 3,834 | Black pepper root, 1-9-2018 | B. velezensis | 79.90 | 96.41 | B. velezensis KCTC 13012 |
| VEWU00000000 | SRR12104957 | A35 | 3,867,857 | 46.3 | 5,469,886 | 93.0 | 20 | 561,718 | 3,896 | Black pepper root, 1-9-2018 | B. velezensis | 79.60 | 96.48 | B. velezensis KCTC 13012 |
| JABSVZ000000000 | SRR12142193 | BP1.2A | 3,872,427 | 46.4 | 7,443,818 | 30.0 | 30 | 471,958 | 3,832 | Sand in Mooc River, 8-5-2019 | B. velezensis | 96.20 | 98.16 | B. velezensis KCTC 13012 |
| JABSVT000000000 | SRR12133237 | BT2.1 | 3,889,067 | 46.4 | 13,578,068 | 50.0 | 33 | 475,102 | 3,855 | Dragon tree stem, 8-5-2019 | B. velezensis | 85.80 | 98.23 | B. velezensis KCTC 13012 |
| JABSVU000000000 | SRR12133171 | BT2.4 | 3,865,088 | 46.4 | 3,355,802 | 12.0 | 46 | 422,236 | 3,851 | Dragon tree stem, 8-5-2019 | B. velezensis | 95.30 | 98.24 | B. velezensis KCTC 13012 |
| JABSVG000000000 | SRR12113939 | CP5.2 | 3,932,265 | 46.4 | 4,591,652 | 17.0 | 34 | 563,859 | 3,876 | Orange plant root, 17-4-2019 | B. velezensis | 80.30 | 97.25 | B. velezensis KCTC 13012 |
| JABSVH000000000 | SRR12114569 | CP6 | 3,825,619 | 46.7 | 6,766,126 | 19.0 | 34 | 482,295 | 3,782 | Orange plant leaf, 17-4-2019 | B. velezensis | 81.10 | 97.46 | B. velezensis KCTC 13012 |
| JABSVI000000000 | SRR12115431 | CP7.1A | 3,874,748 | 46.4 | 4,658,644 | 17.0 | 36 | 475,102 | 3,836 | Orange plant soil, 17-4-2019 | B. velezensis | 96.40 | 98.04 | B. velezensis KCTC 13012 |
| JABSVJ000000000 | SRR12123548 | CP7.1C | 3,871,413 | 46.4 | 4,273,868 | 16.0 | 45 | 339,955 | 3,840 | Orange plant soil, 17-4-2019 | B. velezensis | 96.20 | 98.04 | B. velezensis KCTC 13012 |
| JABSVK000000000 | SRR12123552 | CP7.2A | 3,883,818 | 46.4 | 4,191,800 | 16.0 | 34 | 468,775 | 3,857 | Orange plant soil, 17-4-2019 | B. velezensis | 85.80 | 98.03 | B. velezensis KCTC 13012 |
| JABSVM000000000 | SRR12123706 | DP1.3B | 3,877,050 | 46.4 | 4,778,546 | 18.0 | 24 | 486,178 | 3,827 | Orange plant soil, 17-4-2019 | B. velezensis | 96.70 | 98.03 | B. velezensis KCTC 13012 |
| JABSVL000000000 | SRR12123599 | DP2.2B | 3,871,310 | 46.4 | 4,996,856 | 18.0 | 40 | 471,958 | 3,840 | Orange plant soil, 17-4-2019 | B. velezensis | 96.20 | 98.04 | B. velezensis KCTC 13012 |
| JABSVX000000000 | SRR12134139 | EG5.1A | 4,030,371 | 46.2 | 9,630,302 | 35.0 | 36 | 451,426 | 3,954 | White egg-plant root, 6-12-2018 | B. velezensis | 92.20 | 98.93 | B. velezensis KCTC 13012 |
| JABSVP000000000 | SRR12132923 | HD1.1 | 3,875,639 | 46.4 | 4,494,558 | 16.0 | 33 | 475,102 | 3,834 | Tomato root soil, 23-4-2019 | B. velezensis | 96.50 | 98.03 | B. velezensis KCTC 13012 |
| JABSVN000000000 | SRR12123774 | HD2.2 | 3,874,124 | 46.4 | 3,903,166 | 15.0 | 41 | 475,603 | 3,842 | Tomato root soil, 23-4-2019 | B. velezensis | 96.40 | 98.04 | B. velezensis KCTC 13012 |
