Isolation of the causal agent of blight and wilting in vinca

Samples of diseased plants were taken from urban gardens and ornamental nurseries in Culiacan. Samples of infected tissues were placed in plastic bags and taken to the laboratory to isolate the Phytophthora species related to the disease. In the lab, small portions of infected plant tissue were washed with distilled water, disinfected with sodium hypochlorite at 0.5 % for 1 min and finally placed in Petri dishes with a V8 agar culture medium (840 ml distilled water, 163 ml V8 juice, 3 g of CaCO₃ and 18 g bacteriological agar), which were kept at room temperature for 72 h.

Morphological characterization

The isolations were reactivated in a V8 agar culture medium. Once the mycelium developed, permanent preparations were made using lactophenol and placed for observation under a microscope (Carl Zeiss Axiostar Imager A2). Each isolation was purified with the technique of transference of hyphal tips (^{Donahoo and Lamour, 2008}). To induce the growth of sporangia, cuts were made on the colony under growth and the plate was flooded with sterile distilled water. The plates were incubated for 3 days at room temperature (^{Martin et al., 2012}). The presence of sporangia was observed under a biological microscope adapted with a camera. Once the sporangia were produced, the water was removed and acid fuchsin was added to the plates at 2 % to stain them. Samples were taken after 24 h and observed under the microscope to record the measurements of sporangia and other structures.

Chlamydospores were observed in a carrot-based medium (200 g of carrot, 20 g of bateriological agar, and 1 L of distilled water). The isolates grew in the carrot-based medium and were incubated for 15 days at 25 °C in the dark (^{Mirsoleimani and Mostowfizadeh-Ghalamfarsa, 2013}); after this time, they were observed under the microscope and the presence and size of chlamydospores were recorded.

To determine the type of compatibility and record the formation of structures of sexual reproduction, each isolate was confronted with reference strains A1 and A2 of P. nicotianae in V8 culture medium; an agar disc with mycelia in active growth of each isolate, 0.5 cm in diameter, was placed approximately 1 cm away from agar discs with mycelia in active growth of reference strains A1 and A2 of P. nicotianae. The plates were incubated for 15 days at 25 °C and then the presence/absence of oospores was recorded, as well as the characteristics of oogonia and antheridia (^{Javier-Alva and Mattos, 2006}; ^{Waterhouse, 1963}).

Molecular identification

The mycelia of the isolates was grown in a PD (potato-dextrose) liquid medium, and stored at -20 °C. The DNA was extracted using the protocol proposed by ^{Zelaya-Molina et al. (2011)}, which consists of a series of washings with chloroform-isoamyl alcohol. A group of 10 representative isolates that share morphological characteristics were used to amplify the DNA with the pairs of oligonucleotides ELONGF1 (5´-TCACGATCGACATTGCCCTG-3´) and ELONGR1 (5´-ACGGCTCGAGATGACCAT G-3´) for TEF-1α, TUBUF2 (5´-CGGTAACAACTGGGCCAAGG-3´) and TUBUR1 (5´-CCTGGTACTGCTGGTAC TCAG-3´) for beta tubuline (^{Kroon et al., 2004}) and the oligonucleotides DC6 (5´-GAGGGACTTTTGGGTAATCA-3´) and ITS4 (5´-TCCTCCGCTTATTGATATGC-3´) for the region ITS (^{Bonants et al., 1997}).

The conditions to carry out the PCR using the oligonucleotides for TEF-1α and β-Tub consisted of an initial denaturalization at 95 °C for 2 min, followed by 35 denaturalization cycles at 95 °C for 1 min, alignment at 60 °C for 1 min, elongation at 72 °C for 1 min and a final extension at 72 °C for 10 min. The conditions of amplification with the oligonucleotides for ITS were 94 °C for 3 min, followed by 30 cycles at 94 °C for 1 min, 55 °C for 1 min, 72 °C for 1 min and a final extension at 72 °C for 10 min.

The specific oligonucleotides for the species P. nicotianae, Pn1 (5´-GACTTTGTAAGTGCCACCATAC-3´) and Pn2 (5´-CTCAGCTCTTTTCCTTGGATCT-3´) (^{Meng and Wang, 2010}), were used under the following conditions of PCR: 94 °C for 5 min, 35 cycles at 94 °C for 1 min, alignment at 58 °C for 1 min, elongation at 72 °C for 1 min and a final extension at 72 °C for 10 min.

