Different Responses to Salinity of Pythium spp. Causing Root Rot on Atriplex hortensis var. rubra Grown in Hydroponics

Abstract

Atriplex hortensis var. rubra (red orache, RO) is a halotolerant species rich in nutraceutical compounds, which makes it a valuable crop for human nutrition. This plant could also be exploited for phytoremediation of contaminated soil and wastewater, and for saline aquaponics. A root rot disease was observed on hydroponically grown RO plants, caused by Pythium deliense and Pythium Cluster B2a sp. Identification was based on morphology, molecular analysis (ITS and COI), and phylogenetic analysis. We assessed disease severity in plants grown in a growth chamber with nutrient solutions containing different NaCl concentrations (0, 7, and 14 g L⁻¹ NaCl). In vitro growth at different salinity levels and temperatures was also evaluated. Both Pythium species were pathogenic but showed different responses. Pythium deliense was significantly more virulent than Pythium Cluster B2a sp., causing a steady reduction in root dry weight (RDW) of 70% across all salinity levels. Pythium Cluster B2a sp. reduced RDW by 50% at 0 and 7 g L⁻¹ NaCl while no symptoms were observed at 14 g L⁻¹ NaCl. Pythium deliense grew best at 7 and 14 g L⁻¹ NaCl, while Pythium Cluster B2a sp. growth was reduced at 14 g L⁻¹ NaCl. Both pathogens had an optimum temperature of 30 °C. This is the first report of Pythium spp. causing root rot on RO grown hydroponically. The effective use of halophytic crops must consider pathogen occurrence and fitness in saline conditions.

1. Introduction

Atriplex hortensis, commonly known as orache, is an annual herbaceous plant of the Amaranthaceae family. Due to its tolerance to adverse soil conditions, such as salinity and drought, this plant thrives in arid and semi-arid regions [1]. The red variety A. hortensis var. rubra, also called red orache (RO), is particularly appreciated for its large red-purple leaves. The leaves can be used in salads, soups, and stews, and can also be consumed raw or cooked, just like spinach [2]. In addition, RO is rich in nutraceutical compounds, making it a valuable crop for human nutrition. Among the key compounds are betalains, a natural pigment class including red-violet betacyanins and yellow-orange betaxanthins. Red orache specifically contains betalains that are amaranthin-type pigments, such as amaranthin, celosianin, and argentianin. These compounds play a role in reducing oxidative stress, thereby supporting cardiovascular and immune health, and offering potential benefits in the prevention of chronic diseases. Betalains also exhibit strong growth-inhibitory effects against various cancer cell lines [3,4]. Red orache also represents an interesting source of microelements in the human diet as microgreens and is a good candidate for biofortification practices [2]. The increasing focus on finding alternative crops makes RO a promising novel source of beneficial compounds, especially in regions where conventional crops struggle to grow.

Globally, A. hortensis is an ecologically significant plant, well adapted to salinity and drought conditions; it can grow at relatively high salt concentrations and shows excellent ability to accumulate both sodium and chloride [5,6]. Therefore, A. hortensis can be exploited for phytoremediation to restore degraded or contaminated soil, recover wastewater, and support saline aquaponics programs [7,8,9]. Through the exploitation of halophytic crops, it is possible to reduce the salinity of the soil and improve its fertility, creating more favorable conditions for future agricultural use. This allows plants to mitigate salt stress while maintaining growth and productivity. However, despite its versatility, there is a lack of studies aimed at optimizing RO cultivation methods, including pathogen control. So far, to the best of our knowledge the few available records of pathogens occurring on A. hortensis only describe diseases in nature, and no records deal with cultivated plants [10].

Pythium (Kingdom Straminipila, Phylum Oomycota) is a ubiquitous genus of filamentous fungal-like microorganisms whose lifestyle ranges from plant/animal pathogens to saprophytes. The first studies that tried to unravel the delimitation of the species in the Pythium genus date back to the mid-20th century and the most comprehensive monograph on the Pythium genus was authored by van der Plaats-Niterink [11].

Due to the high variability of morphological features and the frequent coexistence of multiple species within the same environment, the classification of the species in the Pythium genus is challenging. Lévesque and de Cook [12], by phylogenetic analyses based on the nuclear rDNA internal transcribed spacer region (ITS), classified the genus Pythium into 11 clades (A to K). Although ITS is still widely accepted, new molecular markers have been used to better clarify species delimitation within the Pythium genus. Key regions, such as the mitochondrial cytochrome c oxidase subunit I and II (COI and COII) have been used to refine species classification [12,13,14,15].

Ecologically, the Pythium genus is widely distributed throughout the world, inhabiting tropical, temperate, and even cold regions. Pythium species are commonly found in soil, but their ability to produce and release zoospores into water allows them to thrive in aquatic environments, including freshwater lakes, streams, and irrigation systems [16]. Some species are frequently found in greenhouses in temperate climates [17,18]. Although many Pythium species have a saprophytic lifestyle, most are known as plant pathogens. They are generally non-host specific and can affect numerous plant families, commonly causing root, stem and fruit rot, as well as pre- and post-emergence damping-off of seedlings [19].

Pythium species can be a persistent problem, especially in closed systems like greenhouses and hydroponic systems. The pathogens can be introduced via airborne dust, contaminated tools and footwear, irrigation water, and even peat used for rooting media. Transplants also represent an important source of infection in hydroponic vegetable crops, particularly if produced in greenhouses hosting infected soil. Reusing hydroponic materials such as rockwool slabs, coconut fiber, or plumbing systems often allows Pythium spp. inoculum to be conserved between crops, as pathogens persist in biofilms, mucilaginous residues, and root tissue fragments. Furthermore, insect vectors can contribute to the spread by acquiring Pythium structures (e.g., viable oospores) externally or through ingestion [20].

