Susceptibility of common alder ( Alnus glutinosa ) seeds and seedlings to Phytophthora alni and other Phytophthora species

Phytophthora alni is a highly destructive host specific pathogen to alders (Alnus spp.) spreading all over Europe. Recently this pathogen has been reported to cause diseases in common alder (Alnus glutinosa) in Spain. Seeds and seedlings of A. glutinosa were tested in vitro for their susceptibility to alder Phytophthora and other Phytophthora species. Isolates of P. alni ssp. alni, P. cinnamomi, P. citrophthora, P. nicotianae and P. palmivora were used in the experiments. Seeds and seedlings were inoculated with a zoospore suspension and uniform mycelial blocks of agar of the Phytophthora species. Susceptibility was calculated in terms of pathogen virulence on seed germination and seedling mortality 42 and 67 days after inoculation respectively. Seed germination and seedling mortality rates varied differently among the isolates used. Results implied that common alder and its seeds and seedlings are at risk to be infected by P. alni. In addition, other Phytophthora species are able to infect this kind of material showing their relative host non-specificity. This is one important finding concerning alder regeneration in infected areas, and the possibility of disease spread on this plant material.


Introduction
The genus 'Alnus' is characterized by their capability to colonize on bare land and tolerate high groundwater table and flooding (Gibbs et al., 2003).Common alder (Alnus glutinosa (L.) Gaertn) is the most widespread species among alders occurring in Europe which has been used for reforestation and stabilizing river banks.This species is occurring extensively in Spain and is distributed along streams and rivers.
Another Phytophthora species have been found causing damage in a wide tree host range.Among those, Phytophthora nicotianae Breda de Haan (= Phytophthora parasitica Dastur) which is a soilborne pathogen, has been recorded causing dieback in Eucalyptus species in South Africa where Eucalyptus species are planted commercially (Maseko et al., 2001); and damaging several woody ornamental plants in nurseries (Schwingle et al., 2007;Donahoo and Lamour, 2008); but it has been also detected in another 298 plant species (Erwin and Ribeiro, 1996).Phytophthora cinnamomi Rands, which is an invasive soil-borne roots pathogen, has been stated as one of the causal agents of the decline of holm oak (Quercus ilex L.) and cork oak (Q.suber L.) in Southwestern Iberian Peninsula (Brasier et al., 1993;Sanchez et al., 2002).And P. citrophthora (R.E.Sm. and E.H. Sm.) Leonian and P. palmivora (E.J. Butl.) have been recorded in nurseries causing diseases on several woody ornamental plants (Schwingle et al., 2007;Donahoo and Lamour, 2008).
Although few studies have been conducted to examine the susceptibility of alders to P. alni and other Phytophthora species (Brasier and Kirk, 2001;Santini et al., 2003;Santini et al., 2006), the Spanish isolates P. alni have not been tested yet in terms of aggressiveness on common alder.In addition to that, due to the frequent occurrence of other Phytophthora species in the forest ecosystems of Spain (including nurseries), the main objective of the present study was to evaluate the susceptibility of common alder seeds and seedlings to Spanish isolates of P. alni and the other most common Phytophthora species present in nurseries which could prompt the failure of the common alder regeneration.

