Materials and methods

Isolate collection

The Phytophthora spp. isolates were collected from 25 irrigation channels in District 010 of the CuliacánValley in January 2015 (Table 1). The sampling sites were chosen at random; the traps used were organza bags closed with nylon thread in the upper section, which also helped tie the traps to a particular point by the side of the channel (Figure 1). Bags contained 1 pear fruit and 3 azalea leaves, neither one showing any signs of diseases or damages, and 2 bags were placed in each sampling site. Plant material was washed using tap water and disinfected with ethanol at 70 % for 30 s before being placed in the traps. The traps remained in the surface of the irrigation channels for 48 h (^{Bush et al., 2003}). Afterwards, the traps were gathered and taken to the laboratory. The isolation was carried out by placing several sections of infected plant tissue (Figure 2) in a selective medium containing V8 juice (200 mL/L), natamycin 10 µg/L, ampicillin 200 µg/L, rifampicin 10 µg/L, pentachloronitrobenzene 25 µg/L, and hymexazol 50 µg/L (V8-PARPH) (^{Jeffers, 1986}). The Petri dishes were incubated at room temperature (25±2 °C) for 3 to 5 days. The Hyphal tips from developing colonies were transferred to obtain pure cultures (^{Hajebrahimi and Banihashemi, 2011}). Finally, the isolations were preserved placing 5-7 mycelium discs in active growth(5 mm diameter) in corn meal agar (CMA) inmicrofuge tubeswith sterile distilled water, which were then stored at 15 °C (^{Martin et al., 2012}).

Table 1. Sampling sites in irrigation channels in the Culiacán Valley, species identified and accessions of the sequences deposites inNCBI. 

ND= Not determined

* The accession number corresponds to the sequences obtained with the primers ELONGF1/ELONGR1.

Figure 1. Trap with pear fruit placed in an irrigation channel. 

Figure 2. Pear fruits and azalea leaves with lesions from which the isolation was performed. 

Results and Discussion

A total of 29 isolates belonging to the genus Phytophthora were obtained from infection sites in pear fruits and azalea leaves. Out of the 25 sampling points, Phytophthora isolations were obtained from only 10 (Table 1). According to their morphological characteristics, isolates were classified into 2 groups: the first group of 18 isolates that produced sporangia with out papilla, of varying shapes, from oval or globular to obpyriforme with a size of 45.8-57.4 x 32.9-36.9 µm (Figure 3A), persistent, with both internal and external proliferations and long and simple sporangiophores; the isolates produced terminal chlamydospores (Figure 3D) and hyphal swellings of irregular shapes in chains and/or in groups (Figure 3C).

These 18 isolates were heterotallic and formed plerotic oospores with an average diameter of 24.9 µm, and oogonios of 27.6 µm, with amphyginousanteridia (Figure 3B). The sexual reproduction of the isolates took place only in reference isolation P. nicotianae A2, indicating that the isolates belong to the compatibility type A1. The maximum growth temperature of the isolates was 40 °C.

Figure 3. Morphological structure of isolate 3-1F3 of the species P. hydropathica. (A) Non-papillated sporangia, oval-shaped (B) Pleroticoospore with anmphyginousantheridium. (C) Irregular hyphal swellings in groups. (D) Spherical and terminal chlamydospore. Bar = 20 µm. 

These morphological characteristics coincide with those for the species Phytophthora hydropathica described in the key proposed by ^{Martin et al. (2012)}, as well as the description by ^{Hong et al. (2010)}. It is worth mentioning that these two descriptions of P. hydropathica mention that the species Phytophthora parsiana presents very similar morphological characteristics. Both researchers set them apart based on the maximum temperature growth, since P. hydropathicacan grow in temperatures of up to 40 °C, whereas P. parsiana does not grow in temperatures above 37 °C. Also, ^{Martin et al. (2012)} mention that P. parsiana does not present chlamidospores, and P. hydropathica does.

The 18 isolates with chlamidosporeshad a compatibility type A1 when confronted with A1 and A2 strains of P. nicotianae. This coincides with reports by ^{Hong et al. (2010)}, who report the compatibility type A1in P. hydropathica, therefore this species may not be reproducing sexually, and may survive by means of chlamidospores. According to the morphological characteristics observed, the 18 isolatioisolatesns with nonpapillate sporangia belong to the species Phytophthora hydropathica.

