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Article

In Vitro Inhibition of Rhizoctonia oryzae-sativae Using Bacterial Strains as a Sustainable Alternative for Controlling Sheath Blight in Rice

by
Liz Cheril Quiñones-Pezo
1,
Winston Franz Ríos-Ruiz
1,*,
Danny Fran Pompa-Vásquez
1,
Franz Rios-Reategui
2,
Angel David Hernández-Amasifuen
3 and
Mike Anderson Corazón-Guivin
4
1
Laboratorio de Microbiología Agrícola “Raúl Ríos Reátegui”, Departamento Académico Agrosilvopastoril, Facultad de Ciencias Agrarias, Universidad Nacional de San Martín, Tarapoto 22202, Peru
2
Departamento Académico de Ingeniería Electrónica, Facultad de Ingeniería Electrónica y Eléctrica, Universidad Nacional Mayor de San Marcos, Lima 15081, Peru
3
Laboratorio de Biología Molecular de Plantas (LBMP), Instituto de Investigación, Innovación y Desarrollo para el Sector Agrario y Agroindustrial (IIDAA), Universidad Nacional Toribio Rodríguez de Mendoza de Amazonas, Amazonas, Chachapoyas 01001, Peru
4
Laboratorio de Biología y Genética Molecular, Departamento Académico Agrosilvopastoril, Facultad de Ciencias Agrarias, Universidad Nacional de San Martín, Tarapoto 22202, Peru
*
Author to whom correspondence should be addressed.
Int. J. Plant Biol. 2024, 15(4), 988-1000; https://doi.org/10.3390/ijpb15040070
Submission received: 14 August 2024 / Revised: 28 September 2024 / Accepted: 7 October 2024 / Published: 9 October 2024
(This article belongs to the Section Plant–Microorganisms Interactions)
Browse Figures
Versions Notes

Abstract

:
The control of ‘sheath blight’ in rice (Oryza sativa L.), which is caused by the fungus Rhizoctonia oryzae-sativae, has become problematic due to the excessive application of fungicides and their consequent harmful effects. Hence, the search for less contaminating alternatives to conventional chemical products is necessary. This study evaluated the in vitro inhibitory activity of four bacterial strains on the growth of R. oryzae-sativae in both tryptone soy broth (TSB) and mineral medium (MM). The results demonstrated that all evaluated strains (Bacillus tequilensis SMNCT17-02, Priestia aryabhattai SMNCH17-07, Burkholderia vietnamiensis TUR04-01, and Burkholderia vietnamiensis TUR04-03) inhibited the growth of R. oryzae-sativae. Specifically, the activity of B. tequilensis SMNCT17-02 resulted in the smallest area of R. oryzae-sativae growth in both TSB medium (8.54 cm2) and MM (5.53 cm2), suggesting a notable antifungal effect. When evaluating the action of supernatants generated by the growth of the four bacterial strains in TSB and MM culture media, significant inhibition of R. oryzae-sativae growth was only observed for supernatants produced in MM. This inhibition was attributed to the presence of soluble secondary metabolites. These results offer new perspectives in biotechnology, suggesting the possibility of developing effective products based on easily extractable soluble secondary metabolites, thus promoting sustainable agriculture.

1. Introduction

Rice (Oryza sativa L.) is a globally significant staple food. In Peru, it ranks third in terms of size of cultivation area, after potatoes and maize, with the San Martin region contributing to 23.34% (443,881.49 t) of the country’s total rice production and an approximate yield of 8.7 t ha−1 [1]. However, rice crops in San Martin face serious challenges from fungal diseases that thrive in the region’s climatic and soil conditions.
Sheath blight is one of the most significant phytosanitary problems in rice cultivation. Its symptoms include elliptical or irregular spots on the sheaths; lesions with brown margins; and blight bands affecting the leaves, sheaths, and other plant organs, leading to wilting and premature death, as well as the presence of sclerotia, which can serve as inoculum for new infections [2,3]. This disease is caused by three species of the Rhizoctonia genus: Rhizoctonia solani, R. oryzae, and R. oryzae-sativae [4].
R. solani is a fast-growing phytopathogenic fungus that does not produce spores and reproduces vegetatively through mycelial fragments and sclerotia. The optimal growth of R. solani occurs between 25 and 32 °C under high-humidity conditions. R. oryzae, although less aggressive than R. solani, has paler mycelium and produces smaller, more regular sclerotia. R. oryzae contributes to sheath blight by generating lesions that can severely damage leaves and sheaths. On the other hand, R. oryzae-sativae has less pigmented mycelium and produces more numerous sclerotia. Although less aggressive than R. solani, R. oryzae-sativae remains a relevant pathogen in sheath blight growth, with lesions that may coalesce in severe infections [2,5,6].
The presence of these diseases can significantly reduce crop productivity, causing substantial economic losses and raising concerns about food security [7]. Furthermore, the excessive use of chemical products for the control of these diseases has led to environmental damage, affecting soil and water quality, increasing production costs, and posing risks to the health of both producers and consumers [8].
In this context, an alternative strategy for phytopathogen control could involve the use of microorganisms and microbially derived products that promote plant growth, production, and health. These microorganisms can enhance nutrient acquisition, boost physiological and biochemical processes, and help in the inhibition of phytopathogens [9]. Various species, including Bacillus sp., Enterobacter sp., Klebsiella sp., Pseudomonas sp., Burkholderia sp., Pantoea sp., Agrobacterium sp., and Methylobacteria sp. possess the necessary characteristics for this purpose [10]. Specifically, the genera Bacillus and Burkholderia are known for their antagonistic action in suppressing the growth of phytopathogenic microorganisms [11,12], primarily due to their ability to secrete a wide range of secondary metabolites with specific properties against various phytopathogenic fungi, including those affecting rice [13]. These metabolites can be extracted through physical techniques and maintained in aqueous media, making them promising candidates for fungicide production [14].
The objective of this research was to determine the inhibitory activity of the bacterial strains Bacillus tequilensis SMNCT17-02, Priestia aryabhattai SMNCH17-07, Burkholderia vietnamiensis TUR04-01, and Burkholderia vietnamiensis TUR04-03, as well as their soluble secondary metabolites, on the growth of R. oryzae-sativae under laboratory conditions. The results represent a first step toward developing effective products based on easily extractable bacterial secondary metabolites, offering a viable alternative to chemical fungicides in rice cultivation.

