INDUCTION OF SOYBEAN RESISTANCE MECHANISMS TO ANTHRACNOSE BY BIOCONTROL AGENTS

INDUÇÃO DE MECANISMOS DE RESISTÊNCIA DE SOJA À ANTRACNOSE COM AGENTES DE BIOCONTROLE

ABSTRACT

The biological control, thinking about the integrated management, has been inserted with other management techniques to disease control, such as soybean anthracnose. The aims of this work were to verify the action of Trichoderma and Bacillus isolates in the induction of soybean resistance mechanisms to anthracnose as a function of seed treatment. The statistical design was entirely randomised, in a 5 x 2 (agent species x sampling times) factorial scheme with five replicates. Soybean seeds were treated with Bacillus amyloliquefaciens BV03, B. subtilis BV02, Trichoderma asperellum BV10, Carbendazim + Thiram and distilled water (control). Seven days after seedling emergence, 2 μL of 1 x 10⁻⁴ Colletotrichum truncatum spores were inoculated on the cotyledons. Catalase (CAT), peroxidase (POX), phenylalanine ammonia lyase (PAL) and glyceollin (GLY) activities before and after pathogen inoculation, as well as the diameter of the anthracnose lesion on the cotyledons, were evaluated. Data were submitted to analysis of variance and, when significant, the mean values were compared by Fisher’s test (p < 0.05). The treatments did not influence the first sampling time before inoculation. Trichoderma asperellum BV10 increased POX and PAL activities up to 173%, while B. amyloliquefaciens BV03 increased POX activity. Glyceollin was not influenced by the treatments. The T. asperellum BV10 reduces the diameter of the anthracnose lesion by up to 61%. Thus, T. asperellum BV10 has the potential to control soybean anthracnose, improved the response defense against C. truncatum, when performed on seed treatment.

Keywords
Colletotrichum truncatum; Glycine max; Resistance induction

RESUMO

O controle biolósgico, pensando no manejo integrado, tem sido inserido em conjunto com outras técnicas de manejo de doenças, como antracnose da cultura da soja. O objetivo deste trabalho foi verificar a ação de isolados de Trichoderma e Bacillus na indução de mecanismos de resistência da soja á antracnose. O delineamento estatístico foi inteiramente casualizado em esquema fatorial 5 x 2 (agentes de controle biológico x horários de coleta) com cinco repetições. Sementes de soja foram tratadas com Trichoderma asperellum BV10, Bacillus subtilis BV02, B. amyloliquefaciens BV03, Carbendazim + Thiram e água destilada (testemunha). Sete dias após a emergência foi realizada a inoculação de Colletotrichum truncatum sobre os cotilédones com 2 μL de suspensão esporos (5 x 104 mL⁻¹) sobre os cotilédones. A atividade de catalase (CAT), peroxidase (POX), fenilalanina amônia liase (FAL), gliceolina (GLI) foram avaliadas antes e após a inoculação do patógeno. Os dados foram submetidos à análise de variância e as médias comparadas pelo teste de Fisher (p<0,05). Os tratamentos não influenciaram o primeiro horário de coleta. O T. asperellum BV10 incrementou a atividade de POX e FAL em até 173%. B. amyloliquefaciens BV03 aumentou a atividade de POX. A GLI não foi influenciada pelos tratamentos em nenhum horário de coleta e ensaio. T. asperellum BV10 reduziu até 61% do diâmetro da lesão de antracnose. O tratamento de sementes de soja com T. asperellum BV10 possui potencial no controle da antracnose, aumentando a resposta de defesa de plântulas à C. truncatum.

Palavras chaves
Colletotrichum truncatum; Glycine max; Indução de resistência

INTRODUCTION

Soybean is a food source rich in proteins for both humans and animals, and is an important crop in world agribusiness, transforming Brazil into one of the two largest oilseed producers, along with the United States of America, generating jobs and income in producing regions, through the long and organized production chain (CATTELAN; DALL’AGNOL, 2018; CONAB, 2020).

