Chemistry of Coast Live Oak Response to Phytophthora ramorum Infection

Since the mid 1990s, Phytophthora ramorum has been responsible for the widespread mortality of tanoaks, as well as several oak species throughout California and Oregon forests. However, not all trees die, even in areas with high disease pressure, suggesting that some trees may be resistant to the pathogen. The apparent resistance to P. ramorum infection of some individuals within coast live oak populations has been observed in artificial inoculation studies. For example, from artificial branch-cutting inoculation trials, Dodd and others (2005) found significant variation (up to eightfold difference in lesion sizes) in susceptibility to P. ramorum. In addition, apparent resistance has also been observed in naturally infected forests, where a number of coast live oaks have survived for more than seven years despite being infected (McPherson and others 2005 and unpublished data). Elevated levels of secondary metabolites, specifically phenolic compounds in infected tissue, are often associated with resistance to fungal pathogens in angiosperms (Bennett and Wallsgrove 1994; Ostrofsky and others 1984). It is possible that these apparently resistant coast live oaks may have increased amounts of phenolic compounds in the P. ramorum infected tissue, which is inhibiting the growth of the pathogen. However, there are no reports that describe the changes in secondary metabolites of coast live oaks infected with P. ramorum. To date, the majority of studies investigating phenolic chemistry in oak have focused on constitutive wood and foliage chemistry. Three field experiments were carried out in Deer Island and China Camp State Park, CA between December 2004 and September 2005 on large trees (DBH approx. 28 to 69 cm). Trees were either artificially inoculated (experiments 1 and 3) or naturally infected with P. ramorum (experiment 2). Phloem was sampled from the margin of active lesions and also from healthy phloem at least 60 cm away from lesion margin (AFC, away from cankers) of some of the same inoculated trees and from apparently healthy trees. Phenolics were extracted in methanol, identified by HPLC-mass spectrometry or matched to standards and quantified by HPLC-UV analysis. Nine phenolic compounds (gallic acid, catechin, tyrosol, a tyrosol derivative, ellagic acid and four ellagic acid derivatives) were analyzed in this way. Significant differences in phenolic profiles were found between phloem sampled from the active margins of cankers, healthy phloem from asymptomatic trees, or AFC phloem, 1 A version of this paper was presented at the Sudden Oak Death Third Science Symposium, March 5–9, 2007, Santa Rosa, California. 2 Department of Plant Pathology, The Ohio State University, 201 Kottman Hall, 2021 Coffey Road, Columbus, OH 43210, USA. 3 Center for Forestry and Integrated Hardwood Rangeland Management Program, Department of Environmental Science, Policy, and Management, 145 Mulford, University of California, Berkeley, CA 94720, USA. 4 Dept. of ESPM, Div. of Organisms and the Environment, 201 Wellman Hall, University of California, Berkeley, CA 94720, USA. Corresponding author: P. Bonello, [email protected]. GENERAL TECHNICAL REPORT PSW-GTR-214 158 although the magnitude and direction of the responses was not consistent across all experiments. Concentrations of gallic acid, tyrosol, and ellagic acid showed the greatest differences in these different tissues, but varied considerably across treatments. Specifically, significantly greater amounts of ellagic acid and gallic acid were observed in infected phloem than non-infected phloem in experiments 1 and 2. In contrast, significantly higher amounts of tyrosol and ellagic acid were present in infected phloem than in corresponding controls in experiment 3 (tables 1-3). Interestingly, catechin levels were significantly reduced in infected tissue in two of the three experiments, i.e. experiments 1 and 3. Table 1—Effect