Introduction

Oryza sativa, a staple food for more than half of the global population, is cultivated in approximately 308211 hectares in Ecuador, with a total production of 1,546 523 tons and an average yield of 5.05 t ha^-1. In the province of Los Ríos, around 79379 hectares are planted, yielding 358501 tons with an average yield of (4.52 t ha^⁻¹)¹.

Globally, rice cultivation faces significant phytosanitary challenges due to the incidence of biotic agents such as bacteria, fungi, viruses, spiroplasmas, mycoplasmas, nematodes, and protozoa. These pathogens significantly affect crop development, causing considerable yield losses and reducing the profitability for farmers who rely economically on this cereal as a primary source of livelihood². Studies conducted in tropical Asia have shown that yield losses vary depending on the type of disease. In individual cases, losses range from 1 to 10 %, while the simultaneous interaction of multiple diseases, insects, and weeds can lead to average losses of 37.2 %, reaching up to 41 % under severe conditions, depending on the composition of the pathological system and the production environment³.

This cereal crop of great agricultural importance is affected by more than 40 diseases and microbial disorders, which represent a significant threat to its global productivity⁴. The magnitude of economic losses depend both on the vulnerability of the cultivated varieties and on the type of agronomic management applied⁵. In this context, it is essential to validate the resistance of varieties introduced from other regions, considering that their phytosanitary performance may vary depending on agroclimatic conditions.

One of the most aggressive rice pathogens is the necrotrophic fungus Rhizoctonia solani Kühn (teleomorph Thanatephorus cucumeris), which belongs to the family Corticiaceae, order Homenomycetales, and class Basidiomycetes. This phytoparasite secretes a wide range of secondary metabolites, including host-selective toxins and other biologically active molecules⁶. R. solani is a soil-borne pathogen that negatively affects various crops of high economic relevance worldwide. For this reason, it continues to be the subject of research in different regions of the world⁷. The main crops affected belong to the following families: Poaceae (maize, rice, wheat, barley, oat), Fabaceae (soybean, peanut, common bean, alfalfa, lentil, pea), Solanaceae (tobacco, potato), Amaranthaceae (sugar beet), Brassicaceae (canola), Rubiaceae (coffee), Malvaceae (cotton), Asteraceae (lettuce), Araceae (pothos), Moraceae (ficus) and Linaceae (flax)⁸.

R. solani is responsible for the disease known as sheath blight (SB), which is considered the second most important disease in rice cultivation worldwide, after rice blast⁹, and can significantly reduce crop yield¹⁰. In countries such as the Philippines, losses caused by SB range from 10 % under normal conditions to 25-80 % in highly infected areas¹¹. This problem is exacerbated by the use of high-yielding semi-dwarf rice varieties, high planting densities, and excessive use of nitrogen fertilizers. Furthermore, the wide host range of this pathogen and its ability to survive and persist in a latent state under unfavorable conditions make its control difficult¹².

SB primarily affects the leaf sheath and leaf blades, but it can also affect the entire plant, including emerging panicles¹³. Symptoms typically appear during the late tillering stage and early flowering¹⁴. A characteristic symptom of the disease is the formation of water-soaked lesions with a grayish-green color on the leaf sheath, near the waterline. These lesions are initially circular, oblong, or elliptical in shape, measuring about 1 cm in length. They later enlarge and become irregular, with a grayish-white center and brown margins. Lesions can appear anywhere on the sheath and may coalesce to encircle the stem. Under favorable conditions, the infection spreads to upper leaves, causing sheath rot and complete leaf drying¹⁵. In advanced stages, this disease results in poor grain filling¹⁶.

Disease management involves the combination of methods such as genetic resistance, cultural practices, chemical and biological control integrated strategies that promote sustainability, reduce environmental impact, and contribute to strengthening food security¹⁷. These strategies aim to reduce the amount of pathogenic inoculum to tolerable levels¹⁸. One of the most commonly used practices in modern agriculture is the application of chemical fungicides. These products have become essential for efficient food production, often offering a quick, practical, and economically viable solution¹⁹. Although chemical control is one of the most widely employed methods, it poses challenges to sustainability due to increased costs, the development of fungicide resistance, and concerns about residual toxicity^20.21. Nevertheless, the use of triazole fungicides (Propiconazole, Tebuconazole, and others) has remained one of the most common strategies for years to control soilborne fungi^22,23. Additionally, synthetic products cause toxic effects in humans and contaminate the environment^24,25. Therefore, the use of sustainable alternatives such as biological fungicides^26-28, plant extracts²⁹, and microbial antagonists^30-32 has been promoted.

