New Reports of Phytophthora Species in Plant Nurseries in Spain

Abstract

The plant nursery industry has become an ideal reservoir for Phytophthora species and other soilborne pathogens. In this context, isolation from tissues and soil of ornamental and forest plants from nurseries in four regions of Spain was carried out. A high diversity of Phytophthora species was confirmed. Fourteen Phytophthora phylotypes (P. cactorum, P. cambivora, P. cinnamomi, P. citrophthora, P. crassamura, P. gonapodyides, P. hedraiandra, P. nicotianae, P. niederhauserii, P. palmivora, P. plurivora, P. pseudocryptogea, P. sansomeana, and Phytophthora sp. tropicalis-like 2) were isolated from over 500 plant samples of 22 species in 19 plant genera. Nine species were detected in water sources, two of them (P. bilorbang and P. lacustris) exclusively from water samples. P. crassamura was detected for the first time in Spain. This is the first time P. pseudocryptogea is isolated from Chamaecyparis lawsoniana and Yucca rostrata in Spain.

1. Introduction

Different pests and diseases can affect nursery production, which can turn plants into pathogen vectors [1]. Pathogens affect all plant industry sectors across agriculture, horticulture, forestry, and amenity, and they can have a significant impact on yield, market access, sustainability of production, food security, and product integrity [2]. Fungi is considered the kingdom with the largest number of phytopathogenic species [3]. In addition to this, the kingdom Straminipila embraces other important plant pathogens such as Phytophthora and Pythium [3]. Phytophthora species are responsible for large losses of nursery stock throughout the world [4,5,6,7,8,9,10,11].

Phytophthora is one of the most destructive genera which includes currently over 150 known species and about 100 more that are in the process of being described [11,12,13,14]. Almost all Phytophthora species are ecologically and economically important plant pathogens worldwide, some of them with a broad host range. Phytophthora species possess wide environmental adaptation that ranges from terrestrial to aquatic habitats. Some species, such as Phytophthora infestans (Mont.) de Bary, have been responsible for some of the most important epidemics in history, others such as Phytophthora cinnamomi Rands and Phytophthora ramorum Werres, de Cock and Man in ’t Veld, disrupt and diminish biodiversity in natural ecosystems [15,16,17,18,19,20,21]. Others such as Phytophthora citrophthora (R.E. Smith and E.H. Smith) Leonian, Phytophthora nicotianae Breda de Haan, Phytophthora hedraiandra de Cock and Man in ’t Veld, Phytophthora niederhauserii Z.G. Abad and J.A. Abad and P. ramorum, produce major losses in the nursery industry worldwide [7,11,14,22,23,24,25,26,27].

The inocula of Phytophthora spp., which cause foliar as well as root diseases, can increase from low to high levels within a few days or weeks under favourable conditions [28]. Polycyclic diseases can turn into serious epidemics when environmental conditions favour a rapid production of Phytophthora propagules [5]. The movement of plants and plant products between biogeographical zones due to human activity constitutes the leading pathway for the introduction of pathogens and exotic pests [3,29,30].

Phytophthora in its centre of origin does not necessarily constitute an ecological problem or even noticeable because the binomial pathogen–host has co-evolved [28]. Phytophthora native hosts have developed specific defences which confer some tolerance against the pathogen [11,29,31,32]. Nevertheless, when the pathogen is transferred to a new habitat with favourable conditions, it can likely extend to a wide range of new hosts causing serious ecological and economical losses [33,34]. The arrival of new genotypes, lineages, or exempt mating types into a non-native habitat can pose an additional risk to the ecosystem and possibly drive host range expansion for that species [11,23,29,33,34].

Invasive pathogens have been causing damage to native plant communities, woodlands, and landscapes on a global scale for over a century [29,35]. Nursery trade encourages, unintentionally, the dispersal and establishment of invasive and exotic Phytophthora spp. [3,11,36,37]. Even more, the high specialisation and intensification of nursery production favours the reproduction and hybridisation of invasive species enhancing the dispersion and settlement of these on natural ecosystems [29]. The diversity of the genus has increased rapidly in the last decades due to the appearance of new alien species such as Phytophthora alni subsp. alni Brasier and Kirk, Phytophthora austrocedri Greslebin and Hansen, Phytophthora foliorum Donahoo and Lamour, P. hedraiandra, Phytophthora kernoviae Brasier, Beales and Kirk, Phytophthora lateralis Tucker and Milbrath, P. pinifolia Durán, Gryzenh. and Wingf., Phytophthora pluvialis Reeser, Sutton and Hansen or P. ramorum, which requires routine samplings for their early detection, and due to the numerous surveys on unexplored habitats, such as water reservoirs [34,38].

In Spain, since the first report of P. ramorum in 2002 [39,40], surveys have been carried out in ornamental nurseries, garden centres, public gardens, and forest masses to detect and eradicate this pathogen. These surveys have shown that other species of Phytophthora affect many ornamental plants, posing a risk also to nurseries and natural ecosystems [9,41].

Due to the increasing threat of invasive Phytophthora species, and the high risk of hybridisation, a survey was carried out on producing woody and/or ornamental plant nurseries to investigate the presence of Phytophthora and soilborne fungi present and to exclude the presence of quarantine pathogens. Therefore, the aim of this investigation was to identify Phytophthora species and fungal pathogens in ornamental/forest nurseries in different geographical areas of Spain, which could be a threat to the plant nursery industry and to managed and natural ecosystems.