2. Materials and Methods

2.1. Microbiological Material

Four bacterial strains (B. tequilensis SMNCT17-02, P. aryabhattai SMNCH17-07, B. vietnamiensis TUR04-01, and B. vietnamiensis TUR04-03) isolated from rice cultivation areas in the Tumbes and San Martin regions of Peru were used. These strains are part of the native strain bank of the Agricultural Microbiology Laboratory ‘Raúl Ríos Reátegui’ at the Universidad Nacional de San Martín, as reported by Valdez-Nuñez et al. [15] and Ríos-Ruiz et al. [16].
B. tequilensis is a Gram-positive spore-forming bacillus found in soils and environments related to beverage fermentation. This microorganism is characterized by its rapid growth and the formation of smooth colonies, and it is used in agriculture as a biocontrol agent for plant pathogens and as a biofertilizer due to its ability to promote plant growth. Additionally, it produces antimicrobial secondary metabolites, including lipopeptides that have been shown to be effective against various plant pathogenic fungi [17]. On the other hand, P. aryabhattai, also a Gram-positive bacillus, is distinguished by its rapid growth in aerobic conditions and shows promising potential in the biological control of plant diseases, although it has been less studied. This microorganism produces metabolites that contribute to the suppression of phytopathogens and the promotion of plant growth [18]. Similarly, B. vietnamiensis, a Gram-negative and motile bacillus, exhibits remarkable adaptability to various environments. This microorganism is utilized in biological control [15] and is also a nitrogen-fixing bacterium [19]. These isolates, in addition to the aforementioned characteristics, produce siderophores and are resistant to toxoflavin [15]. Table 1 presents the bacterial strains used in this study.
Regarding the fungus R. oryzae-sativae, it was provided by the Universidad Nacional de Tumbes, and its molecular identification is detailed in this article.

2.2. Reviving of Bacterial Strains

Bacterial strains were revived via successive plating on tryptone soy agar (TSA) medium consisting of pancreatic revived digest of casein (15 g), soybean peptone digest (5 g), NaCl (5 g), distilled water (1000 mL), and agar (15 g) with a pH of 7. Using a micropipette, 5 mL of TSA medium was dispensed into sterile 10 mL glass tubes. Subsequently, these tubes were tilted at a 45 °C angle and cooled to room temperature for 12 h. Next, bacterial strains were streaked into the TSA-containing tubes in a zigzag pattern and then incubated at 30 °C for 24 h. After the growth period, the vials were stored at 4 °C. Later, the strains were inoculated into tubes containing tryptone soy broth (TSB) medium composed of the same components as TSA, but without agar, and mineral medium (MM) consisting of glucose (10 g), yeast extract (5 g), KH2PO4 (1 g), MgSO4·7H2O (0.5 g), NaCl (0.01 g), FeSO4·7H2O (0.01 g), and distilled water (1 L) with a pH of 7. The vials were then incubated at 30 °C with constant agitation in a shaker (MRC TOS-4030FD, Holon, Israel) for 24 h, reaching a concentration of 1.0 (OD600 nm) (109 CFU mL−1) measured using a spectrophotometer (Thermo Fisher, Spectronic 200, Suwa, Japan). The fungal strain, R. oryzae-sativae, was revived using potato dextrose agar (PDA, HiMedia, Thane, India) medium by inoculating a small portion of the mycelium in the center of the plate, followed by incubation at 30 °C for 72 h.

