ORIGINAL PAPER

The polyphagous shot hole borer (PSHB) and its fungal symbiontFusarium euwallaceae a new invasion in South Africa

Trudy Paap12amp Z W de Beer1 amp D Migliorini13 amp W J Nel1 amp M J Wingfield1

Received 13 November 2017 Accepted 25 January 2018 Australasian Plant Pathology Society Inc 2018

AbstractThe polyphagous shot hole borer (PSHB) an ambrosia beetle (Coleoptera Curculeonidae Scolytinae) native to Asia togetherwith its fungal symbiont Fusarium euwallaceae has emerged as an important invasive pest killing avocado and other trees inIsrael and the United States The PSHB is one of three cryptic species in the Euwallacea fornicatus species complex thetaxonomy of which remains to be resolved The surge in the global spread of invasive forest pests such as the PSHB has ledto the development of programmes utilising sentinel tree plantings to record new host-pest interactions During routine surveys oftree health in botanical gardens of South Africa undertaken as part of a sentinel project an ambrosia beetlefungal associate wasdetected damaging Platanus x acerifolia (London Plane) in the KwaZulu-Natal National Botanical Gardens PietermaritzburgIdentification of the beetle by sequencing part of the mitochondrial cytochrome oxidase c subunit 1 (COI) gene confirmed itsidentity as PSHB and specifically one of the invasive haplotypes of the beetle The associated fungus F euwallaceae wasidentified based on phylogenetic analysis of elongation factor (EF 1-α) sequences Kochrsquos postulates have confirmed thepathogenicity of fungal isolates to P x acerifolia This is the first report of PSHB and its fungal symbiont causing Fusariumdieback in South Africa This report also represents the first verified case of a damaging invasive forest pest detected in a sentinelplanting project highlighting the importance of such studies Given the potential impact these species present to urban treesnative biodiversity and agriculture both the PSHB and its fungal symbiont should be included in invasive species regulations inSouth Africa

Keywords Invasive pest Fusarium dieback Euwallacea nr fornicatus International Plant Sentinel Network

Introduction

Worldwide there is a growing list of damaging invasive forestpests the introduction of which has largely been precipitatedby international trade and the intentional movement of plant

material (Santini et al 2013 Liebhold et al 2012) Many ofthese introductions involve organisms that were not problem-atic in their native range or were unknown to science prior totheir arrival in a new environment Consequently they couldnot have been regulated against or detected and stopped atcheckpoints (Eschen et al 2015Wingfield et al 2015 Brasier2008) In response to this growing threat there has been amove towards the use of lsquosentinel plantingsrsquo where exoticspecies growing outside of their natural range are utilised toprovide an early warning system to identify new pest andpathogen risks to plants (Vettraino et al 2015 Roques et al2015) Botanical gardens and arboreta host a large range ofexotic plant collections in diverse regions around the worldthus presenting a unique opportunity for sentinel research TheInternational Plant Sentinel Network (IPSN) was launched in2013 as a platform to coordinate information exchange andprovide support for sentinel plant research within botanicalgardens and arboreta (Barham 2016 Britton et al 2010) Inaddition to their value in identification of novel host-pest

Electronic supplementary material The online version of this article(httpsdoiorg101007s13313-018-0545-0) contains supplementarymaterial which is available to authorized users

Trudy Paaptrudypaapfabiupacza

1 Department of Microbiology and Plant Pathology Forestry andAgricultural Biotechnology Institute (FABI) University of PretoriaPretoria 0002 South Africa

2 Kirstenbosch Research Centre South African National BiodiversityInstitute Cape Town 7700 South Africa

3 National Research Council Institute for Sustainable Plant ProtectionSesto Fiorentino Florence Italy

Australasian Plant Pathologyhttpsdoiorg101007s13313-018-0545-0

interactions when they are adjacent to high-risk sites such asports botanical gardens and arboreta can also provide oppor-tunities to detect damaging invasive forest pests during theirinitial stages of establishment (Burgess and Wingfield 2017Tubby and Webber 2010 Paap et al 2017)

The polyphagous shot hole borer (PSHB) an ambrosiabeetle (Coleoptera Curculeonidae Scolytinae) native toAsia has emerged as an important invasive pest in Israeland in California in the United States In these countries it iscausing significant and costly damage to urban trees as wellas presenting a major threat to the avocado industries As adultfemale beetles burrow into trees to establish brood galleriesthey introduce the symbiotic fungus Fusarium euwallaceaewhich colonises gallery walls becoming a food source fordeveloping larvae and adult beetles (Eskalen et al 2012Mendel et al 2012) The fungus then invades tree vasculartissue causing cambial necrosis branch dieback and death ofa broad range of trees (Eskalen et al 2013) The PSHB is oneof three cryptic species in the Euwallacea fornicatus speciescomplex the taxonomy of which remains to be resolved

In 2016 a project was established in South Africa to im-prove surveillance and identification of new and emergingpest risks by using botanical gardens and arboreta as sentinelsites for tree health monitoring During routine surveys mon-itoring tree health in the KwaZulu-Natal National BotanicalGardens (KZN NBG) Pietermaritzburg South AfricaPlatanus x acerifolia (London Plane) trees showing symp-toms of ambrosia beetle attack were observed Removal ofthe bark and cambium exposed galleries with lesions fromwhich wood material was sampled Additionally infestedbranch material was collected from which PSHB and its fun-gal symbiont F euwallaceae were identified We report hereon the first record of PSHB and F euwallaceae causingFusarium dieback in South Africa

Materials and methods

Specimen collection and fungal isolation

Whilst undertaking tree health monitoring surveys in theKwaZulu-Natal National Botanical Gardens (KZN NBG)Pietermaritzburg South Africa Platanus x acerifolia treesshowing symptoms of ambrosia beetle attack were observed(Fig 1a and b) A sterilised chisel was used to remove barkand cambium from suspected beetle entry holes with symp-tomatic tissues frequently observed at a depth beyond thecambium (Fig 1c) An infested branch section was removedfrom one of the trees and double-bagged with heavyweighttrash bags for transport to the laboratory The branch was splitto check for gallery formation presence of adult beetles eggslarvae and pupae (Figs 1d and 2) Fungal isolates (Table 1)were obtained from symptomatic material after surface

disinfestation by briefly flaming with 80 ethanol and plat-ing on 2 Malt Extract Agar (MEA Biolab South Africa)

DNA isolation amplification and sequencing

Fungal isolates

DNAwas extracted from fresh mycelium scraped from active-ly growing cultures as described in Duong et al (2012) Theelongation factor 1-α region was amplified using the primerpair EF1 and EF2 and the PCR thermocycling conditions de-scribed in OrsquoDonnell et al (2010) Each 25 μl PCR reactioncontained 2 μl template DNA 25 μl of 10times PCR buffer200 μM of each dNTP 02 μl of both the forward and reverseprimer 1 U FastStart Taq DNA polymerase (Roche) 25 mMMgCl2 and 164 μl nuclease free water PCR products werepurified by adding 8 μl ExoSap solution (1 U Exonuclease 11 U Shrimp alkaline phosphatase) to 23 μl PCR product andincubating the mixture at 37 degC for 15 min and then at 80 degCfor another 15min Sequencing and contig assembly was donefor both the forward and reverse primers as described byDuong et al (2012) Sequences derived in this study wereadded to GenBank and accession numbers are provided inTable 1

