Morphological and molecular identification of Phytophthora capsici L. in pipiana pumpkin and its greenhouse management

Identificación morfológica y molecular de Phytophthora capsici L. en calabaza pipiana y su manejo en invernadero

José Francisco Díaz-Nájera¹; Mateo Vargas-Hernández¹; Santos Gerardo Leyva-Mir¹; Sergio Ayvar-Serna²; Alejandro Casimiro Michel-Aceves²; Omar Guadalupe Alvarado-Gómez^3*

¹ Universidad Autónoma Chapingo, Departamento de Parasitología Agrícola, Programa de Protección Vegetal. Carretera México-Texcoco km. 38.5, Chapingo, Edo. de México, C.P. 56230, MÉXICO.

³ Universidad Autónoma de Nuevo León, Facultad de Agronomía. Av. Universidad s/n, cd. Universitaria San Nicolás de los Garza, Nuevo León, C.P. 66455, MÉXICO. Correo-e: alvarado@prodigy.net.mx (*Autor para correspondencia).

Received: February 3, 2014.
Accepted: August 10, 2015.

Abstract

The pipiana pumpkin is an important element in the diet of south-central Mexico residents. Its seeds are consumed directly toasted and seasoned with salt, and they are also the main ingredient used for making typical dishes such as green mole or pipian, as well as various traditional sweets. Some soil microorganisms cause severe damage in fruit, which reduces yield. The aim of the study was to identify morphologically and molecularly the oomycete causing rot in pipiana pumpkin fruits, and evaluate options for chemical and biological control in greenhouses. During August and September 2011, in the northern region of the state of Guerrero, pipiana pumpkin fruits with rot symptoms were collected. Morphological identification was performed with the keys proposed by Gallegly and Hong (2008), and molecular identification was by polymerase chain reaction (ITS-PCR). Both tests identified Phytophthora capsici as the causal agent of rot in pipiana pumpkin fruits. The sequences obtained showed 99 % similarity with the GenBank-held sequences for P. capsici in watermelon from the United States and pumpkin from Italy. The active ingredients propamocarb + fosetyl and metalaxyl + chlorothalonil delayed the presence of the pathogen in the fruits by six days, whereas the biocontrol agents delayed it by four days.

Keywords: Cucurbita argyrosperma Huber, ITS, DNA sequencing, chemical disease control.

La calabaza pipiana es importante en la alimentación de la población en el centro-sur de México. Sus semillas se consumen directamente tostadas y aderezadas con sal; además, son el ingrediente principal para elaborar platillos típicos como el mole verde o pipian, así como diferentes dulces tradicionales. Algunos microorganismos del suelo causan severos problemas en frutos, lo que afecta el rendimiento. El objetivo del estudio fue identificar morfológica y molecularmente al oomicete causante de la pudrición de frutos de calabaza pipiana, y evaluar opciones de control químico y biológico en invernadero. Durante agosto y septiembre de 2011, en la zona norte del estado de Guerrero, se colectaron frutos de calabaza pipiana con síntomas de pudrición. La identificación morfológica se realizó con las claves propuestas por Gallegly y Hong (2008), y la molecular fue mediante la reacción en cadena de la polimerasa, ITS-PCR. En ambas pruebas se identificó a Phytophthora capsici como el causante de la pudrición de frutos de calabaza pipiana. Las secuencias obtenidas tuvieron 99 % de similitud con P. capsici en sandía de Estados Unidos y en calabaza de Italia, depositadas en el banco de genes (GenBank). Los ingredientes activos, propamocarb+fosetil y metalaxil+clorotalonil, mostraron control al retrasar seis días la presencia del patógeno en frutos, mientras que los agentes de biocontrol la retardaron cuatro días.

Palabras clave: Cucurbita argyrosperma Huber, ITS, secuenciación de ADN, manejo químico de enfermedades.

INTRODUCTION

The pipiana pumpkin (Cucurbita argyrosperma Huber) is an economically important crop in the state of Guerrero, one of the major state producers of its seed in Mexico. The seed sales price per ton has risen from $ 15,500.00 MXN in 2005 to $ 35,000.00 MXN in 2011, which has led to the area sown in the state increasing from 4,228 to 5,742 ha. However, production in Mexico and in various parts of the world is affected during the summer by the warm conditions and high relative humidity that favor the presence of diseases (Cohen, Burger, Horev, Koren, & Edelstein, 2007; Zitter, Hopkins, & Thomas, 2004).

