Raffaelea quercivora* Scientific Name Raffaelea quercivora Kubono & Shin. Ito Synonyms: none known Common Names Japanese oak wilt Type of Pest Fungus Taxonomic Position Kingdom: Fungi, Phylum: Ascomycota, Order: Ophiostomatales, Family: Ophiostomataceae Reason for inclusion in manual Severe plant pathogen vectored by Platypus quercivorus, a CAPS Priority Pest (FY 2007) Pest Description Fig. 1. White arrows point to oak (Quercus serrata) wilting due to Raffaelea quercivora in Kyoto Prefecture, Japan. [Image courtesy of K. Kuroda, http://cse.ffpri.affrc.go.jp/keiko/hp/oakwilting-overview.html] The genus Raffaelea was named in 1965 by von Arx and Hennebert and amended two years later by Batra (1967) for ambrosia fungi that create sporodochia (small compact * This document is largely excerpted from the report: Kromroy, K.W., and R.C. Venette. 2005. Mini-Risk Assessment: Japanese oak wilt, caused by Raffaelea quercivora Kubono & Shin. Ito. pp 340-371 In R.C. Venette, E.E. Davis, K.W. Kromroy, and S. French. Exotic Pests that Threaten US Forest and Agriculture: Pest Risk Assessments for the Cooperative Agricultural Pest Survey. USDA Forest Service, Internal Report. Available from R.C. Venette ([email protected]). cushion-shaped masses of somatic hyphae covered with conidiophores, Fig. 2). Members of the genus produce terminal conidia acropetally (youngest conidium is at the tip or distal end of the chain) (Ulloa and Hanlin 2000) and sympodially (conidia are produced on a conidiophore that terminates and branches repeatedly, producing a zigzag pattern) (Ulloa and Hanlin 2000). In fungal taxonomy, each species is typified by one specimen (a collection, a culture – dried or otherwise preserved in a metabolically inactive state, microscope slide mount) (Alexopoulus et al. 1996), and a “type species” is the basis for description of a genus. Raffaelea ambrosiae is the type species for Raffaelea (von Arx and Hennebert 1965). Sporodochia Fig. 2. Arrows point to sporodochia (small white pustules) growing on a cross section of a maple. [Reproduced from Batra (1967).] A sexual stage of Raffaelea has not been found. Based on numerous similarities to other asexual forms of fungi for which the sexual form is known, Raffaelea is classified in the Ascomycetes (Kirk et al. 2001). The morphology and growth habit of spore- producing hyphae (called conidiomata); the shape, septation and color of spores; and the processes involved in conidial production are used to classify asexual fungi (Kirk et al. 2001). Molecular tools provide other evidence of taxonomic identity. Results of a cladistic analysis of 18S ribosomal DNA sequences showed that seven of eight species of Raffaelea appear to be phylogenetically related to the genus Ophiostoma (Jones and Blackwell 1998). This work occurred prior to the description of R. quercivora. Formal description of the genus Raffaelea and type species R. ambrosiae. “Sporodochia superficial, effuse, confluent (coalescent), fertile, lush. Conidiophores in fascicles, straight, septate, with a thick base that tapers toward the apex. Conidia originating in succession from the apex of the conidiophore, obovoid, with a conical truncated base, single-celled, hyaline. Aleuriospore solitary, arising from the apex of a septate hypha, globose to even turbinate, single-celled, hyaline, thick covering. Raffaelea ambrosiae is the type species, found in Platypus cylindrus FAB. in Quercus, in England. Type culture in dried agar agaro dessicata CBS 185.64est” (Translated from von Arx and Hennebert (1965)). Conidia Conidiophores Aleuriospore Fig. 3. “Raffaelea ambrosia. Conidiophores with conidia and aleuriospores” [Reproduced from von Arx and Hennebert (1965).] The original description provided by von Arx and Hennebert was based on examination with a light microscope. In a recent examination of R. ambrosia conidial development using scanning electron microscopy, Gebhardt and Oberwinkler (2005) report annellidic percurrent proliferation rather than sympodial production. Raffaelea quercivora. “Colonies on [potato dextrose agar] PDA at 20–25°C [68-77°F] 1 effuse, spreading rapidly, reaching 80 mm [~3 /8 in] diameter in 5 days with an indistinct white margin, appearing water-soaked and mucilaginous; aerial mycelium abundant, floccose, composed of branched, septate, hyaline, smooth hyphae, arranged in fascicles and simulating coremia, reaching 1 cm high; color diffusing and turning pale olive to brown-olive after 2 weeks; odor fragrant, resembling that of ethyl alcohol (Fig. 4). Sporodochia of several fascicles, becoming confluent and mucilaginous. Conidiophores macronematous, mononematous, formed in sporodochia or produced separately, simple or branched, straight, hyaline, septate, smooth, 16.5–22.5 x 0.9– 1.5µm (Fig. 