Potential Application of Rhizobacteria Isolated from the Central Highland of Vietnam as an Effective Biocontrol Agent of Robusta Coffee Nematodes and as a Bio-Fertilizer

Robusta coffee is a major commercial crop in the Central Highland of Vietnam with high economic and export value. However, this crop is adversely affected by various pathogens, particularly nematodes. This study aimed to screen active anti-nematode rhizobacterial strains for sustainable coffee production. Among more than 200 isolates, the isolate TUN03 demonstrated efficient biocontrol with nearly 100% mortality of J2 coffee nematodes Meloidogyne spp. and 84% inhibition of nematode egg hatching. This active strain was identified as Pseudomonas aeruginosa TUN03 based on its 16S rRNA gene sequence and phylogenetic analysis. In greenhouse tests, the strain TUN03 significantly reduced the coffee nematode population in the rhizome-soil with an 83.23% inhibition rate and showed plant growth-promoting effects. Notably, this is the first report of the nematicidal effect of P. aeruginosa against coffee nematodes. This potent strain further showed an antifungal effect against various crop-pathogenic fungi and was found to be the most effective against Fusarium solani F04 (isolated from coffee roots) with a 70.51% inhibition rate. In addition, high-performance liquid chromatography analysis revealed that this bacterial strain also secretes plant growth regulators including indole acetic acid (IAA), gibberellic acid (GA3), kinetin, and zeatin in significant amounts of 100, 2700, 37, and 9.5 µg/mL, respectively. The data from this study suggest that P. aeruginosa TUN03 may be a potential biocontrol agent and biofertilizer for the sustainable production of Robusta coffee and other crops.


Introduction
Coffee is one of the most popular consumed beverages worldwide due to its good taste and numerous health benefits [1]. It is an important industrial crop in many countries, including Brazil, Vietnam, Colombia, Indonesia, Ethiopia, etc. Among these, Vietnam is ranked as the largest coffee producer in Asia, and the 2nd in the world for producing two major coffee species, Robusta and Arabica. Vietnam is the largest producer of Robusta coffee (May 2021 statistics of International Coffee Organization), accounting for 85% of the total production [2], which is mainly cultivated in the Central Highlands.
Coffee crops are seriously affected by plant-parasitic nematodes worldwide; in Vietnam, root-knot nematode Meloidogyne spp. and the lesion nematode Pratylenchus spp. are showing symptoms of nematode infection with yellow leaves was used, and its root system was harvested (B). Sick roots having root-knots (C) were collected to isolate nematode eggs (D), then these eggs were incubated for 3-5 days for their development (E) and hatching (F) to obtain fresh J2 nematodes (G). The images of coffee nematode eggs and J2 nematodes were observed and recorded using an Optical Olympus Microscope (Model CH30RF200, Olympus Co., Tokyo, Japan).  showing symptoms of nematode infection with yellow leaves was used, and its root system was harvested (B). Sick roots having root-knots (C) were collected to isolate nematode eggs (D), then these eggs were incubated for 3-5 days for their development (E) and hatching (F) to obtain fresh J2 nematodes (G). The images of coffee nematode eggs and J2 nematodes were observed and recorded using an Optical Olympus Microscope (Model CH30RF200, Olympus Co., Tokyo, Japan).
Anti-Nematicidal Assay: A hundred microliters of bacterial solution (10 7 CFU mL −1 ) was mixed with 250 µL sterile distilled water containing 30 freshly hatched J2 nematodes. The experiment was performed in a 96-well tissue culture plate at room temperature (around 28 • C), and the dead and live nematodes were counted after 24 h of incubation. The nematodes that did not move on being touched with a fine needle were defined as dead according to the method of Cayrol et al. 1989 [31]. The dead and live nematodes were observed and are illustrated in Figure 2A,B. All the tests were performed in triplicate.
was mixed with 250 µL sterile distilled water containing 30 freshly hatched J2 nematodes. The experiment was performed in a 96-well tissue culture plate at room temperature (around 28 °C), and the dead and live nematodes were counted after 24 h of incubation. The nematodes that did not move on being touched with a fine needle were defined as dead according to the method of Cayrol et al. 1989 [31]. The dead and live nematodes were observed and are illustrated in Figure 2A,B. All the tests were performed in triplicate.
