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Host tree damage inflicted by an invasive ambrosia beetle Euwallacea nr. fornicatus due to interactions

with three symbiotic fungi

Stanley Freeman, Golan Miller, Alex Protasov, Marcel Maymon, Meirav Elazar, and Zvi Mendel

Institute of Plant Protection, ARO, The Volcani CenterRishon LeZion, 7505101, Israel

2. In their original habitat the beetles do not cause damage to healthy tree hosts

Introduction1. Ambrosia beetle pests are often invasive species away

from their original habitat

3. Ambrosia beetles carry their symbiotic fungi in mycangia

4. Each ambrosia beetle species associates with a specific complex of symbiotic fungi

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Described ambrosia fungi (polyphyletic* genera, asexual and clonal)

Raffaelea spp. Ceratocystiopsis spp.Dryadomyces spp.

Fusarium spp.

- Ophiostomatales; Ophiostomataceae

- Sordariomycetes; Nectriaceae

Ambrosiozyma spp.

- Basidiomycota

- Yeasts

Graphium spp. - Microascales

*Polyphyletic group has multiple origins which do not share a common ancestor

Ambrosiella spp. - Microascales; Ceratocystidaceae

Entomocorticium spp.

- NectriaceaeParacremonium spp.

Association between the Ambrosia Fusarium spp. (AF clades) and Euwallacea spp. worldwide

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Euwallacea validus

Euwallacea interjectusFusarium euwallaceae

Fusarium ambrosium

Euwallacea nr. fornicatus

Fusarium sp.

Fusarium oligoseptatum

Euwallacea fornicatus

Euwallacea sp.Fusarium sp.Euwallacea sp.Fusarium sp.Euwallacea sp.Fusarium sp.Euwallacea ? sp. Fusarium cf. ensiformeEuwallacea ? sp. Fusarium pseudoensiformeEuwallacea sp.Fusarium sp.Euwallacea sp.Fusarium sp.

Out-groups

Kasson et al (FGB, 2013);Aoki et al (2018)

PSHB

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Fungal symbionts of Euwallacea fornicatus sensulato*

Polyphagous shot hole borer

PSHB

Fusarium euwallaceae, AF-2

Graphium euwallaceaeParacremonium pembeum

Lynch et al. 2016

California

Freeman et al. 2013; 2016

Israel

Tea shot hole borer Kuroshio shot hole borer

TSHB KSHB

Fusarium sp., AF-8, AF-6, Graphium nr euwallaceae,Paracremonium spp.

Fusarium ambrosium

Campbell, and Geering. 2011

Karunaratne et al. 2008

India

Kumar et al. 1998

Australia

Fusarium ambrosium, AF-1 Graphium sp.

Sri Lanka

Carrillo et al. 2016

Florida

Fusarium kuroshiumGraphium kuroshium

Na et al. 2018

California

*Beetle phylogeny based on Cytochrome oxidase c (CO1) mDNA

Fusarium euwallaceae

Euwallacea nr fornicatus

Graphium euwallaceae

Paracremonium pembeum

After: Gadd & Loos, 1947

The three symbiotic fungi associated with PSHB in Israel and California

Freeman et al, 2013; Mycologia

Lynch et al 2016; Mycologia,

Most of the fungal load of Euwallacea spp. is stored in mandibular mycangia

Freeman et al, 2016; Symbiosis

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Both vertical and horizontal transmission

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Vertical transmission

Horizontal transmission

Biology, feed requirements and specificity

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(ii) Adult beetles of different ages

Occurrences of the symbiotic fungi of Euwallacea nr. fornicatus among different PSHB development stages

(iii) Attempt to understand the roles of each symbiont

(i) Larvae and pupae

Location of the Fusarium in larvae/beetles

Head

Gut

Larva

Freeman et al. (Symbiosis 2016)

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Fusarium euwallaceaeGraphium euwallaceaeParacremonium pembeum

Frequency occurrence of the three symbiotic fungi of the PSHB during the

beetle life cycle

Adult maturation 4-6 days

8 – 15 days since gallery initiation

16-30 days after galley initiation

one day old adult

2-3 days old adult

gallery Dead branch after beetles’ emergence

Deserted galleries

Larvae

mycangia

mycangia mycangia

Freeman et al, 2016; Symbiosis Freeman et al, in preparation

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The frequency of the fungi occurrences in active galleries is similar among different host tree species

1

10

100

1000

10000

100000

1000000

Host plant (location)

Spor

e co

ncen

trat

ion

Freeman et al. (Symbiosis 2015)

