Sampling

The sampling area corresponded to two commercial orchards in the municipal area of Huatusco, Veracruz, Mexico, located in the coordinates 19° 7´23.1” N, 97° 0´ 5.2” W, and 19° 7´ 21.6” N, 97° 0´ 4.9” W, at an altitude of 1,333 masl. The types of soils present in the sampling area were Luvisols and Cambisols. The climate is semiwarm-humid with an average temperature of 18.8ºC; average annual rainfall is 1,763 mm and the characteristic vegetation is of the evergreen subtropics. In December 2014, samples were gathered of chayote plants, var. Ventlali with symptoms of characteristic rotting (loss of turgidity, softening of plant tissue, presence of mycelia, and strong smell) in fruits and the transition area between stem and root. The samples were transported to the lab in plastic bags, which were labeled and placed in a cooler.

Isolation and identification of microorganisms related to the disease

Ten samples were taken of both fruit and of the transition area between stem and root, from each of the two areas of study. In this case, the number of samples taken from each location was standardized, unlike reports by ^{Olguín et al. (2013)}. The infected tissue of chayote plants and fruits was washed with distilled water and cut into 6 sections of approximately 5 mm in length. The result was 60 sections (30 of fruit and 30 of the stem-root transition area). Likewise, 5 asymptomatic samples taken from fruits and the stem-root transition area were analyzed. The sections were disinfected with a commercial sodium hypochlorite solution 1 % for 1 min; they were rinsed several times with sterile distilled water, and dried with sterile paper towels. Later, the sections were cultivated in water-agar plates (acidified with lactic acid at 25 %) and incubated at 28 °C. Once myceliar growth was observed, using a hole puncher, discs of 0.5 cm in diameter were taken from the developed cultures, and there were replanted in a V8 juice-agar (V8) and potato dextrose agar (PDA), and incubated at 28°C for 48 h (^{French and Hebert, 1980}; ^{French-Monar et al., 2006}). The oomycete developed was purified in V8-agar medium plate using monosporangia. The qualitative and quantitative characteristics of the asexual structures (sporangiophores and sporangia) of the oomycete were analyzed from semipermanent preparations with the use of a compound microscope (OLYMPUS. Mod: CX31RBSFA). The sporangia were measured after 10 days of incubation in V8 medium. With the result of 100 measurements, we identified the oomycete using the descriptions by ^{Newhook et al. (1978)}, ^{Stamps (1985)}, ^{O’Donnell (1992)}, and ^{Gallegly and Hong (2008)}. The rest of the fungal isolations present in the samples were purified by transferring cultures, and were identified using the taxonomic keys of ^{Barnett and Hunter (1998)}.

Pathogenicity test of the isolated chayote oomycete

The oomycete with the highest frequency (76 %) was used to carry out the pathogenicity test. The chayote fruits were washed with soap and water, then disinfested with alcohol at 70 %. Using a blade, two cuts were made, each 1 cm deep, inside of which we placed a culture medium disc, of 0.5 cm in diameter, which contained the oomycete. Eighteen chayote fruits were inoculated. The control fruits were only added growth medium without oomycete growth. Afterwards, the inoculated fruit were placed in a wet paper towel on top of a base of styrofoam. The fruits were kept inside sealed plastic bags. The paper towel was added sterile distilled water to maintain moisture saturation conditions in the system.To stimulate the growth of the oomycete, the incubation temperature was kept between 24 and 27 °C, with a photoperoid of 12-12 h. Once the fruits were invaded by the mycelium of the oomycete, the latter was reisolated in V8 medium plates. The morphology of the cultures obtained was compared with that of the original isolation in commercial orchards. The pathogenicity test was performed twice.

DNA amplification and sequencing of the oomycete

After four days’ growth of the oomycete in V8 medium plates, a portion of approximately 5 mm of mycelium was tekan and placed in a 200 μL test tube with30 μL of the lysis solution Lyse N Go (Pierce, USA). The tube was then heated at 95 °C for 5 min and centrifuged for 10 min at 5,000 x g. Finally, 5 μL of supernatant were taken to carry out the amplifications of the ITS by PCR.

