Potential of Novel Sequence Type of Burkholderia cenocepacia for Biological Control of Root Rot of Maize (Zea mays L.) Caused by Fusarium temperatum

In this study, two Burkholderia strains, strain KNU17BI2 and strain KNU17BI3, were isolated from maize rhizospheric soil, South Korea. The 16S rRNA gene and multilocus sequence analysis and typing (MLSA-MLST) were used for the identification of the studied strains. Strain KNU17BI2, which belonged to Burkholderia cenocepacia, was of a novel sequence type (ST) designated ST-1538, while strain KNU17BI3 had a similar allelic profile with the seven loci of Burkholderia contaminans strain LMG 23361. The strains were evaluated in vitro for their specific plant growth promoting (PGP) traits, such as zinc solubilization, phosphate solubilization, ammonia production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, indole acetic acid (IAA) production, siderophore, and hydrolytic enzyme activity. Interestingly, the strains exhibited a positive effect on all of the tested parameters. The strains also showed broad-spectrum antifungal activity against economically important phytopathogens in the dual culture assay. Furthermore, the strains were evaluated under greenhouse conditions for their in vivo effect to promote plant growth and to suppress the root rot of maize that is caused by Fusarium temperatum on four Korean maize cultivars. The results of the greenhouse study revealed that both of the strains were promising to significantly suppress fusarium root rot and enhance plant growth promotion on the four maize cultivars. This study, for the first time, reported in vitro antifungal potential of B. cenocepacia of novel ST against economically important plant pathogens viz., F. temperatum, Fusarium graminearum, Fusarium moniliforme, Fusarium oxysporum f.sp. melonis, Fusarium subglutinans, Phytophthora drechsleri, and Stemphylium lycopersici. This is also the first report of zinc solubilization by B. cenocepacia. Moreover, the present research work reports, for the first time, about the potential of B. cenocepacia and B. contaminans to control the root rot of maize that is caused by F. temperatum. Therefore, we recommend further studies to precisely identify the bioactive chemical compounds behind such activities that would be novel sources of natural products for biological control and plant growth promotion of different crops.


Introduction
Globally, Fusarium species cause several diseases of maize (Zea mays L.), such as seed rot, seedling blight, and stalk rot [1,2]. Fusarium temperatum has been reported to cause seed rot, seedling blight, and stalk rot of maize in different parts of the world [3,4], and it is necessary in the proper monitoring and control of disease in maize-growing areas [5].

