MATERIALS AND METHODS

Soil and infected plant tissue sampling. In 2016 and 2017, samples were collected at the municipalities of Copándaro, Morelia, Queréndaro, Tarímbaro, Vista Hermosa and Yurécuaro, Michoacán; and La Barca, Jalisco (Figure 1). The samples were taken from root, crown, and stem sections (10-20 cm long), and soil from a plot where solanaceae and cucubitaceae had been previously cultivated (Figure 2). In these municipalities, chili pepper is cultivated every year, except in Queréndaro, where rotation with grains and other crops is practiced for 3-5 years, and Morelia, where zucchini-type squash (C. pepo) is mainly grown. The soils collected in Queréndaro plots were loam to loam-sandy; loam-clay-sandy in Copándaro, Morelia and Tarímbaro; and loam-clay in Yurécuaro and La Barca. Drip irrigation was applied to the plots. Poblano is the type of chili most cultivated in the areas where samples were collected, and, to a lesser degree, pasilla, serrano, jalapeño, and güero-type chilis. Zucchini squash samples were collected in the study sites of the Morelia municipality, and tomato samples (S. lycopersicum) in the municipality of Tarímbaro; the rest were samples from serrano-type chili pepper crops.

Preliminary detection of Phytophthora using immunostrips. The samples of infected plant tissue were serologically tested to rapidly detect Phytophthora, including P. capsici, using immunostrips (InmunoStrip, Agdia^®). According to the manufacturer’s instructions, approximately 25 g of necrotic root tissue were taken and placed in a saline solution, gentle friction was applied and the immunostrip was inserted. The positive samples were processed to isolate the pathogen. When isolation was performed in a plot in Queréndaro, where the result had been negative, Fusarium solani was detected (^{Reyes-Tena et al., 2019c}).

Figure 1 Sites where solanaceae and cucurbitaceae showing wilt symptoms were collected. 

Figure 2 Solanaceae and cucurbitaceae crops showing wilt symptoms: a) zuchinni type-squash in Morelia; b) serrano chili in Tarímbaro; c) pasilla chili in Queréndaro; and d) poblano chili in Copándaro, Michoacán; e) poblano chili in La Barca, Jalisco; and f) poblano chili in Yurécuaro, Michoacán. 

Isolation of Phytophthora from soil. The soil samples were taken from the rhizosphere of C. annuum showing wilt symptoms in a plot located in Copándaro municipality. Each sample consisted of 100-150 g of soil collected at 10-15 cm depth; the samples were placed in a cooler and taken to the laboratory. To isolate Phytophthora, a bioassay using Rhododendron leaves as bait was established. For the bioassay, 10 g of soil and 20 mL of sterile distilled water were spread on Petri dishes 16 cm in diameter. The Rhododendron leaves were washed with soap and water, rinsed with sterile distilled water, and placed with the petiole submerged in the soil suspension in order to facilitate the pathogen’s invasion (^{Erwin and Ribeiro, 1996}). The boxes were sealed with plastic paraffin (Parafilm^®) and stored at 24 °C for 36-48 h or until the petiole became necrotic. Later on, the petiole was transferred to a NARPH medium with natamycin (0.02 g L^-1), ampicillin (0.27 g L^-1), rifampicin (0.01 g L^-1), pentachloronitrobenzene (0.10 g L^-1) and hymexazol (0.075 g L^-1) and stored at 24 °C (^{Soto-Plancarte et al., 2017}).

Isolation of Phytophthora using plant tissue. To favor Phytophthora isolation, pieces of root and stem were cut in the pathogen’s active growth zone, that is, pieces with the interface between necrosis and healthy tissue. The tissue pieces were disinfested with a 10% v/v solution of commercial chlorine (0.6% sodium hypochlorite active ingredient), rinsed with sterile distilled water, and placed on sterile blotting paper, according to the procedure described by ^{Soto-Plancarte et al. (2017}). The tissue pieces were placed in NARPH medium. Mycelial growth was observed 48-72 h later.

The obtained isolates were transferred to agar-water medium to be purified using the hyphal tip technique and then placed on an agar-corn meal medium. To determine if the isolates had contaminant bacteria, an agar disk containing mycelium was kept in a test tube with sterile Luria Bertani medium at 24 °C for 24 h; the isolate was considered free of bacteria when no turbidity was observed. When bacteria contamination was observed, the culture was transferred to agar-potato-dextrose medium with 0.14% tartaric acid (^{Soto-Plancarte et al., 2017}). Then, medium disks with 5-7 days-old mycelium were transferred to microtubes containing sterile distilled water and kept at 15 °C. The isolates were deposited in the oomycetes collection (Plant Pathology Collection) of the Plant Pathology Laboratory, Instituto de Investigaciones Agropecuarias y Forestales, Universidad Michoacana de San Nicolás de Hidalgo.

