Genetic diversity and characterization of the growth promotion mechanisms of Burkholderia vietnamiensis isolates from rice cultivars in valleys of the high jungle of Peru.

doi:10.21203/rs.3.rs-4927294/v1

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Genetic diversity and characterization of the growth promotion mechanisms of Burkholderia vietnamiensis isolates from rice cultivars in valleys of the high jungle of Peru.

https://doi.org/10.21203/rs.3.rs-4927294/v1

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Burkholderia is a versatile bacterial genus and from a biotechnological point of view it is a source of various secondary metabolites with enormous application potential, especially in agriculture. The aim of this study was to isolate Gram-negative diazotrophic endophytic bacteria from rice roots, to select and study the genetic diversity of strains of the genus Burkholderia, as well as the growth promotion mechanisms and the effect of their inoculation in two rice cultivars. Seventy-eight Gram-negative diazotrophic isolates were obtained from 132 root samples of different rice varieties, of which only 5.13% was positive for recA gene amplification with specific Burkholderia primers. Sequence analysis showed high similarity with B. vietnamiensis. These strains produced auxin in broth supplemented with tryptophan (up to 13.98 µg mL-1), siderophores (up to 139.52%), phosphate solubilization (up to 15.99 mg PO₄ mL^− 1), so too antibiotic and antagonist capacities against five rice pathogenic fungi. These strains increased the vigour index in two rice cultivars compared to the uninoculated or unfertilised treatment. The accumulation of total biomass was high in one strain, with significant differences observed in the response to inoculation at the cultivar level. The antibiotic and antifungal activities of B. vietnamiensis strains against the two pathogenic fungi Nakataea sigmoidea and Nigrospora oryzae are described for the first time. Due to the taxonomic affinity of our strains within the Burkholderia cepacia complex, their direct use in agriculture is not recommended; however, further research is required to exploit their biotechnological potential for the synthesis of useful metabolites.

recA

Nitrogen fixation

Auxin production

Siderophores

Phosphate solubilization

Antagonism.

Burkholderia sensu lato (s.l.) (Yabuuchi et al. 1992) are a group of Gram-negative bacteria that are ubiquitous in different ecological niches and belong to the subphylum β-proteobacteria. Currently, phylogenetic analyzes of conserved genes and comparative genomics, the Burkholderia s.l. have reformed Burkholderia s.l. into 7 new genera: Burkholderia sensu stricto (s.s.), Caballeronia (Dobritsa et al. 2016), Paraburkholderia (Sawana et al. 2014), Robbsia (Lopes-Santos, 2017), Mycetohabitans and Trinickia (Estrada de los santos et al. 2018), Pararobbsia (Lin et al. 2020). Within the genus Burkholderia, the Burkholderia cepacia complex (Bcc) comprises a group of species from different ecological niches and their metabolic versatility (Jin et al. 2020). It is characterized by mobility, strictly aerobic oxygen requirement and great metabolic versatility due to the diversity of ecological niches it occupies (Adaikpoh et al. 2022). It includes phytobeneficial species that promote plant growth, such as B. ambifaria (Mullins et al. 2019), B. catarinensis (Bach et al. 2017), B. orbicola (Morales-Ruiz et al. 2022) and B. vietnamiensis (Wallner et al. 2022), as antagonists of phytopathogenic fungi and producers of metabolites of biotechnological interest (Depoorter et al. 2016; Elshafie and Camele, 2021). Burkholderia isolates have enormous biotechnological potential, as many strains are producers of hydrolytic enzymes and bioactive substances that promote plant growth and health, and degrade several recalcitrant pollutants (Eberl and Vandamme, 2016; Rojas-Rojas et al. 2019; Alam et al. 2022). On the other hand, some members of the Bcc have also been reported as opportunistic pathogens in humans and animals (Eberl and Vandamme, 2016; Fu et al. 2022), preventing their use as bioinoculant in the field.

Plants have co-evolved with various microorganisms, allowing them to survive in hostile environmental conditions. This association between plants and microorganisms takes place at the level of the rhizosphere, endosphere and/or phyllosphere (Afzal et al. 2019). The relationships between plants and Burkholderia sensu stricto can be mutualistic or pathogenic. Mutualistic or phytobeneficial relationships are usually associated at the rhizosphere level (Lopes-Santos et al. 2017; Aba Regis et al. 2021; Draghi et al. 2021) or at the endosphere level (Fu et al. 2017; Kong et al. 2020; Shinjo et al. 2020). Among the phytobeneficial Burkholderia sensu stricto, B. vietnamiensis (Gillis et al. 1995) is considered a nitrogen-fixing endophyte that is characterized by a mutualistic relationship with Oryza sativa and considered a model bacterium for growth promotion in rice (Wallner et al. 2022; King et al. 2023). Several studies have been reported that B. vietnamiensis strains promote plant growth, through direct mechanisms, such as the production of phytohormones, siderophores, 1-aminocyclopropane-1-carboxylate (ACC) deaminase and nitrogen fixation, and indirect promotion mechanisms, such as biocontrol associated with the production of metabolites. antifungal, antibacterial and quorum quenching (Romero-Gutiérrez et al. 2020; Meng et al. 2023).

B. vietnamiensis has been reported as an endophyte associated with Ficus tikoua (Meng et al. 2023), Ipomoea batatas (Shinjo et al. 2018), Populus trichocarpa (Xin et al. 2009) and very frequently in rice (Govindarajan et al. 2008; da Silva Araújo et al. 2013; Estrada et al. 2013; Ríos-Ruiz et al. 2020; Valdez-Nuñez et al. 2020; Kuang et al. 2024). The interactions between B. vietnamiensis and rice interactions are very competent and persistent in the rhizosphere, synthesising a large number of secondary metabolites and expressing direct and indirect growth mechanisms, which is why it is considered a bacterial model of growth promotion to be studied (Wallner et al. 2022). Recently, Liu et al (2022) demonstrated thath B. vietnamiensis could inhibit root-knot nematode on watermelon by modifying the rhizosphere microbial community.

There is evidence for the growth promoting potential of B. vietnamiensis in rice (Govindarajan et al. 2008; Shinjo et al. 2020; Ríos-Ruiz et al. 2020). Recently, Shinjo et al. 2020, reported that B. vietnamiensis improved rice seedlings growth, enhanced root development and increased nitrogen mobilisation and assimilation. Unfortunately, these phytobeneficial species isolated from the rhizosphere and endosphere of plants could not be used as inoculants on a commercial scale, due to a moratorium imposed in the 1990s on the grounds that they could pose a health risk (Eberl and Vandamme, 2016). However, in recent years, there has been an increase in the number of new Burkholderia species that are beneficial to plants and that are not isolated from clinical sources. For this reason, there is an urgent need to develop new strategies, both to reduce the use of nitrogenous fertilizers, which can be addressed through the study of phytobeneficial Burkholderia associated with rice cultivation in the San Martin region. It is important to study the genetic diversity among Burkholderia species and to resolve their taxonomy, especially to distinguish strains with phytobeneficial potential, from those with opportunistic pathogenic capacity. In addition, knowing the diversity and identity of phytobeneficial Burkholderia strains, we will be able to select strains to study at a level of genomic resolution that allows better decisions of study. The aims of this work were: 1) isolate root endophytic bacteria (potentially phytobeneficial) from rice cultivars in four valleys of the San Martín region, 2) select and identify phytobeneficial Burkholderia sensu stricto strains using specific molecular markers, 3) to study genetic diversity and the phylogenetic relationship among Burkholderia sensu stricto strains, and 4) To evaluate the direct and indirect growth promotion mechanisms of B. vietnamiensis strains and their response to inoculation in two local rice cultivars.

Collection of rice samples

Root samples of different rice varieties were collected from the Bajo Mayo, Altomayo, Alto Huallaga and Central Huallaga valleys in the San Martín-Perú region. Root samples were obtained from plants at the tillering stage according to Ji et al. (2014). Root samples were processed, coded, and transported in falcon tubes (50 mL) at 4°C. Georeferencing data and cultivation history were recorded for each sample.