2. Materials and Methods

2.1. Study Sites

Surveys were conducted in 25 Spanish nurseries located in four geographically different regions during the period 2012–2014 in Catalonia (provinces of Barcelona, Girona, Tarragona, and Lleida), Comunidad Valenciana (provinces of Alicante, Castellón, and Valencia), Extremadura (province of Cáceres), and Basque Country (province of Guipúzcoa) (Figure 1).

In ornamental nurseries, only symptomatic plants were collected, whereas in forest nurseries for habitat restoration non-symptomatic plants were also collected. Foliar symptoms (leaf blotch, blight, chlorosis, defoliation), wilting, dieback, growth reduction, cankers with or without gummosis, rot and presence of dead plants were considered symptoms associated to possible Phytophthora or soilborne pathogen infection (Figure 2). These plants presented in most cases root rot and/or loss of the feeder roots with the presence of necrotic lesions (Figure 2). Plant samples were collected together with their pot media or soil, individually stored in labelled plastic bags, and kept in cold conditions until they were processed in the laboratory at the Instituto Agroforestal Mediterráneo, Universitat Politècnica de València (IAM-UPV). In total, 78 samples were collected from 10 nurseries in Catalonia, 343 samples from 5 nurseries in Comunidad Valenciana, 110 samples from 8 nurseries in Extremadura, and 16 samples from 2 nurseries in Basque Country. Each sample consisted of one plant.

Thirteen water samples from recirculating irrigation ponds were also collected during the survey in Catalonia and from one nursery located in the Comunidad Valenciana. Ten litres of water were filtered using three cellulose membranes (5.0 µm pore diameter, Millipore Corporation), which were placed in sterile Petri dishes, sealed with parafilm, labelled, and stored in a cooler during transport to the laboratory. Furthermore, while water sampling was being performed, two additional samples consisting of five leaves showing Phytophthora-like spots floating in two of the surveyed water ponds, were collected in Catalonia; these were labelled and transported for processing in the laboratory.

2.2. Isolation from Plant Tissues, Soil, and Water

Plant samples (leaves and/or roots) were separated from substrate media, and the roots were washed and kept for 24 h in tap water that was repeatedly renewed for oxygenation. Samples were superficially disinfected spraying alcohol at 70% for oomycete isolation and disinfected for 1 min in a 1.5% sodium hypochlorite solution and washed twice with sterile distilled water for fungi isolation [25,42]. Small fragments from the lesion edge were plated on semi-selective media for isolation of oomycetes (CMA-PARPB supplemented or not with hymexazol [43]). Plates were incubated at 20 °C in the dark for 3–5 days for fungi and up to 7 days for oomycetes. All the colonies grown on isolation media were transferred to PDA plates and incubated at 20 °C in darkness for 7 days for identification. Pure cultures of all putative Phytophthora isolates were obtained by transferring single hyphal tips to PDA plates.

The soil removed from each plant sample was baited using Granny Smith apples targeting oomycetes species isolation [5]. Four 10 mm-diameter and 1–1.5 cm-deep holes were made on the apple fruit with a cork borer, each one was filled with the soil sample, saturated with distilled water, sealed with adhesive tape, and incubated at room temperature until lesions appeared (4–7 days). Small tissue fragments from lesion edges were plated on CMA-PARPB with and without hymexazol and incubated at 20 °C in darkness. Each colony was transferred to PDA and incubated as described above for plant samples.

Oomycetes isolation from filtered water samples was undertaken also by apple baiting; three longitudinal flap-like cuts (one per membrane filter) were made on a Granny Smith apple. In each flap cut, half of the subsample membrane was placed, sealed with parafilm, and incubated at room temperature until symptoms develop (4–7 days). The re-isolation from the apple was performed from the edge of any lesions that developed after incubation following the protocol described above.

2.3. Identification

Molecular Identification

DNA from Phytophthora and Pythium isolates was extracted from pure cultures grown on PDA by scraping the mycelium and mechanically disrupting it by grinding to a fine powder under liquid nitrogen, using the EZNA Plant Miniprep Kit (Omega Bio-tek, Doraville, GE, USA) following the manufacturer’s instructions.

Nuclear ribosomal DNA ITS amplifications were carried out using the universal primers ITS4 and ITS6 that target conserved regions in the 18S and 28S rDNA genes [44,45]. All PCR reactions were performed using HotBegan™ Taq DNA Polymerase (Canvax Biotech SL, Córdoba, Spain), according to the manufacturer’s instructions, in a PTC 200 thermo-cycler (MJ Research, Waltham, MA, USA) with the following parameters: 94 °C for 3 min; 35 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 45 s; and 72 °C for 10 min. Amplified products were purified and sequenced by Macrogen (Amsterdam, The Netherlands).

The isolates were identified to the species level by conducting Basic Local Alignment Search Tool (BLAST) searches with the sequence data on international collection databases (Phytophthora Database, PhyID, and GenBank) and a customised database that included the new Phytophthora species described and segregated from Phytophthora complexes and provisional taxons. An isolate was assigned to a species when the identity was above the 99% cut-off in respect to the ex-type isolates. The ITS sequence did not resolve the identity of 10 isolates. Therefore, for these isolates the mitochondrial cytochrome c oxidase I (COI) region was amplified using the primers OomCoxI-Levup and Fm85mod [46].

The DNA sequences from this study (

Table 1. Phytophthora isolates identified in this study.