In Vitro Plant Growth Promoting (PGP) Activity Assays
With regard to plant growth promoting traits, both of strains showed a promising effect. The results of phosphate solubilization assay revealed that the strains showed the ability to solubilize tricalcium phosphate (TCP) according to the formation of clear solubilization zone around its colony ( Table 2 and Figure 3). Strain KNU17BI2 showed a higher phosphate solubilization index (SI) (2.3) as compared to strain KNU17BI3 (1.8) after 10 days of incubation. The phosphate solubilization index (SI) of both strains increased with the increasing incubation period ( Table 2). The formation of the solubilized halo zone around the bacterial colonies may be due to the ability of the strains to produce phosphatase enzymes or other chemical compounds, such as organic acids and polysaccharides [25]. Similarly, the potentiality of B. cenocepacia to solubilize inorganic phosphates has been previously reported [26]. Phosphorus is the second most important nutrient after nitrogen for plant growth; however, only less than 5% of total soil phosphorus is found in available form to plants [27,28]. Thus, the ability to solubilize the insoluble phosphates is one of the most important features of plant growth promoting bacteria to enhance plant nutrition through an increase in phosphorus uptake by plants [29]. The application of phosphate solubilizing bacteria could contribute to the reduction of excessive fertilizers chemical usage and thereby reduce the harmful effects of fertilizers on the environment and human health [28].  In addition, the Zn solubilization assay result revealed that both strains had the potential to solubilize an insoluble zinc sources, ZnO (1.244 g·L −1 ) and ZnCO3, 1.913 g·L −1 in Pikovskaya (PVK) agar media. The Zn solubilization ability of strain KNU17BI2 was comparatively higher than KNU17BI3 on both tested zinc sources (Table 2 and Figure 3). In addition, both of the strains showed higher solubilization efficiency on ZnO amended PVK agar media than ZnCO3 media. These results comply with the previous studies [14,30] that Burkholderia species have the potential to solubilize the insoluble form of zinc. The solubilization potential may be attributed to the release of different chemical compounds, including gluconic acid and 2 keto gluconic acids [31]. Zn has the propensity to form insoluble complexes in the soil and become unavailable for plant uptake [32]. Thus, the lack of Zn is a continual challenge in crop production, being particularly in high pH soils, and its significant impact on human nutrition has been previously reported [33,34]. Interestingly, the solubilization of an insoluble soil Zn by bacterial inoculations has been reported to significantly increase in the total Zn uptake and thereby increase plant growth [32].
Both the studied strains developed yellow-orange halo around their colonies on blue Chrome Azurol S (CAS) agar medium signifying their ability to produce siderophore. Strain KNU17BI3 exhibited higher (23 mm) siderophore production, while KNU17BI2 exhibited very low siderophore (12 mm) after seven days of incubation ( Figure 3). Similar to our study, previous studies [35,36] reported the potential of Burkholderia species to produce siderophores. A PGPR having siderophore production potential plays an important role in helping plants to acquire iron for optimal growth and it also offers competitive advantages to PGPR by suppressing the proliferation of plant pathogens [37,38].
The studied strains displayed positive results for ammonia production and IAA production. The amount of ammonia that is produced by strain KNU17BI3 was by far higher (14.1 μg·mL −1 ) than strain KNU17BI2 (6.3 μg·mL −1 ) after three days of incubation. In addition, both of the strains were able to produce IAA in LB medium, regardless of amendment of L-tryptophan ( Figure 4). After three days of incubation, the amount of IAA production by strain KNU17BI3 in LB medium without the amendment of L-tryptophan was higher (7.9 μg·mL −1 ) when compared to KNU17B2 (6.1 μg·mL −1 ) ( Figure 4). In L-tryptophan amended LB medium, the amount of IAA was increased as L-tryptophan concentration increased in both strains ( Figure 4). The maximum IAA (26.6 7.9 μg·mL −1 ) was produced by strain KNU17BI3 at higher L-tryptophan concentration (2.0 μg·mL −1 ). Indole-3-acetic acid (IAA) and ammonia are produced by many plant-associated bacteria that can enhance plant growth [39,40] with enhanced water and nutrients uptake [41]. In addition, the Zn solubilization assay result revealed that both strains had the potential to solubilize an insoluble zinc sources, ZnO (1.244 g·L −1 ) and ZnCO 3 , 1.913 g·L −1 in Pikovskaya (PVK) agar media. The Zn solubilization ability of strain KNU17BI2 was comparatively higher than KNU17BI3 on both tested zinc sources (Table 2 and Figure 3). In addition, both of the strains showed higher solubilization efficiency on ZnO amended PVK agar media than ZnCO 3 media. These results comply with the previous studies [14,30] that Burkholderia species have the potential to solubilize the insoluble form of zinc. The solubilization potential may be attributed to the release of different chemical compounds, including gluconic acid and 2 keto gluconic acids [31]. Zn has the propensity to form insoluble complexes in the soil and become unavailable for plant uptake [32]. Thus, the lack of Zn is a continual challenge in crop production, being particularly in high pH soils, and its significant impact on human nutrition has been previously reported [33,34]. Interestingly, the solubilization of an insoluble soil Zn by bacterial inoculations has been reported to significantly increase in the total Zn uptake and thereby increase plant growth [32].
Both the studied strains developed yellow-orange halo around their colonies on blue Chrome Azurol S (CAS) agar medium signifying their ability to produce siderophore. Strain KNU17BI3 exhibited higher (23 mm) siderophore production, while KNU17BI2 exhibited very low siderophore (12 mm) after seven days of incubation ( Figure 3). Similar to our study, previous studies [35,36] reported the potential of Burkholderia species to produce siderophores. A PGPR having siderophore production potential plays an important role in helping plants to acquire iron for optimal growth and it also offers competitive advantages to PGPR by suppressing the proliferation of plant pathogens [37,38].
The studied strains displayed positive results for ammonia production and IAA production. The amount of ammonia that is produced by strain KNU17BI3 was by far higher (14.1 µg·mL −1 ) than strain KNU17BI2 (6.3 µg·mL −1 ) after three days of incubation. In addition, both of the strains were able to produce IAA in LB medium, regardless of amendment of L-tryptophan ( Figure 4). After three days of incubation, the amount of IAA production by strain KNU17BI3 in LB medium without the amendment of L-tryptophan was higher (7.9 µg·mL −1 ) when compared to KNU17B2 (6.1 µg·mL −1 ) ( Figure 4). In L-tryptophan amended LB medium, the amount of IAA was increased as L-tryptophan concentration increased in both strains ( Figure 4). The maximum IAA (26.6 7.9 µg·mL −1 ) was produced by strain KNU17BI3 at higher L-tryptophan concentration (2.0 µg·mL −1 ). Indole-3-acetic acid (IAA) and ammonia are produced by many plant-associated bacteria that can enhance plant growth [39,40] with enhanced water and nutrients uptake [41].
The strains exhibited fast and luxuriant growth colonies on DF-agar plate that was supplemented with 3mM ACC as the unique nitrogen source (data not shown). This signifies ACC deaminase activity of the strains, as they were capable of using ACC as a nitrogen source. Our result complies with previous reports [42,43] that ACC deaminase is the widespread enzyme in diverse Burkholderia species, including B. caryophylli, B. cenocepacia, and B. contaminnans. Furthermore, strain KNU17BI2 exhibited a positive reaction for amylase, while strain KNU17BI3 did not show the activity (Figure 3). Amylase activity can help PGPR to easily hydrolyze the cell wall of phytopathogens [44].
The strains exhibited fast and luxuriant growth colonies on DF-agar plate that was supplemented with 3mM ACC as the unique nitrogen source (data not shown). This signifies ACC deaminase activity of the strains, as they were capable of using ACC as a nitrogen source. Our result complies with previous reports [42,43] that ACC deaminase is the widespread enzyme in diverse Burkholderia species, including B. caryophylli, B. cenocepacia, and B. contaminnans. Furthermore, strain KNU17BI2 exhibited a positive reaction for amylase, while strain KNU17BI3 did not show the activity ( Figure 3). Amylase activity can help PGPR to easily hydrolyze the cell wall of phytopathogens [44].

