Biological Control of Take-All and Growth Promotion in Wheat by Pseudomonas chlororaphis YB-10

Wheat is a worldwide staple food crop, and take-all caused by Gaeumannomyces graminis var. tritici can lead to a tremendous decrease in wheat yield and quality. In this study, strain YB-10 was isolated from wheat rhizospheric soil and identified as Pseudomonas chlororaphis by morphology and 16S rRNA gene sequencing. Pseudomonas chlororaphis YB-10 had extracellular protease and cellulase activities and strongly inhibited the mycelium growth of Gaeumannomyces graminis var. tritici in dual cultures. Up to 87% efficacy of Pseudomonas chlororaphis YB-10 in controlling the take-all of seedlings was observed in pot experiments when wheat seed was coated with the bacterium. Pseudomonas chlororaphis YB-10 was also positive for indole acetic acid (IAA) and siderophore production, and coating wheat seed with the bacterium significantly promoted the growth of seedlings at 107 and 108 CFU/mL. Furthermore, treatment with Pseudomonas chlororaphis YB-10 increased activities of the wheat defense-related enzymes POD, SOD, CAT, PAL and PPO in seedlings, indicating induced resistance against pathogens. Overall, Pseudomonas chlororaphis YB-10 is a promising new seed-coating agent to both promote wheat growth and suppress take-all.


Introduction
Take-all caused by Gaeumannomyces graminis var. tritici (Ggt) is one of the most destructive root diseases of wheat worldwide and significantly reduces wheat grain yield after severe outbreaks [1][2][3]. The fungus G. graminis var. tritici damages wheat by rotting the root and base of the stem, disrupting the uptake and flow of water and nutrients, and eventually causing death of infected plants [2,4,5]. As with other soil-borne plant diseases, take-all is difficult to manage by current approaches such as the development of resistant cultivars, use of chemical pesticides, crop rotation and tillage [6][7][8][9][10]. However, several studies in the past thirty years have demonstrated that antagonistic bacteria and fungi can be applied to wheat to control take-all disease. This is considered to be a more efficient and ecologically friendly alternative management approach [4,[11][12][13][14]. Therefore, the identification of effective antagonistic microorganisms to control take-all disease in wheat is an urgent need for sustainable wheat production. Many studies have revealed that antagonistic bacteria not only play significant roles in controlling different plant diseases as biological control agents (BCAs), but can also increase plant growth as plant growth-promoting rhizobacteria (PGPR) [15][16][17][18][19]. Among them, isolates of Pseudomonas have proven to be capable of producing antibiotic compounds to suppress plant pathogens such as G. graminis var. tritici, and possess plant growth-promoting characteristics such as phosphate solubilization, siderophores, 1-aminocyclopropane-1carboxylate (ACC) deaminase and indole-3-acetic acid (IAA), etc. [20][21][22][23][24]. For example, P. chlororaphis 30-84 produced the antibiotic sphenazine-1-carboxylic acid (PCA) that effectively inhibited take-all [23]; P. fluorescens 2-79 also produced the antibiotics PCA that strongly inhibited G. graminis var. tritici in culture [25]; and P. fluorescens VUPf5 produced siderophores, hydrogen cyanide, protease, phenazine and volatile metabolites that suppressed take-all disease by 85% in pots [26]. Furthermore, isolates of Pseudomonas have been shown to be both BCAs and PGPRs in other plants. For example, P. aeruginosa 2apa produced IAA, salicylic acid and siderophores, and promoted tomato growth along with antimicrobial activity against a wide range of pathogens [27]. Pseudomonas aeruginosa PN1 produced IAA, cyanogens, solubilized phosphorus, siderophore, chitinase and β-1, 3-glucanase and improved the growth of chirpine seedlings along with exhibiting a strong antagonistic property against Macrophomina phaseolina [17]. In addition, inoculations with different Pseudomonas strains have triggered defense-related enzymes in plants, such as peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), phenylalanine ammonia lyase (PAL) and polyphenoloxidase (PPO), indicating that they can produce induced systemic resistance against pathogens [28][29][30][31][32].
In this study, strain YB-10 was isolated from wheat rhizospheric soil and identified as Pseudomonas chlororaphis by morphology and 16S rRNA gene sequencing. It exhibited strong antagonistic activity against G. graminis var. tritici in dual cultures and was shown to have a number of potential growth-promoting and biocontrol traits. When used to coat wheat seeds, P. chlororaphis YB-10 not only promoted the growth of wheat seedlings, but also suppressed take-all caused by G. graminis var. tritici. These results demonstrated that P. chlororaphis YB-10 could be a promising BCA of wheat and PGPR against take-all disease.