The flow dynamics of nutrient solutions heavily influence the dispersal of Pythium propagules in greenhouse hydroponic systems. Pythium species are more likely to establish a robust population in nutrient solutions from the Nutrient Film Technique (NFT) systems than in rockwool and coconut fiber culture [21,22,23]. In NFT, the rapid flow between roots contrasts with areas of stagnation where zoospores tend to aggregate, facilitating localized infection [20]. High humidity and water-saturated conditions allow zoospore-producing pathogens to thrive in hydroponic systems [24]. Pythium root rot in hydroponics is a serious and widespread problem affecting not only food crops but also industrial crops such as hemp [25] and tobacco [26,27].

Although saline hydroponics is a promising method for growing halophytic crops [28], little is known about how salinity influences the virulence of Pythium spp.

In the summers of 2021 and 2022, symptoms of root rot caused by Pythium spp. were found on plants of RO grown hydroponically. Given the high impact of these pathogens on plant growth, the objectives of this study were to evaluate (i) the severity of disease of two Pythium species on RO grown in a floating raft system in both saline and non-saline conditions; (ii) the in vitro growth of the two Pythium species in response to increasing salinity and temperature.

2. Materials and Methods

2.1. Pythium spp. Isolates: Origin and Culture Conditions

Severe symptoms of root rot were observed on seed-propagated RO plants grown in a floating raft system at the University of Pisa. Several Pythium species were isolated from the affected plants, and their pathogenicity was previously confirmed by fulfilling Koch’s postulates through artificial inoculations in hydroponics [29]. In the present study, two different isolates, named 8MC and 1BMR, were chosen for further investigations.

Pythium spp. isolates were grown on V8 agar medium (200 mL V8^® vegetable juice, 3 g CaCO₃, 15 g agar, 800 mL deionized water) amended with streptomycin (0.3 g L⁻¹; Sigma-Aldrich, Saint Louis, MO, USA) at 25 ± 1 °C, to obtain actively growing colonies. Isolates were stored in 2.5 mL vials filled with 1.5 mL of V8 agar medium and kept at 25 ± 1 °C in complete darkness. The medium was autoclaved at 121 °C for 15 min.

2.2. Species Identification

2.2.1. Morphological Observations

To evaluate morphological features, Pythium spp. isolates were grown on clarified V8 agar medium at 25 ± 1 °C. Clarified V8 juice was prepared by dissolving CaCO₃ at a concentration of 1.5% in V8^® vegetable juice under constant stirring. The mixture was centrifuged at 1800 g for 15 min and the supernatant was collected and filtered through two layers of Whatman No. 1 filter paper. The medium was prepared by adding 200 mL of clarified V8 juice to 800 mL of deionized water and 15 g of agar and was autoclaved at 121 °C for 15 min.

The shape and size of both sexual and asexual structures were observed using an optical microscope Dialux 22 (Leitz, Wetzlar, Germany). Images were captured using a Leica DFC 450C digital microscope camera and then measured with the control software Leica Application Suite X Version 3.1.1.17751 (Leica Microsystems Ltd., 146 Heerbrugg, Switzerland). The slide culture technique was performed [30] and at the end of incubation time both lactophenol cotton blue [31] and 0.05% (w/v) acid fuchsin in lactoglycerol (lactic acid–glycerol–water, 14:1:1) are used for staining the different structures of Pythium isolates [32,33]. Measurements of the various structures were determined using water as a mounting medium and were compared with the observations reported by van der Plaats-Niterink [11].

2.2.2. DNA Extraction and Molecular Analysis

Five mycelial discs (6 mm Ø), cut from the edge of actively growing colonies of Pythium spp. on V8 agar plates, were inoculated into 50 mL tubes filled with 25 mL of V8 broth medium. The tubes were kept for 5 days at room temperature (25 ± 2 °C) under constant stirring on the Multi-RS 60 programmable rotator (Biosan, Riga, Latvia) at 60 rpm, with a rotation angle of 75 °.

Mycelium was collected by filtering each liquid culture through a layer of sterile Miracloth (Calbiochem, San Diego, CA, USA), rinsed twice with sterile distilled water, dried on sterile filter paper, and stored at −20 °C for DNA extraction. Genomic DNA was extracted with the Genesig^® Easy DNA/RNA extraction kit (Primer Design Ltd., Chandler’s Ford, UK) using the method described by Spada et al. [34]. DNA was quantified using the Qubit™ DNA BR Assay Kit in a Qubit™ 4 Fluorometer (Invitrogen by Thermo Fisher Scientific Inc., Eugene, OR, USA).

Three barcode regions were amplified by PCR: the internal transcribed spacer (ITS) of rDNA, and the cytochrome oxidases I and II (COI and COII) of mitochondrial DNA.

For ITS amplification, we used the universal primers ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′TCCTCCGCTTATTGATATGC-3′) [35]. PCR was carried out in a 25 µL reaction mix with GoTaq Green Master Mix 2X (Promega Corporation, Madison, WI, USA), 0.1 μM of each primer, 30–50 ng of DNA, and adding nuclease-free water to the volume. PCR conditions were as follows: initial denaturation at 94 °C for 1 min, followed by 30 cycles of 94 °C for 30 s, 54 °C for 1 min, and 72 °C for 4 min and a final extension at 72 °C for 4 min.