Phytophthora isolates and inoculum production
Two isolates of P. alni ssp.alni recovered in 2009 from diseased A. glutinosa trees growing along river Miño (Spain), which were isolated as described by Solla et al. (2010), were used to inoculate seeds and seedlings.Besides, one isolate of each P. cinnamomi, P. citrophthora, P. nicotianae and P. palmivora (Supplied by the Instituto Agroforestal Mediterráneo [Mediterranean Institute of Agroforestry], Universidad Politécnica de Valencia, Valencia, Spain) were used in the experiments.Both, mycelia and zoospores were used in the experiments.For An extensive and rapid mortality of common alder was observed along many rivers of northern Spain (Tuset et al., 2006).Some years later of that observation, Phytophthora alni (Brasier and S.A. Kirk) was associated to cause that mortality in A. glutinosa (Solla et al., 2010;Varela et al., 2010).P. alni is an interspecific hybrid between Phytophthora cambivora (Petri) and an unknown Phytophthora similar to P. fragariae Hickman (Brasier et al., 1995(Brasier et al., , 2004)), and it has been described as a host specific pathogen to alders spreading all over Europe.Three different types have been recognized within P. alni based on morphological characteristics and aggressiveness (Brasier and Kirk, 2001;Brasier et al., 2004): the standard type of the pathogen, which is the most aggressive one, has recently been named as P. alni ssp.alni, the hybrid types collectively known as P. alni ssp.uniformis and P. alni ssp.multiformis, which are locally very damaging and could represent a serious threat to alder population and stability of riparian ecosystems (Brasier et al., 2004).
The association of P. alni in the decline and mortality process of alders has been the center point of several studies in different parts of the world (Gibbs, 1995;Gibbs et al., 1999;Santini et al., 2001;Brasier and Kirk, 2001;Brasier, 2003;Gibbs et al., 2003;Santini et al., 2003;Jung and Blaschke, 2004;Ioos et al., 2005;Cerný and Strnadová, 2010;Solla et al., 2010;Varela et al., 2010).Phytophthora decline of the riparian alder population has recently become an important problem in Spain because of the rapid spreading of the causal pathogen.According to Santini et al. (2003), the way of spreading of alder Phytophthora may be related to its introduction with the planting materials, which may become infected in nurseries where the cross infection between different hosts is frequent due to asymptomatic infections.According to this, alder population may be in danger as they co-exist with Phytophthora contaminated hardwood and ornamental woody species in the same nurseries.In addition to that, container grown seedlings in nurseries may act as a prime carrier facilitating further dispersion of Phytophthora to the natural ecosystems which may hamper alder regeneration.Zoospores of the alder Phytophthora swim freely in water and therefore most likely spread far distance using river system.In young alders, infection and bark killing often start at the collar region where zoospores are attracted.However, the infections in the middle of the trunk in the inner bark might indicate that environmental factors may also play a prominent role in the occurrence of the disease which may be associated with flooding events (Brasier, 2003).Susceptibility of alder seeds and seedlings to Phytophthora alni mycelial agar plugs, colonies of the Phytophthora species were sub-cultured for 1 week at 20 °C in the dark onto 90-mm Petri dishes containing sterilized V8 agar (V8 agar: 100 ml/L V8 Campbell Grocery Products, 3 g/L CaC03, 20 g/L Agar Technical DIFCO, Detroit, MI, USA).For zoospores production, colonies of the Phytophthora species were sub-cultured for 14 days at 20 °C in the dark onto 90-mm Petri dishes containing sterilized V8 agar exposed to 16 h light per day.Sporangia were obtained by flooding the colonies with sterile distilled water, and then transferred into sterile water by rubbing surface of the culture with a sterile bent glass rod.The liquid was poured off the plates and again collected in a sterile beaker which was placed in a refrigerator at 7 °C for 1 h, then returned to room temperature (20 °C) during another 75 min to promote zoospore releasing.Zoospore concentration was determined by using a haemocytometer, and suspension was adjusted to 3 × 10 5 zoospores per ml (Denman et al., 2005).

Plant material
Disease free seeds of common alder were supplied by the Centro Nacional de Mejora Forestal [National Centre for Forest Breeding] El Serranillo (Guadalajara, Spain) in order to be used in the experiments.Before use, seeds were sterilized in the following way: they were firstly washed several times with sterilized distilled water, then dipped into hydrogen peroxide (3%) for 20 minutes.Finally seeds were washed twice with sterilized distilled water to remove excess hydrogen peroxide and dried aseptically.Those sterilized seeds were ready to be used in the seed inoculation experiment.For seedling inoculation experiment, sterilized seed were plated onto water agar Petri dishes and sealed with Parafilm® (American National Can, Greenwich CT, USA) to avoid contamination.Plates were kept into growth chamber at 24 °C and photoperiod (16/8) to promote germination.After that, eight seedlings per plate were aseptically transferred to Petri dishes containing potato-dextrose-agar (PDA) to get the seedling hardening prior to inoculation.

Seed inoculation experiment
Eight sterilized seeds per plate were transferred onto the surface of water agar contained into 90-mm Petri dishes.Each one of the six Phytophthora isolates (two of P. alni ssp.alni, and one of each P. cinnamomi, P. citrophthora, P. nicotianae and P. palmivora) were inoculated in the Petri dishes following two different methods: (1) Individual seed inoculation treatment (noted as IS) when a zoospore suspension of 0.1 ml (3 × 10 5 zoospores per ml) was sprayed at the base of each seed; and (2) central inoculation treatment (noted as CE) when a zoospore suspension of 1 ml was sprayed very precisely at the centre of Petri dishes keeping equal distances from each seed and later shaken gently for the uniform spreading of suspension over the surface to facilitate the zoospore suspension contact with the seeds.In the controls (CO) 0.1 ml of sterile distilled water was sprayed instead the zoospore suspension.Four replications for each Phytophthora species and for each inoculation method were used in the assay.After inoculation, Petri dishes were sealed with Parafilm and kept at 24 °C and photoperiod (16/8).Seeds were inoculated on June 2010.Data of seed germination percentage were taken at every 5 days interval (except the first record which was taken at 7 days) until 42 days.