The second group, composed of 11 isolates, share morphological characteristics that consist of a regular petaloid growth pattern in PDA culture medium, non-papillat esporangia with globular and oval shapes, mainly with conical bases and sizes of 25.3-42.7 x 18.702-31.5 µm (Figure 4A), persistent, with internal an external proliferations (Figure 4B) and with simple sporangiophores; this group of isolates produced hyphal swellings (Figure 4C) and no chlamydospores. The isolates in this group were heterothallic,and their compatibility type was A1, forming pleroticoospores with an average diameter of 25.2 µm and 12.25 µm antheridia (Figure 4D). In Michoacán, Mexico the two mating types of mating were found in this species (^{Mora, 2013}), therefore it is likely that if a broader sampling were to be carried out, the type of compatibility A2 could be found. Another characteristic of this group of isolates is the maximum growth temperature, which fluctuates between 35 and 37 °C.

Figure 4. Morphological structures of isolate 17-1F1 of the species P. drechsleri. (A) Non-papillated sporangia, oval-shaped and with a conical base. Bar = 50 µm. (B) extended proliferation of the sporangium. Bar = 50 µm. (C) hyphal swellings. Bar = 50 µm. (D) pleroticoospore with amphyginousantheridium. Bar = 20 µm. 

When comparing these characteristics with the taxonomical keys by ^{Erwin and Ribeiro (1996)}, and ^{Martin et al. (2012)}, they correspond to the species Phytophthora drechsleri. It is important to point out that both keys mention a relationship between this species and P. cryptogea, although there are also differences between species. One of the most important is the maximum growth temperature, since there are reports indicating that P. cryptogea can not grow in temperatures of 35 °C or above (^{Perlerou et al., 2010}; ^{Kurtbetli, 2014}), whereas reports for P. drechsleri indicate it can grow in temperatures of up to 37 °C. There are other differences between both, such as the size of oospores and antheridia: P. drechsleri produces oospores sized approximately 25.6 µm and the oospores from P. cryptogea are smaller, with sizes of around 22 µm. The antheridia comprise a similar case, in which P. cryptogea presents largerantheridia, reaching up to 19 µm in length, while antheridia in P. drechsleri only reach 14 µm. According to the morphological characteristics and the taxonomic keys, we can infer that the group of 11 isolates belongs to the species P. drechsleri.

The PCR reactions with primers DC6-ITS4 with isolates belonging to the species P. hydropathica amplified a product of approximately 1300 pb (Data not shown) and with the primers ELONGF1-ELONGR1, a product of approximately 960 pb (Figure 5). The consensus sequences showed a similarity of 96-98 % with the species Phytophthora hydropathica (code PD 00342 ITS) in the database specialized in Phytophthora (Phytophthora db.org) for primers ITS, and a similarity of 99-100 % with the species of the type P. hydropathica reported in NCBI for both types of primers (EU583793 and GQ260061). This analysis confirms that these isolations belong to the species Phytophthora hydropathica.

Figure 5. Fragment of DNA amplified with primers ELONGF1/ELONGR1 in isolates obtained from irrigation water. Line 1, 1 Kb molecular marker; Line2, water; Line3, negative control (C. gloeosporioides); Line4, positive control (P. capsici); Lines5-15, isolates of the species P. drechsleri; Lines16-20, isolates of the speciesP. hydropathica; Line21, 1 Kb molecular marker; Lines22-34, isolates of the species P. hydropathica. 

P. hydropathica has been frequently analyzed from superficial waters (Bienaflp and Balci, 2014; ^{Vitale et al., 2014}; ^{Copes et al., 2015}), mainly in the hottest months (^{Copes et al., 2015}) tolerating temperatures up to 40 °C (^{Hong et al., 2010}). It has also been isolated from diseased Japanese andromeda (Pieris japonica) plants (Bienalpf y Balci, 2014) in greenhouses and water used in the irrigation of greenhouses (^{Hulveyet al., 2010}), and from ornamental Viburnumtinus plants with symptoms of shoot dieback (^{Vitale et al., 2014}).

In the P. drechsleri isolation, the reactions of PCR with primers DC6-ITS4 and ELONGF1-ELONGR1 amplified a product of approximately 1300pb (Data not shown) and 970 pb (Figure 5), respectively. The consensus sequences showed a homology of 97 % with isolates reported in the NCBI for the species Phytophthora drechsleri (GU111627 and AY659550), thus confirming the identity of these isolates. P. drechsleri has been found in irrigation water at different depths. For example, ^{Bush et al. (2003)} found this species on the surface of irrigation water and at depths of 1 and 1.5 m.

