Identiﬁcation of Phytophthora spp. isolated from plants and soil samples on strawberry plantations in Poland

Crown and leather rot of strawberry caused by Phytophthora spp. are major soil diseases of cultivated strawberry ( Fragaria 9 ananassa ) in Poland. In this study, in total 45 isolates of Phytophthora spp. from plants of cultivars Elsanta, Honeoye, Florence, Camarosa, Roxana, Onebor, Alba, Elegance, Albion, Senga Sengana and Malwina originating from crowns (20 isolates) and fruits (25 isolates) as well as 25 soil samples from the same fruiting strawberry plantations located in central, north-eastern, and south-eastern regions of Poland were identiﬁed. Among them, 44 isolates from plant organs and eight from soil samples were identiﬁed as Phytophthora cactorum and from one plant and one soil sample as P. citricola ; of those remaining from the soil, one was identiﬁed as P. citrophthora and one as P. cryptogea . Identiﬁcations were based on morphological characteristics and DNA analyses. Species-speciﬁc polymerase chain reaction conﬁrmed the presence of Phytophthora spp. in all positive examined plant and soil samples and conﬁrmed that P. cactorum is predominantly responsible for crown and leather rot diseases of strawberry in monitored regions of Poland. Pathogenicity tests showed that all isolates of Phytophthora spp. originating from soil derived from strawberry plantations were pathogenic both to strawberry and to raspberry.


Introduction
Phytophthora is a genus of plant pathogenic filamentous oomycetes (phylum: Oomycota), which, morphologically, are very similar to the filamentous fungi from the kingdom Fungi [2]. Phytophthora cactorum was first identified on cacti in 1870 by Lebert and Cohn [12], and it currently infects an extremely large number of plant hosts. It can limit production of many economically important crops, such as apple, pear, rhododendron, azalea, and strawberry [13]. Phytophthora cactorum can cause root, collar, and crown rots as well as foliar and fruit damage; thus, it is among the most serious soil-borne pathogens affecting crops worldwide [20]. In Poland, P. cactorum causes serious strawberry yield losses due to plant stunting and collapse, especially on susceptible cultivars, such as Elsanta, Honeoye, Elegance, Onebor. Stunting of whole plants or wilting of young leaves are the first symptoms and may appear at any time during the season. The pathogen infects the underground parts by motile zoospores and then colonises strawberry crown. As a result, a typical uniform brown discoloration is visible after the crown cross section, which may appear at the base or in the middle of the crown [4]. Severe infection of the crown leads to dysfunction of the vascular system and to dieback of roots. Leaf margins begin to turn brown and entire leaves wilt, often quickly, and, finally, the whole plant dies. Symptoms of strawberry crown rot can be similar to those elicited by Colletotrichum acutatum and Verticillium dahliae, which are commonly encountered on strawberry plantations in Poland. Therefore, identification of the causal agents of soil-borne diseases on strawberry plantations is an important issue in preventive and pest control management.
The purpose of this study was to investigate (1) which species of Phytophthora are causing necrosis on strawberry fruits and plants; (2) which species are occurring in soil samples and are involved in the decline of strawberry plants in central, north-eastern, and south-eastern regions of Poland; and (3) the pathogenicity of Phytophthora spp. present in soil for strawberry and raspberry plants.

Sampling and isolation procedure
In 2013-2014, plants with visible disease symptoms of crown rot and/or leather rot were sampled from 25 strawberry fruiting plantations in the central, north-eastern, and south-eastern regions of Poland (Table 1).
Small fragments of infected plant organs (fruit, root crown) were surface-disinfected by dipping in 0.5 % (w/v) sodium hypochlorite for 2-5 min and rinsed with sterile water before they were placed onto potato dextrose agar (PDA, Sigma-Aldrich Sp.z.o.o, Poznań, Poland) containing streptomycin, in 90-mm Petri dishes. For each plant organ, four Petri dishes were used, each with 10 tissue pieces. Subsequently, plates were incubated at 25°C until the mycelial growth was observed. After 3-5 days of growth on PDA at 22°C, isolates were identified by morphological and cultural characteristics [7,12]. Finally, 45 isolates of Phytophthora spp. from fruits and/or crowns of strawberry cultivars Elsanta, Honeoye, Florence, Camarosa, Roxana, Onebor, Alba, Elegance, Albion, Senga Sengana and Malwina were obtained. The severity of leather rot on strawberry fruits was evaluated on three selected plantations by counting all healthy and infected fruits on 26 plants in each of four randomly chosen plots during harvest. The results were analysed statistically by the variance analysis method on per cent data transformed according to the Bliss function. The differences between means were estimated by the Newman-Keuls test at the 5 % level of significance.
Isolations from soil samples were made from the same 25 fruiting strawberry plantations (Table 1). Bulked soil samples (about 1.0 kg) were collected from 10 randomly chosen positions of each plantation, at a depth of  [23] was conducted. Trays were filled with tap water to about 1 cm above soil level, and leaves of Rhododendron cultivar Nova Zembla (8-10 leaves per tray) were floated on the surface. After 5-7 days of incubation at 21-23°C, Rhododendron leaves were harvested, washed under tap and deionised water, and dabbed dry. Then, segments of 5 mm diameter were cut from individual necrotic spots and placed onto PDA in 90-mm Petri dishes, 25 pieces per dish, each in five replicates. Within the next 2 days, hyphae growing from the plated segments were assessed under an optical microscope for the presence of sporangia. Small fragments from the edge of growing microorganisms were transferred onto new PDA plates in order to obtain pure cultures.