| JABSVO000000000 | SRR12132925 | HD2.5 | 3,878,653 | 46.4 | 4,716,380 | 17.0 | 37 | 428,165 | 3,861 | Tomato root soil, 23-4-2019 | B. velezensis | 98.30 | 98.03 | B. velezensis KCTC 13012 |
| JABSVQ000000000 | SRR12132982 | HD3.1B | 3,891,722 | 46.4 | 4,185,344 | 15.0 | 49 | 356,004 | 3,869 | Tomato root soil, 23-4-2019 | B. velezensis | 98.20 | 98.04 | B. velezensis KCTC 13012 |
| JABSVR000000000 | SRR12132999 | HD5.1 | 3,874,949 | 46.4 | 1,960,848 | 14.0 | 48 | 475,102 | 3,852 | Tomato root soil, 23-4-2019 | B. velezensis | 96.40 | 98.06 | B. velezensis KCTC 13012 |
| JABSVS000000000 | SRR12133003 | HD5.2A | 3,878,897 | 46.4 | 6,893,794 | 25.0 | 37 | 428,165 | 3,861 | Tomato leaves, 23-4-2019 | B. velezensis | 98.30 | 98.22 | B. velezensis KCTC 13012 |
| VEWV00000000 | SRR12104919 | KT1 | 3,950,573 | 46.4 | 5,413,752 | 90.0 | 40 | 280,310 | 3,970 | Black pepper root, 1-9-2018 | B. velezensis | 84.70 | 96.13 | B. velezensis KCTC 13012 |
| JABSVY000000000 | SRR12142183 | MR2.1A | 4,027,596 | 46.2 | 6,220,582 | 23.0 | 41 | 451,426 | 3,956 | Maize root, 6-12-2018 | B. velezensis | 92.20 | 98.93 | B. velezensis KCTC 13012 |
| JABSVV000000000 | SRR12133236 | OL1.1 | 3,921,211 | 46.4 | 3,249,582 | 12.0 | 34 | 1,032,611 | 3,850 | Orange plant leaf, 13-6-2019 | B. velezensis | 80.20 | 97.48 | B. velezensis KCTC 13012 |
| JABSVW000000000 | SRR12133982 | OR2.1 | 3,864,359 | 46.4 | 8,863,958 | 30.0 | 33 | 428,160 | 3,834 | Orange plant soil, 13-6-2019 | B. velezensis | 95.30 | 98.25 | B. velezensis KCTC 13012 |
| VEWW00000000 | SRR12104916 | S1 | 3,866,305 | 46.1 | 6,223,108 | 106 | 30 | 486,485 | 3,893 | Coffee rhizosphere, 1-9-2018 | B. velezensis | 85.80 | 97.01 | B. velezensis KCTC 13012 |
| VEWX00000000 | SRR12104917 | S2 | 3,864,301 | 46.1 | 6,033,960 | 103 | 33 | 397,838 | 3,893 | Coffee rhizosphere, 1-9-2018 | B. velezensis | 85.70 | 97.12 | B. velezensis KCTC 13012 |
| VEWY00000000 | SRR12105026 | TK2 | 4,044,692 | 46.2 | 4,395,588 | 71.0 | 48 | 808,279 | 4,144 | Field soil, 1-11-2018 | B. velezensis | 79.80 | 96.26 | B. velezensis KCTC 13012 |
| VEWZ00000000 | SRR12104978 | TL7 | 3,865,047 | 46.4 | 4,381,374 | 77.0 | 29 | 428,384 | 3,879 | Coffee rhizosphere, 1-9-2018 | B. velezensis | 85.70 | 97.94 | B. velezensis KCTC 13012 |
| JABSUV000000000 | SRR12113944 | HB2.2 | 6,346,173 | 47.2 | 5,448,236 | 13.0 | 137 | 133,518 | 5,928 | Orange plant soil, 17-4-2019 | Brevibacillus sp. | 53.70 | 93.46 | Brevibacillus formosus NRRL NRS-863 |
| JABSVA000000000 | SRR12142184 | RS1.1 | 6,246,682 | 47.2 | 4,526,908 | 11.0 | 46 | 810,856 | 5,867 | Maize field soil, 8-5-2019 | Brevibacillus sp. | 54.20 | 93.10 | Brevibacillus formosus NRRL NRS-863 |
| JABSUW000000000 | SRR12132961 | HD1.4A | 6,032,732 | 52.2 | 4,713,548 | 11.0 | 120 | 286,241 | 5,720 | Tomato root soil, 23-4-2019 | Brevibacillus parabrevis | 93.80 | 99.07 | Brevibacillus parabrevis 605 |