Species	Strain Number	Host	Location	GenBank Accession Numbers
				ITS	COI
P. bilorbang	PS-1420	Nursery water pond	Lleida, Spain	MW314333	-
P. cactorum	PS-1463	Photinia x fraseri “Red Robin” (roots)	Barcelona, Spain	MW314274	-
	PS-1411	Nursery water pond	Girona, Spain	MW314275	-
	PS-1417	Nursery water pond	Lleida, Spain	MW314276	-
	PS-1447	Photinia x fraseri “Red Robin” (roots)	Barcelona, Spain	MW314277	-
	PS-1396	Nursery water pond	Barcelona, Spain	MW314278	-
	PS-1395	Nursery water pond	Barcelona, Spain	MW314279	-
	PS-1445	Nursery water pond	Barcelona, Spain	MW314280	-
	PS-1428	Photinia x fraseri “Red Robin” (roots)	Barcelona, Spain	MW314281	-
	PS-1397	Nursery water pond	Barcelona, Spain	MW314282	-
	PS-1400	Nursery water pond	Barcelona, Spain	MW314283	-
P. cactorum	PS-1600	Fagus sylvatica (soil)	Guipúzcoa, Spain	MW314287	-
	PS-1393	Nursery water pond	Barcelona, Spain	MW314286	-
	PS-1399	Nursery water pond	Barcelona, Spain	MW314285	-
	PS-1444	Nursery water pond	Barcelona, Spain	MW314284	-
P. cambivora	PS-1394	Nursery water pond	Barcelona, Spain	MW314353	-
	PS-1392	Nursery water pond	Barcelona, Spain	MW314354	-
	PS-1389	Nursery water pond	Barcelona, Spain	MW314356	-
	PS-1599	Quercus ilex (roots)	Valencia, Spain	MW314355	-
P. cinnamomi	PS-1448	Arbutus unedo (roots)	Barcelona, Spain	MW314360	-
	PS-1470	Arbutus unedo (roots)	Barcelona, Spain	MW314357	-
P. cinnamomi	PS-1602	Pseudotsuga menziesii (roots)	Guipúzcoa, Spain	MW314358	-
	PS-1603	Pinus radiata (roots)	Guipúzcoa, Spain	MW314359	-
P. citrophthora	PS-1419	Nursery water pond	Lleida, Spain	MW314301	-
	PS-954	Quercus faginea (soil)	Castellón, Spain	MW314302	-
	PS-1407	Nursery water pond	Barcelona, Spain	MW314303	-
	PS-1418	Nursery water pond	Lleida, Spain	MW314304	-
	PS-1426	Escallonia sp. (soil)	Barcelona, Spain	MW314305	-
	PS-1429	Picea pungens “Glauca Globosa” (roots)	Girona, Spain	MW314306	-
	PS-955	Quercus faginea (roots)	Castellón, Spain	MW314307	-
	PS-1474	Rosmarinus sp. (roots)	Tarragona, Spain	MW314308	-
	PS-1437	Citrus sinensis (soil)	Tarragona, Spain	MW314309	-
P. crassamura	2308	Pinus pinea (soil)	Cáceres, Spain	MW314336	-
	2309	Pinus pinea (soil)	Cáceres, Spain	MW314335	-
P. crassamura	2310	Pinus pinea (soil)	Cáceres, Spain	MW314334	-
P. gonapodyides	PS-1415	Nursery water pond	Girona, Spain	MW314338	-
	PS-1434	Juniperus hibernicus (roots)	Lleida, Spain	MW314337	-
	PS-1410	Nursery water pond	Girona, Spain	MW314342	-
	PS-1414	Nursery water pond	Girona, Spain	MW314339	-
	PS-1423	Leaves floating in nursery water pond	Barcelona, Spain	MW314341	-
	PS-972	Quercus ilex (soil)	Valencia, Spain	MW314340	-
	PS-1409	Nursery water pond	Girona, Spain	MW314344	-
	PS-1402	Nursery water pond	Barcelona, Spain	MW349609	-
	PS-1413	Nursery water pond	Girona, Spain	MW314343	-
P. hedraiandra	PS-1449	Quercus ilex (soil)	Cáceres, Spain	MW314288	-
	2300	Quercus ilex (soil)	Cáceres, Spain	MW314290	-
	2301	Quercus ilex (soil)	Cáceres, Spain	MW314293	-
P. hedraiandra	PS-1480	Juniperus phoenicia (soil)	Alicante, Spain	MW314289	-
	PS-1427	Viburnum tinus (collar)	Barcelona, Spain	MW314291	-
	PS-1422	Viburnum tinus (roots)	Girona, Spain	MW314292	-
	PS-1421	Viburnum tinus (roots)	Barcelona, Spain	MW314294	-
P. lacustris	PS-1408	Nursery water pond	Barcelona, Spain	MW314345	-
	PS-1424	Leaves floating in nursery water pond	Barcelona, Spain	MW314346	-
	PS-1412	Nursery water pond	Girona, Spain	MW314347	-
	PS-1401	Nursery water pond	Barcelona, Spain	MW314348	-
	PS-1446	Nursery water pond	Girona, Spain	MW314349	-