Beetles

Genomic DNA was extracted and precipitated from beetlesamples using a modified version of the method described inDuong et al (2013) Working concentrations for PCR ampli-fication were prepared by diluting 2 μl of the concentratedDNA solution in 8 μl Tris-HCl (10 mM pH 80) The COIregion was amplified using the primer pair LCO1490 andHCO2198 and the PCR thermocycling conditions as de-scribed in Hebert et al (2003) Each 25 μl PCR reactioncontained 2 μl template DNA 25 μl of 10times PCR buffer200 μM of each dNTP 02 μl of both the forward and reverseprimer 1 U FastStart Taq DNA polymerase 25 mM MgCl2and 164 μl nuclease free water Products were purified byadding 8 μl ExoSap solution (1 U Exonuclease 1 1 UShrimp alkaline phosphatase) to 23 μl PCR product and incu-bating the mixtures at 37 degC for 15 min and then at 80 degC foranother 15min Sequencing and contig assemblywas done forboth the forward and reverse primers using the method de-scribed by Duong et al (2012)

Phylogenetic analysis

Data sets were compiled inMEGA 7026 (Kumar et al 2016)using sequences generated in this study together with se-quences from previous studies obtained from GenBankAlso included in the beetle sequence data set was a singlesequence (BOLD ETKC270ndash13) obtained from a beetle from

T Paap et al

an unknown host near Durban South Africa during alsquoBarcode of Lifersquo project (wwwbarcodeoflifeorg) DNAsequence alignments were done using the online version ofMAFFT 7 (Katoh and Standley 2013)

Pathogenicity tests

Two isolates of F euwallaceae (Table 1) were used in patho-genicity tests Isolates were grown on 2 MEA for 5 daysbefore inoculation of 300 mm long detached healthy woodyshoots of P x acerifolia Xylem tissue was excised from thecentre of each stem (mean diameter 95 mm plusmn 02 mm) with a3 mm cork borer A 3 mm diameter colonised agar plug wastaken from the leading edge of each growing culture placedonto the freshly wounded tissue with the mycelium face

down and the inoculated area wrapped with Parafilm Cleanagar disks were used to inoculate stems as a negative controlStem ends were dipped in wax to prevent desiccation andstems were incubated in moist chambers at 25 plusmn 1 degC for2 weeks At harvest the extent of vascular discolourationwas assessed and re-isolations were made to recover the inoc-ulated fungi The experiment was arranged in a randomiseddesign with 10 replications per isolate and conducted twice

Statistical analysis

Lesion length data from pathogenicity tests were checked fornormality and significant differences among mean valueswere assessed by analysis of variance (ANOVA) and posthoc Least Significant Difference (LSD) test at α = 005

Fig 1 a-b external symptoms of polyphagous shot hole borer attack onPlatanus x acerifolia c removal of bark and cambial tissue exposingsymptoms caused by fungal colonisation associated with beetle entry

hole d longitudinal section through branch showing internal symptomsof discolouration around beetle gallery

The polyphagous shot hole borer (PSHB) and its fungal symbiont Fusarium euwallaceae a new invasion in

an unknown host near Durban South Africa during alsquoBarcode of Lifersquo project (wwwbarcodeoflifeorg) DNAsequence alignments were done using the online version ofMAFFT 7 (Katoh and Standley 2013)

Pathogenicity tests

Two isolates of F euwallaceae (Table 1) were used in patho-genicity tests Isolates were grown on 2 MEA for 5 daysbefore inoculation of 300 mm long detached healthy woodyshoots of P x acerifolia Xylem tissue was excised from thecentre of each stem (mean diameter 95 mm plusmn 02 mm) with a3 mm cork borer A 3 mm diameter colonised agar plug wastaken from the leading edge of each growing culture placedonto the freshly wounded tissue with the mycelium face

down and the inoculated area wrapped with Parafilm Cleanagar disks were used to inoculate stems as a negative controlStem ends were dipped in wax to prevent desiccation andstems were incubated in moist chambers at 25 plusmn 1 degC for2 weeks At harvest the extent of vascular discolourationwas assessed and re-isolations were made to recover the inoc-ulated fungi The experiment was arranged in a randomiseddesign with 10 replications per isolate and conducted twice

Statistical analysis

Lesion length data from pathogenicity tests were checked fornormality and significant differences among mean valueswere assessed by analysis of variance (ANOVA) and posthoc Least Significant Difference (LSD) test at α = 005

Fig 1 a-b external symptoms of polyphagous shot hole borer attack onPlatanus x acerifolia c removal of bark and cambial tissue exposingsymptoms caused by fungal colonisation associated with beetle entry

hole d longitudinal section through branch showing internal symptomsof discolouration around beetle gallery

The polyphagous shot hole borer (PSHB) and its fungal symbiont Fusarium euwallaceae a new invasion in

Statistical analyses were performed using XLSTAT software(Addinsoft Paris France)

Results

Fungal isolation and identification

Isolates resembling Fusarium spp were obtained from symp-tomatic tissues up to 3 cm from beetle entry points (Table 1)DNA sequences of the EF1-α gene region from these isolateswere identical to those of the ex-holotype of F euwallaceaefrom Persea americana (avocado) in Israel as well as isolatesfrom a wide variety of host trees in California USA(Supplementary Figure S1) Fungal sequences generated inthis study have been deposited in GenBank (Table 1)

Recovery and identification of beetles

Splitting of the branch section revealed clear evidence of galleryformation (Fig 1d) However galleries were only up to 3 cmlong with no branching Evidence of reproduction was not ap-parent with eggs andor beetle larvae absent Two adult femalebeetles were located within the galleries Based on morphologi-cal characters and using the taxonomic keys by Rabaglia et al(2006) these were identified as Euwallacea nr fornicatus TheCO1 sequence was obtained for one of the beetles (GenBankaccession number MG642816) and showed a 100 match(Supplementary Figure S2) with haplotype H33 of the PSHBclade of the E fornicatus species complex as defined byStouthamer et al (2017) Other specimens presenting haplotypeH33 came fromDurban South Africa collected during Barcodeof life project Ricinus communis (castorbean) fromVietnam andCalifornia and Persea americana (avocado) in Israel

Table 1 Fusaium euwallaceaeisolates from Platanus xacerifolia in the KwaZulu-NatalNational Botanical Gardens(Pietermaritzburg South Africa)considered in this study andGenBank accession numbers

Species CMW culture numbera GenBank accession number (EF1-α)

F euwallaceae CMW50555b MG642810

F euwallaceae CMW50556 MG642811

F euwallaceae CMW50557 MG642812

F euwallaceae CMW50558 MG642813

F euwallaceae CMW50559b MG642814

F euwallaceae CMW50560 MG642815

a CMW = culture collection of the Forestry and Agricultural Biotechnology Institute (FABI) University ofPretoria South Africab Isolates used in pathogenicity trial

Fig 2 Female polyphagous shothole borer (Euwallacea nrfornicatus)

T Paap et al

Pathogenicity tests

Both isolates of F euwallaceae colonised healthy inoculatedexcised stems of P x acerifolia (Fig 3) The fungus wasrecovered from all of the inoculated stems Lesion lengthsdid not vary significantly between trials and data were conse-quently pooled Mean lesion lengths were significantly differ-ent between fungal inoculation treatments and controls(P lt 00001) although fungal isolates did not differ signifi-cantly in their ability to produce lesions The fungus was suc-cessfully re-isolated from the lesions