In some areas of the state of Guerrero, pipiana pumpkin is grown in flat and poorly-drained soils, which together with the crop's indeterminate and creeping growth habit generate a microclimate with optimal conditions for development and infection by fungi and oomycetes such as Phytophthora spp., causing yield losses (Ayvar-Serna, Mena-Bahena, Durán-Ramírez, Cruzaley-Sarabia, & Gómez-Montiel, 2007) and thereby reducing the incomes of farmers who grow it. Farmers commonly use systemic fungicides for disease control over prolonged periods of time (Gisi & Sierotzki, 2008); their overuse, in turn, has caused environmental pollution and decreased their efficiency due to the constant evolution of pathogens (Bartlett, Clough, Godwin, Hall, Hamer, & Parr-Dobrzanski, 2002).

Recently, the use of non-chemical methods to control plant diseases has increased. In the last 30 years, beneficial microorganisms have been used for biological control of diseases (Lim & Kim, 2010; Yang et al., 2012), mainly fungi such as Trichoderma, Gliocladium, Coniothyrium and Candida, and bacteria such as Streptomyces, Pseudomonas, Bacillus and Agrobacterium (Harman, Obregón, Samuels, & Lorito, 2010; Pliego, Ramos, Vicente, & Cazorla, 2011). The use of isolated strategies has had little impact, thereby necessitating an integrated management program (Antonopoulos, Melton, & Mila, 2010; Bi, Jiang, Hausbeck, & Hao, 2012). Based on the above, the aim of this research was to identify morphologically and molecularly the oomycete causing rot in pipiana pumpkin fruits, and assess options for chemical and biological control in greenhouses.

MATERIALS AND METHODS

During August and September 2011, fruits of pipiana pumpkin (C. argyrosperma), criollo genotype Apipilulco, were collected from a lot in the Guerrero State Agricultural College's Experimental Field, located in the municipality of Cocula, Guerrero, situated at 18° 19' NL, 99° 39' WL, and at 640 meters. The sample size and W-transect type of systematic sampling used in the present study were based on the methodology proposed by Pedroza-Sandoval (2009). Symptoms considered were sunken watery spots with white mycelium growing on the bottom and top of the fruit (Zitter et al., 2004).

Morphological identification

From pumpkin fruits with symptoms and signs of rot, five pieces of 0.5 cm2 tissue were taken from the advancing disease area. Samples were disinfected with 1.5 % sodium hypochlorite for two minutes, washed three times with sterile distilled water and dried. In total 100 tissue samples were sown in Petri dishes containing vegetable juice-Agar (V8-Agar) as culture medium (Singlenton, Mihail, & Rush, 1992). Each developed colony was isolated and purified by monozoosporic culture using the methodology described by Fernández-Herrera, Guerrero-Ruiz, Rueda-Puente, and Acosta-Ramos (2013). To obtain sexual structures, five discs of approximately 1.0 cm in diameter with the medium containing the pathogen were placed in Petri dishes with 20 mL of sterile water. The pathogen was incubated at 25 ± 1 °C, and four days later they produced sporangia. Preparations were made and analyzed in light microscopy at 40 X. The length and width of 30 sporangia were measured. Additionally, scanning electron microscopy was performed. Morphological identification was based on the keys described by Singlenton et al. (1992), Watanabe (2002) and Gallegly and Hong (2008).

Pathogenicity tests

Twenty healthy fruits were disinfected with 1.5 % sodium hypochlorite for two minutes, washed with distilled water and dried on paper towels, both sterile. The increase in the inoculum was in V8-Agar medium. After 15 days of growth, 40 mL of a suspension was prepared at a concentration of 4 × 10⁶ zoospores per mL; in total, 15 fruits were inoculated by spraying with 2.5 mL per fruit. Only sterile distilled water was applied to the control. Inoculated fruits were placed on 60 × 40 cm Styrofoam trays, previously disinfected with 70 % ethanol and incubated under controlled conditions in a glasshouse at 26 °C and 80 % relative humidity. Symptoms of advancing rot were recorded daily for eight days. Ten tissue samples (± 0.5 cm²) were obtained from the forward rot areas, disinfected in the same way as the fruits, and sown on V8-Agar culture medium. The morphological characteristics of the reisolated colony were determined to complete Koch's postulates. The reisolated oomycete was purified and incubated for 15 days in V8-Agar culture medium for DNA extraction.