In a study conducted by Bauzón et al.²⁶, the in vitro efficacy of five biofungicides, a biological agent, a chemical treatment, and an untreated control was evaluated against R. solani. The results indicated that Melaleuca alternifolia + terpenes (3 mL L^-1), Aloe vera + Melaleuca oil (3 mL L^-1), Aloe vera (2 mL L^-1), and Melaleuca alternifolia (2 mL L^-1) were highly effective against the disease, with efficacy comparable to the chemical treatment (Propiconazole + Difenoconazole). The authors recommended validating these findings under in vivo conditions to reduce fungicide use and promote environmental sustainability.

Serenade is a biofungicide based on Bacillus subtilis strain QST 713, which acts through antibiosis and induced resistance. It has been evaluated, along with its filtrates and bacterial suspensions, in the control of clubroot disease in canola (Plasmodiophora brassicae being the causal agent), showing high efficacy when two applications are made, effectively eliminating symptoms of the disease³³. Timorex Gold is a biofungicide derived from tea tree essential oil (Melaleuca alternifolia) and has proven effective against phytopathogenic fungi in various crops. It has shown success in controlling Black Sigatoka and is thus recognized for its potential as a tool in integrated pest management programs³⁴.

Within the integrated management approach, the use of resistant varieties is highly effective for controlling diseases caused by fungi, as serious pathologies such as rusts, vascular wilts, and root rots can be efficiently managed with these varieties. Studies conducted on 10 rice cultivars under greenhouse conditions have reported significant differences among cultivars in terms of susceptibility and resistance to the pathogen, indicating varying levels of incidence and severity depending on the variety evaluated³⁵.

Since some of these varieties are cultivated in neighboring countries, it is crucial to verify whether they are resistant to various diseases when grown in our country, considering that new varieties may exhibit lower disease incidence and severity, which would allow for a reduction in pesticide applications.

In this context, it is essential to adopt technologies such as the use of biological products and new varieties adapted to our study area in order to reduce pesticide use and increase agricultural yields. Therefore, this study focused on evaluating the impact of various fungicides on the control of R. solani in three Peruvian rice varieties cultivated in the Babahoyo canton.

Materials and methods

The research was conducted at kilometer 9.0 on the Babahoyo-Montalvo Road, Ecuador, at the geographic coordinates (UTM) 672845.34 East longitude and 9796946.78 South latitude. The experimental area is located at an altitude of 8 meters above sea level. The region has a humid tropical climate with an average annual temperature of 25.6° C, annual precipitation of 2329.8 mm, relative humidity of 82 %, and an annual average of 998.2 h of sunshine. The soil has flat topography, a clay-loam texture, and regular drainage.

The experiment was carried out during the agricultural cycle from March to July 2021, under field conditions. The plant material consisted of three Peruvian rice varieties known as HP 102 FL-El Valor, Feron, and INIA 515-Capoteña.

The fungicides used in the research were obtained from certified distributors in the country. The products and their respective codes, based on their technical specifications, are listed below: i) Renaste (Epoxiconazole 50 g L^-1 + Pyraclostrobin 133 g L^-1), code: RNST-EPY 133, manufactured by BASF, Germany. ii) Serenade Max (Bacillus subtilis strain QST 713), code: SRND-BSQ 713, registered and distributed by Bayer CropScience, USA. iii) Timorex Gold (Extract of Melaleuca alternifolia 26.8 %), code: TMRX-MEL268, distributed by STK Bio-ag Technologies, Israel. iv) Propiconazole 250 EC, code: PRPC-250EC, commonly used and locally produced, distributed by various local companies. v) Tebuconazole 250 EW, code: TBCN-250, formulated by Adama Agricultural Solutions, Israel, with local distribution under license.

