Effect of elicitors on holm oak somatic embryo development and efficacy inducing tolerance to Phytophthora cinnamomi

Holm oak trees (Quercus ilex L.) mortality is increasing worryingly in the Mediterranean area in the last years. To a large degree this mortality is caused by the oomycete Phytophthora spp., which is responsible for forest decline and dieback in evergreen oak forest areas of the southwestern Iberian Peninsula. This study is based on the possibility of applying chemical elicitors or filtered oomycete extracts to holm oak somatic embryos (SE) in order to induce epigenetic memory, priming, that may increase tolerance to the pathogen in future infections. To this end, we first examined the effect of priming treatments on SE development and its oxidative stress state, to avoid elicitors that may cause damage to embryogenic tissues. Both, the sterile oomycete extracts and the chemical elicitor methyl jasmonate (MeJA) did not produce any detrimental effect on SE growth and development, unlike the elicitors benzothiadiazole (BTH) and p-aminobenzoic acid (PABA) that reduced the relative weight gain and resulted in necrotic and deformed SE when were applied at high concentrations (25 µM BTH or 50 µM PABA) in accordance with their high malondialdehyde content. No significant differences among elicitation treatments were found in dual culture bioassays, although those SEs elicited with 50 µM MeJA increased H2O2 production after challenged against active oomycete indicating the activation of stress response. Since this elicitation treatment did not produce any adverse effect in the embryogenic process we suggest that could be used in further priming experiments to produce holm oak plants adapted to biotic stress.


Scientific RepoRtS
| (2020) 10:15166 | https://doi.org/10.1038/s41598-020-71985-w www.nature.com/scientificreports/ forest protection have to be implemented 13 . Conventional breeding programs to obtain disease tolerant/resistant plants have been scarce probably because traditional propagation is extremely difficult for the species mainly due to the poor rooting ability of cuttings, which is also reduced with the aging of parent plants 14 . Recently, a holm-and cork-oaks genetic improvement program to alleviate the "seca" syndrome has been launched by the Spanish Government (National Rural Development Program 2014-2020) cofunded by the European Agricultural Fund for Rural Development (EAFRD). Biotechnological tools allowing clonal propagation of selected resistant genotypes such as axillary budding 15 or somatic embryogenesis 14,[16][17][18] are available, although acclimazation of generated plants needs to be improved; also, the induction of systemic resistance, triggering natural plant defenses by the use of elicitors, may offer complementary strategies to breeding programs developed to obtain resilient plant material. Plants respond to oomycete infection by multiple defense mechanisms, including strengthening of physical barriers, production of antimicrobial molecules, and programmed cell death 19 . In addition, many biological and chemical elicitors, such as necrotizing pathogens, salicylic acid (SA), benzothiadiazole (BTH), and methyl jasmonate (MeJA), have been reported to activate plant immune response to pathogens [20][21][22][23] . These elicitors cause the induction of a primed state of enhanced defenses which allows plants to display either faster, stronger, or both, activation of the defense responses after further attacks by pathogens or insects, and also under abiotic stress conditions 24 . Thus, exogenous applications of SA and carvacrol to Ulmus minor successfully enhanced the resistance of trees to the fungal pathogen Ophiostoma novo-ulmi 25,26 . Also, foliar sprays of SA or BTH to Pinus radiata significantly decreased plant infections by Diplodia pinea 27 or by Phytophthora cinnamomi 28 . Primed plants present changes at the physiological, molecular, and epigenetic levels that can occur within seconds or hours after stimulation; these changes can be transient or maintained for its lifetime, and can even be inherited by subsequent generations 29 . The most common molecular mechanisms by which a plant is primed involve chromatin modifications, either DNA methylation, histones changes or posttranscriptional variations, which are the basis of inheritable regulation in the expression and gene function that does not involve DNA sequence alterations but epigenetic variations 30 . There are some evidences that biotic stresses exerted on mother plants by herbivores or pathogens can induce transgenerational defenses in progeny when challenging environmental harshness 31,32 . However, most studies of transgenerational induction of defenses to pests and pathogens in plants have focused on short-lived annuals. As an example, applications of β-amino-butyric acid (BABA) as well as virulent or avirulent strains of Pseudomonas syringae pv. tomato to arabidopsis plants displayed enhanced disease resistance in their progenies when were infected with this phytopathogenic bacteria or with the biotrophic oomycete Hyaloperonospora arabidopsidis 33,34 . However, it remains largely unknown whether this type of transgenerational plasticity also occurs in long-lived forest trees 31 . Environmental maternal effects on the tolerance of Pinus pinaster to biotic stress have been reported 32 . In addition, a recent work 35 , demonstrated that chestnut (Castanea sativa) seedlings of ink-diseased mother trees showed increased tolerance to P. cinnamomi; interestingly, this tolerance was not mediated by seed size, but probably as a consequence of seed priming during fruit development. These inheritable variations could open doors for obtaining resistant or tolerant oak genotypes, which would be included in a recovery program in damaged areas, avoiding oak woodlands and dehesa dieback.