For the amplification of the ITS area, we used the universal primers ITS5 (5’-GGAAGTAAAAGTCGTAACAAGG-3’) and ITS4 (5’-TCCTCCGCTTATTGATATGC) (^{White et al., 1990}), which amplify a fragment of 580 pairs of bases, approximately (pb). Another pair of primers used was ITS5 and NL4 (5’-GGTCCGTGTTTCAAGACGG- 3’) (^{O’Donnell, 1992}), to amplify a fragment of 1,100 pb, which included the region of the ITS and ~600 pb of the gene 28S rRNA. The reaction mixture for PCR was prepared in a final volume of 25 μL, containing the enzyme 1X Taq DNA polymerase, 0.8 mM deoxynucleotide triphosphates (0.2 mM each), 100 ng DNA, 20 pmol of each primer, and 2 units of GoTaq DNA (Promega, USA). The amplifications were carried out with an initial denaturation cycle at 95 °C for 2 min; 35 denaturation cycles at 95 °C for 1 min, alignment at 57 °C for 1 min, and a final extension at 72 °C for 2 min; finally, one amplification cycle at 72 °C for 10 min (^{Silva-Rojas et al., 2009}).

All the PCR reactions were carried out in a thermocycler (Model: TECHNER TC-512, brand: Bibby Scientific). The amplifications were verified by electrophoresis in an agarose gel at 1.2 % prepared with the buffer 1X TAE (Tris-AcetatoEDTA) and run at 87 V cm^-3 for 1 h. The gel became stained with ethidium bromide (3 mg L^-1), and the bands were viewed in a UV transilluminator (Model: GL-3120, brand: Scientific). The amplified products were cleaned using the purification kit (Model: QIAquick PCR, brand : Qiagen), following instructions by the manufacturer. These products were sequenced in both directions using an automized system DNA sequencing system (Model 3730XL, brand: Applied BioSystems), to ensure no incorrect nucleotide readings.

Comparing the ITS and LSU sequences

The sequences form both regions were assembled and edited using BioEdit version 7.0.5 (^{Hall, 1999}), used to create a consensual sequence. This sequence was compared with sequences deposited in the GenBank of the National Center for Biotechnology Information (NCBI), with the option BLASTN 2.2.19 (^{Brown et al., 2014}; ^{Zhang et al., 2000}).

Evaluation of the severity of the disease Planting the plant material

For the pregermination of the chayote fruits, we used a 40 x 60 cm black, plastic bag, in which 20 fruits were placed for 20 days. The fruits had previously been washed with soap and rinsed with a 0.6 % chlorine solution. Once the root and stem sprouted, each fruit was placed in a black plastic bag (25 x 25 cm) containing 2,500 cm³ of substrate composed of sand-agrolite-vemicompost (40:40:20), treated twice with steam under 120 pounds of pressure for three hours. The fruits were kept in a growth chamber (Model CEL 37-14, brand: SHERER,) at a temperature of 26±1 °C, with a photoperiod of 14 light hours and 10 of darkness, and a light intensity of 6, 768 lux of fluorescent light. The plants were initially watered at field capacity, and later, irrigated every three days with 300 mL of water per plant until the end of the experiment.

Preparation of the inoculant

The oomycete was cultivated in V8 medium plates for 7 days at 28 °C. Each Petri dish was added an isotonic solution of sodium chloride at 0.9 % (AbbottMR) for 10 min. The isotonic solution was poured out, and using a dissection needle, the dish was divided in four parts. Each part of the medium containing the oomycete was placed in a sterile Petri dish. Sterile distilled water was then added, until it covered the medium, and the dishes were placed under a cold white light at 26 °C for 48 h. The release of the zoospores was then induced, exposing the Petri dishes to two temperatures (4 °C and room temperature) for 30 min. Finally, the number of zoospores was quantified using a cytometer (Marienfeld^®). Inoculation was performed when the plants had an average of 16 leaves (15 days after planting) applying a suspension of 1, 3 and 6 x 10⁵ zoospores to each plant in the transition zone between stem and root. Each treatment (level of inoculant) consisted of four plants and the control. To determine the level of inoculant, the minimum level of of P. capsici used by ^{Trujillo-Viramontes et al. (2005)} in JEP chili plants, was taken into consideration.