Comparative morphology of Phytophthora capsici isolates. The obtained isolates were grown on Agar-V8 medium and stored at 24 °C for 5-7 days. When growth covered the culture medium plates, pieces of approximately 1x1cm were cut. To induce the formation of sporangia, 15-18 mL of sterile distilled water were added, and the water was changed every 24 h for three days. To the isolates that formed few sporangia, non-sterile soil extract was added, and then they were left at 24 °C for 24 h (^{Almaraz-Sánchez et al., 2013}). When the isolates sporulated, the asexual traits were described. The test to determine sporangia expiration was conducted as follows: a drop of water was deposited on a slide and then a piece of agar with mycelium was placed on the drop of water by slightly shaking it. Observations under an optical microscope were made at 40X amplification to record the pedicel caducity or persistence in sporangia. The data recorded were type of sporangiophore, form of sporangia, papilla and pedicel length, as well as the presence or absence of chlamydospores. On the other hand, all the isolates were growth tested on Agar-V8 medium at 35 °C for 48 h.

To determine the mating type, the isolates were crossed with two isolates of known mating type (A1 and A2) on Agar-V8 medium. The cultures were observed 5-10 days after inoculation to detect the formation of oospores. The type of oospore and antheridium was recorded. The results were compared to the interactive key described by ^{Abad et al. (2019}) for Phytophthora of the United States Department of Agriculture (USDA), which is available at http://idtools.org/id/phytophthora/index.php.

Pathogenicity test. The pathogenicity of seven isolates (CPV259, CPV260, CPV267, CPV271, CPV272, CPV277 and CPV279) was determined in a previous study (^{Reyes-Tena et al., 2019a}). In this study, a group of seven isolates (CPV282, CPV290, CPV291, CPV293, CPV296, CPV297 and CPV301) was additionally selected to conduct pathogenicity tests. These 14 isolates corresponded to different hosts, municipalities both of Michoacán and Jalisco, and the two mating types. The production of inoculum and chili pepper seedlings was carried out following the protocol described by ^{Reyes-Tena et al., (2019a)}. Planting and production of seedlings of the California Wonder susceptible variety was carried out in 100 cm³ six-cell trays filled with Mix 3 (Sunshine^®) as substrate. The plants were inoculated with the pathogen 56 days after planting. Six plants per isolate were inoculated with 1 mL of a suspension of 1 x 10^-4 zoospores mL-¹, which was applied at the base of each plant’s stem using a 50 mL dosing syringe (Ape ®). The plants remained saturated with water for 24 h to favor infection. The appearance of symptoms was recorded, and the pathogen was re-isolated in NARPH selective medium.

RESULTS

A total of 41 Phytophthora spp. isolates from cucurbitaceae and solanaceae crops with a range of 20-80% wilt incidence were obtained (Table 1). Seventy-six percent of the isolates were recovered from samples collected in the municipalities of Copándaro, Morelia, Queréndaro and Yurécuaro, Michoacán.

Table 2 shows the description of the different sexual and asexual traits that were observed. The isolates had sporangia arranged in simple sympodia sporangiophores. Forty isolates showed caducous and papillated sporangia, and long pedicel; only the CPV-269 isolate had semi-papillated sporangia with medium-sized pedicel. Regarding the form of sporangia, 10 isolates had irregular or distorted form (Figure 3c). Overall, ellipsoid, ovoid and globose forms prevailed. The presence of globose and terminal chlamydospores (Figure 3d) was detected on the CPV-279 isolate. Eighty-eight percent of the isolates grew at 35 °C, except CPV-280, CPV-285, CPV-294, CPV-295 and CPV-296. All the isolates had plerotic oospores with amphyginous antheridia (Figure 3d). Forty isolates were heterothallic, and one was homothallic. In regard to the mating type, 21 isolates were of A1 type, and 19 of A2 type. Both were recovered from chili pepper plots at the municipalities of Copándaro, Queréndaro, Tarímbaro and Yurécuaro, Michoacán; and La Barca, Jalisco. A ratio of mating types close to 1:1 was found in the municipalities of Copándaro, La Barca, Tarímbaro and Yurécuaro. In Morelia, type A2 isolates were obtained.

In regard to the pathogenicity test, wilt symptoms were observed on the California Wonder variety at day three with the seven isolates that were inoculated; in all the cases, P. capsici was re-isolated to confirm the isolates pathogenicity.

