Revista Mexicana de Ciencias Agrícolas   special publication number 23   September 28 - November 11, 2019

DOI: https://doi.org/10.29312/remexca.v0i23.2020

Article

Microbial antagonists for the biocontrol of wilting and its promoter
effect on the performance of serrano chili

César Alejandro Espinoza-Ahumada1

Gabriel Gallegos-Morales1§

Yisa María Ochoa-Fuentes1

Francisco Daniel Hernández-Castillo1

Reinaldo Méndez-Aguilar2

Raúl Rodríguez-Guerra3

1Antonio Narro Autonomous Agrarian University. Antonio Narro road 1923, Buenavista, Saltillo, Coahuila, Mexico. CP. 25315. (espinozaiap@hotmail.com; yisa8a@yahoo.com.mx; fdanielhc@hotmail.com). 2INIFAP. Mante Tampico Highway km 55, Esperanza, Cuauhtémoc Station, Tamaulipas, Mexico. CP. 89610. (mendez.reinaldo@inifap.gob.mx). 3INIFAP. Highway Montemorelos-China s/n, near the Hacienda Las Anacuas, Cd. General Terán, Nuevo León, Mexico. CP. 67400. (raulrdzg@yahoo.com.mx).

§Corresponding author: ggalmor@uaaan.edu.mx.

Abstract

Fusarium oxysporum and Rhizoctonia solani are causal agents of chili wilt and cause losses up to 80% in the crop in Mexico. The inadequate use of chemical fungicides has generated problems of resistance, this was not observed with microbial agents, so they were evaluated in the biocontrol of wilt of chili in experimental plots where F. oxysporum and R. solani were identified as causal agents of the illness. Trichoderma spp., Bacillus spp., mixture of microbial propagative ferment, thiabendazole and control, were applied on a plot of 800 m2 with six varieties of chili. The treatments Trichoderma and propative ferment showed low percentages of incidence and severity, high yield with Trichoderma in HS-52 and Coloso (15.67 and 13.89 t ha-1). Trichoderma treatments and propagative fermentation promote the biocontrol of chili wilt caused by F. oxysporum and R. solani and increase yield.

Keywords: Trichoderma, Bacillus, chili wilt, propagative ferment.

Reception date: April 2019

Acceptance date: July 2019

Introduction

Chili wilt is one of the main biological limitations in the production of this crop and is caused by Phytophthora capsici, Rhizoctonia solani and Fusarium oxysporum (Albañil et al., 2015). There are reports of chill wilt where it is frequently associated with F. oxysporum, which takes advantage of the mechanical damage and natural openings of the plant to infect, however, P. capsici is considered the most important pathogen due to its capacity to penetrate the epidermis of the roots and invade the vascular bundles (Zapata et al., 2012).

This disease is reported throughout Mexico, estimating losses up to 80% root rot to invade the vascular system of plants (González et al., 2009). Chemical control is the most used method to control the disease, it is common to reduce the inoculum by disinfecting the soil with Metam Sodium, fungicide applications are also made with the active ingredients 2Tiocianomethyl (TCMTB), Metalaxyl, Azoxystrobin and Propanocar to control P. capsici (Pérez et al., 2003), R. solani and Fusarium spp., are controlled with Tebuconazole, Carbendazim, Thiabendazole and Methyl Thiophanate (Yossen and Conles, 2014).

The high genetic variability that exists in the fungal complex associated with the disease and the inadequate use of fungicides has led to resistance problems; in this context, biological control through microbial agents does not report resistance of phytopathogens, which makes them a feasible alternative for the management of the disease (Bardin et al., 2015). Different species of Trichoderma and Bacillus are reported for the management of the genera Phytophthora, Sclerotium, Fusarium, Rhizoctonia, Alternaria, among others, these antagonists promote the production of biomass and the yield of crops (Astorga et al., 2014; Mamani et al., 2016; Arenas et al., 2017); as well as, they also activate the defense response of the plants, where they involve ethylene, jasmonate and salicylic acid (Manganiello et al., 2018), colonize the rhizosphere and produce secondary metabolites that generate antagonism (Fan et al., 2017; Saravanakumar et al., 2017) and parasitism when colonizing and penetrating the phytopathogen (Druzhinina et al., 2018).