Assay for the inhibition of nematode egg hatching: A hundred microliters of bacterial solution (10 7 CFU mL −1 ) was mixed with 250 µL sterile distilled water containing 200 nematode eggs, and then added to a 96-well tissue culture plate. The mixture was kept at room temperature (around 28 °C) for 3 d before the hatched juveniles were counted under a low-power stereoscopic microscope [13]. All the tests were performed in triplicate. Figure 2. Images of the anti-nematicidal and anti-fungal assays of the rhizobacterial strains. The mobilized J2 coffee nematodes (A) became immobilized J2 coffee nematodes (B) after 24 h of treatment with rhizobacterial strain. The pathogenic fungus was grown on potato dextrose agar (PDA) (C), control dish, and also on PDA treated with a rhizobacterial strain (D), experimental dish. After five days of cultivation, the radius (cm) of fungal mycelial spread on the control dish (Rc) and the experimental dish (R) were measured for the calculation of antifungal activity.

The Effect of Rhizobacterial Strains as Anti-Nematodes and the Plant-Promoting Effect for Robusta Trees in Green Houses
Robusta coffee (TR4) seedlings were supplied by the Division of Agro-Forestry System, Western Highlands Agriculture and Forestry Scientific Institute, Buon Ma Thuot 630,000, Vietnam. The initial TR4 seedling sizes, including shoot length (17.41 cm), root length (8.43 cm), shoot dry weight (8.32 g/10 trees), and root dry weight (9.21 g/10 trees), were determined.
The effect of the selected rhizobacterial strain Pseudomonas aeruginosa TUN03 was further evaluated in the greenhouse test. Local Robusta coffee (TR4) seedlings with five leaves and with equal height were used for the tests. The coffee seedlings were randomly categorized into five experimental groups and in triplicate with 30 coffee seedlings per Rc R Figure 2. Images of the anti-nematicidal and anti-fungal assays of the rhizobacterial strains. The mobilized J2 coffee nematodes (A) became immobilized J2 coffee nematodes (B) after 24 h of treatment with rhizobacterial strain. The pathogenic fungus was grown on potato dextrose agar (PDA) (C), control dish, and also on PDA treated with a rhizobacterial strain (D), experimental dish. After five days of cultivation, the radius (cm) of fungal mycelial spread on the control dish (Rc) and the experimental dish (R) were measured for the calculation of antifungal activity.
Assay for the inhibition of nematode egg hatching: A hundred microliters of bacterial solution (10 7 CFU mL −1 ) was mixed with 250 µL sterile distilled water containing 200 nematode eggs, and then added to a 96-well tissue culture plate. The mixture was kept at room temperature (around 28 • C) for 3 d before the hatched juveniles were counted under a low-power stereoscopic microscope [13]. All the tests were performed in triplicate.

The Effect of Rhizobacterial Strains as Anti-Nematodes and the Plant-Promoting Effect for Robusta Trees in Green Houses
Robusta coffee (TR4) seedlings were supplied by the Division of Agro-Forestry System, Western Highlands Agriculture and Forestry Scientific Institute, Buon Ma Thuot 630,000, Vietnam. The initial TR4 seedling sizes, including shoot length (17.41 cm), root length (8.43 cm), shoot dry weight (8.32 g/10 trees), and root dry weight (9.21 g/10 trees), were determined.