Isolation of endophytic bacteria from roots of rice

Endophytic bacteria were isolated from roots according to the method proposed by Baldani et al. 2014. Roots were washed with copious amount of water to remove soil remnants and immersed successively in 70% ethanol solution for 3 min, sodium hypochlorite solution (2.5%) for 5 min, 70% ethanol solution for 30 s, and rinsed five times with sterile distilled water (Sun et al. 2008). To confirm the success of the disinfection process, 100 µL of sterile distilled water from the last rinse of each sample were streaked on the surface on plates containing Tryptone Soy Agar (TSA) and incubated at 28°C for 3 days (Valdez et al. 2020). Only samples with no growth were considered suitable for further analysis. Root tissues were macerated with sterile physiological saline solution (0.85%) and 100 µL were inoculated into JMV nitrogen-free semi-solid medium (Baldani et al. 1996) containing (g L^− 1) mannitol, 5.0; K₂HPO₄, 0.6; KH₂PO₄, 1.8; MgSO₄ 7H₂O, 0.2; NaCl, 0.1; CaCl₂.2H₂O, 0.2; Bromothymol blue (5 g L^− 1 in 0.2 N KOH), 2 mL; FeEDTA (16.4 g L^− 1), 4 mL; micronutrient solution, 2 mL; vitamin solution (1 mL). Distilled water is added to make up 1000 mL, pH is adjusted to 5.2 ± 0.2 with KOH and agar added at a rate of 1.8 g L^− 1. The appearance of a pellicle under the culture medium after 4 days of incubation indicated the presence of diazotrophic endophytic bacteria. This bacterial film was streaked in Petri dishes containing nitrogen-free solid JMV medium (agar, 15 g L^− 1) and incubated at 30°C for 5 days until colonies appeared. Isolates with different morphocolonial characteristics in each sample were purified by the streaking technique in Petri dishes containing TSA until pure cultures were obtained. The preservation of the collection was carried out by cryopreservation at -20°C and − 80°C, as suggested by García and Cotter (2016), and Cui et al. (2021).

Genomic DNA extraction

Genomic DNA was extracted from bacterial cultures incubated overnight in Tryptone Soy Broth (TSB) (25 mL) at 28°C, shaken at 150 rpm, and the cell pellet was harvested by centrifugation at 13,000 rpm for 3 min (Bach, 2017). The commercial GenElute™ Bacterial Genomic DNA kit (Sigma Aldrich, USA) was used according to the manufacturer's instructions. The DNA concentration was measured in a NanoDrop one spectrophotometer (Thermo Scientific, USA) and the quality verified in a 1% agarose gel, using Diamond™ Nucleic Acid Dye (Promega, USA) and visualised in a black light electrophoresis chamber (Cleaver Scientific Ltd., UK). The original DNA was stored at -20°C and − 80°C.

Molecular discrimination of Burkholderia

Strains belonging to the genus Burkholderia were selected using specific primers recA (Spilker et al. 2009). The primers recA-F (forward) 5’-AGGACGATTCATGGAAGAWAGC-3’ and recA-R (reverse) 5’-GACGCACYGAYGMRTAGAACTT-3’, amplify a specific sequence of approximately 704 bp of the recA gene. The polymerase chain reaction was performed in a Biometra thermal cycler (Analytic Jena, Germany). PCR reactions were performed using a 25 µL reaction mix containing 1.0 µL template DNA (50 ng), 12.5 µL KAPA Taq ReadyMix™ (2x) (Sigma-Aldrich, USA) (DNA polymerase, 0.05 units µL^− 1, 3 mM MgCl₂, 400 µM each dNTP), 1.0 µL of each primer (5 pmol each forward and reverse), and 9.5 µL of PCR grade water (Sigma-Aldrich, USA). The PCR cycle was as follows: initial denaturation at 95°C for 2 minutes, 30 cycles of denaturation for 30 s at 94°C, annealing at 58°C for 30 seconds, an extension for 60 seconds at 72°C, and a final extension at 72°C of 5 min. Two µL of each PCR product was visualized on a 1.0% agarose gel in 0.5X TAE buffer according to Mahenthiralingam et al. (2000). Positive amplification of the fragment allowed the discrimination of strains of the genus Burkholderia. The PCR products were purified and sequenced by MACROGEN Inc. (South Korea).

BOX-PCR genomic profiles:

Genomic profiles were obtained by amplifying genomic DNA using the BOX-A1R primer (5'-CTACGGCAAGGCGACGCTGACG-3') (Koeuth et al. 1995) in a Biometra Tone thermal cycler (Analytic Jena), following the cycle program described by Arone. et al. (2014). PCR products were separated on a 1.5% agarose gel running at 60 V for 8 hours and then visualised in a black light electrophoresis chamber (Cleaver Scientific Ltd., UK). The DirectLoad™ 1 kb DNA Ladder (Sigma Aldrich, USA) was used as a molecular weight marker. The genomic profiles of the individual strains generated were photographed, digitised, and converted into a binary matrix of the presence or absence of DNA bands. Cluster analysis allowed the construction of dendrograms using the free programme DendroUPGMA (http://genomes.urv.es/UPGMA/) (García-Vallvé et al., 1999), applying the UPGMA algorithm, (Unweighted Pair-Group Method with Arithmetic Mean) (Sneath and Sokal 1962), and the Jaccard t coefficient (Jaccard 1912), with 2% tolerance.

rec A gene phylogeny

The recA sequences obtained were compared with those from GenBank using the BLASTN algorithm (Altschul et al. 1990). Sequences of all type strains of the genus Burkholderia sensu stricto were obtained from the NCBI database and aligned using ClustalW (Thompson et al. 1994). Phylogenetic trees were reconstructed using a maximum likelihood (ML) approach (Saitou and Nei, 1987), using the MEGA X software (Kumar et al. 2018). Pairwise distances were calculated using MEGA X (Kumar et al. 2018) with the two-parameter Kimura model (Kimura, 1980). The topological robustness of the ML tree was inferred by non-parametric bootstrap analysis based on 100 pseudoreplicates.

Characterization of growth promotion mechanisms:

Standardised inocula of five B. vietnamiensis strains representing each different BOX-PCR group were cultured in TSB broth at 150 rpm and incubated overnight at 28°C, cells were harvested by centrifugation at 13,000 rpm for 3 min (Bach, 2017) and washed at least twice with 0.85% sterile physiological saline. The cell suspension was standardised to an OD600 nm of 1.0 and used to evaluate growth promotion mechanisms.

Auxin production:

Auxin production was assayed by seeding 200 µL of standardised inoculum in 20 mL of TSB broth supplemented with L-tryptophan as precursor (Gravel et al. 2007; Ji et al. 2014) at increasing concentrations (0, 50, 100, 200, 400, 600 µg mL-1). Treatments were incubated for 24 h at 26 ± 2°C with shaking at 150 rpm. The supernatant of each treatment was obtained by centrifugation at 8000 rpm for 15 minutes at 4°C. Salkowski's reagent (150 mL of H₂SO₄ was mixed, 250 mL of distilled water, 7.5 mL of 0.5 M FeCl₃.6H₂O solution) was mixed with the supernatant in equal parts (1:1) and left in the dark for 30 minutes. The absorbance of the mixture was then measured at 535 nm in a spectrophotometer (PG Instruments Ltd, UK). The concentration of IAA was estimated from a standard curve.

Nitrogen fixation:

Asymbiotic nitrogen fixation capacity was tested according to Ríos-Ruiz et al. 2023. The nitrogen fixation capacity was tested in a mineral medium without nitrogen – MM-N (Zapater, 1975) (g L-1) - Mannitol, 10.0 g; Sucrose, 10.0 g; K₂HPO₄, 0.6551 g; KH₂PO₄, 0.15 g; FeCl₃, 0.0034 g; Na₂MoO₄. 2 H₂O, 0.0108 g; NaCl, 0.02 g; CaCl₂. 2 H₂O, 0.01; MgSO₄. 2H₂O, 0.2 g; 1.6 g (semi-solid) or 16 g (solid) agar-agar, 0.5 mL bromothymol blue solution (0.5% in 70% ethanol), pH 7.0 ± 0.2. The standardised inocula were seeded on the surface of the semi-solid MM-N at a rate of 10% (150 µL per tube). The formation of a sub-pellicle under the semi-solid MM-N culture medium and the appearance of colonies in the solid MM-N medium, as well as the turning of the bromothymol blue indicator, indicated nitrogen fixation activity.