In Vitro Antifungal Activity
In the dual-culture assay, the antagonistic activity of strain KNU17BI2 and strain KNU17BI3 were tested against F. temperatum, maize root rot pathogen. The results revealed that both of the strains were effective in inhibiting the mycelial growth of F. temperatum ( Table 3). The strains were further tested for their broad-spectrum activity against eight important phytopathogens ( Table 3). The results showed that both of them had strong antifungal activity against all of the tested phytopathogens (Table 3 and Figure 5). The zone of inhibition around the tested plant pathogens ( Figure 5) by the studied strains may be attributed to the ability of the strains to produce toxin metabolites, proteolytic enzymes, and siderophore [36,45]. Previous studies [14,15] reported the biocontrol potential of Burkholderia species, including B. cepacia and B. contaminans against several phytopathogens. However, the antifungal activity of B. cenocepacia against Fusarium graminearum, Fusarium moniliforme, Fusarium oxysporum f.sp. melonis, Fusarium subglutinans, F. temperatum, Phytophthora drechsleri, and Stemphylium lycopersici has not previously been reported. F. graminearum, F. moniliforme, and F. subglutinans are destructive disease of cereals that cause yield loss and grain quality due to their associated toxic metabolites [46,47]. P. drechsleri is one of the most devastating plant pathogen causing sever root rot in various crops [48,49]. S. lycopersici is an emerging plant pathogen causing leaf spot, stem rot, and fruit rot in various commercial crops [50][51][52]. This generally shows the essence of exploiting B. cenocepacia and B. contaminans for the control of the aforementioned pathogens, which causes huge economic loss in different crops.

