Biocontrol potential of Trichoderma harzianum and Bacillus subtilis against Phytophthora infestans in two potato varieties

Potencial de biocontrole de Trichoderma harzianum e Bacillus subtilis contra Phytophthora infestans em duas varidades de batata

Abstract

The potato is one of the most important crops in the world, due to its nutritional properties. However, Phytophthora infestans causes a significant reduction in potato growth and productivity. The use of chemical fungicides is the main control, consequently, the virulence and aggressiveness of the phytopathogen increases, and the use of biological agents with antagonistic activity is necessary. The objective was to determine the biocontrol effect of Trichoderma harzianum and Bacillus subtilis in a commercial and an improved potato variety as a sustainable strategy for the control of Phytophthora infestans in the highlands of Peru. The experiment was carried out in the field during two production seasons (2022-2023 and 2023-2024), with a 2x2x2 factorial scheme, referring to the variety (commercial variety Yungay and improved variety CIP 302295.32), two biological agents (T. harzianum and B. subtilis at a dose of 1 kg ha^-1 and without application). The results revealed that plants subjected to P. infestans show a significant reduction in potato growth and productivity. However, the combined use of soil-applied T. harzianum and foliar-applied B. subtilis on the improved variety CIP 302295.32 reduced disease severity by 80.78 and 83.78% compared to the control in the two seasons, yielding more than 80% control efficiency of P. infestans. Likewise, this combination produced a significant effect on plant height (59.27 and 60.34%) in both seasons, significant increase in the number of commercial tubers per plant (84.66 and 88.89%), notable improvement in commercial weight per plant (84.88 and 90.11%) and the commercial yield exceeded the control by 85.45 and 90.12% in both seasons. Therefore, this study has clearly demonstrated the antagonistic effect of P. infestans with the combined use of the fungus "T. harzianum" applied to the soil and the endophytic bacterium "B. subtilis" applied via foliar and this effect was more significant in the improved variety CIP 302295.32, in addition, this combination produces a significant effect on the growth and productivity of the potato.

Keywords:
aggressiveness; antagonism; bacteria; fungus; virulence

Resumo

A batata é uma das culturas mais importantes do mundo, devido às suas propriedades nutricionais. No entanto, a Phytophthora infestans causa uma redução significativa no crescimento e na produtividade da batata. O uso de fungicidas químicos é o principal controle e, consequentemente, a virulência e a agressividade do fitopatógeno aumentam, sendo necessário o uso de agentes biológicos com atividade antagônica. O objetivo deste estudo foi determinar o efeito de biocontrole do Trichoderma harzianum e do Bacillus subtilis em uma variedade de batata comercial e em uma variedade melhorada, como estratégia sustentável para o controle da Phytophthora infestans nas regiões andinas do Peru. O experimento foi realizado no campo durante safras produtivas (2022-2023 e 2023-2024), com um esquema fatorial 2x2x2, referente à variedade (variedade comercial Yungay e variedade melhorada CIP 302295.32), dois agentes biológicos (T. harzianum e B. subtilis na dose de 1 kg ha^-1 e sem aplicação). Os resultados revelaram que as plantas submetidas à P. infestans apresentaram uma redução significativa no crescimento e na produtividade da batata. No entanto, o uso combinado de T. harzianum aplicado no solo e B. subtilis aplicado via foliar na variedade melhorada CIP 302295.32 reduziu a gravidade da doença em 80,78% e 83,78%, em comparação com o controle nas duas safras, produzindo mais de 80% de eficiência de controle de P. infestans. Da mesma forma, essa combinação produziu um efeito significativo na altura da planta (59,27% e 60,34%), um aumento significativo no número de tubérculos comerciais por planta (84,66% e 88,89%), uma melhoria notável no peso comercial por planta (84,88% e 90,11%) e o rendimento comercial superou o controle em 85,45% e 90,12% em ambas as safras. Portanto, este estudo demonstrou claramente o efeito antagônico do P. infestans com o uso combinado do fungo T. harzianum aplicado ao solo e da bactéria endofítica B. subtilis aplicada via foliar, sendo esse efeito mais significativo na variedade melhorada CIP 302295.32. Além disso, essa combinação produz um efeito significativo no crescimento e na produtividade da batata.

Palavras-chave:
agressividade; antagonismo; bactérias; fungos; virulência

1. Introduction

Potato (Solanum tuberosum L.) is one of the main crops of major production and consumption worldwide (Almarinez et al., 2023), because the tuber has a high nutritional value and provides organic components beneficial to human health (Djousse et al., 2025). The potato tuber is also rich in starch, vitamins, amino acids and has a wide adaptability to various latitudes (Davis et al., 2024), making it a valuable crop that supports food security for a large part of the human population (Piekutowska and Niedbała, 2025).

World potato production was 360 million tonnes recorded in 150 countries (Southern et al., 2025). Meanwhile, in Peru, production was 6.58 million tonnes, with an average yield of 19.5 t ha^-1, making it the largest potato producer in Latin America (INEI, 2024). Potato yield, however, is modulated by the interaction of genotype and environment, with the temperature factor having the greatest effect on plant growth and development (Southern et al., 2025). It should be noted that potato production and sown area is concentrated in the Peruvian highlands, which are characterized by low temperatures and high relative humidity (Wilches et al., 2022).

However, the main problem in potato productivity is late blight, a disease caused by the oomycete, Phytophthora infestans (Mont de Bary, 1840), which causes large economic losses worldwide (Wei et al., 2024). This disease starts its infection from the initial stages of the crop, since from before sowing, the phytopathogen hibernates in the soil through oospores and infects the tuber seed, starting its infection cycle, or it also infects the plant through asexual zoosporangia, which are transmitted by wind from diseased plants (Lastochkina et al., 2022).

Management of late blight in most potato growers is based on chemical control, which includes the use of fungicides with various chemical active ingredients (Leesutthiphonchai et al., 2018). However, the intensity of infection of P. infestans is so high that the potato grower increases the dosage of fungicides and the frequency of application, so that repeated use of fungicides causes the pathogen to mutate and generate new, more aggressive genotypes, capable of quickly overcoming resistance to the chemical fungicide (Islam et al., 2022).