The effect of the selected rhizobacterial strain Pseudomonas aeruginosa TUN03 was further evaluated in the greenhouse test. Local Robusta coffee (TR4) seedlings with five leaves and with equal height were used for the tests. The coffee seedlings were randomly categorized into five experimental groups and in triplicate with 30 coffee seedlings per group, including two control groups: the negative control group (Group 1), and the positive control group (Group 2), as well as three experimental groups: seedlings infected with nematodes and treated with 100 mL of Pseudomonas aeruginosa TUN03 solution at different densities of 0.5 × 10 7 CFU mL −1 (Group 3), 1.0 × 10 7 CFU mL −1 (Group 4), and 2.0 × 10 7 CFU mL −1 (Group 5). In the negative control group (Group 1), the coffee seedlings were not treated with bacteria and not infected with nematodes, while in the positive control group (Group 2), the seedlings were infected with nematodes without treatment of bacteria.
One coffee seedling was cultivated per pot (prepared according to the previous reports [11,32]) containing 0.6 kg of the mixture of sterilized sand, soil, and organic fertilizer in a ratio of 1:2:1 (v/v), respectively. The distance between each group and each pot were set at 60 cm and 50 cm, respectively. All the experiments were performed in a greenhouse with 75-80% humidity, temperatures between 25-30 • C, and a light intensity of 50-550 µmol m 2 /s −1 (measured from 08 a.m. to 4 p.m.).
Three hundred J2 nematodes were added to each pot of all groups except Group 1. The experimental groups were treated two times with 100 mL of Pseudomonas aeruginosa TUN03 solution each time on days 10 and 30 after nematode infection. Tap water was used for irrigation. The total period of this experiment was three months. The coffee seedlings were removed from the soil and washed. One gram of roots and 10 g soil were used for counting the number of J2 nematodes for calculation of the antinematode activity (%). Some growth parameters including increased shoot, root length, and dry weight (1), and the contents of some photosynthetic pigments, including chlorophyll a, chlorophyll b, and carotenoid contents (2) were also were examined.
(1) Increased shoot, root length, and dry weight were determined by comparing the shoot, root length, and dry weight of the coffee tree after three months (B) to the initial seedling size (A) according to the equation: Increased parameters = B − A. where Ca, Cb (mg g −1 fresh leaf) are content of chlorophyll a and b; and Ccar (mg/g fresh weight) is carotenoid content.

Anti-Fungal Assays
Anti-fungal activity of Pseudomonas aeruginosa TUN03 was assessed according to the method per Ngo et al. 2020 [33]. A total of 12 fungal pathogen strains (supported from the Institute of Biotechnology and Environment, Tay Nguyen University, Buon Ma Thuot, Vietnam), including Purpureocillium lilacinum F01, Fusarium solani F02, F. solani F03, F. solani F04, Colletotrichum gloeosporioides F05, F. incarnatum F06, Gongronella butleri F07, Pestalotiopsis mangiferae F08, F. incarnatum F09, F. oxysporum F10, Neonectria sp. F11, and F. incarnatum F15. A mycelial plug of growing fungal strain was placed in the center of the Petri dish containing PDA (potato D-glucose agar) medium, and three lines of Pseudomonas aeruginosa TUN03 were streaked at a distance of 2 cm from the center of the surface of PDA medium in Petri dishes ( Figure 1). The experiment was maintained at 28 • C for 5 d. The radial growth of fungal mycelium was measured, and the anti-fungal activity was calculated according to the equation: The radius (cm) of fungal mycelium cultivated on the control dish (not treated with TNU3 strain) and experimental dish (treated with TNU3 strain) are presented as Rc and R, respectively ( Figure 1C,D). All the tests were done with triplicate.

High-Performance Liquid Chromatography (HPLC) Analysis of Plant-Promoting Compounds
King B media (Sigma Aldrich) was used for cultivation of Pseudomonas aeruginosa TUN03 and was prepared in distilled water. Twenty grams of King B media powder was dissolved in 1 L distilled water and its initial pH was adjusted to 7, then the medium was sterilized in an autoclave for 30 min at 121 • C.