Siderophores production

The qualitative production of siderophores by B. vietnamiensis strains was detected on chrome azurol-S (CAS) agar (Schwyn and Neilands, 1987). CAS agar consisted of a mixture of a CAS staining solution (solutions 1 and 2) and Succinate medium (SM). For solution 1 (60 mL), 60.5 mg of CAS (Hi-media, India) was dissolved in 50 mL of distilled water (pH 7.0) and then mixed with 10 mL of 1 mM FeCl₃ 6 H₂O (5.4 mg FeCl₃.6 H₂O) dissolved in 10 mM HCl (0.822 mL of 37% HCl and density of 1.19 in 100 mL of distilled water). Using a magnetic stirrer, solution 1 was slowly added to solution 2 (72.84 mg HDMTA (Merck, Germany) dissolved in 40 mL distilled water, pH 7.0). The resulting solution (100 mL) was autoclaved at 121°C for 20 minutes and cooled to 50°C. For the SM culture medium, 720 mL of SM broth was added to 27.45 g of PIPES (Sigma-Aldrich, USA), the pH was reduced to 3.0 and corrected to 6.8 using 30% KOH solution. The final mixture was made up to 810 mL with SM broth and supplemented with 15.0 g of agar-agar, then autoclaved at 121°C for 20 minutes and cooled to 60°C. The CAS agar was completed by mixing 810 mL of SM culture medium and 90 mL of CAS staining solution and then distributing the CAS agar in Petri dishes. Each strain was separately inoculated (20 µL) onto Petri dishes containing CAS agar and incubated at 28 ± 2°C for 72 hours in triplicate. The plates were scored by the colour change from blue to orange, the diameter of the siderophore production halo was evaluated and the efficiency of siderophore production was determined using the formula

E%= [(halo diameter - colony diameter)/colony diameter] x 100. Samples were analysed in triplicate.

Phosphate solubilization

Phosphate solubilisation was evaluated qualitatively according to Ramírez-Bahena et al. (2015) and quantitatively according to Marra et al. (2012) and Song et al. (2008), both performed in agar and National Botanical Research Institute's phosphate (NBRIP) broth, respectively. The NBRIP medium had the following composition: glucose 10 gL-1, Ca₃(PO₄)₂, 5 g; (NH₄)₂SO₄, 0.1 g; MgCl₂.6H₂O, 5 g; MgSO₄.7H₂O, 0.25 g and KCl, 0.2 g (Nautiyal, 1999). The pH was adjusted to 7.0 prior to autoclaving. For qualitative assessment, the NBRIP broth was supplemented with 1.5% agar (Hi-media, India), autoclaved and served in Petri dishes. For each strain, 20 µL of standardised inoculum was inoculated in quadruplicate into a Petri dish containing NBRIP agar. The inoculated plates were incubated at 28°C and the solubilization diameter halo (translucent area around the colony) was measured using an electronic vernier after 15 days of incubation. The solubilisation index (SI) = halo diameter (mm)/colony diameter (mm) was determined as proposed by Marra et al. 2012. For quantitative evaluation, 200 µL of standardised inoculum were inoculated in triplicate into 20 mL of NBRIP broth and incubated at 26 ± 2°C at 150 rpm for 24 h. At the end of this period, the inoculum was discarded. At the end of this period, the supernatant was centrifuged (13,500 rpm for 5 minutes), the pH was measured and the amount of soluble P was quantified using the phosphomolybdate method (Murphey and Riley, 1962). The blank treatment consisted of the NBRIP broth without inoculation and shaking. The strains with phosphate solubilising capacity were those with a soluble P concentration higher than the blank treatment.

Antibiosis and antagonism against phytopathogenic fungi:

The methods used to study the indirect mechanisms related to the biocontrol of phytopathogenic fungi in rice in five strains of B. vietnamiensis were antagonism and antibiosis, as proposed by Castellano-Hinojosa and Bedmar (2017) and Ríos-Ruiz et al. (2020). Several phytopathogenic fungi, i. e. Rhizoctonia solani (RS1), Rhizoctonia oryzae (RO1), Rhizoctonia oryzae-sativae (ROS1), Nakataea sigmoidea (NS1) and Nigrospora oryzae (NO1), were donated by the Plant Disease Diagnostic Centre, Faculty of Agrarian Sciences-Universidad Nacional de Tumbes, and were cultured on potato dextrose agar (PDA).

For antagonism assays, fungi were first grown in PDA medium and used to take 1 cm diameter agar plugs, which were placed in the centre of plates containing PDA medium that had previously been independently inoculated with 100 µL of each of the strains grown in TSB. The cells were incubated for 72 hours at 25°C and the diameter of the inhibition zones was recorded after 7 days. The percentage of inhibition was calculated using the formula: % inhibition = [(R1-R2/R1) x 100, where R1 is the diameter of the fungal mycelium in plates not inoculated with the bacterial culture and R2 is the diameter of the fungal mycelium in plates inoculated with the bacterial culture. For the antibiosis test, the agar plugs containing the fungus were placed in the centre of a plate containing PDA medium, with the strains to be inoculated in the three equidistant quadrants of the plate at a rate of 20 µL for each strain. The inhibitory effect on fungal growth was evaluated after 7 days at 25°C and the percentage of inhibition relative to the control (without bacteria) was evaluated as indicated above. Both assays were performed in triplicate and incubated for 48 h at 25°C.

Evaluation of growth promotion in two rice cultivars:

For the standardisation of inocula, the methodologies proposed by Granada et al. (2014) for germination and vigour index assays and by Wallner et al. (2023) for growth promotion assays were applied. B. vietnamiensis strains were cultured in 3 mL of TSB and incubated at 28°C for 24 h at 170 rpm. The fermented broth was centrifuged at 10,000 rpm for 10 minutes at 4°C to harvest the cells. The cell pellet was washed at least twice with sterile 10 mM MgSO₄ solution at 10,000 rpm for 5 minutes. The cell suspension was adjusted to an OD_{600 nm} of 0.001 (approximately 2 x 10⁶ CFU mL-1) for the germination and vigour index assays and to an OD_{600 nm} of 1.0 (approximately 5 x 10⁹ CFU mL-1) for the growth promotion assay. Finally, the cell count was verified by the drop method in Petri dishes containing TSA agar (Ferreira et al. 2024). The rice varieties used were INIA-510, La Conquista (LC) and INIA 507, La Esperanza (LE), both from the National Rice Programme, El Porvenir-San Martín Experimental Station of the Instituto Nacional de Innovación Agraria (INIA). For the trials, seeds of both varieties were surface disinfected according to the methodology proposed by Ji et al. (2014): seeds were immersed in a 5% NaOCl solution for 8 min, rinsed three times with sterile distilled water, immersed once in a 70% ethanol solution and finally rinsed six times with sterile distilled water.

Germination test and vigour index

To evaluate the germination percentage and the vigour index, a completely randomized experimental design was used, with a factorial arrangement of seven treatments for two rice varieties (LC and LE) and with five replicates of ten seeds each. The inoculated treatments consisted of five strains of B. vietnamiensis (T1 to T5), a treatment inoculated with the control strain Priestia megaterium SMBH14-02 (Ríos-Ruiz et al. 2023) and a blank treatment. Fifty disinfected seeds were placed in a sterile tube (50 mL) and inoculated by immersion with 25 mL of standardised inoculum per strain and 25 mL of 10 mM MgSO₄ solution for the blank treatment. The treatments were incubated for two hours at room temperature under the same conditions. The inoculated seeds were sown on absorbent paper at the bottom of Magenta® boxes (200 mL) previously sterilised at 121°C for 30 minutes and moistened with 4 mL of sterile distilled water. The treatments were incubated in the dark at 25 ± 1°C for 10 days and the following parameters were evaluated: plumule length (cm), root length (cm), germination percentage (%) and vigour index using the formula.