Classification of isolates into the Phytophthora genus
After 5-7 days of growth on PDA at 22°C, isolates were identified by morphological and cultural characteristics [12,18]. The isolates were cultured on PDA in darkness at 25°C for 14 days. Characteristic morphology of Phytophthora spp. mycelial growth and the presence of sporangia and/or oogonia were observed. Additionally, sporangia were produced by flooding round mycelial discs of 6 9 6 mm in diameter taken from growing margins of 3-to 5-day-old colonies on PDA, 1 mm over their surface, with non-sterile soil extract [18] in 90-mm Petri dishes and incubating them in the dark at 18-22°C. Soil extract was replaced with a fresh one every 24 hours. After three days mycelial fragments were taken from the growing edge of submerged PDA plugs and transferred onto microscopic slides. Zoosporangia and oogonia were observed at 4009 magnification under a light microscope Delta Optical Evolution 300 LED (Delta Optical, Mińsk Mazowiecki, Poland).

DNA extraction from Phytophthora spp. cultures
From 2-to 5-day-old Phytophthora spp. cultures of 45 isolates, about 50-100 mg of fresh mycelia were cut out, and total DNA was extracted following the Aljanabi and Martinez [1] protocol for DNA extraction. DNA was suspended in 50 ll of double-deionised water and kept at -20°C for further analyses.
PCR with species-specific primers and sequence analysis The obtained DNA was tested in PCR with the six primer sets ( Table 2) specific for the following Phytophthora species: P. citricola, P. citrophthora, P. cinnamomi, P. cryptogea, P. cactorum, and P. fragariae. Conditions for PCR were as follows: an initial denaturation step of 3 min at 94°C, followed by 30 cycles of 30 s of denaturation at 94°C; 30 s of annealing at 60°C for P. fragariae-specific primers or at 66°C for the other primers; and 60 s of polymerisation at 72°C. The final elongation step was 5 min at 72°C. Each PCR mixture consisted of the following: 10-30 ng of total DNA template, 500 nM of reverse and 500 nM of forward primers of one speciesspecific set, 0.2 mM of each dNTP, 0.45 unit of DreamTaq Green DNA Polymerase (Thermo Scientific, Vilnius, Lithuania), 19 optimised DreamTaq Green Buffer, and double-distilled water in a total volume of 15 ll. All reactions were performed in a Biometra T3000 Thermocycler (Biometra, Göttingen, Germany). Then, PCR  [26] products were separated during electrophoresis on 1.5 % (w/v) gels, stained with ethidium bromide, and visualised using a transilluminator UV (Sigma T1202; Sigma-Aldrich, St. Louis, USA). The obtainment of the propersize PCR products was confirmed by comparison with the DNA of the reference strains, showing positive signals with the species-specific primers. For three selected isolates, GL1, BT1, and WG1 (identified as Phytophthora spp. in the PCR assay), and for those DNA samples derived from Phytophthora-like colonies growing in the baiting test for which no PCR product was obtained in species-specific PCRs, the DNA was amplified with ITS1 and ITS4 primers, specific for ITS (internal transcribed spacer) regions of filamentous fungi and oomycetes [26], and subjected to sequence analysis. The obtained DNA sequences were compared with the sequences available in the GenBank database using the Basic Local Alignment Search Tool (www.ncbi.nlm.nih.gov/BLAST/) and deposited in Gen-Bank (National Center for Biotechnology Information), with accession numbers assigned to them.
Pathogenicity of Phytophthora spp. from strawberry plantations to raspberry and strawberry plants Pathogenicity tests were conducted with isolates derived from soil samples from strawberry plantations: two isolates of P. cactorum (GL1, Serniki, and BT1, Tuszewo) and one of P. citricola (WG1, Serniki). In June 2014, leaf petioles of the strawberry cultivar Elsanta and stems of the raspberry cultivar Polka were taken and washed under tap water, dabbed dry, and transferred to trays onto moist, sterile blotting paper covered with a plastic net. Threemillimetre-diameter discs cut from the growing margins of 5-day-old PDA cultures of the three examined Phytophthora spp. isolates were placed on strawberry leaf petioles and stems of raspberry and covered with parafilm. Control plant material received plain PDA discs. Trays were covered with the foil and incubated at 24°C. For the experiment, each of 10 leaf petioles and canes per isolate-host combination conducted in four replicates was randomised. Length of necrosis was measured on the fifth (raspberry) and eighth days (strawberry) after inoculation. The experiment was repeated twice. In order to fulfil Koch's postulates, the causal agents of observed symptoms were reisolated on PDA plates, identified using molecular tests, and used for reinfection of plants.