In Vitro Antifungal Activity
In the dual-culture assay, the antagonistic activity of strain KNU17BI2 and strain KNU17BI3 were tested against F. temperatum, maize root rot pathogen. The results revealed that both of the strains were effective in inhibiting the mycelial growth of F. temperatum (Table 3). The strains were further tested for their broad-spectrum activity against eight important phytopathogens ( Table 3). The results showed that both of them had strong antifungal activity against all of the tested phytopathogens (Table 3 and Figure 5). The zone of inhibition around the tested plant pathogens ( Figure 5) by the studied strains may be attributed to the ability of the strains to produce toxin metabolites, proteolytic enzymes, and siderophore [36,45]. Previous studies [14,15] reported the biocontrol potential of Burkholderia species, including B. cepacia and B. contaminans against several phytopathogens. However, the antifungal activity of B. cenocepacia against Fusarium graminearum, Fusarium moniliforme, Fusarium oxysporum f.sp. melonis, Fusarium subglutinans, F. temperatum, Phytophthora drechsleri, and Stemphylium lycopersici has not previously been reported. F. graminearum, F. moniliforme, and F. subglutinans are destructive disease of cereals that cause yield loss and grain quality due to their associated toxic metabolites [46,47]. P. drechsleri is one of the most devastating plant pathogen causing sever root rot in various crops [48,49]. S. lycopersici is an emerging plant pathogen causing leaf spot, stem rot, and fruit rot in various commercial crops [50][51][52]. This generally shows the essence of exploiting B. cenocepacia and B. contaminans for the control of the aforementioned pathogens, which causes huge economic loss in different crops. Table 3. Broad spectrum antifungal activity of the study Burkholederia strains.

SEM Analysis
Scanning electron microscope was used to examine any morphological defects from the edges of the inhibitory clear zone of the tested phytopathogens due to the studied strains. The SEM analysis revealed that the studied strains caused serious defects in the hyphal morphology of the selected phytopathogens, while hyphae from the untreated control were intact with normal morphology. The studied strains caused the hyphae of the phytopathogens to shrink, lyse, and deform ( Figure 6). Similar to our study, such morphological defects in an interaction between bacterial strains and fungi in a dual culture have previously been reported [53]. However, to the best of our knowledge, the deleterious effects of B. cenocepacia against F. moniliforme, F. subglutinans, and F. temperatum have not previously been reported.

SEM Analysis
Scanning electron microscope was used to examine any morphological defects from the edges of the inhibitory clear zone of the tested phytopathogens due to the studied strains. The SEM analysis revealed that the studied strains caused serious defects in the hyphal morphology of the selected phytopathogens, while hyphae from the untreated control were intact with normal morphology. The studied strains caused the hyphae of the phytopathogens to shrink, lyse, and deform ( Figure 6). Similar to our study, such morphological defects in an interaction between bacterial strains and fungi in a dual culture have previously been reported [53]. However, to the best of our knowledge, the deleterious effects of B. cenocepacia against F. moniliforme, F. subglutinans, and F. temperatum have not previously been reported.  The influence of KNU17BI2 and KNU17BI3 in reducing the virulence of F. temperatum on the seedlings of four maize cultivars viz., Hikchal, Mibeak-2ho, Chahong-chal, and Oluckdehack-chal was assessed after 30 days of planting. Subsequently, typical symptoms that were caused by the root rot pathogen, F. temperatum, were observed on pathogen-inoculated plants, as indicated by discolored primary and secondary roots [54,55] (Figure 7). The severity data showed that root rot disease was significantly reduced in all of the tested Korean maize cultivars following the soil drenching of the studied strains (Figure 8). To the contrary, non-bacterized plants, but pathogen-challenged (negative control treatment), had the highest F. temperatum-induced root rot severity (Figures 7 and 8). The development of healthy roots on bacterized plants of each cultivar may be due to the direct effect of the Burkholderia strains on pathogen, F. temperatum [56]. Similar to our result, previous studies [7,57] reported the potential of PGPR to suppress maize root diseases. Burkholderia cepacia has been reported to suppress Pythium damping-off of sweet corn through lysis of zoospores and the prevention of cyst germination and germ tube growth [57]. Furthermore, Omar and his co-workers [58] demonstrated the bio-control capacity of Burkholderia cepacia against Fusarium crown and root rot of. However, no previous study has reported the potential of B. cenocepacia and B. contaminans to control root rot of maize that is caused by F. temperatum. Among, the four maize verities tested, root rot severity was comparatively higher on cv. Hikchal than cv. Mibeak-2ho (Figures 7 and 8). This is the first report showing the reaction of Korean maize cultivars to the root rot causing pathogen, F. temperatum.