(Length of aerial part + Length of radicle) * germination (%)

Growth promotion evaluation

To evaluate growth promotion, a completely randomized experimental design was used, with a factorial arrangement of seven treatments for two rice varieties (LC and LE) and with ten replicates each. The inoculated treatments consisted of five strains of B. vietnamiensis (T1 to T5), a treatment inoculated with the control strain P. megaterium SMBH14-02 (Ríos-Ruiz et al. 2023) and a blank treatment. The disinfected seeds were placed in Petri dishes (15 x 150 mm) containing 1% (w/v) sterile water agar and germinated, at 25 ± 1°C for 3 days in the dark until 1 cm radicles were obtained. The germinated seeds were sown in previously disinfected polypropylene pots (9 x 9 cm, 250 mL) with 85 g of fertilised and sterilized greenhouse substrate (Klasmann TS 1®, Lithuania) irrigated with 55 mL of sterile water distilled to reach a capacity of 60% field. Inoculation was carried out at a rate of 1 mL of standardised inoculum per seed and 1 mL of 10 mM MgSO₄ solution for the blank treatment. The treatments were watered at least twice a week with 50% Hoagland nutrient solution supplemented with 1% KNO₃ to 60% field capacity through an irrigation tube installed in each pot. The seedlings were maintained under controlled conditions (80% humidity at 28°C, 16 h light: 8 h dark) and after 21 days the following parameters were evaluated: Height (cm), root length (cm), Shoot Dry Weight (SDW) (mg), Root Dry Weight (RDW) (mg), leaf and root nitrogen content (%).

Statistical analyses

Normality and homoscedasticity of the data were tested. Significant differences between strains and their effects on the growth promotion mechanisms in two rice varieties were studied using an analysis of variance (ANOVA) using the R command package in R studio desktop version 1.3.1093. The Tukey test was used with a significance level of 95% (P ≤ 0.05) when parametric data were available, otherwise the Kruskal-Walli test was used with a significance level of 95% (P ≤ 0.05). Correlations between variables were estimated using Pearson's correlation. The difference in germination rate between the two cultivars was tested with a two-tailed t-test, and the difference in germination rate for each cultivar, whether inoculated with inocula or with MgSO₄ solution, was tested with the empirical rule, where 99.7% of the values are within three standard deviations of the mean.

Isolation and characterization of strains

We collected 132 root samples from cultivated rice varieties in the four rice valleys of Bajo Mayo (30), Alto Mayo (57), Huallaga Central (15) and Alto Huallaga (30) (Table 01). We isolated 211 Gram-negative bacterial isolates from the surface-sterilised roots; however, only 78 strains (36% of the total) showed nitrogen-fixing ability in our tests. As we were focusing on B. vietnamiensis, a nitrogen-fixing species, we only kept these isolated for further analysis. Most (44, 57.90%) of these Gram-negative and diazotrophic isolates came from plants grown in the Huallaga Central Valley, while only 6 came from plants grown in the Alto Mayo Valley.

Molecular discrimination of Burkholderia

Only 4 isolates (5.13%) from our collection of Gram-negative and nitrogen-fixing bacteria, together with two previously isolated strains of B. vietnamiensis la3c3 and la1a4 (Ríos-Ruiz et al. 2020), amplified a fragment of 704 bp corresponding to the recA gene using primers specific to Burkholderia. Sequencing of the recA gene confirmed the taxonomic identity of the isolates in the species B. vietnamiensis. These 4 isolates all originated from the Alto Huallaga valley, and more specifically from the Nueva Esperanza sector of the Uchiza district. The la3c3 and la1a4 strains were originally sampled from the Central Huallaga valley (Ríos-Ruiz et al. 2020)

BOX-PCR genomic profiling

To study the intraspecific diversity of the six B. vietnamiensis strains, genomic DNA profiles were performed using BOX-PCR analysis. The fragments obtained ranged from 500 to 10,000 bp. Using a similarity level of 90% as a cut-off point, 5 different groups were obtained (Figure 01). These groups were named from G1 (BOX group 1) to G5 (BOX group 5). Each group, except G1, contains only one isolate. The strains la3c3 and la1a4 fall into two different BOX-PCR groups. Based on the BOX-PCR dendrogram, one strain per group was selected for amplification, sequencing and phylogenetic study of the recA gene.

Table 1. Geographical location of rices collections and distribution of diazotrophic Gram-negative strains isolated from surface sterilized roots from rice varieties from rice valleys in the San Martin Region
Valley	Province	District/Sector	Rice Cultivar	Geographical coordinates	Amount of samples	Number and code of diazotrophic Gram-negative strains
Bajo Mayo	Lamas	Cacatachi/Rosanaico	La Conquista	6° 28' 11" S, 76° 26' 26" W; 299 m.a.s.l.	30	(3)-B1; B3; B13
Bajo Mayo	San Martín	Juan Guerra/Estación El Porvenir	La Conquista, Valor, La Esperanza, Fedearroz 60	6° 35' 49" S, 76° 19' 32" W; 309 m.a.s.l.	30	(4)-B34; B37; B48; B49
Alto Mayo	Rioja	Awajun/San Francisco de Amayo	Valor, La Esperanza, La Victoria	5° 46' 43" S, 77° 18' 29" W; 916 m.a.s.l.	36	(3)-B51; B52; B74
Alto Mayo	Moyobamba	Moyobamba/La conquista	Valor	5° 52' 41" S, 77° 10' 10" W; 913 m.a.s.l.	21	(3)-B61; B89; B90
Huallaga Central	Bellavista	Bellavista/El Porvenir	Ferón	7° 03' 11" S, 76° 33' 54" W; 235 m.a.s.l.	15	(9)-B91; B92; B94; B102; B103; B104; B105; B106; B106a
	Bellavista	San Rafael/Carhuapoma	La Esperanza	7° 00' 21" S, 76° 30' 08" W; 228 m.a.s.l.		(9)-B126; B129; B130; B131; B135; B138; B139; B140; B142
	Picota	Picota/Santa Rocillo-(E)	Valor	6° 55' 32" S, 76° 22' 23" W; 218 m.a.s.l.		(21)-B107; B108; B109; B110; B111; B113; B114; B115; B118; B119; B121; B122; B124; B154; B155a; B158; B159; B160; B161; B162; B163
	Picota	San Hilarión/San Hilarión	Ferón	6° 59' 55" S, 76° 27' 05" W; 225 m.a.s.l.		(5)-B145; B146; B147; B151; B152
Alto Huallaga	Tocache	Uchiza/Nueva Esperanza	El Valor	8° 15' 39" S, 76° 33' 59" W, 495 m.a.s.l.	15	(7)-B167; B168a; B168b; B169a; B169b; B171a; B172a
		Uchiza/San Juan de Porongo	El Valor, Ferón	8° 23' 27" S, 76° 17' 17" W, 625 m.a.s.l.	15	(10)-B172b; B175; B176; B177; B178; B188; B189; B190; B191; B192
		Tocache/San Miguel del Porvenir	Ferón, Valor	8° 16' 26" S, 76° 32' 25" W; 590 m.a.s.l.	15	(4)-B193; B195; B196; B201

Phylogeny of the recA gene:

Sequencing resulted in partial recA sequences of 625-667 bp. Based on blast and phylogenetic analyses, they confirmed that the 5 isolates representing the BOX-PCR groups belonged to the species B. vietnamiensis, with percentages of similarity of the recA gene between 98.36 and 99.51% with the type strain of B. vietnamiensis LMG 10929^T. The maximum likelihood (ML) phylogenetic tree including sequences of the type strain of the genus Burkholderia sensu stricto is shown in Figure 03. All B. vietnamiensis sequences clustered in the same highly supported (99%BP) clade. The recA gene sequences have been deposited in GenBank under the following accession numbers OQ706314-OQ706318.

Figure 01. Fingerprinting dendrogram of similarity based on the BOX-PCR profiles of the isolates of this study, using the UPGMA algorithm and the Jaccard coefficient with 2% tolerance using the online dendrogram construction utility DendroUPGMA (http:// genomes.urv.es/UPGMA/) (Garcia-Vallvé et al. 1999).