Isolation of StrainYB-10
Soil sample collection and isolation of strain YB-10 from wheat rhizosphere used in this study were as previously described in [12].

Identification of Strain YB-10
A single colony of strain YB-10 was inoculated in Nutrient Broth and grown for 24 h at 30 • C, shaking at 150 rpm. Genomic DNA of YB-10 was extracted by the MiniBEST Bacterial Genomic DNA Extraction Kit Ver. 3.0 (TaKaRa, Beijing, China) following the manufacturer's instructions. The 16S rRNA gene fragment was amplified with primers27F (5 -AGAGTTTGATCCTGGCTCAG-3 ) and 1492R (5 -GGTTACCTTGTTACGACTT-3 ). A phylogenetic tree of the 16S rRNA gene sequences was constructed with MEGA 7.0 using the neighbor joining method [33].

Detection of PGP and Antifungal Traits
IAA production was detected with the Salkowski method as described by Glickmann and Dessaux [34].
Siderophore production was determined on Chrome Azurol S blue agar (CAS) [35]. A single colony of P. chlororaphis YB-10 was inoculated on CAS and cultured at 30 • C in the dark. Siderophore production was indicated by the color change from blue to orange around the colonies.
A single colony of P. chlororaphis YB-10 was inoculated onto carboxymethylcellulose agar and incubated for 7 days at 30 • C. The plates were flooded with 1% (m/v) Congo red Pathogens 2021, 10, 903 3 of 11 and then washed with distilled water. The cellulose activity was detected as clear zones around the colonies [36].
Protease activity was detected on skim milk agar (5 g NaCl, 0.1 g CaCl 2 , 10 g peptone, 10 g skim milk and 18 g of agar per liter (pH 7.2)). A single colony of P. chlororaphis YB-10 was placed on the medium and incubated at 30 • C for 5 days. Protease activity of P. chlororaphis YB-10 was observed by the clear zones around the colonies [37].
2.4. Plant Growth Promotion Assay of P. chlororaphis YB-10 on Wheat Seedlings P. chlororaphis YB-10 was cultured in 100 mL NB broth for 36 h at 30 • C with shaking at 180 rpm. Wheat (Triticum aestivum L.) seeds of cultivar Zhengmai 366 were surfacesterilized in 75% ethanol (v/v) for 30s and then rinsed with sterile water three times. Wheat seeds were air-dried and then soaked with a suspension of P. chlororaphis YB-10 at 10 7 CFU/mL, 10 8 CFU/mL or 10 9 CFU/mL for 2 h. The seeds were then placed in a Petri dish with a diameter of 9 cm in the greenhouse at 25 • C with a 12 h light/12 h dark photoperiod for 10 days. Each treatment had three replicates. Seeds soaked with NB medium were used as control.

Biocontrol Assay of P. chlororaphis YB-10 Against Take-All inWheat
Wheat seeds were soaked with P. chlororaphis YB-10 at 10 7~1 0 8 CFU/mL or sterile NB medium as described above. The seeds were air-dried, and 15 seeds were planted per pot (10 cm high, 10 cm diameter) filled with 350 g potting soil. The pots were placed in the greenhouse at 25 • C. Gaeumannomyces graminis var. tritici GGT-007, isolated from Xiping, Henan Province in 2014, that was previously shown to be highly virulent to wheat (data not shown), was grown on PDA for 10 days at 25 • C. A 5 mm disc of mycelium with agar from the growing edge of the colony was placed at a 1cm depth in a pot and then a wheat seed was attached to the surface of the mycelial disc. This method of inoculation was shown to achieve a 100% infection rate [12]. Four treatments were made:
Seeds not soaked with P. chlororaphis YB-10 and not inoculated with Ggt; 4.
Seeds not soaked with P. chlororaphis YB-10 and inoculated with Ggt.
At 3 weeks post-planting, the treated wheat seedlings were removed from pots and roots were washed free of soil. The symptoms of take-all, disease index and control efficacy were measured as per Yang et al [12]. The experiment was replicated three times.