Discussion

In response to the growing threat posed by invasive forestpests there has been a move towards the use of sentinel plant-ings to identify new host-pest interactions (Tomoshevich et al2013 Roques et al 2015 Vettraino et al 2015) Whilst un-dertaking tree health monitoring as part of a sentinel researchproject established in South Africa we detected the damaginginvasive forest pest PSHB This study provides the first reportof PSHB and its fungal symbiont F euwallaceae causingFusarium dieback on trees in South Africa It also representsthe first case of a damaging invasive forest pest being detectedthrough a sentinel research project highlighting the value andsignificance of investing in such research

Whilst there was no evidence of beetle reproduction on theP x acerifolia branch collected other Platanus spp includingP occidentalis P orientalis and P racemosa have been found

to be reproductive hosts of the insect (Eskalen et al 2013Mendel et al 2017) The lack of observed reproduction inthe collected sample may relate to branch diameter anddistance from the point of branching Mendel et al (2017)found reproductive galleries (in avocado) were largelyfound at the base of the branches close to the branchingpoints The branch section obtained during the presentstudy was several meters away from the branching pointas branch diameter and accessibility precluded removal atthe branch junction

In addition to F euwallaceae Graphium euwallaceae andParacremonium pembeum have been recorded as symbioticfungal species associated with PSHB (Lynch et al 2016)Whilst the latter two species were not observed growing fromnecrotic host tissue in the current study further isolations fromgalleries and beetles may identify their presence

In a recent study Stouthamer et al (2017) included asingle sequence from an unidentified coleopteran specimenobtained in a Barcode of Life project (BOLD ACC9773ETKC270ndash13) from Durban South Africa in their phylog-eny of the E fornicatus species complex The specimenwas identified as haplotype H33 of the PSHB The beetlescollected during the current study from Pietermaritzburg(50 km NW of the BOLD trapping location) were identi-fied as this same haplotype This haplotype has been iden-tified as one of the four invasive haplotypes in theE fornicatus complex (Stouthamer et al 2017) TheBOLD collection was made in 2012 suggesting the beetlehas likely been present but undetected in the region forsome years

Fig 3 Mean lesion length (mm)on Platanus x acerifolia excisedstems 2 weeks after inoculationwith Fusarium euwallaceaeisolates CMW50555 andCMW50559 Vertical linesrepresent standard error of meanValues with the same letter abovethe bar do not differ significantlyat p = 005 according to LSD test

The polyphagous shot hole borer (PSHB) and its fungal symbiont Fusarium euwallaceae a new invasion in

The discovery of PSHB and associated Fusarium diebackin South Africa is significant and of major concern The beetleis native to Asia and appears to be a generalist species withthe appellation lsquopolyphagousrsquo referring to the broad range oftrees attacked (Umeda et al 2016) PSHB has had a majornegative impact on trees in urban landscapes forests and fruitproduction (particularly avocado) where it has invaded inCalifornia and Israel with infestations of susceptible treesresulting in high levels of mortality (Mendel et al 2017Eskalen et al 2013) Eskalen et al (2013) examined the hostrange of the beetle-fungus complex in two heavily infestedbotanical gardens in Los Angeles County determining thatof 335 tree species observed 207 (representing 58 plant fam-ilies) showed symptoms of attack by PSHB These includedseveral species native to southern Africa including Cussoniaspicata (cabbage tree) Calpurnia aurea (common calpurnia)Diospyros lycioides (monkey plum) Erythrina humeana(dwarf coral tree) Erythrina lysistemon (common coral tree)Schotia brachypetala (huilboerboon)Melianthus major (hon-ey flower kruidjie-roer-my-nie) Cunonia capensis (red al-der) Nuxia floribunda (forest elder) and Bauhinia galpinii(red orchid bush) Most of these species showed some levelof susceptibility to Fusarium dieback except the last three thatwere infested by the beetle but did not develop disease Basedon the survey of Eskalen et al (2013) several commercialcrop trees that are planted in South Africa such as Perseaamericana (avocado) Macadamia integrifolia (macadamianut) Carya illinoinensis (pecan) Prunus persica (peach)Citrus sinensis (orange) and Vitis vinifera (grapevine) aresusceptible to PSHB infestation and Fusarium diebackEskalen et al (2013) also listed as susceptible several treesthat while exotic to South Africa are planted as ornamentalsincluding maple holly wisteria oak and Camellia

Many of these potential hosts of the PSHB are present in theKwaZulu-Natal region including the widespread woody weedcastor bean (Ricinus communis) This species is an importantreproductive host in California (Eskalen et al 2013) and couldpotentially act as a corridor for invasion across urban agriculturaland native ecosystem interfaces (Umeda et al 2016) PSHB andFusarium dieback represent a significant new threat to trees inurban and natural areas as well as to avocado orchards in SouthAfrica Both should be considered for listing under SouthAfricarsquos National Environmental Management BiodiversityAct Alien and Invasive Species (NEMBA AIS) Regulationsand immediate surveys to determine the extent of PSHB pres-ence in South Africa should be undertaken to assist in the devel-opment of management actions to reduce the risk of spread

Acknowledgements This work was supported by the South AfricanNational Department of Environment Affairs through the SouthAfrican National Biodiversity Institutersquos Invasive Species ProgrammeWe thank the KZN National Botanical Gardens for allowing us to under-take the survey and we thank Samantha Bush for photographing thebeetle

References

Barham E (2016) The unique role of sentinel trees botanic gardens andarboreta in safeguarding global plant health Plant Biosyst -Int JDealing Aspects Plant Biol 150(3)377ndash380 httpsdoiorg1010801126350420161179231

Brasier CM (2008) The biosecurity threat to the UK and global environ-ment from international trade in plants Plant Pathol 57(5)792ndash808httpsdoiorg101111j1365-3059200801886x

Britton K White P Kramer A Hudler G (2010) A new approach tostopping the spread of invasive insects and pathogens early dectionand rapid response via a global network of sentinel plantings N Z JFor Sci 40109ndash114

Burgess TI Wingfield MJ (2017) Pathogens on the move a 100-yearglobal experiment with planted eucalypts Bioscience 67 (1) doihttpsdoiorg101093bioscibiw146

Duong TA de Beer ZWWingfield BD Wingfield MJ (2012) Phylogenyand taxonomy of species in the Grosmannia serpens complexMycologia 104(3)715ndash732

Duong TA de Beer ZW Wingfield BD Wingfield MJ (2013)Characterization of the mating-type genes in Leptographiumprocerum and Leptographium profanum Fungal Biol 117(6)411ndash421 httpsdoiorg101016jfunbio201304005

Eschen R Roques A Santini A (2015) Taxonomic dissimilarity in pat-terns of interception and establishment of alien arthropods nema-todes and pathogens affecting woody plants in Europe DiversDistrib 21(1)36ndash45 httpsdoiorg101111ddi12267

Eskalen A Gonzalez A Wang DH Twizeyimana M Mayorquin JSLynch SC (2012) First report of a Fusarium sp and its vector teashot hole borer (Euwallacea fornicatus) causing Fusarium Diebackon avocado in California Plant Dis 96(7)1070 httpsdoiorg101094PDIS-03-12-0276-PDN

Eskalen A Stouthamer R Lynch SC Rugman-Jones PF TwizeyimanaM Gonzalez A Thibault T (2013) Host range of Fusarium diebackand its ambrosia beetle (Coleoptera Scolytinae) Vector in SouthernCalifornia Plant Dis 97(7)938ndash951 httpsdoiorg101094PDIS-11-12-1026-RE

Hebert PDN Cywinska A Ball SL deWaard JR (2003) Biological iden-tifications through DNA barcodes Proc R Soc Lond Ser B Biol Sci270(1512)313ndash321 httpsdoiorg101098rspb20022218

