Introduction

In the genus Phytophthora, there are about 124 species described (^{Martin et al., 2014}), which have the capacity to infect hundreds of plant species around the world (^{Gallegly and Hong, 2008}). At present, the presence of Phytophthora species in aquatic environments has increased (^{Zappia et al., 2014}), for example, in recent years the presence of Phytophthora hydropathica stands out mainly in irrigation water (^{Hulvey et al., 2010}; ^{Hüberli et al., 2013}; ^{Bienalfp and Balci, 2014}; this is to be expected, since etymologically, the name of this microorganism derives from the Greek words ‘hydro’ which refers to its aquatic nature and ‘pathica’ to its pathogenic nature (^{Hong et al., 2010}). Most of the reports of P. hydropathica are related to infections in ornamental plants, such as foliar necrosis in azalea (Rhododendron catawbiense), neck rot in mountain laurel (Kalmia latifolia) (^{Hong et al., 2010}), as well as wilting and regressive death of wild laurel (Viburnum tinus) (^{Vitale et al., 2014}).

Before being formally described as P. hydropathica, ^{Hong et al. (2008)} showed that this species is capable of causing ‘damping off’ in cucumber seedlings, while in tomato and pepper plants it caused root infections; In addition, they mention that P. hydropathica penetrates in fruits of tomato and chili by means of wounds. In Mexico, the presence of P. hydropathica was reported in irrigation water in the Valley of Culiacan and was shown to cause necrosis in tomato and chili leaves (^{Álvarez et al., 2017}).

To control diseases caused by Phytophthora, the chemical ingredient metalaxyl, which is a phenylamide-type fungicide that protects plants systemically is mainly used for the control of oomycetes (^{Urech et al., 1977}). Metalaxyl acts on specific sites of the pathogen by preventing protein biosynthesis through interference in the synthesis of ribosomal RNA (^{Nunninger et al., 1995}); however, excessive use of this product can cause pathogens to generate resistance (^{Damicone, 2004}). An example is the rapid development of resistance in populations of P. infestans, which was detected in the 80s in Europe (^{Davidse et al., 1981}) and in the 90s in the USA, Canada and Mexico (^{Matuszak et al., 1994}; ^{Power et al., 1995}).

Resistant isolates are equal to or more aggressive than susceptible isolates, thus converting metalaxyl resistance into an important agronomic characteristic in the integrated management of diseases caused by Phytophthora, especially in late potato blight caused by P. infestans (^{Forbes et al., 1998}); in addition, in vitro bioassays have been used to characterize and classify Phytophthora isolates and other oomycetes according to the level of susceptibility to metalaxyl (^{Peters et al., 2001}; ^{Fontem et al., 2005}; ^{Tian et al., 2016}).

The objectives of the present study were: 1) to determine the pathogenic potential of isolates of P. hydropathica in leaves and fruits of tomato, cucumber and zucchini; and 2) determine the sensitivity or resistance of said isolates to metalaxyl.

Materials and methods

Pathogenic potential. To determine the pathogenic potential, three representative strains of P. hydropathica were used [13F2 (KX298864), 16-1F2 (KX298868) and 18-2F1 (KX298873). Strains were reactivated in potato dextrose agar medium (PDA). In the pathogenicity experiments three fruits of tomato (Saladette), cucumber (SFPP) and pumpkin (Nurizeli) were used, as well as three leaves of pumpkin (Nurizeli) and cucumber (SFPP) of 1 month of age. The plant materials were washed with running water and disinfested with 70% ethanol. Both leaves and fruits were wounded with a sterile dissection needle and inoculated by placing 5 mm diameter discs of PDA medium with active growth of the three strains. The inoculated leaves and fruits were placed in a humid chamber for 120 h (Orlikowski et al., 2012). As control, fruits and leaves were used in which wounds were made and discs of culture medium were placed.

Metalaxyl sensitivity. To evaluate the susceptibility of the strains of P. hydropathica to metalaxyl, an in vitro study was carried out, which consisted of adding 10 mg L^-1 of metalaxyl to the PDA culture medium (^{Shattock, 1988}; ^{Rekanovic et al., 2011}), the commercial formula Ridomil Gold 480 EC (480 g of active ingredient of metalaxyl) was used, which was added to the culture medium after sterilization and just before emptying it in Petri dishes. From the border of the colonies of each strain, 5 mm diameter discs of culture medium with mycelial growth were taken and placed in Petri dishes with PDA medium added with metalaxyl and without metalaxyl (control). The in vitro growth of each isolate was evaluated after six days at 25 °C (^{Paez et al., 2001}).

The growth was determined by measuring the diameter of the colony perpendicularly. To determine the relative growth, the formula PC= (DMC-5mm/DMCA)100 was used; where PC= percentage of growth, DMC= diameter of the colony with metalaxyl and DMCA= diameter of the colony without metalaxyl. A percentage of growth in diameter of the colony equal to or greater than 60% is considered to be resistant isolate, a growth of the colony between 10-60% is classified as intermediate and when the colony is less than 10% it is considered as susceptible (^{Shattock, 1988}; ^{Deahl et al., 1995}; ^{Riveros et al., 2003}).

To determine the CE₅₀ values in each strain, four concentrations of metalaxyl 0.1, 1, 5 and 10 mg L^-1 were used; the CE₅₀ was calculated by a linear regression between the log₁₀ of the relative radial growth and the log10 of the concentration of the fungicide (^{Paez et al., 2001}). Three repetitions were performed for each isolate and concentration of metalaxyl.

