Introduction

The organisms of the genus Phytophthora, belong to the kingdom Cromista or Stramenopila, are inhabitants of aquatic and terrestrial environments, with high capacity for reproduction, dissemination and survival, particularities that facilitate their establishment in agricultural or forestry fields (^{Erwin and Ribeiro, 1996}; ^{Chen et al., 2017}, ^{Hon, 2018}; ^{Judelson et al., 2019}). This genus includes species with a wide range of hosts such as P. cinnamomi which is capable of parasitizing around 5 000 species of plants (^{Hardham et al., 2018}) and P. capsici, known for causing economic losses in solanáceae and cucurbitaceae plantations grown around the world (^{Vega et al., 2017}).

About 30 species of Phytophthora were collected in bodies of water such as ponds, rivers, canals, reservoirs and hydroponic crops (^{Aram and Curl, 2018}) in addition, the genus Phytophthora, like other phytopathogenic oomycetes, strictly requires water for reproduction which begins with the formation of sporangiophores and sporangia, within which zoospores are formed for further release and dissemination; these zoospores, are able to infect susceptible plants and start the cycle of a disease (^{Kang et al., 2017}).

Zoospores are the main infective propelling spread by free water, so it is possible to recover them regularly from surface water (^{Hon, 2018}). Examples of isolated water species are often: P. cactorum, P. parasitica, P. citrícola, P. gonapodyides, P. cambivora, P. hydropathica and P. drechsleri, among others (^{Loyd, 2014}; ^{Redekar and Park, 2018}; ^{Ristvey et al., 2019}). In Mexico, there are about 17 species belonging to the genus Phytophthora reported as parasites of plants of agricultural importance, the most common are: P. capsici, P. infestans and P. cinnamomi; however, information on the attribution of surface water as a source of inoculum and dispersal of organisms of the genus Phytophthora is scarce.

Thus, ^{Álvarez et al. (2016)} were able to isolate and identify the species P. hydropathica and P. drechsleri from different water channels for agricultural use in Culiacán, Sinaloa, Mexico. On the other hand, the identification of oomycete genus and species was commonly based on their morphological and cultural characteristics; however, erroneous identifications were common, because some species share these characteristics; currently, these practices are complemented by biotechnological techniques based on the extraction and sequencing of nucleic acids, which allows the identification of organisms effectively and concretely (^{Trzewik et al., 2016}). Therefore, the objective of this study was to determine the incidence of species of the genus Phytophthora in surface waters for agricultural use in Culiacán, Sinaloa and to determine their pathogenic potential in plants grown in Mexico.

Materials and methods

Obtaining, purifying and conservation of isolates Phytophthora spp.

Using a map of the location of dams, dikes, ponds, rivers and water storage and distribution of the water used in agricultural activities, of the area of influence of irrigation district number 010 of the Culiacán Valley, Sinaloa, granted by the National Water Commission (CONAGUA) (Figure 1), 37 sampling sites were randomly selected in 28 locations (Table 1).

Figure 1 Main sampling sites in the municipality of Culiacán, Sinaloa. Image provided by CONAGUA. 

Table 1 Geographic location of the sampling sites to obtain isolates of Phytophthora spp., Culiacán, Sinaloa, 2018-2019. 