Effect of Strain KNU17BI2 and Strain KNU17BI3 on Virulence of F. temperatum
The influence of KNU17BI2 and KNU17BI3 in reducing the virulence of F. temperatum on the seedlings of four maize cultivars viz., Hikchal, Mibeak-2ho, Chahong-chal, and Oluckdehack-chal was assessed after 30 days of planting. Subsequently, typical symptoms that were caused by the root rot pathogen, F. temperatum, were observed on pathogen-inoculated plants, as indicated by discolored primary and secondary roots [54,55] (Figure 7). The severity data showed that root rot disease was significantly reduced in all of the tested Korean maize cultivars following the soil drenching of the studied strains ( Figure 8). To the contrary, non-bacterized plants, but pathogen-challenged (negative control treatment), had the highest F. temperatum-induced root rot severity (Figures 7 and 8). The development of healthy roots on bacterized plants of each cultivar may be due to the direct effect of the Burkholderia strains on pathogen, F. temperatum [56]. Similar to our result, previous studies [7,57] reported the potential of PGPR to suppress maize root diseases. Burkholderia cepacia has been reported to suppress Pythium damping-off of sweet corn through lysis of zoospores and the prevention of cyst germination and germ tube growth [57]. Furthermore, Omar and his co-workers [58] demonstrated the bio-control capacity of Burkholderia cepacia against Fusarium crown and root rot of. However, no previous study has reported the potential of B. cenocepacia and B. contaminans to control root rot of maize that is caused by F. temperatum. Among, the four maize verities tested, root rot severity was comparatively higher on cv. Hikchal than cv. Mibeak-2ho (Figures 7 and 8). This is the first report showing the reaction of Korean maize cultivars to the root rot causing pathogen, F. temperatum.

Plant Growth Promotion Effect of Strain KNU17BI2 and Strain KNU17BI3
The result of our study revealed that F. temperatum highly affected the negative control plants, thereby significantly reducing the shoot growth and amount of total chlorophyll content ( Figure 9). On the other hand, plants that were treated with either of the strains had a highly significant positive effect on all plant growth parameters that were tested viz., total chlorophyll content, plant height, shoot dry weight, and root dry weight in all maize cultivars (Figures 9 and 10). Similar to our study, previous studies [1,4] reported that seedling chlorosis, reduction in shoot growth, and fresh weight were observed on maize that was growing in F. temperatum inoculated soil. Furthermore, previous reports [1,59,60] explained that the degree of virulence by F. temperatum was defined as the inhibition of shoot elongation and chlorosis symptoms. More importantly, the increase in all tested plant growth parameters may be due to the plant growth promoting traits that the strains possess. In addition, the plant growth promotion effect of Burkholderia species on maize plant has been previously reported [14,61]. It is worth to note that B. cenocepacia has been reported as plant-beneficial endophytic bacterium to control Fusarium wilt of banana [62,63]. However, this is the first report demonstrating the bio-control potential of B. cenocepacia and B. contaminans against root rot of maize that is caused by F. temperatum.