The contents of plant growth-promoting compounds (PGPCs), including IAA, GA3, kinetin, and zeatin were determined by using the high-performance liquid chromatography (HPLC; UHPLC-UV Ultimate 3000, Thermo, Germering, Germany) technique. The residues and bacterial biomass in culture broths of Pseudomonas aeruginosa TUN03 were removed by centrifugation at 8000 rpm in 10 min and the harvested culture supernatants were used to detect and determine the concentration of some PGPCs. Five microliters of culture supernatants were injected into the HPLC system and separated via a C18 column (BDS Hypersil C18 (250 × 4.6 × 5)). The PGPCs were detected at a wavelength of 254 nm under the following analysis conditions: mobile phase 60 methanol in water adjusted at pH 5.8 using 10 mM ammonium acetate with a flow rate of 0.8 mL, at a column temperature of 30 • C for 10 min. Commercially available substances, including IAA, GA3, kinetin, and zeatin (Merck KGaA, Darmstadt, Germany) with purities of 98%, 97%, 99%, and 95%, respectively, were used as standards for calculating the concentration of compounds in the culture supernatants as following equations: where x is concentration of PGPCs and y is the peak area of commercial PGPCs. In addition, all the tests were done in triplicate.

Statistical Analysis
All the experiments were of a randomized design. The experimental data on antinematodes, anti-fungi, and plant growth-promoting effects were obtained and analyzed via simple variance (ANOVA) followed by Duncan's multiple range test (when the experiment contains ≥6 items that need to be compared) and Fisher's LSD test (when the experiment contains ≤5 items that need to be compared) at p = 0.05. Statistical Analysis Software (SAS-9.4) purchased from SAS Institute Taiwan Ltd. (Taipei, Taiwan) was used for statistical analysis.

Isolation, Evaluation, and Identification of the Most Active Anti-Nematodes Rhizobacterium via In Vitro Tests
More than 200 rhizobacterial strains were isolated from the rhizosphere soil samples collected from Robusta coffee fields in the Central Highland of Vietnam. These isolated strains were evaluated for their anti-nematode activity. The experimental results indicated that 24 strains demonstrate a high anti-nematicidal activity, with a J2 nematode mortality value of ≥60%; of these, some strains (TUN03, TUN67, and TUN85) displayed the most potent effect against J2 nematodes, with mortality value in the range of 92.36-98.26% (Table 1). For further evaluation of the anti-nematode efficacy of these 24 strains, the coffee nematode egg hatching inhibitory assay was also performed. The data were recorded and are presented in Table 1. These isolates showed nematode egg hatching inhibition in the Agronomy 2021, 11, 1887 7 of 18 range of 35.80-84.03%, and the strain TUN03 was also found to be the most effective strain, with the highest egg hatching inhibition value of 84.03%. Thus, this potential bacterium was chosen for further investigation. Based on the 16S rDNA gene sequence analysis, this bacterial strain was identified as Pseudomonas aeruginosa, with a similarity rate of 100%. The phylogenetic analysis of the identified strain is illustrated in Figure 3, and the 16s rRNA gene sequence of this identified strain was submitted in DDBJ/EMBL/Genbank (accession number: LC645701). Rhizobacterial bacterial strains were cultivated in TSB medium with shaking speed (150 rpm) at 28 • C for 48 h. Bacteria were harvested by centrifuging the cultured medium at 8000 rpm for 10 min. The supernatant was removed by centrifugation at 8000 rpm for 10 min to settle the bacterial biomass, and then each bacterial mass was suspended in the saline buffer to adjust the density to 10 7 CFU mL −1 for antinematode activity assay. All the experiments were performed in triplicate. The data were analyzed via simple variance (ANOVA), and then Duncan's multiple range test at p = 0.05 was performed. Values in the same column with the different letters are significantly different.
