Irradiation of Pinus radiata seedlings with ultraviolet-C (UV-C) radiation or spray application of methyl jasmonate (MeJA) each resulted in induced resistance to subsequent wound inoculation with Diplodia pinea. Induced resistance was expressed by lower incidence of disease and by reduced size of stem lesions than in untreated seedlings. UV-C was more effective than MeJA and the induced resistance was greatest in seedlings that were irradiated with UV-C for 60 min, 1 week before pathogen inoculation. Induced resistance in the UV-C treated stems was concomitant with increases in peroxidase (POX) and polyphenol oxidase (PPO) activity but showed no correlation with concentrations of α-pinene and β-pinene or total phenolics. Furthermore, POX induction in stem tissue was cumulative and was greatest after three repeat treatments with UV-C at 6, 3 and 1 week before inoculation. In contrast to UV-C, MeJA induced a significant increase in β-pinene concentration in stem tissue but did not affect PPO activity. POX activity was induced by MeJA in stems, although to a lesser extent than by UV-C, but was not affected in needles. This appears to be the first report demonstrating the use of UV-C radiation to induce resistance to fungal infection in a coniferous species. The implications of the underlying differences between UV-C- and MeJA-mediated resistance to D. pinea are discussed.
The solar ultraviolet (UV) spectrum is continuous but may be divided into three bands of different wavelength: UV-A (320–400 nm), UV-B (290–320 nm) and UV-C (200–290 nm). UV radiation is highly energetic and can be harmful to living organisms because of its damaging effects on DNA, proteins and lipids (Hollosy, 2002; Trombert et al., 2007). Short-wavelength UV-C radiation is the most energetic and is sometimes described as ‘germicidal UV’ because of its potency against a broad range of microorganisms (Mercier et al., 2001; Fredericks et al., 2011). Plants too are vulnerable to UV radiation and mitigate its damaging effects by accumulating UV-absorbing phenolics and flavonoids in epidermal cells to reduce light penetration and by activating antioxidant defences to limit photo-oxidative damage (Hollosy, 2002; Kasim et al., 2008).
Paradoxically, irradiation with UV-C can be beneficial and studies have shown that exposure to low doses of UV-C (<10 kJ m−2) can suppress postharvest decay in fruits and vegetables including capsicum (Mercier et al., 2001), tomatoes (Charles et al., 2009; Obande et al., 2011), strawberries (Marquenie et al., 2002) and pear (Li et al., 2010). Disease suppression in these cases was attributed to direct germicidal effects of UV-C on surface pathogens and/or to the elevation of host resistance as a result of UV-induced ‘stress’ responses in epidermal tissues. Indeed, many of the protective measures adopted by plants against UV, including phenolics, flavonoids and oxidative enzymes, are commonly associated with plant defence to pathogen attack (Dixon & Paiva, 1995).
In conifers, research on biological effects of short-wavelength UV have focused mainly on acclimatization responses to UV-B (Turunen & Latola, 2005), with apparently no published studies describing the specific effects of UV-C. Conifers exhibit a high degree of tolerance to UV-B (Turtola et al., 2006) and attenuate its penetration by altering needle chemistry, especially the content of UV-absorbing compounds (Turunen & Latola, 2005; Kozlowska et al., 2007; Brzezinska & Kozlowska, 2008). Oxidative stress responses are important to protect cells from reactive oxygen species (ROS) that can be generated when UV radiation penetrates the epidermis, because the photosynthetic apparatus is particularly sensitive to photo-oxidative damage (Laakso & Huttunen, 1998; Laakso et al., 2001). Biochemical antioxidants such as glutathione (Laakso et al., 2001) and oxidative enzymes such as peroxidases and polyphenol oxidase (Bogdanovic et al., 2007; Zu et al., 2011) have been reported to contribute to protection against photo-oxidative stress by quenching of ROS in conifers. Terpenes are highly abundant in conifers and have been identified as potent antioxidants (Graβmann, 2005) and as having a protective role against the harmful effects of UV in other woody species (Zavala & Ravetta, 2002). Concentrations of terpenes in the current-year needles of Scots pine (Pinus sylvestris) and Norway spruce (Picea abies) were not significantly affected by UV-B radiation and it was inferred from this that they may play only a minor role in UV-protection (Turtola et al., 2006). However, further experimental evidence is necessary to better establish the relationship between terpenes and UV stress in conifers.