2.3. Determination of the Inhibitory Effect of Volatile Secondary Metabolites on the Growth of R. oryzae-sativae

The determination of the effect of volatile secondary metabolites was carried out using the ‘sealed plate’ method following the methodology proposed by Fernando et al. [20]. This procedure involved diluting the inoculum (bacterial strain) to the sixth solution (10−6). To achieve this, 6 microtubes were prepared, each containing 900 µL of NaCl. An amount of 100 µL of the inoculum was added to the first microtube, which was shaken for 20 s to obtain a 1:10 dilution. Next, 100 µL of this first dilution was transferred to the second microtube, which was shaken to achieve a 1:100 dilution. This procedure was repeated consecutively until a 10−6 dilution was reached. When the dilutions were completed, 100 µL of the 10−6 dilution was inoculated in the center of a plate with TSA medium, by spreading it with a Drigalsky spatula from the center to the edge of the plate. Subsequently, a 6 mm agar disc containing the fungus R. oryzae-sativae was placed in the center of a plate with PDA medium. Finally, the TSA plate containing the bacteria was inverted over the PDA plate containing the fungus to directly expose the latter to the antagonistic environment produced by the bacteria; the plate was then sealed with parafilm.

2.4. Determination of the Inhibitory Effect of the Isolated Bacterial Strains on the Growth of R. oryzae-sativae

First, an amount of 20 µL of inoculum from each of the four bacterial strains was grown in TSB and MM culture media on three equidistant sides of a Petri dish containing PDA medium. A disk containing the R. oryzae-sativae fungus (previously cultured on PDA medium) was extracted from another plate using a sterile hole puncher and placed in the center of the Petri dish. Plates containing only PDA medium inoculated with the R. oryzae-sativae fungus were used as controls. The area of fungal growth was determined by considering its surface growth using the digital imaging program ImageJ (version 1.52p) to identify which bacteria inhibited the growth of R. oryzae-sativae.

2.5. Evaluation of the Inhibitory Activity of Supernatants (Soluble Secondary Metabolites) Produced by Bacterial Strains on the Growth of R. oryzae-sativae

The evaluation of the bacteria’s ability to produce soluble secondary metabolites was conducted using the agar well diffusion method, following the methodology proposed by Memenza-Zegarra and Zúñiga-Dávila [21]. Bacterial strains B. tequilensis SMNCT17-02, P. aryabhattai SMNCH17-07, B. vietnamiensis TUR04-01, and B. vietnamiensis TUR04-03 were cultured in Erlenmeyer flasks containing 20 mL of TSB and MM culture media, and incubated at 30 °C with constant shaking at 180 rpm for 36 h. Subsequently, 1 mL of each inoculated medium was centrifuged (Rotofix 32 A Hettich, Tuttlingen, Germany) at 13,000 rpm for 15 min to obtain two phases: the solid phase (bacterial biomass) and the liquid phase (supernatant). The latter was used to evaluate the production of soluble secondary metabolites. Sequentially, three 6 mm diameter wells were made in a Petri dish containing PDA medium using a sterile hole puncher and then 20 μL of the supernatant obtained in the previous step was placed in each well. Additionally, a 6 mm agar disk containing the R. oryzae-sativae fungus was placed in the center of the same plate. Another plate containing PDA medium was used as the control, following the same procedure as the previous test, except that 20 μL of sterile water was placed in each well. The area of fungal growth was determined using the ImageJ 1.52p digital imaging program.

2.6. Molecular Characterization of R. oryzae-sativae

Genomic DNA was extracted from ≈100 mg of R. oryzae-sativae mycelium using the CTAB method established by Kalendar et al. [22]. The isolated DNA was quantified using spectrophotometry NanoDrop One (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −20 °C. Fragments of the ribosomal DNA (rDNA) gene ITS1-5.8S-ITS2 were amplified using the primer pairs ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) for forward and reverse reactions, respectively [23]. PCR reactions consisted of 35 cycles, each with 30 s of denaturation at 95° C, 45 s of annealing at 58 °C, and 1 min of extension at 72 °C. Initial denaturation for 2 min at 95 °C was followed by a final extension for 5 min at 72 °C. The amplicons were run using 1% agarose gel and visualized using a gel documentation system omniDOC (Cleaver Scientific, Rugby, UK). Selected PCR products were purified and sent to Macrogene (Santiago, Chile/Seoul, Republic of Korea) for bidirectional sequencing. The sequences’ noisy sections were removed from the obtained chromatograms using the BioEdit sequence alignment editor to generate consensus sequences, which were compared with sequences deposited in GenBank using the BLAST+ 2.15.0 tool. For phylogenetic analysis, multiple nucleotide alignments and a neighbor-joining (NJ) phylogenic tree construction were generated using the Tamura three-parameter genetic distance model (T92) with 1,000 bootstraps. All analyses were performed using MEGA11 software [24]. R. oryzae (teleomorph: Waitea circinata) (GenBank accession number MH861519.1) was used as the outgroup [25].

