Bacillus velezensis tolerance to the induced oxidative stress in root colonization contributed by the two-component regulatory system sensor ResE.

Efficient root colonization of plant growth-promoting rhizobacteria is critical for their plant-beneficial functions. However, the strategy to overcome plant immunity during root colonization is not well understood. In particular, how Bacillus strains cope with plant-derived reactive oxygen species (ROS), which function as the first barrier of plant defence, is not clear. In the present study, we found that the homolog of flg22 in Bacillus velezensis SQR9 (flg22SQR9 ) has 78.95% identity to the typical flg22 (flg22P.s. ) and induces a significant oxidative burst in cucumber and Arabidopsis. In contrast to pathogenic or beneficial Pseudomonas, live B. velezensis SQR9 also induced an oxidative burst in cucumber. We further found that B. velezensis SQR9 tolerated higher H2 O2 levels than Pst DC3000, the pathogen that harbours the typical flg22, and that it possesses the ability to suppress the flg22-induced oxidative burst, indicating that B. velezensis SQR9 may exploit a more efficient ROS tolerance system than DC3000. Further experimentation with mutagenesis of bacteria and Arabidopsis showed that the two-component regulatory system, ResDE, in B. velezensis SQR9 is involved in tolerance to plant-derived oxidative stress, thus contributing to root colonization. This study supports a further investigation of the interaction between beneficial rhizobacteria and plant immunity.

Microbulbifer flavimaris sp. nov., a halophilic Gammaproteobacteria isolated from marine sediment of the Yellow Sea, China.

A Gram-stain-negative, aerobic, non-flagellated, non-motile bacterium, designated strain WRN-8T, was isolated from marine sediment of the Yellow Sea, China (36° 5' 33'' N, 121° 20' 37'' E). Colonies of strain WRN-8T were 0.2-0.3 µm wide, 2.1-2.8 µm long, catalase-positive and oxidase-positive. Colonies on marine agar solid media were circular, wet, smooth, light yellow and approximately 1.3 mm in diameter. Growth occurred optimally at 33-37 °C, pH 7.0-7.5 and in the presence of 2-4 % NaCl (w/v). Phylogenetic analysis of the 16S rRNA gene indicated that strain WRN-8T is a member of the genus Microbulbifer within the family Microbulbiferaceae, and the closest described neighbour in terms of 16S rRNA gene sequence identity is Microbulbifer aestuariivivens KCTC 52569T (98.1 %). The major respiratory quinone of strain WRN-8T is Q-8, its predominant fatty acids are iso-C15 : 0, iso-C17 : 0, C16 : 0, iso-C11 : 03-OH and summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), and its major polar lipids are phosphatidylethanolamine, phosphatidylglycerol, glycolipid, an unidentified phospholipid and an unidentified lipid. The draft genome obtained in this study was 3 643 020 bp, and the G+C content was 59.2 mol%. DNA-DNA hybridization (<46.3 %) and average nucleotide identity (<86.7 %) values between strain WRN-8T and the closest-related recognized Microbulbifer species confirmed the novelty of this new species. Therefore, we propose a novel species in the genus Microbulbifer to accommodate the novel isolate: Microbulbifer flavimaris sp. nov. (type strain WRN-8T=KCTC 42989T=ACCC 19926T).

Exploring Elicitors of the Beneficial Rhizobacterium Bacillus amyloliquefaciens SQR9 to Induce Plant Systemic Resistance and Their Interactions With Plant Signaling Pathways.

Beneficial rhizobacteria have been reported to produce various elicitors that induce plant systemic resistance, but there is little knowledge concerning the relative contribution of multiple elicitors from a single beneficial rhizobacterium on the induced systemic resistance in plants and the interactions of these elicitors with plant signaling pathways. In this study, nine mutants of the plant growth-promoting rhizobacterium Bacillus amyloliquefaciens SQR9 deficient in producing the extracellular compounds, including fengycin, bacillomycin D, surfactin, bacillaene, macrolactin, difficidin, bacilysin, 2,3-butandiol, and exopolysaccharides, were tested for the induction of systemic resistance against Pseudomonas syringae pv. tomato DC3000 and Botrytis cinerea and the transcription of the salicylic acid, jasmonic acid, and ethylene signaling pathways in Arabidopsis. Deficiency in producing any of these compounds in SQR9 significantly weakened the induced plant resistance against these phytopathogens. These SQR9-produced elicitors induced different plant defense genes. For instance, the enhancement of 1,3-glucanase (PR2) by SQR9 was impaired by a deficiency of macrolactin but not surfactin. SQR9 mutants deficient in the lipopeptide and polyketide antibiotics remained only 20% functional for the induction of resistance-related gene transcription. Overall, these elicitors of SQR9 could act synergistically to induce plant systemic resistance against different phytopathogens through different signaling pathway genes, and the bacterial antibiotics are major contributors to the induction.