Among the limiting factors of soybean production, phytopathological losses are considered important, and can be caused by viruses, bacteria, nematodes, and fungi, agents that cause diseases such as anthracnose, which together can damage up to 32% of crop production (ALMEIDA; FERREIRA; YORINORI, 2005; SAVARY; WILLOCQUET; PETHYBRIDGE, 2019).

Anthracnose, caused by the fungi Colletotrichum truncatum, C. plurivorum, C. musicola, and C. sojae (ROGÉRIO et al., 2020), is considered one of the most frequent and harmful, especially in the Brazilian Cerrado, which may cause loss of up to 100% when control measures are not adopted (DIAS, PINHEIRO; CAFÉ-FILHO, 2016; DIAS; DIAS-NETO; SANTOS, 2019; ROGÉRIO; GLADIEUX; MASSOLA JUNIOR, 2019). At high temperatures and humidity, especially in rainy years, the pathogen causes the failure of the seeds to germinate or reduces their vigor promoting seedling death, compromising the final soybean stand. It can also affect petioles and leaf ribs, in addition to stems and pods at any phenology stage of the crop, causing necrosis. Severe seed abortion, loss of pods and grain deterioration are responsible for the highest losses in culture when climatic conditions are favorable to the development of the pathogen (ALMEIDA; FERREIRA; YORINORI, 2005;; DIAS, PINHEIRO; CAFÉ-FILHO, 2016; ROGÉRIO; CIAMPI- GUILLARDI,; BARBIERI, 2016; BORAH, 2019; NATARAJ et al., 2020).

The main control strategies for soybean anthracnose are the use of healthy seeds, crop rotation, limiting plant population to reduce microclimate formation, proper soil management, balanced fertilization, seed treatment, chemical treatment with chemical or biological agents (GODOY; ALMEIDA; SOARES, 2014; SILVA; SANTOS; AMARAL, 2020). Seed treatment and aerial spraying with fungicides are the main measures to control soybean anthracnose (PEREIRA; OLIVEIRA; ROSA, 2009; PESQUEIRA; BACCHI; GAVASSONI, 2016). However, isolated control measures lose efficiency in the short or long term, such as chemical control, with the recorded appearance of populations of C. truncatum resistant to the fungicide Carbendazim, used in seed treatment (GODOY; BUENO; GAZZIERO, 2015; POTI; MAHAWAN; CHEEWANGKOON, 2020). Despite the efficiency of chemical treatment, other tools should be included as an alternative for managing this phytopathogen for successful integrated disease management. An example is induced resistance in plants.

Innate immune resistance has been reported as an important defense mechanism against diseases and can be triggered during pathogen-host interaction and/ or when induced by eliciting agents that may be of microbial origin, such as biocontrol agents (DUBERY; SANABRIA; HUANG, 2012; KUSHALAPPA; YOGENDRA; SHAILESH, 2016; LOLLE; STEVENS; COAKER, 2020; WANG et al., 2020). During the immune response, the plant activates mechanisms that include peroxidase, catalase, polyphenoloxidase, phenylalanine ammonia lyase, and phytoalexins activity (DUBERY; SANABRIA; HUANG, 2012; YANG; CAO; RUI, 2017).

In induced resistance, the elicitor plays a key role in controlling diseases and can be classified as a biotic due to its source material, such as plant extracts, algae, bacteria, and fungi (DUBERY; SANABRIA; HUANG, 2012; THAKUR; SOHAL, 2013; XING et al., 2015; SOLINO et al., 2016; MALIK, KUMAR; NADARAJAH, 2020). When dealing with bacteria and fungi, the microbial genera Bacillus and Trichoderma have been used for plant disease biocontrol as well as to increase agricultural crop productivity (BETTIOL; MORANDI, 2009; TAHIR; GU; WU, 2017; GLICK, 2015; PENHA et al., 2020). In this context, the objective of this study was to evaluate the induction of soybean plant resistance mechanisms to anthracnose by biocontrol agents through seed treatment.