Seedling inoculation experiment
Seedling were inoculated on April 2010 with the six Phytophthora isolates following two different methods: (1) individual seedling inoculation (noted as IT) when a 2-mm size mycelial agar plug was put at the base of each seedling; and (2) central inoculation method (noted as CE) when a 2-mm size mycelial agar plug was placed precisely at the centre of Petri dishes keeping equal distances from each seedling.In the controls, seedlings were inoculated with sterile V8 agar plugs.Four replications for each isolate and method were used in the experiment.After inoculation, plates were sealed with Parafilm and kept at 24 °C and photoperiod (16/8).Data of seedling mortality percentage were taken at every 5 days interval (except the first record which was taken at 7 days) until 67 days.

Statistical analysis
Seed germination and seedling mortality were analyzed by repeated measures ANOVAs (p < 0.05) to examine significant differences among the Phytophthora isolates and inoculation methods, as well as the time period when different measurement data were taken.The differences between means were considered significant (p < 0.05) according to Tukey multiple range test.All statistical analyses have been performed with the software Statistica 6.0 for Windows (StatSoft Inc., Tulsa, Oklahoma, USA).

Seed inoculation experiment
Repeated measures ANOVA applied to seed germination percentage (Table 1) showed that both the isolate of Phytophthora species and the treatments used in the experiment produced significant effect on seed germination.Interactions between the factors as source of variation were significant with the exception of time.Seeds started to germinate during the first week, regardless of the treatment, although in the controls a higher percentage was obtained after 7-12 days after the in-oculation.In case of seeds inoculated, comparatively a low germination was achieved 22-27 days after the inoculation depending on the isolates of Phytophthora species and pursued almost a steady rate up to 37-42 days (Figure 1).The complete progression of seed germination after the inoculation with the isolates of Phytophthora species with respect to number of days has been indicated in Figure 1.Forty two days after inoculation, all the isolates of the Phytophthora species tested hampered significantly germination regardless of the method used.When zoospore suspension was applied at the centre of the plate (method CE), no differences were found among the isolates, but when suspension was applied in each seed (method IS), P. cinnamomi caused a significant lower reduction of the germination percentage than that caused by rest of the species (Figure 2).On average, seed germination percentage when inoculation was made for each seed, was   26.04%; and when zoospore suspension was applied at the centre, the percentage was 36.97%.In controls, germination percentage was higher than 80% in all cases (Figure 2).

Seedling inoculation experiment
Repeated measures ANOVA (Table 2) revealed that the seedling mortality percentage was significantly influenced by the Phytophthora isolates, the inoculation methods, the time period, and by their interactions.Most of the seedlings inoculated with the different isolates of Phytophthora started to die 7 days after inoculation, with a progressive mortality increment, reaching the maximum after 15-20 days of the inoculation (Figure 3).However this maximum was achieved earlier by both isolates of P. alni and P. citrophthora.At the end of the experiment all the isolates, excepting that of P. cin-  namomi, produced a seedling mortality rate higher than 90%, regardless of the inoculation method (Figure 4).P. cinnamomi isolate caused a seedling mortality percentage of 46.9% when it was inoculated in the center of the plate (method CE), and 78.1% when it was inoculated in each seedling (method IT).In controls were not observed any seedling mortality (Figure 4).Between inoculation methods, differences were found mainly at the beginning of the experiment (in the first 2-5 measurements depending of the isolate).In those first records, the inoculation of the isolate plugs in each seedling (method IT) caused a greater seedling mortality than the inoculation in the centre of the plate (method CE) (Figure 3).The exception was found for the P. alni isolate 2; in this case the method CE was more aggressive than the method IT in the first record interval.At the end of the experiment, no differences were found between inoculation methods, except in the P. cinnamomi isolate which caused a significant higher mortality when was inoculated separately in each seedling (Figure 4).