The efficiency of microorganisms for the management of diseases has been shown to obtain a greater effect with formulations of B. subtilis and T. asperellum compared with chemical products of active ingredients such as Propanocarb, Aluminum Fosetil and Etridiazol (Villanueva, 2018). It was proposed to evaluate the effectiveness of microbial agents as biocontrol of the wilt of the chili crop and effect on the yield of different varieties of serrano chili.

Materials and methods

Location of the experiment

The research work was established in the year 2017 in the El Bajío Experimental Field, where there is a history of the presence of wilt of chili, also worked in the phytopathology laboratory of the Agrarian Autonomous University Antonio Narro (UAAAN) of Saltillo, Coahuila, Mexico.

Experimental establishment

The materials of serrano chili HS-52, Coloso, HS-44, Centauro, Paraiso and Tampiqueño 74 were evaluated, provided by the Experimental Field ‘Las Huastecas’ INIFAP of Villa Cuauhtémoc, Tamaulipas, Mexico. The plant was developed in polystyrene trays of 200 cavities, which were disinfected with 3% sodium hypochlorite, packed with a mixture of peatmoss-perlite in a ratio of 2-1 and maintained in greenhouse conditions for 40 days. Planting in the field was done with seedlings of 10 cm in height, transplanted in beds at 1.5 m to double row.

Microbial control agents were provided by the Department of Parasitology of the UAAAN, as a research product, Trichoderma asperellum, T. harzianum and T. yunnanense (Osorio et al., 2011; Jimenez et al., 2018), Bacillus amyloliquefaciens, B. liqueniformis and B. subtilis (Hernandez et al., 2014) and a mixture of microbial propagative ferment (MFPM) based on Trichoderma spp. -  Bacillus spp. The mixture of three species of Trichoderma (1x108) (treatment 1), MFPM (treatment 2), mixture of three species of Bacillus (1x108) (treatment 3), Thiabendazole (60%) (treatment 4) and an absolute control (treatment 5), at a dose of 1 L ha-1 for treatments 1, 2 and 3, while for thiabendazole they were 0.5 kg ha-1.

The application was made to the drench with a manual sprayer at 7, 28 and 49 days after the transplant (DDT). Subsequently to 85, 105, 125 and 145 ddt the yield per block (4.5 m2) was determined and transformed to t ha-1, from samples of 10 fruits the fruit weight (g) and size (mm) were determined. In the first and last cut the incidence transformed to percentage was evaluated, the severity was evaluated through the visual scale shown in Figure 1, where 0= no visible symptoms; 1= initial. Light chlorosis, presence of flowers and fruits; 2= intermediate. Partial withering, severe chlorosis, premature ripening of fruits and 3= advanced. Total withering without recovery, the leaves and fruits stay stuck to the stem.

Figure 1. Photographs used to determine the severity of chili wilt.

The data of the scales obtained in the assessments of the degree of severity of the disease were transformed by using the following formula cited by Carrión (2016):

Where: s= severity; Σ(a*b)= summation of the degree of affectation (0, 1, 2, 3); n= number of plants evaluated; k= greater degree of the scale (3).

Isolation of phytopathogenic fungi

Plants with apparent symptoms of the disease were collected and transferred to the laboratory, where their roots were washed with running water and cross sections were made at the base of the stem. Subsequently, they were superficially disinfected with a 1% sodium hypochlorite solution, where the stem sections were immersed for three minutes and rinsed in three sterile water passes. The tissues were dried on sterile paper and deposited in Petri dishes with the culture medium potato dextrose agar (PDA), incubated seven days for the development of fungi and colonies with different characteristics were isolated.

The purification was performed at 24 h by the technique of tip of hypha, were incubated for 15 days to make observations of the growth in PDA and under the microscope the characteristics of the mycelium, structures of reproduction and resistance, were used taxonomic keys of Sneh et al. (1991); Barnett and Hunter (1998); Leslie and Summerell (2006).