Molecular identification

Evaluation of chemical and biological control

The effect of different active ingredients on the pathogen isolated in the Department of Agricultural Parasitology's Plant Pathology glasshouse at the Universidad Autónoma Chapingo, Chapingo, state of Mexico was compared. The plant material was the pipiana pumpkin (criollo genotype Apipilulco), using only tender and healthy fruits weighing about 150 g.

A monozoosporic isolate was obtained from fruits with different symptoms and levels of damage and then sown in V8-Agar culture medium for growth (Singlenton et al., 1992). To induce zoospore production, V8-Agar medium discs containing the pathogen were sown in Petri dishes containing 20 mL of sterile distilled water and incubated at 25 °C for three days (Fernández-Herrera et al., 2013). Subsequently, the zoospore suspension was adjusted to a concentration of 4 x 10⁶ mL^-1. First, the treatments were applied (Table 1) using a 2-liter hand sprayer (RL FLO MASTER); the surface of the fruits was sprayed with a water expenditure of 300 liters∙ha^-1. Five hours were allowed to elapse to allow reentry of the products; once the time had elapsed, 2.5 mL of the adjusted pathogen concentration were sprayed.

Variable evaluated

The variable evaluated was days to the presence of the pathogen, which was the number of days it took, after applying the treatments and inoculating the pathogen, for colonies to begin appearing on the 0.5 cm² samples.

Experimental design and data analysis

A completely randomized design with four replications was used. The experimental unit consisted of four fruits; due to the characteristics of the experiment, the entire unit was considered as useful plot. An analysis of variance was performed with the data obtained from the study variable. Similarly, a multiple comparison test was performed using the Tukey method with a significance level of 1 %, and orthogonal contrasts were used to compare the chemical vs. biological treatment groups. Statistical analyzes were performed using the SAS statistical package (Statistical Analysis System [SAS], 2009).

RESULTS AND DISCUSSION

Morphological identification

The original isolate colonies showed white, cottony mycelial growth with unipapillate and bipapillate sporangia from 20-50 × 15 -42.5 μm, and papilla from 6.02-7.05 μm wide. The morphological characteristics observed are consistent with those described by Singlenton et al. (1992), Wantanabe (2002) and Gallegly and Hong (2008) for P. capsici (Figure 1).

Pathogenicity test

All inoculated fruits showed symptoms of sunken watery rot, abundant white mycelium typical of the oomycete and sunken, brownish-gray to brown watery lesions (Figure 2A), whereas the control fruits showed no signs of rot (Figure 2B).

Molecular identification

The ITS-PCR products were bands of 779 bp (base pairs). The two directions sequenced per isolate had 99 % similarity. The sequence of the isolate identified by morphology was aligned with two sequences of the same species in the NCBI GenBank, and the alignment was with the highest identity value. The obtained sequence was deposited in GenBank and the access number KJ652220 was obtained.

Evaluation of chemical and biological control

The presence of the pathogen in fruits is an important characteristic; since once it is identified in tender fruits, they are completely damaged and lost, affecting the crop's most valuable product: the seeds (Trigos, Ramírez, & Salinas, 2008; Díaz-Nájera, 2013). This variable showed highly significant differences (P < 0.0001), since in the mean comparisons the active ingredients, propamocarb + fosetyl and metalaxyl + chlorothalonil, delayed the appearance of P. capsici to the sixth day, while the control differed from all the treatments as the pathogen appeared at 2.8 days after inoculation (Figure 3).

The inhibitory effect of the oomycete was due to the fact that the active ingredients used are specific to oomycetes. Propamocarb belongs to the carbamate group, and in oomycetes it affects lipids and membrane synthesis (FRAC, 2011). The phosphonate group includes fosetyl-Al, which is very selective to oomycetes in in vivo conditions. Fosetyl-Al causes effects in various metabolic sites in the mycelial phase of the pathogen's life cycle and inhibits sporulation at low concentrations (Erwin & Ribeiro, 2005). In this regard, Hu, Hong, Stromberg, and Moorman (2007) note that propamocarb and fosetyl-Al have good protective and curative action against oomycetes, which supports what was found in the present study. For their part, Reiter, Wenz, Buschhaus and Buchenauer (1995) state that the active ingredient propamocarb inhibits the formation of oospores in P. infestans. However, it is reported that fosetyl-Al has a high degree of systemic activity and efficacy that is generally superior against oomycetes, exerting good control (Gent, Ocamb, & Farnsworth, 2010).