2.7. Statistical Analysis

To determine the areas of R. oryzae-sativae growth, 4 bacterial strains and 2 culture media were used, resulting in 8 treatments, each with 3 replicates. The normality of data was checked using the Shapiro–Wilk test and standard deviations and experimental standard errors were calculated. These results were subjected to statistical analyses, which included analysis of variance and correlation coefficients. Data were processed and analyzed using InfoStat/L Version 2020 statistical software. The Tukey test (p < 0.05) was used to assess the significance of the studied factors, which included fungal growth in TSB and MM culture media as a result of both the bacteria’s direct action and the effect of the supernatant obtained after bacterial growth in MM.

3. Results

3.1. Inhibitory Activity of Volatile Secondary Metabolites on R. oryzae-sativae Using the ‘Sealed Plate’ Method

Figure 1a shows the ‘sealed plate’ bacterium B. tequilensis SMNCT17-02 and the growth of the fungus R. oryzae-sativae. The ‘sealed plate’ method was used to determine whether the bacteria produced volatile secondary metabolites that could affect fungal growth. Figure 1b,c show bacterial and fungal growth, respectively, in open plates, indicating no inhibition of fungal growth. All other strains tested showed the same behavior.

3.2. Inhibitory Effect of the Isolated Bacterial Strains on the Growth of R. oryzae-sativae

The results revealed that all four studied strains, B. tequilensis SMNCT17-02, P. aryabhattai SMNCH17-07, B. vietnamiensis TUR04-01, and B. vietnamiensis TUR04-03, showed a remarkable ability to inhibit the growth of R. oryzae-sativae. This inhibition was consistently observed in both culture media (TSB and MM) (Figure 2).
Strains of B. tequilensis SMNCT17-02 and P. aryabhattai SMNCH17-07 exhibited a stronger inhibitory effect compared with the Burkholderia strains in both TSB and MM culture media. Specifically, the B. tequilensis SMNCT17-02 strain demonstrated the highest inhibition capacity, measuring 8.54 cm2 in TSB medium and 5.53 cm2 in MM, as detailed in Table 2.

3.3. Inhibitory Activity of Supernatants (Soluble Secondary Metabolites) Produced by Bacterial Strains on the Growth of R. oryzae-sativae

When evaluating the action of supernatants generated by the growth of the four bacterial strains in TSB and MM culture media, significant inhibition of R. oryzae-sativae growth was only observed for supernatants produced in MM. B. tequilensis SMNCT17-02 exhibited the highest pathogen inhibition (44.07 cm2) compared with P. aryabhattai SMNCH17-07 (47.67 cm2), B. vietnamiensis TUR04-01 (55.18 cm2), and B. vietnamiensis TUR04-03 (50.07 cm2) (p < 0.05) (Figure 3 and Table 3).

3.4. Identity of R. oryzae-sativae Through rDNA-ITS Sequencing and Phylogenetic Analysis

The rDNA-ITS sequence of our isolate (GenBank accession number PP754447) had 100% identity with three reference sequences (OR471265.1, AB122144.1, and FJ231391.1), 99.9% with the FJ667257.1 sequence, 99.7% with MH861282.1, and 99.4% with AJ000194.1, confirming the isolate’s identity as R. oryzae-sativae (teleomorph: Ceratorhiza oryzae-sativae). The phylogenetic tree constructed using the NJ method based on the rDNA-ITS sequence evidenced the clustering of our isolate with the reference sequences with R. oryzae (teleomorph: Waitea circinata) as an outgroup (Figure 4).

4. Discussion

4.1. Inhibition of R. oryzae-sativae Growth by Volatile Secondary Metabolites

This study’s results regarding the inhibitory activity of volatile secondary metabolites did not follow the trend found by Loor et al. [26] who determined that strains of Bacillus spp. produced volatile compounds that inhibited the mycelial growth of Moniliophthora roreri. Similarly, Patel et al. [27] observed the action of volatile secondary metabolites produced by Bacillus spp. against phytopathogenic fungi. Several factors can prevent volatile secondary metabolites from effectively controlling phytopathogenic fungi, including an insufficient concentration of these metabolites produced by the bacteria and the interaction of volatile secondary metabolites with other fungi-produced compounds, which can neutralize their inhibitory effect [28]. These factors might have influenced the lack of action of the metabolites produced in this study.

