e-Xtra* Host Range of Fusarium Dieback and Its Ambrosia Beetle (Coleoptera: Scolytinae) Vector in Southern California Akif Eskalen, Department of Plant Pathology and Microbiology, Richard Stouthamer, Department of Entomology, Shannon Colleen Lynch, Department of Plant Pathology and Microbiology, and Center for Conservation Biology, Paul F. Rugman-Jones, Department of Entomology, and Mathias Twizeyimana and Alex Gonzalez, Department of Plant Pathology and Microbiology, University of Califor- nia, Riverside, 92521; and Tim Thibault, Huntington Library, Art Collections, and Botanical Gardens, San Marino 91108 Abstract Eskalen, A., Stouthamer, R., Lynch, S. C., Twizeyimana, M., Gonzalez, A., and Thibault, T. 2013. Host range of Fusarium dieback and its ambrosia beetle (Coleoptera: Scolytinae) vector in southern California. Plant Dis. 97:938-951. The polyphagous shot hole borer (PSHB) is an invasive ambrosia bee- with attack by PSHB. The Fusarium sp. was recovered from 54% of tle that forms a symbiosis with a new, as-yet-undescribed Fusarium the plant species attacked by PSHB, indicated by the presence of the sp., together causing Fusarium dieback on avocado and other host Fusarium sp. at least at the site of the entry hole. Trees attacked by plants in California and Israel. In California, PSHB was first reported PSHB included 11 species of California natives, 13 agriculturally im- on black locust in 2003 but there were no records of fungal damage portant species, and many common street trees. Survey results also until 2012, when a Fusarium sp. was recovered from the tissues of revealed 19 tree species that function as reproductive hosts for PSHB. several backyard avocado trees infested with PSHB in Los Angeles Additionally, approximately a quarter of all tree individuals planted County. The aim of this study was to determine the plant host range of along the streets of southern California belong to a species classified as the beetle–fungus complex in two heavily infested botanical gardens in a reproductive host. These data suggest the beetle–disease complex Los Angeles County. Of the 335 tree species observed, 207 (62%), potentially may establish in a variety of plant communities locally and representing 58 plant families, showed signs and symptoms consistent worldwide. Since the beginning of 2012, an emerging plant disease, referred referred to as Euwallacea sp. and the common name polyphagous to as Fusarium dieback (FD), has been detected on several host shot hole borer (PSHB) is used. plants in Los Angeles and Orange Counties, CA (7). FD is caused In California, PSHB was first collected in 2003 from several tree by the combined effects of a tiny (<2.5 mm) invasive ambrosia species (20) but there were no reports of fungal damage before beetle (Scolytinae) and its symbiotic fungal partner, a currently 2012. Preliminary survey data in 2012 showed that backyard avo- undescribed Fusarium sp. (7,15). Adult female beetles burrow into cado trees and many other woody plants in the urban landscape, a tree, creating brood galleries beyond the cambium, which are especially castor bean (Ricinus communis), were heavily infested concurrently inoculated with the Fusarium sp. The fungus colo- with this beetle–fungus disease complex in Los Angeles and nizes the gallery walls and becomes the sole source of food for Orange counties (A. Eskalen, unpublished data). Some of the most developing larvae and adult beetles (7,15). The fungus invades the heavily infested areas in southern California are home to two of its vascular tissue of the tree, blocking water and nutrients from the biggest botanical gardens, the Los Angeles (LA) Arboretum and roots to the rest of the tree, eventually causing branch dieback and The Huntington Library, Art Collections and Botanical Gardens, tree death (Fig. 1) (7,15). which are relatively close to the original 2003 find of the beetle. The specific identity of the beetle is currently unknown. It is These botanical gardens harbor a wide range of plant species that morphologically indistinguishable from the tea shot hole borer represent unique and common ecosystems from all over the world, (TSHB), Euwallacea fornicatus, a serious pest of tea (Camellia and contain all the host species planted throughout urban forests in sinensis) in Sri Lanka and India (5) (Fig. 2A–C). However, there are significant differences in nuclear and mitochondrial DNA se- quences between the California beetles and those from tea planta- tions in Sri Lanka and other Asian collection sites (P. F. Rugman- Jones and R. Stouthamer, unpublished data). This indicates that the California beetle is most likely a separate, congeneric species (rep- resentative mitochondrial sequences for the two species are availa- ble in GenBank [1], accession numbers JX912723-JX912725). Species within the genus Euwallacea have been subject to much revision and synonymization (28); therefore, establishing the exact identity of this beetle is likely to require extensive taxonomic re- search. Thus, for the purpose of this study, the beetle is simply Corresponding author: A. Eskalen, E-mail: [email protected] *The e-Xtra logo stands for “electronic extra” and indicates that Figures 1 to 5, 7, and 8 appear in color in the online edition. Accepted for publication 24 January 2013. http://dx.doi.org/10.1094/PDIS-11-12-1026-RE Fig. 1. Wood discoloration and xylem vessel plugging caused by Fusarium sp. on © 2013 The American Phytopathological Society Albizia julibrissin. 