Many of the biochemical responses affected by short-wavelength UV radiation are also considered to be important defensive components against pathogenic attack; however, no study has specifically investigated the impact of UV-mediated changes on disease resistance in conifers. The aims of the current study were to establish whether irradiation of Pinus radiata seedlings with UV-C (254 nm) would affect their susceptibility to infection by the fungal pathogen Diplodia pinea, and, if so, to determine whether this could be correlated with alterations in the endogenous concentrations of α-pinene, β-pinene and total phenolics, and/or the activities of the antioxidative enzymes peroxidase (POX) and polyphenol oxidase (PPO). A foliar spray with methyl jasmonate (MeJA) was included for comparative purposes because MeJA was previously shown to activate defence mechanisms and to induce resistance to D. pinea in P. radiata (Gould et al., 2008, 2009).
Materials and methods
Pinus radiata seedlings (GF Plus™; Olsen Seeds, PF Olsen and Company Ltd) were propagated in vermiculite in a controlled-environment room at 22°C with a 16/8-h light/dark cycle (250 μmol m−2 s−1). Four weeks after emergence the seedlings were potted into 0·5-L pots containing bark-based potting mix amended with a controlled-release fertilizer (Osmocote Plus®, Daltons) and then grown on for a further 4 months in a controlled-environment room before experimentation.
Seedling treatment and inoculation
In the first experiment, P. radiata seedlings (each 15–20 cm in height) were exposed to UV-C (1·2 kJ m−2) for 15, 30 or 60 min. There were 28 seedlings per treatment and also 28 untreated seedlings. Seven days after treatment the seedlings were inoculated by placing 5 μL suspension of D. pinea (synonym = Sphaeropsis sapinea morphotype A), adjusted to 20 000 spores mL−1, onto a fresh fascicle wound located 2–3 cm from the shoot tip, as described by Gould et al. (2008). The inoculated seedlings were incubated for 48 h under high humidity to encourage infection and maintained thereafter at 22°C with a 16/8-h light/dark cycle (250 μmol m−2 s−1). The incidence of D. pinea infection (visible stem lesion and/or tip dieback) was recorded 7 and 21 days after inoculation. Stem lesion length was recorded using digital callipers.
In the second experiment, P. radiata seedlings were treated with UV-C (1·2 kJ m−2) for 60 min on one occasion, at 1, 3 or 6 weeks before inoculation, or on three occasions at 1, 3 and 6 weeks before inoculation with D. pinea. In addition, another group of seedlings was untreated and one group was sprayed until run-off with a solution containing 4·5 mm MeJA (Sigma-Aldrich, Inc.) in 0·05% Du-Wett® (Elliot Chemicals) 1 week before inoculation. Application of MeJA was performed in a well-ventilated area physically apart from the other treatment groups to avoid cross-contamination. The seedlings were kept separate for 6 h before being regrouped in a randomized block design. Each of the six treatment groups consisted of 46 seedlings: 30 for inoculation and 16 for biochemical analyses. Treatments were scheduled so that all seedling inoculations were carried out on the same day (T = 0) using the method described above. Incidence of D. pinea infection and lengths of stem lesions were recorded 7, 14 and 21 days after inoculation.
Seedlings were harvested at T = 0, as defined above, in order to investigate seedling biochemistry at the time of pathogen challenge. The top 10 cm of each seedling, as measured from the growing tip, was used for analysis. There were four seedlings per replicate and four replicates per treatment. Needle and stem tissues were separated before being snap-frozen in liquid nitrogen and stored at −80°C until analysis.
Frozen tissues were ground in liquid nitrogen and then transferred to Eppendorf tubes for enzyme extraction at a ratio of 150 mg tissue to 750 μL buffer. The extraction buffer contained 100 mm potassium phosphate (pH 7·0) buffer, 7% w/v polyvinylpolypyrrolidone and 2·5% (v/v) Triton X-100. The homogenates were extracted on ice for 2 h, and then centrifuged at 10 500 g for 10 min. The supernatant was used as crude enzyme extract for determination of soluble POX activity and PPO activity.
Enzyme activity was measured in multiwell plates using a Biotek PowerWaveXS plate reader. Guaiacol POX activity was measured using a modification of the method described by Allison & Schultz (2004). The POX substrate, containing 40 mm guaiacol and 10 mm H2O2 in 50 mm phosphate buffer (pH 5·5), was equilibrated in a water bath at 40°C for 30 min before use. The enzyme reaction was initiated by adding 4 μL crude extract to 196 μL POX substrate. The multiwell plates were then shaken for 5 s and POX activity determined by measuring the change in absorbance at 470 nm over a 5-min period at 40°C. Polyphenol oxidase activity (PPO) was measured using a modification of the method described by Bogdanovic et al. (2007). The PPO substrate, containing 40 mm catechol in 50 mm phosphate buffer (pH 6·5), was equilibrated in a water bath at 40°C for 30 min before use. The enzyme reaction was initiated by adding 7 μL crude extract to 193 μL PPO substrate. The multiwell plates were then shaken for 5 s and PPO activity determined by measuring the change in absorbance at 410 nm over a 5-min period at 40°C. The extraction buffer plus the appropriate substrate was used as the blank for each enzyme assay.
