Antagonistic activity of GL18 to pathogenic fungi. The antagonistic activity test showed that GL18 inhibited the growth and expansion of F. graminearum, F. acuminatum, F. oxysporum and A. niger to varying degrees. The average diameters of the inhibition zones were 20, 24, 18 and 21 mm, respectively; the inhibition rates to the four pathogenic fungi ranged from 40 to 54%. Among them, GL18 had the most significant inhibitory effect on F. acuminatum with an inhibitory rate of 53.3%, and the inhibitory rates on F. graminearum, F. oxysporum and A. niger were 44.4, 40.0 and 46.7%, respectively, which significantly inhibited the growth and expansion of mycelia and showed strong antagonistic activity (Fig. 1a, Table 1).
Bacteriostatic-related hydrolase activity of GL18. The activities of cellulase, protease, pectinase and β-1, 3-glucanase secreted by GL18 were detected by hydrolase detection media. The transparent zones indicated that the strain could produce antibacterial hydrolase, and the size of the transparent zone indicated its enzyme production ability. GL18 formed a transparent zone on different hydrolase detection media, indicating that it secreted different hydrolases of cellulase, protease, pectinase and β-1, 3-glucanase, which degraded the cell walls of the pathogens and inhibited their growth. The average diameters of the transparent zones on the detection media for cellulase, protease, pectinase and β-1, 3-glucanase were 42, 54, 38 and 32 mm, respectively, showing strong cellulase and protease production ability. It was speculated that GL18 could inhibit growth and reduce the activity of pathogenic bacteria by degrading the cell wall of pathogenic bacteria, thus antagonizing plant pathogens (Fig. 1b, Table 1).
Table 1
Plant growth-promoting traits of strain GL18
Test
|
Antagonistic to fungal pathogens
|
|
|
F. graminearum
|
F. acuminatum
|
F. oxysporium
|
Aspergillus niger
|
|
Qualitative
|
+++
|
++++
|
++
|
+++
|
|
Inhibition zone (mm)
|
20 ± 1.500c
|
24 ± 2.179ab
|
18 ± 1.000c
|
21 ± 1.323bc
|
|
Antibacterial ratio
|
44.4%
|
53.3%
|
40.0%
|
46.7%
|
|
Test
|
Ectoenzyme
|
|
|
Cellulase
|
Protease
|
Pectinase
|
β1, 3glucanase
|
|
Qualitative
|
+++
|
++++
|
+++
|
++
|
|
Transparent zone (mm)
|
42 ± 2.000b
|
54 ± 2.500a
|
38 ± 2.000b
|
32 ± 1.000c
|
|
± means standard deviation, different letters indicate a significant difference at the level of 0.05 (P < 0.05).
Low-temperature resistance. At 14 and 18°C, strain GL18 grew and formed a colony after 1 d. At 10°C, it grew and expanded quickly, forming a colony after 2 d. At 4°C, GL18 could hardly grow, therefore, GL18 has a certain tolerance to a low-temperature environment.
Salt resistance. The concentration of bacterial liquid is usually measured by absorbance at OD600, which reflects the relative salt tolerance of the strain under salt stress. The OD600 value of GL18 in the 2% NaCl concentration was higher than that in the control, indicating that the 2% NaCl concentration was beneficial to GL18 growth. With the increase in NaCl concentration, the OD600 value of GL18 showed a downward trend. The concentration of bacterial liquid decreased, indicating that the growth of GL18 was gradually inhibited. When the NaCl concentration reached 11%, a small amount of GL18 survived but grew slowly. When the NaCl concentration reached 12%, GL18 could not survive. Therefore, GL18 grew at a NaCl concentration between 2% and 11% and had strong salt tolerance (Fig. 2a).
Drought resistance. The drought resistance of GL18 was determined by simulating drought stress with different PEG-6000 concentrations. GL18 was not seriously affected by drought stress when the PEG-6000 concentration was below 9%. With the increase in the PEG-6000 concentration, the OD600 value of GL18 decreased, and the bacterial concentration of the strain liquid decreased, indicating that the growth of GL18 was affected to a certain extent. The maximum PEG-6000 concentration that GL18 could tolerate was 15%. GL18 had strong drought tolerance and a certain drought tolerance to arid habitats (Fig. 2b).
Growth-promoting effect on Avena sativa under cold stress. A GL18 bacteria suspension (cell concentration 1×107 cfu/mL) was used to irrigate the roots of Avena sativa seedlings twice at 10°C, and plant height, root length and fresh weight increased significantly compared to CK1 (Fig. 3a). The average plant height and root length of the seedlings were 8.03 and 4.46 cm, respectively, increasing by 51.80% and 38.94%, compared with CK1 (Fig. 3b). The average fresh weight was 0.11 g, with an increase of 74.04% (Fig. 3c). Strain GL18 had a significant growth-promoting effect on Avena sativa seedlings under cold stress.