938 Plant Disease / Vol. 97 No. 7 southern California. In addition, the collections receive more con- confirming the presence of eggs, larvae, pupae, or teneral females sistent and even care as opposed to lands with multiple manage- or males in the galleries. Trees that did not show signs of heavy ment strategies. Therefore, the botanical gardens provided a perfect infestation by PSHB were not cut to examine and confirm as a natural laboratory to investigate the plant host range of the beetle– reproductive host because species of these trees were limited in fungus complex. An understanding of the host range of the beetle– number within the botanical gardens’ collections. fungus complex is important in determining the present disease Fusarium sp. and PSHB confirmation. To carry out fungal distribution and to make predictions about its future distribution. isolations, gross contamination was removed before bringing At present, the beetle–fungus complex appears to be limited to plant samples into a clean area by briefly flaming samples and the urban forest. In itself, this will likely present economic prob- removing the outer surface with a sterilized paring knife. All lems for households and cities that must bear the cost of removing samples were then split in half and pieces excised from the dead or dying trees. However, the same disease complex was also leading margin of clean necrotic tissue were plated onto potato found in Israel in 2009 in commercial avocado orchards, where it dextrose agar (Difco) amended with 100 µg of tetracycline causes significant damage to avocado trees (15). Thus, the presence (Fisher Scientific). Plates were incubated at 25°C, and fungal of FD in urban landscapes throughout southern California is a growth was subcultured for identification and long-term storage concern for the California avocado industry because it may eventu- after 7 to 10 days. ally spread to commercial avocado groves. Similarly, the wide host Total genomic DNA was extracted from the mycelium of each range of this beetle may ultimately threaten native trees in the natu- isolate using the method described by Cenis (2). Portions of the ral forests of southern California. following four gene fragments were chosen for multilocus se- The objectives of this study were to (i) determine the plant host quence typing based on published genetic analyses (3,16–18,29): range of the PSHB and Fusarium sp. and (ii) characterize their the internal transcribed spacer (ITS) region, translation elongation symptoms on different host plants. factor 1-α (EF1-α), the second-largest subunit of RNA polymerase (RPB2), and DNA-directed RNA polymerase subunit 1 (RPB1). Materials and Methods Each 25-µl polymerase chain reaction (PCR) mixture contained Host identification and symptom characterization. Trees 19.4 µl of PCR-grade water, 2.5 µl of standard Taq reaction buffer, were visited and identified to species at the LA Arboretum and The 0.5µl of dNTPs (10 mM), 0.125µl of NEB Taq DNA polymerase Huntington Library, Art Collections and Botanical Gardens in Los (5,000 U/ml; New England BioLabs), 0.5 µl of each primer at 10 Angeles County; specific locations of sampled trees were noted mM, and 1.5 µl of template DNA. PCR was carried out in a ther- using global positioning system equipment (Garmin 60CSx, 5- to mal cycler (Bio-Rad Laboratories, Inc.) using published cycling 10-m accuracy). Each botanical garden covers approximately 48 ha conditions (14,17,25). Amplification products were separated by and, in general, the species collections housed in each are different. electrophoresis in 1.5% agarose gels in 1.0× Tris-boric acid-EDTA Although the gardens contain a large number of plant species, the buffer and visualized under UV light after staining with SYBR criteria in host plant selection included (i) common street trees found Green (Invitrogen). PCR products were purified using an Exo in southern California (4), (ii) agriculturally important trees and SAP-IT kit (Affymetrix). ITS, EF1-α, RPB1, and RPB2 regions vines, (iii) species of tree families, and (iv) trees randomly were sequenced in both directions at the Institute for Integrative encountered in the search for species outlined using the criteria i–iii. Genome Biology at the University of California, Riverside. Se- Signs and symptoms of FD (presence or absence; see Results), quences of isolates from the present study were confirmed as severity of beetle colonization (0 = none, 1 = minor [1 to 10 entry Fusarium sp. after a BLAST search comparison in GenBank, holes], 2 = moderate [10 to 