The oomycete-specific degenerate primers OomCoxI-Levup (5′-TCAWCWMGATGGCTTTTTTCAAC-3′) and OomCoxI-Levlo (5′-CYTCHGGRTGWCCRAAAAACCAAA-3′) were used to amplify a 680 bp region of the COI mitochondrial gene [15]. PCR was carried out in a 25 μL reaction mix containing: 1X DreamTaq Buffer, 3.5 mM MgCl₂, 0.1 mM of dNTPs mix, 0.1 mM of each primer, 0.125 μL of DreamTaq DNA Polymerase (5U μL⁻¹; Thermo Fisher Scientific Inc., Eugene, OR, USA), and 1–10 ng of DNA. PCR conditions were those described by the authors [15].

For COII amplification, we used the forward primer Cox2-F (5′-GGCAAATGGGTTTTCAAGATCC-3′) [36] and the reverse primer Cox2-RC4 (5′-TGATTWAYNCCACAAATTTCRCTACATTG-3′) [37]. PCR was carried out in a 25 μL reaction mix containing: 1X DreamTaq Buffer, 2 mM of MgCl₂, 0.2 mM of dNTPs mix, 0.1 mM of each primer, 0.1 μL of DreamTaq Polymerase (5U μL⁻¹), and 1–10 ng of DNA. PCR conditions were as follows: initial denaturation at 95 °C for 4 min, followed by 36 cycles of 95 °C for 40 s, 50 °C for 40 s, and 72 °C for 1 min, and a final extension at 72 °C for 5 min.

Amplicons were visualized by electrophoresis in 0.5 x TBE (45 mM Trizma base, 44 mM boric acid, 1 mM EDTA, pH 8.4) buffer with 1% (w/v) agarose and detected by UV fluorescence after GelRed™ (Biotium, Inc., Fremont, CA, USA) staining. The 100 bp DNA ladder (Promega, Madison, WI, USA) was used as a molecular size marker. Amplicons were purified with the QIAquick PCR purification kit (QIAGEN, Milan, Italy), according to the manufacturer’s instructions and sent to BMR Genomic (Padua, Italy) for Sanger sequencing. Amplicons were sequenced in both directions using the same set of primers. Consensus sequences were edited with BioEdit v7.7 [38] and used as queries in BLASTn searches of the GenBank database hosted by NCBI.

2.2.3. Phylogenetic Analysis

Phylogenetic analysis was conducted using MEGA-X software [39] on the concatenated sequences of ITS and COI. The sequences used in this study (Table S1) were retrieved from GenBank using those described by Robideau et al. [15], and aligned using the MUSCLE algorithm implemented in MEGA-X. The alignments were edited with BioEdit v7.7 [38] to obtain the concatenated file. The analysis involved 15 concatenated nucleotide sequences, with a total of 1581 positions in the final dataset.

The phylogenetic tree was inferred by using the Maximum Likelihood method and the General Time Reversible model [40]. The strength of the internal branches of the resulting trees was tested by bootstrap analysis using 1000 replicates. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites [5 categories (+G, parameter = 0.3808)]. The tree was rooted using Achlya bisexualis as the outgroup.

2.3. Pathogenicity Assay

The pathogenicity of the Pythium isolates 8MC and 1BMR was assessed in a floating raft system under saline conditions (at 0, 7, and 14 g L⁻¹ NaCl) towards A. hortensis var. rubra plants. The salinity levels (7 and 14 g L⁻¹) were chosen based on the results of preliminary experiments in which different NaCl concentrations (0, 7, 14, 21, and 28 g L⁻¹) were tested. Indeed, we found that (i) at 7 and 14 g L⁻¹ NaCl the growth rate of the two Pythium isolates was equal to or even greater than the control grown without NaCl (the no-salt control); (ii) the growth of RO was severely impaired at 21 and 28 g L⁻¹ NaCl.

Red orache seeds (De Bolster Organic Seeds, Epe, Gelderland, The Netherlands) were sown on polystyrene trays filled with rockwool plugs (Grodan, Roermond, The Netherlands), covered with vermiculite, and kept in a greenhouse for germination. After emergence, corresponding to 15 days after sowing (das), RO seedlings were removed from the trays and transferred to polystyrene rafts capable of accommodating 20 plants each. Each raft was placed on a 1 L, dark-colored, polypropylene tank filled with a nutrient solution containing: N-NO₃ 14.0 mM, N-NH₄ 2.0 mM, P-H₂PO₄ 2.0 mM, K 10.0 mM, Ca 4.5 mM, Mg 2.0 mM, S-SO₄ 5.0 mM, Fe 40.0 μM, B 40.0 μM, Cu 3.0 μM, Zn 10.0 μM, Mn 10.0 μM, Mo 1.0 μM. The nutrient solution in each hydroponic tank was continuously aerated to provide oxygen to the plants. Plants were moved to a growth chamber (25 ± 1.5 °C, PAR 150 μmol m² s⁻² by LED tubes) for 10 days to promote root development. At 25 das, a progressive salinization phase began. Salinization was achieved by adding one-quarter of the final concentration of NaCl into the nutrient solution for four days. At 30 das, the nutrient solution was replaced with a new solution (with or without salt) and RO plants were inoculated with zoospores of Pythium spp.

For the inoculum production, we followed the protocol of Jack and Nelson [41], with some modifications. Mycelial discs (2 cm Ø) were cut with a cork borer from 3-day-old colonies of Pythium spp. grown on V8 plates. The discs were placed on 6 cm Petri plates and flooded with 10 mL of sterile distilled water. The plates were incubated statically overnight at 25 ± 1 °C in complete darkness to promote the formation of sporangia. After incubation, the liquid was replaced with another 10 mL of sterile distilled water and further incubated for 7 h, as described above. Finally, to estimate the inoculum concentration, aliquots of the zoospore suspensions were briefly vortexed and quantified with a hemocytometer (Bürker, LO—Laboroptik Ltd., Lancing, UK). The zoospore suspensions were properly diluted to obtain the same concentration for both isolates. Hence, the suspensions were poured into the tanks with a final concentration of 10⁴ zoospores mL⁻¹ for each isolate. The same methodology was applied for control plants using a non-inoculated V8 agar plug.