Statistical analyses
Results from the different pathogenicity trials were analysed using analysis of variance (ANOVA) followed by the Tukey HSD test for multiple comparisons (STATISTICA 10; StatSoft Polska, Kraków, Poland).

Results and discussion
Classification of Phytophthora spp. isolates on the basis of morphological traits On the basis of morphological trait estimation during Phytophthora spp. growth in pure cultures (colony characters, conidial and setal morphology, and production of sporangia and/or oogonia), 44 isolates derived from examined plant tissues were classified as P. cactorum and one as a P. citricola. Among cultures growing in baiting tests, typical Phytophthora-like morphology was observed for 10 soil samples. For fast detection of Phytophthora species in strawberry plants, culture-based isolation methods are widely used. On PDA media, P. cactorum usually forms a white, loosely matted colony. Sporangia are distinctively papillate and are usually borne terminally. Each sporangium may contain more than 50 zoospores [10]. According to Waterhouse [25], P. citricola can be readily differentiated from P. cactorum var. applanata on the basis of some morphological features. In culture, P. citricola tends to grow in a petaloid manner, while P. cactorum grows uniformly fluffy. Moreover, the apex of a sporangium of P. citricola is flat and wide with shallow thickening, while in P. cactorum prominent papillae with deep thickenings can be observed. Described differentiating features were also observed for these species in our study (Fig. 1a, b, e, f). Sporangia and oogonia produced by P. cactorum isolates on PDA media were observed after 10 days of cultivation, whereas P. citricola growing on PDA produced only oogonia; sporangia of the latter species were observed on the third day of cultivation in non-sterile soil extract. The oogonia of P. citricola were similar in size to those of P. cactorum (Fig. 1i, h). Our observations of growing cultures obtained in baiting tests were similar to those previously described for Phytophthora spp.; therefore, it was also possible to preliminarily classify the oomycetes as P. citricola and P. cactorum and, additionally, to P. citrophthora and P. cryptogea. Phytophthora citrophthora showed stellate, rosettal growth on PDA medium (Fig. 1c) and was able to produce sporangia in non-sterile soil extract (Fig. 1g), but oogonia were not observed on PDA medium, nor in soil extract. Colonies of P. cryptogea were fairly fluffy and cottony, with lobate margins and a slight floral pattern (Fig. 1d), but they did not produce sporangia nor oogonia on PDA or in soil extract. The lack of the production of oogonia by P. cryptogea and P. citrophthora is consistent with the previous observations [12,19] that these species are heterothallic and require crossing with an opposite mating type to form oogonia and oospores.