	PS-961	Nursery fountain	Valencia, Spain	MW314350	-
	PS-1404	Nursery water pond	Barcelona, Spain	MW314351	-
P. lacustris	PS-1403	Nursery water pond	Barcelona, Spain	MW314352	-
P. nicotianae	PS-1441	Pistacia lentiscus (roots)	Barcelona, Spain	MW314298	-
	PS-1425	Escallonia sp. (roots)	Barcelona, Spain	MW314299	-
	PS-1439	Citrus sinensis (roots)	Tarragona, Spain	MW314297	-
	PS-1435	Rosmarinus sp. (roots)	Tarragona, Spain	MW314295	-
	PS-1442	Myrtus communis “Tarentina” (roots)	Barcelona, Spain	MW314296	-
	PS-1443	Buxus sempervirens (roots)	Barcelona, Spain	MW314300	-
P. niederhauserii	PS-1606	Arbutus unedo (soil)	Cáceres, Spain	MW314361	-
P. palmivora	PS-1465	Pistacia lentiscus (roots)	Barcelona, Spain	MW314330	-
	PS-1475	Cupressus sempervirens (roots)	Tarragona, Spain	MW314331	-
	PS-1416	Nursery water pond	Lleida, Spain	MW314332	-
P. plurivora	1978	Quercus faginea (roots)	Castellón, Spain	MW314325	-
P. plurivora	PS-1405	Nursery water pond	Barcelona, Spain	MW314314	-
	PS-1432	Chamaecyparis lawsoniana “Elwoodii” (soil)	Girona, Spain	MW314326	MW314042
	PS-1476	Cupressus sempervirens (roots)	Tarragona, Spain	MW314327	MW314043
	PS-957	Quercus faginea (soil)	Castellón, Spain	MW314315	-
	PS-959	Quercus faginea (roots)	Castellón, Spain	MW314316	-
	PS-1431	Chamaecyparis lawsoniana “Elwoodii” (soil)	Girona, Spain	MW314317	-
	PS-1390	Nursery water pond	Barcelona, Spain	MW314318	-
	PS-1391	Nursery water pond	Barcelona, Spain	MW314319	-
	PS-958	Quercus faginea (roots)	Castellón, Spain	MW314320	-
	PS-960	Quercus faginea (roots)	Castellón, Spain	MW314321	-
	PS-969	Juniperus sp. (soil)	Valencia, Spain	MW314322	-
P. plurivora	PS-971	Quercus ilex (roots)	Valencia, Spain	MW314323	-
	2213	Juniperus chinensis “Expansa Variegata” (roots)	Barcelona, Spain	MW314324	-
P. pseudocryptogea	PS-1469	Ilex x meserveae “Blue Maid” (roots)	Barcelona, Spain	MW314362	-
	PS-1450	Chamaecyparis obtusa “Nana gracilis” (roots)	Barcelona, Spain	MW314363	-
	PS-1430	Chamaecyparis lawsoniana “Elwoodii” (roots)	Girona, Spain	MW314364	-
	PS-967	Quercus ilex (soil)	Valencia, Spain	MW314365	-
	PS-966	Quercus ilex (roots)	Valencia, Spain	MW314366	-
	PS-1406	Nursery water pond	Barcelona, Spain	MW314367	-
	PS-1436	Yucca rostrate (collar)	Tarragona, Spain	MW314368	-
	PS-1466	Chamaecyparis lawsoniana “Elwoodii” (roots)	Girona, Spain	MW314369	-
P. pseudocryptogea	PS-970	Quercus ilex (soil)	Valencia, Spain	MW314370	-
	PS-964	Quercus ilex (roots)	Valencia, Spain	MW314371	-
	PS-978	Quercus ilex (soil)	Valencia, Spain	MW314372	-
	PS-973	Quercus ilex (soil)	Valencia, Spain	MW314373	-
	PS-962	Quercus ilex (roots)	Valencia, Spain	MW314374	-
	PS-965	Quercus ilex (roots)	Valencia, Spain	MW314376	-
	PS-968	Quercus ilex (soil)	Valencia, Spain	MW314377	-
	PS-1398	Nursery water pond	Barcelona, Spain	MW314375	-
	PS-1605	Quercus ilex (soil)	Cáceres, Spain	MW314378	-
P. sansomeana	PS-974	Quercus ilex (soil)	Valencia, Spain	MW314379	-
	PS-979	Quercus ilex (soil)	Valencia, Spain	MW314380	MW314044
P. sansomeana	PS-980	Quercus ilex (roots)	Valencia, Spain	MW314381	MW314045
Phytophthora sp. tropicalis-like 2	PS-1433	Juniperus hibernicus (soil)	Lleida, Spain	MW314329	MW314040
	PS-1604	Arbutus unedo (roots)	Cáceres, Spain	MW314328	MW314041
Phytophthora sp. 1 clade 2	PS-1477	Citrus sinensis (soil)	Tarragona, Spain	MW314310	MW314036
	PS-1460	Citrus sinensis (soil)	Tarragona, Spain	MW314311	MW314037
	PS-1478	Citrus sinensis (roots)	Tarragona, Spain	MW314312	MW314038
	PS-1479	Citrus sinensis (roots)	Tarragona, Spain	MW314313	MW314039

