The Use of qPCR Reveals a High Frequency of Phytophthora quercina in Two Spanish Holm Oak Areas

The struggling Spanish holm oak woodland situation associated with Phytophthora root rot has been studied for a long time. Phytophthora cinnamomi is considered the main, but not the only species responsible for the decline scenario. This study verifies the presence and/or detection of Phytophthora species in two holm oak areas of Spain (southwestern “dehesas” and northeastern woodland) using different isolation and detection approaches. Direct isolation and baiting methods in declining and non-declining holm oak trees revealed Phytophthora cambivora, Phytophthora cinnamomi, Phytophthora gonapodyides, Phytophthora megasperma, and Phytophthora pseudocryptogea in the dehesas, while in the northeastern woodland, no Phytophthora spp. were recovered. Statistical analyses indicated that there was not a significant relationship between the Phytophthora spp. isolation frequency and the disease expression of the holm oak stands in the dehesas. Phytophthora quercina and P. cinnamomi TaqMan real-time PCR probes showed that both P. cinnamomi and P. quercina are involved in the holm oak decline in Spain, but P. quercina was detected in a higher frequency than P. cinnamomi in both studied areas. Thus, this study demonstrates that molecular approaches complement direct isolation techniques in natural and seminatural ecosystem surveys to determine the presence and distribution of Phytophthora spp. This is the first report of P. pseudocryptogea in Europe and its role in the holm oak decline should be further studied.


Introduction
Holm oak (Quercus ilex L.) grows spontaneously throughout the Mediterranean basin, from the Iberian Peninsula to Turkey in the North and from Morocco to Tunisia in the South, having its optimum growing conditions in the Western Mediterranean regions [1].Holm oak is a low nutrient demanding species, which prefers dry soils situated in Spain from sea level up to 2000 m high, although the most dense holm oak forests' altitude ranges from 200 to 800 m.This species is well adapted to Mediterranean xeric conditions, with an early active taproot development and little branching at the expense of shoot development [2].
In Spain, holm oak is the most abundant evergreen Fagaceae tree species, covering almost all Spanish provinces except the Canary Islands and Galicia regions, where it is scarce [1].About 2.8 M ha of the Spanish forestry surface are holm oak woodlands and 2.4 M ha are oak rangelands (henceforth called dehesas) (which consist mainly of holm oaks mixed with cork oaks (Quercus suber L.), and even a deciduous oak (Quercus faginea Lam.)) [3].
Holm oak constitutes a fundamental pillar of the Spanish dehesa, an agro-silvo-pastoral system, benefiting from the use of its fruit mainly for livestock during the autumn season and the grass growing underneath the canopy for grazing.Its wood it is also a valuable asset.In addition, it hosts migrant birds from Central and Northern Europe during the winter season.This complex system is suffering a significant decline due to biotic and abiotic factors [4,5].The Spanish dehesas' decline associated with Phytophthora root rot has been studied since the end of the 20th century [6][7][8][9][10][11]. Phytophthora cinnamomi Rands. is considered the main pathogen responsible for the decline of this ecosystem [4,[7][8][9][10]12,13], but it is not the only Phytophthora species infecting holm oaks [14][15][16].
On the other hand, Spanish natural oak woodlands are also undergoing this decline caused by Phytophthora spp.[17,18].Several studies across Europe demonstrate the association of declining oak woodlands with Phytophthora quercina T. Jung, among other species, causing root infections [19][20][21][22][23][24].In addition, abiotic factors, such as increasing temperatures and water stress, are being enhanced by climate changing conditions, which have a negative impact on the tree health status, weakening the stands and making holm oaks more susceptible to Phytophthora and Pythium infection [9,[24][25][26][27].Moreover, in view of the lack of regeneration of the stands, reforestations and afforestations are conducted with nursery material, with the consequent risk of introducing alien Phytophthora species to natural ecosystems [14,[28][29][30][31].
Some Phytophthora species infect plants without causing external symptoms and this plant material is transported worldwide, allowing pathogens to be disseminated without generating any alert at the inspection points [30,32].Denman et al. [33] reported that leaves from holm oak and rhododendron saplings remained asymptomatic when they were infected with Phytophthora ramorum Werres, De Cock, and Man in't Veld and Phytophthora kernoviae Brasier, Beales, and S.A. Kirk, two invasive species affecting ornamental and natural ecosystems.In 2006, imported ornamental Grevillea plants, which were asymptomatic, were found to be infected with Phytophthora niederhauserii Z.G.Abad and J.A. Abad [30].Thus, visual screening for monitoring Phytophthora without complementary tests is not an appropriate management tool.The direct isolation of Phytophthora species on semiselective media from affected tissue or baiting techniques do not always generate quick and sensitive results, making it difficult to accurately monitor forest areas [5,20].Economic and environmental losses caused by Phytophthora worldwide [31,34,35] require the use of all available techniques to detect and identify invasive species as quickly as possible.Combining direct isolation and baiting techniques with molecular tools, such as quantitative real-time PCR, increases the specificity, reproducibility, and sensitivity of the assessments, adding efficiency and accuracy to the diagnosis, an essential part of forest management strategies.
The aim of this study was to verify the presence of Phytophthora species in the holm oak rhizosphere in southwestern Spanish dehesas and in a northeastern Spanish holm oak woodland.In addition, the association between the Phytophthora species and the symptomatology of the holm oaks was studied in the dehesas by taking samples from declining and non-declining stands.For this purpose, different Phytophthora spp.isolation and detection approaches were performed: Direct isolation on semiselective media and apple and soil baiting using leaf material.Moreover, as P. cinnamomi and P. quercina are considered among the main pathogens associated with holm oak decline, their presence and relative abundance were studied in the samples using specific TaqMan real-time PCR probes.