Katoh K Standley DM (2013) MAFFT multiple sequence alignmentsoftware version 7 improvements in performance and usabilityMol Biol Evol 30(4)772ndash780 httpsdoiorg101093molbevmst010

Kumar S Stecher G Tamura K (2016) MEGA7 Molecular EvolutionaryGenetics Analysis Version 70 for bigger datasets Mol Biol Evol33(7)1870ndash1874 httpsdoiorg101093molbevmsw054

Liebhold AM Brockerhoff EG Garrett LJ Parke JL Britton KO (2012)Live plant imports the major pathway for forest insect and pathogeninvasions of the US Front Ecol Environ 10(3)135ndash143 httpsdoiorg101890110198

Lynch SC Twizeyimana M Mayorquin JS Wang DH Na F Kayim MKasson MT Thu PQ Bateman C Rugman-Jones P Hulcr JStouthamer R Eskalen A (2016) Identification pathogenicity andabundance of Paracremonium pembeum sp nov and Graphiumeuwallaceae sp novmdashtwo newly discovered mycangial associatesof the polyphagous shot hole borer (Euwallacea sp) in CaliforniaMycologia 108(2)313ndash329 httpsdoiorg10385215-063

Mendel Z Protasov A Sharon M Zveibil A Yehuda SB OrsquoDonnell KRabaglia RWysokiM Freeman S (2012)AnAsian ambrosia beetleEuwallacea fornicatus and its novel symbiotic fungus Fusarium sppose a serious threat to the Israeli avocado industry Phytoparasitica40(3)235ndash238 httpsdoiorg101007s12600-012-0223-7

Mendel Z Protasov A Maoz Y Maymon M Miller G Elazar MFreeman S (2017) The role of Euwallacea nr fornicatus

T Paap et al

Pathogenicity tests

Both isolates of F euwallaceae colonised healthy inoculatedexcised stems of P x acerifolia (Fig 3) The fungus wasrecovered from all of the inoculated stems Lesion lengthsdid not vary significantly between trials and data were conse-quently pooled Mean lesion lengths were significantly differ-ent between fungal inoculation treatments and controls(P lt 00001) although fungal isolates did not differ signifi-cantly in their ability to produce lesions The fungus was suc-cessfully re-isolated from the lesions

Discussion

In response to the growing threat posed by invasive forestpests there has been a move towards the use of sentinel plant-ings to identify new host-pest interactions (Tomoshevich et al2013 Roques et al 2015 Vettraino et al 2015) Whilst un-dertaking tree health monitoring as part of a sentinel researchproject established in South Africa we detected the damaginginvasive forest pest PSHB This study provides the first reportof PSHB and its fungal symbiont F euwallaceae causingFusarium dieback on trees in South Africa It also representsthe first case of a damaging invasive forest pest being detectedthrough a sentinel research project highlighting the value andsignificance of investing in such research

Whilst there was no evidence of beetle reproduction on theP x acerifolia branch collected other Platanus spp includingP occidentalis P orientalis and P racemosa have been found

to be reproductive hosts of the insect (Eskalen et al 2013Mendel et al 2017) The lack of observed reproduction inthe collected sample may relate to branch diameter anddistance from the point of branching Mendel et al (2017)found reproductive galleries (in avocado) were largelyfound at the base of the branches close to the branchingpoints The branch section obtained during the presentstudy was several meters away from the branching pointas branch diameter and accessibility precluded removal atthe branch junction

In addition to F euwallaceae Graphium euwallaceae andParacremonium pembeum have been recorded as symbioticfungal species associated with PSHB (Lynch et al 2016)Whilst the latter two species were not observed growing fromnecrotic host tissue in the current study further isolations fromgalleries and beetles may identify their presence

In a recent study Stouthamer et al (2017) included asingle sequence from an unidentified coleopteran specimenobtained in a Barcode of Life project (BOLD ACC9773ETKC270ndash13) from Durban South Africa in their phylog-eny of the E fornicatus species complex The specimenwas identified as haplotype H33 of the PSHB The beetlescollected during the current study from Pietermaritzburg(50 km NW of the BOLD trapping location) were identi-fied as this same haplotype This haplotype has been iden-tified as one of the four invasive haplotypes in theE fornicatus complex (Stouthamer et al 2017) TheBOLD collection was made in 2012 suggesting the beetlehas likely been present but undetected in the region forsome years

Fig 3 Mean lesion length (mm)on Platanus x acerifolia excisedstems 2 weeks after inoculationwith Fusarium euwallaceaeisolates CMW50555 andCMW50559 Vertical linesrepresent standard error of meanValues with the same letter abovethe bar do not differ significantlyat p = 005 according to LSD test

The polyphagous shot hole borer (PSHB) and its fungal symbiont Fusarium euwallaceae a new invasion in

The discovery of PSHB and associated Fusarium diebackin South Africa is significant and of major concern The beetleis native to Asia and appears to be a generalist species withthe appellation lsquopolyphagousrsquo referring to the broad range oftrees attacked (Umeda et al 2016) PSHB has had a majornegative impact on trees in urban landscapes forests and fruitproduction (particularly avocado) where it has invaded inCalifornia and Israel with infestations of susceptible treesresulting in high levels of mortality (Mendel et al 2017Eskalen et al 2013) Eskalen et al (2013) examined the hostrange of the beetle-fungus complex in two heavily infestedbotanical gardens in Los Angeles County determining thatof 335 tree species observed 207 (representing 58 plant fam-ilies) showed symptoms of attack by PSHB These includedseveral species native to southern Africa including Cussoniaspicata (cabbage tree) Calpurnia aurea (common calpurnia)Diospyros lycioides (monkey plum) Erythrina humeana(dwarf coral tree) Erythrina lysistemon (common coral tree)Schotia brachypetala (huilboerboon)Melianthus major (hon-ey flower kruidjie-roer-my-nie) Cunonia capensis (red al-der) Nuxia floribunda (forest elder) and Bauhinia galpinii(red orchid bush) Most of these species showed some levelof susceptibility to Fusarium dieback except the last three thatwere infested by the beetle but did not develop disease Basedon the survey of Eskalen et al (2013) several commercialcrop trees that are planted in South Africa such as Perseaamericana (avocado) Macadamia integrifolia (macadamianut) Carya illinoinensis (pecan) Prunus persica (peach)Citrus sinensis (orange) and Vitis vinifera (grapevine) aresusceptible to PSHB infestation and Fusarium diebackEskalen et al (2013) also listed as susceptible several treesthat while exotic to South Africa are planted as ornamentalsincluding maple holly wisteria oak and Camellia

Many of these potential hosts of the PSHB are present in theKwaZulu-Natal region including the widespread woody weedcastor bean (Ricinus communis) This species is an importantreproductive host in California (Eskalen et al 2013) and couldpotentially act as a corridor for invasion across urban agriculturaland native ecosystem interfaces (Umeda et al 2016) PSHB andFusarium dieback represent a significant new threat to trees inurban and natural areas as well as to avocado orchards in SouthAfrica Both should be considered for listing under SouthAfricarsquos National Environmental Management BiodiversityAct Alien and Invasive Species (NEMBA AIS) Regulationsand immediate surveys to determine the extent of PSHB pres-ence in South Africa should be undertaken to assist in the devel-opment of management actions to reduce the risk of spread