5). Conidiogenous cells gradually narrowed toward the apex, polyblastic, sympodial, indeterminate, discrete or integrated, terminal, hyaline, smooth, with a series of flat, scarcely protruding scars situated toward the apex (Figs. 6B and 7C). Conidia short-clavate slimy, borne in acropetal order, hyaline, aseptate, straight, smooth, eguttulate, obovoid to pyriform, tapered markedly toward the truncate base, apex obtuse, frequently yielding sprout cells, formed in droplets, 3.1–4.7 x 2.0–2.4µm” (Figs. 6A and 6C, 7A and 7B)” (Kubono and Ito 2002). Aleuriospores were not described for this species. Fig. 4. “Raffaelea quercivora colony Fig. 5. “Raffaelea quercivora with sporulation on potato dextrose conidiophores and conidia on PDA.” agar (PDA) at 20º C after 10 days.” [Photo and legend from Kubono and Ito [Photo and legend from Kubono and Ito (2002)]. (2002).] Fig. 6. “Raffaelea quercivora. A. Conidia. B. Conidiogenous cell with holoblastic proliferation and cicatricial scars. C. Conidia and conidiophores; one at the left and one at the center showing conidia in droplet. Bars A, B are 1 µm, C is 5 µm.” [Drawings and legend from Kubono and Ito (2002).] A B C Fig. 7. “Raffaelea quercivora on PDA observed by SEM. A. Conidiogenous cell with holoblastic proliferation and conidia. Bar 1µm. B. Conidia and sprout cell (arrow) produced from conidia. Bar 1µm. C. Conidiogenous cell with a series of flat cicatricial scars (arrows). Bar 10 1µm.” [Photos and legend from Kubono and Ito (2002).] Biology and Ecology Raffaelea quercivora is one of many species of ambrosia fungi, so-called because of their symbiotic relationships with some species of wood boring Scolytidae and Platypodidae. Most often associated with recently logged timber and trees of low vitality, ambrosia fungi line the tunnels and galleries created in the sapwood and heartwood by their associated insects with a continuous layer of hyphae and conidia- bearing conidiophores (Batra 1967). The insect relies on the fungus for nourishment during at least some its life stages, meanwhile providing the fungus with protection and means of dispersion and inoculation (Baker 1963, Kinuura 2002). Similar to other ambrosia fungi, R. quercivora has not been found apart from the insect, Platypus quercivorus (Coleoptera: Platypodidae). Thus, the life cycle of the fungus in nature is intimately related to the life cycle of this insect (Fig. 8). The reciprocal is not necessarily true. Conidia of R. quercivora are carried by P. quercivorus and contact the plant host when the insect bores into the sapwood and heartwood of the tree (Kinuura 2002). Conidia may germinate directly, or produce sprout cells which germinate and elongate into septate hyphae (Fig. 8A) (Kubono and Ito 2002). Hyphae grow in the tunnels and galleries excavated by the beetle (Fig. 8B), and eventually line the entire surface (Kinuura 2002). Hyphae may aggregate into bundles, called fascicles (Fig. 8C), which may further aggregate into sporodochia (see ‘Pest Description’) from which conidiophores develop (Fig. 8D) and produce conidia (Fig. 8E). While providing nourishment for the insect, the fungus continues to grow, and hyphae extend into the wood adjacent to the tunnel. The creation of insect galleries and the presence of the fungus stimulate defense responses from the tree, which include the production of tyloses (parenchyma cell overgrowths) that extend into the xylem and prevent the ascent of water (Yamada and Ichihara 2003). In the meantime, the female insect lays eggs, which hatch in about a week. Larvae feed on the fungus until they pupate. Young adults may emerge in autumn (October, November), allowing for the possibility of a second generation that year, or remain in the tree over the winter and emerge in the spring (Sone et al. 1998). Young adults consume and acquire the fungus in their mycangia. When they emerge from the tree, they carry the fungus. The insects move on to another tree and bring the pathogen to a new host. The fungus may go through many life cycles from the time it is introduced into the tree until the time the next generation of insects emerges with the fungus. Adults emerge from pupa, Adult beetles carrying conidia bore into a tree, feed on the fungus, then creating tunnels in the sapwood and heartwood emerge from the tree carrying conidia Final instar pupates Conidiophores Conidia produce sprout cells which E) produce conidia germinate into hyphae A) Some hyphae Hyphae grow on the surface of become beetle tunnels and into the wood conidiophores D) B) Eggs hatch and larvae feed on the fungus in Hyphae may Adult beetles feed on the galleries through aggregate into the fungus while all instars bundles making galleries in (fascicles), and the wood form sporodochia C) Adult females lay eggs in the