Study Sites and Sampling
Studies were conducted in autumn 2012 and 2013 at 10 and 15 mature dehesas, respectively, located in the Extremadura region (southwestern Spain) (Table 1).This region has siliceous soils with Pyro bourgaeanae-Querceto rotundifoliae sigmetum vegetation series, and calcareous soils with Paeonio coriaceae-Querceto rotundifoliae sigmetum vegetation series, within an altitude ranging from 300 to 600 m [36].At each site, two different areas were studied: A declining area where three symptomatic trees were randomly selected and a non-declining area with three randomly selected asymptomatic trees.Trees severely affected by aerial pathogens or insect pests were discarded.In the 2012 survey, one soil sample including fine roots from the rhizosphere around the base of each tree was collected (60 samples in total) by making three 20-30 cm deep holes at approximately 1 m distance from the trunk and bulked, obtaining a representative 0.5 kg sample, as described by Pérez-Sierra et al. [18] (Table 1).In the 2013 survey, sites 1 to 5 were sampled as described above, but in the remaining ten sites, two pooled samples per site were collected (Table 1).In sites 6 to 15 from 2013, one pooled sample from 3 declining trees and one pooled sample from 3 non-declining trees were collected at approximately 1 m distance from the trunk of each tree at 20-30 cm depth.Fifty samples in total were collected in 2013.As differences in the Phytophthora spp.have been identified in eastern Spanish holm oak surveys [18], a study area located in northeastern Spain was included in the study.Montseny mountains is a 31,063 ha area located in Catalonia that since 1978 has been a biosphere reserve.It is made up of primarily siliceous rocks, with limestone rocks located on the western slopes of the mountains [37,38].Quercus ilex is located in the lower altitudes among a Quercetum-ilicis-galloprovinciale vegetation series [38,39].Fifteen holm oak declining stands showing defoliation, dead branches, and dieback symptoms and whose altitude ranged from 293 to 868 m were sampled as described above for 2012 just after a precipitation period in autumn 2013 (Table 1).
All the samples from the different surveys were transported to the laboratory, where roots were separated from soil for processing and soil was conserved at 5 • C until processing.