Discussion
Pathogenicity of Phytophthora alni ssp.alni, P. cinnamomi, P. citrophthora, P. nicotianae and P. palmivora on common alder (Alnus glutinosa) seeds and seedling was examined in vitro.This is the first study of this type performed so far in Spain with the aim to examine the interaction between A. glutinosa and host specific pathogen P. alni along with non-host specific pathogens P. cinnamomi, P. citrophthora, P. nicotianae and P. palmivora under laboratory conditions.Both inoculation methods applied have confirmed their ability to infect seeds and seedlings.Our findings have revealed that all the Phytophthora spp.tested in the present assay could represent a serious threat to A. glutinosa which may cause failure of the alder regeneration.
In our present inoculation tests, P. alni ssp.alni has appeared as a highly aggressive pathogen on seeds and seedlings of A. glutinosa.The high aggressiveness of P. alni has been already stated by other authors in several European countries (Brasier et al., 1995;Gibbs, 1995;Szabó et al., 2000;Santini et al., 2001;Streito et al., 2002;Nagy et al., 2003;Jung and Blaschke, 2004;Cerný and Strnadová, 2010;Solla et al., 2010;Varela et al., 2010).Pathogenic ability of P. alni on seeds of A. glutinosa has been revealed from a similar laboratory test made by Schumacher et al. (2006), where seeds were inoculated with a zoospore suspension.The finding has strengthened the speculation that P. alni could infect alder seeds under natural conditions.Results of seedling inoculation test are consistent with a laboratory test conducted by Santini et al. (2003) where seedlings of A. glutinosa, A. cordata and other hosts were inoculated with one isolate of P. alni.In that study, the maximum seedling mortality was observed in Alnus species.Infecting ability of P. alni has been revealed from baiting tests which confirmed the involvement of the pathogen on A. glutinosa seedlings at nurseries in Germany (Jung and Blaschke, 2004).In Italy, P. alni was found infecting A. cordata both in young plantations and nurseries (Santini et al., 2003).Several other authors have also reported the association of P. alni with alder seedlings in plantations and nurseries (Santini et al., 2001;Gibbs et al., 2003;Schumacher et al., 2006).So, we assume that P. alni ssp.alni is able to infect and contaminate seeds and seedlings of A. glutinosa both in artificial and natural conditions.
Another Phytophthora species tested is P. cinnamomi, which has not been detected naturally in alders yet, but in our study it has proved its ability to reduce seed germination and cause seedling mortality under in vitro conditions.Similar results were obtained from a previous laboratory test by Santini et al. (2003) where an isolate of P. cinnamomi caused mortality of A. glutinosa seedlings after its inoculation using two different methods.In general, P. cinnamomi has been described as a soilborne pathogen and it has been reported causing root rot in Quercus suber and Q. ilex under Mediterranean conditions and stem canker of Q. rubra in France (Brasier et al., 1993;Marcais et al., 1993;Linde et al., 1999;Sanchez et al., 2002).Hardy and Sivasithamparam (1988) recorded the involvement of P. cinnamomi and several other Phytophthora species with root rot of container grown seedlings from 14 nurseries in Western Australia.In contrast to our study, Australian and South African isolates of P. cinnamomi showed a considerable variation in virulence when were inoculated into seedlings of Eucalyptus (Dudzinski et al., 1993;Linde et al., 1999).Robin and Desprez-Loustau (1998) observed a wide range in aggressiveness of P. cinnamomi isolates to Q. rubra.Host range of P. cinnamomi was examined by Tippet et al. (1985) who assessed differences in susceptibility of P. cinnamomi to the hosts examined in inoculation tests.On the basis of the findings, we draw conclusion that the alders are under risk to be infected as sharing same environmental conditions to grow with other plants hosting P. cinnamom.
Among the other pathogen species tested, P. citrophthora has shown its virulence on A. glutinosa seeds and seedlings.P. citrophthora has been described as a pathogen of Citrus and it has been reported causing crown rot of peach, plum and cherry rootstocks after artificial inoculations (Thomidis, 2001).Association of P. citrophthora in nurseries of woody ornamental plants has been surveyed (Donahoo and Lamour, 2008).Although P. citrophthora is non-host specific to alders, in our present inoculation tests, the pathogen has showed a great aggressiveness on A. glutinosa seeds and seedlings.In support to our findings, a separate inoculation test conducted by Santini et al. (2006), showed a certain seedling mortality of Alnus spp.after inoculation with P. citrophthora, although a lower mortality was obtained.In contrast, P. citrophthora was the most aggressive pathogen out of eleven Phytophthora species tested in a pathogenicity experiment conducted in vivo on cherry (Thomidis and Sotiropoulos, 2003).Being an aggressive pathogen, we could assume that alders growing in nurseries and mixed plantations may be at risk to be infected by P. citrophthora.P. nicotianae and P. palmivora are occurring frequently in nurseries with container grown seedlings.Donahoo and Lamour, (2008) reported the presence of P. nicotianae and P. palmivora in nurseries of woody ornamentals.In addition, Hardy and Sivasithamparam (1988) found the presence of P. nicotianae in 9 nurseries out of 14 surveyed in Western Australia.Besides, a baiting bioassay detected P. nicotiana