Pathogenicity test

It was carried out using the technique proposed by Sánchez et al. (1975), with slight modifications, using seeds of serrano chili of the variety Tampiqueño 74. The seeds were disinfected for three minutes in a solution of sodium hypochlorite at 1%, rinsed in three sterile water passes, and then dried in sterile paper and transfer them to Petri dishes containing water agar (AA) at 2% and incubated at 28 °C with 12-hour light cycles. They were checked every 24 h for three days to select seeds free of the growth of phytopathogens, to then transfer them in groups of three germinated seeds to Petri dishes with the same medium where they were inoculated near the root system with a fragment of the fungi of 0.5 cm of diameter approximately. They were incubated at 28 °C, for 7 days and the mortality of the seedlings was evaluated.

Statistical analysis

It was carried out by means of a completely randomized design with three repetitions, through an analysis of variance (Anva) and a comparison test of means according to Fisher’s supported by LSD test (p< 0.05), using the statistical program R (R Development Core Team, 2007).

Results and discussion

Two phytopathogenic fungi associated with the wilting of the chili culture of the experimental plot established in Saltillo, Coahuila, Fusarium oxysporum and Rhizoctonia solani were recovered. The growth of F. oxysporum in PDA showed white color and microscopically unicellular and bicellular microconidy were observed, from ovoid to ellipsoid on short fialides grouped in falas heads and microconidy of three septa with slightly curved apical cells. R. solani in PDA presented growth of light brown mycelium, under the microscope the hyphae tended to branch at right angles with a slight constriction near the point of formation of the produced angle.

The recovered fungi were confirmed as causative agents by means of the pathogenicity tests, where a smaller amount of absorbent hairs and necrosis were observed in the root. R. solani was more aggressive observing death of the seedlings at 8 days and F. oxysporum until 12 days. In the symptomatology of the disease, yellowness, flower abortion, wilting, necrosis in the root system and premature maturity of fruits were observed, which were adhered to the plant (Figure 2).

Figure 2. Symptomatology of wilting in field conditions. a) Healthy plant; b) Yellowing in the foliar stratum of the plant; c) Yellowing, flower abortion and premature ripening of fruits; and d) Fruits ripe and attached to plants killed by drought.

As in the present investigation, Albañil et al., (2015) reported F. oxysporum and R. solani as causal agents of chili wilt in the Bajio and southwest of Guanajuato. The symptomatology in the severity scale agrees with the report in the states of Aguascalientes and Zacatecas, where these phytopathogens were found in the greater percentage of the isolations causing wilting, defoliation, changes in foliage color, root rot, among other symptoms (Velásquez et al., 2001).

In the Table 1 shows the incidence of the disease, where statistical differences were observed (p≤ 0.05) in the materials HS-52 and Coloso. The Trichoderma treatment with the variety HS-52 has an incidence of the disease of 10.67% that represented a decrease with respect to the control of 21.16%.

The Coloso variety showed low percentages of incidence with thiabendazole where 6.83% existed, this shows excellent response of the variety to the application of the chemical. The varieties HS-44, Centauro, Tampiqueño 74 and Paraíso did not present significant statistical differences, placing them in the same statistical group.

Table 1. Incidence of the disease (%) in serrano chili with respect to each treatment.

Average values in the same column with different lowercase letters significant statistical difference (p< 0.05) according to Fisher’s supported by the LSD test; NS= not significant. Mean values in the same column with equal letters.

In the Figure 3 showed significant statistical differences (p≤ 0.05) in percentages of incidence of the disease between treatments in the six evaluated materials, where Trichoderma and MFPM had low percentages of incidence (14.39 and 16.39%), being control and Bacillus the who presented high levels of the presence of symptoms (24.08 and 23.36%).

Figure 3. Incidence of the disease in the treatments with respect to all varieties of serrano chili.

The severity of the disease transformed to percentage (Table 2) showed statistical differences (p≤ 0.05), where the MFPM treatment with the material HS-52 expressed the best results with 8.33%, in this same variety the Trichoderma treatment presented low percentage of incidence and severity of 11.30%. In the variety Tampiqueño 74 the application of thiabendazole had 6.54% severity, followed by Trichoderma with 7.83% and control with 17.96%, observing response of the material to the application of the treatments.