Growth promotion mechanisms detected in Burkholderia vietnamiensis

All the strains studied were able to express the direct mechanisms of growth promotion, auxin and siderophore production, phosphate solubilisation and diazotrophic capacity in free life (Table 02), as well as the capacity for antibiosis and antagonism against phytopathogenic rice fungi as indirect growth promotion mechanisms (Table 03). All strains were able to produce auxin in the basal TSB medium (0 µg mL^-1) and in the TSB media supplemented with tryptophan (100 ppm to 600 ppm). There was also a strong positive correlation between auxin production and tryptophan supplementation in TSB broth (r²= 0.99). Under all conditions, B. vietnamiensis strain B169b produced a significantly higher amount of auxin compared to the other strains.

Figure 02. Maximum-Likelihood phylogenetic tree based on recA gene sequences (650 positions) showing the relationships among phytobeneficial Burkholderia isolated in this study and closely related species of the genus Burkholderia sensu stricto. The significance of each branch is indicated by a bootstrap value (as percentage) calculated for 1000 subsets (only values greater than 50 % are indicated). Bar, 5 substitution per 100 nucleotide positions. The recA sequence of Bradyrhizobium vignae 7-2^T was used as outgroup.

Table 02. Direct growth promotion mechanisms evaluated in five endophytic Burkholderia vietnamiensis strains isolated from rice roots

Strains

Auxins production (µg mL^-1)

Siderophores production

Phosphate solubilization

Free-living nitrogen fixation

0 ppm

100 ppm

200 ppm

400 ppm

600 ppm

EPS (%)

qualitative

quantitative

Solubilization Index

Solubilization Efficiency

Solubilized P (mg PO₄ mL^-1)

la1a4

8.10 (±0.15) B

8.86 (±0.21) B

8.72 (±0.16) B

11.79 (±0.35) A

10.97 (±0.20) B

93.38 (±1.85) B

2.63 (±0.04) A

162.73 (±3.54) A

3.89

11.62 (±0.22) A

la3c3

0.25 (±0.25) D

0.70 (±0.03) D

0.15 (±0.08) D

1.27 (±0.10) C

2.76 (±0.05) D

52.32 (±1.18) D

2.64 (±0.03) A

163.88 (±3.46) A

3.93

12.18 (±0.77) A

B169b

9.08 (±0.07) A

9.70 (±0.17) A

13.98 (±0.31) A

11.76 (±0.07) A

13.90 (±0.22) A

139.52 (±3.30) A

2.58 (±0.04) A

157.80 (±4.33) A

3.81

15.99 (±0.58) A

B168a

0.10 (±0.05) D

1.89 (±0.09) C

1.41 (±0.05) C

3.79 (±0.10) B

4.60 (±0.44) C

72.3 (±11.91) C

2.59 (±0.04) A

159.31 (±3.66) A

3.84

15.26 (±0.28) A

B171a

2.82 (±0.05) C

1.41 (±0.05) C

0.62 (±0.03) D

3.30 (±0.07) B

3.33 (±0.38) B

47.72 (±1.87) DE

2.53 (±0.01) A

153.25 (±0.69) A

3.69

17.63 (±0.48) A

C.V (%)

5.94

5.01

5.68

4.76

7.09

10.03

2.65

4.24

19.30

Values followed by different letters indicate significant differences. Tukey test, p = 0.05.

All strains were efficient in the production of siderophores, forming orange halos around the bacterial colonies of smaller or larger diameter depending on the strain (Figure 03), highlighting B. vietnamiensis strain B169b as significantly the most efficient strain (139.52%). Regarding phosphate solubilisation, all strains were able to solubilise tricalcium phosphate from NBRIP agar, as evidenced by the appearance of a transparent halo around the bacterial colonies. This activity is reflected in the solubilisation indices and the solubilisation efficiency obtained, although no statistically significant differences were observed between them. The strains also showed no statistically significant differences in the concentration of phosphate released into the culture medium. A negative correlation was observed between the final pH of the culture medium and the difference in soluble phosphate in the culture medium with the white treatment (r²= -0.92). Finally, all strains were able to form a subfilm in the semi-solid JMV medium and to grow on the JMV agar, both nitrogen-free, indirectly demonstrating their ability to fix atmospheric nitrogen.

Figure 03. Production of siderophores by Burkholderia vietnamiensis B168a on CAS agar

Antagonism and antibiosis capacity of B. vietnamiensis strains.

In terms of antibiosis capacity, all strains inhibited the growth of the five phytopathogenic fungi, but strain la3c3 showed the greatest antibiosis activity, inhibiting 100% of the growth of R. solani, R. oryzae, N. oryzae and N. sigmoidea. All five strains were able to inhibit 100% of the growth of N. oryzae, the most susceptible phytopathogenic fungus, while the least susceptible was Rhizoctonia oryzae-sativae, with radial growth inhibition ranging from 76.47% (la1a4) to 86.04% (B171a) (Table 03 and Figure 04).

Table 03. Antibiosis and antagonism of five strains of Burkholderia vietnamiensis on five species of rice phytopathogenic fungi.
Strains	Rhizoctonia solani (RS1)	Rhizoctonia oryzae (RO1)	Rhizoctonia oryzae-sativae (ROS1)	Nigrospora oryzae (NO1)	Nakataea sigmoidea (NS1)
ANTIBIOTIC (%)
B168a	78.59 (±0.12) C	100.00 (±0.00) A	82.23 (±1.94) AB	100.00 (±0.00) A	100.00 (±0.00) A
B171a	100.00 (±0.00) A	100.00 (±0.00) A	86.04 (±0.35) A	100.00 (±0.00) A	90.86 (±0.24) B
B169b	100.00 (±0.00) A	77.93 (±2.04) B	79.72 (±1.53) BC	100.00 (±0.00) A	87.80 (±0.50) C
la3c3	100.00 (±0.00) A	100.00 (±0.00) A	84.82 (±0.69) A	100.00 (±0.00) A	100.00 (±0.00) A
la1a4	80.39 (±0.61) B	75.80 (±3.49) B	76.47 (±0.80) C	100.00 (±0.00) A	100.00 (±0.00) A
C.V(%)	0.39	3.45	2.56	0	0.45
ANTAGONISM (%)
B168a	56.22 (±2.60) A	59.03 (±2.36) A	73.85 (±5.87) A	41.98 (±1.92) A	73.63 (±1.46) A
B171a	61.20 (±2.64) A	57.54 (±1.47) A	54.39 (±5.27) B	44.96 (±1.20) A	72.79 (±2.00) A
B169b	58.71 (±1.00) A	55.06 (±2.27) A	65.87 (±2.89) AB	47.50 (±3.07) A	77.56 (±1.45) A
la3c3	64.68 (±2.13) A	61.51 (±0.99) A	64.87 (±2.00) AB	44.72 (±1.61) A	59.94 (±0.92) B
la1a4	56.22 (±1.42) A	57.54 (±0.99) A	61.87 (±2.40) AB	49.21 (±3.17) A	63.75 (±2.73) B
C.V(%)	8.52	7.26	15.3	12.5	6.4
Values followed by different letters indicate significant differences. Tukey test, p = 0.05.

All strains inhibited the growth of the five phytopathogenic fungi, with strain B169b showing the highest range of antagonistic capacity against the five phytopathogenic fungi. The most susceptible fungus was N. sigmoidea with a range of 59.94% (la3c3) to 77.56% (B169b) antagonism and the least susceptible was N. oryzae with a range of 41.98% (B168a) to 49.21% (la1a4) growth inhibition. (Table 03 and Figure 04).

All B. vietnamiensis strains increased length, germination and vigour index in both rice cultivars under gnotobiotic conditions, although there was no clear significant difference among isolates. While there were significant differences between the strains for root length in the LE cultivar, we found none for LC. The same result was observed for the length of aerial parts. The highest growth promotion effects compared to the uninoculated reached 50% and 35.6% for root length (LE/B169b and LC/lac3c, respectively) and 32.2% and 33.4% for shoot length (LE/B168a and LC/B168a, respectively).

All inoculation conditions resulted in a significant increase in the total length or vigour index of the plant, irrespective of the cultivar, but with no significant difference between the cultivars. (Table 04). While the germination rate differed between the two varieties (p<0.04), the effects of inoculation on this rate were highly significant compared to the uninoculated conditions (p<0.003), with a higher germination rate for inoculated seeds.