4.2. Assessment of the Inhibitory Activity of Isolated Bacterial Strains on the Growth of R. oryzae-sativae

The results obtained indicate that the four bacterial strains cultivated in TSB and MM culture media showed inhibition effects on R. oryzae-sativae. Moreover, the B. tequilensis SMNCT17-02 and P. aryabhattai SMNCH17-07 strains exhibited higher levels of inhibition compared with the B. vietnamiensis TUR04-01 and B. vietnamiensis TUR04-03 strains. Several studies have addressed the potential of endophytic microorganisms to combat various types of stress, both biotic and abiotic, in rice production. These include research regarding growth promotion and phytopathogen control [29], with an emphasis on the genera Bacillus and Burkholderia [11,30]. Iqbal et al.’s comprehensive review [31] highlighted antimicrobial peptides produced by B. subtilis and their numerous beneficial applications in various fields.
Kumar et al. [4] isolated and characterized endophytic bacteria from six rice varieties grown in central, eastern, and north-eastern India, and found that the B. subtilis isolate exhibited antibacterial and antifungal activity. Similarly, Devi et al. [32] identified various growth-promoting Bacillus strains, such as B. licheniformis MNNITSR2 and B. velezensis MNNITSR18, which strongly inhibited the growth of rice phytopathogens R. solani and Fusarium oxysporum. Heo et al. [12] isolated B. subtilis GYUN-2311 from the rhizosphere of apple trees, which showed antagonistic activity against 12 pathogenic fungi. Both volatile organic compounds and the culture filtrate of B. subtilis exhibited antifungal activity. Regarding the biocidal effect of the Burkholderia genus, Song et al. [33] reported the action of Burkholderia cepacia, which suppressed the growth of R. solani through biofilm formation. Although the Burkholderia complex comprises a group of bacteria of significant agrobiotechnology value, using this genus is limited to laboratory assays due to its opportunistic pathogenic nature. Burkholderia’s action mechanism is poorly understood, representing an unexplored field in the search for new metabolites produced by bacteria. In this context, Meng et al. [11] isolated more than 50 endophytic bacteria from the roots of the medicinal plant Ficus tikoua Bur and analyzed their antifungal activity; the Burkholderia vietnamiensis C12 strain showed the highest inhibitory activity against the rice sheath blight causal agent, R. solani. Additionally, Saxena et al. [34] noted that due to the genetic and metabolic diversity of the Bacillus genus, it responds better to phytopathogen biocontrol than the Burkholderia genus does. Their result corroborates this study’s finding that the B. tequilensis SMNCT17-02 and P. aryabhattai SMNCH17-07 strains demonstrated greater inhibition capacity than the B. vietnamiensis TUR04-01 and B. vietnamiensis TUR04-03 strains, in both TSB and MM culture media.

4.3. Perspectives on the Effects of Secondary Metabolites on the Growth of R. oryzae-sativae

Figure 3 and Table 3 show the inhibitory effects of supernatants (soluble secondary metabolites) produced by bacterial strains cultivated in MM and subjected to the agar well diffusion assay; a procedure that ensured the direct action of secondary metabolites on the growth of the R. oryzae-sativae fungus. Conversely, after conducting the same test using TSB medium, there was no evidence of the supernatant’s inhibitory action. This result is probably because the TSB medium contained nitrogen and carbon concentrations in a proportion that suppressed the formation of secondary metabolites, which did not favor the production of soluble compounds in the TSB medium’s supernatant [35]. The TSB and MM culture media used in this study contained different nitrogen and carbon sources, such as peptone and glucose, respectively. According to Mohanrasu et al. [36], the production of secondary metabolites is conditioned by the presence of excess carbon and a scarcity of nitrogen sources. These authors observed that the bacterium Bacillus megaterium (MK386891) accumulated a greater amount of the secondary metabolite poly (3-hydroxybutyrate) (PHB) under limited nitrogen. On the other hand, Robles-Huízar et al. [37] evaluated seven culture media with different components and C/N ratios and determined that the maximum biosynthetic expression of secondary metabolites by Pseudomonas fluorescens MR-IB66 occurred when using a medium with minimal salt content and 20% glucose as a carbon source. In this research, antagonistic activity was evident in the MM, resulting in inhibited R. oryzae-sativae growth. Additionally, other physicochemical factors, such as temperature, humidity, oxygen, and the presence of lipopeptides as carbon and nitrogen sources, may influence bacteria’s production of antibiotics [21]. Jawan et al. [38] studied the production of antibacterial peptides by strains of lactic acid bacteria and found that organic nitrogen sources were more favorable than inorganic ones. Furthermore, they observed a significant increase in bacteriocin production when fructose was substituted for carbon sources. Similarly, Memenza-Zegarra and Zúñiga-Dávila [21] found that supernatants obtained from Bacillus sp. cultures showed inhibitory effects on the growth of R. solan. Xue et al. [39] evaluated the antifungal activity of Burkholderia sp. BV6 isolated from rice roots and determined its biocontrol potential against rice blast caused by Magnaporthe oryzae-sativae. They found small molecular hydrophilic compounds with inhibitory effects on the growth of the fungus in the bacterial culture’s supernatant.
Alviz et al. observed a similar inhibitory effect on bacteria [40] and determined the inhibitory action of soluble metabolites produced by endophytic strains in MM on the growth of Burkholderia glumae. Valdez-Nuñez et al. [10] determined the inhibitory effect of B. subtilis SMNCT17-02 (now B. tequilensis SMNCT17-02) [16] grown in TSB medium on the Burkholderia glumae strain THT. Additionally, Andric et al. [13] examined the potential of Bacillus velezensis to modulate its secondary metabolome after detecting the presence of a bacterial competitor. They observed that the microorganism mobilizes a significant portion of its secondary metabolites upon chemically detecting the presence of Pseudomonas, triggering a response in the form of antibacterial agent production. Moreover, Wang et al. [41] analyzed the genomic characteristics of Bacillus halotolerans Q2H2 and identified gene clusters associated with the synthesis of antibacterial peptides, hydrolases, phosphate solubilization, nitrogen fixation, siderophore production, and indoleacetic acid (IAA) synthesis.
In this study, a differentiated result was observed when evaluating the action of TSB-cultivated bacterial strains compared with the effect of the supernatants obtained from the same medium. Bacterial inoculum treatments showed phytopathogen inhibition, whereas cell-free supernatants lost their antagonistic potential. On the other hand, the antagonistic power of the inoculum containing MM-cultivated bacterial strains was superior compared with the action of the supernatants (Table 2 and Table 3).
Figure 5 shows a correlation analysis of the action of bacterial strains and their supernatants (soluble secondary metabolites) on R. oryzae-sativae grown in TSB and MM culture media. When interacting with MM-cultivated bacterial strains, the B. tequilensis SMNCT17-02 and P. aryabhattai SMNCH17-07 treatments had the smallest areas of R. oryzae-sativae growth (5.53 cm2 and 13.89 cm2, respectively) and the B. vietnamiensis TUR04-01 treatment had the largest area of growth (32.83 cm2). Areas of fungal growth against the supernatant and soluble secondary metabolites produced by MM-cultivated bacteria exhibited the same pattern: the B. tequilensis SMNCT17-02 and P. aryabhattai SMNCH17-07 treatments had the smallest areas of R. oryzae-sativae growth (44.07 cm2 and 47.67 cm2, respectively), whereas B. vietnamiensis TUR04-01 had the largest area of growth (55.18 cm2).