Acknowledgements This work was supported by the South AfricanNational Department of Environment Affairs through the SouthAfrican National Biodiversity Institutersquos Invasive Species ProgrammeWe thank the KZN National Botanical Gardens for allowing us to under-take the survey and we thank Samantha Bush for photographing thebeetle

References

Barham E (2016) The unique role of sentinel trees botanic gardens andarboreta in safeguarding global plant health Plant Biosyst -Int JDealing Aspects Plant Biol 150(3)377ndash380 httpsdoiorg1010801126350420161179231

Brasier CM (2008) The biosecurity threat to the UK and global environ-ment from international trade in plants Plant Pathol 57(5)792ndash808httpsdoiorg101111j1365-3059200801886x

Britton K White P Kramer A Hudler G (2010) A new approach tostopping the spread of invasive insects and pathogens early dectionand rapid response via a global network of sentinel plantings N Z JFor Sci 40109ndash114

Burgess TI Wingfield MJ (2017) Pathogens on the move a 100-yearglobal experiment with planted eucalypts Bioscience 67 (1) doihttpsdoiorg101093bioscibiw146

Duong TA de Beer ZWWingfield BD Wingfield MJ (2012) Phylogenyand taxonomy of species in the Grosmannia serpens complexMycologia 104(3)715ndash732

Duong TA de Beer ZW Wingfield BD Wingfield MJ (2013)Characterization of the mating-type genes in Leptographiumprocerum and Leptographium profanum Fungal Biol 117(6)411ndash421 httpsdoiorg101016jfunbio201304005

Eschen R Roques A Santini A (2015) Taxonomic dissimilarity in pat-terns of interception and establishment of alien arthropods nema-todes and pathogens affecting woody plants in Europe DiversDistrib 21(1)36ndash45 httpsdoiorg101111ddi12267

Eskalen A Gonzalez A Wang DH Twizeyimana M Mayorquin JSLynch SC (2012) First report of a Fusarium sp and its vector teashot hole borer (Euwallacea fornicatus) causing Fusarium Diebackon avocado in California Plant Dis 96(7)1070 httpsdoiorg101094PDIS-03-12-0276-PDN

Eskalen A Stouthamer R Lynch SC Rugman-Jones PF TwizeyimanaM Gonzalez A Thibault T (2013) Host range of Fusarium diebackand its ambrosia beetle (Coleoptera Scolytinae) Vector in SouthernCalifornia Plant Dis 97(7)938ndash951 httpsdoiorg101094PDIS-11-12-1026-RE

Hebert PDN Cywinska A Ball SL deWaard JR (2003) Biological iden-tifications through DNA barcodes Proc R Soc Lond Ser B Biol Sci270(1512)313ndash321 httpsdoiorg101098rspb20022218

Katoh K Standley DM (2013) MAFFT multiple sequence alignmentsoftware version 7 improvements in performance and usabilityMol Biol Evol 30(4)772ndash780 httpsdoiorg101093molbevmst010

Kumar S Stecher G Tamura K (2016) MEGA7 Molecular EvolutionaryGenetics Analysis Version 70 for bigger datasets Mol Biol Evol33(7)1870ndash1874 httpsdoiorg101093molbevmsw054

Liebhold AM Brockerhoff EG Garrett LJ Parke JL Britton KO (2012)Live plant imports the major pathway for forest insect and pathogeninvasions of the US Front Ecol Environ 10(3)135ndash143 httpsdoiorg101890110198

Lynch SC Twizeyimana M Mayorquin JS Wang DH Na F Kayim MKasson MT Thu PQ Bateman C Rugman-Jones P Hulcr JStouthamer R Eskalen A (2016) Identification pathogenicity andabundance of Paracremonium pembeum sp nov and Graphiumeuwallaceae sp novmdashtwo newly discovered mycangial associatesof the polyphagous shot hole borer (Euwallacea sp) in CaliforniaMycologia 108(2)313ndash329 httpsdoiorg10385215-063

Mendel Z Protasov A Sharon M Zveibil A Yehuda SB OrsquoDonnell KRabaglia RWysokiM Freeman S (2012)AnAsian ambrosia beetleEuwallacea fornicatus and its novel symbiotic fungus Fusarium sppose a serious threat to the Israeli avocado industry Phytoparasitica40(3)235ndash238 httpsdoiorg101007s12600-012-0223-7

Mendel Z Protasov A Maoz Y Maymon M Miller G Elazar MFreeman S (2017) The role of Euwallacea nr fornicatus

T Paap et al

The discovery of PSHB and associated Fusarium diebackin South Africa is significant and of major concern The beetleis native to Asia and appears to be a generalist species withthe appellation lsquopolyphagousrsquo referring to the broad range oftrees attacked (Umeda et al 2016) PSHB has had a majornegative impact on trees in urban landscapes forests and fruitproduction (particularly avocado) where it has invaded inCalifornia and Israel with infestations of susceptible treesresulting in high levels of mortality (Mendel et al 2017Eskalen et al 2013) Eskalen et al (2013) examined the hostrange of the beetle-fungus complex in two heavily infestedbotanical gardens in Los Angeles County determining thatof 335 tree species observed 207 (representing 58 plant fam-ilies) showed symptoms of attack by PSHB These includedseveral species native to southern Africa including Cussoniaspicata (cabbage tree) Calpurnia aurea (common calpurnia)Diospyros lycioides (monkey plum) Erythrina humeana(dwarf coral tree) Erythrina lysistemon (common coral tree)Schotia brachypetala (huilboerboon)Melianthus major (hon-ey flower kruidjie-roer-my-nie) Cunonia capensis (red al-der) Nuxia floribunda (forest elder) and Bauhinia galpinii(red orchid bush) Most of these species showed some levelof susceptibility to Fusarium dieback except the last three thatwere infested by the beetle but did not develop disease Basedon the survey of Eskalen et al (2013) several commercialcrop trees that are planted in South Africa such as Perseaamericana (avocado) Macadamia integrifolia (macadamianut) Carya illinoinensis (pecan) Prunus persica (peach)Citrus sinensis (orange) and Vitis vinifera (grapevine) aresusceptible to PSHB infestation and Fusarium diebackEskalen et al (2013) also listed as susceptible several treesthat while exotic to South Africa are planted as ornamentalsincluding maple holly wisteria oak and Camellia

Many of these potential hosts of the PSHB are present in theKwaZulu-Natal region including the widespread woody weedcastor bean (Ricinus communis) This species is an importantreproductive host in California (Eskalen et al 2013) and couldpotentially act as a corridor for invasion across urban agriculturaland native ecosystem interfaces (Umeda et al 2016) PSHB andFusarium dieback represent a significant new threat to trees inurban and natural areas as well as to avocado orchards in SouthAfrica Both should be considered for listing under SouthAfricarsquos National Environmental Management BiodiversityAct Alien and Invasive Species (NEMBA AIS) Regulationsand immediate surveys to determine the extent of PSHB pres-ence in South Africa should be undertaken to assist in the devel-opment of management actions to reduce the risk of spread

Acknowledgements This work was supported by the South AfricanNational Department of Environment Affairs through the SouthAfrican National Biodiversity Institutersquos Invasive Species ProgrammeWe thank the KZN National Botanical Gardens for allowing us to under-take the survey and we thank Samantha Bush for photographing thebeetle

References

Barham E (2016) The unique role of sentinel trees botanic gardens andarboreta in safeguarding global plant health Plant Biosyst -Int JDealing Aspects Plant Biol 150(3)377ndash380 httpsdoiorg1010801126350420161179231