MATERIALS AND METHODS

The assays were conducted at the phytopathology laboratory of the University of Rio Verde (UNIRV). The Colletotrichum truncatum isolate CPA002 was supplied by the company Campos Pesquisa Agrícola, kept on potato dextrose agar (PDA) growth medium at 25 ± 2 °C and 12 hours light photoperiod until sporulation and used to prepare the spore suspension for inoculation. DNA extraction and SSR genotyping were previously carried out by Campos Pesquisa Agricola to confirm the identification of C. truncantum isolates. Further, morphological structures were analyzed by microscope to confirm the purity of the material.

The bacterium Bacillus subtilis (type BV-02), registered as the Ministério da Agricultura e Pecuaria e Abastecimento - MAPA: 43418, is a commercial product (c. p.) with a concentration suspension formulation of 3 x 10⁹ CFU mL⁻¹. The bacterium Bacillus amyloliquefaciens (type BV-02), registered as MAPA: 34518, has a c. p. concentration suspension formulation of 3 x 10⁹ CFU mL⁻¹. The fungus Trichoderma asperellum (type BV-10), registered as MAPA: 34018, has a c. p. concentration suspension formulation of 1 x 10¹⁰ viable conidia mL⁻¹.

The trials were carried out in a completely randomized design, in a 5 x 2 factorial arrangement design with five replicates. Factor A was represented by three species of biocontrol agents, namely T. asperellum (1 mL c. p. kg seed⁻¹, Tricho-Turbo®), B. subtilis (2 mL c.p. kg seed⁻¹, Bio-Immune®), and B. amyloliquefaciens (2 mL c. p. kg seed⁻¹), in addition to a control treatment (distilled water) and a chemical fungicide, Carbendazim (150 g L⁻¹) + Thiram (350 g L⁻¹) at the concentration 200 mL c.p. 100 kg of seeds⁻¹. Factor B corresponded to the sampling times before (0 hrs) and after (24 hours) pathogen inoculation. The samples were evaluated destructively considering 25 replicates per treatment. Thereby, five replicates were used to measure glyceollin at the first sampling time (before inoculation) and five replicates for the second sampling time (after inoculation). A similar procedure was carried out for enzymatic activity quantification. The anthracnose severity was analyzed in the last 5 replicates.

The microorganisms B. amyloliquefaciens (BV-03) and B. subtilis (BV-02) were identified and characterized by Empresa Brasileira de Pesquisa Agropecuaria - Embrapa and deposited in the “Diazotrophic and Plant Growth Promotion Rhizobacteria Culture Collection of Embrapa Soja”, Word Federation Culture Collection #1213, and World Data Center for Microorganisms #1054, respectively. Sequence analysis of the 16S RNA region was performed by BOX-PCR. Strain BV-02 (CNPSo 3219) was classified as the B. subtilis operational group within the B. subtilis species complex and strain BV-03 (CNPSo 3418) was classified as the B. amyloliquefaciens operational group within the B. subtilis species complex.

The fungus T. asperellum (BV-10) was identified and characterized by the Fungus Detection and Identification Service of the Fungi Systematics and Ecology Laboratory, Department of Phytopathology DFP of the Federal University of Lavras. The filamentous fungus sample, type BV- 10, was submitted to microscopic preparation and phylogenetic analysis of the RPB2 gene region (second-largest subunit of RNA polymerase), and grouped with the reference isolate of Trichoderma asperellum species, with a posterior probability of 100% and a bootstrap value of 72%. The isolate was deposited at the institution under the code CML 3751.