Figure 04. Evaluation of antagonism (A) and antibiosis (B) in five strains of Burkholderia vietnamiensis against five strains of phytopathogenic rice fungi, RS1 (Rhizoctonia solani), ROS1 (Rhizoctonia oryzae-sativae), RO1 (Rhizoctonia oryzae), NS1 (Nakataea sigmoidea) and NO1 (Nigrospora oryzae)

Table 04. Evaluation of the vigor index of two rice varieties inoculated with five strains of Burkholderia vietnamiensis grown under gnotobiotic conditions.

Strains

Germination

Vigor Index

LE¹

LC¹

LE¹

LC¹

LE¹

LC¹

LE¹

LC¹

la1a4

58.63 (±1.42) DE

60.41 (±0.92) A

38.16 (±1.42) C

45.36 (±1.09) A

94.40 (±2.57) B

105.77 (±1.71) A

8582.06 (±199.35) B^b

10365.66 (±194.20) A^a

la3c3

68.13 (±1.02) C

60.52 (±2.19) A

44.97 (±0.94) AB

44.73 (±2.25) A

112.71 (±2.02) A

105.25 (±3.54) A

100

11310.27 (±193.25) A^a

9892.84 (±414.10) A^b

B169b

75.27 (±1.97) A

58.74 (±1.88) A

44.22 (±1.23) B

44.20 (±1.46) A

115.18 (±2.79) A

102.94 (±3.18) A

11470.50 (±305.63) A^a

9675.80 (±308.90) A^b

B168a

65.22 (±1.62) CD

58.79 (±1.52) A

49.75 (±1.33) A

45.62 (±2.20) A

111.12 (±2.01) A

104.41 (±4.09) A

100

11496.67 (±294.38) A^a

10023.49 (±355.84) A^b

B171a

72.8 (±1.80) B

57.89 (±1.43) A

43.47 (±0.96) B

42.65 (±1.40) A

112.08 (±2.26) A

100.54 (±2.49) A

100

11626.93 (±272.51) A^a

9450.57 (±264.82) A^b

Priestia megaterium SMBH14-02

67.81 (±0.82) CD

60.68 (±1.31) A

42.28 (±0.91) BC

40.36 (±1.10) AB

108.21 (±1.60) A

101.04 (±2.16) A

100

11008.73 (±172.29) A^a

10103.97 (±236.04) A^b

Non-inoculated control

49.97 (±1.11) E

44.63 (±1.19) B

37.61 (±0.70) DE

34.19 (±1.22) B

88.32 (±1.59) B

78.82 (±2.01) B

8235.35 (±176.77) B^a

6620.43 (±201.69) B^b

CV (%)

12.13

14.73

14.83

20.63

11.17

15.76

12.31

16.94

Values followed by different uppercase letters indicate significant differences between strains, and values followed by different lowercase superscript letters indicate significant differences between cultivars.
RL: Root length; SL: Aerial part length; TL: Total length of the plant; LE: INIA 507 “La Esperanza” rice cultivar; LC: INIA 510 "La Conquista" rice cultivar
¹Kruskal Wallis test, p = 0.05.

Table 05. Evaluation of growth promotion of two rice varieties inoculated with five strains of Burkholderia vietnamiensis grown for 21 days under gnotobiotic conditions.
Parameter		Burkholderia vietnamiensis					Priestia megaterium SMBH14-02	Non-inoculated control	CV (%)
Parameter		la1a4	la3c3	B169b	B168a	B171a	Priestia megaterium SMBH14-02	Non-inoculated control	CV (%)
RL-LE²	mm.	103.89 (±3.22) B	103.90 (±5.02) B	115.78 (±5.15) B	142.04 (±1.90) A	116.04 (±2.98) B	106.10 (±7.64) B	97.51 (±2.98) B	13.28
RL-LC²		73.94 (±4.94) AB	77.36 (±4.83) AB	79.68 (±3.04) A	82.93 (±3.00) A	88.55 (±2.14) A	81.77 (±1.64) A	63.86 (±3.10) B	13.90
SL-LE²		167.64 (±2.59) B	192.27 (±3.76) A	175.77 (±4.91) AB	121.85 (±2.72) C	169.79 (±4.50) B	172.00 (±7.61) B	159.81 (±2.68) B	8.47
SL-LC¹		196.73 (±5.44) AB	201.12 (±7.77) AB	195.07 (±3.84) AB	211.53 (±4.89) A	199.70 (±3.27) AB	205.49 (±5.23) AB	180.50 (±9.57) B	9.67
TL-LE¹		268.29 (±3.42) ABC^a	285.26 (±5.30) Ab^a	288.06 (±8.96) A^a	258.98 (±4.28) BC^b	281.65 (±5.50) A^a	263.82 (±8.74) ABC^b	251.13 (±4.81) C^a	7.22
TL-LC²		275.69 (±8.02) A^a	281.12 (±10.43) A^a	265.25 (±5.17) A^b	289.96 (±8.96) A^a	284.90 (±4.73) A^a	290.04 (±4.63) A^a	232.43 (±13.65) B^a	9.41
RDW-LE²	mg	11.27 (±0.18) A	11.35 (±0.29) A	11.28 (±0.40) AB	11.25 (±6.05) A	11.57 (±0.20) A	9.81 (±0.27) B	9.31 (±0.18) B	7.84
RDW-LC¹		10.58 (±0.32) A	9.98 (±0.30) ABC	10.01 (±0.27) ABC	10.56 (±0.19) AB	11.21 (±0.26) A	9.69 (±0.47) BC	8.77 (±0.20) C	9.36
SDW-LE²		16.85 (±0.61) AB	17.55 (±0.34) AB	17.69 (±0.60) AB	16.47 (±0.81) B	19.20 (±0.64) A	13.37 (±0.48) C	13.39 (±0.35) C	11.01
SDW-LC¹		14.79 (±0.54) AB	13.95 (±0.91) ABC	14.36 (±0.41) ABC	13.78 (±0.34) ABC	16.47 (±0.75) A	12.60 (±0.49) BC	11.68 (±0.86) C	14.76
TDW-LE²		27.85 (±0.68) A^a	28.61 (±0.50) A^a	28.78 (±1.04) A^a	27.47 (±0.90) A^a	30.49 (±2.54) A^a	22.47 (±0.75) B^a	23.05 (±0.49) B^a	8.91
TDW-LC¹		24.62 (±0.43) AB^b	22.16 (±0.61) BC^b	23.44 (±0.43) AB^a	24.72 (±0.72) AB^b	26.20 (±1.01) A^b	22.73 (±0.62) BC^a	20.44 (±0.59) C^b	8.84
% NPA-LE	%	4.91	5.67	4.21	4.64	4.76	4.56	4.29	---
%NPA-LC		4.63	4.87	5.03	4.06	6.24	4.39	4.18	---
% NPR-LE		1.80	1.29	1.39	1.31	1.76	1.34	1.05	---
% NPR-LC		1.54	1.31	1.44	1.78	0.93	1.25	1.54	---
%NT-LE		6.70	6.96	5.60	5.95	6.52	5.90	5.34	---
%NT-LC		6.17	6.19	6.46	5.83	7.17	5.64	5.71	---
Values followed by different capital letters indicate significant differences and values followed by different lowercase superscript letters indicate significant differences between cultivars. RL: Root length; SL: Shoot part length; TL: Total length of the plant; RDW: Root dry weight; SDW: Shoot dry weight; TDW: Total dry weight; %NPA: Percentage of nitrogen in the aerial part; %NPR: Percentage of nitrogen in root part; %NT: Percentage of total nitrogen. LE: INIA 507 “La Esperanza” rice cultivar; LC: INIA 510 Cultivate “La Conquista” ¹Tukey test, p = 0.05; ²Kruskal Wallis test, p = 0.05.

Finally, in the 21-day test, there was no significant difference between the strains of B. vietnamiensis with P. megaterium SMBH14-02 for length, dry weight and nitrogen accumulation. Similarly, non-significant differences were observed in the accumulation of TDW inoculated with B. vietnamiensis strains in the LC cultivar. Differences were observed in the accumulation of total nitrogen, which was higher in the LE cultivar inoculated with B. vietnamiensis la3c3 (6.96%) and with B. vietnamiensis B171a (7.17%) in the LC cultivar (Table 05).