Locality	Coordinates of the sampling site	Isolation
Adolfo López Mateos Prey (The Varejonal)	25°10'08'' N 107°26'10'' W 25°09'10'' N107°26'11'' W 25°09'56'' N 107°26'30'' W	PV1, PV2, PV3, PV4, PV5, PV6, PV7, PV8, PV9, PV10, PV11, PV12
La Presita	24°55'49'' N 107°25'39'' W	PR13, PR14, PR15, PR16
Empaque Castro	24°55'28'' N 107°31'59'' W	EC17, EC18, EC19, EC20
The Tamarindo	24°55'07'' N 107°33'32'' W  24°55'11'' N 107°32'57'' W	TR21, TR22, TR23, TR24
Empaque Valle del sol	24°53'20'' N 107°29'17'' W	EVS25, EVS26, EVS27, EVS28
Sanalona Prey	24°48'37'' N 107°08'11'' W 24°48'46'' N 107°08'14'' W 24°48'49'' N 107°08'30'' W	PS33, PS34, PS35, PS36, PS37, PS38, PS39, PS40, PS41, PS2, PS43
Tamazula River	24°49'00'' N 107°11'16'' W 24°48'53'' N 107°21'29'' W	RT44. RT45, RT46, RT47, RT48, RT49
Humaya River	24°51'48'' N 107°24'25'' W	RH50, RH51, RH52, RH53, RH54, RH55
Botanical garden	24°49'23'' N 107°23'06'' W 24°49'24'' N 107°23'04'' W	JB56, JB57, JB58, JB59, JB60, JB61
Imala	24°51'21'' N 107°13'11'' W	IMA62, IMA63, IMA64, IMA65
Three Rivers	24°48'43'' N 107°24'24'' W 24°48'24'' N 107°24'33'' W	3R66, 3R67, 3R68, 3R69, 3R70, 3R71
Bellavista	24°48'45'' N 107°27'56'' W	BELL74 and BELL75
Bachigualato	24°46'59'' N 107°26'48'' W	BACH76 and BACH77
Aguaruto	24°47'49'' N 107°30'24'' W	AGUA78 and AGUA79
Batan Field	24°46'10'' N 107°30'19'' W 24°45'40'' N 107°30'45'' W	CB80 and CB81
Moroleón Field	24°41'14'' N 107°31'09'' W	CM82 and CM83
Cardenal Field	24°45'10'' N 107°28'39'' W	CC84 and CC85
Primavera Dam	24°43'48'' N 107°24'10'' W	DP86 and DP87
Hazera Station	24°43'32'' N 107°27'24'' W	HAZ88 and HAZ89
Enza Zaden Station	24°40'58'' N 107°28'47'' W	EZ90 and EZ91
Monsanto Station	24°40'37'' N 107°28'50'' W	MON92 and MON93
Divemex Field	24°39'46'' N 107°26'58'' W	DIV94 and DIV95
US Agriseeds Station	24°38'11'' N 107°26'38'' W	US96 and US97
Syngenta Station	24°37'08'' N 107°26'41'' W	SYN98 and SYN99
Rijk Zwaan Station	24°36'12'' N 107°26'41'' W	RZ100 and RZ101
Bayer Station	24°36'14'' N 107°27'22'' W	BAY102 and BAY103
Costa Rica Dam	24°35'44'' N 107°18'33'' W	CR104 and CR105
Faculty of Agronomy	24°37'28'' N 107°26'34'' W	PP106 and PC107

The sampling period from September 2018 to January 2019, by sampling site was placed a trap made of polypropylene net bag, closed from the front with a rope of the same material, together, that rope was used to fix the trap at some point in the slope (Figure 2).

Figure 2 Floating sampling trap (A) infected pear fruits recovered from the traps (B). 

These traps, inside, contained two injury-free pear fruits, washed with simple water and superficially disinfested with 96° ethanol (^{Soto et al., 2017}). The traps remained suspended for 48 hours, then collected and transported to the phytopathology laboratory of the Faculty of Agronomy of the Autonomous University of Sinaloa. The fruits with obvious lesions were washed with sterile distilled water, dried with sterile brown paper and disinfected with 96° degree ethanol.

The insulations were made from cuts of 1 cm² taken from the brown lesions on the fruits (Figure 2) and every five fragments were placed equidistantly in petri boxes containing selective culture medium made with corn-agar flour (HMA, 17 g L^-1), penicillin (10 µg L^-1), ampicillin (200 µg L^-1), rifampicin (10 µg L^-1), pentachloronitrobenzene (PCNB, 25 µg L^-1) (PARP) (^{Jeffers, 2006}; ^{Soto et al., 2017}).

Petri boxes were left two days at room temperature (26 ±2 °C). In order to purify and conserve individual strains from the developed colonies of the sick tissue, it was transferred to HMA culture medium using the hypha tip technique, placed 48 h at room temperature, once the colonies were developed, with tweezers, five pieces of 0.5 cm diameter of culture medium with mycelial growth were taken individually from each isolation to later place them in microvial tubes (5 ml) containing sterile distilled water and were stored at 15 °C for further studies (^{Jeffers, 2006}; Abbot et al., 2012; ^{Soto et al., 2017}).

Results and discussion

17 isolates belonging to the genus Phytophthora were obtained, which were classified into two groups taking into account their morphological similarity (Table 3): the first group consisted of 14 isolations that developed simple sporangiophores, non-papillate ovoid sporangia with average length and width dimensions 47.5-60 x 26.25-32.5 µm (TR21, PS36, 3R68, CR104 and AGUA79), 47.5-57.5 x 26.25-32.5 µm (PV1, PV11, JB61, CC84 and CC85), 45-60 x 22.5-32.5 µm (RT45 and RT47) and 45-57.5 x 25-32.5 µm (JB61 and 3R70), with nested and extended proliferation, simple sporangiophores and irregular swellings present (Figure 3). The type of compatibility of these insulations with A1 and A2 affinity strains of P. drechsleri and P. capsici could not be identified.