A factorial design was used with two factors (salt concentration and inoculum) and each factor had three levels: 0, 7, and 14 g L⁻¹ NaCl; no inoculum, inoculum of Pythium isolate 1BMR, inoculum of Pythium isolate 8MC. Forty plants were used for each of the nine treatments for a total of 360 plants.

The trial lasted up to 45 das, corresponding to 15 days after inoculation (dai). At the end of the trial, the roots of A. hortensis var. rubra plants were gently separated and dried in a ventilated oven at 70 °C until a constant weight.

The virulence of the two isolates was assessed by measuring the root dry weight (RDW) of each plant. Additionally, an empirical qualitative ordinal scale of symptoms was used to assess foliage damage. The scale was based on five levels: 0—no chlorosis, 1—few chlorotic spots, 2—diffuse chlorotic spots, 3—partially dried leaf, 4—fully dried leaf (Figure 1). For each plant, the McKinney Index (MKI) [42] was calculated according to the following formula:

$$\mathrm{M}\mathrm{K}\mathrm{I}= {\displaystyle \frac{\sum (d \times f)}{N \times D}} \times 100$$

(1)

where d represents the level of disease infection scored, f is the disease frequency, N is the total number of observations, and D is the highest level of disease infection that occurred on the empirical qualitative ordinal scale.

Furthermore, at 15 dai, plant mortality and colonization rate of isolates were recorded. The colonization rate was determined by re-isolating the Pythium species from crowns of infected RO plants. Briefly, crowns were cut and surface-sterilized with sodium hypochlorite (NaOCl; 1% available chlorine) for 2 min, rinsed twice in sterile distilled water, and plated on V8 agar plates amended with streptomycin (0.3 g L⁻¹, Sigma-Aldrich, Saint Louis, MO, USA). The plates were incubated at 25 ± 1 °C and checked daily. Identification of isolates was carried out based on morphological observations.

The pathogenicity assay was repeated twice with the same methodology, yielding comparable results. Here, we report the results obtained from the second experiment.

2.4. In Vitro Growth of Pythium spp. at Different Temperatures and Salt Concentrations

We tested the ability of the two Pythium isolates to grow in response to different temperatures and salinities. Mycelial discs (6 mm Ø) were cut from actively growing colonies and inoculated onto Corn Meal Agar (CMA, Sigma-Aldrich, Saint Louis, MO, USA) Petri plates supplemented with 0, 7, 14, 21, and 28 g L⁻¹ NaCl. Plates were incubated at 10, 15, 20, 25, 30, 35, and 40 °C in complete darkness. Mycelial discs were placed at the edge of the plate, and linear growth was recorded daily until the mycelium reached the opposite side. In cases where salt was present, an additional 0.5% of agar was added to aid in solidification of the medium [43]. The trial consisted of five biological replicates for each temperature and salinity. Daily growth rate (mm day⁻¹) was calculated using consecutive measurements in the linear phase of the growth curve.

2.5. Statistical Analysis

Data from RDW and in vitro growth assays were analyzed by two-way ANOVA using the aov function in RStudio (v. 2024.9.1.394) [44]. The normality of the data was assessed with the Shapiro–Wilk test, performed using the Shapiro Test function, and homoscedasticity was tested using Levene’s test, which was performed using the Levene Test function in the car package (Tables S2–S4).

For RDW data, to assess the effect of inoculation across different salinity levels, means were separated by Tukey’s honestly significant difference (HSD) post-hoc test (p ≤ 0.05) using salinity as a factor. Pairwise comparisons were conducted using the contrast function from the emmeans package.

The in vitro growth data for each isolate were separated by Tukey’s HSD test, using both temperature and salinity as factors. The test was performed using the Tukey HSD function.

For MKI data, each isolate was individually analyzed using the Kruskal–Wallis test and means were separated using the Dunn test with the Bonferroni method. Prior to analysis, MKI data were arcsine square root-transformed (arcsin√%) to meet the assumptions of the test.

3. Results

3.1. Species Identification: Morphological Observations

3.1.1. Pythium sp. Isolate 8MC

The colony morphology on V8 agar medium of Pythium sp. isolate 8MC was characterized by sparse, colorless mycelium. After a few days of colony growth, abundant oogonia and antheridia were observed. Oogonia were smooth, spherical, and terminal with a diameter of 37.2 ± 0.9 μm (n = 40). Only one antheridium was observed per oogonium. The antheridia were terminal and occasionally intercalary. They were primarily monoclinous, though occasionally diclinous, often arising as side branches of the oogonial stalk and establishing apical contact with the oogonium. Oogonia stalks bent towards the antheridium. Oospores were aplerotic and measured 28.4 ± 0.6 µm (n = 40), with a wall thickness of 4.1 ± 0.1 μm (n = 40) (Figure 2a–c). Sporangia were mostly terminal and rarely intercalary, consisting of typical filamentous, inflated (toruloid) structures with swollen side branches (Figure 2d–g). Encysted zoospores measured 15.6 ± 0.4 µm (n = 40) in diameter.