The dependent variables of the research were disease incidence and severity, fungicide efficacy, and rice crop yield, while the independent variables included the rice varieties and fungicide treatments (Table 1).

The experiment consisted of 12 treatments arranged in a Randomized Complete Block Design (RCBD) with a factorial arrangement A (varieties) × B (fungicides), and three replications. Factor A corresponded to the rice varieties, while Factor B included the fungicide treatments with their respective doses. Each experimental unit (EU) consisted of an area of 4 m² (2 m × 2 m), with a separation of 1.0 m between blocks and 0.5 m between plots. To evaluate the effects of the treatments, the following parameters were measured: disease incidence and severity, fungicide efficacy, number of tillers, percentage of grains per panicle, weight of 1000 grains, and grain yield.

Disease incidence and severity were determined at 60 days after sowing (das). Incidence refers to the proportion of diseased individuals; for this, the number of diseased plants in the useful area was recorded and divided by the total number of plants in the same area, then multiplied by 1003³⁶.

Severity was determined through visual observation of the area affected by the disease, and the data obtained were applied to the equation proposed by Ivancovich et al.³⁷.

Fungicide efficacy was calculated using Abbott’s formula³⁸:

Where: E = Efficacy of the fungicide, IT = Infection in the control y It = Infection in the treatment.

To calculate the number of tillers in the useful area of each experimental plot, a 1 m² frame was used once the rice reached physiological maturity. Tillers present within this frame were counted, using the same procedure as for counting the number of panicles in each EU.

To determine the percentage of unfilled grains per panicle, five panicles were randomly selected from each EU, and the percentage of filled and unfilled grains was recorded.

The weight of 1000 grains were obtained by selecting grains from each experimental plot, ensuring they were free from insect or disease damage. The results were expressed in grams.

Grain yield was calculated based on the weight of grains collected from the useful area of each experimental plot. This weight was adjusted to 14 % moisture content and converted to kg ha^-1. The weights were standardized using the formula by Azcon-Bieto & Talon³⁹:

Where: Pu is the standardized weight, Pa is the actual weight, ha is the actual moisture content, and hd is the desired moisture content.

For the statistical analysis, the collected data were organized in electronic spreadsheets. The Shapiro-Wilk normality test was applied to each evaluated variable. Subsequently, to analyze the effect of the treatments, Tukey’s test was used with a 95 % confidence level. The statistical analyses were performed using SigmaPlot software⁴⁰. Pearson's correlation test (r) was applied to determine the relationships among the evaluated variables.

Results

Incidence of R. solani. The varieties A₁, A₂, and A₃, when combined with specific fungicides and doses, reduced the incidence of R. solani compared to the untreated control. The incidence ranged from 39.42 to 41.38 %, with variety A₃ showing the lowest number of affected plants, while variety A₁ showed the highest value. The highest incidence observed was 50.56 %. In treated plots, incidence values ranged between 36.61 and 37.17 %. No significant differences were observed in incidence for the interaction between varieties, fungicides, and doses.

In plots treated with fungicides and different doses, incidence fluctuated between 35.00 and 38.67 %, with no significant differences between treatments. In contrast, the three untreated varieties showed higher incidence values between 49.33 and 51.33 %, exceeding those observed with the application of treatments and doses, as well as their interactions. Table 2 shows the incidence of R. solani in response to the applied treatments.

Severity of R. solani. No significant differences were observed in severity among the fungicide treatments and doses, although a slight reduction in damage was noted compared to the untreated control. Severity ranged from 38.25 to 39.75 %, with the highest value recorded in variety A₁. Treated plots showed severity values between 34.11 and 39.56 %. The lowest severity was observed with treatment B₃, commonly used by local farmers. However, severity differences among treatments were not significant.

In contrast, plants without fungicide treatment (B₄) had the highest severity percentage, 43.67 %. No significant differences were observed in severity for the interactions between varieties, fungicides, and doses. Varieties A₂ and A₃ treated with B₃ showed the lowest severity percentage (33.00 %), which was lower than that of other fungicides, including untreated plots (Table 2).