Plant Growth Promotion Effect of Strain KNU17BI2 and Strain KNU17BI3
The result of our study revealed that F. temperatum highly affected the negative control plants, thereby significantly reducing the shoot growth and amount of total chlorophyll content ( Figure 9). On the other hand, plants that were treated with either of the strains had a highly significant positive effect on all plant growth parameters that were tested viz., total chlorophyll content, plant height, shoot dry weight, and root dry weight in all maize cultivars (Figures 9 and 10). Similar to our study, previous studies [1,4] reported that seedling chlorosis, reduction in shoot growth, and fresh weight were observed on maize that was growing in F. temperatum inoculated soil. Furthermore, previous reports [1,59,60] explained that the degree of virulence by F. temperatum was defined as the inhibition of shoot elongation and chlorosis symptoms. More importantly, the increase in all tested plant growth parameters may be due to the plant growth promoting traits that the strains possess. In addition, the plant growth promotion effect of Burkholderia species on maize plant has been previously reported [14,61]. It is worth to note that B. cenocepacia has been reported as plant-beneficial endophytic bacterium to control Fusarium wilt of banana [62,63]. However, this is the first report demonstrating the bio-control potential of B. cenocepacia and B. contaminans against root rot of maize that is caused by F. temperatum.   In conclusion, MLSA-MLST revealed that strain KNU17BI2, which belongs to B. cenocepacia, was of a novel sequence type (ST) designated ST-1538. On the other hand, strain KNU17BI3, identified as B. contaminans, had similar allelic profile when compared to all seven loci of B. contaminans strain LMG 23361 with a sequence type 102. The current study reported two multi-trait bacterial strains viz., B. cenocepacia strain KNU17BI2 and B. contaminans strain KNU17BI3 possessing promising in vitro PGP traits, such as zinc solubilization, phosphate solubilization, ammonia production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, indole acetic acid (IAA) production, siderophore, and hydrolytic enzyme activity. This study is the first report on the strong in vitro antifungal activity of B. cenocepacia strain KNU17BI2 of novel ST against several economically important plant fungal pathogens. These include, against F. temperatum, F. graminearum, F. moniliforme, F. oxysporum f.sp. melonis, F. subglutinans, P. drechsleri, and S. lycopersici. In our study, root drenching of plants of four maize cultivars with the studied strains lead to an increase in the growth of the four maize cultivars and a significant positive effect in the control of root rot of maize seedlings on four different maize cultivars. The present research work reports, for the first time, the potential of B. cenocepacia and B. contaminans to control root rot of maize that is caused by F. temperatum. Hence, further studies are needed to precisely identify the prevailing bioactive chemical compounds behind such activities that would be novel sources of natural products for plant growth promotion as well as biological control.

Microbial Sources
In this study, two bacterial strains (strain KNU17BI2 and strain KNU17BI3) were isolated from maize rhizosphere soil that was located in Gangwon-do province (37°86′ N, 127°75′ E), South Korea. For subsequent experiments, inoculum suspensions of each strain were grown in a tryptic soy broth (TSB) medium on a shaking incubator at 150 rpm in the dark at 28 ± 2 °C for 48 h. To harvest the bacterial cells, the growing media was centrifuged (6000 rpm, 5 min) and the bacterial cells were washed five times using phosphate-buffered saline solution (PBS; 5 mM K2HPO4, 150 mM NaCl, pH 7.0). Subsequently, the inoculum concentration of each strain was adjusted to 10 8 cells·mL −1 . In conclusion, MLSA-MLST revealed that strain KNU17BI2, which belongs to B. cenocepacia, was of a novel sequence type (ST) designated ST-1538. On the other hand, strain KNU17BI3, identified as B. contaminans, had similar allelic profile when compared to all seven loci of B. contaminans strain LMG 23361 with a sequence type 102. The current study reported two multi-trait bacterial strains viz., B. cenocepacia strain KNU17BI2 and B. contaminans strain KNU17BI3 possessing promising in vitro PGP traits, such as zinc solubilization, phosphate solubilization, ammonia production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity, indole acetic acid (IAA) production, siderophore, and hydrolytic enzyme activity. This study is the first report on the strong in vitro antifungal activity of B. cenocepacia strain KNU17BI2 of novel ST against several economically important plant fungal pathogens. These include, against F. temperatum, F. graminearum, F. moniliforme, F. oxysporum f.sp. melonis, F. subglutinans, P. drechsleri, and S. lycopersici. In our study, root drenching of plants of four maize cultivars with the studied strains lead to an increase in the growth of the four maize cultivars and a significant positive effect in the control of root rot of maize seedlings on four different maize cultivars. The present research work reports, for the first time, the potential of B. cenocepacia and B. contaminans to control root rot of maize that is caused by F. temperatum. Hence, further studies are needed to precisely identify the prevailing bioactive chemical compounds behind such activities that would be novel sources of natural products for plant growth promotion as well as biological control.