The phenylamide group includes metalaxyl, which in oomycetes affects the synthesis of nucleic acids; therefore, its control begins when the mycelium starts growing (FRAC, 2011); However, despite being a very effective fungicide, it has a high risk of developing resistance due to its specific and unique mode of action (Elliott, Shamoun, & Grace, 2015); it is recommended to use it in a mixture with contact fungicides such as chlorothalonil whose multi-site effect makes it effective against pathogens such as oomycetes, and it also has a lower risk of resistance (FRAC, 2011; Elliott et al., 2015).

There are reports that metalaxyl has reduced the progress of diseases caused by oomycetes (Álvarez-Romero, García-Velasco, Mora-Herrera, González-Díaz, & Salgado-Siclán, 2013), since it is effective at all stages of the pathogen's life cycle (Qi, Wang, Zhao, Li, Ding, & Gao, 2012). Studies have shown that metalaxyl is efficient in reducing infection by P. capsici (Fernández-Herrera, Acosta-Ramos, & Pinto, 2007). The multi-site fungicide chlorothalonil proved to be one of the most effective treatments within the range evaluated in a mixture with metalaxyl, which was due to it being a compound with multi-site action on oomycetes, as reported by Gisi and Sierotzki (2008). Amrutha, Eswara-Reddy, Bhasakara-Reddy, and Prasanthi (2014) point out that the key factors contributing to the antagonistic effect of Trichoderma are its rapid growth, production of antimicrobial metabolites and physiological characteristics (El-Katatny & Emam, 2012).

The result obtained with T. virens strain G-41 agrees with that reported by different authors (Harman et al., 2010; El-Katatny & Emam, 2012) who mention the biocontrol capabilities of the genus Trichoderma. In general, Trichoderma spp. has been extensively studied as a biocontrol agent of many plant pathogens. Osorio-Hernández, Hernández-Castillo, Gallegos-Morales, Rodríguez-Herrera, and Castillo-Reyes (2011) reported a suppressor and inhibitory effect of Trichoderma spp. against P. capsici.

There is reported evidence that microorganisms such as Bacillus subtilis, Streptomyces and Trichoderma spp., included in the product PHC^® Biopak-F^® individually and in combination, are very promising biocontrol agents in the management of soil-borne pathogens (Köberl et al., 2013). Different authors report good control in soil-borne pathogens similar to those of this research, such as P. capsici (Lim & Kim, 2010).

The fundamental purpose of all agricultural activity is the optimal use of available resources, thereby maximizing profitability; for this reason, it is important to carry out economic analyses to determine the feasibility of treatments applied to this crop. The benefit-cost ratio of applying chemical and biological fungicides has been studied by Díaz-Nájera (2013), who reports earnings per peso invested ranging from $ 0.02 MXN to $ 0.39 MXN, obtaining the greatest profit with the native strain CSAEGro T. asperellum. The consequences of possible residuality in the seeds have not been studied; however, it may be ignored because the applications are made preventively at the beginning of fructification (40-45 days after sowing) and the fruits are harvested once they reach physiological maturity (uniform yellow color) at 120 days after sowing, when 75-80 days have already elapsed since application (Díaz-Nájera, 2013).

Orthogonal contrast test

This test indicated that there are highly significant differences (P < 0.0001) between the biological and chemical products. The biological treatment group delayed the appearance of P. capsici by an average of 4.22 days, while the chemical treatment group delayed it by an average by 5.80 days; the chemical group, therefore, was better because it delayed the pathogen's appearance by an additional 1.58 days (Figure 4).

CONCLUSIONS

Morphological and molecular identification, plus pathogenicity tests, confirmed that the causal agent of rot in pipiana pumpkin fruits was P. capsici.

The fungicides propamocarb + fosetyl-Al and metalaxyl + chlorothalonil delayed the presence of the oomycete by six days, whereas the biological control agents delayed it by about four days.

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