Paracoccus salipaludis sp. nov., isolated from saline-alkaline soil.

A Gram-stain-negative, short ovoid- to coccus-shaped, aerobic, non-flagellated, and nonmotile strain, designated WN007T, was isolated from the natural saline-alkali wetland soil. Growth occurred at 10-45 °C (optimum 33-37 °C), pH 6.5-10.0 (optimum, pH 7.5-8.0) and with 0-15 % (w/v) NaCl (optimum, 2-4 % NaCl). Catalase- and oxidase-positive. A comparison of the 16S rRNA gene sequence of WN007T showed the highest sequence similarities to Paracoccus chinensis (97.5 %) and Paracoccus niistensis (97.4 %). The major respiratory quinone of strain WN007T was Q10 and the fatty acid profile of strain WN007T contained a predominant amount of summed feature 7 and small quantities of C10 : 0 3OH, C16 : 00 and C18 : 00. The major polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, glycolipid, aminolipid and lipid. The genome revealed that the G+C content was 63.9 mol% and the DNA-DNA relatedness values between strain WN007T and the type strains of P. chinensis CGMCC 1.7655Tand P. niistensis KCTC 22789T were 46.9±2.3 and 42.4±1.7 %, respectively. This was also confirmed by the low average nucleotide identity values (<83.5 %) between strain WN007T and the most closely related recognized Paracoccus species. According to these results, strain WN007T represents a novel species of the genus Paracoccus, for which the name Paracoccussalipaludis sp. nov. is proposed. The type strain is WN007T (=KCTC 52851T=ACCC 19972T).

Identification of Root-Secreted Compounds Involved in the Communication Between Cucumber, the Beneficial Bacillus amyloliquefaciens, and the Soil-Borne Pathogen Fusarium oxysporum.

Colonization of plant growth-promoting rhizobacteria (PGPR) is critical for exerting their beneficial effects on the plant. Root exudation is a major factor influencing the colonization of both PGPR and soil-borne pathogens within the root system. However, the tripartite interaction of PGPR, plant roots, and soil-borne pathogens is poorly understood. We screened root exudates for signals that mediate tripartite interactions in the rhizosphere. In a split-root system, we found that root colonization of PGPR strain Bacillus amyloliquefaciens SQR9 on cucumber root was significantly enhanced by preinoculation with SQR9 or the soil-borne pathogen Fusarium oxysporum f. sp. cucumerinum, whereas root colonization of F. oxysporum f. sp. cucumerinum was reduced upon preinoculation with SQR9 or F. oxysporum f. sp. cucumerinum. Root exudates from cucumbers preinoculated with SQR9 or F. oxysporum f. sp. cucumerinum were analyzed and 109 compounds were identified. Correlation analysis highlighted eight compounds that significantly correlated with root colonization of SQR9 or F. oxysporum f. sp. cucumerinum. After performing colonization experiments with these chemicals, raffinose and tryptophan were shown to positively affect the root colonization of F. oxysporum f. sp. cucumerinum and SQR9, respectively. These results indicate that cucumber roots colonized by F. oxysporum f. sp. cucumerinum or SQR9 increase root secretion of tryptophan to strengthen further colonization of SQR9. In contrast, these colonized cucumber roots reduce raffinose secretion to inhibit root colonization of F. oxysporum f. sp. cucumerinum.

Beneficial Rhizobacterium Bacillus amyloliquefaciens SQR9 Induces Plant Salt Tolerance through Spermidine Production.

The inoculation of plants with plant-growth-promoting rhizobacterium has been an effective strategy for enhancing plant salt tolerance to diminish the loss of agricultural productivity caused by salt stress; however, the signal transmitted from bacteria to the plant under salt stress is poorly understood. In this study, the salt tolerance of Arabidopsis thaliana and Zea mays was enhanced by inoculation with Bacillus amyloliquefaciens SQR9. Using dialysis bags with different molecular weight cutoffs, we sorted through the molecules secreted by SQR9 and found that spermidine is responsible for enhancing plant salt tolerance. An SQR9 ΔspeB mutant deficient in spermidine production failed to induce plant salt tolerance. However, the induction of plant salt tolerance was disrupted by mutating genes involved in reduced glutathione (GSH) biosynthesis and the salt overly sensitive pathway in Arabidopsis. Using quantitative real-time polymerase chain reaction, this study demonstrated that spermidine produced by SQR9 leads to increased glutamine synthetase and glutathione reductase gene expression, leading to increased levels of GSH, which is critical for scavenging reactive oxygen species. SQR9-derived spermidine also upregulates the expression of NHX1 and NHX7, which sequesters Na+ into vacuoles and expels Na+ from the cell, thereby reducing ion toxicity.