In addition, the aggressiveness of Phytophthora infestans in potatoes is due to the favorable climatic conditions where they are being grown, such as constant rainfall and low temperatures, which influence the rapid spread and germination of this phytopathogen (Wilches et al., 2022). It should be noted that sporangium maturation on diseased plants occurs in the afternoon, coinciding with dew on the foliage, low light and low temperatures, which consequently release zoospores (Bassani et al., 2020). These zoospores are wind-borne and with the use of their flagella swim a few centimeters’ through moist soil towards the seed tuber, healthy or diseased plants, being favored if rainfall is constant or by irrigation water (Zhang et al., 2025), encyst in them, subsequently germinate and enter the stem tissue or leaves of the plant (Grove et al., 1985) and initiate the infection process (Evenhuis et al., 2024).

At the end of the potato phenological cycle, they produce oospores (sexual spore) and hibernate in the soil, until they detect the growth of seed tuber sprouts and initiate a new cycle of infection (Legrifi et al., 2025). As a result, P. infestans is considered a foliar and root colonizer in potato, demonstrating its ability to overcome control strategies, including suppression of host defenses and application of chemical fungicides (Leesutthiphonchai et al., 2018).

Despite this, potato growers resort to chemical control of late blight because it works quickly and is readily available, which implies indiscriminate and excessive use of chemical fungicides, i.e. the farmer increases doses and application frequencies (Tirado-Malaver et al., 2024), resulting in a negative impact on the environment and on the health of both the farmer and consumers (Ivanov et al., 2021). Thus, the use of chemical fungicides can be ineffective, due to environmental conditions that influence the development and spread of the phytopathogen and its resistance to the fungicide (Enciso-Rodríguez et al., 2018). Therefore, innovative and effective control measures are required to minimise or eliminate the use of chemical fungicides and suppress the effect of P. infestans on potato (Zhu et al., 2024).

In the face of these problems, the best strategy will be biological control, which is based on the use of beneficial microorganisms, characterized by their antifungal capacity and environmental friendliness (Rigobelo et al., 2024). However, in the agricultural market, there is a great diversity of these beneficial microorganisms, which have different control efficiency rates (El-Hasan et al., 2022). Previous studies carried out in the field confirmed that, among the bio-controllers, Trichoderma harzianum and Bacillus subtilis reduce the attack of P. infestans on potato due to their anti-oomycete activity (Chinheya et al., 2023).

Numerous investigations have shown that the application of endophytic strains of Bacillus subtilis reduces the incidence and severity of late blight disease through mechanisms of action such as the synthesis of endosporic components, mycotoxins and enzymes that degrade the oomycete cell wall (Kumbar et al., 2019). In addition, this microorganism promotes plant growth (Cochard et al., 2022). In a laboratory experiment, Zhang et al. (2023) reported that the application of a commercial preparation of B. subtilis in a dual confrontation effect in vitro inhibited mycelial growth of P. infestans by more than 80%. While Sorokan et al. (2020) reported that treating potato foliage with B. subtilis resulted in up to 20% leaf area with late blight symptoms compared to the control which had 70% leaf area with the disease.

On the other hand, several studies indicate that the application of Trichoderma harzianum controls late blight through the production of antimicrobial secondary metabolites, mycoparasitism, competition for space and the induction of systemic resistance in the plant (Li et al., 2023). In this regard, Alfiky et al. (2023) have found in dual culture confrontation trials that T. harzianum significantly inhibited mycelial growth and viability of P. infestans zoospores from 53 to 95%. In contrast, Napolitano et al. (2024), in field studies, showed that the application of T. harzianum on potato reduced late blight disease by less than 54.4% compared to the control. In addition, it promoted plant growth and increased tuber number and yield (Napolitano et al., 2024).

These findings suggest that applying Bacillus subtilis and Trichoderma harzianum have a control effect on late blight disease under in vitro conditions, whereas under field conditions, these microorganisms tend to vary in their control effect. This lack of consistency in the biocontrol activity of these micro-organisms should include the study of individual and joint applications and the effect of the genetic composition of an improved potato variety on the plant pathogen. Bearing in mind that the effectiveness of integrated management is the result of the interaction between pathogen, plant and environment (Campos et al., 2021).

Thus, the genetic composition of the plant plays an important role in the control of the phytopathogen (Manuela, 2018). However, commercial potato varieties in Peru have lost their phytosanitary properties over time, making them susceptible to late blight, which is why genetic improvement has been initiated through germplasm based on native potatoes, which have a high level of genetic resistance, which have accumulated over time and the high pressure of blight in the Peruvian highlands, being an important plant genetic resource for transferring these resistance genes and developing new improved varieties (Tirado-Malaver et al., 2021). However, the virulence of P. infestans has the ability to suppress the genetic resistance (major genes) of improved potato varieties, necessitating the development of varieties with partial resistance (Vásquez-Castillo et al., 2022). Thus, breeding new varieties with partial resistance could reduce the massive and excessive use of chemical fungicides (Berindean et al., 2024).

Therefore, the present study aims to determine the biocontrol effect of Trichoderma harzianum and Bacillus subtilis on a commercial and an improved potato variety as a sustainable strategy for the control of Phytophthora infestans in the northern highlands of Peru.

2. Materials and Methods

The experiment was carried out in the experimental plots of ACOMEBIO (Asociación para la conservación del medio ambiente y la biodiversidad), located in the locality of El Pargo in the province of Cutervo, Cajamarca, geographically located at 6°22'53" N & 78°49'06" W, and at an altitude of 2617 m above sea level.

2.1. Methodology

The research was carried out using a completely randomised block experimental design with a 2 x 2 x 2 factorial scheme, which consisted of the effect of three factors (variety, biocontrolling beneficial fungus and biocontrolling endophytic bacteria) with two levels each. In the first factor, two potato varieties were studied; an improved variety (clone CIP 302295.32), this genotype comes from CIP and was selected for its agronomic attributes through a series of tests in different locations and years, in addition to presenting partial resistance to late blight (Tirado-Malaver et al., 2020). Likewise, the commercial variety Yungay was used, as this variety is one of the most widely consumed in Peru. As for the second factor, two levels of the beneficial biocontrol fungus "Trichoderma harzianum" strain 1123 were used (with and without application) and the third factor consisted of two levels of an endophytic biocontrol bacterium "Bacillus subtilis" strain 2233 (with and without application), thus obtaining eight treatments with three replicates.