Microbial Sources
In this study, two bacterial strains (strain KNU17BI2 and strain KNU17BI3) were isolated from maize rhizosphere soil that was located in Gangwon-do province (37 • 86 N, 127 • 75 E), South Korea. For subsequent experiments, inoculum suspensions of each strain were grown in a tryptic soy broth (TSB) medium on a shaking incubator at 150 rpm in the dark at 28 ± 2 • C for 48 h. To harvest the bacterial cells, the growing media was centrifuged (6000 rpm, 5 min) and the bacterial cells were washed five times using phosphate-buffered saline solution (PBS; 5 mM K 2 HPO 4 , 150 mM NaCl, pH 7.0). Subsequently, the inoculum concentration of each strain was adjusted to 10 8 cells·mL −1 .
The study phytopathogens were obtained from Korean agricultural culture collection (KACC), South Korea. The phytopathogens were: A. alternate (KACC43921), F. graminearum (KACC47499), F. moniliforme (KACC41032), F. oxysporum f.sp. melonis (KACC47669), P. drechsleri (KACC40190), and S. lycopersici (KACC40967). F. temperatum and F. subglutinans were kindly provided by Prof. Kim Kyoung Su, Kangwon National University, South Korea. The tested phytopathogens were maintained on potato dextrose agar (PDA) plates at 4 • C for further experimental use and mycelia colonized PDA plugs (8 mm) of the pathogens from the edge of culture plate were used in all of the experiments.

16S rRNA Gene Sequencing
Almost the full-length 16S rRNA gene sequences were PCR amplified with the universal primers 27f (5 -AGAGTTTGATCATGGCTCAG-3 ) and 1492R (5 -TACGGYTACCTTGTTACGACTT-3 ) [64]. Sequencing was carried out at Macrogen Inc. (Seoul, South Korea) using a 3730XL DNA sequencer (Applied BioSystems, Waltham, CA, USA). The search for sequence similarity was carried out using the BLAST server (http://www.ncbi.nlm.nih.gov/BLAST/). The phylogenetic tree of 16S rRNA gene was constructed by the neighbor-joining method using Kimura's two-parameter model [65] that was implemented in the MEGA 6 software [66]. A bootstrap confidence analysis was performed with bootstrap 1000 replicates.

Genbank Accession Numbers
The nucleotide sequences of each allelic profiles and the sequence type of strain KNU17BI2 are found at the Burkholderia cepacia complex PubMLST database (http://pubmlst.org/bcc) with sequence type (ST) 1538. In addition, the nucleotide sequences of 16S rRNA gene and each MLSA-MLST loci of strain KNU17BI2 and strain KNU17BI3 have been deposited in GenBank/EMBL/DDBJ (Table 4).

In Vitro Antifungal Activity
The broad-spectrum inhibitory activity of the studied strains was tested in vitro against seven different economically important plant fungal pathogens using dual culture technique on PDA. These were: F. temperatum, A. alternate, F. graminearum, F. moniliforme, F. oxysporum f.sp. melonis, P. drechsleri, and S. lycopersici. After five days of incubation at 28 ± 2 • C, the inhibitory activity of the strains was determined as the length of the zone of mycelial growth inhibition between the bacterial colony and the test phytopathogen. The percent inhibition of mycelial growth of the tested phytopathogens was calculated based on the formula: PI = (C − T)/C × 100, where PI is the inhibition of mycelial growth in percent, C is the radial growth of the phytopathogen in control, and T is the radial growth of the phytopathogen in dual culture. The experiment was carried out in three replications and the experiment was repeated three times.

Scanning Electron Microscope (SEM) Analysis
To study the interaction of the selected phytopathogens (F. moniliforme, F. subglutinans, and F. temperatum) and the studied strains, the mycelia of the phytopathogens from the edge of the halo zone in the dual culture Petri plates were taken and then observed under scanning electron microscope (SEM). The mycelia samples were prepared by thin coating with gold and palladium (60:40). The coated mycelia samples were observed under SEM (LEO Model 1450VP Variable Pressure Scanning Electron Microscope; Carl Zeiss, Cambridge, MA, USA).