Activities of Defense Enzymes in Wheat Seedlings
At 3 weeks post-planting, the shoots of the treated wheat seedlings were harvested and stored at −80°C. In brief, 0.5 g shoots were ground in liquid nitrogen, and a 1 mL extraction buffer was added. The buffer was then centrifuged, and the supernatant was removed and used for enzyme assays. The activities of SOD, POD, CAT, PAL and PPO were measured following the procedures of the manufacturer (Solarbio, Beijing, China) using an SOD assay kit (Cat. No. BC0175), POD assay kit (Cat. No. BC0095), CAT assay kit (Cat. No. BC0205), PAL assay kit (Cat. No. BC0215) and PPO assay kit (Cat. No. BC0195), respectively. Absorbance was determined by a plate reader (Tecan Spark, Tecan, Switzerland).

Statistical Analysis
Statistical analysis was performed using SPSS v21.0, one-way analysis of variance (ANOVA). Means were compared with Duncan's multiple range tests at a probability of p ≤ 0.05.

Screening and Identification of Strain YB-10
Strain YB-10 isolated from wheat rhizospheric soil was milky-white, non-pigmented on NA and Gram-negative ( Figure 1A,C). Dual cultures of strain YB-10 and G. graminis var. tritici on NA displayed strong growth inhibition of G. graminis var. tritici by YB-10 ( Figure 1B). A search using the 16S rDNA sequence of YB-10 with the Basic Local Alignment Search Tool (BLAST) against the NCBI nr database demonstrated that strain YB-10 had 100% sequence homology to P. chlororaphis. The sequence data were submitted to GenBank with the accession number MW093066. The phylogenetic tree of the 16S rRNA gene sequences revealed that strain YB-10 was in a cluster of P. chlororaphis isolates ( Figure 2). Statistical analysis was performed using SPSS v21.0, one-way analysis of variance (ANOVA). Means were compared with Duncan's multiple range tests at a probability of p ≤ 0.05.

Screening and Identification of Strain YB-10
Strain YB-10 isolated from wheat rhizospheric soil was milky-white, non-pigmented on NA and Gram-negative ( Figure 1A,C). Dual cultures of strain YB-10 and G. graminis var. tritici on NA displayed strong growth inhibition of G. graminis var. tritici by YB-10 ( Figure 1B). A search using the 16S rDNA sequence of YB-10 with the Basic Local Alignment Search Tool (BLAST) against the NCBI nr database demonstrated that strain YB-10 had 100% sequence homology to P. chlororaphis. The sequence data were submitted to GenBank with the accession number MW093066. The phylogenetic tree of the 16S rRNA gene sequences revealed that strain YB-10 was in a cluster of P. chlororaphis isolates ( Figure  2).

Screening and Identification of Strain YB-10
Strain YB-10 isolated from wheat rhizospheric soil was milky-white, non-pigmented on NA and Gram-negative ( Figure 1A,C). Dual cultures of strain YB-10 and G. graminis var. tritici on NA displayed strong growth inhibition of G. graminis var. tritici by YB-10 ( Figure 1B). A search using the 16S rDNA sequence of YB-10 with the Basic Local Alignment Search Tool (BLAST) against the NCBI nr database demonstrated that strain YB-10 had 100% sequence homology to P. chlororaphis. The sequence data were submitted to GenBank with the accession number MW093066. The phylogenetic tree of the 16S rRNA gene sequences revealed that strain YB-10 was in a cluster of P. chlororaphis isolates ( Figure  2).

Effect of P. chlororaphis YB-10 Against Take-All in Wheat Seedlings
For wheat seedlings at 21 days after G. graminis var. tritici inoculation without seed treatment of P. chlororaphis YB-10 in pots under greenhouse conditions (Figure 4), symptoms of take-all, including blackened or chocolate brown lesions on the stembase and root, were observed ( Figure 4D,H). Limited typical disease symptoms were observed in wheat seedlings without G. graminis var. tritici inoculation and without seed treatment of P. chlororaphis YB-10, indicating a low level of G. graminis var. tritici in the potting soil ( Figure  4C,G). However, no typical disease symptoms were observed with or without G. graminis var. tritici inoculation with wheat seed treatment of P. chlororaphis YB-10 ( Figure 4A and 4E, 4B and 4F, respectively).The disease index of wheat seedlings was 7.14 ± 0.11 and 6.67 ± 0.28 with seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively, but the disease index was 78.57 ± 0.47 and 51.59 ± 1.11 without seed treatment of P. chlororaphis YB-10 with or without G. graminis var. tritici inoculation, respectively (Table 1). Disease incidence with seed treatment of P. chlororaphis YB-10 with or without G. graminis var. tritici inoculation was only 0.07%. However, disease incidence was 92.86% ± 0.62% and 64.29% ± 0.72% without seed treatment of P. chlororaphis YB-10 with or without G. graminis var. tritici inoculation, respectively. The take-all control efficacy in wheat seedlings was 86-87% with seed treatment of P. chlororaphis YB-10.