5. Conclusions

The four bacterial strains evaluated in this study (Bacillus tequilensis SMNCT17-02, Priestia aryabhattai SMNCH17-07, Burkholderia vietnamiensis TUR04-01, and Burkholderia vietnamiensis TUR04-03) demonstrated significant in vitro inhibitory activity against the growth of Rhizoctonia oryzae-sativae, with B. tequilensis SMNCT17-02 showing the most substantial reduction in fungal growth. Given the substantial losses caused by phytopathogenic fungi and the environmental concerns associated with chemical fungicides, microbial biofungicides, particularly those from the Bacillus and Priestia genera, present a sustainable alternative for controlling these pathogens. This study highlights the potential of native bacterial strains isolated from Peruvian rice soils as effective biocontrol agents, emphasizing the unique properties of their soluble metabolites, which significantly inhibit fungal growth, especially in mineral media. Additionally, B. tequilensis demonstrates exceptional antifungal capabilities across various culture media, indicating promising avenues for commercial applications. The utilization of supernatants for fungal inhibition eliminates the need for live cells, streamlining production and application processes. Finally, the precise molecular characterization of R. oryzae-sativae confirms its identity, thus bolstering the validity and rigor of this study.

Author Contributions

Conceptualization: W.F.R.-R.; methodology: L.C.Q.-P., D.F.P.-V., F.R.-R., A.D.H.-A. and M.A.C.-G.; validation: W.F.R.-R.; formal analysis: W.F.R.-R. and M.A.C.-G.; investigation: L.C.Q.-P.; data curation: W.F.R.-R., A.D.H.-A. and M.A.C.-G.; writing—original draft preparation: L.C.Q.-P.; writing—review and editing: W.F.R.-R., F.R.-R. and M.A.C.-G.; visualization: W.F.R.-R. and F.R.-R.; supervision: W.F.R.-R.; project administration: W.F.R.-R.; funding acquisition: W.F.R.-R. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Universidad Nacional de San Martín through the Institute of Research and Development, according to Resolution No. 438-2020-UNSM/CU-R.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The molecular datasets generated during the study are available in NCBI GenBank under accession numbers MK449440, MK449444, MK449433, MK449435, and PP754447. Other datasets used during the current study are available from the corresponding author upon reasonable request.