Soybean seeds were packaged in a 100 g container and coated to avoid excessive moisture uptake, using 600 mL of commercial product per 100 kg of seeds (LUDWIG; LUCCA FILHO.; BAUDET, 2011). Subsequently, the IPRO 7739 treated soybean seeds were sown in a 250-mL polyethylene container with a mixture of sand and commercial organic fertilizer, in the proportion of 1:1, respectively.

Seven days after sowing, seedlings with the first pair of leaves and extended cotyledons were inoculated with 2 μL of a 5 x 10⁴ mL⁻¹ spore suspension of C. truncatum; subsequently, the plants were kept in a partially controlled relative humidity chamber for 72 h (adapted from WRATHER; EROLD, 1990).

The evaluated variables were anthracnose severity in soybean cotyledons, catalase, peroxidase, phenylalanine ammonia lyase activity, and glyceollin accumulation.

To assess the size of anthracnose lesions in soybean cotyledons, the lesion diameter (ALD) was measured using images of the lesions and processed by ImageJ® version 1.8.0 software, seven days after inoculation and data were expressed in cm. For enzymatic quantification, cotyledons were collected 7 days after sowing, before C. truncatum inoculation (first sampling time) and 24 hours after pathogen inoculation (second sampling time). The samples were weighed, immediately placed in aluminum foil envelopes and stored at -20 °C.

Enzyme extract was obtained by macerating and homogenizing plant tissue samples in 4 mL of 0.01 M sodium phosphate buffer (pH 6.0), followed by centrifugation at 6,500 g for 30 minutes at 4°C. The supernatant was collected and considered the enzymatic extract; it was used in the determination of total protein content and for the enzymatic activity determination of peroxidase, catalase, and phenylalanine ammonia lyase (FAN et al., 2011).

Total protein quantification was performed through the Bradford method (1976). Briefly, absorbance values were plotted on a standard curve of bovine serum albumin (BSA) concentrations, and protein concentrations were expressed in μg protein mL⁻¹.

Catalase activity (CAT) was quantified viaGoth’s method (1991), modified by Tománková et al. (2006). The determination was performed using the direct spectrophotometric method at a wavelength of 405 nm, and the results were expressed in μmol min⁻¹ mg⁻¹ protein. The reaction that determines CAT activity in the oxidative process is shown as Reaction (1):

(1) $$\underset{\begin{array}{l}\text{Hydrogen}\\ \text{Peroxide}\end{array}}{2{\text{H}}_2{\text{O}}_2}\stackrel{\text{Catalase}}{\to }\underset{\text{Water}}{2{\text{H}}_2\text{O}+}\underset{\text{Oxygen}}{{\text{O}}_2}$$

Guaiacol peroxidase (POX) activity was quantified by spectrophotometry at 470 nm, and the results were expressed in absorbance min⁻¹ mg⁻¹ protein (LUSSO; PASCHOLATI, 1999). The reaction that determines POX activity in oxidative process is shown as Reaction (2):

(2)

Phenylalanine ammonia lyase (PAL) activity was determined by colorimetric quantification of the trans-cinnamic acid released from the phenylalanine substrate. Absorbance was determined at 290 nm, and enzymatic activity was expressed in μg of transcinnamic acid h⁻¹ mg⁻¹ protein (UMESHA, 2006).

For glyceollin build-up determination (GLY), cotyledons were collected at 7 days after sowing, before inoculation (6 plants and 12 cotyledons), and 24 hours after inoculation (6 plants and 12 cotyledons). Cotyledons were kept in Petri® dishes on filter paper moistened with distilled water. The plates were capped and kept in the dark at 26°C. After 20 hours, the cotyledons were removed from the plates, cut in half and added into a Falcon tube containing 5 mL of distilled water and stirred for 1 hour. The extracted glyceollin was measured by spectrophotometry at 285 nm (AYERS et al., 1976). Results were expressed in unit absorbance per gram of fresh weight (ABS gfw⁻¹).

The assays were performed twice. The obtained data were subjected to analysis of variance (ANOVA), and joint analyses were performed between the assays. For significant ANOVA results, data were run in a factorial scheme and means were compared by Fisher’s test (p < 0.05).

