Phytophthora capsici Epidemic Dispersion on Commercial Pepper Fields in Aguascalientes, Mexico

Chili pepper blight observed on pepper farms from north Aguascalientes was monitored for the presence of Phytophthora capsici during 2008–2010. Initially, ELISA tests were directed to plant samples from greenhouses and rustic nurseries, showing an 86% of positive samples. Later, samples of wilted plants from the farms during the first survey were tested with ELISA. The subsequent survey on soil samples included mycelia isolation and PCR amplification of a 560 bp fragment of ITS-specific DNA sequence of P. capsici. Data was analyzed according to four geographical areas defined by coordinates to ease the dispersal assessment. In general, one-third of the samples from surveyed fields contained P. capsici, inferring that this may be the pathogen responsible of the observed wilt. Nevertheless, only five sites from a total of 92 were consistently negative to P. capsici. The presence of this pathogen was detected through ELISA and confirmed through PCR. The other two-thirds of the negative samples may be attributable to Fusarium and Rhizoctonia, both isolated instead of Phytophthora in these areas. Due to these striking results, this information would be of interest for local plant protection committees and farmers to avoid further dispersal of pathogens to new lands.


Introduction
Chili pepper blight due to Phytophthora capsici and Verticillium spp. [1] has been present in commercial pepper fields in Aguascalientes and neighboring states in Mexico [2]. Nevertheless, there is a lack of detailed data on the degree of incidence related to spatial distribution from Mexican areas where this Phytophthora is prevalent. Some approaches have been developed for Aguascalientes [3], Zacatecas [2], and Chihuahua [4,5]. From these studies only one [4] deals with some indicators of incidence of Phytophthora capsici according to one region. Since pepper blight is so devastating for pepper farms, it is worthy to study its incidence through a survey [6]. Therefore, the purpose of this work is to add more information on the spatial distribution of this pathogen on the northern part of Aguascalientes and describe its actual incidence on chili pepper farms.

Plant and Soil
Sampling. Whole plants showing severe wilting, collapsed still-green leaves, and root rot were collected from commercial fields. All of the plants collected in the field showed flowers and fruits. Additionally, 36 plantlet samples from two greenhouses and 16 rustic nurseries were included due to suspicious contamination only for ELISA testing (not for fungi isolations). Collection of plant samples in the field was done five times (once each month) on the same fields (92 geographical positions) from April to September 2008. Some data was missing for the first time of sampling (26 sites) due to late planting. Data

ELISA Detection of Phytophthora on Wilted Chili Plants.
Immunological detection through ELISA was performed as described by the supplier of the reagent kit for Phytophthora detection in plants (CAB 92600/0500 and ACC00948 from AGDIA). Stems of plants showing root rot (wilted) and controls with normal appearance were sliced with a knife and ground with a mortar. After grinding, 100 μL of sap was placed in Eppendorf tubes plus 1 mL of the extraction buffer GEB2 from the kit and mixed gently. At the same time, mycelia from Phytophthora capsici cultures were used as positive control together with the positive control included in the kit. A scrap from cultures about 1 cm 2 and 500 μL GEB2 were placed inside an Eppendorf tube and ground with a crystal bar. After grinding, tubes were left in vertical position for 10 min to allow particles to sink and reach the bottom. The rest of the protocol was followed according to the directions of the supplier.

Fungal Isolates from Wilted Chili Plants.
Fungi isolates were obtained from crown segments of wilted plants with rot roots and lesions on the stem that retained leaves and fruits. Plants were taken fresh from commercial fields (22 during 2010) and washed using tap water and commercial soap. Segments (5-10 mm) from the stem having visible lesions were cut with a razor blade and disinfected with 1% sodium hypochlorite (5-10 min) and rinsed with sterile distilled water for 1-2 min; then, the segments were dried on sterile paper napkins. In order to check for the microorganisms present in the plants, samples were placed on V8-agar (200 mL V8 + 800 mL distilled water + 0.2 g CaCO 3 + 20 g agar + 4 g sucrose) for 4-7 days in the darkness. In order to obtain Phytophthora, selective medium PARP (V8-agar + 5 mg Pimaricin + 250 mg Ampicillin sodium + 10 mg Rifamycin sodium + 50 mg PCNB) [7][8][9].

