Phytoparasitica (2012) 40:235–238 DOI 10.1007/s12600-012-0223-7

An Asian ambrosia and its novel symbiotic fungus sp. pose a serious threat to the Israeli industry

Z. Mendel & A. Protasov & M. Sharon & A. Zveibil & S. Ben Yehuda & K. O’Donnell & R. Rabaglia & M. Wysoki & S. Freeman

Received: 10 November 2011 /Accepted: 27 January 2012 /Published online: 15 February 2012 # Springer Science+Business Media B.V. 2012

Abstract The , Euwallacea fornicatus The ambrosia beetle, Euwallacea fornicatus Eichhoff Eichhoff, was first recorded in Israel in 2009. The (Coleoptera: , Scolytinae, ) symbiotic fungus Fusarium sp., carried in mycacangia (Fig. 1), was first recorded in Israel in 2009. Damage located in the anterior region of the female head, is inflicted on avocado (Persea americana Miller) trees is responsible for the typical wilt symptoms inflicted on caused by a novel, yet unnamed, symbiotic species avocado (Persea americana Miller) trees. The beetle– within Clade 3 of the Fusarium solani species complex fungus complex has become a serious threat to the (FSSC; O’Donnell et al. 2008) which is vectored by the future of the avocado industry in Israel. beetle. In the present study, seven Fusarium sp. isolates (NRRL 54722–54728) from live and dead and Keywords First report . Fusarium solani species symptomatic trees, appeared to be clones based on complex . Persea americana multilocus sequencing of the following four genes: nuclear ribosomal ITS rDNA region and 5′ end of the nuclear large subunit (LSU rDNA; 1010 bp alignment), ! ! Z. Mendel (*) : A. Protasov : M. Wysoki translation elongation factor 1 (EF-1 ; 694 bp align- Department of Entomology, ARO, The Volcani Center, ment), the largest subunit of RNA polymerase (RPB1; Bet Dagan 50250, Israel 1607 bp alignment), and the second largest subunit of e-mail: [email protected] RNA polymerase (RPB2; 1665 bp alignment) M. Sharon : A. Zveibil : S. Freeman (GenBank accessions JQ038007-JQ038034, submit- Department of Plant Pathology and Weed Research, ARO, ted). Maximum parsimony (Swofford 2002) and maxi- The Volcani Center, mum likelihood (D. J. Zwicki, 2006, Ph.D. thesis, Univ. Bet Dagan 50250, Israel of Texas, USA) bootstrapping of the individual and S. B. Yehuda combined 4-gene datasets strongly supported the gene- Ministry of Agriculture, Extension Service, alogical exclusivity of the novel Fusarium sp. on avo- Bet Dagan 50250, Israel cado in Israel and indicated that it is a reciprocally monophyletic sister of F. ambrosium, a symbiont devel- K. O’Donnell Bacterial Foodborne Pathogens and Mycology Research ops in the galleries of Chinese tea ( Unit, NCAUR-ARS-USDA, (L.) Kuntze) in Sri Lanka (Gadd and Loos 1947). The Peoria, IL 61604, USA adult and larva beetles feed on the fungal mycelium which is inoculated into the xylem and develops on R. Rabaglia USDA Forest Service, Forest Health Protection, the walls of the galleries tunneled in the sapwood by Arlington, VA 22209, USA the colonizing adult females. 236 Phytoparasitica (2012) 40:235–238

Fig. 1 Euwallacea fornica- tus mature female (right) and young male (left). The females (2.5 mm in length) are larger than the males

The beetle is native to Southeast Asia, where it tunneling and fungal infection of the wood have develops on dying and dead trees (Hulcr et al. been observed in both latter tree species. The beetle 2007; Jiri Hulcr, North Carolina State University, also tries to penetrate several ornamental tree spe- NC, USA, personal communication). This species cies and persimmon (Diospyros kaki Creveld). appears to be a generalist and has been recorded However, beetle galleries and fungal infection were from over 100 plant species comprising 36 families not observed in those cases. Trees under attack are in Asia (Browne 1961; Danthanarayana 1968). The characterized by a diagnostic gum exudation, which beetle has also been collected recently from several is specific for each tree species. species of trees in Florida and (Rabaglia The first symptomatic avocado trees were observed et al. 2006). It is sporadically collected from avo- in Israel in 2005. Newly infested trees exhibited few cado in Florida, but does not appear to cause any external symptoms. The most obvious was discolor- damage (Jorge Peña, Univ. of Florida, FL, USA, ation of an area of the outer bark surrounding the personal communication). E. fornicatus is a major penetration spot which was covered by a large amount pest of tea (Camellia sinensis) in Sri Lanka, of the white powdery exudate (Fig. 2) termed ‘persei- where it was first described nearly 100 years ago tol’ (e.g. Liu et al. 2002). While there was no visible (Danthanarayana 1968). However, avocado has not injury to the cortex at this stage of colonization, ex- beenfoundtobeahostofE. fornicatus in Sri amination of the wood under the infested spot bored Lanka (Keerthi Mohotti, Tea Research Institute of by the beetle, revealed a brownish staining of the Sri Lanka, personal communication). Besides avo- xylem and necrosis caused by the fungus. Preliminary cado, box elder (Acer negundo Linnaeus) and the observations suggest that the fungus may spread from castor oil plant (Ricinus communis Linnaeus) are the infestation point to a distance of at least 150 cm also recorded as suitable hosts in Israel. The beetle along the tracheids. Three additional typical symptoms were observed in affected avocado trees, including: (i) wilting of branches and discoloration of the leaves; (ii)