3.1.2. Pythium sp. Isolate 1BMR

On V8 agar medium, the colony of Pythium sp. isolate 1BMR appeared dense and whitish, and exhibited abundant aerial mycelium. Oogonia and antheridia were rarely observed. Sexual structures were produced only after 20 days of culture, although to a lesser extent than isolate 8MC. Oogonia are terminal or intercalary, non-ornamented, with an average diameter of 26.0 ± 0.4 µm (n = 24). Oospores were aplerotic and measured 26.8 ± 0.4 µm (n = 20) (Figure 3a,b). Antheridia were consistently diclinous, i.e., originated from a different hypha than the oogonial stalk. Appressoria were cylindrical or club-shaped (Figure 3c). Sporangia were filamentous and occasionally slightly inflated (Figure 3d). Encysted zoospores had a mean diameter of 12.4 ± 0.2 μm (n = 24).

3.2. Species Identification: DNA Barcoding and Phylogenetic Analysis

To determine the species of our Pythium spp. isolates, we sequenced three regions: ITS, COI, and COII. Consensus sequences were generated and used as queries for BLASTn search. GenBank accession numbers for the most significant matches are indicated in brackets.

For isolate 8MC, sequencing yielded fragments of 864, 666, and 619 bp for ITS, COI, and COII barcodes, respectively. The sequences were deposited in GenBank under accession numbers PV203520, PV235405, and PV235407.

For ITS, the Pythium sp. isolate OPU809 showed the highest match (99.7% identity; AB543064), followed by various P. deliense isolates, with identities ranging from 98.1% to 98.4%. However, the BLASTn search for ITS provided ambiguous results, as strong matches were also found with two P. aphanidermatum isolates (98.1% identity; OR437986 and PP859007).

In the COI analysis, a 100% identity match was obtained with P. deliense (OP622276), followed by the Pythium sp. isolate OPU809 (LC224108) with 99.8% identity. Similarly, BLASTn search for the consensus sequence of the COII gene identified P. deliense (AF196589) as the best match, with 99.0% identity. As observed with ITS, the COI and COII analyses revealed strong matches with multiple P. aphanidermatum isolates, with 95–96% identity. However, the highest identity was consistently found with a single isolate of P. deliense.

In the case of isolate 1BMR, we obtained fragments of 866, 677, and 630 bp for ITS, COI, and COII barcodes, respectively. The sequences were deposited in GenBank under accession numbers PV203521, PV235406, and PV235408.

The BLASTn search output for ITS showed a 100% match with species belonging to the Pythium Cluster B2a [15]: P. coloratum (MK813919), P. diclinum (JQ898459), P. dissotocum (KM061658), and P. lutarium (JQ898467).

For both COI and COII, the best match was P. dissotocum (identity 99.0%, MZ562299 and identity 99.8%, PQ349319). However, strong matches were also found for other species within the Pythium Cluster B2a. For COI, these included P. coloratum (99.0%, MT996498) and P. diclinum (99.0%, PQ231532). For COII, the strongest matches included P. diclinum (98.3%, MK425693) and P. lutarium (98.2%, KJ595359).

Phylogenetic relationships among isolates were inferred by constructing a tree with concatenated sequences of ITS and COI (Table S1). The Maximum Likelihood method and the General Time Reversible model were applied. The tree with the highest log likelihood (−7563.32) is shown in Figure 4. The percentage of trees in which the associated taxa clustered together is indicated next to the branches.

Phylogenetic analysis confirmed the DNA barcoding results, as both isolates of this study clustered as expected. Pythium isolate 8MC strongly clustered with both P. aphanidermatum and P. deliense (clade A) [15], but it is more closely related to the latter species, supported by a high node value. For isolate 1BMR, the phylogenetic tree did not resolve its position within the species complex referred as Pythium Cluster B2a which includes P. coloratum, P. diclinum, P. cf. dictyosporum, P. dissotocum, P. lutarium, P. sp. ‘Group F’ and P. sp. ‘tumidum’ [15] (Figure 4). Therefore, based on morphological traits, DNA barcoding and phylogenetic analysis, we concluded that isolate 8MC corresponds to P. deliense Meurs and isolate 1BMR belongs to Pythium Cluster B2a sp.

3.3. Pathogenicity Assay

The susceptibility of RO to Pythium spp. was evaluated in a floating raft system in saline and non-saline conditions. Three salinity levels (NaCl) 0, 7, and 14 g L⁻¹ were tested. Fifteen days after inoculation, the roots of RO plants were dried and the virulence of the isolates was assessed based on the reduction in root dry weight (RDW).

Overall, the presence of salt led to a progressive decrease in RDW, even in the absence of pathogen inoculum. For non-inoculated plants, RDW was 60.9 ± 1.0 mg, 47.8 ± 0.9 mg, and 25.7 ± 0.8 mg at 0, 7, and 14 g L⁻¹ NaCl, respectively (Figure 5).

Pythium deliense induced the lowest RDW in all three salinity levels (i.e., 16.7 ± 0.7, 10.8 ± 0.7, and 6.4 ± 0.5 mg), while isolate Pythium Cluster B2a sp. showed an RDW of 35.0 ± 1.0 mg at 0 g L⁻¹ NaCl, and 25.4 ± 0.92 mg at 7 g L⁻¹ NaCl. At the highest salinity level, the RDW of plants inoculated with Pythium Cluster B2a sp. (27.3 ± 0.7 mg) was not significantly different from control (Figure 5).

The two-way ANOVA revealed a significant (p ≤ 0.001) effect of both inoculum and salinity and their interaction (Table S5) indicating a strong influence of both factors, as well as their interaction, on RDW. The significant interaction highlights the collective impact of the two factors, when considered together, on root growth of RO.

Within the same salinity level, when P. deliense was inoculated, all mean differences were statistically significant, for both control and Pythium Cluster B2a sp. inoculated plants. In contrast, when Pythium Cluster B2a sp. was inoculated, there was no significant effect on RDW at 14 g L⁻¹ NaCl compared to control plants (p = 0.385; Table S6).