30 entry holes], 3= [>31 entry holes]), which showed they were 100% homologous to known sequences and branch dieback (0 = none, 1 = minor [1 to 25% of branches of isolates of Fusarium sp. from Eskalen et al. and Mendel et al. showed dieback], 2 = moderate [25 to 50%], and 3 = severe (7,15). The ITS, EF1-α, RPB1, and RPB2 sequences used in this [>50%]) were also recorded. For each tree species showing symp- study were deposited into GenBank. toms characteristic of FD (one tree per species), tissue samples PSHB collection and confirmation. When present, live beetles were collected by extracting symptomatic tissue from the trunk were collected directly into 95% ETOH and returned to the labora- with a sterilized knife to a depth beyond the cambium. Samples tory for identification. In the field, beetles were collected from the were returned to the laboratory (University of California, River- surface of the trees using forceps. If they were in a gallery, the side) for morphological and molecular identification of the beetle, beetles were first coaxed to the entrance by spraying 95% ethanol the fungus, or both. If visible, beetles were collected and put di- in the gallery and then removed with the forceps (11). rectly into 95% ethanol for later molecular identification (see be- In the laboratory, DNA was extracted from individual beetles by low). The data were analyzed using JMP 9.0.0 statistical software grinding two to three tibia in 2 µl of proteinase-K (>600 mAU ml–1; (SAS 2012). Logistic regression was used to determine the pres- Qiagen) and 35 µl of Chelex 100 resin (Bio-Rad Laboratories) ence and absence of Fusarium sp., gumming, and severity of beetle followed by incubation for 1 h at 55°C, then for 10 min at 99°C. A attack as contributing factors to branch dieback >50% at α = 0.05. section of the cytochrome c oxidase subunit I gene commonly used The suitability of a tree species to act as a reproductive host for in barcoding studies (10) was amplified using the primers the PSHB was evaluated, where possible, by cutting into trees and LCO1490 (5′-GGTCAACAAATCATAAAGATATTGG-3′) and Fig. 2. Polyphagous shot hole borer (PSHB) (Euwallacea sp.): A, female; B, male; C, size comparison of both male (left) and female (right) on a penny. Plant Disease / July 2013 939 Table 1. Hosts of the polyphagous shot hole borer and their susceptibility to Fusarium dieback (FD)a Family, speciesb Common name Nc Cropd Origin FDe S P G Aceraceae s.s. (Sapindaceae s.l.) *Acer buergerianum Trident maple 4 … East China, Taiwan Y … … … A. davidii Pere David’s maple … … China … Y … … A. caudatifolium (syn. A. kawakamii) Kawakami maple … … Taiwan … Y … … *A. macrophyllum Big leaf maple … … Western North America Y Y … … *A. negundo Box elder … … North America Y Y … … *A. palmatum ‘Bonfire’ Bonfire Japanese maple … … East Asia Y … … … *A. paxii Evergreen maple 3 … Southwest China Y Y … … A. × freemanii Freeman maple … … Eastern North America … Y … … Alangiaceae s.s. (Cornaceae s.l.) Alangium chinensis … … … Tropical Africa to China Y Y … … Anacardiaceae Pistacia chinensis Chinese pistache 74 … China … … … Y Schinus terebinthifolius Brazilian pepper tree 333 … Brazil Y Y … … Apocynaceae Thevetia thevetioides Yellow oleander … … Mexico … Y … … Aquifoliaceae Ilex aquifolium English holly … … Western and Southern Europe, Northwest Africa, Southwest Asia … … … … *I. cornuta Chinese holly … … China and Korea Y Y … Y I. latifolia Tarajo holly … … Japan and China Y Y Y … Araliaceae Cussonia spicata Spiked cabbage tree … … South Africa Y Y … Y Fatsia japonica Japanese aralia … … Japan Y Y … Y Arecaceae Brahea armata Mexican blue palm 1 … Western Mexico … … … … Butia capitata Pindo palm … … Brazil, Uruguay, Argentina … Y … Y Livistona chinensis Chinese fan palm … … Australia, Southeast Asia Y … … Y Washingtonia filifera Desert fan palm 54 … Southwest United States, Northwest Mexico Y … … Y Asparagaceae Draceana draco Canary islands dragon tree … … Canary Islands, Cape Verde, Madeira, Morocco … Y … … Asteraceae Verbesina gigantea Crownbeard … … Tropical America … … … Y Betulaceae … … Alnus incana Grey alder … … Europe, Caucasus … Y … Y A. rhombifolia white alder 72 … Western United States … Y … … Betula pendula Silver birch 12 … Europe, Asia Minor Y … … … Corylus colurna Turkish hazelnut … Y Southeast Europe, Asia Minor, Western Asia Y … … … Bignoniaceae Catalpa speciosa Northern catalpa 8 … Central and Eastern United States Y Y … … Handroanthus impetiginosus (syn. Tabebuia impetiginosa) Brazilwood … … Mexico to South America … … Y … Bombacaceae s.s. (Malvaceae s.l.) Bombax ceiba Cotton tree … … India, China, Southeast Asia … … … Y Ceiba speciosa Silk floss tree 14 … Brazil, Argentina Y Y … Y Pseudobombax ellipticum Shaving brush tree … … Mexico and Central America … … … Y Buddlejaceae Nuxia floribunda Forest elder … … Africa … … … … Burseraceae Bursera hindsiana … … … Western Mexico … Y … … Casuarinaceae Casuarina cunninghamiana She-oak … … Northeast Australia … Y … … Cunoniaceae Cunonia capensis