Growth-promoting effect on Avena sativa under salt stress. Avena sativa seedlings were irrigated twice with a GL18 bacteria suspension (cell concentration 1×107 cfu/mL) and then irrigated with 30 mL of 11% NaCl solution. On the second day after salt stress, plant height, root length and fresh weight increased significantly compared with CK2 (Fig. 3a). The average plant height and root length of the Avena sativa seedlings were 6.89 and 2.97 cm, respectively, increasing by 22.82% and 45.59%, respectively, compared with CK2 (Fig. 3b). The average fresh weight was 0.05 g, with an increase of 70.07% (Fig. 3c). GL18 had a significant growth-promoting effect on Avena sativa seedlings under salt stress.
Growth-promoting effect on Avena sativa under drought stress. Avena sativa seedlings were irrigated twice with the GL18 bacteria suspension (cell concentration 1×107 cfu/mL) and then irrigated with 30 mL of 15% PEG-6000 solution. On the second day after drought stress, plant height, root length and fresh weight increased significantly compared with CK3 (Fig. 3a). The average plant height and root length of the Avena sativa seedlings were 10.44 and 3.49 cm, respectively, increasing by 39.39% and 57.74%, compared with CK3 (Fig. 3b). The average fresh weight was 0.12 g, with an increase of 12.5% (Fig. 3c). GL18 had a significant growth-promoting effect on Avena sativa seedlings under drought stress.
Basic features of the genome. According to the analysis of assembly results, the genome of GL18 was composed of a chromosome with a DNA sequence of 3,915,550 bp, with 272,699 reads, a total base length of 2,249,346,685 and a total base number of 1,276,942,372. The GC content was 46.47%, the number of CDS was 3726, and the total length of coding genes was 3,462,321 bp, with an average length of 929.23 bp. GL18 contained 27 rRNA, comprising nine 16S rRNA, nine 23S rRNA and nine 5S rRNA. The number of tandem repeats was 38, and the total length was 7570. The accession number of the GL18 genome sequence in NCBI is CP096033, and Fig. 4 shows the genome circle diagram.
Functional gene annotation of GL18. COG annotation results indicated that the genome sequence of GL18 was compared with the eggNOG database, and COG function annotation was performed. A total of 3088 genes were annotated, accounting for 82.88% of all genes. Among them, the known predicted functional genes were more related to amino acid transport and metabolism, transcription, carbohydrate transport and metabolism, general function prediction, translation, ribosomal structure, and biogenesis (Fig. 5a).
GO annotation results indicated that the genome sequence of GL18 was compared with the gene ontology database to perform GO gene annotation. A total of 2869 functional genes were annotated, accounting for 77% of all genes, which were divided into biological process (BP), cellular component (CC) and molecular function (MF). Among them, 1411 genes related to biological processes were mainly involved in the regulation of transcription and DNA-templated, translation, carbohydrate metabolic process, and transmembrane transport, as well as in the biosynthetic process of fatty acid, amino acid transport and other processes. In cellular components, 1383 functional genes were closely related to integral components of membrane, plasma membrane, cytoplasm and ribosome. In molecular function, 2319 functional genes were mainly associated with ATP binding, DNA binding, metal ion binding, hydrolase activity and transmembrane transporter activity (Fig. 5b).
KEGG annotation results indicated that the genome sequence of GL18 was annotated with related genes of the pathway in the KEGG database. A total of 2357 genes were found to be involved in functional annotation, including 174 pathways involved in cellular processes, 3033 pathways involved in metabolism, 215 pathways involved in genetic information processing, and 302 pathways involved in environmental information processing, which were mainly related to carbohydrate metabolism, amino acid metabolism, and metabolism of cofactors and vitamins (Fig. 5c).