Earlier reports prove that Pseudomonas aeruginosa has a wide range of important applications in environment management [34][35][36], in industry for the production of various secondary metabolites (Rhamnolipid, Vanillin, enzymes, and pigments) [36][37][38][39][40][41], as well as its potential use in agriculture [42][43][44][45][46][47][48][49][50][51]. This bacterial genus has been proven to have potential use as a plant growth promoter [42] and produces an anti-plant virus agent [43]. In particular, Pseudomonas aeruginosa and its secondary metabolites have been widely used for controlling numerous plant-pathogen fungal strains [44][45][46][47][48]. However, there are several reports on the assessment of nematicidal activity of Pseudomonas aeruginosa against several nematodes, including banana, tomato, and okra root-knot nematodes [49][50][51]. There is also no report of the nematicidal effect of this genus against coffee nematodes. Thus, this study shows for the first time the potential nematicidal effect of Pseudomonas aeruginosa against coffee nematodes J2 and its egg hatching and adds to the existing knowledge of the biological activities of Pseudomonas aeruginosa. as its potential use in agriculture [42][43][44][45][46][47][48][49][50][51]. This bacterial genus has been proven potential use as a plant growth promoter [42] and produces an anti-plant virus ag In particular, Pseudomonas aeruginosa and its secondary metabolites have been wid for controlling numerous plant-pathogen fungal strains [44][45][46][47][48]. However, there eral reports on the assessment of nematicidal activity of Pseudomonas aeruginosa several nematodes, including banana, tomato, and okra root-knot nematodes There is also no report of the nematicidal effect of this genus against coffee nem Thus, this study shows for the first time the potential nematicidal effect of Pseu aeruginosa against coffee nematodes J2 and its egg hatching and adds to the knowledge of the biological activities of Pseudomonas aeruginosa.

Assessment of the Potential Biocontrol of Robusta Coffee Nematodes and Plant-Prom Effect of Pseudomonas aeruginosa TUN03 under Greenhouse Conditions
Pseudomonas aeruginosa TUN03 was chosen for further experimental study i house tests. Robusta coffee TR4 strain is the most cultivated in the Central Hig Vietnam; therefore, the TR4 coffee seedlings were used as a plant model to test ac to the protocol presented in the Methods Section 2.3. A summary of the nematicid of Pseudomonas aeruginosa TUN03 in the greenhouse is presented in Table 2.
After three months of infection with nematodes, the density of J2 nematode and roots of coffee seedlings in group 2 were much higher than those of group result indicated the seedlings were successfully infected with nematodes, and th ment data are qualified enough for analysis. Furthermore, all the three expe groups treated with Pseudomonas aeruginosa TUN03 showed a

Assessment of the Potential Biocontrol of Robusta Coffee Nematodes and Plant-Promoting Effect of Pseudomonas aeruginosa TUN03 under Greenhouse Conditions
Pseudomonas aeruginosa TUN03 was chosen for further experimental study in greenhouse tests. Robusta coffee TR4 strain is the most cultivated in the Central Highland of Vietnam; therefore, the TR4 coffee seedlings were used as a plant model to test according to the protocol presented in the Methods Section 2.3. A summary of the nematicidal effect of Pseudomonas aeruginosa TUN03 in the greenhouse is presented in Table 2.