Total phenolics and terpenoid analysis
Frozen needle and stem samples were ground separately in liquid nitrogen and then stored at −80°C until analysis. Total soluble phenolics were extracted by placing 100 mg powdered tissue into an Eppendorf tube containing 1 mL 95% ethanol. Samples were mixed thoroughly using a vortex and then stored 24 h in the dark at 4°C. Solid material was removed by centrifugation at 10 500 g and 5 μL supernatant was mixed with 545 μL milli-Q water and 100 μL Folin-Ciocalteu reagent (Sigma-Aldrich). After 5 min, 100 μL saturated solution of sodium carbonate was added and the reaction mix was incubated for 90 min at room temperature before determination of absorbance at 640 nm using a plate reader. Phenolics content was quantified against a standard curve prepared using gallic acid.
Terpenoid extraction was performed using 3 : 1 n-hexane:diethyl ether containing 0·1 mg mL−1 anethole as an internal standard as described by Gould et al. (2009). The target analytes, α-pinene, β-pinene and anethole, in sample extracts were analysed by gas chromatography–mass spectrometry (GCMS) using an Agilent 6890 gas chromatograph coupled to an Agilent 5975 MSD. The injector was held at 250°C and 1 μL sample extracts and calibration standards were injected by autosampler using split injection and a split ratio of 1 : 50. Chromatographic separation was achieved using a J&W DB5-MS glass capillary column (30 m × 0·250 mm internal diameter × 0·25 μm film thickness). Helium carrier gas was maintained at a flow rate of 1 mL min−1. The GC oven was programmed at 50°C (1 min), then increased to 120°C at 4°C min−1, followed by 50°C min−1 to 200°C (with 5·0 min hold at final temperature). The GCMS interface was held at 280°C and the mass spectrometer source and quadrupole temperatures at 230°C and 150°C, respectively. Calibration standards for each compound, ranging from 10 to 1000 μg mL−1, were analysed together with the sample extracts. The three target analytes were identified by comparison of retention times against certified standards and comparison of total ion mass spectra against the NIST mass spectral database. The concentrations of the three target analytes were determined by extracting compound-specific mass ions and comparing the relative abundance of the four mass ions against those obtained from pure compound standards. Quantification of target analytes was completed by internal standard quantitation, with anethole as the internal standard, and analysis was performed using Agilent MSD Enhanced Chemstation software. All chemicals and reagents were obtained from Sigma-Aldrich unless otherwise stated.
The data were analysed in GenStat, v. 13 (VSN International). No transformations of the data were required. Lesion, score and biochemistry data at each time point were analysed with a randomized block analysis of variance. The proportions of plants showing incidence of disease were compared using a regression analysis of binomial data.
Induction of resistance to Diplodia pinea in Pinus radiata seedlings by UV-C radiation
Effect of exposure time
The irradiation of P. radiata seedlings with UV-C resulted in a dose-dependent induction of resistance to D. pinea (Table 1). By 7 days after inoculation, the seedlings treated with UV-C exhibited a lower incidence of disease (P <0·05) and smaller stem lesions than the untreated seedlings. However, by 21 days after inoculation these differences remained only for seedlings that had been exposed to UV-C for 60 min. Seedlings that had been treated with UV-C for 30 min had smaller stem lesions but a D. pinea incidence similar to seedlings in the control group, whilst those treated for 15 min no longer exhibited any significant differences (P >0·05).
Table 1. Development of stem lesions and dieback incidence, caused by Diplodia pinea, in Pinus radiata seedlings irradiated once with UV-C 1 week before pathogen inoculation
Values followed by the same letter are not significantly different (P <0·05).