of artificial P. ramorum inoculation on the concentration of nine phenolic compounds extracted from the phloem of coast live oak in experiment 1 Experimental factors* Compound Infected (N = 5) Healthy (N = 5) Gallic Acid 1.97 (0.93) a 0.09 (0.03) b Tyrosol 0.91 (0.35) 1.01 (0.18) TY1 2.88 (0.77) 4.08 (0.69) Catechin 0.63 (0.16) a 2.87 (0.35) b EA1 0.11 (0.05) 0.39 (0.16) EA2 0.03 (0.01) a 0.37 (0.17) b Ellagic acid 1.47 (0.69) a 0.02 (0.01) b EA3 0.11 (0.06 0.35 (0.09) EA4 0.09 (0.06) 0.10 (0.02) *All concentrations expressed as mg/g fresh weight (SE). x Compound quantified in terms of tyrosol equivalents. y Compounds quantified in terms of ellagic acid equivalents. Values in each row followed by different letters are significantly different (P < 0.05). Table 2—Effect of natural P. ramorum infection on the concentration of nine phenolic compounds extracted from the phloem of coast live oak in experiment 2 *All concentrations expressed as mg/g fresh weight (SE). Compound quantified in terms of tyrosol equivalents. Compounds quantified in terms of ellagic acid equivalents. Values in each row followed by different letters are significantly different (P < 0.05). Experimental factors* Compound Infected (N = 7) AFC (N = 7) Healthy (N = 5) Gallic Acid 0.94 (0.29) a 0.09 (0.02) b 0.07 (0.01) b Tyrosol 0.82 (0.25) 1.13 (0.20) 1.26 (0.17) TY1 2.89 (0.56) 2.71 (0.34) 2.58 (0.23) Catechin 3.52 (0.59) 3.32 (0.54) 2.07 (0.19) EA1 0.36 (0.13) 0.43 (0.15) 0.32 (0.06) EA2 0.15 (0.05) 0.31 (0.16) 0.14 (0.05) Ellagic acid 0.16 (0.05) a 0.04 (0.02) b 0.04 (0.02) ab EA3 0.21 (0.10) 0.20 (0.12) 0.10 (0.10) EA4 0.13 (0.06) 0.15 (0.07) 0.09 (0.08) Proceedings of the Sudden Oak Death Third Science Symposium 159 Table 3—Effect of artificial P. ramorum inoculation on the concentration of nine phenolic compounds extracted from the phloem of coast live oak in experiment 3 Experimental factors* Compound Infected AFC Gallic Acid 0.42 (0.30) 0.47 (0.21) Tyrosol 2.23 (0.33) a 0.75 (0.57) b TY1 2.19 (0.87) 4.25 (1.32) Catechin 1.18 (0.34) a 3.84 (0.88) b EA1 0.21 (0.13) 0.40 (0.16) EA2 0.18 (0.12) 0.37 (0.18) Ellagic acid 0.53 (0.16) a 0.04 (0.01) b EA3 0.07 (0.03) 0.23 (0.10) EA4 0.14 (0.06) 0.12 (0.05) *All concentrations expressed as mg/g fresh weight (SE). x Compound quantified in terms of tyrosol equivalents. y Compounds quantified in terms of ellagic acid equivalents. Values in each row followed by different letters are significantly different (P < 0.05). The soluble phenolic compounds identified in the phloem extracts of infected coast live oak have been implicated as playing key roles in defense against fungi and herbivores in many woody species, including other Quercus spp. (Feucht and Treutter 1999; Malterud and others 1985, Pearce 1996). For example, the durability of some hardwood species against microbes has been directly attributed to the elevated presence of hydrolysable tannins (Barry and others 2001, Hillis 1999, Klumpers and others 1994, Vivas and others 2004). The antifungal activities of gallic acid (2, 3, 6 and 10 mg/mL) and tyrosol (1, 2, 3 and 6 mg/mL) were tested against P. ramorum, P. cinnamomi, P. citricola, and P. citrophthora in vitro. Both compounds showed strong dose-dependent inhibitory effects against all four species (fig. 1). In conclusion, this study demonstrated clear host secondary metabolite responses that may be implicated in resistance of coast live oak to attack by P. ramorum. Further studies involving correlation of compound concentrations with disease resistance in planta will be necessary to establish a potential defensive role for any of these compounds. If such a role is established, then some of these compounds could be used as biomarkers in the selection of resistant coast live oak genotypes. These studies, however, are contingent on developing reproducible techniques that can be used routinely to obtain quantitative measures of host resistance (e.g. Blodgett and others. 2007), which are lacking at present.