Design of experiments. In the assay with PDA culture medium with 10 mg L^-1 of metalaxyl, a totally random factor design was used, where the factor was the 18 isolates. An analysis of variance and comparisons of means were made using the Tuckey test. For the in vitro assay in which the resistance or sensitivity of the isolates to metalaxyl was determined, a completely randomized two-factor design was used; one factor consisted of the isolates with 18 levels and the other factor in the concentration of metalaxyl. The response variable was the growth of the colony of the strains in millimeters.

Results and discussion

Pathogenic potential. In cucumber and zucchini leaves, symptoms of disease were visible at 48 h after inoculation (hdi). In cucumber leaves, the three inoculated isolates induced symptoms of necrosis with wrinkling and darkening of the affected tissues (Figure 1). There were some differences between the three inoculated isolates, the main one occurred in the leaf inoculated with strain 18-2F1, in which signs of the pathogen (whitish mycelium) were observed (Figure 1B).

Figure 1 Cucumber leaf lesion caused by P. hydropathica 48 hdi. A) isolated 16-2F1; B) isolated 18-2F1; C) isolated 13-F2; and D) control leaf. 

In zucchini leaves, the isolates also caused symptoms of necrosis (Figure 2). In zucchini there are no reports of studies that consider it as a host of P. hydropathica. In contrast, cucumber P. hydropathica has been reported to cause damping-off in seedlings (^{Hong et al., 2008}).

Figure 2 Symptoms on zucchini leaves caused by P. hydropathica 48 hdi. A) isolated 13-F2; B) isolated 18-2F1; C) isolated 16-2F1; and D) control leaf. 

In zucchini fruits showed symptoms of necrosis; while, in tomato and cucumber there were symptoms of softening (Figures 3, 4 and 5). Strain 13F2 was the most aggressive in cucumber and tomato fruits, even at 72 hdi, signs of the pathogen were observed (Figures 3B and 3C). In contrast, strain 16-2F1 caused symptoms of barely visible necrosis in zucchini fruit and symptoms of softening in tomato (Figures 4A and 4B). Strain 18-2F1 was the most aggressive in zucchini fruits (Figure 5A), where as in tomato fruits, at 72 hdi signs of the pathogen were observed (Figure 5B), these differences observed between the different isolates were can attribute to the different degree of virulence of each strain.

Figure 3 Symptoms in fruits caused by P. hydropathica isolated 13-F2 72 hdi. A) zucchini; B) tomato; and C) cucumber. 

Figure 4 Symptoms in fruits caused by P. hydropathica isolated 16-2F1 72 hdi. A) zucchini; B) tomato; C) cucumber. 

Figure 5 Symptoms in fruits caused by P. hydropathica isolated 18-2F1 72 hdi. A) zucchini; B) tomato; C) cucumber. 

Metalaxyl sensitivity. The 18 strains used in the trial showed a reduction in their growth when exposed to 10 mg L^-1 of metalaxyl compared to growth in medium without metalaxyl (Figure 6); no resistant strains were found; this means that no isolate grew more than 60% in the medium added with 10 mg L^-1 of metalaxyl. Seven strains showed intermediate resistance and 11 were susceptible according to the values proposed by ^{Shattock, (1988)} (Table 1). Of the 11 sensitive strains, six of them were totally inhibited at the concentration of 10 mg L^-1 of metalaxyl, these data are similar to those obtained in tests conducted by ^{Riveros et al. (2003)}, where the isolate used as control (sensitive to metalaxyl) was completely inhibited at the concentration of 10 mg L^-1. In some cases, in vitro sensitivity results have been presented in oomycetes, which do not accurately predict the sensitivity they will have to chemicals in vivo conditions (^{Moorman and Kim, 2004}).

Figure 6 Growth of P. hydropathica isolates in culture medium with 10 mg L^-1 of metalaxyl and in a metalaxyl-free culture medium after 6 days of incubation. Different letters indicate significant differences (Tuckey p< 0.05). 

For example, in P. infestans there was no good correlation between the results obtained in tuber discs with the results in culture medium (^{Straub et al., 1979}). Despite this, the high degree of sensitivity presented by P. hydropathica isolates may be due to the fact that these isolates have not been exposed to this chemical, as there are no reports of this species causing damage to agricultural crops in the region.

The CE₅₀ of the 18 strains were very low, fluctuating in concentrations less than 0.000013 μg L^-1 and up to 1 μg L^-1 (Table 1), similar CE₅₀ were determined in strains of Phytophthora infestans considered sensitive to metalaxyl where the lowest concentration was 0.02 μg L^-1 (^{Power et al., 1995}), whereas, in a study with the species P. nicotianae, maximum CE₅₀ concentrations of 0.04 μg mL^-1were obtained for isolates classified as sensitive. (^{Hu et al., 2008}), higher concentrations of CE₅₀ have also been found in isolates classified as sensitive, such is the case of those obtained by ^{Paez et al. (2001)} where they determined minimum concentrations of 1 000 μg mL^-1 for isolates of P. infestans.

Conclusion

P. hydropathica has the potential to infect leaves and fruits of tomato, cucumber and zucchini, plants that are important in the horticultural sector in Sinaloa. The 18 strains of Phytophthora hydropathica are sensitive to metalaxyl.