Brasier CM (2008) The biosecurity threat to the UK and global environ-ment from international trade in plants Plant Pathol 57(5)792ndash808httpsdoiorg101111j1365-3059200801886x

Britton K White P Kramer A Hudler G (2010) A new approach tostopping the spread of invasive insects and pathogens early dectionand rapid response via a global network of sentinel plantings N Z JFor Sci 40109ndash114

Burgess TI Wingfield MJ (2017) Pathogens on the move a 100-yearglobal experiment with planted eucalypts Bioscience 67 (1) doihttpsdoiorg101093bioscibiw146

Duong TA de Beer ZWWingfield BD Wingfield MJ (2012) Phylogenyand taxonomy of species in the Grosmannia serpens complexMycologia 104(3)715ndash732

Duong TA de Beer ZW Wingfield BD Wingfield MJ (2013)Characterization of the mating-type genes in Leptographiumprocerum and Leptographium profanum Fungal Biol 117(6)411ndash421 httpsdoiorg101016jfunbio201304005

Eschen R Roques A Santini A (2015) Taxonomic dissimilarity in pat-terns of interception and establishment of alien arthropods nema-todes and pathogens affecting woody plants in Europe DiversDistrib 21(1)36ndash45 httpsdoiorg101111ddi12267

Eskalen A Gonzalez A Wang DH Twizeyimana M Mayorquin JSLynch SC (2012) First report of a Fusarium sp and its vector teashot hole borer (Euwallacea fornicatus) causing Fusarium Diebackon avocado in California Plant Dis 96(7)1070 httpsdoiorg101094PDIS-03-12-0276-PDN

Eskalen A Stouthamer R Lynch SC Rugman-Jones PF TwizeyimanaM Gonzalez A Thibault T (2013) Host range of Fusarium diebackand its ambrosia beetle (Coleoptera Scolytinae) Vector in SouthernCalifornia Plant Dis 97(7)938ndash951 httpsdoiorg101094PDIS-11-12-1026-RE

Hebert PDN Cywinska A Ball SL deWaard JR (2003) Biological iden-tifications through DNA barcodes Proc R Soc Lond Ser B Biol Sci270(1512)313ndash321 httpsdoiorg101098rspb20022218

Katoh K Standley DM (2013) MAFFT multiple sequence alignmentsoftware version 7 improvements in performance and usabilityMol Biol Evol 30(4)772ndash780 httpsdoiorg101093molbevmst010

Kumar S Stecher G Tamura K (2016) MEGA7 Molecular EvolutionaryGenetics Analysis Version 70 for bigger datasets Mol Biol Evol33(7)1870ndash1874 httpsdoiorg101093molbevmsw054

Liebhold AM Brockerhoff EG Garrett LJ Parke JL Britton KO (2012)Live plant imports the major pathway for forest insect and pathogeninvasions of the US Front Ecol Environ 10(3)135ndash143 httpsdoiorg101890110198

Lynch SC Twizeyimana M Mayorquin JS Wang DH Na F Kayim MKasson MT Thu PQ Bateman C Rugman-Jones P Hulcr JStouthamer R Eskalen A (2016) Identification pathogenicity andabundance of Paracremonium pembeum sp nov and Graphiumeuwallaceae sp novmdashtwo newly discovered mycangial associatesof the polyphagous shot hole borer (Euwallacea sp) in CaliforniaMycologia 108(2)313ndash329 httpsdoiorg10385215-063

Mendel Z Protasov A Sharon M Zveibil A Yehuda SB OrsquoDonnell KRabaglia RWysokiM Freeman S (2012)AnAsian ambrosia beetleEuwallacea fornicatus and its novel symbiotic fungus Fusarium sppose a serious threat to the Israeli avocado industry Phytoparasitica40(3)235ndash238 httpsdoiorg101007s12600-012-0223-7

Mendel Z Protasov A Maoz Y Maymon M Miller G Elazar MFreeman S (2017) The role of Euwallacea nr fornicatus

T Paap et al

(Coleoptera Scolytinae) in the wilt syndrome of avocado trees inIsrael Phytoparasitica 45(3)341ndash359 httpsdoiorg101007s12600-017-0598-6

OrsquoDonnell K Sutton DA RinaldiMG Sarver BAJ Balajee SA SchroersH-J Summerbell RC Robert VARG Crous PW Zhang N Aoki TJung K Park J Lee Y-H Kang S Park B Geiser DM (2010)Internet-accessible DNA sequence database for identifying fusariafrom human and animal infections J Clin Microbiol 48(10)3708ndash3718 httpsdoiorg101128jcm00989-10

OrsquoDonnell K Sink S Libeskind-Hadas R Hulcr J Kasson MT PloetzRC Konkol JL Ploetz JN Carrillo D Campbell A Duncan RELiyanage PNH Eskalen A Na F Geiser DM Bateman C FreemanS Mendel Z Sharon M Aoki T Cosseacute AA Rooney AP (2015)Discordant phylogenies suggest repeated host shifts in theFusariumndashEuwallacea ambrosia beetle mutualism Fungal GenetBiol 82277ndash290 httpsdoiorg101016jfgb201410014

Paap T Burgess TI Wingfield MJ (2017) Urban trees bridge-heads forforest pest invasions and sentinels for early detection BiolInvasions httpsdoiorg101007s10530-017-1595-x

Rabaglia RJ Dole SA Cognato AI (2006) Review of AmericanXyleborina (Coleoptera Curculionidae Scolytinae) occurring northof Mexico with an illustrated key Ann Entomol Soc Am 99(6)1034ndash1056 httpsdoiorg1016030013-8746(2006)99[1034ROAXCC]20CO2

Roques A J-t F Courtial B Y-z Z Yart A Auger-Rozenberg M-ADenux O Kenis M Baker R J-h S (2015) Planting sentinelEuropean trees in eastern asia as a novel method to identify potentialinsect pest invaders PLoS One 10(5)e0120864 httpsdoiorg101371journalpone0120864

Santini A Ghelardini L De Pace C Desprez-Loustau ML Capretti PChandelier A Cech T Chira D Diamandis S Gaitniekis T Hantula

J Holdenrieder O Jankovsky L Jung T Jurc D Kirisits T Kunca ALygis V Malecka M Marcais B Schmitz S Schumacher J SolheimH Solla A Szabograve I Tsopelas P Vannini A Vettraino AMWebber JWoodward S Stenlid J (2013) Biogeographical patterns and deter-minants of invasion by forest pathogens in Europe New Phytol197(1)238ndash250 httpsdoiorg101111j1469-8137201204364x

Stouthamer R Rugman-Jones P Thu PQ Eskalen A Thibault T Hulcr JWang L-J Jordal BH Chen C-Y Cooperband M Lin C-S KamataN Lu S-S Masuya H Mendel Z Rabaglia R Sanguansub S ShihH-H Sittichaya W Zong S (2017) Tracing the origin of a crypticinvader phylogeography of the Euwallacea fornicatus (ColeopteraCurculionidae Scolytinae) species complex Agric For Entomolhttpsdoiorg101111afe12215

TomoshevichM Kirichenko N Holmes K Kenis M (2013) Foliar fungalpathogens of European woody plants in Siberia an early warning ofpotential threats For Pathol 43(5)345ndash359 httpsdoiorg101111efp12036

Tubby KV Webber JF (2010) Pests and diseases threatening urban treesunder a changing climate Forestry 83(4)451ndash459 httpsdoiorg101093forestrycpq027