| JABSUX000000000 | SRR12132995 | HD3.3A | 6,074,516 | 52.1 | 4,338,330 | 11.0 | 178 | 190,049 | 5,781 | Tomato root soil, 23-4-2019 | Brevibacillus parabrevis | 93.60 | 99.05 | Brevibacillus parabrevis 605 |
| JABMIV000000000 | SRR12105150 | HB1.1 | 6,317,805 | 47.2 | 6,867,506 | 28.0 | 44 | 376,218 | 6,010 | Orange plant soil, 17-4-2019 | Brevibacillus porteri | 82.70 | 97.36 | Brevibacillus porteri B-41110 |
| JABMIU000000000 | SRR12105325 | HB1.2 | 6,342,770 | 47.1 | 6,815,744 | 17.0 | 55 | 468,289 | 6,077 | Orange plant soil, 17-4-2019 | Brevibacillus porteri | 82.80 | 97.38 | Brevibacillus porteri B-41110 |
| JABSUT000000000 | SRR12113913 | HB1.4B | 6,377,995 | 47.2 | 12,209,194 | 29.0 | 41 | 493,935 | 6,133 | Orange plant soil, 17-4-2019 | Brevibacillus porteri | 82.20 | 97.24 | Brevibacillus porteri B-41110 |
| JABSUU000000000 | SRR12123671 | DP1.3A | 6,579,985 | 47.1 | 5,839,560 | 13.0 | 62 | 512,617 | 6,114 | Orange plant soil, 17-4-2019 | Brevibacillus sp. | 59.10 | 93.65 | Brevibacillus porteri B-41110 |
| JABMIT000000000 | SRR12105327 | HB1.3 | 6,066,817 | 47.3 | 4,162,250 | 17.0 | 44 | 476,614 | 5,750 | Orange plant soil, 17-4-2019 | Brevibacillus sp. | 53.80 | 93.00 | Brevibacillus formosus DSM 9885 |
| JABSUY000000000 | SRR12141636 | M2.1A | 6,216,907 | 47.3 | 4,162,250 | 10.0 | 53 | 477,247 | 5,898 | Maize field soil, 6-12-2018 | Brevibacillus sp. | 56.50 | 93.95 | Brevibacillus formosus DSM 9885 |
| JABSUZ000000000 | SRR12141690 | MS2.2 | 6,273,578 | 47.3 | 1,594,872 | 10.0 | 72 | 144,852 | 5,898 | Maize field soil, 8-5-2019 | Brevibacillus sp. | 55.50 | 93.30 | Brevibacillus brevis DSM 30 |
| VEXA00000000 | SRR12104985 | CD3.6 | 4,369,550 | 36.8 | 6,766,126 | 25.0 | 13 | 533,701 | 4,321 | Brassica juncea rhizosphere, 1-11-2018 | Lysinibacillus sp. | 31.10 | 85.47 | Lysinibacillus varians GY32 |
For biomass production, colonies of a fresh culture grown on Luria-Bertani (LB) agar plates were selected. Genomic DNA was extracted using the DNeasy blood and tissue kit (Qiagen, Hilden, Germany) after growth on LB agar plates for 24 h at 37°C. The sequencing was conducted at LGC Genomics (Berlin, Germany) with an Illumina HiSeq system using paired-end 150-bp reads. Default parameters were used for all software unless otherwise specified. Reads were trimmed and filtered using fastp v0.20.1 (https://github.com/OpenGene/fastp) with default settings. De novo assemblies were generated by using the short-read assembler SPAdes v3.13.0 (3) (http://cab.spbu.ru/software/spades) without read correction and with normal bridging. The quality of assemblies was assessed by determining the proportion of falsely trimmed proteins by using Ideel (https://github.com/phiweger/ideel). The complete pipeline results were saved as a Snakemake file (4) and