Fungal Isolates from Soil
Media Used. Acidified Potato Dextrose Agar (PDA + 16 g agar + 250 μL lactic acid) and a PARP variant (V8-agar + 5mg Pimaricin + 250 mg Ampicillin sodium + 10 mg Rifamycin sodium + 30 mg Cefalexin) were fixed. In order to count fungi CFUs, one gram of soil was dissolved on sterile water (10 mL final volume) and vortexed. Once the soil particles descended by gravity, a volume of the supernatant was taken and diluted 1/10. From that dilution 1 mL was spread over the acidified PDA and left for 48 h at room temperature. Colonies were randomly chosen by marking dots with permanent marker, placing a 25-point grid (as a guide) on the bottom of the Petri dish. Microscopic view (4x objective lens) was centered on each dot for colony quantification. Fungi genera were determined as previously described [10] using a 40x objective lens.
Phytophthora capsici was isolated using autoclaved fresh peanuts as trap. First, 16 g of soil samples plus 10 mL sterile water was placed in Petri dishes. Then, peanut halves were placed in the Petri dishes together with the soil samples and incubated at room temperature during 48 h. After incubation, peanut halves were placed on the PARP variant for at least 48 h, and once a culture was identified as Phytophthora, monosporal or hyphal tip cultures were transferred to a fresh PARP medium, as many times as required, until isolated. Half-an-inch agar dishes containing mycelia from the isolates were cultivated on acidified PDA Petri dishes covered with ≈6 g ground peanut dried at 36 • C plus 6 mL sterile water with antibiotics (30 mg/L Cefalexin + 30 mg/L Rifampicin + 5 mg/L Pimaricin) for 24 h.

PCR Test of Isolates from
Soil. DNA from soil isolates was extracted as described elsewere [11]. Briefly, mycelia desiccated at 36 • C for 24 h were ground on a mortar with liquid nitrogen. Then, 100 mg of ground mycelia plus 800 μL of extraction buffer (0.2 M Tris, 0.05 M EDTA pH 7.5, 0.2 M NaCl, 2% CTAB and 60 mL 5% Sarcosyl) was placed into a 1.5 mL Eppendorf tube. Tubes were incubated at 65 • C during 15 min under continuous gently shaking. After that, 600 μL phenol : chloroform : iso-amyl alcohol (24 : 25 : 1) was added to the tubes and incubated at −20 • C for 10 min. Tubes were centrifugated at 13,000 g during 15 min. The supernatant was placed in a fresh tube in order to repeat the previous step. Next, 600 μL of 70% ethanol was added and incubated overnight at −20 • C. After that, tubes were centrifugated at 13,000 g during 15 min and the supernatant was discarded. Pellets were washed with 70% ethanol and dried at room temperature. DNA was resuspended in 40 μL TE. PCR was performed as described by [12]. The primers used amplify a 560 bp fragment from specific Phytophthora capsici ITS sequences (PC-1: 5 -GTCTTG TACCCTATCATGGCG-3 and PC-2: 5 -CGCCACAGCAG GAAAAGCATT-3 ). Reaction mixture included 12.5 μL 2x GoTaq Green Master Mix (Promega 2800, Woods Hollow Road Madison WI 53711 USA), 2.5 μL from both primers (10 μM PC-1 and PC-2), 3 μL of genomic DNA, and 7 μL of nuclease-free water (Promega) for a 25 μL final volume. Temperatures of the thermocycler were set as follows: preheat at 94 • C during 4 min; 35 cycles including 30 sec at 94 • C for denaturing, 30 sec at 70 • C for annealing, and 1.5 min at 72 • C for polymerization; a final extension at 72 • C for 7 minutes. Once the PCR was concluded, samples were run in 1.5% agarose SB gel, with 240 V during one hour.
2.6. Statistical Analysis. ELISA results from greenhouse and nurseries were combined and analyzed using chi-square for an equal chance of a positive/negative response (χ 2 , 1 df, e 1 and e 2 = 50%). ELISA results from four geographical zones were analyzed as a contingency table. In both cases, the observed significance level (P) was recorded.