Non-inoculated plants did not show any symptoms at the root level (Figure 6a–c). When Pythium spp. were inoculated, the main symptom was root rot, consisting of root browning, reduced root biomass, and stunting. The development of root rot symptoms and disease severity were particularly evident in the case of P. deliense, appearing within a few days after inoculation, while symptom development for Pythium Cluster B2a sp. was slower.

In the case of Pythium Cluster B2a sp., roots showed moderate browning and some reduction in root biomass at 15 dai (Figure 6d–f). The decrease in RDW was influenced by salinity. In the absence of salt and at 7 g L⁻¹ of NaCl, Pythium Cluster B2a sp. caused a reduction in RDW of about 50% compared to the control, while this effect was absent at 14 g L⁻¹ (Figure 6f).

Pythium deliense caused severe root rot, showing marked browning of the roots and a drastic reduction in root biomass at 15 dai (Figure 6g–i). The fitness of P. deliense was not affected by the presence of salt, as the pathogen induced a 70% decrease in RDW compared to the control plants across all salinity levels.

The effects of root rot were also evident in the aerial parts of the plants. An empirical symptoms scale was used to assess foliage damage. The symptoms scale was based on five levels (Figure 1), and the MKI was calculated for each plant. Initial symptoms included poor growth, leaf chlorosis and wilting. In later stages, affected plants showed leaf necrosis and complete foliage desiccation.

Foliage damage was more severe in plants inoculated with P. deliense (Figure 7). Disease severity, reported as MKI (%), was 64.5 ± 4.3, 58.3 ± 3.0 and 68.6 ± 3.5 at 0, 7, and 14 g L⁻¹ NaCl, respectively. On the other hand, Pythium Cluster B2a sp. was less virulent, and inoculation caused less damage to foliage at all salinity levels (Figure 7). In this case, MKI (%) was 42.7 ± 1.8, 35.9 ± 3.1, and 25.1 ± 1.5 at 0, 7, and 14 g L⁻¹ NaCl, respectively.

Interestingly, at the highest salinity level, Pythium Cluster B2a sp. did not reduce RDW but still caused foliar symptoms. However, the severity of these symptoms was significantly lower than under non-saline or moderate saline conditions (Table S7). Thus, similar to its effect on RDW, Pythium Cluster B2a sp. was less effective at 14 g L⁻¹ NaCl. In contrast, P. deliense was not affected by salinity, with no significant differences in symptom severity across the three salinity levels (Table S7).

Finally, at 15 dai, plant mortality and the rate of colonization of RO crowns were recorded. Inoculation with Pythium Cluster B2a sp. did not cause any plant death. In contrast, the mortality rates of P. deliense were 27.5%, 22.5%, and 30% at 0, 7, and 14 g L⁻¹ NaCl, respectively. Pythium Cluster B2a sp. was recovered from 52.5% (21/40), 47.5% (19/40), and 27.5% (11/40) of the crowns at 0, 7, and 14 g L⁻¹ NaCl, respectively. P. deliense was recovered from 77.5% (31/40), 70% (28/40), and 67.5% (27/40) of the crowns at 0, 7, and 14 g L⁻¹ NaCl, respectively.

3.4. In Vitro Growth of Pythium spp. at Different Temperatures and Salt Concentrations

The ability of the two Pythium spp. isolates to grow in response to salinity and temperature was tested on CMA plates (Figure 8, Figure 9, Figure 10 and Figure 11). No growth was observed at 40 °C. In both Pythium species, a significant (p ≤ 0.001) effect of temperature and salinity, as well as their interaction, was observed (Tables S8 and S9).

For both isolates, the highest daily growth rate was recorded at 7 g L⁻¹ (16.6 ± 1.3 and 22.3 ± 2.3 mm day⁻¹ for Pythium Cluster B2a sp. and P. deliense, respectively; Figure 8 and Figure 9) and at 30 °C (17.9 ± 1.3 and 28.5 ± 1.1 mm day⁻¹ for Pythium Cluster B2a sp. and P. deliense, respectively; Figure 10 and Figure 11).

Pythium Cluster B2a sp. was more sensitive to salt and high temperatures than P. deliense. In fact, the daily growth rate of Pythium Cluster B2a sp. decreased with increasing salt concentration (Figure 8) and at the highest temperature (35 °C) (Figure 10). However, at 10 °C, daily growth of Pythium Cluster B2a sp. was nearly double that of P. deliense (4.12 ± 0.46 and 2.15 ± 0.25 mm; Figure 10 and Figure 11).

On the other hand, P. deliense was more tolerant of salt. Although it showed the highest daily growth rate at 7 g L⁻¹, higher salt concentrations did not negatively affect its growth. At 14 g L⁻¹, daily growth rate remained significantly higher (20.5 ± 2.1 mm day⁻¹) than in salt-free conditions. Daily growth rate at 0 g L⁻¹ (15.5 ± 1.8 mm day⁻¹) and 21 g L⁻¹ (16.0 ± 1.8 mm day⁻¹) was comparable (Figure 9). Furthermore, this isolate showed a faster growth at 35 °C (27.7 ± 1.5 mm day⁻¹) than at 25 °C (22.9 ± 0.9 mm day⁻¹) (Figure 11).

4. Discussion

In this work, we present a study in which we examined the effect of salt (0, 7, and 14 g L⁻¹ NaCl) on the severity of a root rot of RO grown in hydroponics in a floating raft system after inoculation with two pathogenic isolates belonging to the genus Pythium.