Phytophthora spp. Isolation
Roots from each sample were carefully washed under tap water and blotted on filter paper and direct isolation was performed on CMA-PARPB, as described by Jeffers and Martin [40], with and without the addition of hymexazol.Green apple baits were used for soil isolation.Granny Smith apples were surface disinfested with 95% ethanol.Four perpendicular 1 cm 2 holes were cut, filled with soil and remains of fine roots, and moistened with sterile water.These filled holes were sealed with tape and incubated in covered trays at 20 • C. The apples were examined daily until lesions developed.Small tissue fragments from the edge of the lesions were plated on CMA-PARPB with and without hymexazol and incubated at 20 • C in the dark.Oomycete-like colonies grown both from root and soil samples were transferred to potato dextrose agar (PDA) (Biokar-Diagnostics, Beauvais, France) and incubated at 20 • C in the dark for 7 days for further identification.Pure cultures of all putative Phytophthora isolates were obtained by transferring single hyphal tips to PDA plates.
Additionally, in the 2013 surveys, soils were also baited using leaflets of Camellia sp., Rhododendron sp., and Viburnum sp., following the methods described by Jung et al. [41,42].Isolations were made using CMA-PARBPH as the selective agar medium [40] and processed as described above.

Culture DNA Extraction, Sequencing, and Statistical Analyses
DNA was extracted from pure cultures of putative Phytophthora grown on PDA by scraping the mycelium and grinding to a fine powder under liquid nitrogen, using the commercial kit EZNA Plant Miniprep Kit (Omega Bio-Tek, Doraville, GA, USA) following the manufacturer's instructions.Ribosomal DNA ITS amplifications were carried out using the universal primers ITS6 and ITS4 [43,44].The PCR reaction final volume was 25 µL: PCR buffer 1×, 2.5 mM MgCl 2 , 200 µM each dNTP, 0.4 µM of each primer, 1 U of DNA Taq polymerase (Dominion MBL, Córdoba, Spain), and 1 µL of template DNA.All PCR reactions were performed in a PTC 200 thermocycler (MJ Research Inc., 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 sequenced at Macrogen Europe (Amsterdam, The Netherlands).The isolates were identified to the species level by conducting Basic Local Alignment Search Tool (BLAST) and comparing with the sequence data on international collection databases (Phytophthora Database, https://www.phytophthoradb.organd GenBank, https://www.ncbi.nlm.nih.gov/genbank/).
The total number of Phytophthora spp.isolates (Phytophthora pool) obtained and the number of isolates from each Phytophthora species were converted into frequencies relative to the total number of Phytophthora isolates recovered in the dehesas surveys.An analysis of variance (ANOVA) was performed with the 2012 and 2013 dehesas' data using a general linear model (GLM) in SAS version 9.0 (SAS Institute, Cary, NC, USA), in order to study the relationship between the frequency and diversity of Phytophthora spp.and the symptomatology shown by the trees in the dehesas.Mean values were compared using the Fischer's least significant difference (LSD) procedure at p-value = 0.05.

Environmental Samples: DNA Extraction and P. cinnamomi and P. quercina qPCRs
Roots and soil from both types of holm oak stands were tested with specific TaqMan probes for the main two oak Phytophthora pathogens, P. cinnamomi and P. quercina [16,45].Each soil sample was passed through a 2 mm sieve to remove the organic matter and gravel.Once it was homogenized, 50-80 g per sample was lyophilized overnight and pulverized using FRITSCH Variable Speed Rotor Mill-PULVERISETTE 14 (ROSH, Oberstein, Germany).DNA was extracted in duplicate with the Power Soil DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA) following the manufacturer's protocol.The root samples were first ground using a mortar and pestle under liquid nitrogen and then extraction was performed from 60 to 80 mg using the Power Plant Pro DNA Isolation Kit (MO BIO Laboratories, Carlsbad, CA, USA).