2.2. Description of treatments

The treatments used in this study are the results of combining the levels of each of the three factors, as shown in Table 1.

Table 1
Description of treatments.

Treatments	Variety	Trichoderma harzianum	Bacillus subtilis	Description
		......L ha-1......	...kg ha-1...
T0	Yungay	Not applicable	Not applicable	(Yungay + not T. harzianum + not B. subtilis)
T1			With application	(Yungay + not T. harzianum + B. subtilis)
T2		With application	Not applicable	(Yungay + T. harzianum + not B. subtilis)
T3			With application	(Yungay + T. harzianum + B. subtilis)
T4	Clon CIP 302295.32	Not applicable	Not applicable	(CIP 302295.32 + not T. harzianum + not B. subtilis)
T5			With application	(CIP 302295.32 + not T. harzianum + B. subtilis)
T6		With application	Not applicable	(CIP 302295.32 + T. harzianum + not B. subtilis)
T7			With application	(CIP 302295.32 +T. harzianum + B. subtilis)

2.3. Conduct experiment

The experiment was conducted in two potato production seasons (2022-2023 and 2023-2024). A total of 24 experimental units (3 m x 4 m) were established, with each plot containing 40 plants, 10 of which were in each furrow. Irrigation was carried out using the gravity system. Fertilization was carried out in two stages, at the beginning of sowing with 50% nitrogen (N), 100% phosphorus (P) and potassium (K), and the second stage was carried out with the application of the remaining 50% of N. The sowing was carried out with two different fertilizers. Sowing was carried out using two different fertilizers and two potato varieties: an improved variety (clone CIP 302295.32) and a commercial variety (Yungay). Microbial inoculation was carried out through the preparation of each biocontroller as described below:

T. harzianum strain 1123 (1.5x1010 conidia g^-1) was applied by making a solution of 250 g of the commercial strain with 0.5 kg of molasses, chlorine-free water at pH 5.57 and homogenizing it for 30 min in a tank of 36 L volume, allowing activation of the biocontrol fungus, then 50 mL of the solution was applied at the foot of the plant according to Bader et al. (2020), this application was carried out in four moments, from sowing to the hilling of the potato (32 days after sowing), reaching an accumulated dose of 1 kg ha^-1 of the commercial strain.

B. subtilis strain 2233 was applied by preparing a solution of 250 g the commercial strain and a bacterial activator in water without chlorine in tank of 36 L volume, then it was applied at the foot of the plant and by foliar application according to Efthimiadou et al. (2020), this bacterium was applied in four moments reaching an accumulated dose of 1 kg ha^-1 of commercial product.

While the joint application of these two beneficial microorganisms, once each microorganism was activated, T. harzianum strain 1123 was applied at the foot of plant at a cumulative dose of 1 kg ha^-1 the commercial strain and B. subtilis strain 2233 was applied by foliar application at a cumulative dose of 1 kg ha^-1 of commercial strain. In the control, sterile distilled water was used instead of bio-controllers. Noted that no chemical fungicides were applied during the experiment.

2.4. Inoculation of Phytophthora infestans

The experiment was carried out in the field, because in laboratory conditions (in vitro), by means of the confrontation test between the biocontrollers and the phytopathogen on potato dextrose agar (PDA) plates, they do not provide relevant information, since the aggressiveness and virulence of P. infestans depends on the climatic conditions and the farmer's management, which implies that the phytopathogen presents the suppression of host resistance, resistance to fungicides or evasion of any control management (Islam et al., 2022), whereas in vitro bio-controllers have high percentage of blight inhibition (Chinheya et al., 2023). Therefore, the field trial was conducted.

Phytophthora infestans, provided by the ACOMEBIO laboratory, was collected from organs of diseased plants with late blight symptoms, then cultured for five days at 28°C on PDA plates, and mycelia were extracted from the colonies formed with the use of a 0 punch 5cm in diameter, then inoculated into a 250 mL flask with potato dextrose broth and shaken at 180rpm for three days to obtain sufficient P. infestans hyphae for inoculation according to Zhang et al. (2023).

Inoculation of P. infestans was prior to planting by immersing the seed tubers in a suspension of sporangia dissolved in water for 30 minutes according to Tirado-Malaver et al. (2020). Foliar application to the plants was not necessary, because this field, according to its history, has been continuously cultivated with potatoes for years and has been constantly contaminated with this phytopathogen and even high severity and dead plants have been reported, since this field is in an area with a favorable climate for its development (Tirado-Malaver et al., 2024).

2.5. Assessment of disease and biometric parameters in potato

The severity of late blight was evaluated in plants after the application of the treatments, taking readings every seven days, starting from day 35, 42, 49, 56, 63, 70, 77 and 85 days after sowing (DDS) of the potato during two growing seasons, calculating the severity by using a numerical scale from 0 to 9 of the International Potato Centre (Henfling, 1987), assigning the scores according to the percentage of the infected leaf area: 1= < 10%, 2= 1 - 10%, 3= 11 - 25%, 4= 26 - 40%, 5= 41 - 60%, 6= 61 - 70%, 7= 71 - 80%, 8= 81 - 90% y 9= > 90%. The area under the disease progress curve (AUDPC) was also assessed using the area of damaged tissue, using the Equation 1 proposed by Fry (1978).

$AUDPC={\displaystyle \sum }_{i=1}^n(\frac{X_{i+1}+X_i}{2})\left[t_{i+1}-t_i\right]$ (1)

Where xi+1 and xi determines the lesion size over time from ti+1 and ti, n is the total number of observations over time, using the severity value of the commercial variety Yungay used as susceptible control, calculating with the number of days from the last reading of the assessment minus the first reading times 100, this value standardized so its value has no units, as proposed by Fry (1978). The percentage of control efficacy of each treatment was evaluated by subtracting the highest severity scale and the value of the severity scale of the treatment evaluated from the highest severity scale by 100, the result was expressed in %, as established by the Forbes et al. (2014).

Regarding the biometric parameters of the plant, the following were evaluated: plant height, number of non-commercial tubers per plant and commercial tubers per plant, weight of commercial tubers per plant and commercial yield.