Effect of P. chlororaphis YB-10 Against Take-All in Wheat Seedlings
For wheat seedlings at 21 days after G. graminis var. tritici inoculation without seed treatment of P. chlororaphis YB-10 in pots under greenhouse conditions (Figure 4), symptoms of take-all, including blackened or chocolate brown lesions on the stembase and root, were observed ( Figure 4D,H). Limited typical disease symptoms were observed in wheat seedlings without G. graminis var. tritici inoculation and without seed treatment of P. chlororaphis YB-10, indicating a low level of G. graminis var. tritici in the potting soil ( Figure 4C,G). However, no typical disease symptoms were observed with or without G. graminis var. tritici inoculation with wheat seed treatment of P. chlororaphis YB-10 ( Figure 4A,E,B,F, respectively).The disease index of wheat seedlings was 7.14 ± 0.11 and 6.67 ± 0.28 with seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively, but the disease index was 78.57 ± 0.47 and 51.59 ± 1.11 without seed treatment of P. chlororaphis YB-10 with or without G. graminis var. tritici inoculation, respectively (Table 1). Disease incidence with seed treatment of P. chlororaphis YB-10 with or without G. graminis var. tritici inoculation was only 0.07%. However, disease incidence was 92.86% ± 0.62% and 64.29% ± 0.72% without seed treatment of P. chlororaphis YB-10 with or without G. graminis var. tritici inoculation, respectively. The take-all control efficacy in wheat seedlings was 86-87% with seed treatment of P. chlororaphis YB-10.

Growth Promotion of Wheat Seedlings after P. chlororaphis YB-10 Inoculation
For wheat seeds soaked with P. chlororaphis YB-10 at different doses, P. chlororaphis YB-10 at 10 7 and 10 8 CFU/mL significantly promoted the growth of wheat seedlings, but P. chlororaphis YB-10 at 10 9 CFU/mL had an inhibitory effect on the growth of wheat seedlings ( Figure 5). The fresh weights of shoots of wheat seedlings inoculated with P. chlororaphis YB-10 at 10 7 and 10 8 CFU/mL were 1.68 ± 0.06 g and 1.72 ± 0.04 g, respectively, compared to 1.18 ± 0.07 g without inoculation with P. chlororaphis YB-10. The fresh weights of roots of wheat seedlings inoculated with P. chlororaphis YB-10 at 10 7 and 10 8 CFU/mL were 1.53 ± 0.03 g and 1.51 ± 0.05 g, respectively, compared to 0.91 ± 0.02 g without inoculation with P. chlororaphis YB-10. The lengths of shoots of wheat seedlings inoculated with P. chlororaphis YB-10 at 10 7 and 10 8 CFU/mL were 12.41 ± 0.52 cm and 12.43 ± 0.67 cm, respectively, compared to 8.21 ± 0.42 cm without inoculation with P. chlororaphis YB-10. Overall, seed treatment with P. chlororaphis YB-10 at 10 7 and 10 8 CFU/mL caused a significant improvement (p < 0.05) in fresh weights of shoots and roots and in the lengths of shoots of wheat seedlings, but seed treatment with P. chlororaphis YB-10 at 10 9 CFU/mL did not differ significantly (p > 0.05) compared with non-inoculated treatment ( Table 2).