RESULTS AND DISCUSSION

Anthracnose severity in seedling cotyledons receiving T. asperellum seed treatment was 61% and 24% lower than that of the control in the first and second experimental assays, respectively. The other treatments did not differ from the control (Figure 1).

Figure 1
Diameter of anthracnose lesions in soybean cotyledons in assay I and assay II treated with biocontrol agents 7 days after C. truncatum inoculation. T. asperellum = Trichoderma asperellum BV10 (Tricho-Turbo®); B. subtilis = Bacillus subtilis BV02 (Bio-Immune®). Means followed by the same lowercase letter over the bar do not significantly differ from each other by the Fisher test (p < 0.05).

When analyzing the CAT data (Table 1), it was noted that enzyme activity was not influenced by the treatment, a fact evidenced by the absence of significant differences between treatments and control at the first sampling time (prior to inoculation with C. truncatum) for both assays performed (I and II). Enzyme activity evaluated 24 hours after pathogen inoculation (second time) was also unchanged in assay I, but in assay II, CAT activity was lower in the B. subtilis BV02 treatment compared to the control. Despite the non-significant difference by Fisher’s test in assay I, it is possible to observe that the value obtained before incubation with C. truncatum (0.57) was higher compared to the time after incubation with the pathogen (0.26), similar to assay II. The fact of not detecting significant differences is related to the higher random error obtained in assay I.

Table 1
Catalase specific activity (CAT) in assay I and assay II treated with biocontrol agents 7 days after C. truncatum inoculation.

Treatment	Assay I			Assay II
	1st time	2nd time	Mean	1st time	2nd time	Mean
	μmol min−1 mg−1 protein
Control	0.55 aA	0.47 aA	0.51	4.85 aA	3.97 abB	4.41
Trichoderma asperellum	0.56 aA	0.48 aA	0.52	4.52 aA	4.48 aA	4.50
Bacillus subtilis	0.57 aA	0.26 aB	0.42	4.52 aA	3.57 bB	4.05
Bacillus amyloliquefaciens	0.56 aA	0.45 aA	0.50	4.94 aA	4.16 abA	4.55
Carbendazim + Thiram	0.55 aA	0.48 aA	0.47	4.43 aA	3.95 abA	4.19
Mean	0.56	0.41		4.65	4.03
CV (%)		42

Trichoderma asperellumTrichoderma asperellumBacillus subtilisBacillus subtilis

When analyzing sampling times within the treatments, it was observed that the seeds treated with B. subtilis promoted reduction of CAT activity at the second sampling time in assays I and II. In assay II, there was a reduction in CAT activity at the second sampling time compared to the first from the control treatment (Table 1).

POX activity did not differ as a function of seed treatment (first time) for both assays (I and II), except for the fungicide treatment in assay II, which promoted a reduction of seed enzyme activity in relation to the control (Table 2). At the second time (after inoculation), seed treatment with T. asperellum BV10 promoted a 173% and 126% increase in POX activity when compared to control, in assays I and II, respectively.

Table 2
Specific guaiacol peroxidase (POX) activity in assay I and assay II treated with biocontrol agents 7 days after C. truncatum inoculation.

Treatment	Assay I			Assay II
	1st time	2nd time	Mean	1st time	2nd time	Mean
	Absorbance min−1 mg−1 protein
Control	1.62 aA	1.85 bA	1.74	4.78 aA	5.56 bcA	5.17
Trichoderma asperellum	1.17 aB	3.21 aA	2.19	4.50 aB	7.03 aA	5.76
Bacillus subtilis	1.58 aA	1.89 bA	1.73	4.28 aA	4.40 cA	4.34
Bacillus amyloliquefaciens	1.39 aA	2.39 bA	1.80	4.11 abB	6.68 abA	5.40
Carbendazim + Thiram	1.27 aA	1.75 bA	1.46	2.98 bB	5.45 bcA	4.22
Mean	1.39	2.22		4.13	5.82
CV (%)		52			19

Trichoderma asperellumTrichoderma asperellumBacillus subtilisBacillus subtilis

There was an increase in POX activity from the second sampling time compared to the first time, in assays I and II, when treating soybean seeds with T. asperellum BV10 (Table 2). The B. amyloliquefaciens BV03 treatment promoted an increase of POX at the second sampling time compared to the first, in assay II.