Since somatic embryo (SE) development mimics zygotic embryogenesis, we hypothesize that priming SEs during the proliferation phase may induce transgenerational defense mechanisms that would produce resistance to P. cinnamomi in subsequent infections. It is of practical interest to determine first if elicitor molecules released during the early stages of the plant-pathogen interaction could be directly applied to embryogenic lines without altering their development, and second, whether these elicitors may activate priming signals in order to suppress the deleterious effects of fungal diseases in plants. Because of this, the objectives of this work were to assay the effect of some elicitors on holm oak SE development, and to study the preliminary responses of elicited SEs after infection with P. cinnamomi.

Results
Effect of Phytophthora filtered extract (PFE) and chemical elicitors on holm oak SE growth and development. The transference of elicited material to proliferation medium produced secondary embryogenesis and some of the globular embryos underwent maturation up to the early cotyledonary stage. Some elicited-SEs turned necrotic and therefore were subtracted from total well-formed embryos.
After 60 days of elicitation significant differences in SE growth (p < 0.001) among elicitation treatments were found. Fresh weight increase was higher on globular SE elicited with MeJA at 5, 10 or 25 µM, as well as for those elicited with 5 or 10 µM BTH, but reduced on those SEs elicited with 25 µM BTH (Fig. 1a). However, SE growth rates achieved in these treatments did not significantly differ to that of controls (2.4 ± 0.9 g), although on average, we observed higher growth rate for MeJA-elicited SEs (2.7 ± 0.6 g) as compared to BTH and PABA treatments (means 1.9 ± 0.8 and 1.9 ± 0.6 g, respectively, Fig. 1a) and these differences were statistically significant (p = 0.01). Growth rate of PFE-elicited SEs was on average similar to that of non-elicited embryogenic material (2.3 ± 0.7 g, Fig. 1a).
The analysis of variance showed significant differences on holm oak somatic embryo development among the 14 elicitation treatments tested (p < 0.001, Fig. 1b). On average, PABA-elicitation treatments significantly reduced the percentage of well-developed embryos as compared to controls (40.2 ± 19.1% vs. 72.5 ± 7.8%, p = 0.047), particularly at the highest concentration (50 µM). This detrimental effect was also observed for BTH-elicitation treatments at 25 and 50 µM. In contrast, PFE-elicited embryogenic material produced, on average, similar rates of well-developed SEs (57.4 ± 13.0%) to that of controls, as occurred with MeJA treatments (47.7 ± 16.0%). These results would indicate a more detrimental effect on holm oak SEs development of BTH and PABA elicitation treatments, especially when higher concentrations of these compounds are used, as compared to application of MeJA or PFE. Phytophthora cinnamomi growth in dual cultures with elicited holm oak SEs. When Phytophthora cinnamomi mycelia were inoculated in plates containing control and elicited holm oak globular SEs, similar hyphae growth rates were observed towards both types of material, (ratios r control /r elicited around 1). As it is  www.nature.com/scientificreports/ (7.50 ± 2.00 µg mL −1 ), although values did not differ significantly from that found in non-elicited material. In contrast, H 2 O 2 production was significantly repressed in holm oak SEs elicited with 5 or 25 µM PABA as compared to controls (2.34 ± 0.61 and 2.86 ± 0.92 µg mL −1 , respectively, vs 5.31 ± 0.33 µg mL −1 ).