Growth Promotion of Wheat Seedlings after P. chlororaphis YB-10 Inoculation
For wheat seeds soaked with P. chlororaphis YB-10 at different doses, P. chlororaphis YB-10 at 10 7 and 10 8 CFU/mL significantly promoted the growth of wheat seedlings, but P. chlororaphis YB-10 at 10 9 CFU/mL had an inhibitory effect on the growth of wheat seedlings ( Figure 5). The fresh weights of shoots of wheat seedlings inoculated with P. chlororaphis YB-10 at 10 7 and 10 8 CFU/mL were 1.68 ± 0.06 g and 1.72 ± 0.04 g, respectively, compared to 1.18 ± 0.07 g without inoculation with P. chlororaphis YB-10. The fresh weights of roots of wheat seedlings inoculated with P. chlororaphis YB-10 at 10 7 and 10 8 CFU/mL were 1.53 ± 0.03 g and 1.51 ± 0.05 g, respectively, compared to 0.91 ± 0.02 g without inoculation with P. chlororaphis YB-10. The lengths of shoots of wheat seedlings inoculated with P. chlororaphis YB-10 at 10 7 and 10 8 CFU/mL were 12.41 ± 0.52 cm and 12.43 ± 0.67 cm, respectively, compared to 8.21 ± 0.42 cm without inoculation with P. chlororaphis YB-10. Overall, seed treatment with P. chlororaphis YB-10 at 10 7 and 10 8 CFU/mL caused a significant improvement (p < 0.05) in fresh weights of shoots and roots and in the lengths of shoots of wheat seedlings, but seed treatment with P. chlororaphis YB-10 at 10 9 CFU/mL did not differ significantly (p > 0.05) compared with non-inoculated treatment ( Table 2).

Effect of P. chlororaphis YB-10 on Activities of Defense Enzymes in Wheat Seedlings
POD activity of wheat shoots was 9406.51 ± 200.20 U/g and 12,925.14 ± 998.21 U/g with seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation,respectively, whereas the POD activity was 7261.47 ± 251.01 U/g and 10,517.07 ± 234.12 U/g without seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively ( Figure 6). PPO activity of wheat shoots was 85.46 ± 6.16 U/g and 166.95 ± 4.87 U/g with seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively, whereas PPO activity was 70.16 ± 5.18 U/g and 128.78 ± 5.67 U/g without seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively. CAT activity of wheat shoots was 201.73 ± 7.18 U/g and 387.59 ± 10.94 U/g in seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively, whereas CAT activity was 161.97 ± 7.40 U/g and 241.73 ± 5.76 U/g without seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively. PAL activity of wheat shoots was 39.78 ± 3.46 U/g and 57.38 ± 4.11 U/g with seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively, whereas PAL activity was 30.87 ± 2.33 U/g and 45.61 ± 2.47 U/g without seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively. SOD activity of wheat shoots was 881.57 ± 25.97 U/g and 1514.12 ± 54.61 U/g with seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively, whereas SOD activity was 748.87 ± 16.23 U/g and 1178.64 ± 62.32 U/g without seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively. In general, wheat seeds soaked with P. chlororaphis YB-10 resulted in significant increases in POD, SOD, CAT, PAL and PPO activities of wheat shoots regardless of G. graminis var. tritici inoculation.

Effect of P. chlororaphis YB-10 on Activities of Defense Enzymes in Wheat Seedlings
POD activity of wheat shoots was 9406.51 ± 200.20 U/g and 12,925.14 ± 998.21 U/g with seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively, whereas the POD activity was 7261.47 ± 251.01 U/g and 10,517.07 ± 234.12 U/g without seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively ( Figure 6). PPO activity of wheat shoots was 85.46 ± 6.16 U/g and 166.95 ± 4.87 U/g with seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively, whereas PPO activity was 70.16 ± 5.18 U/g and 128.78 ± 5.67 U/g without seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively. CAT activity of wheat shoots was 201.73 ± 7.18 U/g and 387.59 ± 10.94 U/g in seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively, whereas CAT activity was 161.97 ± 7.40 U/g and 241.73 ± 5.76 U/g without seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively. PAL activity of wheat shoots was 39.78 ± 3.46 U/g and 57.38 ± 4.11 U/g with seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively, whereas PAL activity was 30.87 ± 2.33 U/g and 45.61 ± 2.47 U/g without seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively. SOD activity of wheat shoots was 881.57 ± 25.97 U/g and 1514.12 ± 54.61 U/g with seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively, whereas SOD activity was 748.87 ± 16.23 U/g and 1178.64 ± 62.32 U/g without seed treatment of P. chlororaphis YB-10 without or with G. graminis var. tritici inoculation, respectively. In general, wheat seeds soaked with P. chlororaphis YB-10 resulted in significant increases in POD, SOD, CAT, PAL and PPO activities of wheat shoots regardless of G. graminis var. tritici inoculation.