When PAL specific activity was quantified (Table 3), it was noticed that the seeds treated with T. asperellum BV10 showed an increase in enzyme activity in the first hour, by 65%, in relation to the control in assay I. In the second assay, PAL activity increased by 137% in response to the treatment of soybean seeds with B. amyloliquefaciens BV03 compared to the control, not differing from T. asperellum BV10.

Table 3
Specific activity of phenylalanine ammonia lyase (PAL) in assay I and assay II treated with biocontrol agents 7 days after C. truncatum inoculation.

Treatment	Assay I			Assay II
	1st time	2nd time	Mean	1st time	2nd time	Mean
	μg of trans-cinnamic acid h−1 mg−1 protein
Control	0.44 aB	5.12 bA	2.78	0.45 aA	0.61 bA	0.53
Trichoderma asperellum	0.28 aB	8.49 aA	4.38	0.39 aB	0.75 abA	0.57
Bacillus subtilis	0.29 aB	3.56 bA	1.92	0.0 aA	0.34 bA	0.37
Bacillus amyloliquefaciens	0.26 aB	5.10 bA	2.68	0.45 aB	0.84 aA	0.65
Carbendazim + Thiram	0.20 aB	4.71 bA	2.46	0.47 aA	0.61 bA	0.54
Mean	0.29	5.40		0.43	0.63

Trichoderma asperellumTrichoderma asperellumBacillus subtilisBacillus subtilis

When analyzing the unfolding of sampling times within the treatments, increased PAL activity was observed in all treatments at the second sampling time when compared to the first, in assay I (Table 2). In assay II, the treatments with B. amyloliquefaciens BV03 and T. asperellum BV10 promoted an increase in PAL at the second time when compared to the first sampling time.

When quantifying glyceollin (GLY) accumulation in soybean cotyledons, phytoalexin accumulation in response to seed treatment alone was lower than in the control in response to B. amyloliquefaciens BV03 in the first hour of assays I and II (Table 4). The GLY accumulation was not influenced by sampling time in assay I, that is, even after inoculation, there were no stimuli for phytoalexin synthesis. However, in assay II, there was a reduction in GLY accumulation at the second sampling time when compared to the first in all treatments, except for B. amyloliquefaciens BV03, when no difference was observed between the first and second sampling times (Table 4).

Table 4
Glyceollin (GLY) accumulation in assay I and assay II treated with biocontrol agents 7 days after C. truncatum inoculation.

Treatment	Assay I			Assay II
	1st time	2nd time	Mean	1st time	2nd time	Mean
	ABS g fw−1
Control	1.92 abA	1.68 aA	1.80	2.03 aA	0.90 aB	1.46
Trichoderma asperellum	2.11 abA	1.68 aA	1.90	2.24 aA	1.29 aB	1.76
Bacillus subtilis	2.29 aA	1.88 aA	2.08	1.95 aA	1.20 aB	1.58
Bacillus amyloliquefaciens	1.61 bA	1.67 aA	1.64	1.56 bA	1.41 aA	1.48
Carbendazim + Thiram	1.99 abA	1.57 aA	1.78	2.13 aA	1.27 aB	1.70
Mean	1.98	1.70		1.98	1.21
CV (%)		24			17

Trichoderma asperellumTrichoderma asperellumBacillus subtilisBacillus subtilis

Table 5 shows, in green, the results that are higher than the control. Notably, T. asperellum BV10 was the treatment that stood out regarding the induction of resistance mechanisms and anthracnose control, as it promoted an increase in POX activity at the second sampling time (24 hours after C. truncatum inoculation) in assays I and II. Also, T. asperellum BV10 promoted an increase in PAL at the second sampling time in the assay, in addition to reducing the size of the anthracnose lesion diameter.