After three months of infection with nematodes, the density of J2 nematodes in soils and roots of coffee seedlings in group 2 were much higher than those of group 1. This result indicated the seedlings were successfully infected with nematodes, and the experiment data are qualified enough for analysis. Furthermore, all the three experimental groups treated with Pseudomonas aeruginosa TUN03 showed a significant effect on reducing nematodes in soils and in roots compared to the positive control group (Group 2). The treatment bacterial density of 1.0 × 10 7 CFU mL −1 (Group 4) and 2.0 × 10 7 CFU mL −1 (Group 5) demonstrated great nematicidal effect with a high reduction of 78.8% and 83.1% of J2 nematodes in rhizosphere soils and 42.7% and 52.1% in coffee roots, respectively. In addition, the death rate of coffee seedlings in these treated groups and the negative control group (Group 1) was low, in the range of 7.7-8.3%, while the death rate of coffee seedlings Agronomy 2021, 11, 1887 9 of 18 in the positive group (Group 2) and Group 3 was found to be rather high, at 37.3% and 18.3%, respectively. Thus, treatment with all densities of Pseudomonas aeruginosa TUN03 did not harm the coffee seedlings in the assays. In addition, these experimental groups treated with Pseudomonas aeruginosa TUN03 (Groups 4 and 5) showed positive plant promoting effect on coffee seedlings (Table 3), including the dramatically increased shoot length (47.7-48.1 cm), and increased shoot dry weight (32.6-34.1 g/10 trees) compared to those in other groups. The roots of these coffee trees in Groups 4 and 5 seemed stronger and healthier ( Figure 4) than those in control groups, as evidenced by measuring the root dry weight. As shown in Table 3, all the groups treated by Pseudomonas aeruginosa TUN03 showed a much higher root dry weight (30.2-30.4 g/10 trees) than those in the control groups (20.6-22.5 g/10 trees).   The effect of Pseudomonas aeruginosa TUN03 on the content of photosynthetic pigments, including chlorophyll a, chlorophyll b, and carotenoid, in the leaves of the coffee seedlings cultivated under greenhouse conditions was also observed and the results are recorded in Table 4. This bacterial strain enhanced photosynthetic pigment content in the leaves of coffee seedlings. Among the treated groups, Group 4 showed a slightly higher content of photosynthetic pigments than in Group 5 and much higher than those in other groups. Based on the experimental results, the rhizobacterium Pseudomonas aeruginosa TUN03 was found to be a potential candidate for biocontrol of coffee nematodes as well as a good plant growth enhancer for this crop.

Potential Application of P. aeruginosa TUN03 as a Potent Biofertilizer Agent Related to Large-Scale Production of Plant Growth Promoting Compounds (PGPCs)
For a further understanding of the potential plant-promoting effect of Pseudomonas aeruginosa TUN03 on the coffee seedlings in the previous experiments (Section 3.2), we used the HPLC technique to explore its ability to synthesize PGPCs. This bacterium was cultivated in King B medium broth at 28 • C for two days. Analysis of the fermented culture broth supernatant revealed the production of some major PGPCs including GA3, IAA, zeatin, and kinetin through HPLC fingerprinting as major peaks at the retention times of 3.172, 3.218, 3.963, and 4.662 min, respectively ( Figure 5). Of these, the highest peak was that of GA3, indicating that this compound was produced at the highest yield among the PGPCs in the culture broth. To clarify this result, the concentrations of these four PGPCs were determined using the equations (presented in Section 2.5) created by using the commercial PGPCs as standard compounds. The yields of these PGPCs were 231 µg/mL (IAA), 2702 µg/mL (GA3), 36.8 µg/mL (kinetin), and 7.71 µg/mL (zeatin). In addition, the culture broth contained a dark green pigment, which is well-known to be Pyocyanin, produced by Pseudomonas aeruginosa [46,52]. Pyocyanin is a potential biocontrol compound against a vast array of plant-pathogen diseases [53], and as such, it may be a potent plant-promoting agent via its indirect mode of action [11,13,[54][55][56][57]. Pyocyanin was found to be produced at 16 µg/mL, which was determined using the method published by Devnath et al. (2017) [58]. Because of its high anti-pathogenic activity (anti-nematode effect) and biosynthesis of multi PGPCs at a high level, these data may prove Pseudomonas aeruginosa TUN03 to be a potential biofertilizer for use in agriculture.
The comparison of the yield of PGPCs produced by different Pseudomonas aeruginosa strains in different reports is summarized in Table 5. The level of cytokinin compounds produced by Pseudomonas aeruginosa TUN03 (7.71-36.8 µg/mL) was comparable to those produced by earlier reported strains (1.32-30 µg/mL); however, IAA was produced by TUN03 at a much higher yield (231 µg/mL) than that reported earlier (4.9-74.54 µg/mL). Notably, GA3 was biosynthesized by Pseudomonas aeruginosa TUN03 at a remarkable concentration of 2702 µg/mL (based on HPLC determination), which is a new record of high biosynthesis of GA3 by Pseudomonas aeruginosa.