Phytophthora spp. Isolation
In the 2012 survey, Phytophthora spp.were detected in three dehesas, which represented 30% of the sampled sites.Three isolates of Phytophthora were recovered through the apple baiting method, one of each of: Phytophthora cambivora (Petri) Buisman (from a non-declining site), P. cinnamomi, and Phytophthora gonapodyides (H.E.Petersen) Buisman (from declining sites) (Table 2).
In 2013, the dehesas were surveyed and the soils baited in addition to the other methods already described for 2012.Phytophthora spp.were recovered in the 2013 dehesas survey from 21 holm oak samples, which represented 42% of the total samples, with 20% from declining samples and the remaining 22% from non-declining samples.A total of 165 Oomycetes isolates were obtained in 2013: 59 Phytophthora spp.isolates (clustered into four species) and 107 Pythium spp.isolates.In 2013, 39% of the Phytophthora spp.isolates were recovered from declining sites, and 61% were recovered from non-declining sites (Table 3).Regarding the isolation method, 13.4% of the Phytophthora spp.isolates were isolated directly from roots, 5.1% from apple baits, and 81.3% from leaf baits.As for the diversity of species obtained in 2013, P. cinnamomi, P. gonapodyides, Phytophthora megasperma Drechsler and Phytophthora pseudocryptogea Safaiefarahani, Mostowfizadeh, G.E. Hardy, and T.I.Burgess were isolated (Table 3).The range of abundance according to isolation was 39% P. cinnamomi, 35.6% P. gonapodyides, 20.3% P. megasperma, and 5.1% P. pseudocryptogea.Twenty-three isolates of P. cinnamomi were isolated from eight samples in the dehesas in 2013 (Table 3); percentages from declining and non-declining samples are shown in Figure 1.Twenty-one cultures of P. gonapodyides were isolated from five samples, with most of the samples from non-declining sites (Table 3, Figure 1).Twelve P. megasperma isolates were isolated from five samples (Table 3), and most of these samples were from non-declining trees (Figure 1).Three P. pseudocryptogea cultures were isolated from two samples (Table 3, Figure 1).The statistical analysis showed that the factors' symptomatology (p-value = 0.3626) and dehesa (p-value = 0.3087) were not significant for the frequency of the different Phytophthora species present in the dehesas in 2013.Considering the different species isolated separately, only the presence of P. gonapodyides was significantly higher in non-declining samples (p-value = 0.0366).The presence of either one species or another was not significantly associated with the dehesa factor (P. cinnamomi pvalue = 0.2277, P. gonapodyides p-value = 0.9176 and P. megasperma p-value = 0.7029).P. pseudocryptogea was only isolated in one dehesa, but from both declining and non-declining sites.

Site Symptomatology
No Phytophthora isolates were recovered from the 15 samples of the Montseny Biosphere Reserve by direct isolation on semiselective media from affected tissues and/or the baiting.The statistical analysis showed that the factors' symptomatology (p-value = 0.3626) and dehesa (p-value = 0.3087) were not significant for the frequency of the different Phytophthora species present in the dehesas in 2013.Considering the different species isolated separately, only the presence of P. gonapodyides was significantly higher in non-declining samples (p-value = 0.0366).The presence of either one species or another was not significantly associated with the dehesa factor (P. cinnamomi p-value = 0.2277, P. gonapodyides p-value = 0.9176 and P. megasperma p-value = 0.7029).P. pseudocryptogea was only isolated in one dehesa, but from both declining and non-declining sites.
No Phytophthora isolates were recovered from the 15 samples of the Montseny Biosphere Reserve by direct isolation on semiselective media from affected tissues and/or the baiting.

Environmental Samples: Hydrolysis Probes-P. cinnamomi and P. quercina qPCRs
The Phytophthora quercina standard curve plot showed that the correlation between the Cq-value and the DNA concentration was high (r 2 = 0.99966), with an efficiency of 0.90389.For P. quercina, the limit of detection (LOD) was established at a DNA concentration of 2 fg/µL.
Phytophthora quercina was detected in all the surveyed dehesas in 2012 (65.1% of the samples).Of these, 31.8%came from declining holm oak trees and 33.3% from non-declining trees (Table 2).In 2013, P. quercina was detected in all the surveyed dehesas (79.6% of the samples) (Table 3).A total of 66.7% of the soil samples were positive for P. quercina: 27.8% were from declining soil samples, while 38.9% were from non-declining soil samples.A total of 55.6% of the root samples were positive for P. quercina: 24.1% were from declining holm oak fine roots, while 31.5% were from non-declining holm oak roots.In the survey conducted in Montseny Biosphere Reserve in 2013, 66.7% of the samples were positive for P. quercina, of which 40% were from root samples and 53.3% from soil samples (Table 4).The Phytophthora cinnamomi standard curve revealed a high correlation between the Cq-value and the DNA concentration (r 2 = 0.99731), with a reaction efficiency of 0.92014.The LOD was established at 4 fg/µL.
Phytophthora cinnamomi was detected in seven out of the ten surveyed dehesas in 2012 (19.7% of the samples) (Table 2).A total of 12.1% of the soil detections came from declining holm oak trees and 7.6% from non-declining.In 2013, P. cinnamomi was detected in 11 dehesas (57.4% of the samples) (Table 3).A total of 38.9% of the soil samples were positive for P. cinnamomi; 22.2% were from declining trees, while 16.7% were from non-declining trees.A total of 33.9% of the root samples were positive for P. cinnamomi, with 22.2% from declining oak fine roots and 14.8% from non-declining oak roots.In Montseny Biosphere Reserve, 53.3% of the samples were positive for P. cinnamomi, of which 46.7% were from root samples and 33.3% from soil samples (Table 4).