Discussion
The design of an effective strategy to induce tolerance to P. cinnamomi in holm oak must consider the effect of elicitation treatments on the embryogenic material, to avoid a decrease in somatic embryos proliferation that impede the plant regeneration 36 . In our study, abnormally developed embryos were mainly observed in embryogenic material elicited with high concentrations (25 or 50 µM) of BTH or PABA. Other authors also reported that high concentrations of some elicitors could negatively influence the biologic development of plant material 37 . On the contrary, MeJA application produced a recovery in the differentiation capacity of an embryogenic line of Quercus ilex 36 . Then, possible detrimental effects of the elicitation treatments tested herein were also assessed by determining MDA content of holm oak SEs. MDA production indicates high lipid peroxidation in plant cells [38][39][40][41] . In our experiments this compound was detected in higher concentration in SEs from all elicitation treatments, although MDA contents were not significantly different from that of controls. Interestingly, 7 d after elicitation we observed lower MDA levels in SEs treated with the highest BTH or PABA concentration (50 µM), in contrast to the higher MDA content determined in material elicited with 5 µM PABA. However, after 30 days we observed a recovery of cell membranes integrity in this material, and also in those elicited with PFE or MeJA at the higher concentrations, while MDA content increased in material elicited with 25 or 50 µM PABA or with 50 µM BTH. These results suggest a negative effect of PABA-elicitation on cell membrane integrity of holm oak SEs that could be related to the lower embryo development frequencies observed on average in these treatments. Chong et al. 42 suggested that damages in cell membranes during the first days after elicitation of Morinda elliptica cell cultures with MeJA would trigger the biosynthesis of antioxidant enzymes, therefore inducing a subsequent decrease in MDA. Experiments using dual cultures have shown that the stimulation or inhibition of mycelium growth in these conditions could give useful information about plant susceptibility [43][44][45] , and therefore could be used to select efficient elicitation treatments 36,42 . In our study, elicited holm oak somatic embryos did not inhibit Phytophthora cinnamomi mycelium growth in dual culture tests, being the plates with somatic embryos elicited with PFE-50, 50 µM BTH, 5, 10 or 50 µM MeJA and 5 µM PABA those showing ratios higher than 1 between growth towards control and growth towards elicited material at the end of the experiment. Treatments with cinnamomin have been reported to inhibit oomycete root colonization in holm oak 21 ; foliar applications of a BTH derivate also reduced the incidence of oomycete root infection in several forest 28 ; MeJA have been referred to induce resistance in conifers seedlings to a pathogenic oomycete 46 and pests [47][48][49] , as well as resistance to pathogens in plants such as arabidopsis 50 , grapevine 51 or tomato 37 , and in harvested fruits as bayberries 52 and grapes 53,54 .
Wang et al. 54 reported that induced resistance to Botrytis cinerea of grape fruits treated with MeJA was tightly associated with increased H 2 O 2 production in this plant material. This biotic stress response was characterized in holm oak elicited somatic embryos, and we found higher H 2 O 2 contents in samples elicited with 50 µM MeJA In spite of significant differences in all parameters were not found, an overview of our results concerning SE growth and development, MDA production, inhibition of mycelia growth and H 2 O 2 production suggest that in our conditions 50 µM MeJA seems to be the most adequate elicitation treatment in holm oak embryogenic material. These results contribute to the scarce available information about elicitation of somatic embryogenic material of Quercus species.

Material and methods
Plant and oomycete material. The experiments were undertaken with the embryogenic holm oak (Quercus ilex L.) line Ha13. This line was obtained from male catkins of a 50-100-year-old holm oak located in La Hunde mountain (Ayora, Valencia, Spain) as described in 16 and has being maintained in vitro by monthly subcultures on proliferation MS 55 medium supplemented with 20 µM silver thiosulfate and 4 g L −1 activated charcoal (named after MS/STS/AC medium as described in 56 ). On this medium, Ha13 line produces secondary embryogenesis and somatic embryos (SEs) at different developmental stages (Fig. 5) from which material consisting of embryogenic calli and globular SEs was employed for elicitation experiments.