Figure 3 Sexual and asexual structures of oomycetes. A-D) ovoid sporangia Phytophthora spp.; E) papillated sporangia of P. capsici (isolation PC107); F) release of zoospores (isolation TR21); G) nested proliferation (PV11 isolation); H) extended proliferation (isolation JB61); I) swelling of the intercalary mycelium (isolation RT45) and J) oospore of P. capsici (isolation MPV31). 

The second group consisting of three isolations (PC107, MJB72 and MPV31), those who developed papillate ovoid sporangia, long and wide average dimensions of 40-68 x 17-30 µm, simple sporangiophores, simple sporangia (without proliferation) and papillates; without the presence of swellings in the hyphae. Sexual reproduction with A1 affinity of P. capsici, with formation of pleroticas oospores, without ornaments and average diameter of 22-31 µm (Figure 3).

Comparison of these characteristics with those described in the taxonomic keys of ^{Erwin and Ribeiro (1996)} and ^{Abad et al. (2012)}, coincide with the species Phytophthora parsiana, P. virginiana, P. lagoariana, P. hydropathica and P. capsici. PCR reactions with ITS initiators (4-6) amplified a product approximately 941 bp, with COX 972 pb initiators and NADH 897 pb initiators.

The consensus sequences showed results with percentages of similarity to Phytophthora virginiana: 99.42 (isolation PV1), 99.47 (isolation PV11), 99.52 (isolation JB61), 99.60 (isolation CC84) and 99.78 (isolation CC85), for Phytophthora parsiana: 99 (isolation RT45) and 99.22 (isolation RT47), for Phytophthora lagoariana: 98 (isolated JB61) and 98.52 (isolated 3R70), for Phytophthora hydropathica. 97 (isolation TR21), 98 (isolation PS36), 98.5 (isolation 3R68), 99 (isolation CR104) and 99.64 (isolation AGUA79) and for Phytophthora capsici of 99.48 (isolation PC107), 99.70 (isolation MJB72 and 100% (isolation MPV31) (Table 4).

The species identified in this research are regularly found in surface water (^{Zappia et al., 2014}, ^{Yang and Hong, 2015}). The 17 isolations obtained were pathogenic, because after inoculation they all caused symptoms of seedling withering and root necrosis; in fruits caused symptoms of rot (Figure 4).

Figure 4 Pathogenicity tests of the species P. hydropathica. A-C) tomato, chili and cucumber plants, left inoculated plants and right witness plants. D-F) stems and roots of tomato, chili and cucumber plants, left inoculated plants and right witness plants. G-I) inoculated fruits of tomato, chilli and cucumber. 

The isolations of P. capsici originated withering plants three days after inoculation, while those same symptoms appeared 10 days after inoculation with the rest of the species. These results corroborate the pathogenicity report of Phytophthora parsiana, P. virginiana, P. lagoariana, P. hydropathica and P. capsici on tomato, chili and cucumber plants described by ^{Cline (2008)}; ^{Lamour et al. (2012)}; ^{Álvarez et al. (2016)}.

This research verifies the existence of unreported phytopathogenic species that represent a risk to the health of horticultural crops in the region. The extraction, amplification and sequencing of regions of the DNA of organisms using the molecular techniques used in this research (Its, Cox and Nadh), allowed their identification when determining homology levels between 97 and 100% in the NCBI database and with that, greater efficiency than with the traditional identification morphological characterization.

This surface water study demonstrates the presence in that habitat of Phytophthora virginiana, Phytophthora parsiana, Phytophthora lagoariana, Phytophthora hydropathica and Phytophthora capsici; also, the importance of water as an inoculum source and dispersion of these pathogens. In turn, it expands the range of studies for future research on the survival of such pathogens, discovering new natural hosts in the area, determining other possible means of dissemination, finding new hosts of economic importance, identifying whether there is the presence of other types of pathogens of agricultural importance in water, and in this way consider the potential risk that this type of organisms represents for the agriculture in the region.

Conclusions

In surface waters for agricultural use of irrigation district number 10 of the Culiacán Valley, Sinaloa, 17 isolations were collected that corresponded to the organisms P. virginiana, P. parsiana, P. lagoariana and Phytophthora capsici, all with the capacity to infect plants and fruits of tomato, chili and cucumber.