Table 5
Response of the variables catalase (CAT), guaiacol peroxidase (POX), phenylalanine ammonia lyase (PAL), glyceollin (GLY), and anthracnose lesion diameter (ALD) compared to the control in assay I and assay II treated with biocontrol agents 7 days after C. truncatum inoculation.

Treatment	Assay I
	Analyzed variables
	CAT		POX		PAL		GLY		ALD
	1st T	2nd T	1st T	2nd T	1st T	2nd T	1st T	2nd T
Trichoderma asperellum
Bacillus subtilis
Bacillus amyloliquefaciens
Carbendazim + Thiram
Treatment	Assay I
	Analyzed variables
	CAT		POX		PAL		GLY		ALD
	1st T	2nd T	1st T	2nd T	1st T	2nd T	1st T	2nd T
Trichoderma asperellum
Bacillus subtilis
Bacillus amyloliquefaciens
Carbendazim + Thiram

Trichoderma asperellumTrichoderma asperellumBacillus subtilisBacillus subtilis

Treatment with T. asperellum BV10, B. subtilis BV02, B. amyloliquefaciens BV03 and Carbendazim + Thiram did not influence the activity of antioxidant enzymes, CAT and POX, and a key enzyme pf the metabolism of phenylpropanoids, PAL in seedling cotyledons 7 days after germination, not yet inoculated with C. truncatum (first sampling time), indicating that they do not continuously or systemically activate defense mechanisms in s o ybean seedling cotyledons when seed treatment is performed, which favors the use of biocontrol agents, since from the point of view of inducing resistance, the continuous activation of defense mechanisms generates an energy cost to the plant (MATYSSEK, et al., 2012).

In the second sampling time, 24 hours after the inoculation of C. truncatum, an increase of 173% and 126% of the POX enzyme was observed, in assays I and II, respectively, in addition to the 65% increase in FAL, after inoculation of the pathogen, in assay I, when applying the treatment of soybean seeds with T. asperellum BV10. The enzyme activity increase after inoculation of the pathogen did not stop the infection by C. truncatum, however, it suggests a containment of the fungal disease, because there was a reduction of 61% and 24% in the diameter of the anthracnose lesion in assays I and II, respectively.

The genus Trichoderma has been described as a potential biocontrol agent for plant pathogens in agriculture, such as the genus Colletotrichum (BEGUM et al., 2008; JAGTAPA; GAVATEA; DEYA, 2012; KUCHLAN; KUCHLAN; ANSARI, 2019). This acts through mechanisms of competition for space and nutrients, antibiosis, parasitism, and induction of the defense mechanisms of the plant against pathogens (CONTRERAS-CORNEJO et al., 2020; SOOD et al., 2020; VINALE; SIVASITHAMPARAM, 2020).

When triggering the plant defense response against pathogens, the genus Trichoderma, acts by preventing the formation of the infection or slowing its growth, through the accumulation of reactive oxygen species (ROS), phenols, phytoalexins, pathogeneses related to enzymes, oxidative cycle enzymes, phenylpropanoids cycle enzymes, increased thickness, density, and incorporation of secondary compounds, such as phenols in the cell wall, or programmed death of tissue adjacent to infection caused by pathogens (CONTRERAS-CORNEJO et al., 2020; SOOD et al., 2020; VINALE; SIVASITHAMPARAM, 2020). This response can be intensified as this biocontrol agent can act by altering carbohydrate metabolism and photosynthesis activities, physiological activities in enzymes related to plant growth and development (PÉREZ-GARCÍA; VICENTE, 2011; JAHAN et al., 2015; PASCHOLATI; DALIO, 2018; MILJAKOVI’C; MARINKOVI’C; BALESEVEC-TUBI’C, 2020).