Standard error of difference
Duration of UV-C-induced resistance
Pinus radiata seedlings that were irradiated with UV-C for 60 min at 1, 3 and/or 6 weeks before inoculation with D. pinea expressed a slower onset of infection than untreated seedlings. The most effective treatment (P <0·05), UV-C at 1 week before pathogen inoculation, recorded the lowest incidence (Fig. 1) and the smallest stem lesions (Fig. 2) on each assessment date. By 3 weeks after pathogen inoculation, D. pinea incidence in this UV-C group was 50% compared with 97% in the untreated seedlings, whilst stem lesion length was 5 and 41 mm for the two groups, respectively. The irradiation of seedlings with UV-C at 3 weeks before D. pinea inoculation significantly reduced diplodia incidence for at least 2 weeks post-inoculation and reduced stem lesion growth (P <0·05) for at least 3 weeks when compared with untreated seedlings. Exposure of seedlings to UV-C radiation 6 weeks before D. pinea inoculation did not affect disease incidence but did reduce stem lesion growth for up to 3 weeks post-inoculation (P <0·05) when compared with untreated controls. Disease incidence and lesion length in seedlings that were exposed to UV-C at 6, 3 and 1 week before inoculation was not different to that observed in seedlings after a single treatment with UV-C at 1 week before inoculation. Foliar spray application of MeJA promoted resistance to D. pinea in P. radiata seedlings, but disease incidence was greater (P <0·05) than in seedlings that were irradiated with UV-C 1 week before pathogen inoculation.
Peroxidase and polyphenol oxidase activity in treated seedlings
Peroxidase activity was significantly elevated (P <0·05) in the stems, but not in the needles, of seedlings 1, 3 or 6 weeks following UV-C irradiation, compared with the untreated control (Fig. 3). Similarly, spray application of MeJA induced an increase in POX activity in stems (P <0·05) but not in needles 1 week after treatment compared with the untreated control. The strongest induction in both needles and stems occurred after repeated UV-C exposure at 1, 3 and 6 weeks before measurement, resulting in significantly greater POX activity (P <0·05) than that determined in all other treatments.
Polyphenol oxidase activity was elevated (P <0·05) in stems 1 and 3 weeks after UV-C irradiation, compared with untreated controls, but was not significantly different by 6 weeks after treatment (Fig. 4). Repeat treatment with UV-C did not have a cumulative effect on PPO activity in stems. None of the treatments affected PPO activity in needles.
Concentrations of α-pinene, β-pinene and total phenolics in treated seedlings
None of the UV-C treatments produced statistically significant differences in the concentrations of α-pinene, β-pinene and total phenolics in the stems or needles of P. radiata seedlings (Table 2). Spray application of MeJA resulted in a significant increase (P <0·05) of β-pinene in the needles and stems, and of total phenolics in needles, compared with the untreated control.
Table 2. Effects of UV-C radiation on concentrations of α-pinene, β-pinene and total phenolics in needles and stems of Pinus radiata seedlings
Irradiation with UV-C has been used successfully to extend shelf life and to reduce postharvest disease in fruits and vegetables (Vicente et al., 2005; Charles et al., 2009; Artes-Hernandez et al., 2010; Li et al., 2010; Obande et al., 2011). These beneficial effects arise via two different mechanisms: the direct germicidal activity of UV-C on pathogenic surface microbes, and the induction of host resistance as a result of UV-induced stress responses in treated tissues (Mercier et al., 2001; Stevens et al., 2006; Obande et al., 2011). In the current study, irradiation of P. radiata seedlings with UV-C resulted in a dose-dependent enhanced resistance to wound inoculation with D. pinea, whereby treated seedlings exhibited smaller stem lesions and a reduced incidence of tip dieback than untreated seedlings. Because the pathogen was not directly exposed to UV-C, it is proposed that the response was attributable solely to induced resistance.
UV-C induced resistance in fruits and vegetables has been correlated with the activation of various host defences, including the accumulation of phenolic and flavonoid compounds and the stimulation of antioxidant mechanisms (Stevens et al., 2006; Kasim et al., 2008; Obande et al., 2011). These biochemical defence responses, although offering cross-resistance to pathogenic attack, are primarily stress-induced reactions that alleviate damage to cells by absorbing UV radiation and by scavenging ROS (Dixon & Paiva, 1995). Effects of UV-C on conifers are not well described; however, several studies have demonstrated that exposure to UV-B can alter the content of UV-absorbing compounds, including phenolics, flavonoids and terpenes, in epidermal tissues. It is difficult to generalize about UV-B-induced effects in conifers because they can vary depending upon species, age and environment. For example, supplemental UV-B radiation promoted phenolics accumulation in needles of Scots pine and Norway spruce grown under glass (Lavola et al., 2003) but did not affect concentrations of phenolics or terpenes in outdoor plants (Turtola et al., 2006). Conversely, outdoor western yellow pine (Pinus ponderosa) accumulated phenolics in response to UV-B radiation (Warren et al., 2002), whereas flavonoid content decreased in glasshouse-grown Korean pine (Pinus koraiensis) following exposure to UV-B radiation (Zu et al., 2011).