2.6. Statistical analysis

The data obtained from the variables related to the disease and the biometric parameters of the potato were processed using the statistical package SAS (Statistical Analysis System). To detect differences between treatment means, analysis of variance and comparison of means using Tukey's test at 5% significance was used. Additionally, regression analysis was performed to measure the effect of the microorganisms plus the improved variety on late blight control. Data were expressed as mean (µ) ± standard deviation (σ).

3. Results and Discussion

3.1. Variables associated with the disease

Table 2 shows the results of the mean squares of the analysis of variance for the variables associated with late blight and its control. Highly significant differences (p<0.01) were observed for the main factors variety, T. harzianum (TH) and B. subtilis (BS) in all variables, during the two seasons. Regarding the interaction of factors, the variables AUDPC, severity and control efficiency showed significant effects in the interaction between variety x TH, variety x BS and TH x BS in both seasons, indicating that the varieties were affected by the application of TH and BS, as well as, TH and BS. Likewise, the interaction of three factors showed statistical significance for the variables in both seasons, indicating that as time passes, the application of TH and BS on potato varieties has a significant effect on late blight disease progress, severity and control efficiency.

Table 2
Analysis of variance for the area under the disease progress curve (AUDPC), severity scale and the percentage of efficiency control.

Sources of variation	MS
	DF	AUDPC-2022-2023	AUDPC-2023-2024	Severity -2022-2023	Severity -2023-2024	Effic 2022-2023	Effic 2023-2024
Block	2	900.78ns	30830.47ns	0.05ns	0.65ns	6.47ns	80.35ns
Variety (V)	1	1850926.04**	2653350.00**	12.41**	16.04**	1530.72**	1978.90**
T. harzianum (T)	1	9362504.17**	11302537.50**	62.73**	66.80**	7736.09**	8241.77**
B. subtilis (B)	1	4120959.38**	4330751.04**	27.52**	25.05**	3400.31**	3094.69**
V x T	1	338437.50**	478837.50*	2.26**	2.82*	279.14**	346.48*
V x B	1	223301.04*	455126.04*	1.47*	2.68*	182.11*	331.60*
T x B	1	297037.50**	307134.38*	2.02**	1.90*	248.52¨¨	235.69*
V x T x B	1	192604.27*	291501.04*	1.27*	1.59*	156.32*	198.09*
Error	14	29397.81	62516.18	0.21	0.37	26.1	45.49
Total	23
CV (%) =		8.69	12.56	9.01	12.54	11.79	14.59

• Note: area under the disease progress curve (AUDPC), severity scale (Severity); percentage efficiency control (Effic); CV (%)= Coefficient of Variation, GL= Degrees of Freedom, MS= Mean Squares; ns= not significant (p>0.05).
• ^*
  = significant (p<0.05).
• ^**
  = highly significant (p<0.01).

The results of the comparison of means (Table 3) show that the treatment T7 (CIP 302295.32 + TH + BS) presented statistically lower values than the other treatments for the variables; AUDPC and disease severity, in the 2022-2023 season, indicating that T7 significantly halted the advance of late blight disease by less than 80.74% compared to the control (T0: Yungay + without TH + without BS). Whereas, for the second season, T7 and T3 (Yungay + TH + BS), significantly arrested late blight disease progress by minus 83.74 and 74.92% relative to the control respectively.

Table 3
Multiple comparison of means according to Tukey's test for comparison of treatment means for area under the disease progress curve, relative area under the disease progress curve, severity scale and percentage efficiency.

Treat	AUDPC-2022-2023	AUDPC-2023-2024	Severity -2022-2023	Severity -2023-2024	Effic 2022-2023	Effic 2023-2024
	…….n° ……..		……. scale ……..		……. % ……..
T7	670.83 ± 114.48 a*	603.33 ± 90.19 a	1.73 ± 0.26 a	1.47 ± 0.24 a	80.79 ± 2.93 a	83.68 ± 2.63 a
T3	975.00 ± 103.46 b	930.83 ± 110.72 a	2.52 ± 0.23 a	2.27 ± 0.32 a	72.04 ± 2.60 a	74.81 ± 3.52 a
T6	1708.33 ± 39.60 c	1624.17 ± 319.26 b	4.41 ± 0.08 b	3.92 ± 0.65 b	50.95 ± 0.89 b	56.39 ± 7.25 b
T2	2040.00 ± 56.16 d	2061.67 ± 123.16 bc	5.27 ± 0.22 c	5.03 ± 0.55 c	41.40 ± 2.42 c	44.14 ± 6.11 c
T5	2084.17 ± 72.87 d	2140.00 ± 127.10 cd	5.39 ± 0.18 c	5.20 ± 0.37 c	40.16 ± 2.00 c	42.20 ± 4.08 c
T4	2318.33 ± 260.91 de	2267.50 ± 186.42 cd	5.99 ± 0.70 cd	5.50 ± 0.27 cd	33.41 ± 7.81 cd	38.94 ± 3.05 cd
T1	2505.00 ± 179.83 e	2591.67 ± 296.13 d	6.48 ± 0.56 d	6.34 ± 1.03 d	27.98 ± 6.25 d	29.62 ± 11.44 d
T0	3483.33 ± 59.49 f	3710.83 ± 190.28 e	9.00 ± 0.00 e	9.00 ± 0.00 e	0.00 ± 0.00 e	0.00 ± 0.00 e

• Note: area under the disease progress curve (AUDPC), severity scale (Severity); percentage efficiency (Effic); Treat = Treatments, %= Percentage.
• ^*
  = different letters indicate significance according to Tukey's test at p<0.05.

Likewise, T7 and T3 in the first season obtained the lowest severity values, with 1.73 (1 to 10% of the infected leaf area) and 2.52 (11 to 25% of the infected leaf area), respectively. This indicates that applications of the microorganisms together on the two varieties reduced disease severity by 80.78 and 72% compared to the control. While in the second season T7 and T3 reported less than 10% of the leaf area infected, reducing severity by less than 83.67 and 74.78% compared to the control respectively (Table 3).