The identification of the isolates to the species level involved both a morphological and molecular approach. For isolate 8MC, a key feature in the identification is the habitus of sexual reproductive structures. In particular, the bending of the oogonial stalk towards the antheridium, a typical feature of Pythium deliense [11], was frequently observed (Figure 2a–c).

Pythium deliense is closely related to P. aphanidermatum; both species belong to the Pythium Clade A [12,15]. Both species share many morphological features, such as swollen filamentous sporangia, smooth terminal oogonia, and aplerotic oospores, making them nearly indistinguishable based on morphology only. Much larger oogonia and oospores were observed than the diameters recorded by van der Plaats-Niterink [11] and Meurs [45]. Variable size of oogonia and oospores has also been reported by other authors for both structures [46,47], indicating a degree of intraspecific variability.

In our work, DNA barcoding using ITS, COI, and COII sequences revealed that ITS was not informative, as several strong matches were found with P. aphanidermatum and P. deliense. Indeed, ITS barcoding for Oomycota is known to be less discriminative than COI or COII [15,37]. BLASTn search with the COI sequence of isolate 8MC yielded a 100% identity match with a P. deliense isolate (OP622276) recently found in the United States, causing root rot on sugar beet [48]. Phylogenetic analysis with the concatenated sequences of ITS and COI placed isolate 8MC within the Pythium Clade A, but more closely related to P. deliense than P. aphanidermatum (Figure 4). Therefore, based on the combined results, we identified isolate 8 MC as P. deliense.

Morphological observations did not reveal any distinctive features in the recognition of Pythium isolate 1BMR, mainly due to the poor differentiation of sexual reproductive structures. BLASTn search for ITS, COI, and COII sequences showed high similarity with several species, such as P. coloratum, P. diclinum, and P. dissotocum, which all belong to the Pythium Cluster B2a [15]. Particularly, ITS was 100% identical to all the above-mentioned species. Similar to isolate 8MC, oogonia were larger (26.0 ± 0.4 µm) than the mean size measured for P. coloratum (22.7 µm), P. diclinum (20.5 µm), and P. dissotocum (22.5 µm) [11], although for P. coloratum, the size of oogonia reached up to 26 µm, which corresponded to the mean size measured in 1BMR. In addition, the diameter of encysted zoospores of isolate 1BMR (12.4 ± 0.2 µm) was closer to that of P. coloratum (10–12 µm) than that of P. diclinum (6–7 µm) and P. dissotocum (8–9 µm) [11]. All three species produced aplerotic oospores, like isolate 1BMR. However, the colony pattern on CMA of P. coloratum is often yellowish or slightly violet [11], which was not observed in our case.

Phylogenetic analysis placed isolate 1BMR within the Pythium Cluster B2a (Clade B) (Figure 4) [15]. However, combining morphological and molecular analyses, an unambiguous species identification of isolate 1BMR was not possible. We concluded only that this isolate belongs to the Pythium Cluster B2a, a species complex that has remained unresolved in several studies [49,50,51,52,53,54].

Both Pythium species are polyphagous, soil-borne pathogens [11]. Pythium deliense is restricted to warmer regions and it has been reported not only in soil but also in recirculating water systems and hydroponic cultivations [55,56,57]. Pythium Cluster B2A, which includes P. coloratum, P. diclinum, P. cf. dictyosporum, P. dissotocum, P. lutarium, P. sp. ‘Group F’ and P. sp. ‘tumidum’, is particularly important under warm temperatures on lettuce and other vegetables. This species complex has been reported in soil and irrigation water, but occurs predominantly in hydroponics [49,50,51,52,53,54,58,59,60].

Pythium spp. are among the most well-known and damaging pathogens of all Oomycota in soilless cropping systems. Root rot diseases are a major problem in hydroponic systems because infections produce motile zoospores that spread through the nutrient solution [61,62]. The emergence of Pythium root rot caused by thermophilic Pythium species in hydroponically-grown RO plants could be the result, in part, of rising temperatures associated with climate change [63].

Pathogenicity assays revealed differences between the two Pythium species. Both factors (inoculum and salinity) had a significant effect on root dry weight (RDW) of RO plants (Figure 5). P. deliense caused a dramatic reduction of root biomass, and the impact of inoculum was significant at all the salinity levels tested. Thus, the virulence of P. deliense was not influenced by salinity, and it was recovered from 67.5% of RO crowns at 14 g L⁻¹. In contrast, Pythium Cluster B2a sp. was not effective at the highest salinity tested (14 g L⁻¹) (Figure 5), indicating increased salt sensitivity, although it was still isolated in 27.5% of RO crowns. Data on disease severity caused by the two Pythium species (MKI) followed a similar trend to the RDW data, for both tested factors (inoculum and salinity) (Figure 7).

These results indicate that Pythium Cluster B2a sp. is less virulent and more sensitive to high doses of NaCl than P. deliense. Root rot from zoospore inoculum of Pythium Cluster B2a sp. did not occur at 14 g L⁻¹ even if the pathogen retained the ability to partially colonize the plant tissues. These findings suggest that zoospores of Pythium Cluster B2a sp. are more sensitive to salinity compared to those of P. deliense and that the effects of salinity on pathogen fitness vary greatly among species [43,64,65].

In vitro, P. deliense grows more rapidly at 7 and 14 g L⁻¹ NaCl than in the absence of salt, further supporting its higher salt tolerance (Figure 9). Pythium Cluster B2a sp., on the other hand, slowed its in vitro growth at 14 g L⁻¹ (Figure 8). One hypothesis is that Pythium Cluster B2a sp. may exhibit reduced virulence at the highest salt concentration tested, although it did not lose the ability to colonize host tissues, since it was recovered from 30% of RO crowns.