Acknowledgments

To the Universidad Nacional de San Martín for the support provided to Liz Cheril Quiñones Pezo for the development of her thesis titled ‘Actividad inhibitoria in vitro de metabolitos secundarios de Bacillus spp. y Burkholderia vietnamiensis sobre Rhizoctonia sp.’ from which this article is derived. To Miguel Garrido Rondoy, Head of the Phytopathology Laboratory, Faculty of Agricultural Sciences, Universidad Nacional de Tumbes, Tumbes, Peru, and Eybis José Flores García, Head of the Phytopathology Laboratory, Faculty of Agricultural Sciences, Universidad Nacional de San Martín, San Martin, Peru, who provided us with the Rhizoctonia sp. strain used in this research. Special thanks to Beatriz Reátegui Ruiz for her support in writing and editing the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. ‘Sealed plate’ (a) containing the strain Bacillus tequilensis SMNCT17-02 on the upper plate and the fungus Rhizoctonia oryzae-sativae on the lower plate. To assess the potential action of volatile secondary metabolites produced by the bacterial strain, the plate was inverted. Plate (b) shows the growth of the bacterium, and plate (c) shows the growth of the fungus.
Figure 1. ‘Sealed plate’ (a) containing the strain Bacillus tequilensis SMNCT17-02 on the upper plate and the fungus Rhizoctonia oryzae-sativae on the lower plate. To assess the potential action of volatile secondary metabolites produced by the bacterial strain, the plate was inverted. Plate (b) shows the growth of the bacterium, and plate (c) shows the growth of the fungus.
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Figure 2. Petri dishes containing PDA culture medium showing the growth of bacterial strains in triplicate, with one replicate indicated by an arrow. Bacteria were cultured in TSB medium (plates a–d) and mineral medium (plates f–i) to assess the inhibition of Rhizoctonia oryzae-sativae, seeded at the center of the plates. (a,f): Burkholderia vietnamiensis TUR04-01; (b,g): Bacillus tequilensis SMNCT17-02; (c,h): Priestia aryabhattai SMNCH17-07; (d,i): Burkholderia vietnamiensis TUR04-03; (e,j): Control plates containing PDA medium with R. oryzae-sativae growth, without bacterial strains.
Figure 2. Petri dishes containing PDA culture medium showing the growth of bacterial strains in triplicate, with one replicate indicated by an arrow. Bacteria were cultured in TSB medium (plates a–d) and mineral medium (plates f–i) to assess the inhibition of Rhizoctonia oryzae-sativae, seeded at the center of the plates. (a,f): Burkholderia vietnamiensis TUR04-01; (b,g): Bacillus tequilensis SMNCT17-02; (c,h): Priestia aryabhattai SMNCH17-07; (d,i): Burkholderia vietnamiensis TUR04-03; (e,j): Control plates containing PDA medium with R. oryzae-sativae growth, without bacterial strains.
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Figure 3. Petri dishes containing PDA culture medium, with agar wells in triplicate (one marked by an arrow), each filled with supernatant (soluble secondary metabolites) from MM cultures of the four bacterial strains (plates a–e). The inhibitory effect of the strains on the growth of R. oryzae-sativae, seeded at the center of the plates, is visible. (a): Bacillus tequilensis SMNCT17-02; (b): Priestia aryabhattai SMNCH17-07; (c): Burkholderia vietnamiensis TUR04-03; (d): Burkholderia vietnamiensis TUR04-01; and (e): Control plate with PDA medium, R. oryzae-sativae growth, and distilled water in the agar wells.
Figure 3. Petri dishes containing PDA culture medium, with agar wells in triplicate (one marked by an arrow), each filled with supernatant (soluble secondary metabolites) from MM cultures of the four bacterial strains (plates a–e). The inhibitory effect of the strains on the growth of R. oryzae-sativae, seeded at the center of the plates, is visible. (a): Bacillus tequilensis SMNCT17-02; (b): Priestia aryabhattai SMNCH17-07; (c): Burkholderia vietnamiensis TUR04-03; (d): Burkholderia vietnamiensis TUR04-01; and (e): Control plate with PDA medium, R. oryzae-sativae growth, and distilled water in the agar wells.
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Figure 4. Phylogenetic tree generated using the NJ method showing the genetic relationship of the rDNA-ITS region between our Rhizoctonia oryzae-sativae isolate (indicated by a black dot ●) and those of other R. oryzae-sativae isolates available in GenBank (NCBI). R. oryzae was used as an outgroup.
Figure 4. Phylogenetic tree generated using the NJ method showing the genetic relationship of the rDNA-ITS region between our Rhizoctonia oryzae-sativae isolate (indicated by a black dot ●) and those of other R. oryzae-sativae isolates available in GenBank (NCBI). R. oryzae was used as an outgroup.
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Figure 5. Correlation of the effect of bacterial strains and supernatants grown in TSB and MM culture media on the growth of Rhizoctonia oryzae-sativae. SMNCT17-02 = Bacillus tequilensis; SMNCH17-07 = Priestia aryabhattai; TUR04-01 = Burkholderia vietnamiensis; TUR04-03 = Burkholderia vietnamiensis.
Figure 5. Correlation of the effect of bacterial strains and supernatants grown in TSB and MM culture media on the growth of Rhizoctonia oryzae-sativae. SMNCT17-02 = Bacillus tequilensis; SMNCH17-07 = Priestia aryabhattai; TUR04-01 = Burkholderia vietnamiensis; TUR04-03 = Burkholderia vietnamiensis.
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Table 1. Endophytic rice bacterial strains used in the different treatments.
Table 1. Endophytic rice bacterial strains used in the different treatments.
Rice Endophytic BacteriaStrainsOriginAccession Numbers
Bacillus tequilensisSMNCT17-02San MartinMK449440
Priestia aryabhattaiSMNCH17-07San MartinMK449444
Burkholderia vietnamiensisTUR04-03TumbesMK449433
Burkholderia vietnamiensisTUR04-01TumbesMK449435
Adapted from Valdez-Nuñez et al. [15] and Ríos-Ruiz et al. [16] where it is noted that B. subtilis has been reclassified as B. tequilensis.
Table 2. Inhibition area (cm2) of Rhizoctonia oryzae-sativae on PDA plates inoculated with bacterial strains grown in TSB and MM.
Table 2. Inhibition area (cm2) of Rhizoctonia oryzae-sativae on PDA plates inoculated with bacterial strains grown in TSB and MM.
Bacterial StrainsGrowth Area of Rhizoctonia oryzae-sativae
TSB (cm2)MM (cm2)
Bacillus tequilensis SMNCT17-028.54 ± 0.28 b5.53 ± 0.26 a
Priestia aryabhattai SMNCH17-0711.76 ± 0.14 c13.89 ± 0.02 d
Burkholderia vietnamiensis TUR04-0130.20 ± 0.07 f32.83 ± 0.03 g
Burkholderia vietnamiensis TUR04-0348.88 ± 0.10 h26.87 ± 0.64 e
The growth areas were determined using the ImageJ 1.52p program. Different letters in each column denote statistically significant differences based on the Tukey test (p < 0.05).
Table 3. Growth area (cm2) of Rhizoctonia oryzae-sativae on PDA plates treated with supernatants from bacterial strains cultured in MM.
Table 3. Growth area (cm2) of Rhizoctonia oryzae-sativae on PDA plates treated with supernatants from bacterial strains cultured in MM.
Treatments (Bacterial Strains)Growth Area of R. oryzae-sativae in MM (cm2)
Bacillus tequilensis SMNCT17-0244.07 ± 0.01 a
Priestia aryabhattai SMNCH17-0747.67 ± 0.02 b
Burkholderia vietnamiensis TUR04-0155.18 ± 0.00 e
Burkholderia vietnamiensis TUR04-0350.07 ± 0.00 c
The growth areas were determined using the ImageJ 1.52p program. Different letters in each column denote statistically significant differences based on the Tukey test (p < 0.05).
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Quiñones-Pezo, L.C.; Ríos-Ruiz, W.F.; Pompa-Vásquez, D.F.; Rios-Reategui, F.; Hernández-Amasifuen, A.D.; Corazón-Guivin, M.A. In Vitro Inhibition of Rhizoctonia oryzae-sativae Using Bacterial Strains as a Sustainable Alternative for Controlling Sheath Blight in Rice. Int. J. Plant Biol. 2024, 15, 988-1000. https://doi.org/10.3390/ijpb15040070