To prepare the oomycete extract, flasks containing 40 mL liquid ESM medium (Elicitin Secretion Medium 57 ) were inoculated with eight 0.5 cm 2 P. cinnamomi mycelium cuts from outer edge of 1630 strain P. cinnamomi mycelium that had been growing in PDA medium, at 25 °C and darkness during 10 days. ESM medium consisted of 500 mg L −1 KH 2 PO 4 , 250 mg L −1 MgSO 4 ·7H 2 O, 1,000 mg L −1 asparagine, 1 mg L −1 thiamine, 20 mg L −1 glucose   For elicitation, the embryogenic line containing SE at the globular stage was cultured for 3 days in 40 mL ESM medium supplemented with the above-mentioned elicitors in agitation at 100 rpm, 25 °C and darkness; after this period, the embryos were recovered on Whatman filter paper, using Büchner funnels, and transferred to MS/ STS/AC proliferation medium. The effect of elicitation treatments on holm oak embryogenesis was determined as SE growth (fresh weight increase) and SE development (percentage of well-formed immature cotyledonary SEs) 60 days after elicitation in triplicate.

Determination of malondialdehyde.
To assess the potential damage of elicitors on cell membrane integrity, we determined malondialdehyde (MDA) concentration in control and elicited embryos 7 and 30 days after elicitation, as described in 59 . To this end, 0.1 g of embryogenic material was homogenized in 1 mL of 50 mM extraction solution (0.07% NaH 2 PO 4 ·2H 2 O and 1.6% Na 2 HPO 4 ·12H 2 O) and centrifuged at 13,000 rpm for 25 min at 4 °C. After that, 200 µL of the supernatant were added to 800 μL of a solution containing 20% trichloroacetic acid (TCA) and 0.5% thiobarbituric acid (TBA) (both from Sigma-Aldrich, USA). The mixture was heated at 95 °C for 30 min, quickly cooled in an ice bath and centrifuged at 13,000 rpm for 10 min. The absorbance of the supernatant was read at 532 and at 600 nm in an Eppendorf BioSpectrometer. The value for the non-specific absorption at 600 nm was subtracted from the reading at 532 nm. MDA concentration was calculated using the MDA extinction coefficient of 155 mM −1 cm −1 according to 60 . Each treatment was tested in triplicate.
Growth of Phytophthora cinnamomi in dual cultures with elicited holm oak SEs. Globular SEs (0.3 g) from elicited and control material maintained in MS/STS/AC medium for 90 days were incubated in the presence of P. cinnamomi mycelium (0.5 cm 2 ) on 90 mm petri dishes containing SH medium 61 without plant growth regulators. The section of P. cinnamomi mycelium was placed in the middle of the dish (Fig. 7) and the ratio of mycelium growth towards the control and the elicited material was evaluated daily until oomycete hyphae reached SEs (3 days). Therefore, mycelia growth ratios > 1 indicate that the oomycete mycelium spread faster to control samples than to elicited material. Material from each elicitation treatment was tested in triplicate.
Determination of hydrogen peroxide in plant material during dual cultures. As a measure of primary defense response to oomycete's infection, we determined hydrogen peroxide (H 2 O 2 ) formation in both control and elicited SEs after 3 days of dual culture with P. cinnamomi mycelium, according to 42 . Briefly, 0.1 g of elicited or control and infected SEs was homogenized in 1 mL 0.1% (w/v) TCA. The homogenate was centrifuged at 13,000 rpm at 4 °C for 15 min and 250 µL of the supernatant was added to 250 µL 10 mM potassium phosphate buffer (pH 7.0) and 500 µL 1 M potassium iodide. Absorbance of each sample was read at 390 nm and treatments were tested in triplicate.
Statistical analysis. Data were analyzed by one-way analysis of variance (ANOVA), and are presented as mean ± standard deviation of three independent replications. When appropriate, treatment means were separated using Tukey's HSD (honestly significant difference 62 ). Data that not followed a normal distribution were analyzed by the Kruskal-Wallis one-way non-parametric test (k independent samples) or U-Mann-Whitney test (2 independent samples). The arcsine transformation was applied to percentage data prior to analysis. All statistical analyses were performed using SPSS for Windows, version 26 (SPSS Inc., Chicago, IL, USA). License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.