The ability to produce IAA, especially GA3, by Pseudomonas aeruginosa TUN03 isolated in this study was different from that of other previously reported strains, possibly due to various factors, such as variation in fermentation conditions, different carbon/nitrogen sources, and particularly, detection by different methods and types of equipment. HPLC analysis (the most widely accepted for quantity and concentration determination of various chemical compounds) was used in this study for the detection and concentration determination of these active compounds. Thus, we suggest that the bacterial strain isolated in this study possesses a significant characteristic of IAA and GA3 biosynthesis at a high level.

Novel Potential Anti-Fungal Effect of Pseudomonas aeruginosa TUN03
To further investigate the potential use of this strain in agriculture, we explored its antifungal activities against various plant-pathogen fungal strains that seriously damage some crops, such as coffee, black pepper, Durio, Persea, Dimocarpus, knotweed, citrus, and sweet potato, cultivated in the Central Highland of Vietnam. A total of 12 pathogen fungal strains were used for the tests. The data are summarized and recorded in Table 6 and also illustrated in Figure 6. The HPLC profiles of production of plant-promoting compounds (PGPCs) biosynthesized by Pseudomonas aeruginosa TUN03. This bacterial strain was cultivated in King B medium at 28 °C for two days. The fermented culture broth was centrifugated at 8000 rpm for 10 min to remove the residues and bacterial biomass and the supernatant was collected to be used for the detection of PGPCs. Five microliters of culture supernatant were injected into the HPLC system and separated on a C18 column, the PGPCs were detected at the wavelength of 254 nm under the following analysis conditions: mobile phase 60% methanol in water adjusted to pH 5.8 using 10 mM ammonium acetate with a flow rate of 0.8 mL, and a column temperature of 30 °C for 10 min. The HPLC profiles of production of plant-promoting compounds (PGPCs) biosynthesized by Pseudomonas aeruginosa TUN03. This bacterial strain was cultivated in King B medium at 28 • C for two days. The fermented culture broth was centrifugated at 8000 rpm for 10 min to remove the residues and bacterial biomass and the supernatant was collected to be used for the detection of PGPCs. Five microliters of culture supernatant were injected into the HPLC system and separated on a C18 column, the PGPCs were detected at the wavelength of 254 nm under the following analysis conditions: mobile phase 60% methanol in water adjusted to pH 5.8 using 10 mM ammonium acetate with a flow rate of 0.8 mL, and a column temperature of 30 • C for 10 min.   [44,45,47]. In this study, we first evaluated the antifungal activity of Pseudomonas aeruginosa TUN03 against the pathogen various plant-pathogen fungal strains which seriously damaged some crops (listed in Table 6) cultivated in the Central Highland of Vietnam. Interestingly, this strain displayed a high inhibitory effect against F. solani F04 which was isolated from the roots of sick coffee (yellow leaves) plants. This finding proved that Pseudomonas aeruginosa TUN03 could be a potential candidate for biocontrol and biofertilizer of coffee and other crops. This strain needs to be examined for its potential application on Robusta coffee plants under field conditions.