Phytophthora spp. identification in PCR assays
In PCRs with six Phytophthora species-specific primer sets, the presence of the proper-size products of reactions was observed on agarose gel. For 45 tested DNA samples obtained from Phytophthora spp. isolates from diseased plant tissues, positive signals for P. cactorum were observed (44 isolates) but only one for P. citricola (Table 3). From 25 soil samples, only in 20 % (nine soil samples) were Phytophthora spp. strains trapped and recognised in the baiting test. In the PCR assay, isolates from eight plantations were identified as P. cactorum, from one as P. cryptogea (Zarzecze), from one as P. citrophthora (Tuszewo), and from one as P. citricola (Serniki) ( Table 3). This may suggest that Phytophthora spp. propagules are transferred between plantations mostly by strawberry stocks. It has been also reported that Phytophthora spp. can survive in infected host tissue [14,22].
In the case of those Phytophthora spp. isolates for which sequencing of the ITS rDNA region and its comparison with reference sequences in GenBank were performed, the species classification was consistent with the results of the PCR assay. BLAST analysis of obtained DNA sequences of GL1 (GenBank Acc. No. KT361202) and BT1 (Gen-Bank Acc. No. KT361201) showed 100 % similarity with the corresponding region of reference of the P. cactorum strain (GenBank Acc. No. GU111587.1), while the sequence of the WG1 isolate (GenBank Acc. No. KT361200) was 100 % similar to the sequence of P. citricola E strain (GenBank Acc. No. AF266788.1).
For other microorganisms whose colonies were obtained in the baiting test, the sequence analysis of the ITS rDNA region demonstrated the presence of fungi or oomycetes from the genera Pythium, Fusarium, Rhizopus, and Mortierella. Species identification based on morphological and physiological characters is a primary approach, although it is mostly laborious and time-consuming. Due to high intra- species variability and small inter-species differences between Phytophthora species, additional confirmation is often required. Techniques based on the PCR method offer rapid, simple, and reliable identification of fungal and oomycetal species. Not only can species be identified in pure cultures, but it is also possible to detect pathogens directly in plant material and soil, which highly facilitates the possibility for specific monitoring of these pathogens.
On the basis of some species-specific regions in the genome of Phytophthora, Ersek et al. [11] developed speciesspecific primers for P. parasitica and P. citrophthora detection. The variability in the DNA regions of ITS regions of rRNA of Phytophthora spp. has been also examined by Schubert et al. [21] and Boersma et al. [5]; thus, it became possible to discriminate species within the Phytophthora genus using species-specific primers designed by those authors. Because of recent changes in the P. citricola complex-the new species P. plurivora sp. nov. was designated [18]-there was a need to additionally confirm the species affiliation of the WG1 P. citricola isolate by comparing its ITS1-5.8S-ITS2 rDNA region with the sequence of reference strains from this complex, available in GenBank database.

Association of Phytophthora spp. presence with yield loss
Excessive rainfall during the 2013 growing season was associated with heavy losses in strawberry crop due to fruit rot. Leather rot caused by P. cactorum accounted for up to 11 % yield loss depending on cultivar (Fig. 2) within three examined plantations. Propagules of P. cactorum are readily dispersed from infected strawberry fruit by  splashing water [16]. Gerlach et al. [14] showed that zoospores of P. citrophthora were readily dispersed to potted Pieris japonica by water. Outbreaks of fruit rot on apple caused by P. syringae in the UK were related to the heavy rains, and inoculum was splashed with infested leaf debris on the orchard floor [24]. Phytophthora spp. spread mainly through the movement of infested soil, water, and infected plants or plant material; however, there are species that are transmitted aerially [8,15]. Especially substantial as a source of inoculum are those plants that are infected but do not show symptoms either because the disease has not yet progressed to the stage where symptoms are evident. Therefore, the major challenge in preventing the spread of Phytophthora is detection of the pathogen in asymptomatic tissue.
Pathogenicity of Phytophthora spp. from soil samples to raspberry and strawberry The average length of necrosis on raspberry cane ranged from 8.0 to 19.7 mm, while on strawberry leaf petioles it ranged from 32.0 to 49.5 mm, depending on the isolate of Phytophthora spp. (Table 4). The longest necrosis was observed for isolate WG1 (P. citricola), and the smallest, for isolate BT (P. cactorum); however, this difference was not significant. All isolates of Phytophthora were successfully reisolated from symptomatic tissues and identified using molecular tests, fulfilling Koch's postulates. This study shows that two P. cactorum and one P. citricola strains isolated from soil samples collected in the strawberry plantations where disease symptoms were observed were pathogenic both to strawberry plants and to raspberry plants, causing necrotic lesions on wounded leaf petioles and raspberry canes. The differentiation in P. cactorum isolates according to host preference was found in studies of Cooke et al. [9] and Hantula et al. [17]. Bielenin [3] reported that isolates of P. cactorum both from strawberry crowns and from fruit caused distinctly smaller necrosis symptoms on apple shoots than isolates from apple trees.
In conclusion, we have shown on the basis of morphology and DNA sequence analysis that the P. cactorum species is the most common causal agent of strawberry infections in Poland. Under favourable weather conditions for development and spread of the pathogen (high humidity and temperatures above 20°C), it causes significant losses in yield of strawberries as well as decay of whole plants. The results of pathogenicity trials revealed that the filamentous parasitic organisms Phytophthora spp. (class Oomycetes) occurring in the soil can be pathogenic for both strawberry and raspberry plants, showing no host specificity.