Endophytic bacteria are an important part of the phytomicrobiome, not only because they represent a selected fraction of the soil microbiota, but also because of the growth-promoting mechanisms they express in close association with plants, especially in the Poaceae family. There are several reports of bacteria being selected for their endophytic character and also for their diazotrophic capacity in rice cultivation (Ji et al. 2014; de Oliveira et al. 2015; Shabanamol et al. 2018; Banik et al. 2019; Madhaiyan et al. 2021; Kuang et al. 2024).

De Oliveira et al. (2015), in a bioprospecting study of diazotrophic endophytic bacteria in the rice cultivar BRS Tropical, reported that 18% of the isolates obtained were similar to those in the genus Burkholderia, and diverse species of Burkholderia have been repeatedly isolated from healthy rice roots (see Wallner et al. 2023 and refins). In our study, the proportion of Burkholderia was much lower (5.26%) and was isolated only from the rice variety "El valor", grown in the Alto Huallaga valley. It cannot be excluded that the primers used for the detection of Burkholderia missed some isolates and did not amplify them. However, when searching the complete or draft genomes of B. vietnamiensis available in NCBI, we did not find any example of genomes (among the 208 genomes available) where the primers would not have amplified the recA fragment. This explanation therefore seems unlikely. The low frequency of Burkholderia isolates in our study could also be due to the selection of isolates for which we attempted to amplify a recA fragment. We focused on isolates that could fix nitrogen because all previous studies and available genomes have shown that B. vietnamiensis strains are free-living nitrogen fixers. This choice may have excluded some other Burkholderia species that are not free-living nitrogen fixers, thus reducing the number of Burkholderia isolates, although several have been shown to act as plant-associated nitrogen fixers (Estrada de los Santos et al. 2001). Finally, the low frequency of B. vietnamiensis isolates in our sampling may simply reflect their low frequency in soil, although this species has been shown to be frequently recovered from rice roots in different geographical locations, and although Burkholderia s. l. is a highly conserved microbial group of the endophytic microbiota in rice, regardless of soil type and cultivar (Samuel et al. 2023).

Quite surprisingly, the box PCR profiles showed diversity among the different isolates and among the four recovered in this study. One might have expected, or feared, that a very small number of isolates recovered from a single site would end up as a single identical clone, which is not the case, since we detected at least three different profiles (together with the two other strains isolated in a previous study, but from a different site in Peru). The recA gene, as a reliable phylogenetic marker for assigning taxonomic identity within the genus Burkholderia (Payne et al. 2005; Spilker et al. 2009; Bach et al. 2017; Depoorter et al. 2020; Velez et al. 2023; Valdez-Nuñez et al. 2024), confirmed that the 4 strains we isolated belonged to the species B. vietnamiensis, but showed less diversity than the Box-PCR approach (as expected). In fact, the 3 representative isolates of the 3 groups showed the same recA sequences, while the other 2 showed 2 nucleotide differences with our isolates. The Box-PCR diversity, with 3 different profiles among the 4 isolates obtained, however suggests that we may indeed have missed some B. vietnamiensis diversity in our sampling and that we should possibly intensify our efforts to recover more isolates.

Based on these results and the genetic differences detected between the isolates, we expected some discrepancies in their growth promoting abilities.

While several studies have previously demonstrated beneficial effects of B. vietnamiensis on rice growth and possibly protection against pathogens, we investigated whether and how these strains might actually play a role as growth promoters. Among bacterial auxins, indoleacetic acid (IAA) is one of the most studied, influencing root architecture, nutrient uptake and tolerance to abiotic factors. Bacterial IAA concentrations are influenced by intrinsic factors such as strain type, presence of complete biosynthetic pathway genes, or extrinsic factors such as precursor concentration, pH, carbon source, among others (Etesami and Glick, 2024). It has been reported that IAA production is common in plant-associated strains of B. vietnamiensis (Govindarajan et al. 2006; Xin et al. 2009; Estrada et al. 2013; de Oliveira et al. 2015; Ríos-Ruiz et al., 2020; Shinjo et al. 2020; Nguyen et al. 2022). Our results showed that there is a wide range of variation in auxin production among our isolates. At 600 ppm L-tryptophan, strain B169b produced 5 times more auxin than strain la3c3 (13.90 vs 2.76 µg mL-1). Most of the auxin found in the rhizosphere is believed to come from the biosynthesis by microorganisms (Kamilova et al. 2006). Variations in auxin production between strains present in the rhizosphere can therefore have a major impact on plant development, and, from the point of view of artificial inoculation, our results proved that there is a basis for bacterial selection of the most productive strains.

B. vietnamiensis is also characterised by the production of siderophores (Gillis et al. 1995; Meyer et al. 1995; Conway and Greenberg, 2002; Nguyen et al. 2022). Among the functions performed by siderophores, biocontrol stands out due to their high affinity for Fe³⁺, which limits their access to phytopathogens and promotes plant growth under iron-limiting conditions (Afzal et al. 2019). As for auxin production, we also found large differences in the production of siderophores among the isolates, ranging from 47.72% (B171a) to 139.52% (B169b), leaving as for auxin the possibility of strain selection for their ability to produce highest amount of siderophore that can play a major role in agriculture (Timofeeva et al. 2022).

Regarding the other two activities, we did not detect significant differences in phosphate solubilisation, although all strains showed qualitative and quantitative evidence of P solubilisation. The ability of B. vietnamiensis to solubilise insoluble phosphates has been widely reported previously (Park et al. 2010; Estrada et al. 2013; Liu et al. 2022; Nguyen et al. 2022; Kuang et al. 2024). Estrada et al (2013) reported that B. vietnamiensis isolates from rice achieved solubilisation index values similar to those reported in this study (2.34–2.95). However, while Bashal et al. (2013) suggested a low correlation between halo formation and the ability to solubilise phosphates released in liquid medium, we observed in our study that all strains that formed a solubilisation halo (qualitatively) also solubilised phosphate in liquid medium (quantitatively).

Finally, all isolates showed the ability to fix nitrogen as free-living bacteria, but we could not measure this activity precisely and therefore could not estimate significant differences between them.

Given these results, we were expected not only an effect of inoculation on plant growth, but also possibly differences between strains for plant phenotypic traits in response at least to auxin production levels differences. The beneficial interaction between B. vietnamiensis and rice has been described previously (Gillis et al. 1995; Tran Van et al. 2000; Govindarajan et al. 2008; Ríos-Ruiz et al. 2020; Shinjo et al. 2020; Wallner et al. 2022), but with sometimes conflicting results. King et al. (2019) reported that Nipponbare rice (japonica) plants inoculated with B. vietnamiensis TVV75 (LMG10929^T) had no significant effect on biomass production compared to the non-inoculated control at 7 and 14 days after inoculation under gnotobiotic conditions. In our study, we found significant differences between the strains and the non-inoculated control evaluated at 10 and 21 days after inoculation. The LE genotype showed significant differences for root and shoot length at 10 days between isolates, but with a weak correlation between the two sets of measurements (r² = 0.38), while the LC genotype showed almost no significant differences, but with a higher r² value (0.75). The low correlation value for LE resulted in a lack of significant differences between the strains when considering the total length (shoot + aerial), except for isolate la1a4, which is not different from the uninoculated condition, although it was one of the higher auxins and siderophore producers in our tests. Many previous studies have demonstrated the effect of bacterial auxin on plant growth, in terms of height, root length, dry weight shoot and dry weight root, plant nutrient content, chlorophyll content, leaf area and yield (Pal et al. al. 2022), with an effect being concentration dependent (Gholami et al. 2012). Quite disappointingly, we did not detect any relation between the level of auxin production of each strain and their impact on either root or shoot length. There was either no significant difference among strain (for variety LC) or no correlation for variety LE. The other putative growth-promoting activities we measured in our isolates do not appear to have an effect on rice growth either. This lack of correlation may be due to the absence of a dose effect, which would be rather surprising given the different production levels. More likely the growing conditions may not have allowed auxin-related effects to be fully expressed. In our study, we chose to place our plants in non-limiting growing conditions, i. e. with a rich growth medium, where Fe or phosphate is not limiting and therefore the capacity of the bacteria to recover these elements does not play a major role. The increase in plant height and biomass is associated with an increase in the expression of in planta genes involved in iron storage, siderophore biosynthesis and nutrient transport (Zhao et al. 2020).