Buttetspoon tree … … South Africa … Y … … Cornaceae Cornus controversa Giant dogwood … … Himalayas to Japan Y … … … Cupressaceae Juniperus chinensis Chinese juniper 26 … Northeast Asia Y … … Y J. virginiana Eastern red-cedar … … Eastern North America … … … Y Ebenaceae Diospyros kaki Japanese persimmon 2 Y Asia … Y … … D. lycidioides … … Y South Africa Y Y … … Elaeocarpaceae Crinodendron patagua Lily of the valley tree … … Chile … Y … … Ericaceae Arbutus unedo Strawberry tree … … Southwest Ireland to Asia Minor … Y … … (continued on next page) a S = staining, P = powder, G = gumming, and Y = “yes”. b Asterisks: * indicates reproductive hosts and ** indicates critically endangered tree. c Number of times planted along streets of City of Orange (4). d Agriculturally important crop. e FD susceptible. 940 Plant Disease / Vol. 97 No. 7 Table 1. (continued from preceding page) Family, speciesb Common name Nc Cropd Origin FDe S P G Euphorbiaceae Aleurites fordii Tung oil tree … … Asia … … … Y Bischofia javanica Bishop wood … … Tropical Asia Y Y … … Jatropha cf. cinerea Limberbush … … Arizona, Western Mexico Y … … … Manihot esculenta Cassava … … Brazil … … … … *Ricinus communis Castor bean … … Mediterranean basin, East Africa, India Y Y … … Sapium sebiferum Chinese tallow tree … … China, Japan Y Y … … Fabaceae Acacia caven Roman cassie … … South America … Y … … A. stenophylla Shoestring acacia … … Australia … … … Y A. visco Arca … … Argentina Y … … … Albizia kalkora Kalkora mimosa … … Southern Asia … Y … … *A. julibrissin Silk tree … … Southwest to east Asia Y Y … … Bauhinia × blakeana Orchid tree 1 … China Y … … Y B. galpinii Red orchid bush … … South Africa … Y Y … Calpurnia aurea … … … Southern India, Africa Y Y … Y Cassia brewsteri Cassia pea … … Northeast Australia Y Y … Y *Castanospermum australe Black bean … … Northeast Australia, New Caledonia Y Y … Y *Cercidium floridum Blue palo verde … … Southwest United States, Western Mexico Y Y … … *C. × sonorae Brea … … Northwest Mexico Y Y … … Cladrastris sinensis Chinese yellow wood … … China … Y … Y Erythrina folkersii Coral tree … … Central America Y … Y … E. coralloides Flame coral tree … … Arizona and Mexico … Y … … *Erythrina corallodendron Coral tree … … West Indies Y Y … … E. crista-galli Ceibo … … South America Y Y … Y E. humeana Dwarf coral tree … … South Africa, Mozambique Y Y Y … E. lysistemon Common coral tree … … South Africa Y Y Y … E. × sykesii Coral tree … … Horticultural origin Y Y … … Inga feuilleii Pacay … … South America Y Y … … Lysiphylllum carronii Queensland ebony … … Australia Y … … Y *Parkinsonia aculeata Palo verde … … Tropical America Y Y … … Pithecellobium sp. … … … … … … … Y Schotia brachypetala Huilboerboon … … Southern Africa Y Y … Y Senna racemosa Limestone senna … … Central and South America … Y … … Senna racemosa var. liebmanni … … … Central and South America Y Y … … S. spectabilis var. spectabilis … … … … … Y Y Y Styphnolobium japonicum (syn. Sophora japonica) Pagoda tree … … East Asia … … … Y Tipuana tipu Tipu tree 1 … Brazil, Argentina, Bolivia … Y … … Wisteria sinensis Chinese wisteria … … China Y … … … W. floribunda ‘Macrobotyrus’ Japanese wisteria … … Japan Y Y … … Zenia insignis … … … South China and Vietnam … Y … … Fagaceae Fagus sylvatica European beech … … Central Europe to Caucasus Y Y … … F. sylvatica ‘Riversii’ Riversii European Beech … … Horticultural origin … … Y … Quercus acutissima Sawtooth oak … … China, Korea and Japan … … … … *Q. agrifolia Coast live oak 13 … California, Baja California Y Y … … Q. alba White oak … … Eastern North America … … … … Q. chrysolepis Canyon live oak … … Western North America Y Y … … Q. engelmannii Engelmann Oak … … Southwest California … Y … … Q. ilex Holly oak 306 … Mediterranean Y Y … … Q. lobata Valley oak … … California Y Y … … Q. macrocarpa Bur oak … … Central and Eastern North America Y Y … … Q. mexicana Cozahuatl … … Mexico Y Y … Y *Q. robur English oak … … Europe, Anatolia, and North Africa Y Y … … Q. rubra Northern red oak 7 … Eastern North America … Y Y Y Q. suber Cork oak … … Southwest Europe and Northwest Africa Y Y … … Q. virginiana Southern live oak … … Southeastern United States Y Y … … Hamamelidaceae s.l. (Altingiaceae s.s.) Liquidambar formosana Chinese sweet gum 12 … South China, Taiwan Y Y … … *L. styraciflua American sweetgum 4,916 … Eastern North America Y Y … … Hydrophyllaceae s.s. (Boraginaceae s.l.) Wigandia urens … … … Peru … Y … … Juglandaceae Carya illinoinensis Hickory 36 … South-Central North America Y Y … Y Jugans mandshurica (syn. Juglans formosana) Hayata … … China, Taiwan … … Y … J. nigra Eastern black walnut 5 Y Eastern North America … Y … … Pterocarya sp. Wingnut tree … … Asia … Y … Y (continued on next page) Plant Disease / July 2013 941 Table 1. (continued from preceding page) Family, speciesb Common name Nc Cropd Origin FDe S P G Lauraceae Cinnamomum glanduliferum False camphor tree … … China … … … … C. camphora Camphor tree 690 … Japan to Tropical Asia Y Y Y … Nothaphoebe cavalieri … … … South China … Y … … *Persea americana Avocado 70 Y Mexico, Guatamala, West Indies Y Y Y … Machilus thunbergii (syn. Persea thunbergii) Asian avocado … … East Asia Y Y … … Umbellularia californica California bay laurel … … Western North America Y Y … … Magnoliaceae Magnolia ‘Columbus’ Columbus magnolia … … Horticultural origin … Y … … M. delavayi Chinese evergreen magnolia … … Southwest China … Y … … M. doltsopa Sweet michelia … … Southwest China, Himalayas … Y … … M. grandiflora Southern magnolia 2,174 … Southeast United States Y Y … … M. guatemalensis Mamey … … Central America … Y … Y M. × soulangeana Saucer magnolia 8 … Horticultural origin … Y … Y M. × veitchii Veitch’s magnolia … … Horticultural origin Y Y … … Malvaceae Dombeya cacuminum Strawberry tree … … Madagascar Y … Y … Brachychiton acerifolius Illawarra flame tree 46 … Northeast Australia Y Y … Y B. australis Kurrajong … … Northern Australia Y Y … … B. discolor lacebark tree … … Northeast Australia Y … … … B. rupestris Queensland bottle tree … … Northeast Australia Y … … Y Chiranthodendron pentadactylon Devil’s hand tree … … Mexico and Central America … Y … Y Firmiana simplex Chinese parasol tree … … East Asia Y … … … Meliaceae Melia azedarach Cape lilac 11 … India … Y … … Swietenia chickrassa (syn. Chukrasia tabularis) … … … India and China … … … Y Melianthaceae Melianthus major Giant honey flower … … South Africa Y … … … Menispermaceae Cocculus laurifolius Laurel-leaved snail tree … … Himalayas to Japan Y Y … … C. orbiculatus (syn. C. trilobus) Korean moonseed … … East and Southeast Asia Y Y … Y Monimiaceae Peumus boldus Boldo … … Chile … Y … … Moraceae Broussonetia papyrifera Paper mulberry … … Eastern Asia … Y … … Ficus benjamina Weeping fig 1,182 … Southeast Asia and Australia … Y … … F. macrophylla Moreton Bay fig 2 … Northeast Australia Y Y … Y F. maxima Fig … … Tropical America … … … … F. platypoda Desert rock fig … … Australia and Indonesia Y Y … Y Morus alba White mulberry 43 Y China Y Y … … Myrtaceae Callistemon salignus Willow bottlebrush … … Australia … … … … C. viminalis Weeping bottlebrush 91 … Australia … Y … … Eucalyptus camaldulensis River red gum 2 … Australia … … … … E. cinerea Argyle apple … … Southeast Australia … Y … … E. ficifolia Red flowering gum 33 … Western Australia … Y … Y E. froggattii … … … Southeast Australia … Y … Y E. kitsoniana Gippsland mallee … … Southeast Australia … Y … … E. perriniana Spinning gum … … Southeast Australia … Y … Y E. polyanthemos Red box 6 … Southeast Australia Y Y … … E. torquata Coral gum … … Western Australia Y Y … … Nyssaceae s.s. (Cornaceae s.l.) Camptotheca acuminata Happy tree … … China and Tibet Y Y … … Oleaceae Chionanthus retusus Chinese fringetree … … East Asia Y Y … … Fraxinus sp. Ash … … … Y … … F. uhdei Shamel ash 48 … Central America Y Y … Y F. velutina Velvet ash 57 … Southwestern North America … Y … Olea europaea Olive 29 Y Mediterranean, Asia and Africa Y Y … Y Osmanthus fragrans Sweet osmanthus … … Himalayas to Japan … Y Y … Onagraceae Hauya microcerata … … … Central America … … … … Pinaceae Cedrus atlantica Atlas cedar 1 … Algeria and Morocco … Y … Y Keteleeria delavayi (syn. K. evelyniana) … … … South China, Laos and Vietnam … Y … … Pinus densiflora Japanese red pine … … Japan, Korea, China … Y … Y P. douglasiana … … … Western Mexico … … … Y Pittosporaceae Hymenosporum flavum Native frangipani 20 … Australia and New Guinea Y … … Y Pittosporum undulatum Victorian box … … Eastern Australia Y Y … Y (continued on next page) 942 Plant Disease / Vol. 97 No. 7 HCO2198 (5′-TAAACTTCAGGGTGACCAAAAAATCA-3′) (8). translated (http://www.ebi.ac.uk/Tools/emboss/transeq/index.html) PCR and sequencing was performed as described by Rugman- to confirm the absence of nuclear pseudogenes (26). All specimens Jones et al. (23). Sequences were aligned manually using BioEdit, were confirmed as PSHB (and not TSHB) by comparison with version 7.0.9.0 (9). Primers were removed and each sequence was GenBank accession numbers JX912723–JX912725. Table 1. (continued from preceding page) Family, speciesb Common name Nc Cropd Origin FDe S P G Platanaceae Platanus mexicana Mexican sycamore … … Northeast Mexico Y Y … … P. occidentalis American sycamore 19 … Eastern North America Y Y … … *P. racemosa California sycamore 68 … California, Baja California Y Y … … P. wrightii Arizona sycamore … … Southwest United States, Northwest Mexico Y … … … Poaceae Bambusa sp. Bamboo … … … … Y … … Podocarpaceae Afrocarpus gracilior (syn. Podocarpus gracilior, Decussocarpus gracilior) Fern pine … … East Africa … … … … Proteaceae Banksia saxicola Grampians banksia … … Southeast Australia Y Y … … Macadamia integrifolia Macadamia nut … Y Eastern Australia Y Y … … Rhamnaceae Rhamnus californica Coffee berry … … Western North America … … … Y Ziziphus jujuba Chinese date … … Temperate Asia Y Y … … Rosaceae Eriobotrya japonica Loquat 4 Y China, Japan Y … … … Malus floribunda Japanese crabapple 2 Y East Asia … Y … Y Prunus cerasoides Wild Himalayan cherry … … South Asia … … … Y P. caroliniana Wild mock orange … … Southeastern United States … Y … Y P. mume Chinese plum … … Southern Japan Y … … Y P. persica Peach 4 Y China Y Y … Y P. serrulata Japanese cherry … … Japan, Korea and China Y … … Y Chaenomeles sinensis (syn. Cydonia sinensis) Chinese quince … … China … … … … Pyrus kawakamii Evergreen pear 156 … China, Taiwan Y Y … … Rutaceae Citrus sinensis Orange 6 Y Southeast Asia Y Y … Y Salicaceae Salix babylonica Babylon willow 1 … Asia … Y Y Y Salix sp. … … … … Y Y Y Y Xylosma congestum Dense logwood … … China Y Y … … Sapindaceae Alectryon excelsus Tītoki … … New Zealand Y Y … Y Harpullia arborea Tulip-wood tree … … Tropical Southeast Asia Y Y Y H. pendula Tulipwood … … Northeast Australia, Tropical Southeast Asia … Y Y Y Koelreuteria bipinnata Chinese flame tree 30 … Southwest China … Y … Y K. elegans Chinese rain tree … … Taiwan and Southern China … Y … Y K. elegans subsp. formosana (syn. K. henryi) … … … Taiwan … Y … Y Ungnadia speciosa Mexican buckeye … … South United States, North Mexico Y Y … … Simaroubaceae Ailanthus altissima Tree of heaven 2 … China and Taiwan … … … Y Taxodiaceae s.s. (Cupressaceae s.l.) Metasequoia glyptostroboides** Dawn redwood … … Western China Y … … Y Theaceae Camellia japonica Japanese camellia … … Japan, Korea and China … … Y … Camellia ‘Apple Blossom’ Apple Blossom camellia … … Horticultural origin … Y … … Camellia ‘Pink Sparkle’ Pink Sparkle camellia … … Horticultural origin … Y … … C. reticulata Camellia … … Southwest China Y Y … … *C. semiserrata Camellia … … … Y … … … Cleyera japonica Sakaki … … East Asia Y Y … … Tiliaceae s.s. (Malvaceae s.l.) Heliocarpus donnellsmithii … … … Mexico and Central America Y Y … … Luehea divaricata Açoita-cavalo … … Brazil Y Y Y Y Tilia americana Basswood 1 … Central and Eastern North America Y Y Y … Ulmaceace Ulmus alata Winged elm … … Central and Eastern United States … Y … … U. americana American elm 1 … Eastern North America Y … … … U. parvifolia Chinese elm 183 … East Asia Y Y … … Zelkova serrata Ju shu 1 … East Asia Y … … … Urticaceae Pipturus argenteus … … … Australia, Tropical Asia Y Y Y … Verbenaceae Aloysia virgate Sweet almond bush … … South America … … … … Vitaceae Vitis vinifera Grapevine … Y Mediterranean, Europe Y … … Y Plant Disease / July 2013 943 Table 2. Apparent nonhosts of polyphagous shot hole borer Family, species Common name Origin N planteda Aceraceae s.s. (Sapindaceae s.l.) Acer rubrum Red maple Eastern North America 1 Adoxaceae Sambucus mexicana Mexican elderberry Western North America … Sambucus nigra Black elderberry Europe, North Africa, Southwest Asia … Agavaceae s.s. (Asparagaceae s.l.) Beaucarnea recurvata (syn. Nolina recurvata) … Mexico … Aloeaceae s.s. (Xanthorrhoeaceae s.l.) Aloe bainesii … Southern Africa … Aloe dichotoma … Southern Africa … Anacardiaceae Rhus lancea African sumac South Africa 18 Apocynaceae Acokanthera oppositifolia … Tropical and Southern Africa … Nerium oleander Oleander Mediterranean, Middle and Central Asia 68 Arecaceae Archontophoenix cunninghamiana King palm Eastern Australia 15 Syagrus romanzoffianum (syn. Arecastrum romanzoffianum) Queen palm South America 426 Chamaerops humilis Mediterranean fan palm Mediterranean 2 Phoenix canariensis Canary Island date palm Canary Islands 34 P. dactylifera Date palm Unknown origin 46 P. reclinata Senegal date palm Tropical Africa … Trachycarpus fortunei Chinese windmill palm Asia 80 Asteraceae Montanoa guatemalensis … Central America … Betulaceae Betula nigra ‘Cully’ Heritage river birch Horticultural origin … Carpinus caroliniana America hornbeam Eastern North America, Mexico, Central America … Ostrya virginiana American hophornbeam Eastern North America … Bignoniaceae Jacaranda mimosifolia Jacaranda South America 753 Handroanthus chrysotrichus (syn. Tabebuia chrysotricha) Golden trumpet tree Brazil 1 Boraginaceae Ehretia latifolia (syn. E. austin-smithii) … Mexico, Central America … Burseraceae Bursera odorata … Mexico … Cactaceae Pereskia grandiflora … Brazil … Caprifoliaceae Weigela coraeensis Japanese weigela Eastern Asia … Celastraceae Euonymus bungeanus … China, Korea … Maytenus boaria Mayten Chile 5 Cercidiphyllaceae Cercidiphyllum japonicum Katsura tree China, Japan … Clethraceae Clethra macrophylla Mexican clethra Mexico … Corynocarpaceae Corynocarpus laevigatus Karaka New Zealand … Cupressaceae Calocedrus decurrens California incense cedar Western North America 22 Juniperus bermudiana Bermuda juniper Bermuda … Ericaceae Erica × darleyensis … Horticultural origin … Fabaceae Ceratonia siliqua Carob Mediterranean, North Africa, Middle East and Western Asia 15 Millettia reticulata Evergreen wisteria South China, Taiwan … Pithecellobium dulce Huamuchil Mexico, Central America … Sophora secundiflora Mountain laurel Southwest United States, Mexico … Fagaceae Lithocarpus glaber … Japan, East China … Quercus emoryi Emory oak Southwest United States, Mexico … Q. variabilis Chinese cork oak China, Japan, Korea … Fouquieriaceae Fouquieria macdougalii Mexican ocotillo Northwest Mexico … Garryaceae Garrya wrightii … Southwest United States, Northwest Mexico … Ginkgoaceae Ginkgo biloba Maidenhair tree China 231 (continued on next page) a Number of times planted along streets of City of Orange (4). 