Effi cacy of Heat-based Treatments in Eliminating the Recovery of the Sudden Oak Death Pathogen (Phytophthora ramorum) from Infected California Bay Laurel Leaves

Phytophthora ramorum (Oomycota) (Werres et al., 2001) is the plant pathogen responsible for the lethal disease of several oak species in California known as sudden oak death. The pathogen also causes a foliar disease on Umbellularia californica (bay laurel or simply bay). Bay leaves have been identifi ed as the major source of natural inoculum in California coastal woodlands. Because of the epidemiological relevance of bay leaves, their movement needs to be regulated. Our study shows that P. ramorum is highly heat tolerant and can be reisolated from artifi cially inoculated bay laurel leaves placed at 55 °C for up to 1 week. The pathogen cannot be recovered after 2 weeks at 55 °C. Prolonged heat treatments, however, are impractical for bay leaves intended to be sold commercially as a spice, since they negatively impact the quality of the leaves. Here we describe a treatment involving a progressive and gradual heating process combined with the application of moderate vacuum. This method can be completed in 22 hours and is shown here to eliminate the recovery of P. ramorum without having a negative impact on the quality of the bay leaves. The worldwide trade of agricultural crops and ornamental plants represents an increasing threat to natural ecosystems. Traded plants or even plant parts represent an ideal medium for the long-distance transport of pathogens, potentially resulting in the introduction of exotic microbes in new environments, where their aggressiveness on native plants may be extremely high due to the lack of coevolution (Wingfi eld et al., 2001). The Oomycete Phytophthora ramorum (Werres et al., 2001) is a major pathogen of native plant communities in California and southern Oregon, and causes the disease commonly referred to as sudden oak death (SOD). This pathogen was recently identifi ed as the primary agent causing SOD (Garbelotto et al., 2001; Rizzo et al., 2002). The same organism was also recently described as a pathogen of rhododendron and viburnum in European nurseries (Werres et al., 2001). The origins of this pathogen are still unknown. The low genetic diversity identifi ed in the U.S. population by amplified fragment-length polymorphism (AFLP) analyses (Garbelotto et al., 2003a), and the limited geographic distribution of the pathogen, which is signifi cantly smaller than the natural range of distribution of its plant hosts, suggest it may be an introduced organism (Rizzo and Garbelotto, 2003). The host range of P. ramorum is extremely broad and includes species native not only to California, but to many world regions (Davidson et al., 2003). The high tree mortality caused by P. ramorum in coastal California, combined with the limited knowledge on this pathogen’s biology and epidemiology, have prompted regulatory agencies from the European Union, South Korea, Canada and the U.S. to implement quarantines against the movement of plant material or soil that may be at risk of harboring the pathogen (Garbelotto et al., 2003a; USDA, 2002) Umbellularia californica (Lauraceae) (bay laurel, bay, oregon myrtle, pepperwood tree) is a widespread and abundant tree native to California and southern Oregon. This species is the most common host for P. ramorum in California (Rizzo and Garbelotto, 2003). The symptoms caused by P. ramorum on bay laurel are limited to a foliar blight (i.e., dead spots or dark blotches on the leaves), and there have been no reports of death of infected trees (Davidson et al., 2002; Garbelotto et al., 2002). Nevertheless, infected bay leaves play a key epidemiological role in the spread of SOD (Davidson et al., 2002). Two studies have shown a signifi cant relationship between the presence of bay laurel and the presence of SOD on neighboring oak trees (Kelly and Meentemeyer, 2002; Swiecki and Bernhardt, 2002). Sporulation of P. ramorum on bay leaves is extraordinarily prolifi c. Even after several weeks dry leaves may support sporulation if rehydrated (Rizzo and Garbelotto, 2003) Sporangia and chlamydospores are produced by P. ramorum either on the surface of bay leaves or embedded within the leaf’s mesophyll (Jennifer Parke, personal communication). P. ramorum sporangia are microscopic ovoid structures that can be airborne (rain splash, wind-driven rain). Upon landing on wet surfaces, mature sporangia may germinate and directly infect plant tissue or release a swarm of motile zoospores which will infect plant tissue (Davidson et al., 2002). The role played by the round-shaped thick-walled microscopic chlamydospores is still unclear, but they are thought to serve as resting propagules, allowing the pathogen to survive during climatically unfavorable spells similar to other Phytophthora spp. (Erwin and Ribeiro, 1996). The leaves of bay laurel are used for culinary purposes as a spice. Dried plant material is distributed locally or exported nationally and internationally. Since leaf collecting occurs in California and southern Oregon, infected leaves could potentially be placed on the market. An excess of 5000 t of california bay laurel leaves, worth several million dollars, is placed on the market yearly (James Lewis, personal communication). Because of the diffi culty in diagnosing early symptoms, visual screening of processed leaves will not prevent trading of infected, but apparently healthy, leaves. An alternative approach to the visual screening of plant materials involves the generalized application of a treatment that is capable of eliminating the recovery of P. ramorum from infected leaves. Leaves used as spices are normally preserved through drying. The drying process is critical not only to prevent molding, but also to inhibit chemical reactions that would otherwise reduce the shelf life of the product (King et al., 2000, Madamaba and Liboon, 2001). The purpose of this paper is to test whether a treatment already in use by a commercial provider of bay leaves (here defi ned as Treatment 3) may be considered as a viable sanitation tool for infected bay leaves. Three additional treatments were also tested and are discussed in this article. Materials and Methods Heat treatments. Asymptomatic bay leaves were collected from 10 different trees on the campus of the University of California at Berkeley so as to account for differences in individual tree susceptibility. Leaves were inoculated in the lab using an agar slurry previously infected with the pathogen strains Pr 1 (coast live oak, Marin county), Pr 2 (Tanoak, Marin county) and Pr 5 (Tanoak, Marin county). Leaves were scratched with a needle before being inoculated to favor infection by HORTSCIENCE 39(7):1677–1680. 2004. Received for publication 23 Dec. 2003. Accepted for publication 15 Mar. 2004. The critical comments on the manuscript by D. Rizzo and W. Schweigkofl er were much appreciated. We also thank G. S. Biging for advice on the statistical analysis. This work is dedicated to the memory of Jamie Westoby. Part of this study was funded by the USDA award 02-JV11272138-066 granted through the Pacifi c Southwest Forest/Range Experiment station. E-mail [email protected]. Bayseng Spice Company, 21 Old Tunnel Road,