Phenolics and terpenes are considered to be important defence compounds in conifers (Krokene et al., 2008; Eyles et al., 2010) and yet few attempts have been made to correlate UV-mediated changes in tissue chemistry with disease resistance (Manning & Vontiedemann, 1995; Witzell & Martin, 2008). In the current study, UV-C irradiation promoted strong resistance to D. pinea but did not affect the concentrations of total phenolics or α-pinene and β-pinene in the needles or stems of treated seedlings. This may suggest that increases in the constitutive concentrations of these compounds are not necessary for UV-C-induced resistance to D. pinea. The role of phenolics in induced resistance to forest pathogens has been shown to be quite variable depending upon the pathogen and the environment (Witzell & Martin, 2008). However, results from studies on Austrian pine (Pinus nigra) indicate that phenolics do not play a major role in induced resistance to D. pinea (Bonello & Blodgett, 2003; Blodgett et al., 2007). In keeping with this, foliar spray application of MeJA in the current study, like UV-C, promoted resistance to D. pinea without affecting the concentrations of total phenolics in the stems. However, in contrast to UV-C, induced resistance in the MeJA-treated seedlings was accompanied by an increase in the concentration of β-pinene in stems. This confirms previous results in P. radiata (Gould et al., 2009; Reglinski et al., 2012) and is consistent with evidence that MeJA is a potent elicitor of terpene-based defences, particularly in relation to resin flow at wound sites in conifers (Hudgins et al., 2004; Moreira et al., 2009). The data reveal an interesting divergence in the underlying modes of action of UV-C-induced resistance and MeJA-mediated resistance. Further analyses of the enantiomeric profiles of α-pinene and β-pinene in treated seedlings are warranted because changes in enantiomer ratios have been shown to accompany D. pinea infection in Italian stone pine (Pinus pinea) (Bonello et al., 2008).
Oxidative enzymes can protect plant cells against photo-oxidative damage by scavenging ROS that are generated by UV-induced stress. In conifers, POX and PPO have been reported to protect photosynthesizing cells in needles by quenching ROS generated by UV-B (Laakso & Huttunen, 1998; Laakso et al., 2001; Zu et al., 2011). These enzymes can also catalyse the oxidation of monolignols, a key step in lignin biosynthesis, and hence play an important role in lignification processes at wound sites (Whetten et al., 1998). In the current study, irradiation with UV-C did not significantly affect the activity of PPO in needles, whilst needle POX activity was significantly increased only after three repeat treatments, suggesting a cumulative response to UV-C irradiation. However, there was no observable sign of photo-oxidative damage in treated seedlings after the repeat treatments. In stems, there was a significant increase in both POX and PPO activities after UV-C irradiation and, in the case of POX, the effect was cumulative and was greatest after three repeat treatments. These data indicate a stronger response to UV-C in stem tissues than in needles and this may be because needles are naturally better equipped to shield out harmful UV; indeed the needles contained higher constitutive concentrations of soluble phenolics than stems, which would be consistent with this hypothesis. Moreover, it may be that the greater vulnerability of stem tissues to UV-C radiation resulted in a stress-induced stimulation of POX and PPO activity, thereby facilitating more rapid and extensive lignification at the wound site to restrict the ingress and spread of D. pinea. In contrast, MeJA promoted only a slight increase in POX in stems and did not affect PPO activity in either tissue. Curiously, the MeJA-induced responses recorded in this study were weaker than those reported previously, where, for example, MeJA promoted a two- to threefold increase in enzyme activity over the same period (Gould et al., 2009). However, it is worth noting that Gould et al. (2009) raised concerns that volatiles from wounded seedlings in that study were causing defence elicitation in untreated controls; therefore, it is conceivable that the MeJA-treated seedlings were similarly affected and so the measured enzyme induction was in fact an additive response to MeJA and airborne volatiles.
In conclusion, this study has shown that irradiation of P. radiata seedlings with UV-C resulted in enhanced resistance to stem inoculation with D. pinea. The UV-C-induced resistance was concomitant with increased activities of POX and PPO in stems, which, it is proposed, catalysed more rapid lignification around the inoculation site to restrict pathogen entry and spread. Exposure to UV-C radiation did not significantly affect concentrations of phenolics or terpenes. Conversely, MeJA-induced resistance was associated with β-pinene accumulation and a moderate increase in POX in treated stems.
Thanks to Catherine Cameron for statistical analyses and to Drs Philip Elmer, Stephen Hoyte and Nick Gould for helpful comments. Thanks also to NZ Foundation for Research, Science and Technology (LINX0804 ‘Ecosystem Bioprotection’) for financial support.