On the other hand, Paraíso with application of the mixture of MFPM and Trichoderma showed low levels of severity (6.46 and 6.93%), representative and inferior to the control, which allowed a better harvest. When testing with different Bacillus species in the chili culture Guillén et al. (2006) reported that the application of these microorganisms increased plant biomass and yield, this is possibly related to the type of clay-loam soil where good results were obtained, such effect was not found in this investigation where the control of the disease and yields were low.

Table 2. Severity of the disease (%) in serrano chili with respect to treatments.

Average values in the same column with different lowercase letters significant statistical difference (p< 0.05) according to Fisher’s supported by the LSD test; NS= not significant. Mean values in the same column with equal letters.

The biofungicidal efficiency of Trichoderma spp. it is attributed to antagonism (Reyes et al., 2012) and mycoparasitism to phytopathogenic fungi, which is appreciable in this investigation where the incidence and severity of wilt of the chili culture was reduced (Atanasova et al., 2013).

In Table 3 it was observed that the fruit size was statistically higher (p≤ 0.05) in the Centauro variety with the MFPM (73.44 mm), Bacillus (67.94 mm) and Trichoderma (66.44 mm) treatments, with an increase of 28, 23 and 21%, with respect to the witness. In the variety Tampiqueño 74, Bacillus, MFPM and Trichoderma treatments had sizes of 78.74, 74.73 and 73.63 mm, respectively. For the fruit weight variable, the material HS-44 was found to be statistically different, with a better response in Bacillus (10.83 g), MFPM (10.5 g) and Trichoderma (10.00 g) with respect to the control.

In the crop yield (Table 3) the statistical difference (p≤ 0.05) was studied in varieties HS-52, Paraiso and Centauro, in this context it was observed that the Trichoderma treatment presented a better behavior with 15.67 and 13.22 t ha-1 in the variety HS-52 and Centauro. The application of Trichoderma increases the production of 62% and 61% in the varieties mentioned above, with respect to the control. The application of the MFPM reports high yields in the variety Centauro (11.52 t ha-1), Paraíso (10.59 t ha-1) and HS-52 (10.37 t ha-1), the control was the lowest yield.

It was analyzed that the application of the MFPM increased 76% the yield with respect to the control, with statistically significant difference (p≤ 0.05) regarding the treatments Tiabendazol and control (Table 3). It was observed that Trichoderma increased the yield, this has been shown by different Trichoderma species in habanero chili plants (Capsicum chinense) (Candelero et al., 2015), lettuce (Lactuca sativa), radish (Raphanus sativus) (Ortuño et al., 2013) and pea (Pisum sativum) (Camargo and Avila 2014). In the same context, Cubillos et al. (2009) tested with Trichoderma harzianum in the cultivation of passion fruit (Passiflora edulis) where they could determine that it is an antagonist to F. oxysporum and F. solani, besides stimulating germination, increase of biomass and root length.

Table 3. Length, fruit weight and yield with the different evaluated treatments of the serrano chili culture.

Average values in the same column with different lowercase letters significant statistical difference (p< 0.05) according to Fisher’s supported by the LSD test; NS= not significant. Mean values in the same column with equal letters.

The positive interaction between Trichoderma and the host plant is attributed to a complex chemical activity of volatile and diffusible secondary metabolites, release of phytohormones and antibiotics in the rhizosphere, which promote the development of the root and a greater absorption of nutrients, which help control phytopathogens and increase yield (López et al., 2015), which explains the effect produced in this investigation. Microbial extracts as biofertilizers have the capacity to generate hormones that stimulate the development and increase the yield (Martínez et al., 2017), which could be verified with the application of the MFPM of the growth of Trichoderma spp., and Bacillus spp., which showed an effect in the control of the disease and in the development of the crop in equal or better percentage than when using the microorganisms.

Conclusions

The application of Trichoderma and the MFPM are excellent alternatives for the control of the wilt of the chili caused by Fusarium oxysporum and Rhizoctonia solani, besides increasing the yield of the different materials of serrano chili under the conditions of the Experimental Field El Bajío of the UAAAN in Saltillo Coahuila.

Acknowledgments

To the National Council of Science and Technology (CONACYT) for the financial support through a student scholarship, to MC Moisés Ramírez Meraz for providing serrano chili seeds used in this experiment and to MC Fidel Maximiliano Peña Ramos for the support in the statistical analysis.

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