F. euwallaceae G. euwallaceae P. pembeum

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Interim summary

1. All three fungi are carried by adult females

3. Graphium euwallaceae is the main food source for the larvae andcallow adults

4. Paracremonium is not required as a food source, role ?

5. All three fungi were recovered from galleries

2. The mycangia of mature females contain mainly Fusarium euwallaceae that acts as a pseudo-pathogen in host plant tissue

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Damage to the host tree xylem

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External bark symptoms due to the ambrosia beetle and symbiotic fungal infections

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Internal xylem symptoms due to the ambrosia beetle and symbiotic fungal infections

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Staining of xylem tissue

Avocado OakCastor bean 17

0

10

20

30

40

50

60

70

Sam

ples

with

Fus

ariu

m(%

)

Xylem

(48)

(11)

Stained

Isolation of Fusarium euwallaceae from randomly sampled xylem points (stained and healthy) of beetle colonized

branches

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Occurrence of Fusarium euwallaceae in avocado xylem

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Total ion chromatogram of xylem EtOH

Stained xylem

Healthy xylem

Overlay chromatogramsStained xylem samples ----------------Healthy xylem samples ---------------

Putative Taxifolin

Putative Quercetin

Putative Naringenin

Putative Epicatechin

ND

ND

Putative flavonoids

Courtesy, E. Lewinsohn

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Tree – fungi relationships: studied survival of the symbiotic fungi in live host tree xylem - artificial inoculation and

natural infestations

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Inoculation treatments

Symbiotic ambrosia fungi: 1) Fusarium euwallaceae (isolate 3-2) 2) Graphium euwallaceae (isolate G-1) 3) Paracremonium pembeum (isolate

ACR-1)

Control treatment: 4) Sterilized water (control)

Tree – fungal relationship: artificial inoculation of the symbiotic fungi

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Inoculation of English oakInoculation of castor bean

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Two weeks after inoculation

Six weeks after inoculation

Typical sugar exudation after fungal inoculation in avocado

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Natural infections

Incidence (%) of the symbiotic fungi under artificial inoculation vs. natural infection conditions

0

20

40

60

80

100

0 5 10 15 20 25

Fusarium euwallaceae

Graphium euwallaceae

Paracremonium pembeum

Artificial inoculations

Months after inoculation

Live, activexylem tissue

0

20

40

60

80

100

1 4 8 12 16 20 24

Weeks after beetle infestations

Natural infections

Live, activexylem tissue

Dry, deadxylem tissue

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The effect of constant temperature on Fusarium euwallaceae, Graphium euwallaceae and Paracremonium pembeum

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The Fusarium rate of development is much faster than the other two symbionts, but it also more sensitive to extreme high temperature

Survival of the symbiotic fungi in the xylem of live hosts under artificial inoculation and natural

conditionsInterim summary

1. Inoculation of the fungi into healthy xylem indicates that Graphiumeuwallaceae and Paracremonium pembeum disappear rapidly, while Fusarium euwallaceae survives for much longer periods, perhaps as long as the xylem tissue remains viable

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2. Survival of Graphium euwallaceae and Paracremonium pembeum in dry, dead xylem tissues may allow survival of the beetle during long distance transport

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Multi-funnel trap

Long distance transport

Discussion and conclusions (few)

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1. The beetle is capable of re-arranging the quantitative proportional load of the fungal symbionts in its mycangia to ascertain successful attack, development and survival

2. As revealed in our previous studies, survival of Fusarium euwallaceae in healthy xylem induces further attacks of stem sections or branches after previous unsuccessful attacks by the beetle

3. Fusarium euwallaceae acts as a pseudo- pathogen and enables the beetle colonization of xylem of live trees, while Graphiumeuwallaceae and Paracremonium pembeum develop and survive on weakened xylem tissue

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Discussion and conclusions (cont.)

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4. We suggest that the specific interaction of Fusarium euwallaceaewith the xylem is the key to understanding the difference between attacked hosts which are unsuitable for reproduction as opposed to suitable reproductive hosts

5. Survival of Graphium euwallaceae and Paracremonium pembeum in dead xylem tissues allows the survival of the beetle during long distance transport

Acknowledgements

Funding: Avocado growers organization and Chief Scientistof the Israeli Ministry of Agriculture

Collaborators from abroad:R. Rabaglia, USDA, VA, USAK. O'Donnell, USDA, IL, USAA. Eskalen, UCR, CA, USAT. Aoki, NIAS, Ibaraki, JapanK. Mohotti, TRI, Sri Lanka

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Thank you for your attention

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