The progress of the damage induced by P. capsici was measured in cm from the transition area between stem and root. Apart from the length of the necrosis, we determined the number of wilted leaves and guides, as well as the presence of mycelia in the seed 3, 5 and 7 days after inoculation (ddi). Based on the symptoms and measurements observed, as in the case of other crops (^{Navarrete and Acosta, 1999}), we designed a scale of severity to evaluate the damage of P. capsici in chayote plants in growth chambers under controlled conditions. The pathogenicity evaluation test was set up on three occasions with similar results.

Statistical analysis

The data of each variable underwent an analysis of variance and comparison of average, using the Tukey test (p≤ 0.05) (^{SAS Institute, 2002-2003}).

Detecting Phythopthora in diseased chayote plants

Out of the 60 sections (30 fruit and 30 stem sections) planted in V8 agar, in 46 (76 %) Phythopthora sp grew, in 9 (15 %) Fusarium, and in 3 (4 %), Alternaria. The rest of the sections corresponded to saprophytic fungi and bacteria. ^{Babadoost and Pavon (2013)} indicate that the high frequency of P. capsici, as well as its virulence, determine its role as a causative agent of the disease. However, pathogenicity tests are required for each host plant. ^{Rivera et al. (1992)} and ^{Olguín (2010)} initially reported Pythium sp., Fusarium oxysporum and F. sambucinum as causative agents of rotting of chayote in Costa Rica and Mexico, respectively. However, the low frequency of Fusarium found in this investigation could indicate that the wilting of the chayote plants is not induced by this fungus.

Morphological characterization of the rotcausing oomycete

The cultures developed in V8 medium plates presented an abundance of aerial hyphae, of considerable length, cotton-like, little radial, and compact. The mycelium was cenocytic, hyaline, branched, and with an irregular diameter. The sporangia produced in V8 and sterile water medium plates (Figure 1a) presented different sizes and shapes: ovoid, starchy (on average, 55 x 32 µm), with a prominent papilla, expired, mainly with only one papilla (seldom were there any with two found) (Figure 1b), long and persistent peduncle, sometimes symmetrical and other times, assymetrical and with clamidospores (Figure 1c). The morphometric data of the sporangia indicate the presence of Phytophthora capsici in the transition area between stem and roots and the fruits of the chayote, which agrees with reports by ^{Olguín et al. (2013)}, who indicated that this oomycete is the causative agent of rotting in chayote plants.

Figure 1. Micrographies of the isolated chayote oomycetes. a) Phytophthora capsici sporangia; b) Bipapilate sporangium; and c) Chlamidiospore, observed using a light microscope. 

Pathogenicity test for Phytophthora sp. in plantlets in growth chambers with a controlled environment

The oomycete inoculated in the chayote fruits indicates the same characteristic rotting of the fruits it was isolated from: loss of turgidity, rotting and the presence of white mycelia 5 days after inoculation (Figure 2a). Under humid conditions (80 to 100 %) the growth of the pathogen was conspicuous (Figure 2b). In pieces of fruits with mycelia placed in sterile distilled water and under continuous white light (Figure 2c), we observed abundant sporangia, irregularly spread out (Figure 2d).

Figure 2. a) Chayote fruits with white mycelia, loss of turgency, and symptoms of rotting; b) Fruits under conditions of humidity, between 80 and 100 %, with a conspicuous growth of the pathogen; c) pieces of fruit with mycelia, placed in sterile distilled water under continuous white light; and d) sporangia irregularly spread out. 

Analyses of DNA sequences

The four sequences obtained in this study were placed in the GenBank-NCBI under the accession numbers KM087089, KM087090.1, KM087091, and KM087092. DNA sequencing confirmed that the isolated strain of the necrotic chayote tissue samples gathered in the area of study belonged to Phytophthora capsici, with an identity of 99 and 100 % with respect to the sequences of different isolations of the GenBank. It is worth mentioning that the strain with accession number KM087090.1, which showed an identity of 100 % (Table 1), was used in the severity test.