Umeda C Eskalen A Paine TD (2016) Polyphagous Shot Hole Borer andFusarium Dieback in California In Paine TD Lieutier F (eds)Insects and diseases of mediterranean forest systems SpringerInternational Publishing Cham pp 757ndash767 httpsdoiorg101007978-3-319-24744-1_26

Vettraino A Roques A Yart A Fan JT Sun JH Vannini A (2015)Sentinel trees as a tool to forecast invasions of alien plant pathogensPLoS One 10(3)15 httpsdoiorg101371journalpone0120571

WingfieldMJ Brockerhoff EGWingfield BD Slippers B (2015) Plantedforest health the need for a global strategy Science 349(6250)832ndash836 httpsdoiorg101126scienceaac6674

The polyphagous shot hole borer (PSHB) and its fungal symbiont Fusarium euwallaceae a new invasion in

EF JX891842 Inga feuillei USA CALIFORNIAJX891843 Jatropha cf cinerea USA CALIFORNIAJX891841 Hymenosporum flavum USA CALIFORNIAJX891840 Heliocarpus donnellsmithii USA CALIFORNIAJX891839 Harpullia arborea USA CALIFORNIAJX891838 Fraxinus uhdei USA CALIFORNIAJX891837 Firmiana simplex USA CALIFORNIAJX891836 Ficus platypoda USA CALIFORNIAJX891835 Ficus macrophylla USA CALIFORNIAJX891834 Fatsia japonica USA CALIFORNIAJX891833 Fagus sylvatica USA CALIFORNIAJX891832 Eucalyptus torquata USA CALIFORNIAJX891831 Eucalyptus polyanthemos USA CALIFORNIAJX891830 Erythrina x sykesii CALIFORNIAJX891829 Erythrina lysistemon USA CALIFORNIAJX891828 Erythrina humeana USA CALIFORNIAJX891827 Erythrina folkersii USA CALIFORNIAJX891826 Erythrina crista-galli USA CALIFORNIAJX891825 Erythrina corallodendron USA CALIFORNIAJX891824 Eriobotrya japonica USA CALIFORNIAJX891823 Dombeya cacuminum USA CALIFORNIAJX891822 Diospyros lycioides USA CALIFORNIAJX891821 Cussonia spicata USA CALIFORNIAJX891820 Corylus colurna USA CALIFORNIAJX891819 Cornus controversa USA CALIFORNIAJX891818 Cocculus orbiculatus USA CALIFORNIAJX891817 Cocculus laurifolius USA CALIFORNIAJX891816 Cleyera japonica USA CALIFORNIAJX891815 Citrus sinensis CALIFORNIAJX891814 Cinnamomum camphora USA CALIFORNIAJX891813 Ceiba speciosa USA CALIFORNIAJX891812 Chionanthus retusus USA CALIFORNIAJX891811 Cercidium floridum USA CALIFORNIAJX891810 Cercidium sonorae USA CALIFORNIAJX891809 Catalpa speciosa USA CALIFORNIAJX891808 Castanospermum australe USA CALIFORNIAJX891807 Cassia brewsteri USA CALIFORNIAJX891806 Carya illinoinensis USA CALIFORNIAJX891805 Camptotheca acuminata USA CALIFORNIAJX891804 Camellia semiserrata USA CALIFORNIAJX891803 Camellia reticulata USA CALIFORNIAJX891802 Calpurnia aurea USA CALIFORNIAJX891801 Brachychiton rupestris USA CALIFORNIAJX891800 Brachychiton discolor USA CALIFORNIAJX891799 Brachychiton australis USA CALIFORNIAJX891798 Brachychiton acerifolius USA CALIFORNIAJX891797 Bischofia javanica USA CALIFORNIAJX891796 Betula pendula USA CALIFORNIAJX891795 Bauhinia blakeana USA CALIFORNIAJX891794 Banksia saxicola USA CALIFORNIAJX891793 Alectryon excelsus USA CALIFORNIAJX891792 Albizia julibrissin USA CALIFORNIAJX891791 Alangium chinense USA CALIFORNIAJX891790 Acer buergeranum USA CALIFORNIAJX891789 Acer paxii USA CALIFORNIAJX891788 Acer palmatum USA CALIFORNIAJX891787 Acer negundo USA CALIFORNIAJX891786 Acer macrophyllum USA CALIFORNIAJX891785 Acacia visco USA CALIFORNIAJQ723760 Persea americana USA CALIFORNIAJQ038007 Persea americana ISRAEL ex-holotypeCMW50560 Platanus SOUTH AFRICACMW50559 Platanus SOUTH AFRICACMW50558 Platanus SOUTH AFRICACMW50557 Platanus SOUTH AFRICACMW50555 Platanus SOUTH AFRICACMW50556 Platanus SOUTH AFRICAJX891844 Juniperus chinensis USA CALIFORNIA

F euwallaceae

KC691533 Acer negundo USA FLORIDAKC691534 Acer negundo USA FLORIDAKC691535 Acer negundo USA FLORIDA

Fusarium sp AF3

KM406629 Platanus racemosa USA CALIFORNIAKM406630 Platanus racemosa USA CALIFORNIAKC691537 Ailanthus altissima USA PennsylvaniaKC691538 Ailanthus altissima USA PennsylvaniaKC691539 Ailanthus altissima USA Pennsylvania

KC691542 Hevea brasiliensis MALAYSIAKC691543 Hevea brasiliensis MALAYSIA

Fusarium sp AF5

Fusarium sp AF4

Fusarium sp AF10KM406626 Unknown host SINGAPOREKM406627 Camellia sinensis SRI LANKAKM406628 Unknown host COSTA RICA

Fusarium sp AF11KC691545 Persea americana USA FLORIDAKC691546 Persea americana USA FLORIDA

Fusarium sp AF6KC691547 Persea americana AUSTRALIAKC691548 Persea americana AUSTRALIA Fusarium sp AF7

AF178332 Camellia sinensis INDIAFJ240350 Camellia sinensis INDIAKC691531 Camellia sinensis SRI LANKAKM406624 Camellia sinensis SRI LANKA

F ambrosium

KM406625 Delonix regia USA FLORIDANRRL22643 Unknown host COSTA RICA Fusarium sp AF9KC691549 Persea americana USA FLORIDAKC691550 Persea americana USA FLORIDAKC691553 Persea americana USA FLORIDA

Fusarium sp AF8Fneocosmosporiellum AF178349 Arachis hypogum GUIANA

Fneocosmosporiellum EF452940 USA100

100

99

96

96

96

81

80

98

82

70

80

0005

Fusarium sp AF12

CO1Haplotype 33

ETKC270-13 BOLDACC9773 SOUTH AFRICA KZNF0005 Platanus SOUTH AFRICA KZNJX912724 H33 Ricinus communis USA CALIFORNIAKM406722 H33 Persea americana ISRAELKU727021 H33 Ricinus communis VIETNAMKU727022 H34 Ricinus communis VIETNAMJX912723 H35 Ricinus communis USA CALIFORNIAKM406726 H35 Quercus robur USA CALIFORNIAKM406727 H35 Acer USA CALIFORNIAKU727023 H35 Ricinus communis USA CALIFORNIAKU727020 H32 Ricinus communis VIETNAM

KU727024 H36 Persea americana TAIWANKU727025 H37 Persea americana TAIWANKU727026 H38 Persea americana TAIWANKU727027 H39 Unknown host JAPANKU727030 H42 Oreocnide pedunculata TAIWANKU727028 H40 Oreocnide pedunculata TAIWANKU727029 H41 Oreocnide pedunculata TAIWAN