Among the biochemical reactions observed when treating soybean seeds with T. asperellum BV10 and later inoculating with C. truncatum, there is an increase in the activity of POX, which is an important biochemical mechanism in disease control (NASERINASAB; SAHEBANI; ETEBARIAN, 2011; SINGH et al., 2011; YOUSSEF; TARTOURA; ABDELRAOUF, 2016; MOHAPATRA; MITTRA, 2017). This enzyme is involved in the hydrogen peroxide (H2O2) dismutation reaction in water and oxygen, in this way, reducing the excess of reactive oxygen species (ROS), formed during oxidative stress caused by the pathogen is recognize of the plant (KUSHALAPPA; YOGENDRA; SHAILESH, 2016; YANG; CAO; RUI, 2017). Thus, the increase of POX, as observed in the treatment of soybean seeds with T. asperellum BV10, in this work, suggests that it might cause an indirect increase in H₂O₂, since this is the main substrate of the enzyme.

The accumulation of ROS’s, such as H₂O₂, are reported in reactions in incompatible interactions between plants-pathogen, that is, resistance of the plant, when it accumulates at sites of infection, activating localized cell death and induction of defense genes in adjacent cells (LEVINE et al., 1994; MEHDY, 1994). In addition, peroxidase is also involved in catalyzing the oxidation of various substrates such as phenolic compounds and lignin precursors, along with hydrogen peroxide reduction (COSIO; DUNAND, 2009).

The role of peroxidases in lignin polymerization is the most widely studied function of this class of enzymes leading to lignin formation and subsequent cell wall deposition, which will later act in the defense of the plant against pathogens (VANHOLME et al., 2010; THAKUR; SOHAL, 2013; KUSHALAPPA; YOGENDRA; SHAILESH, 2016; BEGOVIC et al., 2017), which may explain the delay in the development of anthracnose damage when using seed treatment with T. asperellum BV10, in this work.

The increase in phenylalanine ammonia lyase activity observed after C. truncatum inoculation (second time of assay I and the trend on the second time of assay II) and potentiated in seedlings that received T. asperellum BV10 seed treatment may also be involved in containing the growth of the pathogen lesion. The transient increase of the activity of phenylalanine ammonia lyase is related to acquired and induced resistance (MAUCH-MANI; SLUSARENKO, 1996; SMITH-BECKER et al., 1998; SHINE et al., 2016). The enzyme is a precursor to the phenylpropanoid pathway, originating several other compounds from benzoic acid, coumarins, precursors of lignin, ammonia, and others, used by plants for their defense (LORENZETTI et al., 2021).

Considering the increase in POX and PAL observed after the pathogen inoculation in seedlings from seeds treated with T. asperellum BV10 observed in this work, the consequent reduction in the diameter of anthracnose lesion in soybean cotyledons may have occurred in the first response due to the accumulation of ROS’s and later deposition of lignin and phenols on the cell wall, mainly on the secondary walls, which become thicker and denser, giving resistance to pathogen attack (MIEDES et al., 2014; PASCHOLATI; DALIO, 2018).

CONCLUSIONS

Treatment of soybean seeds with Trichoderma asperellum BV10 promoted a reduction of anthracnose lesion diameter under controlled conditions.

The application of T. asperellum BV10 activated the latent defense mechanisms, promoting increased guaiacol peroxidase and phenylalanine ammonia lyase activities after inoculation with C. truncatum.

ACKNOWLEDGEMENTS

Acknowledgements to the University of Rio Verde and Biovalens for financial support to perform part of this work.

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Publication Dates

• Publication in this collection
  13 May 2022
• Date of issue
  Apr-Jun 2022

History

• Received
  06 Aug 2020
• Accepted
  16 Aug 2021