Table 1. E-value, % of identity and access number in the GenBank-NCBI of the Phytophthora capsici strains isolated from fruits and the transition area between the stem and roots of Sechium edule chayote plants. 

Severity of P. capsici in chayote plants

No significant differences (α=0.05) were observed between the three levels of P. capsici inoculant for the variebles number of wilted leaves, necrosis in the transition area between stem and roots, and wilting of chayote plants (Figure 3a, b, and c). When the plants were inoculated with P. capcisi, differences were observed with the control plants (not inoculated), like in reports by ^{Uribe-Lorio et al. (2014)} in their work with chili pepper plants. The control plants remained healthy throughout the experiment. As in the present study, other authors (^{Trujillo-Viramontes et al., 2005}) report that inoculation of 3 x 10⁵ zoospores of the oomycete is enough to induce a response of the chili plant. Although no significant differences were found in the three levels of P. capsisci inoculant used, there were numerical differences. Probably, 3 x 10⁵ zoospores per plant is the top threshold, above which damage in chayote plants may not be statistically significant.

Figure 3. Number of wilted leaves (a), length of the necrosis in the transition area between the stem and the roots (b) and percentage of wilted chayote plants (c) inoculated with 1, 2, and 6 x 10⁵ P. capsici zoospores. Each dot represents the average of 4 repetitions, Tukey (p≤ 0.05). 

Using the data obtained, we built the disease progress curve. Regardless of the level of zoospores inoculated, at three ddi, we observed 50 % of wilted leaves, and 2.3 to 3.0 cm of necrosis in the transition area between stem and roots (Figure 4). At five ddi, wilting of leaves was 80% with the progress of necrosis at 2.5 to 3.15 cm and the presence of mycelia on the seed (the fruit). At seven ddi leaves presented 100 % wilting, necrosis from 2.67 to 3.3 cm, and the presence of mycelia in guides, seed, and roots. Therefore, the three levels of P. capsici inoculant were equally effective for the induction of wilting of chayote plants. Comparatively, the time of appearance of symptoms of the disease was lower than that reported by ^{Olguín et al. (2013)}. The level of inoculation was crucial, while ^{Olguín et al. (2013)} used 11,500 sporangia mL^-1, this investigation used 1, 3 and 6 x 10⁵ zoospores mL^-1.

Figure 4. Progress of the necrosis in the transition area between the stem and the roots of chayote plantlets three days after inoculation with 300,000 P. capsici zoospores. 

The necrosis in the transition area between stem and roots was observed starting on day 3 after inoculation, regardless of the level of inoculant. Early necrosis may have accelerated plant wilting

At five ddi, plant wilting was observed to be above 50 %, also regardless of the level of inoculant, which shows the pathogenicity of the isolation studied, according to reports by ^{Olguín et al. (2013)}. At seven ddi, necrosis development was, on average, 3.0 cm, which contributed to the wilting of plants at 100 %. Probably, the degradation of cell walls and middle lamella of the parenchyma tissue of the vascular system, typically caused by P. capsici, appeared in the transition area between stem and roots, which prevented the flow of water and nutrients for the adequate development of the plant (^{Li, et al., 2011}; ^{Wang et al., 2011}).

The substrate used was crucial in the experiment, since it provided high humidity conditions which favored the development and spreading of P. capsici. High humidity levels favored the mobility of zoospores, ensuring the infection by the oomycete. ^{Silva-Rojas et al. (2009)} indicate that, indeed, this environmental factor is crucial for the spreading of P. capsici.

The progress curve of the disease (Figure 4) was the base from which to create a Severity Scale (Table 2), which can be used to evaluate damages produced by P. capsici in chayote under controlled conditions. The scale was validated on three occasions. The variables used to create the scale were: percentage of wilted leaves, length of necrosis in the transition area between stem and roots, percentage of wilted plant, and the presence of mycelia. This attempts to become a contribution for the basis of a system for the control of the disease induced by P. capsici in chayote plants.

Table 2. Scale of severity to evaluate the damages produced by P. capsici in chayote plants grown under controlled conditions.