AMA Style

Quiñones-Pezo LC, Ríos-Ruiz WF, Pompa-Vásquez DF, Rios-Reategui F, Hernández-Amasifuen AD, Corazón-Guivin MA. In Vitro Inhibition of Rhizoctonia oryzae-sativae Using Bacterial Strains as a Sustainable Alternative for Controlling Sheath Blight in Rice. International Journal of Plant Biology. 2024; 15(4):988-1000. https://doi.org/10.3390/ijpb15040070

Chicago/Turabian Style

Quiñones-Pezo, Liz Cheril, Winston Franz Ríos-Ruiz, Danny Fran Pompa-Vásquez, Franz Rios-Reategui, Angel David Hernández-Amasifuen, and Mike Anderson Corazón-Guivin. 2024. "In Vitro Inhibition of Rhizoctonia oryzae-sativae Using Bacterial Strains as a Sustainable Alternative for Controlling Sheath Blight in Rice" International Journal of Plant Biology 15, no. 4: 988-1000. https://doi.org/10.3390/ijpb15040070

APA Style

Quiñones-Pezo, L. C., Ríos-Ruiz, W. F., Pompa-Vásquez, D. F., Rios-Reategui, F., Hernández-Amasifuen, A. D., & Corazón-Guivin, M. A. (2024). In Vitro Inhibition of Rhizoctonia oryzae-sativae Using Bacterial Strains as a Sustainable Alternative for Controlling Sheath Blight in Rice. International Journal of Plant Biology, 15(4), 988-1000. https://doi.org/10.3390/ijpb15040070

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