KU727018 H30 Unknown host TAIWANKU727013 H25 Acer buergerianum CHINA YunnanKU727019 H31 Cinnamomum cassia VIETNAM

KU727015 H27 Acacia mangium VIETNAMKU727016 H28 Persea americana ThailandKU727017 H29 Platanus sp CHINA Yunnan

KU727011 H23 Acacia mangium VIETNAMKU727012 H24 Acacia mangium VIETNAM

KU727014 H26 Acacia mangium VIETNAM

PSHB

KU727010 H22 Ricinus communis TAIWANKU727005 H17 Unknown host JAPANKU727004 H16 Unknown host JAPAN

KU727006 H18 Unknown host TAIWANKU727009 H21 Unknown host TAIWANKU727007 H19 Unknown host TAIWANKU727008 H20 Unknown host TAIWAN

KSHB

HM064086 Exanthopus TAIWANKU727034 Exanthopus trap SOUTH AFRICA Eastern Cape

KU727032 Euwallacea sp TAIWANKU727033 Euwallacea sp Acacia UGANDA

E xanthopus

KU726991 H1 Cinnamon SINGAPOREKU726993 H3 Citrus sp PNG

KU726992 H2 Unknown host AUSTRALIAKM406729 H2 Unknown host AUSTRALIA

KM406733 H4 Camellia sinensis SRI LANKAKU726994 H4 Camellia sinensis SRI LANKA

KJ866509 H5 Camellia sinensis INDIAKJ866508 H6 Camellia sinensis INDIAKU726995 H7 Durio zibethinus THAILAND

KM406728 H8 Persea americana USA FLORIDAKU726996 H8 Persea americana USA FLORIDAKM406731 H8 Persea americana USA FLORIDA

KU726997 H9 Durio sp THAILANDKU726998 H10 Durio sp THAILAND

KU726999 H11 Unkown host THAILANDKU727000 H12 Durio sp THAILANDKU727001 H13 Durio zibethinus THAILANDKU727002 H14 Durio zibethinus THAILANDKU727003 H15 Unknown host THAILAND

TSHB

Eu

wa

llac

ea

forn

ica

tus

sp

ec

ies

co

mp

lex

KU727039 Eandamanensis THAILANDHM064083 Eandamanensis THAILAND

KU727041 Einterjectus VIETNAMKM406716 Einterjectus USA FLORIDAKP313802 Einterjectus USA FLORIDA

KM406719 Evalidus USA MARYLANDKP313803 Evalidus USA OHIOHM064087 Evalidus USA

HM064084 Eandamanensistalumalai PNGKU727040 Eandamanensistalumalai PNG

KP313799 Eandamanensis PNGKU727038 Euwallacea sp VIETNAM

KU727037 Esimilis VIETNAMKU727036 Esimilis THAILANDKU727035 Esimilis THAILAND

HM064085 Esubemarginatus BORNEOGU808698 Erussulus PNG

GU808707 Xylosandrus compactus GHANAKU727031 Xylosandrus compactus VIETNAM

99

99

98

99

99

99

99

99

96

70

98

86

99

98

99

91

96

88

99

96

71

93

99

70

71

76 82

93

99

75

77

005

Haplotype 35

Haplotype 8

Haplotype 20

• paap1
• paap2
• paap3

CO1Haplotype 33

ETKC270-13 BOLDACC9773 SOUTH AFRICA KZNF0005 Platanus SOUTH AFRICA KZNJX912724 H33 Ricinus communis USA CALIFORNIAKM406722 H33 Persea americana ISRAELKU727021 H33 Ricinus communis VIETNAMKU727022 H34 Ricinus communis VIETNAMJX912723 H35 Ricinus communis USA CALIFORNIAKM406726 H35 Quercus robur USA CALIFORNIAKM406727 H35 Acer USA CALIFORNIAKU727023 H35 Ricinus communis USA CALIFORNIAKU727020 H32 Ricinus communis VIETNAM

KU727024 H36 Persea americana TAIWANKU727025 H37 Persea americana TAIWANKU727026 H38 Persea americana TAIWANKU727027 H39 Unknown host JAPANKU727030 H42 Oreocnide pedunculata TAIWANKU727028 H40 Oreocnide pedunculata TAIWANKU727029 H41 Oreocnide pedunculata TAIWAN

KU727018 H30 Unknown host TAIWANKU727013 H25 Acer buergerianum CHINA YunnanKU727019 H31 Cinnamomum cassia VIETNAM

KU727015 H27 Acacia mangium VIETNAMKU727016 H28 Persea americana ThailandKU727017 H29 Platanus sp CHINA Yunnan

KU727011 H23 Acacia mangium VIETNAMKU727012 H24 Acacia mangium VIETNAM

KU727014 H26 Acacia mangium VIETNAM

PSHB

KU727010 H22 Ricinus communis TAIWANKU727005 H17 Unknown host JAPANKU727004 H16 Unknown host JAPAN

KU727006 H18 Unknown host TAIWANKU727009 H21 Unknown host TAIWANKU727007 H19 Unknown host TAIWANKU727008 H20 Unknown host TAIWAN

KSHB

HM064086 Exanthopus TAIWANKU727034 Exanthopus trap SOUTH AFRICA Eastern Cape

KU727032 Euwallacea sp TAIWANKU727033 Euwallacea sp Acacia UGANDA

E xanthopus

KU726991 H1 Cinnamon SINGAPOREKU726993 H3 Citrus sp PNG

KU726992 H2 Unknown host AUSTRALIAKM406729 H2 Unknown host AUSTRALIA

KM406733 H4 Camellia sinensis SRI LANKAKU726994 H4 Camellia sinensis SRI LANKA

KJ866509 H5 Camellia sinensis INDIAKJ866508 H6 Camellia sinensis INDIAKU726995 H7 Durio zibethinus THAILAND

KM406728 H8 Persea americana USA FLORIDAKU726996 H8 Persea americana USA FLORIDAKM406731 H8 Persea americana USA FLORIDA

KU726997 H9 Durio sp THAILANDKU726998 H10 Durio sp THAILAND

KU726999 H11 Unkown host THAILANDKU727000 H12 Durio sp THAILANDKU727001 H13 Durio zibethinus THAILANDKU727002 H14 Durio zibethinus THAILANDKU727003 H15 Unknown host THAILAND

TSHB

Eu

wa

llac

ea

forn

ica

tus

sp

ec

ies

co

mp

lex

KU727039 Eandamanensis THAILANDHM064083 Eandamanensis THAILAND

KU727041 Einterjectus VIETNAMKM406716 Einterjectus USA FLORIDAKP313802 Einterjectus USA FLORIDA

KM406719 Evalidus USA MARYLANDKP313803 Evalidus USA OHIOHM064087 Evalidus USA

HM064084 Eandamanensistalumalai PNGKU727040 Eandamanensistalumalai PNG

KP313799 Eandamanensis PNGKU727038 Euwallacea sp VIETNAM

KU727037 Esimilis VIETNAMKU727036 Esimilis THAILANDKU727035 Esimilis THAILAND

HM064085 Esubemarginatus BORNEOGU808698 Erussulus PNG

GU808707 Xylosandrus compactus GHANAKU727031 Xylosandrus compactus VIETNAM

99

99

98

99

99

99

99

99

96

70

98

86

99

98

99

91

96

88

99

96

71

93

99

70

71

76 82

93

99

75

77

005

Haplotype 35

Haplotype 8

Haplotype 20

• paap1
• paap2
• paap3