Functional genes related to antagonistic activity. AntiSMASH software [30] was used to predict the gene cluster of secondary metabolite synthesis of GL18, and 13 gene clusters of secondary metabolites were predicted (Table 2). Five types of substances with antibacterial activity were related to non-ribosomal peptide synthase or polyketone synthase: surfactin, bacillaene, fengycin, iturin and bacillibactin. Cluster 1 was 91% similar to the known gene cluster and contained 41 coding genes, including srfAA, srfAB, srfAC and srfATE, which encode surfactin synthesis. Surfactin is a lipopeptide biosurfactant with antibacterial, antiviral and anti-mycoplasma activities [31]. Cluster 6 was 100% similar to the known gene cluster and contained 52 coding genes, which contain genes of the pks family encoding bacillaene synthesis, including pksC, pksE, pksG, pksI, pksJ, pksH, pksL, pksM, pksN and pksR. Bacillaene is a polyketone antibiotic that can effectively inhibit the growth of pathogenic bacteria [32]. Bacillibactin can also absorb and accumulate iron ions under iron-limited conditions and compete with other microorganisms to exert its antibacterial effect [33]. Cluster 7 was found to be 100% similar to a known gene cluster containing 64 coding genes, including ituA, ituB and ituC, which encode the key genes of iturin synthesis, and ppsA, ppsB, ppsC, ppsD and ppsE, which encode the key genes of fengycin synthesis. Iturin is a non-ribosomal synthesized cyclolipidic peptide that can inhibit fungal activity by changing the permeability of the fungal cell membrane [34]. Fengycin is a circular lipopeptide that acts as an antagonist against fungi by damaging the cell membranes of the target cells [35]. Cluster 12 was found to be 100% similar to the known gene cluster and contained 45 coding genes, including dhbF, a key gene involved in non-ribosomal peptide synthase. Cluster 13 was found to be 100% similar to the bacilysin gene cluster and contained 43 coding genes.
In addition, clusters 4, 8, 9 and 11 with unknown functions were found in the genome of GL18, including two terpene, one T3PKS and one NRPS, speculating that the GL18 genome may have gene clusters of synthesizing other antibacterial substances. Cluster 3 was only 7% similar to the known gene cluster and contained 41 coding genes, suggesting that the gene cluster might synthesize new antibacterial substances (Fig. 6). Therefore, we speculated that Bacillus GL18 could promote plant growth by synthesizing antibacterial substances to inhibit the growth of pathogens indirectly through the expression of the functional genes of bacteriostatic-related substances.
Table 2
Annotation statistics of secondary metabolites
Cluster ID
|
Type
|
Start
|
End
|
Similar Cluster
|
Similarity (%)
|
Gene No.
|
cluster1
|
NRPS
|
318207
|
383067
|
surfactin
|
91
|
41
|
cluster2
|
LAP
|
718153
|
740336
|
plantazolicin
|
91
|
19
|
cluster3
|
PKS-like
|
935681
|
976926
|
butirosin A / butirosin B
|
7
|
41
|
cluster4
|
terpene
|
1062550
|
1079780
|
−
|
−
|
22
|
cluster5
|
transAT-PKS
|
1366839
|
1454621
|
macrolactin H
|
100
|
43
|
cluster6
|
transAT-PKS
|
1676753
|
1786365
|
bacillaene
|
100
|
52
|
cluster7
|
NRPS
|
1846486
|
1983924
|
fengycin
|
100
|
64
|
cluster8
|
terpene
|
2010878
|
2032762
|
−
|
−
|
22
|
cluster9
|
T3PKS
|
2099247
|
2140348
|
−
|
−
|
47
|
cluster10
|
transAT-PKS-like
|
2256753
|
2362930
|
difficidin
|
100
|
59
|
cluster11
|
NRPS
|
2845913
|
2900807
|
−
|
−
|
43
|
cluster12
|
NRPS
|
2997798
|
3049590
|
bacillibactin
|
100
|
45
|
cluster13
|
other
|
3572815
|
3614234
|
bacilysin
|
100
|
43
|
− means none |
Functional genes related to growth promotion. Key genes of trpC, trpB and trpA encoding proteins related to the synthesis of growth hormones exist in the GL18 genome, which are involved in the tryptophan-dependent IAA synthesis pathway. It is speculated that GL18 can promote plant growth directly by synthesizing IAA. Moreover, the functional genes of miaB and idi are related to cytokinin synthesis in the GL18 genome. Cytokinin is involved in plant growth and development, thus improving the tolerance of plants to abiotic stress [36]. In addition, the GL18 genome contains a variety of functional genes related to the growth promotion of Bacillus, including glnB [37], trkH, trkA and kch [38], which are involved in the synthesis of nitrogen or potassium required for plant growth and development. GL18 may improve the soil environment and promote plant growth through nitrogen fixation and phosphorus hydrolysis. Functional genes YclQ and fetB are related to the synthesis and transport of siderophores [39], which can improve the ferritin content of plants through iron chelation and indirectly promote plant growth (Table 3).