Induced maize salt tolerance by rhizosphere inoculation of Bacillus amyloliquefaciens SQR9.

Salt stress reduces plant growth and is now becoming one of the most important factors restricting agricultural productivity. Inoculation of plant growth-promoting rhizobacteria (PGPR) has been shown to confer plant tolerance against abiotic stress, but the detailed mechanisms of how this occurs remain unclear. In this study, hydroponic experiments indicated that the PGPR strain Bacillus amyloliquefaciens SQR9 could help maize plants tolerate salt stress. After exposure to salt stress for 20 days, SQR9 significantly promoted the growth of maize seedlings and enhanced the chlorophyll content compared with the control. Additional analysis showed that the involved mechanisms could be the enhanced total soluble sugar content for decreasing cell destruction, improved peroxidase/catalase activity and glutathione content for scavenging reactive oxygen species, and reduced Na(+) levels in the plant to decrease Na(+) toxicity. These physiological appearances were further confirmed by the upregulation of RBCS, RBCL, H(+) -PPase, HKT1, NHX1, NHX2 and NHX3, as well as downregulation of NCED expression, as determined by quantitative reverse transcription-polymerase chain reaction. However, SQR9 counteracted the increase of abscisic acid in response to salt stress. In summary, these results show that SQR9 confers plant salt tolerance by protecting the plant cells and managing Na(+) homeostasis. Hence, it can be used in salt stress prone areas, thereby promoting agricultural production.

Transcriptomic and QTL Analysis of Seed Germination Vigor under Low Temperature in Weedy Rice WR04-6.

Low temperature is one of the major factors affecting rice germination, and low temperature germination (LTG) is an important agronomic trait. Although significant progress has been made in the study of rice LTG, the molecular mechanism of LTG remains poorly understood. To explore more rice LTG gene resources, we first demonstrated that weedy rice WR04-6 (Oryza sativa f. spontanea) had significantly higher LTG ability at 10 °C than the cultivated rice Qishanzhan (QSZ Oryza sativa L. ssp. indica). RNA-seq was used to investigate the gene expression of WR04-6 and QSZ at 10 °C for 10, 12 and 14 days after imbibition (DAI) of seed germination. The results of Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment revealed that the differentially expressed genes (DEGs) between WR04-6 and QSZ were mainly concentrated on the response to starch catabolic processes and the response to abscisic acid (ABA). This is consistent with the results of α-amylase activity, ABA and gibberellins (GA) treatment. A recombinant inbred line (RIL) population derived from a cross between WR04-6 and QSZ and its high-density SNP genetic map were used to detect quantitative trait loci (QTL) for LTG rates. The results showed that two new QTLs were located on chromosome 3 and chromosome 12. Combined with the mapped QTLs and RNA-seq DEGs, sixteen candidate genes potentially associated with LTG were identified. Validation of the expression of the candidates by qRT-PCR were consistent with the RNA-seq data. These results will enable us to understand the genetic basis of LTG in weedy rice and provide new genetic resources for the generation of rice germplasm with improved LTG.

A High-Density Genetic Linkage Map of SLAFs and QTL Analysis of Grain Size and Weight in Barley (Hordeum vulgare L.).

Grain size is an important agronomic trait determines yield in barley, and a high-density genetic map is helpful to accurately detect quantitative trait loci (QTLs) related to grain traits. Using specific-locus amplified fragment sequencing (SLAF-seq) technology, a high-density genetic map was constructed with a population of 134 recombinant inbred lines (RILs) deriving from a cross between Golden Promise (GP) and H602, which contained 12,635 SLAFs with 26,693 SNPs, and spanned 896.74 cM with an average interval of 0.07 cM on seven chromosomes. Based on the map, a total of 16 QTLs for grain length (GL), grain width and thousand-grain weight were detected on 1H, 2H, 4H, 5H, and 6H. Among them, a major QTL locus qGL1, accounting for the max phenotypic variance of 16.7% was located on 1H, which is a new unreported QTL affecting GL. In addition, the other two QTLs, qGL5 and qTGW5, accounting for the max phenotypic variances of 20.7 and 21.1%, respectively, were identified in the same region, and sequencing results showed they are identical to HvDep1 gene. These results indicate that it is a feasible approach to construct a high-quality genetic map for QTL mapping by using SLAF markers, and the detected major QTLs qGL1, qGL5, and qTGW5 are useful for marker-assisted selection (MAS) of grain size in barley breeding.