Phylogenetic analysis and sequencing

The PCR products obtained from the genes ITS, TEF-1α and β-Tub were purified using the kit Wizard SV Gel and PCR Clean-Up System (Promega), following the specifications described by the supplier. Sequencing was carried out by capillary electrophoresis with an automatic sequencer ABI PRISM 310 (Genetic Analizer PE Applied Biosystems), in the Unit of Molecular Biology of the Cell Physiology Institute of the UNAM. The sequences were edited using the program CHROMAS Pro 1.6 (Technelysium Pty Ltd, South Brisbane, Qld, Australia), and compared with sequences of the GenBank of the National Center for Biotechnology Information (NCBI) using the Nucleotide Basic Local Alignment Search Tool (BLAST-N) and the Megablast algorithm. For the multiple alignment and phylogenetic analysis of four sequences of representative isolations, we used the software MEGA 6.06 (^{Tamura et al., 2013}). The sequences were aligned with another 22 belonging to different species of Phytophthora (Table 1) using the program MUSCLE integrated in MEGA (^{Edgar, 2004}), using the default values for the penalization for opening and extension of gaps and with the later manual correction of the alignment. The alignments underwent the nucleotide substitution model selection program in order to select the evolutionary model that best fit the data. For the phylogenetic reconstruction we used the maximum verisimilitude model and the Tamura-Nei model. The variation of rates between sites was modeled by a gamma distribution plus invariable sites (4 categories). The support of the internal topology of the dendrograms was determined by bootstrap analysis with 1000 re-sampling (^{Felsenstein, 1985}). Linked alignments were carried out on the 3 markers using the software DAMBE 6.1.23 (^{Xia, 2013}) and a phylogenetic tree was constructed as described earlier.

Table 1. Species, strains, and accession numbers of sequences used for the phylogenetic analysis. 

* isolates of this study.

Pathogenicity evaluation

Ten-week old vinca plants were inoculated by spraying a suspension of zoospores of 1 x 10⁴ zoospores/ml of 10 representative isolates from different sampling sites. Three pots containing 5 plants each were inoculated with isolate. Plants inoculated with only sterile distilled water were used as a control treatment. All plants were kept in a shade mesh area at 25 °C and 80-90 % relative humidity. Once the symptoms developed in the inoculated plants, the pathogen was reisolated and the incidence of the disease was quantified.

Morphological identification

Thirty-nine Phytophthora sp. isolates were obtained from vinca plants with symptoms of blight and wilting (Figure 1A); 77 % came from ornamental nurseries and 23 % came from urban areas. The isolations produced coenocytic mycelia and white cotton-like colonies in V8 agar; they grew between 7 and 30 °C (optimum temperature 25 °C); they developed persistent sporangia, oval or lemon shaped terminals, mainly papillate (Figure 1B) with a average of 34.16 μm in length and 26.35 μm in width (n=24). ^{Waterhouse (1963)} mentions that the range of the size of P. nicotianae sporangia is around 25-50 x 20-40 μm; meanwhile, ^{Erwin and Ribeiro (1996)} reported ranges of 11-60 x 20-45 μm. A proportion in length/width of 1.29 μm (n=24) was observed in the sporangia, similar to the proportion of 1.3 described by ^{Erwin and Ribeiro (1996)} for P. nicotianae. All isolations displayed intercalary and terminal chlamydospores (Figure 1D), which is a characteristic of P. nicotianae (^{Waterhouse, 1963}; ^{Erwin and Ribeiro, 1996}).

Figure 1. Blight symptoms in vinca and P. nicotianae reproductive structures. A. Blight in a vinca plant, B. Papillate sporangium, C. Oospore, D. Chlamydospores. 

Sexual compatibility

The isolations were heterothallic, 21 of a compatibility type A1 and 18 of compatibility type A2 (Table 2). The interaction between compatibility types A1 and A2 produced anaplerotic oospores (Figure 1C), with spherical and flat oogonia, and amphiginous antheridia. These characteristics are consistent with P. nicotianae according to ^{Waterhouse (1963)}, and ^{Erwin and Ribeiro (1996)}. In some sampling sites, both types of compatibility were found, which suggests the presence of sexual recombination; this would increase the genetic variability of the population and could cause the appearance of highly pathogenic isolates (^{Rodríguez-Tovar et al., 2004}; ^{Blaya et al., 2015}). An important observation was the presence of both types of compatibility in three out of the four nurseries observed, which means that recombination could take place in this site, and that if plants with initial infections were to be sold, they would be a factor of dispersion in the region.