uploaded on GitHub (https://github.com/CptChiler/snakeGenome). Genome coverage of the contigs obtained was 50× on average (Table 1). Contigs were submitted to GenBank for gene annotation, which was implemented using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP) v4.11 (5). The Genome-to-Genome Distance Calculator (GGDC) v2.1 provided by DSMZ (http://ggdc.dsmz.de) was used for genome-based species delineation via estimated digital DNA-DNA hybridization (dDDH) values against a reference genome. Formula 2, which is especially appropriate for analyzing draft genomes, was used (6). In addition, JSpeciesWS (http://jspecies.ribohost.com/jspeciesws) was used to determine average nucleotide identity based on BLAST+ (ANIb) values by pairwise genome comparisons (7). Accession numbers and characteristics of the genomes, including their ANIb values, are summarized in Table 1.
According to their draft genome sequences, we have assigned 49 of the isolates with potential to control plant pathogens as representatives of Bacillus altitudinis (strain BT2.2), Bacillus cereus (strains A8, A22, A24, A31, A42, HB3.1, HD1.4B, HD2.4, M2.1B, SN4.3, and TK1), Bacillus pacificus (strain SN4-1), Bacillus subtilis subsp. subtilis (strain GR2.1), Bacillus tequilensis (strain DL2.1), Bacillus tropicus (strains CD3.2 and SN1), Bacillus velezensis (strains A25, A35, BT2.1, BT2.4, CP5.2, CP6, CP7.1A, CP7.1C, CP7.2A, DP1.3B, DP2.2B, EG5.1A, HD1.1, HD2.2, HD2.5, HD3.1B, HD5.1, HD5.2A, KT1, MR2.1A, OL1.1, OR2.1, S1, S2, TK2, TL7, and BP1.2A), Brevibacillus parabrevis (strains HD1.4A and HD3.3A), and Brevibacillus porteri (strains HB1.1, HB1.2, and HB1.4B).
Ten strains, i.e., Bacillus sp. strains HD1.3, CD3.1A, and CD3.5, Brevibacillus sp. strains HB2.2, RS1.1, DP1.3A, HB1.3, MS2.1A, and MS2.2, and Lysinibacillus sp. strain CD3.6, could not be identified to the species level since their estimated taxonomic values and values were below the species cutoff values (GGDC, <70%; ANIb, <96%). Further research in order to characterize these novel biocontrol strains and their secondary metabolites is in progress.
Data availability.
These whole-genome shotgun projects have been deposited in GenBank under the accession numbers listed in Table 1.
ACKNOWLEDGMENTS
This work was supported through project ENDOBICA (Bundesministerium für Bildung und Forschung [BMBF] grant no. 031B0582A), the National Foundation for Science and Technology Development (NAFOSTED; code no. 106.03-2017.28), and the Ministry of Science and Technology (MOST) in Vietnam (code no. NDT.40.GER/18).
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Associated Data
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Data Availability Statement
These whole-genome shotgun projects have been deposited in GenBank under the accession numbers listed in Table 1.