Fig. 2 Bark of stem and branches of avocado tree with typical lesions formed around beetles’ entrance spots. Exudation of a large amount of white powdery ‘persein’ is typical at these Fig. 3 Galleries constructed by adult female beetles at a typical points of attack during the early phase of tree colonization breaking point of an avocado branch Phytoparasitica (2012) 40:235–238 237

Fig. 4 Koch postulates performed on avocado at The Volcani 54726, 2 - NRRL 54725, 3 - NRRL 54723 and 4 - NRRL 54722) Center experimental farm (A- 13 days post-inoculation; B-6weeks isolated from galleries of infected avocado, dead and live beetles, post-inoculation) with four isolates of Fusarium sp. (1 - NRRL and water control (C), respectively branches with heavy yield break down often in heavily trees that served for the Koch postulate tests were not infested plantations; branches are frequently broken at attacked by the beetles or any other pest during the the section where the beetle galleries were established course of the observations. (Fig. 3); and (iii) death of young and mature trees. The beetle attacks the major avocado cultivars in Trees further weakened by the beetle–fungus assault Israel: namely, ‘Haas’, ‘Pinkerton’ and ‘Ettinger’, with became more susceptible. However, new beetle pene- ‘Haas’ appearing to be the most susceptible. To date, trations were not associated with the typical ‘persein’ the symbiotic pathogen has been isolated from these exudation. Adult brood emergence was accompanied avocado cultivars in several avocado growing areas, by small tubes of compacted sawdust. Stems and from at least 15 different growers in the coastal plain branches of various diameters (from 2 to >30 cm, of central Israel, and from box elder. The avocado corresponding to 1- to 30-yr-old growth) may become plantations in Israel cover approximately 7,000 ha; infested by the beetle. about two-thirds of the total production is exported. Koch postulate tests were initiated with the symbi- Thus, the beetle and its symbiotic fungus have become otic fungus in an experimental plot of a 20-yr-old a serious threat to the future of this industry in Israel. avocado orchard at The Volcani Center, Bet Dagan on 6th January 2011. Trees of cv. ‘Haas’ were artifi- Acknowledgments The authors would like to thank Prof. cially inoculated with four different isolates (NRRL Randy C. Ploetz and Prof. Jorge Peña from the Univ. of Florida, 54723, NRRL 54723, NRRL 54725 and NRRL Homestead, Florida, USA, and Michael Noi, Ministry of Agri- 54726) of the Fusarium sp. (isolated from live and culture, Israel, for helpful discussions, and the Israeli avocado ’ dead beetles, and from infected avocado trees) at a growers organization for partial funding of this research. We 6 −1 also thank Stacy Sink for generating the DNA sequence data concentration of 2×10 conidia ml by pipetting a reported in this study and Nathane Orwig for running the DNA 40 μl suspension into mock-punctured bore holes of sequences in NCAUR’s DNA core facility. The mention of firm 3 mm diameter, 1 cm deep. Each isolate was inoc- names or trade products does not imply that they are endorsed or ulated at five different locations on five different recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned. The USDA is an equal branches ranging in age from 3 to 20 years, while opportunity provider and employer. each site also included a mock-inoculated sterile water negative control. Symptom development was observed at all inoculation spots 13 days post- References inoculation (p.i.) and reisolations were conducted 6 weeks p.i. from within the necrotic bark of infected tissue displaying typical disease symptoms (Fig. 4). The Browne, F. G. (1961). The biology of Malayan Scolytidae and – resulting isolates were identical to those originally used, Platypodidae. Malayan Forest Records No. 22, 1 255. Danthanarayana, W. (1968). The distribution and host-range of thus completing Koch postulates. The plantation was the shot-hole borer ( fornicatus Eichh.) of tea. only slightly infested by the ambrosia beetle and the Tea Quarterly, 39,61–69. 238 Phytoparasitica (2012) 40:235–238

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