Regarding control efficiency (Table 3), the results show that T7 and T3 reached the highest values, with 80.79% and 72.04% control efficiency in the first season and in the second season T7 and T3 obtained statistically higher values than the other treatments with 83.68 and 74.81 ± 3.2% control efficiency of late blight respectively. It can be observed that as time passes, the joint application of T. harzianum and B. subtilis on potato varieties controls late blight disease. It is also observed that the single application of T. harzianum on the improved variety narrowly exceeded 50% control efficiency in both seasons, while the single application of B. subtilis on the improved variety did not reach 50% control efficiency.

In Figure 1A and B, it can be seen that as time goes by, without the application of the microorganisms, the advance of the late blight disease is significant, reaching 100% of the leaf area affected by P. infestans in both seasons. Likewise, when T. harzianum is applied individually to both varieties, it relatively reduces the advance of the disease at the beginning of the season, but as time goes by, the advance of the disease increases in both varieties, exceeding 50% of the leaf area affected by P. infestans from 70 and 77 DDS in both seasons. As for the individual application of B. subtilis in the Yungay variety, from 56 DDS the plants show more than 50% of area affected by blight, while in the improved variety, it reaches more than 50% of symptoms from 70 DDS in both seasons.

Figure 1
Percentage of leaf area affected by P. infestans on potato in the campaign 2022-2023 (A) and campaign 2023-2024 (B).

Meanwhile, the joint application of T. harzianum via soil and B. subtilis via foliar, shows that in the commercial variety Yungay it reduced the advance of the disease from the beginning of the season, reaching 30% of leaf area affected by late blight at the end of the season in both seasons, while in the improved variety CIP the joint application of both microorganisms reduced the advance of the disease during the whole season, reaching 25% of leaf area affected by P. infestans at the end of the season in both seasons.

3.2. Biometric parameters of potato

The analysis of variance of the variables associated with potato agronomic performance (Table 4) showed that the main factors and the interaction between factors had a significant effect (p<0.05) on plant height (PH) and number of commercial tubers per plant (NCTP) in both seasons. This indicates that the varieties responded to the application of the micro-organisms, increasing plant growth and the number of tubers per plant. While the variable number of non-commercial tubers per plant (NNCTP) shows no significance (p>0.05) in the main factors "variety" and "B. subtilis" and in the interactions between two and three factors, in the two seasons. With respect to the variables commercial tuber weight per plant (CTWP) and commercial yield (CY), highly significant differences were observed (p<0.01) in the main factors, but for the interaction between variety x T. harzianum and variety x B. subtilis, it was not significant (p>0.05) in both seasons and the interaction of T. harzianum x B. subtilis, shows significance, which indicates that the joint applications of these microorganisms produce significant effects on the potato. However, in the three-factor interaction, significance is observed for CTWP and CY in the second season, indicating that as time passes, both potato varieties show significant effects to the combined application of T. harzianum and B. subtilis.

Table 4
Analysis of variance for plant height (PH), number of non-commercial per plant (NNCTP), number commercial of tubers per plant (NCTP), commercial tubers weigh per plant (CTWP) of potatoes and commercial yield (CY).

SV	MS
	DF	PH	PH	NNCTP	NNCTP	NCTP	NCTP	CTWP	CTWP	CY	CY
		2022-2023	2023-2024	2022-2023	2023-2024	2022-2023	2023-2024	2022-2023	2023-2024	2022-2023	2023-2024
Block	2	2.77ns	1.09ns	3.50ns	1.04ns	1.04ns	2.17ns	16753.76ns	33464.67ns	18.61ns	74.40*
V	1	1407.06**	1132.14**	0.04ns	1.04ns	24.00**	20.17**	386842.04**	338247.53**	429.85**	375.88**
T	1	1908.88**	1135.34**	5.04**	12.04**	42.67**	73.50**	681077.04**	1167944.64**	756.79**	1297.72**
B	1	1308.92**	1657.85**	0.38ns	0.04ns	32.67**	37.50**	523626.04**	615168.24**	581.84**	683.52**
V x T	1	107.10**	823.80**	1.04ns	2.04ns	1.50*	1.50*	19722.67ns	23387.53ns	21.91ns	26.00ns
V x B	1	77.54**	467-20**	1.04ns	1.04ns	1.50*	1.40*	19722.67ns	26880.43ns	19.90ns	29.88ns
T x B	1	292.18**	177.94**	0.38ns	9.37*	4.17*	4.17*	70850.67*	67161.93*	78.74ns	74.62*
V x T x B	1	58.91*	34.01*	1.04ns	0.37ns	0.67ns	0.17*	12927.04ns	6441.93*	14.37ns	7.15*
Error	14	10.28	7.05	0.26	0.57	1.04	0.5	15882.26	8243.95	17.65	9.16
Total	23
VC (%) =		5.98	4.71	19.5	20.94	20.08	13.91	19.84	14.17	19.81	14.17

• Note: SV=Source of variation, VC= Coefficient of Variation, DF= Degrees of Freedom, MS= Mean Squares, V= Variety, T=T. harzianum, B=B. subtilis; ns= not significant (p>0.05).
• ^*
  = significant (p<0.05).
• ^**
  = highly significant (p<0.01).

Since no interaction between the three factors was found for the variables NNCTP (both seasons), NCTP, CTWP and CY in the first season, main effects analysis was performed, as shown in Figure 2. When analyzing the NNCTP (Figure 2A), the levels of the variety factor were statistically similar, with the T. harzianum factor showing significance at both levels, which implies that the number of non-commercial tubers is greater when the microorganism is not applied and as time goes by there is an increase of 12% with respect to the previous season, whereas with the levels of the B. subtilis factor no significant differences were found.

Figure 2
Simple effect of varieties, Trichoderma harzianum, Bacillus subtilis, for number of non-commercial tubers per plant (A), number of commercial tubers per plant (B), commercial weight per plant (C) and commercial yield (D) from two seasons. Note: Means with a common letter are not significantly different according to Tukey's test (p < 0.05).

With respect to the NCTP (Figure 2B), significance is observed for varieties, being the improved variety the one that obtained the highest number of commercial tubers per plant, exceeding the commercial variety in 32.89% in the first season and 30.5% in the second season, T. harzianum factor shows that in the first season with application of the microorganism the increase is 41.59% with respect to the level without application and in the following season there is an increase of 6% in relation to the previous season and with B. subtilis, the level with application is 37.28 and 39.49% higher than the level without application in both seasons.