), together with those of reference species of each clade retrieved from Genbank, were aligned using the ClustalW algorithm [47] contained within the MEGA X software package [48]. The sequences of the reference isolates were selected from ex-type or well-authenticated Phytophthora species recommended in IDphy: molecular and morphological identification of Phytophthora (https://idtools.org/id/phytophthora/molecular.php (accessed on 15 February 2021).). The alignments were inspected and corrected manually. Incomplete portions at either end of the alignments were excluded prior to analyses.

Phylogenetic analyses were based on Bayesian inference (BI), maximum likelihood (ML), and maximum parsimony (MP). Bayesian analyses were performed using MrBayes v 3.2.6 on the NGPhylogeny.fr web service [49]. Four simultaneous analyses were run for 100,000 generations, sampling every 10,000, with four Markov chain Monte Carlo (MCMC) chains. The first 25% of saved trees were discarded and posterior probabilities were determined from the remaining trees. The ML analyses were completed with the tool Randomized Axelerated Maximum Likelihood (RAxML) implemented on the T-REX web server (http://www.trex.uqam.ca/ (accessed on 11 July 2022).) [50]. ML tree searches were performed under the generalised time-reversible with gamma correction (GTR + Γ) nucleotide substitution model using 1000 pseudoreplicates. The other parameters were used as default settings. MP analyses were performed in MEGA X [48] with the Tree Bisection and Reconnection (TBR) algorithm, where gaps were treated as missing data. The robustness of the topology was evaluated using 1000 bootstrap replications [51]. Measures for the maximum parsimony such as tree length (TL), consistency index (CI), retention index (RI), and rescaled consistency index (RC) were also calculated.

2.4. Conservation of Phytophthora and Pythium Isolates

Pure cultures obtained by hyphal tipping were maintained in the oomycete culture collection at the IAM-UPV. Each isolate was grown on V-8 Juice Agar and incubated at 20 °C for 7 days in darkness. A total of 15 mycelium plugs (6 mm diameter) from the border of the colony were extracted and placed into a 12 cm³ glass flask which contained 1.5% sterile soil extract solution for long-term conservation at 14 °C. The sterile soil extract solution was prepared mixing 100 g of soil with 900 mL distilled water. The mixture was stirred and allowed to stand for 24 h. Subsequently, 50 mL of the supernatant was taken and added to 950 mL of distilled water to be autoclaved.

Phytophthora isolates were also conserved in tubes with Oat Agar medium (72.5 g L⁻¹ oatmeal agar, Sigma Aldrich, Steinheim, Germany) for long-term storage. A single 6 mm-diameter agar disk was placed in each OA tube, incubated at 25 °C until mycelium growth was observed, and then it was sealed with parafilm for conservation at 14 °C.

3. Results

3.1. Symptomatology

In all nurseries, a broad range of symptoms was observed: cankers (with or without gummosis exudates), collar rot, dead plants, dieback (partial dieback or the whole plant), foliar symptoms (chlorosis, defoliation, leaf spots, irregular shaped blotches in the leaf margins or starting at the leaf apex or petiole, necrotic spots), growth reduction, and wilting (Figure 2). Figure 3 shows the percentage distribution of symptoms observed in the sampled plants collected in the nurseries referred to the total number of plants collected (in blue colour) and to the number of plants on which Phytophthora was isolated (in green colour). The most frequent symptoms among the total number of collected samples were dieback (37.1%), followed by foliar symptoms (29.4%) and growth reduction (13.3%).

A total of 547 samples were collected and oomycetes were identified in 30.7% of the plant samples. The most frequent symptoms observed in samples positive for oomycetes were dieback (43.5%), foliar symptoms (28.6%), and growth reduction (11.3%).

Phytophthora was isolated from 59 plants (

Table 2. Phytophthora species isolated from plant tissues, floating leaves from two nursery ponds, and water samples taken from the irrigation system in the surveyed nurseries.

Phytophthora spp.	Host	Nursery	Source	N. Samples	Region
BIL		13		1
CAC	Fagus sylvatica, Photinia “Red Robin”, Pinus pinea	6, 7, 9, 10, 12, 13, 19	R, S, W	14	Cat., Com. Val., Bas. C.
CAM	Quercus ilex	3, 7, 9	R, W	9	Com. Val.
CIN	Arbutus unedo, Pinus radiata, Pseudotsuga menziesii	9, 18	R, S	4	Cat., Bas. C.
CIP	Citrus sinensis, Escallonia sp., Picea pungens “Glauca Globosa”, Quercus faginea, Rosmarinus officinalis	4, 10, 11, 13, 15, 16	R, S, W	9	Cat., Com. Val.
CRA	Pinus pinea	17	S	3	Ext.
HED	Juniperus phoenicea, Q. ilex, Viburnum tinus	5, 7, 8, 10, 17	R, S	7	Cat., Com. Val., Ext.
GON	Juniperus communis “Hibernica”, Q. ilex	2, 8, 9, 10, 12, 13	R, S, L, W	9	Cat., Com. Val.
LAC		2, 8, 10	L, W	8	Cat.
NIC	Buxus sempervirens, Citrus sinensis, Escallonia sp., Myrtus communis “Tarentina”, Pistacia lentiscus, Rosmarinus sp.	7, 10, 15, 16	R	6	Cat.
NIE	Arbutus unedo	24	S	1	Ext.
PAL	Cupressus sempervirens, Pistacia lentiscus	7, 13, 16	R, W	3	Cat.
PLU	Chamaecyparis lawsoniana “Elwoodii”, Cupressus sempervirens, Juniperus chinensis “Expansa”, Q. faginea, Q. ilex	2, 4, 9, 10, 12, 16	R, S, W	14	Cat., Com. Val.
PSC	Chamaecyparis lawsoniana “Elwoodii”, Q. ilex, Yucca rostrata	2, 8, 10, 12,15, 23	R, S, W	17	Cat., Com. Val., Ext.
SAN	Quercus ilex	2	R, S	3	Com. Val.
TRO	Arbutus unedo, Juniperus communis “Hibernica”	13, 23	R	2	Cat., Ext.
SP. 1	Citrus sinensis	16	R	3	Cat.