Fungicide efficacy. The control percentage of R. solani according to the treatments analyzed, (Figure 1), revealed that B₃ achieved an efficacy of 27.60 %. B₂ and B₁ showed 26.80 and 26.46 %, respectively. These values did not differ significantly from each other. The coefficient of variation was 22.08 %. Among the evaluated varieties, none of the fungicide treatments exceeded 30 % efficacy for controlling R. solani.

The interaction between varieties and fungicides was not statistically different. However, Figure 2 shows that for variety A₂, treatment B₁ achieved a control of 29.49 %. This result was similar to those obtained with 28.99 and 27.98 % (B₃) in the same variety. On the other hand, for varieties A₁ and A₃, the fungicide treatments reported efficacy values ranging from 24.05 to 29 %. These values were not significantly different. Moreover, the persistence period of the fungicides exceeded 20 days.

Number of tillers. According to Table 3 and the evaluated treatments, variety A₁ had the highest number of tillers, with an average of 293.92, followed by A₃ with 286.08. Plots without fungicide application (B₄) had an average of 306.67 tillers m^-2. Treatments B₂ and B₁ resulted in 289.67 and 280.56 tillers m^-2, respectively, while B₃ produced 277.67 tillers m^-2. The latter did not differ from the number observed in plots without fungicide application.

When considering the interaction between varieties and fungicides, the highest number of tillers per square meter was recorded in A₁ without fungicide treatment, with 316.33 tillers m^-2, followed by A₂ with B₃ (309.67), and A₃ without fungicides (306.00). The lowest number of tillers per square meter was recorded in A₂ with B₂ (261.33 tillers), which was similar to the values observed in A₃ with B₃ (261.67), A₁ with B₃ (261.67), A₃ with B₁ (267.67), and A₂ with B₁ (275.00).

Yield. Table 3 shows the grain yield in kg ha^-1 for the different treatments analyzed. The yields of the varieties ranged from 5192.32 to 6395.67 kg ha^-1, with A₃ standing out as the highest-yielding variety per unit area. The yield of A₃ was significantly different from that of A₂ and A₁, whose yields were statistically similar to each other.

Figure 1 Fungicide efficacy and dosage in controlling R. solani 

Figure 2 Interaction of varieties with fungicides in the control of R. solani 

Regarding the fungicide and dose factor, B₃ and B₁ achieved the highest yields, with 5968.98 and 5786.79 kg, respectively, with no significant differences between them. However, the yield in B₃ (5968.98 kg) was significantly different from B₂ (5231.15 kg) and the untreated control (5500.44 kg).

In the interaction between varieties and fungicide treatments with different doses, variety A₃ combined with B₃, B₁, and B₂ showed the highest yields per hectare, with 6891.73, 6749.49, and 6257.44 kg, respectively. However, the yield of LAP-003-2020 with B₁ did not differ significantly from the absolute control (5684.00 kg).

On the other hand, the lowest yields per hectare were obtained with the combinations A₁ y B₂, A₂ y B₂, and A₁ y B₁, which reached 4692.05, 4743.95, and 4804.05 kg, respectively. These values were not significantly different from their respective untreated controls.

Unfilled grains. In Figure 3A, variety A₃ presented the lowest number of unfilled grains per panicle, with 16.14 %. Figure 3B shows the percentage of unfilled grains in response to different fungicide treatments and their respective doses. In this variable, the percentage of empty grains per panicle ranged from 18.79 to 23.85 %. The lowest value was recorded in B₃, which was significantly different from B₁, which reported 23.85 % unfilled grains. The absolute control recorded 22.17 % empty grains, but this value did not differ significantly from the others.

In the interaction between varieties and fungicide treatments with different doses (Figure 3C), A₃ without fungicides presented 14.68 % unfilled grains, followed by A₃ with B₂ (15.66 %), A₃ with B₁ (16.26 %), and A₁ with B₃ (16.28 %). The percentages observed in these treatments did not show significant differences among them.