Discussion
In recent years, many studies have demonstrated that Pseudomonas strains from the plant rhizosphere could control diseases caused by a variety of pathogens [21,[38][39][40]. For example, Pseudomonas fluorescens CV6 had broad antimicrobial activity against 12 pathogens [41], and P. fluorescens VUPf5 strongly inhibited G. graminis var. tritici in vitro and greenhouse, suppressing take-all disease by 85% [26]. Many of these strains have antifungal attributes, such as β-1,3-glucanase, cellulase, protease and chitinase activities [42,43]. It is also well known that a number of Pseudomonas strains from the plant rhizosphere can promote plant growth [18,20,40,41]. These strains show multiple growth-promoting properties, including IAA production, siderophore production, hydrogen cyanide production and phosphate solubilization [42,43]. Further screening of new Pseudomonas isolates is desirable for creating new options for controlling plant diseases and promoting plant growth in sustainable agriculture systems.
In this study, P. chlororaphis strain YB-10 was isolated from wheat rhizosphere, which had cellulose and protease activities and suppressed take-all caused by G. graminis var. tritici with very high efficacy. In addition, P. chlororaphis YB-10 was positive for IAA and siderophore production and significantly promoted the growth of wheat seedlings at 10 7 and 10 8 CFU/mL. However, the growth promotion effect was dose-dependent as wheat seedlings soaked with P. chlororaphis YB-10 at 10 9 CFU/mL did not differ significantly compared with non-inoculated treatment. Therefore, P. chlororaphis YB-10 showed itself to be an effective PGPR for wheat seedlings and BCA against take-all of wheat.
In nature, plants are attacked by various pathogens inducing the production of different defense-related enzymes that play important roles in disease resistance [44,45]. The level of defense-related enzymes, such as POD, PPO, CAT, PAL and SOD, have been correlated with plant disease resistance [46,47]. Many studies have reported that biocontrol agents such as P. fluorescens, Pseudomonas sp. 23S, Bacillus subtilis, P. chlororaphis ToZa7

Discussion
In recent years, many studies have demonstrated that Pseudomonas strains from the plant rhizosphere could control diseases caused by a variety of pathogens [21,[38][39][40]. For example, Pseudomonas fluorescens CV6 had broad antimicrobial activity against 12 pathogens [41], and P. fluorescens VUPf5 strongly inhibited G. graminis var. tritici in vitro and greenhouse, suppressing take-all disease by 85% [26]. Many of these strains have antifungal attributes, such as β-1,3-glucanase, cellulase, protease and chitinase activities [42,43]. It is also well known that a number of Pseudomonas strains from the plant rhizosphere can promote plant growth [18,20,40,41]. These strains show multiple growth-promoting properties, including IAA production, siderophore production, hydrogen cyanide production and phosphate solubilization [42,43]. Further screening of new Pseudomonas isolates is desirable for creating new options for controlling plant diseases and promoting plant growth in sustainable agriculture systems.
In this study, P. chlororaphis strain YB-10 was isolated from wheat rhizosphere, which had cellulose and protease activities and suppressed take-all caused by G. graminis var. tritici with very high efficacy. In addition, P. chlororaphis YB-10 was positive for IAA and siderophore production and significantly promoted the growth of wheat seedlings at 10 7 and 10 8 CFU/mL. However, the growth promotion effect was dose-dependent as wheat seedlings soaked with P. chlororaphis YB-10 at 10 9 CFU/mL did not differ significantly compared with non-inoculated treatment. Therefore, P. chlororaphis YB-10 showed itself to be an effective PGPR for wheat seedlings and BCA against take-all of wheat.
In nature, plants are attacked by various pathogens inducing the production of different defense-related enzymes that play important roles in disease resistance [44,45]. The level of defense-related enzymes, such as POD, PPO, CAT, PAL and SOD, have been correlated with plant disease resistance [46,47]. Many studies have reported that biocontrol agents such as P. fluorescens, Pseudomonas sp. 23S, Bacillus subtilis, P. chlororaphis ToZa7 and Clonostachys rosea IK726 induced the expression of defense-related enzymes including SOD,