Two species of Phytophthora were isolated from the surface of 10 irrigation channels in the Culiacán Valley. In the channels where Phytophthora was found, P. hydropathical was found in only 40 %, in 30 % only P.drechsleri, and in 30 %, both species (Figure 6). P. hydropathica is considered a native species to river ecosystems (^{Hong et al., 2010}), therefore it is very common to find, in this type of studies, a high frequency of isolates of this species (^{Bienapfl and Balci, 2014}; ^{Brazee et al. 2016}).

Figure 6. Distribution of P. hydropathica and P. drechsleri in irrigation channels in the Culiacán Valley. 

The sequences obtained with primers ELONGF1-ELONGR1 that correspond to the sequences of the gene that codifies for elongation factor 1-α in both species were deposited in the NCBI database (Table 1).

In the pathogenicity tests, representative P. hydropathica isolates 13F2, 15-1F1, 3-2F1, 18-2F1, 16-2F1, 20-2F1, 15-2F3, 3-1F3, 16-1F2, and 16-1H2 caused visible necrosis symptoms 48 h after inoculation in tomato leaflets and chilli pepper leaves (Table 2; Figure 7), as opposed to the leaves used as a control, where there were no symptoms present. The isolations were more aggressive on chilli leaves, causing necrosis on the entire leaf (45 mm), 120 h after inoculation, while in tomato leaves, the largest lesion measured 26 mm (Table 2). ^{Hong et al. (2010)} mention chilli pepper, tomato, and cucumber plants as possible hosts, which was confirmed in this study for chilli and tomato. These studies show that P. hydropathica is a threat for tomato and chilli crops, which are extremely important in this region. Similar studies (^{Bienapfl and Balci, 2014}; ^{Brazee et al., 2016}), mention that although P. hydropathica rarely has been found to have a relation to plant diseases, it does have the potential to cause diseases.

Table 2. Length of the lesion caused by P. hydropathica and P. drechsleri in chili pepper leaves and tomato leaflets. 

hdi= hours after inoculation; 45 mm= complete leaf.

Figure 7. Pathogenicity of P. hydropathica 3-1F3 in chili pepper leaves and tomato leaflets 48 h after inoculation. (A) Inoculated chili leaf. (B) Inoculated tomato leaflet. (C) and (D) Control chili leaf and tomato leaflet. 

Isolates16-1F3, 1-2H2, 17-1F1, 15-1F2, 23-1F1, 1-1H2, 11F2, 23-2F1, 23-1F3, 23-2F4 y 23-2F2 of the species P. drechsleri only infected tomato leaf lets (Table 2; Figure 8), in which symptoms of necrosis were observed approximately 48 h after inoculation. Lesions in tomato leaf lets measured up to 45 mm after 120 h, while chilli leaves showed no apparent symptoms, possibly due to the hybrid used for the bioassay may be tolerant to the isolates used. ^{Erwin and Ribeiro (1996)} mention that P. drechsleri has been reported as the causal agent of fruit rot in tomato and chili. In Mexico, P. drechsleri has been reported to infects a fflower, chili pepper, gerbera, lettuce, poinsettia, and tomato (^{Mills et al., 1991}; ^{Romero-Cova and Solis-Aragon, 1996}; ^{Fernandez-Pavía et al., 2013}).

Figure 8. Pathogenicity of P. drechsleri 1-2H2 in tomato leaflets 48 h after inoculation. (A) Inoculated tomato leaflet. (B) Control tomato leaflet. (C) Inoculated chili leaf. (D) Control chili leaf. 

This first study in Mexico in the irrigation channels in the CuliacánValley shows the presence of P. hydropathica and P. drechsleri and highlights the importance of irrigation water as a way of spreading both pathogens. At the same time, it lays the foundations for future studies, such as knowing the population dynamics through out the agricultural cycle, determining whether these species only use irrigation channels as a way of dissemination or if they complete their life cycle in these bodies of water, as well as to determining the presence of P. hydropathica and P. drechsleri in natural infections of horticulturally important plants in the region. This is the first report on Phytophthora hydropathica in irrigation channels in Mexico. These results indicate that it is necessary to continue research on natural environments, whether in superficial waters or in soils, to know the diverse species of Phytophthora present in Mexico, and therefore consider the potential risk these microorganisms represent for Mexican agriculture.

Conclusions

This study detected the presence of Phytophthora hydropathica and Phytophthora drechsleri in the irrigation channels of the Culiacán Valley. These species showed differential pathogenicity in chili pepper leaves and tomato leaf lets.