Novel Potential Anti-Fungal Effect of Pseudomonas aeruginosa TUN03
To further investigate the potential use of this strain in agriculture, we explored its antifungal activities against various plant-pathogen fungal strains that seriously damage some crops, such as coffee, black pepper, Durio, Persea, Dimocarpus, knotweed, citrus, and sweet potato, cultivated in the Central Highland of Vietnam. A total of 12 pathogen fungal strains were used for the tests. The data are summarized and recorded in Table 6 and also illustrated in Figure 6. Among these tested fungal strains, Pseudomonas aeruginosa TUN03 demonstrated the most efficient inhibition against F. solani F04 isolated from coffee roots with a high inhibition value of 70.51%, and this value was ranked as excellent (level a) based on Duncan's multiple range test. The strain TUN03 also showed inhibition against F. solani F02, F. incarnatum F06, and G. butleri F07, which were isolated and identified from Durio roots, citrus roots, and pepper roots, respectively, with potent inhibition values in the range of 41.67-50%, and ranked at levels b-d. Pseudomonas aeruginosa TUN03 showed moderate inhibitory effect against several fungal strains, including F. solani F03, C. gloeosporioides F05, Pestalotiopsis mangiferae F08 (30-35%), and weak inhibition of F. incarnatum F09, and Neonectria sp. F11 at a rate of 15.09-20.5%. Pseudomonas aeruginosa TUN03 was not effective against F. oxysporum F10 isolated from pepper roots. As shown in Figure 2A, Pseudomonas aeruginosa TUN03 significantly reduced the appearance of fungal colonies of Purpureocillium lilacinum F01, indicating more than 70% inhibition of this pathogenic fungus (the calculation was based on counting the colonies in experiment and control dishes). The spread of spores of this fungal strain around the surface of the Petri dish and the quick appearance of the colonies ( Figure 1A) made it difficult to calculate the standard inhibition values based on the protocol presented by Ngo et al. 2020 [32] in the methods section. Among these tested fungal strains, Pseudomonas aeruginosa TUN03 demonstrated the most efficient inhibition against F. solani F04 isolated from coffee roots with a high inhibition value of 70.51%, and this value was ranked as excellent (level a) based on Duncan's multiple range test. The strain TUN03 also showed inhibition against F. solani F02, F. incarnatum F06, and G. butleri F07, which were isolated and identified from Durio roots, citrus roots, and pepper roots, respectively, with potent inhibition values in the range of 41.67-50%, and ranked at levels b-d. Pseudomonas aeruginosa TUN03 showed moderate inhibitory effect against several fungal strains, including F. solani F03, C. gloeosporioides F05, Pestalotiopsis mangiferae F08 (30-35%), and weak inhibition of F. incarnatum F09, and Neonectria sp. F11 at a rate of 15.09-20.5%. Pseudomonas aeruginosa TUN03 was not effective against F. oxysporum F10 isolated from pepper roots. As shown in Figure 2A, Pseudomonas aeruginosa TUN03 significantly reduced the appearance of fungal colonies of Purpureocillium lilacinum F01, indicating more than 70% inhibition of this pathogenic fungus (the calculation was based on counting the colonies in experiment and control dishes). The spread of spores of this fungal strain around the surface of the Petri dish and the quick appearance of the colonies ( Figure 1A) made it difficult to calculate the standard inhibition values based on the protocol presented by Ngo et al. 2020 [32] in the methods section.

Conclusions
In this study, we first assessed the potential use of rhizobacteria for biocontrol of Robusta coffee nematodes in the Central Highland of Vietnam. Pseudomonas aeruginosa TUN03 was screened and identified as the most effective strain. This selected bacterium significantly reduced the number of nematodes in soils and roots of coffee seedlings and was significantly effective in plant growth promotion in greenhouse tests. Pseudomonas aeruginosa TUN03 was found to produce multiple plant-promoting compounds and showed anti-fungal effects against various plant pathogen fungi that seriously damage some crops. Notably, this is the first report of the potent nematicidal effect of Pseudomonas aeruginosa against coffee nematodes J2 and its egg hatching, and the production of multiple plantpromoting compounds with high yield. Among these, the yield of GA3 was the highest, at 2702 µg/mL. TUN03 also displayed potency in biocontrol of various tested pathogen fungal strains. This work suggests, for the first time, that Pseudomonas aeruginosa TUN03 could be a potential biocontrol agent and a biofertilizer. Institutional Review Board Statement: Not applicable.