One exception is related to nitrogen. It has been reported that inoculation of rice with B. vietnamiensis increases the efficient use of mineral nitrogen (Ríos-Ruiz et al. 2020; Shinjo et al. 2020). Consequently, the five treatments with B. vietnamiensis and the control strain P. megaterium SMBH14-02, inoculated in the two rice varieties and supplemented with mineral nitrogen (KNO3 1%), increased the nitrogen content in the aerial and root parts from 11 to 19% on average. Although nitrogen fixation is an intrinsic capacity of B. vietnamiensis (Gillis et al. 1995; Shinjo et al. 2018; Bach et al. 2022), recent studies have concluded that it would not be the main growth promoting mechanism in rice. Shinjo et al. (2020) inoculated B. vietnamiensis RS1 into seedlings of rice cultivar Nipponbare (japonica) and concluded that the promotion of root growth occurred through hormonal regulation and increased nitrogen uptake through the overexpression of genes related to the uptake, transport and assimilation of mineral nitrogen. This ability is intrinsic to endophytic Burkholderia, as it improves the acquisition and transport of nutrients in tissues at the transcriptional level (Zhao et al. 2020).

Finally, with regard to plant genotype, it is well known that plant genotype influences the response to inoculation of specific bacterial strains (Sharma et al. 2014; Belimov et al. 2015; Ahmed et al. 2021). Wallner et al. (2022) inoculated B. vietnamiensis LMG10929 into two rice cultivars, Nipponbare (japonica) and IR64 (indica); although they observed similar colonisation patterns between the two genotypes, they found that there was a strong influence of rice genotype, particularly in relation to the indica group more than the japonica group. The IR64 (indica) cultivar inoculated with B. vietnamiensis increased the expression of the nitrate transporter NRT1.1B, affecting its capacity for assimilation and uptake of nitrate from the nutrient solution. In our study, the results suggested, once again as in other studies, that selecting bacterial strains for inoculation to improve plant growth can only be done by taking into account the diversity of cultivated varieties, but also by integrating into breeding programmes the ability of varieties to interact with micro-organisms.

Inoculation of B. vietnamiensis and protection against pathogens

Recently, Wang et al. (2023), reported that the inoculation of B. vietnamiensis strain B23 in Citrus plants increased the expression of genes related to the use and uptake of nutrients from the rhizosphere and increased antagonistic activities against competing bacteria and fungi, as well as resistance to competitor-derived metabolites. Meng et al. (2023) reported the isolation of B. vietnamiensis C12 as an antifungal endophyte of the medicinal plant Ficus tikoua and demonstrated the production of siderophores with bactericidal activity, ornibactin C-4 and C-8. Similarly, Wang et al. (2022) demonstrated the ability of B. vietnamiensis YQ9 to produce hydroxamate type siderophores.

All B. vietnamiensis strains in our study showed antagonistic and antibacterial activity against three members of the Rhizoctonia complex, including Rhizoctonia solani, the causal agent of rice downy mildew (Cuong et al. 2011; Zhang et al. 2012; Sivaji et al. 2016; Ríos-Ruiz et al. 2020; Meng et al. 2023). The antifungal activity of B. vietnamiensis against Nakataea sigmoidea and Nigrospora oryzae, the etiological agents of stem rot (Garrido and Vilela, 2019) and panicle branch rot (Liu et al. 2021), respectively, is reported for the first time. Meng et al. (2023) reported the antifungal activity of B. vietnamiensis C12 against several phytopathogenic fungi, reaching 94.78% inhibition against R. solani, a higher range than that reported in our study (56.22 to 64.68%). The authors conclude that this antifungal activity against R. solani is due to the production of several secondary metabolites, highlighting the siderophores ornibactin C4 and C8, the antifungal peptides burkholdin 1097, 1213, 1215 and 1119, and the monoterpenoid phenol carvacrol. Here we show that all B. vietnamiensis strains have a strong effect on reducing fungal growth on artificial media in Petri dishes. Such ability of Burkholderia isolates to directly suppress the growth of phytopathogenic fungi has been described previously and is important (Pal et al. 2022). Antifungal metabolites produced by Burkholderia that are effective against the Rhizoctonia complex include pyrrolnitrin, phenazines, 1-phenazine carboxylic acid, volatile indolic compounds (Cartwright et al. 1995), siderophores such as ornibactin (Rojas-Rojas et al. 2018), some quinolone antibiotics such as hydroxymethyl-alkylquinoline (Saalim et al. 2020), and occidiofungins A-D (Lu et al. 2009). Searches for these compounds in B. vietnamiensis genomes have detected some of the genes involved in the production of these compounds (such as ornibactin), while others were not detected (such as pyrrolnitrin or occidiofungins A-D).

There is still a long way to go to isolate and characterise the antifungal compounds produced by B. vietnamiensis, but we can expect new metabolites to be found. It is also clear that there is a huge gap from Petri dish tests to the use of either live bacteria or metabolites extract in the field to combat rice diseases, but even if Burkholderia s.s. is still a problematic genus for use as a bioinoculant, and B. vietnamiensis species in particular, it remains an extremely interesting and promising genus in the search for solutions and alternative compounds to fight against rice diseases.

Among the endophytic strains of Burkholderia vietnamiensis there is genetic diversity, as well as a wide metabolic diversity in terms of direct and indirect growth promotion mechanisms in both rice varieties, at the level of aerial biomass and foliar nitrogen accumulation. Although, no significant differences are observed in growth promotion between the inoculated treatments, nor is superiority observed against harmless and beneficial strains, such as P. megaterium. On the other hand, our tests were only conducted at the greenhouse level, so there must be other variables at play at the field level. An important factor is that B. vietnamiensis adapts very well to the endophytic style of rice, which is undoubtedly a great advantage over other strains, so it is necessary to evaluate a balance between benefits and risks, and this opens the door to new studies on the possibility or not of using this strain, which is in the cepacia complex. For future studies, the biosafety involved in the use of these strains should be evaluated, considering the opportunistic pathogenic potential of members of the Burkholderia cepacia complex. To do this, virulence genes associated with pathogenicity must be searched in the genomes of the respective strains.

Author Contribution

Conceptualization: R.A.V.N.; J.C.Ch.G., and G.B.; Methodology: R.A.V.N and G.B.; Validation: P.P.M.C. and N.R.A.S.; Formal analysis: R.A.V.N., A.W.O.R, and G.B.; Research: P.P.M.C., N.R.A.S and R.A.V.N.; Resources: R.A.V.N.; Writing-original draft preparation: R.A.V.N. and P.P.M.C.; Writing-review and editing: G.B. and J.C.Ch.G.; Visualization: R.A.V.N.; Supervision: R.A.V.N and G.B.; Funding acquisition: R.A.V.N. and J.C.Ch.G. All the authors read and approved the final manuscript.

Acknowledgement

The authors thank Edson Torres Chávez, leader of the rice program of the National Institute of Agrarian Innovation (INIA), for providing the rice varieties tested in this study, and they also thank Professor Miguel Garrido Rondoy, research professor at the National University of Tumbes, for donating the strains of phytopathogenic fungi to carry out this study.

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Genetic diversity and characterization of the growth promotion mechanisms of Burkholderia vietnamiensis isolates from rice cultivars in valleys of the high jungle of Peru.

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Abstract

Figures

Introduction

Materials and methods

Collection of rice samples

Isolation of endophytic bacteria from roots of rice

Genomic DNA extraction

BOX-PCR genomic profiles:

Characterization of growth promotion mechanisms:

Auxin production:

Nitrogen fixation:

Siderophores production

Phosphate solubilization

Antibiosis and antagonism against phytopathogenic fungi:

Evaluation of growth promotion in two rice cultivars:

Germination test and vigour index

Growth promotion evaluation

Statistical analyses

Results

Discussion

Conclusions

Declarations

Author Contribution

Acknowledgement

References

Additional Declarations

Status:

Version 1