944 Plant Disease / Vol. 97 No. 7 Table 2. (continued from preceding page) Family, species Common name Origin N planteda Hernandiaceae Hernandia bivalvus Grease nut Australia … Hippocastanaceae s.s. (Sapindaceae s.l.) Aesculus hippocastanum Horse chestnut Southeast Europe 2 Aesculus × carnea Red horse chestnut Horticultural origin … Iteaceae Itea yunnanensis … Southwest China … Lamiaceae Vitex agnus-castus Vitex Southern Europe … Lauraceae Cryptocarya rubra … Chile … Lythraceae Lagerstroemia indica Crape myrtle Asia 1,979 L. subcostata Asia … Punica granatum Pomegranate Eastern Mediterranean to Himalayas 1 Magnoliaceae Liriodendron tulipifera Tulip tree Eastern North America 232 Magnolia figo Banana shrub China … Malvaceae Brachychiton populneus Bottle tree tree Northeast Australia 129 Lagunaria patersonii Cow itch tree Northeast Australia, South Pacific 1 Moraceae Ficus carica Edible fig Mediterranean to Near East 2 Myrtaceae Acmena smithii Lilly pilly Eastern Australia 1 Agonis flexuosis Willow myrtle Western Australia … Angophora costata Smooth-bark apple Eastern Australia … Callistemon citrinus Lemon bottlebrush Southeast Australia 433 Eucalyptus cladocalyx Sugar gum South Australia 8 E. deglupta Rainbow gum New Guinea to Philippines … E. erythrocorys Red cap gum Western Australia 5 E. globulus Blue gum Southeast Australia, Tasmania 43 E. maculata Spotted gum Eastern Australia … E. microtheca Coolibah Australia … E. robusta Swamp mahogany Northeast Australia … E. saligna Sydney blue gum Eastern Australia … E. sideroxylon Red ironbark Eastern Australia 9 E. torelliana Cadagah Northeast Australia … Acca sellowiana (syn. Feijoa sellowiana) Pineapple guava Brazil, Argentina … Lophostemon confertus Brush box Eastern Australia 92 Melaleuca lanceolata Moonah South Australia … M. linariifolia Snow-in-summer Northeast Australia … M. quinquenervia Cajeput Eastern Australia 53 Metrosideros excelsa New Zealand Christmas tree New Zealand … Syzygium buxifolium … Chile … S. paniculatum Brush cherry Northeast Australia … Nyctaginaceae Bougainvillea Bougainvillea … … Nyssaceae s.s. (Cornaceae s.l.) Nyssa sylvatica Sour gum Eastern North America … Oleaceae Ligustrum lucidum Chinese privet East Asia 100 Papaveraceae Bocconia arborea Tree poppy Central America … Pinaceae Pinus canariensis Canary Islands pine Canary Islands 724 P. halepensis Aleppo pine Mediterranean 35 P. pinea Italian stone pine Mediterranean 11 P. thunbergiana Japanese black pine Japan, Korea 42 P. torreyana Torrey pine California 1 Pittosporaceae Pittosporum tobira Japanese pittosporum China, Japan 3 Poaceae Phyllostachys aurea Golden bamboo Southeast China … Podocarpaceae Podocarpus elatus Rockingham podocarp Eastern Australia … P. macrophyllus Bigleaf podocarp South China, Japan 2 Nageia nagi (syn. Podocarpus nagi) Nagi China, Taiwan, Japan 7 P. totara Totara New Zealand … Polygalaceae Polygala apopetala Dwarf milkwort Baja California … Proteaceae Grevillea robusta Silky oak Eastern Australia 9 Stenocarpus sinuatus Firewheel tree Eastern Australia … (continued on next page) Plant Disease / July 2013 945 Table 2. (continued from preceding page) Family, species Common name Origin N planteda Rhamnaceae Hovenia dulcis Raisin tree East Asia … Ziziphus spina-christi … East Africa, Middle and Near East … Rosaceae Crataegus laevigata Midland hawthorn Europe, North Africa, India … Eriobotrya deflexa Bronze loquat China, Taiwan … Malus syvestris Apple Central Europe 1 Photinia × fraseri Fraser’s photinia Horticultural origin 14 Pyrus calleryana Ornamental pear East Asia 2173 Rutaceae Calodendrum capense Cape chestnut South Africa 12 Casimiroa edulis Cochitzapotl Mexico and Central America … Citrus limon Lemon Unknown origin 5 C. sinensis ‘Moro’ Moro blood orange Horticultural origin … Tetradium danieli (syn. Evodia danieli) China, Korea … Geijera parviflora Australian willow Australia 147 Melicope elleryana Pink flowered soughwood Australia … × Citrofortunella … Horticultural origin … Salicaceae Dovyalis hebecarpa Ketembilla Sri Lanka, Southern India … Olmediella betschleriana Guatemalan holly Mexico, Central America … Oncoba spinosa Snuff-box tree Africa, Arabia … Sciadopityaceae Sciadopitys verticillata Japanese umbrella-pine Japan … Scrophulariaceae Eremophila bignoniflora Emu bush Australia … Myoporum laetum Myoporum New Zealand 1 Solonaceae Brugmansia ‘Charles Grimaldi’ Charles Grimaldi angel’s trumpet Horticultural origin … Lycianthes rantonnei Blue potato bush Argentina, Paraguay 1 Taxodiaceae s.s. (Cupressaceae s.l.) Sequoia sempervirens Coast redwood Western North America … Taxodium mucronatum Mexican swamp cypress Mexico … Theaceae Camellia hiemalis ‘Kanjiro’ Kanjiro camellia Horticultural origin … C. sinensis Tea plant Western China … Gordonia axillaris … Southeast Asia … Ulmaceae Ulmus parvifolia Chinese elm