Discussion

The results obtained demonstrate that the integration of biological and chemical fungicides, combined with the appropriate selection of rice varieties, can play a key role in reducing the incidence and severity of R. solani under field conditions. Although no treatment exceeded 30 % efficacy, B₁ and B₃ stood out compared to the untreated control, supporting their inclusion as complementary strategies within integrated management programs. This moderate efficacy may be attributed to factors such as high inoculum pressure in tropical environments, intensive use of nitrogen fertilizers, and varietal susceptibility, which highlights the need for multifactorial approaches^41-44. Therefore, the implementation of comprehensive disease management measures is essential. These strategies include the use of resistant or tolerant materials, optimal planting times, balanced nutrition, crop rotation, incorporation of antagonistic microorganisms, and the rational application of fungicides^45-48. Collectively, these measures have been associated with a reduction in R. solani symptom incidence, approximately 8 % lower than chemical control, especially during the early growth stages and vegetative development of the crop⁴⁹.

The lowest incidence and severity of the disease were observed with B₁ and B₃, consistent with previous studies. The efficacy of Bacillus subtilis and Melaleuca alternifolia extract highlights their ability to control pathogens through antibiosis, induction of systemic resistance, and competition for ecological niche^26,33-34.

Several studies have confirmed that the use of B. subtilis as a biofungicide can significantly improve rice resistance to soilborne pathogens. For example, Lahlali et al.³³ reported that Serenade Max, based on B. subtilis strain QST 713, induces systemic resistance and reduces disease severity in canola. Similarly, Boukaew et al.²⁸ indicated that combining biocontrol agents with chemical fungicides can enhance control efficacy against Rhizoctonia solani without causing environmental harm or inducing resistance. In another study, Rashid et al.²⁷ reported that biopesticides based on plant extracts and beneficial bacteria were effective in reducing the incidence of sheath rot disease in rice in Bangladesh, with results comparable to conventional treatments.

Figure 3 Empty grains (%) and full grains (%) in varieties (A), fungicide treatments and doses (B) and interaction between varieties and fungicides and doses (C). Bars with a letter in common are not significantly different (p>0.05) according to the Tukey test at 5 % probability 

Additionally, Yang et al.²⁹ noted that bacterial strains isolated from bovine manure had potential as biological control agents against R. solani, highlighting the feasibility of using native microorganisms as a sustainable and local solution. This body of evidence strengthens the notion that an integrated management approach based on biological products and their interaction with plant material constitutes a solid strategy to reduce pathogen impact and improve productive stability in tropical rice systems.

Variety A₃ exhibited the least damage, and when combined with treatment B₁, it achieved the highest yield (6749.49 kg ha^-1) and the lowest percentage of unfilled grains (16.14 %), demonstrating a favorable interaction between the plant material and the phytosanitary strategy used. Similar findings have been previously reported, emphasizing the importance of integrating management practices, including the appropriate selection of tolerant varieties and complementing their management with fungicides having different modes of action to substantially reduce disease incidence in the field⁵⁰.

This suggests that the selection of rice variety, along with the appropriate biological fungicide, is crucial to maximizing control of R. solani. The interaction between host, pathogen, and biological agent requires further investigation to better understand these mechanisms, which aligns with the observations of Quiroz Ojeda et al.⁷, who noted that R. solani interacts with its hosts through biological, genetic, and pathogenic pathways fundamental to understanding host pathogen relationships across different pathosystems.

The percentage of unfilled grains affects crop yield by compromising photosynthesis, nutrient translocation, and grain filling⁵¹. Variety A₃ showed the lowest percentage of unfilled grains regardless of the treatment applied. A similar trend was observed in variety A₁ with the application of Propiconazole and Tebuconazole, which reported 16.28 % of unfilled grains per panicle. This suggests that the percentage of unfilled grains could be influenced by biotic factors (pathogens, insect pests, and genetic traits) and abiotic factors (low temperature, relative humidity, and cloud cover)^45,52.

On the other hand, synthetic fungicides such as Propiconazole and Tebuconazole continue to show a certain level of effectiveness, as evidenced by studies conducted by Pérez Vicente et al.⁵³. However, their continued use presents risks of resistance development in pathogens and may lead to undesirable environmental impacts. Therefore, the results obtained in this study support the progressive transition from synthetic molecules to bioproducts, in line with agroecological approaches. This research stands out for integrating biological products with rice varieties currently used by farmers in the field, generating valuable information and strengthening the decision-making process for producers interested in more sustainable agricultural practices in the rice-growing areas of the Ecuadorian coast.