BIL: P. bilorbang. CAC: P. cactorum. CAM: P. cambivora. CIN: P. cinnamomi. CIP: P. citrophthora. CRA: P. crassamura. HED: P. hedraiandra. GON: P. gonapodyides. LAC: P. lacustris. NIC: P. nicotianae. NIE: P. niederhauserii. PAL: P. palmivora. PLU: P. plurivora. PSC: P. pseudocryptogea. SAN: P. sansomeana. TRO: Phytophthora sp. tropicalis-like 2. SP. 1: Phytophthora sp. 1 clade 2. R: roots. S: soil. L: leaves. W: water from irrigation ponds in the nursery. Cat.: Catalonia. Com. Val.: Comunidad Valenciana. Ext: Extremadura. Bas. C.: Basque Country. Each nursery was coded with a number.

), which means 10.8% of total plant samples collected in this survey. On plants affected by Phytophthora, dieback was the most frequent symptom observed (59.3%), followed by foliar symptoms (27.1%), wilting, and growth reduction (both 6.8%) (Figure 3). In almost all positive Phytophthora plants, the aerial symptomatology corresponded with a damaged root system. Nevertheless, in some plants, the damage was limited to the aerial part, with no visible root symptoms. Furthermore, in only two non-symptomatic plants Phytophthora was isolated.

3.2. Phytophthora Species Isolated in the Study

Seventy-one isolates of Phytophthora were recovered from 18 nurseries from the four locations (Table 2). These isolates were obtained from infected tissues (roots) and/or the rhizosphere soil of 547 plant samples belonging to 22 species included in 19 plant genera (Table 2). Thirty-six Phytophthora isolates were isolated from water samples collected in Catalonia region from the irrigation ponds (Table 2).

Molecular identification of the isolates revealed the presence of 17 Phytophthora phylotypes (Figure 4). The ITS alignment consisted of 887 positions including gaps. Of these, 568 were constant and 270 were parsimony-informative characters. The heuristic search using MP generated the 10 most parsimonious trees (TL = 603, CI = 0.651, RI = 0.934, RC = 0.608), from which one was selected (Figure 4). The topology of the phylogenetic tree inferred by MP analysis was identical to those obtained by the BI and ML analyses; therefore, only the MP tree is presented with MP and ML bootstrap support values and BI posterior probability scores at the nodes (available on request). Sequences from this study were deposited in Genbank (Table 1).

The species isolated were Phytophthora bilorbang Aghighi and Burgess, Phytophthora cactorum (Lebert and Cohn) J. Schr., Phytophthora cambivora (Petri) Buisman, P. cinnamomi, P. citrophthora, Phytophthora crassamura Scanu, Deidda and Jung, P. hedraiandra, Phytophthora gonapodyides (H.E. Petersen) Buisman, Phytophthora lacustris Brasier, Cacciola, Nechwatal, Jung and Bakonyi, P. nicotianae, P. niederhauserii, Phytophthora palmivora E.J. Butler, Phytophthora plurivora T. Jung and T.I. Burgess, Phytophthora pseudocryptogea Safaiefarahani, Mostowfizadeh, Hardy and Burgess and Phytophthora sansomeana Hansen and Reeser. Two Phytophthora isolates recovered from the roots of Arbutus unedo and Juniperus communis were identified as the informally designated taxon Phytophthora sp. tropicalis-like 2 [52]. There were four Phytophthora isolates that could not be identified to the species level, so they were tentatively named as Phytophthora sp. 1 clade 2.

The ITS of Phytophthora sp. 1 clade 2 was closely related to Phytophthora meadii McRae showing differences in two positions with the ex-type, whereas the COI results placed these isolates close to P. citrophthora. Therefore, these isolates were designated as Phytophthora sp. 1 clade 2.

Among plant and soil samples, P. pseudocryptogea was the species with the highest incidence (21.1%), followed by P. plurivora (15.5%), P. hedraiandra (9.9%), P. citrophthora and P. nicotianae (8.5% each species), P. cactorum, P. cinnamomi, and Phytophthora sp. 1 clade 2 (5.6% each species), P. crassamura and P. sansomeana (4.2% each species), P. gonapodyides, P. palmivora, and Phytophthora sp. tropicalis-like 2 (2.8% each species). Two other species, P. cambivora and P. niederhauserii, had the lowest incidence values (1.4% each species). Some plants were co-infected with more than one Phytophthora species. Mixed infections occurred on Chamaecyparis lawsoniana (P. plurivora–P. psedocryptogea), Citrus sinensis (P. citrophthora–Phytophthora sp. 1 clade 2), Cupressus sempervirens (P. palmivora–P. plurivora), Escallonia sp. (P. citrophthora–P. nicotianae), Juniperus communis (P. gonapodyides–Phytophthora sp. tropicalis-like 2), Pistacia lentiscus (P. nicotianae–P. palmivora), Quercus ilex (P. plurivora–P. pseudocryptogea and P. pseudocryptogea–P. sansomeana), and Rosmarinus officinalis (P. citrophthora–P. nicotianae).