DISCUSSION

P. capsici was identified as the causal agent in a total of 14 isolates (seven correspond to a pathogenicity test conducted in a previous study (^{Reyes-Tena et al., 2019a}) and 27 are associated to wilt disease in cucurbitaceae and solanaceae crops in the Jalisco and Michoacán municipalities where samples were collected. The morphological identification was in agreement with the molecular identification in a sample of isolates (^{Reyes-Tena et al., 2019a}; ^{Reyes-Tena et al., 2019b}). The isolates of this pathogen had the two mating types (A1 and A2) in a ratio close to 1:1 in four municipalities: Copándaro, La Barca, Tarímbaro and Yurécuaro. This result suggests that sexual reproduction could occur (^{Castro-Rocha et al., 2016}). A similar result regarding the ratio of both mating types was reported in P. capsici populations recovered from crops in Aguascalientes, Chihuahua, Mexico City, State of Mexico, Guanajuato, Michoacán, Querétaro, and Zacatecas (Castro-Rocha et al., 2016; ^{Fernández-Pavía et al., 2007}; ^{Pérez-Moreno et al., 2003}; ^{Silva-Rojas et al., 2009}). A ratio of mating types close to 1:1 was also found in populations from China, the United States and South Africa (^{Bi et al., 2014}; ^{Fernández-Pavía et al., 2004}; ^{Gevens et al., 2007}; ^{Meitz et al., 2010}; ^{Yin et al., 2012}). The presence of a single mating type (A2) in Morelia is comparable to that reported by Pérez-Moreno et al. (2003) with isolates obtained in Salvatierra, Guanajuato, where only A2 was found, as well as to studies using populations from Argentina, Spain, and Peru, where clonal populations were detected (^{Gobena et al., 2012}; ^{Hurtado-Gonzáles et al., 2008}; ^{Silvar et al., 2006}). This suggests that the presence of the pathogen is recent and that no isolates with both mating types have been introduced. Eighty-eight percent of the isolates grew at 35 ^oC, a result that was similar to that obtained by ^{Pons-Hernández et al., 2020}, who reported 96% for isolates from Guanajuato.

Figure 3  Phytophthora capsici sexual and asexual structures: a) simple sympodia sporangiophore; b) papillated and ellipsoid sporangia; c) sporangia with irregular forms; d) plerotic oospores with amphyginous antheridium; e) globose and terminal chlamydospores; and f) expired caducous sporangium. 

The sexual reproduction observed on P. capsici populations in cucurbitaceae and solanaceae crops in Jalisco and Michoacán could favor the survival and emergence of greater genetic variability within the pathogen’s populations in this Mexican region and thus hinder the management programs efforts (^{Babadoost and Pavon, 2013}; ^{Lamour and Hausbeck, 2000}). Although there is information about a high level of genetic diversity in populations from central Mexico (^{Castro-Rocha et al., 2016}), it is necessary to consider additional genetic factors that could cause variability, since the high level of P. capsici polymorphism could be mediated by other processes that produce variability, such as mutations, genetic recombination, epigenetic processes, and horizontal gene and chromosome transfer (^{Raffaele and Kamoun, 2012}). Another unfavorable factor in wilt management is the practice of susceptible crops rotation, such as C. pepo and S. lycopersicum. Crop rotation with non-susceptible hosts in 3-5-year periods is preferred in order to reduce the levels of the pathogen’s inoculum (^{Barchenger et al., 2018}).

The form of sporangia observed on this study agrees with the description for P. capsici, which develop varied forms, ranging from ovoid, ovo-ovoid, sub-globose, globose, ellipsoid, fusiform, piriform, to irregular forms, as well as the frequent presence of bi-papillated sporangia (^{Li et al., 2007}; ^{Soto-Plancarte et al., 2017}). The C. pepo isolates had sporangia with irregular or distorted forms, a fact that reflects the phenotypic plasticity of this species (^{Iribarren et al., 2015}). On the other hand, the CPV-269 isolate had semi-papillated sporangia with pedicel medium in size, which is a typical characteristic of P. capsici, and short, medium, and large pedicels (^{Granke et al., 2011}; ^{Martin et al., 2012}). Semi-papillated sporangia have been previously reported in P. capsici isolates (^{French-Monar et al., 2006}). In this study, the CPV-279 isolate that was obtained from C. annuum had apical globose chlamydospores; this isolate was previously identified as P. capsici at the molecular level by sequencing the cytochrome oxidase genes 1 and 2 (^{Reyes-Tena et al., 2019a}). The presence of chlamydospores is an unusual characteristic in P. capsici and is not considered in the description of the interactive key for this species (^{Aragaki and Uchida, 2001}; ^{Bowers et al., 2007}; ^{Donahoo and Lamour, 2008}; Martin et al., 2012). However, there are several reports of P. capsici forming these structures in the United States and Malaysia (^{Farhana et al., 2013}; Granke et al., 2011; ^{Islam et al., 2004}). For this reason, the high level of phenotypic variability that this pathogen shows could be associated with the location and the host from which they were obtained, but more studies need to be conducted to confirm this fact.

CONCLUSIONS

The results provide information about the presence and distribution of P. capsici in solanaceae (35 isolates) and cucurbitaceae (six isolates) producing areas in Michoacán and Jalisco municipalities. Thirty-three isolates from C. annuum, six from C. pepo, and two from S. lycopersicum were obtained. Since no previous studies have been conducted in these production areas, the information provided in this research will help horticultural producers to adopt preventive measures and apply specific products to combat this pathogen.