and several other Phytophthora in naturally infested container mixes from South Carolina nurseries in United States (Ferguson and Jeffers, 1999).Up to date, no report was available on the pathogenic ability of P. nicotianae and P. palmivora on Alnus but, in our tests it has been revealed their capacity to show virulence on A. glutinosa seeds and seedlings.That's why it is assumed that container grown seedlings of Alnus spp.may be vulnerable to P. nicotianae and P. palmivora if there is enough inoculum as it occurs with other plants in nurseries.In support of the hypothesis, several non-host specific pathogens P. citricola, P. cactorum and P. gonapodyides were reported to be present in a nursery soil in the vicinity of alders (Oszako et al., 2007).Several other Phytophthora species such as P. cambivora, P. cactorum, P. citricola, P. megasperma and P. quercina were baited from flooding alder plants (Jung et al., 1999(Jung et al., , 2003)).All these observations may suggest that under suitable conditions, P. nicotianae and P. palmivora could be potentially harmful to A. glutinosa.The phenomenon gives a serious implication for future establishment of forest stands with infested nursery stock.
Production of zoospores in the presence of water significantly contributes to the dispersal of Phytophthora via irrigation (Yamak et al., 2002;Hong et al., 2006).Transportation of nursery stock and use of natural rivers and other water courses to irrigate nurseries give ideal opportunities to Phytophthora to infect and spread in those nurseries.Out-planting with Phytoph-thora infected nursery stock have added further more to the dissemination process of Phytophthora to natural ecosystems.Falling of seeds, young shoot or leaves onto the contaminated water or soil and later disseminating far distances through water ways.For the alder seeds and seedlings, which are vulnerable to P. alni and other Phytophthora species, is certainly a new threat.In addition to that, the emergence of a new Phytophthora species provides additional threat, as it has been the case of Alnus by a hybrid pathogen P. alni ssp.alni (Brasier et al., 2004).For instance, a new Phytophthora species, P. polonica, was isolated from rhizosphere soil samples when surveys conducted in declining alder stands grown naturally by the riversides in Poland (Belbahri et al., 2006).New pathogens can be brought into nurseries and distributed with seeds and seedlings to plantations or riparian ecosystems.In Great Britain, spreading of alder Phytophthora has occurred through watercourses (Gibbs et al., 1999) whereas in Bavaria, Germany P. alni has been introduced into many places either by planting infected nursery stock or by irrigation water (Jung and Blaschke, 2004).
Artificial inoculation methods applied in an artificial environment may not provide precise information on the real interactions between hosts and pathogens.Besides, it may not possible to get exact results on the virulence of the Phytophthora species on hosts.Nevertheless, experiments done in an artificial environment may avoid interactions with other organism or minimize host natural defense mechanism.In this sense, the present study provides consistent clue of how seed germination and seedling mortality are affected by different Phytophthora isolates.Extrapolation of laboratory results to natural environment may not always be valid.However, results obtained from those in vitro inoculation methods can be useful for the identification of potential hosts and contribute to pathogens risk assessment.The results of the present study provided us a rough estimation on the susceptibility of an important tree species towards Phytophthora.As other Phytophthora spp.are able to infect alder seeds and seedlings, it is important to apply further controls on nursery management and irrigation system to avoid dissemination of Phytophthora.As a whole, additional dissemination of inoculum with diseased nursery stock will hamper control measures of the diseases on alders.There is an immediate need of a molecular-based detection protocol and safer conditions for A. glutinosa to grow in nurseries and plantations.It is important to apply effective and immediate actions to prevent spread and transfer of Phytophthora spp.Susceptibility of alder seeds and seedlings to Phytophthora alni In order to reduce the spread of Phytophthora diseases in plantations and nurseries the following few measures could be adapted: (a) careful selection of plant material (seeds and seedlings) free of diseases, (b) avoidance of frequent seedling transportation with soils from nurseries to planting sites, (c) monitoring of plant health growing in nurseries at a regular interval, (d) careful inspection and testing of nursery soil and water reservoir used to irrigate nurseries, (e) sterilization of nursery tools before performing silvicultural treatments, (f) planting of Alnus in the stands where nursery plants have not been planted for a long period of time, and (g) development of molecular based detection protocol for a rapid and effective identification of Phytophthora species.
alder seeds and seedlings to Phytophthora alni

Table 1 .
Repeated measures analysis of variance for seed germination (%) that considers Phytophthora species, treatment and time, and their interactions as source of variation

Table 2 .
Repeated measures analysis of variance for seedling mortality (%) that considers Phytophthora species, treatment and time, and their interactions as source of variation SS = sum of square; df = degree of freedom; MS = Mean square.