Discussion
This study provides evidence that molecular approaches complement direct isolation methods of Phytophthora species from fine roots from holm oak in natural (Montseny Biosphere Reserve) and seminatural (dehesas) ecosystems, confirming that it is not only P. cinnamomi that is involved in the holm oak decline in Spain, but P. quercina is also present.Moreover, this is the first report of P. pseudocryptogea in Europe.
Regarding traditional isolation methods, an increase in Phytophthora isolation was observed in the dehesas in 2013 compared with the sampling conducted in the dehesas in 2012.This was probably not only due to the implementation of the leaf baiting technique, but also because in 2013, isolation of Phytophthora spp.from fine roots was more successful.This could be explained by the fact that under favorable environmental conditions, Phytophthora spp.infected the tree root systems and rotted fine roots containing the pathogens detached from the plant, so the pathogens can establish again in the soil [47].Phytophthora spp.dispersion requires warm temperatures and free water to produce infective zoospores; if not, they remain as resistant structures in the soil [34].Furthermore, the efficiency of Phytophthora isolation techniques can be compromised by the climatic conditions suffered during the period previous to the survey and by the presence of other microorganisms [8,34].In fact, the dehesa regions where the surveys were conducted received less precipitation in 2012 than in 2013 [48].Thus, according to this, the environmental conditions for Phytophthora spp.isolation were more favorable in 2013 than in 2012 in the southwestern Spanish dehesas, as they were recovered in 2013 from fresh lesions [49].Another possible explanation for the low efficiency of Phytophthora recovery in Montseny Biosphere Reserve is the presence of other fast-growing species in the samples, such as Pythium spp., making the isolation difficult.Pythium spp.were recorded in very low numbers in dehesas in 2012 (explained by the absence of favorable environmental conditions), but their presence was very relevant in the 2013 dehesas and in the Montseny Biosphere Reserve surveys.The genus Pythium is present in almost all soils and, as the isolation medium used for Phytophthora isolation is semiselective [34,40], Pythium spp.were also isolated with a high frequency in our study and were able to mask Phytophthora spp.presence.
Oak decline, associated with abiotic and biotic factors, has been occurring across Europe during the past decades [4,11,22,28,42,50].Among the several Phytophthora species that have been associated with this decline, P. cinnamomi has been considered the main biotic factor responsible for oak mortality in Spain since the 1990s [8,9,51].In 2013, P. cinnamomi was the most frequent species isolated in the infested dehesas, as was expected according to previous studies [7,52].In addition to P. cinnamomi, Corcobado et al. [15] reported that P. gonapodyides was also involved in oak decline in this region.In our surveys, P. gonapodyides, P. megasperma, and P. pseudocryptogea were recovered at low frequencies, and these species may play an important role as causal agents of the disease, as reported in other studies, where they were also recovered at low frequencies [14,25,28,52].There is no statistical evidence to support a differential distribution of Phytophthora species among the dehesas in 2013.Moreover, statistical results indicated that there was not a significant relationship between the Phytophthora spp.isolation frequency and the symptomatology of the holm oak stands.Our results showed a higher percentage of Phytophthora spp.recovery in 2013 from non-declining sites than from declining sites.Phytophthora cinnamomi, which was found in six dehesas, either from declining or from non-declining trees, is a primary root pathogen of woody species, considered a hemibiotrophic organism with life strategies which can change from biotrophic to necrotrophic, according to the environmental conditions [53][54][55].This species is also present in plant reservoirs and, depending on its behavior, will determine if the plant remains asymptomatic or not [54][55][56][57].Furthermore, P. cinnamomi is highly aggressive to holm oaks, as demonstrated previously [11,12,25,[58][59][60].Tsao [61] stated that a certain percentage of lost roots is required for symptoms to emerge and our results in the 2013 survey provide evidence that a tree symptomatology is not always an indication about the conditions of its root system.Statistical analyses showed that P. gonapodyides is more frequent in non-declining stands, in agreement with the results of Vettraino et al. [28], while the other species found did not show any statistical pattern.Phytophthora gonapodyides is known to attack the small or fine feeder roots [62] and to produce a wilting toxin [41].Nevertheless, Brasier et al. [62] stated that P. gonapodyides is often in balance with the unstressed oak root system, but this can change under stress conditions, contributing to a rapid decline.Hansen et al. [63] suggested that some Phytophthora spp.from clade 6 ecologically related to Phytophthora chlamydospora could cause limited root damage with no above ground disease symptoms contributing to the oak decline.Phytophthora megasperma isolated in the present study had been previously associated with oak decline [28,42].Although it is considered a pathogen of herbaceous plants and agricultural trees, it can become a serious problem when the oak balance is broken due to other factors, such as droughts or waterlogging [34].A similar behavior has been indicated for other Phytophthora spp.such as P. gonapodyides [10].Phytophthora megasperma, P. quercina, P. psychrophila, Phytophthora drechsleri, and Phytophthora syringae have also been associated with oak decline [18,19,25], although these species were not found in our samples.Nevertheless, P. pseudocryptogea in the present study was isolated for the first time in Europe and from a holm oak-rangeland in Spain.This species was described by Safaiefarahani et al., who re-evaluated the P. cryptogea complex [64].Although it was isolated from three soil samples in the present study, from three soil samples, the role of P. pseudocryptogea in holm oak decline remains unknown.The pathogenicity of this species in holm oak should be further studied.
The results obtained with the P. quercina probe are relevant, since it has always been thought that the holm oak decline in acidic soils in Spain is caused primarily by P. cinnamomi.Phytophthora cinnamomi was present in a high number of samples in both study locations, as was expected, but surprisingly it was not the most frequent species detected.Phytophthora quercina was shown as the most frequent species in this study, and the number of positive samples was higher in both studied areas.Molecular diagnoses provide faster and more sensitive detection of Phytophthora spp.[16,45,[65][66][67][68][69][70][71][72].