As for the CTWP variable (Figure 2C), the improved variety outperforms the commercial variety by 33.31 and 31.26% in both seasons, for the T. harzianum factor, in the first season, with application there is an increase of 41.92% in comparison with the level without application and in the following season, the application of the microorganism presented an increase of 6.69% in relation to the previous season, likewise, with application of B. subtilis the commercial weight per plant is higher by 37.73 and 39.98% than without application, during the two seasons.

Regarding the CY (Figure 2D), it is observed that the improved variety outperforms the commercial variety by 33.29% in the first season and 31.28% in the second season. The application of T. harzianum produced an increase of 41.92% compared to no application of this microorganism and the following season the increase was 6.69% compared to the previous season. With respect to B. subtilis, the level with application was 37.74 and 40% higher than without application in both seasons.

Table 5 shows that T7 and T3 were significantly superior to the other treatments for PH, with 75.57 and 71.21 cm respectively, outperforming the control by 59.27 and 56.78% in the first season, in the following season no significant changes were observed. Regarding the NNCTP, it is observed that all the treatments obtained a range of 2 to 4.33 non-commercial tubers.

Table 5
Multiple comparison of means according to Tukey's test for plant height (PH), number of non-commercial per plant (NNCTP), number commercial of tubers per plant (NCTP), commercial tubers weigh per plant (CTWP) of potatoes and commercial yield (CY).

Treat	PH	PH	NNCTP	NNCTP	NCTP	NCTP	CTWP	CTWP	CY		CY
	2022-2023	2023-2024	2022-2023	2023-2024	2022-2023	2023-2024	2022-2023	2023-2024	2022-2023		2023-2024
	..................µ ± σ..............						...........g plant-1...........			............t ha-1.............
T7	75.57±1.62a*	72.16 ± 1.41a	2.33 ± 0.47b	1.33 ± 0.47 c	8.67 ± 0.47 a	9.0 ±0.82a	1098.67 ± 51.17 a	1144.8 ± 108.73a	36.62 ± 1.71a		38.16 ± 87.63a
T3	71.21 ±1.77 a	71.23 ± 0.81a	2.00 ± 0.82b	1.67± 0.47 c	7.33 ± 1.25 ab	8.0 ±0.82a	913.1 ± 155.28 ab	1004.3 ± 102.47a	30.43 ± 5.18ab		33.47 ± 122.58a
T6	60.55 ±3.00 b	62.06 ± 1.29b	2.33 ± 0.47b	2.67 ± 0.47 bc	5.67 ± 0.47 bc	6.0 ±0.82b	705.5 ± 58.69 bc	753.1 ± 102.47b	23.52 ± 1.96bc		25.10 ± 100.08b
T5	58.12 ±3.22 b	60.54 ± 1.46b	2.67 ± 0.47ab	4.33 ± 0.47 a	5.33 ± 0.94 cd	5.0±0.82bc	664.0 ± 117.38 cd	627.5 ± 102.47bc	22.13 ± 3.91cd		20.92 ± 147.21bc
T4	50.79 ±3.25 c	58.09 ± 1.77b	3.00 ± 0.82ab	3.67 ± 0.47 ab	4.67 ± 1.25 cd	4.3±0.82cd	581.2 ± 155.28 cd	543.83 ±104.67cd	19.37 ± 5.18cd		18.13 ± 32.12cd
T2	42.73 ±2.91 d	53.13 ± 2.31c	2.00 ± 0.82b	2.67 ± 0.94 bc	4.00 ± 0.82 cd	4.0±0.47cd	498.1 ± 101.65 cd	512.8 ± 59.16cd	16.60 ± 3.39cd		17.09 ± 98.54cd
T1	39.04 ±2.06 d	44.85 ± 4.26d	4.00 ± 0.82a	4.00 ± 0.82 ab	3.67 ± 0.47 d	3.3 ±0.47d	456.5 ± 58.89 d	427.3 ± 123.56 d	15.22 ± 1.96d		14.24 ± 123.56d
T0	30.78 ±1.16 e	28.62 ± 0.78e	2.67 ± 0.47ab	2.00 ± 0.82 c	1.33 ± 0.47 e	1.0 ± 0.0e	166.1 ± 58.89 e	113.2 ± 86.91 e	5.33 ± 1.92e		3.77 ± 86.91 e

• Note: Treat = Treatments, %= Percentage.
• ^*
  = different letters indicate significance according to Tukey's test at p<0.05.

Meanwhile, in the NCTP and CTWP variables for the first season, treatments T7 and T3 reached the highest values, with 8.67 and 7.33 tubers per plant and 1098.67 and 913.1 g plant^-1, exceeding the control by more than 84.66 and 81.86% for NCTP and 84.88 and 81.81% for CTWP respectively. However, the following season, treatments T7 and T3 produced increases of 3.67 and 2.88% for NCTP and 4.03 and 9.08% for CTWP compared to the previous season (Table 5).

Regarding commercial yield (Table 5), in the first season, T7 reached the highest value, with 36.62 ± 1.71 t ha^-1, exceeding the control by 85.46%, while in the second season the treatments T7 and T3 registered increases of 4.04 and 9.08% in relation to the previous season, which means that the improved and commercial varieties showed a good development when T. harzianum was applied jointly to the soil and B. subtilis to the foliage.

3.3. Regression

Figure 3A and B shows the relationship between control efficiency and potato varieties with and without the application of the microorganisms, which were fitted to a linear function, indicating that as T. harzianum and B. subtilis are applied together in the improved potato variety, a higher control efficiency is registered and in the following season the control efficiency increases, the same happens with the commercial variety Yungay. This model demonstrates that the improved variety combined with the joint application of both microorganisms significantly reduces the severity and increases the control efficiency of late blight.

Figure 3
Regression for control efficiency (A) and (B), plant height (C) and (D), number of commercial tubers per plant (E) and (F), commercial weight per plant (G) and (H) and commercial yield (I) and (J) from two seasons. Note: Th= Trichoderma harzianum, Bs= Bacillus subtilis.