ELISA Detection of Phytophthora on Wilted Chili Plants.
Plantlets from greenhouse and nursery: initially, 36 samples were selected from plantlets showing either wilting or lesions from two greenhouses (10 samples from cultivars: Caloro, Hungaro, Guajillo, Pasilla, and Puya), and 16 rustic nurseries from poor farmers (26 samples from the same cultivars but Caloro) were tested for the presence of the pathogen. Only one sample out of six from the first greenhouse was positive to the presence of Phytophthora while the four samples from the second greenhouse were all positives. Furthermore, all of the samples from nurseries were positive to the test. This was a possible indication that these 86% positive samples may be the starting point for soil contamination of the Aguascalientes chilli fields. Since previous field trips (in 2007) have shown that there was no field free of wilted plants with root rot (5-90%) and information of Phytophthora capsici dissemination in chilli fields of Aguascalientes is rather scarce, the expected values of positives were at least 50% from either the nurseries or the greenhouses (data combined as G + N in Table 1). Plants from the fields: data from the five dates of sampling was combined within each cluster or zone. Zones are described from north to south. In the first zone, located within 22  (Table 1). Zone 4 had the highest percentage of positives to Phytophthora. Nevertheless, this data does not reflect the damage observed on the fields because they were not surveyed for the number of wilted plants or production of commercial fruits. In some cases, the damage was so intense that the whole field was plowed before the crop cycle was completed. Taking together the results of the four zones, only five out of 92 geographical sites consistently did not show the presence of the pathogen on the successive sampling. This is an alarming result, since most of the farmers move to new fields that were not used for chilli pepper, leaving contaminated patches with the pathogen in the northern Aguascalientes.

Fungal Isolates from Wilted Chili Plants.
Plants for fungi isolation during 2010 were collected from the same geographical area (176 plants from 22 different fields) as the previous two years; the criteria for previous plant sampling prevailed. Since this type of wilting is due to the attack of fungal complexes, Phytophthora was not the only genera isolated. Actually, its counts appear to be lower, probably because the others are aggressive at the time of colonizing the media. Fusarium and Rhizoctonia have their own contributions for the wilting and root rot as previously described [1]. The rest may be saprophytes or contaminants of the rotten areas ( Table 2).
The results of isolation not necessarily reflect all of the events of the process from infection to death of chilli plants due to Phytophthora. González-Chavira [13] initially tried to establish a relationship in Guanajuato (Mexico) of this kind but the number of Phytophthora isolates was rather low (2%) as compared to Fusarium spp. (65%) and Rhizoctonia solani (33%). Later, this author moved to PCR for detection of Phytophthora from fresh plants [14]. Some of the same pathogens were found as a complex on plants showing similar symptoms (brown-black discolored collar with root rots causing permanent wilting and plant death) in Europe. A three-year survey on pepper showing wilting and discoloration (755 plants from 120 farms, Spain) showed that Phytophthora capsici (18%), Sclerotium rolfsii (83%), Rhizoctonia solani, Verticillium dahliae (33%), and other potential pathogens were present [6].    were not recorded due to their low number. Specific trapping with peanut and Phytophthora capsici isolation on PARP, allowed confirming its presence through PCR ( Figure 1; Table 3). In this case, it was difficult to obtain positives without trapping Phytophthora, since only 1.8% was evident from 164 samples. Trapping with peanut helped in the detection, since 28% of a subsample (25 soil samples) was positive.

Conclusions
Plantlets positive to Phytophthora taken either from greenhouse or nursery were recorded as 86% (31/36); this figure indicates that they may be the primary source for the local spreading of the pathogen in pepper fields of Aguascalientes.
ELISA test showed that one-third of pepper blight samples from the field were positive to the presence of P. capsici. Nevertheless, most of the farms showed at least one positive during successive sampling of the same geographical site. Exemptions to the rule were five farms, corresponding to five geographical sites from a total of 92. Therefore, most farms from north Aguascalientes during 2008-2010 were detected as contaminated with Phytophthora capsici, distributed approximately the same on three out of four geographical zones. Only zone 4 showed about 50% of ELISA positives. Confirmation on the presence of Phytophthora through pathogen isolation and PCR supports ELISA data, although they were done one year later.
Wilting of the two-thirds not positive samples to Phytophthora may be attributable to fungus Fusarium and Rhizoctonia isolated alone or as a complex in this work. Other work includes to Verticillium and Pythium in the complex [1]. Since our surveys covered most of the pepper farms on the north Aguascalientes and all of the surveyed farms had wilted spots with similar symptoms (wilting with hanging still-green leaves and root rot), attention and preventive procedures should be devoted to avoid spreading of more than one pathogen associated to the blight to new farms. Additionally, detailed data on local dynamics of the disease are necessary [5,15] to support the work of the local committee for plant protection CESAVEA.