In Vitro Plant Growth Promoting (PGP) Activity Assays
The strains were also tested for important traits of plant growth promotion activity. The traits were: phosphate solubilization, zinc solubilization, indole-3-acetic acid (IAA) production, ammonia production, and siderophore production. For each trait, the experiments were conducted in triplicates and the experiment was repeated three times.
Phosphate solubilizing ability of the studied strains was assessed on national botanical research institute's phosphate growth (NBRIP) medium that was supplemented with tricalcium phosphate (TCP). In addition, the zinc solubilizing potential of the strains was determined on PVK medium supplemented with an insoluble zinc sources containing 1% zinc (ZnO, 1.244 g·L −1 and ZnCO 3 , 1.913 g·L −1 ). After seven days of incubation at 28 ± 2 • C, the diameter of the clear zone around the bacterial colonies was measured. The solubilization index (SI) of phosphate and zinc by the studied strains was determined as the ratio between the halo zone diameter and the colony diameter.
The potential of the studied strains to produce ammonia was determined following the method of Cappuccino and Sherman [67]. The amount of ammonia produced was spectrophotometrically determined (UV-1800, Shimadzu Corporation, Kyoto, Japan) from the standard curve of ammonium sulfate (0-10 µmol·mL −1 ). The method of Gordon and Weber [68] was employed to determine the potential of the tested strains to produce indole acetic acid (IAA). The concentration of IAA in culture was determined while using a standard curve of IAA that was prepared by diluting pure IAA (Sigma-Aldrich, St. Loise, MO, USA) in LB medium at various concentrations (0 to 2 mg·mL −1 ). The strains were also qualitatively tested for the aminocyclopropane-1-carboxylate (ACC) deaminase activity on Dworkin and Foster (DF) minimal salt medium amended with 3 mM filter sterilized ACC as the sole nitrogen source [69]. Amylase activity was determined on starch agar plates [67].

Greenhouse Experiments
Strain KNU17BI2 and strain KNU17BI3 were further evaluated for biological control of maize root rot on four Korean maize cultivars under greenhouse conditions. The cultivars were Hikchal, Mibeak-2ho, Chahong-chal, and Oluckdehack-chal. In this study, four mycelia colonized PDA plugs (8 mm) of F. temperatum were mixed with the top 5 cm of soil in each plastic pot (10 cm diameter) [70]. After 24 h of pathogen inoculation, the seeds of each cultivar were planted at a depth of approximately 4 cm (one seed per pot). Subsequently, one milliliter of bacterial inoculum adjusted at a concentration of 10 8 cells·mL −1 was applied to seeds of each cultivar prior to covering with soil. Non-treated plants (neither pathogen, nor Burkholderia strains) were served as non-treated control and non-bacterized plants, but the pathogen-challenged were served as negative control. In this experiment, treatments (single Burkholderia strains, non-treated control, and negative control) were arranged in a completely randomized design (CRD) with three replications with 10 plants per replication, and the experiment was repeated twice.

Assessment of Effect of Burkholderia Strains on Virulence of F. temperatum
The effect of strain KNU17BI2 and strain KNU17BI3 in controlling the root rot of maize due to F. temperatum on the four Korean maize cultivars was assessed after 30 days of planting. Disease scaling of root rot that is caused by F. temperatum was done based on a 0-4 scale, where 0 = 0, healthy root; 1, less than 25% of the root spoiled due to rotting; 2, 25-50% of the root spoiled, evident from dropping of the leaves during daytime; 3, up to 75% of the root damaged, as evident from starting of wilt and drying of leaves from bottom to top; and, 4, complete rotting of the root, completely wilted, dead, and dry plants [57]. For analysis, the scale was converted into the percentage severity index (PSI) [71].

PSI =
∑ of all numerical ratings × 100 Total number of observations × maximum score on scale Furthermore, based on previous studies [37,38], the degree of virulence that is caused by F. temperatum was defined as the inhibition of shoot elongation and chlorosis symptoms. Hence, leaf chlorosis that was based on total chlorophyll content (SPAD unit) was measured using chlorophyll-meter SPAD 502 (Konica Minolta, Tokyo, Japan) from the middle leaf of each plant. Morphological data, including shoot and root dry weight (oven dried at 65 • C for 72 h), were also recorded.

Statistical Data Analysis
Analysis of variance (ANOVA) of all experimental data was carried out using SAS software version 9.2 [72]. All of the experiments of in vitro and greenhouse were conducted in three replications and were repeated twice. The results of the experiments were expressed as mean ± standard error. Mean separation was carried out using the multiple comparison procedure and Duncan's multiple range test (DMRT) at p ≤ 0.05.