Likewise, Figure 3C and D show a positive relationship between PH and potato varieties with and without the application of the microorganisms, which adjusted to a linear function, indicating that as T. harzianum is applied with B. subtilis, the plant shows a linear growth, being the improved variety the one that reached a greater height, in both seasons. As for the NCTP (Figure 3E and F), an ascending regression line is observed from left to right, indicating that as the micro-organisms are applied together, the plant produces more commercial tubers per plant in both varieties. Likewise, the CTWP (Figure 3G and H) increases as T. harzianum and B. subtilis are applied together, with the improved variety showing the best response. Figure 3I and J show the CY fitted to a linear function, where the highest value was achieved by the variety improved with T. harzianum and B. subtilis in both seasons. In contrast, the combined application of microorganisms increases the potential of the potato varieties, showing higher plant growth, tuber formation and tuber weight.

4. Discussion

The results show that plants subjected to Phytophthora infestans show a significant reduction in growth and productivity of potato, because the plant once infected with the phytopathogen, produces damage in the leaf area and as time passes, the severity increases, losing its photosynthetic activity (Lastochkina et al., 2022), weakening the plant (Evenhuis et al., 2024) and even causing death (Wei et al., 2024). However, our study showed that the combined use of the antagonistic fungus "Trichoderma harzianum" applied to the soil and the endophytic strain "Bacillus subtilis" applied via foliar on an improved potato variety "CIP 302295.32", increases the control of Phytophthora infestans and produces a significant effect on the biometric parameters of potato in both seasons, thus minimizing the use of chemical fungicides (Gleń-Karolczyk et al, 2022) and thus minimizing the risk to human health and the environment (Southern et al., 2025).

In our study, it has been observed that plants of the commercial variety Yungay subjected to P. infestans tend to show an accelerated progress of late blight disease and reach 91 to 100% severity from 63 and 70 DDS in both seasons, while the improved variety CIP 302295.32, the progress was significantly lower, however, the highest severity occurred at 85 DDS. These results are consistent with the findings of Vásquez-Castillo et al. (2022) who tested the genetic resistance of improved varieties on P. infestans, indicating that varieties with partial resistance tend to reduce the incidence and severity of the phytopathogen, if environmental conditions are favorable, disease progress increases from the middle of the phenological cycle of the potato, so fungicide applications may be more effective in controlling P. infestans.

Considering that disease resistance is a genetic characteristic of the potato variety and the environment, in contrast to the genetic composition of the improved variety, the temperature and relative humidity in the study area favored the development of P. infestans, causing the virulence and aggressiveness of the potato to be higher (Purwantisari et al., 2021).

Our results showed that soil treatment of T. harzianum individually reduced late blight disease progress up to 63 DDS compared to the control by 61.11%, but from 77 DDS onwards the disease progress increased to more than 50% severity in the improved variety CIP 302295.32 during the two seasons. On the other hand, in the commercial variety Yungay, a slow progress of the disease was also observed, less than the control by 50%, but at 70 DDS the disease progress covered more than 50% of the leaf area in both seasons. This was corroborated by Khatun et al. (2021) who, when applying T. harzianum via soil, found that the microorganism proliferates the root system and initiates its action, where it releases substances with antifungal activity, delaying the symptoms of the disease; however, the zoospores that infect the plant through the wind, added to the growth of the foliage and the climatic conditions that favor the development of the phytopathogen, reduce the antagonistic action of Trichoderma.

It was also found that the application of B. subtilis to the soil and via foliar, from the initial stage of the crop, there was a rapid advance of the late blight disease, taking into account that more than 50% of foliar damage was registered at 56 DDS in the commercial variety and at 70 DDS in the improved variety, after which the advance slowed down, demonstrating that the endophytic strain presents 40 to 44% efficiency of control of P. infestans in both varieties during the two seasons. These observations are very similar to the data of Yan et al. (2020) who found that applying the endophytic strain Bacillus velezensis in the field reduced the severity of late blight by 40.79 and 36.67% in two seasons, and when combined with low concentrations of chemical fungicides, the control efficiency on P. infestans increases.

These results are also in agreement with data from Kumbar et al. (2019) who applied B. subtilis to soil and foliage in the field, found that effectiveness varies depending on environmental conditions, although it slightly delays disease symptoms due to the production of functional metabolites including Iturin which causes suppression of P. infestans. However, infestation of the phytopathogen due to favorable climatic conditions increases the infection rate which reduces the antifungal efficiency of the bacterium.

Therefore, the study revealed that the combined application of T. harzianum and B. subtilis, shows a high control efficiency of P. infestans, due to the independent action of each biological agent, highlighting that T. harzianum applied to the soil colonizes the root system and the foliar application of B. subtilis colonizes the root system. subtilis colonizes the foliage, which significantly halted the advance of the disease, presenting slight damage at the end of the season, with the improved variety CIP 302295.32 reducing the severity of the disease by 34.21% compared to the Yungay variety.

This effect is due to the application of T. harzianum from transplanting and in early stages of the plant, which colonizes the root system and through it, releases metabolites with antibiotic action, hydrolytic enzymes (proteases, cellulases, chitinase and gluconase) that degrade the cell wall of the oospores (Xiao et al., 2023). While the endophytic bacterium B. subtilis applied via the foliar route, produces cellulases that degrade the cell wall of the plant leaf tissue, which allows it to enter and colonize the host tissue and through it, secretes secondary metabolites with antifungal activity, hydrolytic enzymes and stimulates plant defense’s, which inhibits the mycelial growth of P. infestans in the area of the plant (Wang et al., 2022).

The study showed that the combined use of T. harzianum and B. subtilis produced more than 80% control efficiency of P. infestans in the improved variety during the two seasons and 75.69 and 72% with the Yungay variety in both seasons. This useful finding may be since the effect of T. harzianum and B. subtilis had independent actions on the plant organs where they colonized, which halted the progress of the disease and inhibited the mycelial growth of the phytopathogen (Rigobelo et al., 2024). Our results were consistent with the data of Islam et al. (2022) who found that the combined use of bacterial bioagents B. subtilis and fungal T. harzianum were effective in controlling P. infestans, achieving a 99% severity reduction up to 60 DDS and 89.16% severity reduction up to 71 DDS under field conditions, making these biological agents an alternative for control of late blight in potato, allowing the use fungicides to be reduced by half.