In the aquatic habitats, P. cactorum, P. lacustris, and P. gonapodyides were the most abundant species (26.3%, 21.1%, and 18.4% respectively), followed by P. cambivora, P. citrophthora, and P. plurivora (7.9% each species), P. pseudocryptogea (5.3%). The lowest value of presence in water was 2.6%, shared by P. bilorbang and P. palmivora.

3.3. Pythium and Phytopythium Species Isolated in the Study

In total, 6 Phytopythium species (Pp. chamaehyphon, Pp. helicoides, Pp. litorale, Pp. mercuriale, Pp. montanum, and Pp. vexans) and 11 Pythium species (Py. sterilum, Py. intermedium, Py. attrantheridium, Py. rostratifingens, Py. oopapillum, Py. irregulare, Py. ultimum, Py. undulatum, Py. sylvaticum, Py. Pleroticum, and Py. diclinum) were isolated in the survey.

4. Discussion

This study provides evidence of Phytophthora’s wide spread in ornamental and forest nurseries, since the pathogen was isolated from plant material and water samples in the large majority of surveyed nurseries.

In the surveyed nurseries, the sampled plants showed crown symptoms that could be associated with Phytophthora infection, such as dieback, shoot blight, chlorosis, defoliation, irregular leaf blotches, wilting, and cankers with gummosis. The symptomatology of aerial plant parts was generally associated with root damage such as change in colour, lesions, absence, and/or rot of the feeder roots. This set of observed symptoms agree with the symptomatology described in the literature [9,11,25,27,53,54,55,56]. It should be noted that disease symptoms may be suppressed due to prophylactic fungicide treatments or the natural lag period between root and crown rots and the development of foliar symptoms [26].

Seventy-one Phytophthora isolates from clades 1, 2, 4, 6, 7, and 8 were recovered from 22 species included in 19 plant genera. From some of the plants more than one species of Phytophthora was isolated, revealing mixed infections as in previous nursery surveys [9,10,33,54,57]. Some species were very frequent (P. pseudocryptogea, P. plurivora, P. hedraiandra, P. citrophthora, and P. nicotianae) and others were rare (P. cambivora and P. niederhauserii, as well as the informally designated taxon, Phytophthora sp. tropicalis-like 2).

Four Phytophthora species represented a significant finding for the Spanish nursery sector. This study is the first report of P. crassamura in Spain; Phytophthora crassamura sp. nov. was described by Scanu et al. in Sardinia (Italy) [58] and since then it has been isolated from other hosts in Italy and in California [59,60]. As our isolates were baited from the P. pinea nursery substrate, we cannot state P. pinea as a new P. crassamura host, even the three seedlings showed a highly diminished root system with no secondary feeder roots. This finding suggests that probably P. pinea seedlings are susceptible to P. crassamura. As initially these isolates were misidentified as Phytophthora megasperma Dreschsler, no pathogenicity tests were performed. Phytophthora pseudocryptogea [61] was reported on Quercus ilex in 2018 in different regions of Spain and the present study not only confirms its presence in the nurseries from those regions [62,63] but also it was isolated for the first time in Spain on Chamaecyparis lawsoniana and Yucca rostrata. Moreover, this is the first time P. sansomeana was isolated in Europe and in Q. ilex worldwide. Phytophthora sansomeana was segregated from the P. megasperma complex in 2009 and until now it was only in the United States and in China, from diverse forest and agricultural hosts, such as Douglas-fir nursery seedlings, weeds, and soybean [64,65,66]. Since it is not the first time that the species has been identified in nursery material, its pathogenicity on holm oak should be tested to understand the risk it poses to this fundamental tree species of forest ecosystems and landscapes of Mediterranean Europe. Lastly, two isolates from our study clustered with Phytophthora sp. tropicalis-like 2 described by Jung et al. in 2020 based on ITS blast-assigned identity with the isolate VN830 [52]. This is a provisional first report of Phytophthora sp. tropicalis-like 2 on Arbutus unedo and Juniperus communis.

In previous nursery surveys in Spain, P. cactorum, P. cinnamomi, P. citricola, P. citrophthora, P. cryptogea, Phytophthora drechsleri Tucker, Phytophthora hibernalis Carne, Phytophthora multivora Scott and Jung, P. nicotianae, P. niederhauserii, P. palmivora, P. plurivora, Phytophthora syringae (Kleb.) Kleb., Phytophthora tentaculata Kröber and Marwitz, and P. tropicalis have been reported [9,25,67]. According to Moralejo et al., P. cinnamomi and P. cryptogea (probably P. pseudocryptogea) have escaped from nurseries and are currently spreading in Q. ilex forests, and infect associated shrubs such as Arbutus unedo and Cistus monspeliensis in the lowlands of northern Mallorca [25]. Other studies in nurseries worldwide recovered almost the same species which demonstrates that global nursery trade is the main pathway for Phytophthora dispersion [6,9,11,25,26,27,29,33,53,54,56,57,60,68,69,70,71,72].

In Europe, a very extensive analysis of incidence of Phytophthora spp. was conducted, based on data from 23 countries between 1972 and 2013, in order to study the pathway of Phytophthora from nurseries into natural, semi-natural, and horticultural ecosystems [11]. From nursery plant material, 49 Phytophthora taxa were identified, being P. plurivora, P. cinnamomi, P. cactorum, P. nicotianae, P. ramorum and P. citrophthora the most commonly sampled species, considered all alien pathogens in Europe. From forest and landscape plantings, 56 Phytophthora taxa were recovered, and invasive species with wide host ranges, such as P. plurivora, P. cinnamomi, P. nicotianae, P. cryptogea, and P. cactorum, were the most common. This large-scale study demonstrates that Phytophthora infect nursery stock across Europe and the spread of these pathogens through infested nursery stock into natural ecosystems.