East Asia 175 Verbenaceae Lippia umbellata (syn. L. torresii) … Mexico, Central America … Table 3. Polyphagous shot hole borer (PSHB)–Fusarium sp. complex collected from tree species found in two botanical gardens located in a heavily infested part of Los Angeles County Symptoms Number of tree species Fraction of all species (%) Fraction of attacked species (%) Total number 335 … … Attacked by PSHB 207 62 … Host for Fusarium sp. 112 33 54 Weeping spots on bark 147 44 71 Powder depositions 22 7 11 Gumming on bark 69 20 33 Reproductive hosts for the beetle 19 6 9 Results tive known sequences of isolates of Fusarium sp. from Mendel et al. (15) (GenBank accessions JQ038007 to -038034) and Eskalen Host identification. Of 335 tree species (in 83 families) exam- et al. (7) (GenBank accessions JQ723753 to -723756 and ined (Tables 1 and 2), 207 (62%) had symptoms consistent with JQ723760 to -723763). These findings indicate the susceptibility of attack by PSHB (small entry holes in tree bark, wet spots, gum- the host tree to the growth of Fusarium sp., at least at the site of the ming, white powdery exudate, and frass; Tables 1 and 3; Figs. 3A– entry hole. Fungal sequences generated in the current study have K and 4A–C). The 207 species belonged to 58 families, with ori- been deposited in GenBank under the following accession num- gins representing all the continents except Antarctica. Among the bers: ITS, JX891673 to -JX891784; EF1-α, JX891785 to attacked species were 11 species native to California, and 13 that -JX891896; RPB1, JX891897 to -JX892008; and RPB2, JX892009 are agriculturally important crops in California (see below). There to -JX892120. Fusarium sp. was isolated from 113 tree species in were 128 tree species in 58 families without obvious signs of at- 40 families. Families with the highest number of infected species tack by PSHB (Table 2). Thirty-three plant families examined con- were the Aceraceae, Fabaceae, and Fagaceae. tained species that were both attacked and not attacked by PSHB. Of the tree species susceptible to varying degrees of attack by Fungal DNA was extracted from Fusarium sp. isolates recovered PSHB, 19 were suitable for beetle reproduction. Suitability for from symptomatic tissues of 113 of the 207 tree species (54%) reproduction was confirmed by either the presence of eggs, larvae, attacked by PSHB. In all cases, sequences of the ITS, EF1-α, pupae, or teneral females or the presence of males in the galleries RPB1, and RPB2 genes of the fungus were identical to the respec- (Table 3; Fig. 5). This number might be an underestimate, because 946 Plant Disease / Vol. 97 No. 7 Fig. 3. A–D, External symptoms of staining; E–H, gumming; and I–K, branch dieback for polyphagous shot hole borer (PSHB)–Fusarium dieback disease on A, Quercus agrifolia; B, Liquidambar styraciflua; C, Platanus racemosa; D, Senna specatibilis var. excelsa (syn. Cassia excelsa); E, Alectryon excelsus; F, Acacia melanoxylon; G, Alianthus altissima; H, Koelreuteria elegans; and I–K, Persea americana. confirmation requires cutting into trees, which was not logistically inner bark layer when the beetle entry hole penetrated through the possible at the time due to the limited number of trees that repre- cambium and into the xylem (Figs. 7 and 8). On various hosts, sent each species within the botanical gardens’ collection. PSHB attack resulted in the deposition of an amber colored gum All tree species considered to be reproductive hosts had severe that exuded through the entry holes and varied in consistency be- branch dieback (Fig. 3I–K), and the death of mature reproductive tween spongy and pliable to solid and hard, depending on the host hosts that were infested with FD was observed for Quercus robur, (Table 1, Fig. 3E–H). After peeling back the outer bark beneath the Acer negundo, and R. communis. A. negundo and Q. robur also gum deposition, tissue discoloration was observed and subsequent exhibited leaf wilting and discoloration on the branches prior to recovery of Fusarium sp. occurred on 46% of the tree species dieback and tree death. showing a gumming response (Table 2). Otherwise, the beetle was Symptom characterization. Symptoms surrounding the beetle found embedded within the gum deposit, and there was no dis- entry hole included staining (71% of hosts), gum deposition (33% coloration of the plant tissue. A white powdery exudate was ob- of hosts), and white powder-like exudates (11% of hosts) (Figs. 3, served on some species (Table 1). On avocado, it characteristically 4, and 6, Tables 1 and 3). Staining was characterized by a moist emerged from entry holes and formed a white “sugar volcano” discoloration of the outer bark that oozed out of the entry holes in approximately 1 to 15 cm in diameter and 0.5 to 5 cm in height various amounts, depending on the host (Fig. 3A–D). Discolora- (Fig. 4A). Frass was occasionally observed, particularly on trees in tion caused by fungal colonization was observed underneath the the Aceraceae (Fig. 4B). Plant Disease / July 2013 947