Our results agree with several studies on the interactions between salinity and other pathogenic Pythium species, which have shown that disease severity increases with increasing salinity. Examples include P. aphanidermatum [43,64] and P. ultimum [66]. Pythium spp. have been reported to cause increased disease in soybean and rice seedlings with increasing soil salinity [65].

Studies on several Pythium species have shown that they are tolerant to salinity. However, the effects of salinity varied both between and within species. Pythium aphanidermatum, P. spinosum, and P. splendens exhibited increased or unaffected growth under saline conditions [43]. Rasmussen and Stanghellini [64] observed a slight increase in mycelial growth rate of P. aphanidermatum, P. dissotocum, and P. catenulatum at low salinity levels, while mycelial growth decreased at higher salt concentrations.

Biochemical and physiological responses of halophytic plants to salt stress may enhance their defense mechanisms against pathogens. The production of reactive oxygen species (ROS) is one of the major factors overlapping the salt and biotic stress response pathways [67]. Under abiotic stress conditions, such as salt stress, the apoplast and cell membranes often undergo localized bursts of ROS release [68]. This phenomenon, like the oxidative burst during pathogen infection, may act as a first line of defense against invading pathogens by directly damaging microbial cells. Reactive oxygen species trigger modifications of cell wall components, creating a physical barrier that may limit pathogen entry [69], such as callose deposition [70,71]. Some halophytes such as Atriplex lentiformis can switch from C3 to C4 carbon assimilation pathways during salt stress, an adaptation likely aimed at reducing ROS damage [72]. Furthermore, salt stress activates hormonal pathways that are also involved in plant immune responses, such as the production of salicylic acid, which promotes systemic acquired resistance in plants [73,74,75,76]. Salicylic acid is involved in the salt-resistance of the halophyte plant species Solanum chilense [77].

The combination of salt stress and pathogen attacks in glycophytes (i.e., salt-susceptible plants) often exacerbates symptom development [78]. Salinity stress increased the severity of Phytophthora root rot by promoting zoospore attachment to roots and/or inhibiting host defenses. Root rot of citrus caused by Phytophthora parasitica increased with increased soil salinity [79,80]. Consistent with our results, Sánchez-Montesinos et al. [81] found no differences in symptom development when P. ultimum was inoculated on melon seedlings under both saline and non-saline conditions. On the other hand, in the halophyte Cakile maritima, a reduced infection by Alternaria alternata was observed under saline conditions [82].

Temperature is an important factor in the incidence and severity of root rot diseases in hydroponic vegetable production [64,83]. Once root rot diseases are established, farmers have few options for controlling the pathogens responsible for these diseases [59,83]. In hydroponic farms, Pythium root rot diseases were found to be prevalent in warm climates when the nutrient solution temperature was 20 °C or higher but were not observed when temperatures remained below 18 °C [84].

Our two Pythium species, regardless of salinity, exhibited different temperature-dependent growth patterns. Pythium Cluster B2a sp. grew more rapidly than P. deliense at lower temperatures (10, 15, and 20 °C). At a temperature of 25 °C, both showed a comparable daily growth rate. In contrast, at higher temperatures (30 and 35 °C), P. deliense demonstrated significantly faster growth than Pythium Cluster B2a sp. (Figure S1). The severity of root rot disease could be partially explained by the optimal growth temperatures of the Pythium species involved in the study, as described elsewhere [65].

In addition, the development of root rot symptoms and the severity of the disease were more pronounced in the case of P. deliense, with symptoms appearing within a few days after inoculation. This Pythium species caused plant mortality rates of 27.5%, 22.5%, and 30% at 0, 7, and 14 g L⁻¹ NaCl, respectively. In contrast, disease progression for Pythium Cluster B2a sp. was slower and did not cause any plant death (Figure S2).

A similar pattern was observed in a study on hydroponically grown spinach: plants died within 3–4 days after inoculation with P. aphanidermatum at root zone temperatures above 23 °C. When spinach plants were inoculated with P. dissotocum at nutrient solution temperatures below 23 °C they wilted but did not die [59].

Given the importance of Pythium root rot in hydroponic crops, it is strategically important to understand the biology of these pathogens and the environmental conditions that promote disease outbreaks. This study on Pythium root rots of RO offers useful insights for future research focused on the management of Pythium root rot diseases in hydroponic cultivation of halophytic species.

5. Conclusions

To the best of our knowledge, this study represents the first report of Pythium spp. affecting RO (A. hortensis var. rubra). Understanding the behavior of plant pathogens under saline conditions is essential for the proper exploitation of halophytic crops. The presence of salt emerged as a critical factor in determining whether halophytes exhibit tolerance or susceptibility to pathogens, an aspect that warrants greater attention.

The results presented here clearly demonstrated that Pythium root rot in RO grown hydroponically was more severe under saline conditions. The observed increase in disease severity may have resulted from increased tissue susceptibility and/or inhibition of root growth and regeneration. These effects, combined with the ability of Pythium spp. to tolerate high levels of salinity, could undermine the salt tolerance traits of RO.

Given that temperature plays a critical role for both the pathogen and the host plant, the ability to regulate environmental temperature to the minimum threshold required for plant growth could serve as a valuable disease management strategy for mitigating damage caused by Pythium root rot pathogens in RO.

Our experiments were conducted under controlled conditions in a growth chamber, which however was designed to reproduce a greenhouse scenario as closely as possible, thus providing important information that can be used in future greenhouse experiments. A holistic approach to the performance of RO grown hydroponically in greenhouses with saline water will require studying the influence of other factors, including dynamic environmental parameters, the composition of nutrient solution, and natural inoculum of Pythium spp.