In California, Sims et al. [60] reported P. cactorum as the most frequent species in restoration nurseries but P. hedraiandra, P. multivora, P. occultans, P. crassamura, P. thermophila, and P. pseudocryptogea were also isolated. Rooney-Latham et al. [72] reported P. tentaculata, P. cactorum, P. cryptogea complex, P. cambivora, P. cinnamomi, P. citricola, P. hedraiandra, P. megasperma, P. multivora, P. nicotianae, P. niederhauserii, P. parvispora, P. pini, P. plurivora, and P. riparia in Californian nurseries.

Regarding water surveys, the nine species reported in this study once again agree with Phytophthora spp. recovered from irrigation water, waterways, or riparian ecosystems published in other studies [73,74,75,76,77,78,79,80,81,82]. It is not surprising that as Phytophthora is adapted for aquatic dispersal, multiple Phytophthora spp. have been recovered from waterways or irrigation waters. Indeed, several novel species have been detected in the last decade from water fluxes or riparian ecosystems such as Phytophthora lateralis (clade 8) causing Chamaecyparis lawsoniana decline [83], Phytophthora alni (clade 7) causing Alnus spp. decline [84], and P. ramorum (clade 8) causing sudden oak death on Quercus spp. and Notholithocarpus densiflorus [17]. Detection of Phytophthora taxa belonging to clade 6 has increased in recent years as riparian systems have grown in attention [13,77,85,86]. Phytophthora spp. from clade 6 are thought to be adapted to survive in rivers due to their rapid colonisation of leaves and plant debris [87,88]. Jung et al. consider the possibility that species from clade 6 are probable saprotrophs, as these Phytophthora spp. depend on their ability to rapidly colonise fresh plant material (such as fallen leaves) in order to outcompete other saprotrophic organisms [88]. There is a significant gap in understanding waterborne plant pathogens, particularly in open irrigation systems [78,82,89].

Among other plant pathogens that were also isolated, the most important genera were Pythium and Phytopythium. The percentage of recovered Pythium and Phytopythium species highlights the importance of sanitary measures in the nursery industry. Pythium and Phytopythium are also among the most frequent plant pathogens in nurseries (seed rot and damping-off), Pythium species require free water to complete their cycle but compared with Phytophthora, they have a quicker development and growth. Most Pythium and Phytopythium species used to be considered saprotrophs but nowadays the pathogenicity of some species has been demonstrated [90,91,92,93].

The impact that plant pathogens can have on the plant industry can extend into billions of dollars, but the worst is the environmental risk, which biodiversity, forestry, and agriculture are currently experiencing [11,33,94,95]. Biosecurity needs to be the cornerstone of the global nursery trade to avoid the possibility of Phytophthora spp. spreading to new habitats where they may be exposed to compatible species and potentially form new hybrids [29,96,97].

The exclusion of nursery pathogens from forested areas is a critical issue for forest health [60,98]. Monitoring the pathogen zone, restricting vehicle movement from infested to uninfested areas, cleaning vehicles before entering uninfested areas, preventing infested and uninfested soil mixing, preventing water draining from infested to uninfested areas, and education of public and forestry workers are some of the exclusion measures that should come into full force and effect [60,98].

A high priority should be placed on the production of pathogen-free propagating material by appropriate sanitary practices [99]. The microbial community plays an important role in the protective effect against Oomycetes. Organic soils in the form of compost have long been found to supress a number of Phytophthora and Pythium spp. [99]. Nursery sanitation measures such as the following ought be implemented in all nurseries and garden centres: use of new seedling containers, container media pasteurised; irrigation water Phytophthora-free (sand filters or chlorine interventions); water splash kept off leaves and wetness time minimised; containers kept off the ground; suppressive composts or fungicides avoided; sustained heat treatment to kill resting structures in plant or soil material via composting, solarisation, oven treatment or autoclaving, heating installation in greenhouses, correct aeration between seedling benches and plantations, pH control (a low pH [3.5–4.5] to avoid spore liberation), moderate nitrogen fertilisation, and routine tool disinfection [98].

It has long been known that nursery stock is the most common pathway for the introduction of new Phytophthora species into natural habitats worldwide [11]. Supplying healthy plants should be the fundamental principle of nursery production. Implementing molecular detection through the most recent, effective, and specific assays for Phytophthora [33,62,100] will facilitate early detection and the application of control measures to minimise the risk of spread through plant trade.

5. Conclusions

This study confirms the widespread presence of pathogens in plant nursery stocks and the risk posed by the plants for the planting pathway. Seventeen Phytophthora phylotypes were isolated from tissues and rizosphere soil of 22 plant species in 19 genera and from water samples. The presence of Phytophthora mixed infections is noteworthy. It is also relevant reporting, for the first time, the presence of P. crassamura, P. pseudocryptogea, P. sansomeana, and Phytophthora sp. tropicalis-like 2 in the Spanish nursery industry. The need for preventing Phytophthora dispersion to natural ecosystems must be translated in implementing new policies at the global scale. Good biosecurity practices in nurseries and early detection are critical to mitigate the risk of spread of these pathogens.