The main mode of action of antagonistic biological agents is due to their effect on the cell wall of the plant pathogen, through the production of lipopeptides, biosurfactants and hydrolytic enzymes (protease, cellulose, chitinase and gluconase) that degrade the cell wall, other mechanisms such as competition for space and nutrients, which significantly inhibit mycelial growth and mycoparasite oospora thus reducing the infective inoculum in soil (Lastochkina et al., 2022), in addition, these antifungal compounds reduce the germination of sporangia and activate the plant's natural defense mechanisms, which inhibits zoospores on the foliage (Zhang et al., 2023).

In our study, it is observed that the improved variety CIP 302295.32 combined with both biological agents, is superior to the commercial variety Yungay in 8.80 and 10.81% of control efficiency in the two seasons, this effect is due to the fact that the resistance induced by the microorganism increases the genetic resistance of the plant and the control is significantly increased on P. infestans, in contrast, this effect was absent with the individual use of the biological agents, making this combination a sustainable strategy for the control of late blight and among other soil pathogens that threaten potato tubers. This minimizes the massive and excessive use of chemical fungicides (Berindean et al., 2024).

Also, the results of our study show significant effects of the application of biological agents on the improved variety CIP 302295.32 on the biometric parameters of potato. However, in the treatments without application of microorganisms, the severity was high, in effect, the plant did not show a normal development, but it was less pronounced after the individual use of each microorganism. In the study reported by Purwantisari et al. (2021) revealed that plants subjected to P. infestans plant growth was arrested in the early phenological stages, whereas with the application of T. harzianum the plant showed improved growth and development of the potato.

In terms of agronomic characteristics of the potato, the combined applications of the biological agents with the improved variety CIP 302295.32 increase by 59.27 and 60.34% compared to the control in plant height in the two seasons. We suggest that the results found in the combined activity of the improved variety CIP 302295.32 with application of T. harzianum and B. subtilis, are the result of the interaction of the induced resistance of the microorganisms and the genetic composition of the variety, reducing the biotic stress allowing the plant to show good growth and development.

This is also confirmed by the data of Wang et al. (2022) who found that the combination of these beneficial microorganism’s increases potato plant growth. This effect is because T. harzianum colonizing the root system also secretes phytohormones, such as gibberellins and auxins, resulting in increased rooting (Xiao et al., 2023), and also releases organic compounds that solubilize ions such as phosphorus, potassium, ammonium and micronutrients, making them available for plant uptake, thus improving growth (Woo et al., 2023). On the other hand, B. subtilis, through its mechanisms, produces phytohormones that allow a better flow of carbohydrates to the foliage during vegetative growth, resulting in greater leaf expansion.

The has been observed in this study that P. infestans causes significant reductions in the number of marketable tubers and marketable weight per plant. This is corroborated by many researchers who indicate that P. infestans is a phytopathogen that alters the metabolism of the plant, reducing photosynthetic activity and if the environment is favorable for this phytopathogen, the severity is greater, even causing the death of the plant (Wilches et al., 2022). However, in our research, the individual application of each micro-organism during the two seasons produced a significant effect on the response of the number of marketable tubers and marketable weight per plant. This effect is due to the dual action of the beneficial microorganisms studied.

The effect was more pronounced when T. harzianum was applied via soil and B. subtilis via foliar and became even more significant when applied in combination with the improved variety CIP 302295.32, reporting an increase of 84.66 and 88.89% compared to the control in NTCP in both seasons and for PCP the increase was 84.88 and 90.11% higher than the control in both seasons. This effect is due to the fact that T. harzianum proliferates the root system by improving nutrient uptake and stimulating endogenous hormones (gibberellins and auxins) in the plant, which are key to the continuous flow of photosynthates and water into the root system, resulting in a higher number of marketable tubers and greater tuber fill, thus achieving a higher marketable weight (Duan et al., 2024). While B. subtilis also produces hormones and enhances the formation of foliage during the vegetative stage and in the reproductive stage it directs the accumulated foliage for tuber filling, thus achieving a higher number and weight of tubers (Amin et al., 2023).

With respect to commercial potato yield, our study showed that the use of the improved and commercial variety combined with T. harzianum and B. subtilis significantly reduces the severity of late blight disease and as such, the plant does not show biotic stress and we can assume that, in this case, the increase in commercial yield is due to the dual action of the biological agents.

These findings revealed that the improved variety CIP 302295.32 combined with the biological agents outperformed the control by 85.45 and 90.12% in the two seasons and in the Yungay variety which is rated as sensitive to P. infestans, leading to continuous and excessive use of chemical fungicides and consequently causing negative impact on the environment and human health (Berindean et al., 2024). On the other hand, with the combined application of the two biological agents on the commercial variety Yungay, the increase in commercial yield was 82.38 and 88.74% compared to the control (Yungay variety without application), in both seasons.

Recent studies demonstrated significant effects on potato yield using these two biological agents, reporting commercial yields ranging from 30 to 40 tha^-1 (Chinheya et al., 2023). In the study reported by Rigobelo et al. (2024) indicate that T. harzianum triggers protection and biofertilization mechanisms, since, as mentioned above, this microorganism in the soil produces ion solubilizing compounds, stimulates the production of phytohormones, releases siderophores and the B. subtilis, produces double action, therefore, both microorganisms increase photosynthetic activity and accumulate photosynthates in the leaf area and then direct it towards the root system, resulting in an increase in the number of tubers per plant and a higher tuber filling (Rigobelo et al., 2024).

5. Conclusions

Therefore, the results found confirm that P. infestans significantly reduces potato growth and productivity. However, this study has clearly demonstrated the antagonistic effect of P. infestans with the combined use of the fungus "Trichoderma harzianum" applied to the soil and the endophytic strain "Bacillus subtilis" applied via foliar and this effect was more significant in the improved potato variety CIP 302295.32, achieving more than 80% efficiency of late blight control in the two seasons. It has also been found that the joint application of T. harzianum with B. subtilis significantly increased plant height, number of tubers and tuber weight per plant, which effect is due to the dual action of these biological agents, resulting in increased marketable potato yields.

Data Availability Statement

Data is available upon request.

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Edited by

• Editor:
  Takako Matsumura Tundisi

Publication Dates

• Publication in this collection
  03 Oct 2025
• Date of issue
  2025

History

• Received
  14 May 2025
• Accepted
  09 July 2025