Functional genes related to stress tolerance. The GL18 genome contains gene clusters encoding key genes related to inducing plant resistance, in which functional genes lytE, ykfC and cwlO are related to ABA biosynthesis [40]. ABA induces plant resistance by regulating the physiological response process of plants to abiotic stress. The GL18 genome also contains genes that encode pheT, pheS and menF, which are key genes involved in SA biosynthesis, and SA can respond to the antioxidant defense system of plants and improve the stress resistance of plants [41]. Meanwhile, the GL18 genome has functional genes encoding osmotic regulatory substances involved in response to abiotic stress. Gene clusters of the encoding Na+/H+ antiporter, including mnhA, mnhB, mnhC, mnhD, mnhE, mnhF and mnhG [42], can regulate intracellular pH, maintain ion homeostasis and promote cell growth under stress. It also has genes proV, proW and opuA encoding osmoprotein synthesis of proline and betaine [43], which can increase the tolerance to osmotic stress. In addition, the GL18 genome also contains the key gene cspA encoding cold shock proteins CspC, CspB and CspD [44], which participate in the low-temperature response, maintain the normal growth and metabolism of cells, and enhance the low-temperature resistance of cells (Table 3).
Table 3
Related function gene analysis of GL18
Gene name
|
Location
|
Length
|
Function
|
trpC
|
2 178 057 − 2 177 305
|
753
|
Indole-3-glycerol phosphate synthase, involved in the tryptophan-dependent IAA synthesis pathway, associated with defense responses
|
trpA
|
2175471 − 2174674
|
798
|
Tryptophan synthase subunit alpha gene
|
trpB
|
2176666 − 2175464
|
1 203
|
Tryptophan synthase subunit beta gene
|
miaB
|
1 686 372 − 1 687 901
|
1 530
|
Isopentenyl adenine gene, considered as the main cytokinin
|
idi
|
2 196 713 − 2 195 664
|
1 050
|
Isopentenyl adenine gene, considered as the main cytokinin
|
glnB
|
3 483 211 − 3 483 561
|
351
|
Involved in the regulation of nitrogen fixation
|
trkH
|
1 283 441–1 284 793
|
1 353
|
TrkH family potassium uptake protein, involved in absorption and utilization of K+
|
trkA
|
1 378 966 − 1 379 631
|
666
|
Ktr system potassium transporter gene KtrC
|
kch
|
2 956 934 − 2 957 920
|
987
|
Potassium channel family protein gene
|
yclQ
|
401 740 − 402 684
|
945
|
Siderophore synthase gene, involved in growth-promoting gene expression
|
fetB
|
1 197 756 − 1 196 995
|
762
|
Ferrous iron transport protein B
|
lytE
|
942 569 − 943 393
|
825
|
ABA biosynthesis gene, induced stress resistance
|
ykfC
|
1 243 194 − 1 244 093
|
900
|
ABA biosynthesis gene, induced stress resistance
|
cwlO
|
3 314 292 − 3 312 880
|
1 413
|
ABA biosynthesis gene, induced stress resistance
|
pheT
|
2 690 097 − 2 687 683
|
2 415
|
SA biosynthesis gene, induced stress resistance
|
pheS
|
2 691 145 − 2 690 111
|
1 035
|
SA biosynthesis gene, induced stress resistance
|
menF
|
2 915 727 − 2 914 312
|
1 416
|
SA biosynthesis gene, induced stress resistance
|
mnhA
|
2 984 830 − 2 987 232
|
2 403
|
Na+/H + antiporter subunit A, maintained normal growth under high salt stress
|
mnhB
|
2 987 229 − 2 987 660
|
432
|
Na (+) / H (+) antiporter subunit B, maintained normal growth under high salt stress
|
mnhC
|
2 987 660 − 2 988 001
|
342
|
Na (+) / H (+) antiporter subunit C, maintained normal growth under high salt stress
|
mnhD
|
2 987 985 − 2 989 475
|
1 491
|
Monovalent cation/H + antiporter subunit D, maintained normal growth under high salt stress
|
mnhE
|
2 989 481 − 2 989 957
|
477
|
Na+/H + antiporter subunit E, maintained normal growth under high salt stress
|
mnhF
|
2 989 957 − 2 990 241
|
285
|
Na (+) /H (+) antiporter subunit F1, maintained normal growth under high salt stress
|
mnhG
|
2 990 225 − 2 990 599
|
375
|
Monovalent cation/H (+) antiporter subunit G, maintained normal growth under high salt stress
|
proV
|
289 900 − 291 156
|
1 257
|
Involved in proline or betaine synthesis, alleviated cell apoptosis
|
proW
|
291 157 − 292 005
|
849
|
Involved in proline or betaine synthesis, alleviated cell apoptosis
|
opuA
|
3 214 816 − 3 213 671
|
1 146
|
Betaine/proline/choline family ABC transporter ATP-binding protein gene
|
cspA
|
516 907 − 517 110
|
204
|
Cold shock protein CspC
|
cspA
|
909 691 − 909 491
|
201
|
Cold shock-like protein CspB
|
cspA
|
2 110 200 − 2 110 400
|
201
|
Cold-shock protein CspD
|