Table 2. Sampling sites and type of compatibility in P. nicotianae isolates, source of blight and wilting in Vinca rosea plants in Culiacan. 

Molecular identification

A fragment of approximately 1300 pb was obtained when oligonucleotides DC6/ITS4 were used in the ten isolates selected. The products were sequenced and compared with sequences deposited in the NCBI. Homology percentages ranged between 98 and 100 % with the species P. nicotianae. Oligonucleotides ELONGF1/ ELONGR1 generated a fragment of approximately 1000 pb; the products were sequenced and when the sequences were compared, a homology was obtained between 99 and 100 % with P. nicotianae. Oligonucleotides TUBUF2/TUBUR1 generated a fragment of approximately 1000 pb, and its sequences displayed a homology between 98 and 100 % with P. nicotianae. The sequences of the different amplified regions were deposited in the NCBI (Table 3).

Table 3. Accesions of sequences of three loci deposited in the GenBank of P. nicotianae isolates obtained from V. rosea in urban areas in Culiacan and incidence of infection in pathogenicity experiment. 

Β-Tub = beta tubulin, TEF 1-α = elongation factor, ITS= internal transcribed spacer region, ND= not determined.

The oligonucleotides Pn1/Pn2 specific to P. nicotianae amplified a fragment of approximately 400 pb in all the isolates (Figure 2). These results were expected for P. nicotianae (^{Meng and Wang, 2010}). The use of these oligonucleotides can be a quick strategy to identify this species.

Figure 2. Amplified DNA fragment with the oligonucleotides Pn1-Pn2 specific to P. nicotianae. Line 1, Molecular marker 100 bp; Line 2, water; Line 3, negative control (P. capsici); Line 4, P. nicotianae positive control, Lines 5-43 P. nicotianae isolated from Vinca rosea. 

The phylogenetic reconstruction resulting from linking the three markers showed that the four isolates analyzed in this study were grouped in the same clade as the reference strains of P. nicotianae (Figure 3; 100 % bootstrap), belonging to clade 1 of species of Phytophthora (^{Cooke et al., 2000}; ^{Kroon et al., 2004}; ^{Blair et al., 2008}). The analyses of the individual partial sequences of each marker showed low percentages of support of the internal topology of the dendrograms compared with the values resulting from linking the 3 loci under analysis. With the sequences obtained in this study, individual alignments were carried out for each marker used (ITS, β Tub and TEF 1-α). Later, the 3 alignments were linked (1589 pb) and a similarity was found of 99.6 to 100 % between sequences, therefore not all sequences are equal; 11 differences were found between nucleotides and 2 gaps per insertions of nucleotides (Data not shown). Future studies could estimate thte genetic diversity of Phytophthora nicotianae populations by analyzing a greater number of isolates from different regions and years of collection in order to enrich the study and correlate it with the phenotypical characteristics of the pathogen.

Figure 3. Phylogenetic tree diagram of representative isolates of this study (OV2R, OV5N, OV11, and OVH) and 22 reference strains of Phytophthora derived from the partial sequences of three loci (ITS, β Tub, and TEF 1-α). The tree was created using the software MEGA 6.06 (bootstrap=1000), using the Tamura-Nei substitution model (TN93 G+I). The bootstrap values are shown on the branches. 

Pathogenicity evaluation

The 10 isolates evaluated in the pathogenicity tests caused the symptoms of the disease in V. rosea plants. The initial symptoms of the disease were observed 4 days after inoculation and included leaf blight. Afterwards, plants developed symptoms of wilting and died 12 days after inoculation. All isolations were virulent, they infected healthy vinca plants, although there was a variation in the incidence level, since the percentage of plants with symptoms fluctuated between 13 and 66.7 % (Table 3). Inoculated strains were reisolated from inoculated plants.

It is important to mention that P. nicotianae infects more than 300 different hosts, including Solanum lycopersicum and Capsicum annuum (^{Erwin and Ribeiro 1996}). For this reason, we cannot discard that the species P. nicotianae found in vinca plants in urban areas is also causing some infections in the vegetable-growing area of the Culiacan valley, which is based largely on the production of tomatoes and peppers. Although diverse species of Phytophthora have been reported as causal agents of blight and wilting in vinca (^{McGovern and Begeman, 1996}; ^{McMillan and Garofalo, 2004}; ^{Orilowski et al., 2011}; ^{Hao et al., 2010}), the morphological, phylogenetic, and pathogenic analysis performed in this investigation shows that P. nicotianae is responsible for the damages inflicted on this ornamental plant in the samples gathered in the city of Culiacan.