Conclusions
Different Phytophthora species were detected and identified in the study areas, regardless of whether they cause symptoms of decline or not.Further research is needed to clarify the effect of these pathogens in combination and abiotic factors in the oak stands.The implementation of the different direct and baiting isolation techniques for the isolation of Phytophthora spp., along with the available molecular detection techniques, allows a better diagnosis and understanding of the role of Phytophthora spp. in the holm oak forest areas.

Figure 1 .
Figure 1.Percentage of each Phytophthora species cultures isolated in the dehesas 2013 survey according to whether the holm oaks were declining or non-declining.

Figure 1 .
Figure 1.Percentage of each Phytophthora species cultures isolated in the dehesas 2013 survey according to whether the holm oaks were declining or non-declining.

Table 1 .
Description of the survey conducted in 2012 and 2013 in the dehesas of the Extremadura region and in 2013 in the oak woodland of Montseny Biosphere Reserve.

Table 2 .
Number of isolates of Phytophthora spp.obtained from Quercus ilex roots and soil in the 2012 survey in the dehesas of the Extremadura region according to the symptomatology of the sampled trees and results of the TaqMan real-time PCR assays.Results obtained from samples in each site are grouped according to whether samples were from declining or non-declining trees.

Table 3 .
Number of isolates of Phytophthora spp.obtained from Q. ilex roots and soil in the 2013 survey in the dehesas of the Extremadura region according to whether samples were from declining or non-declining trees and results of the TaqMan real-time PCR assays.PCR results obtained from samples in sites 1 to 5 are grouped according to whether samples were from declining or non-declining trees.

Table 4 .
Results of the TaqMan real-time PCR assays obtained from Q. ilex roots and soil in the 2013 survey in the oak woodland of Montseny Biosphere Reserve.