insects and fungi: ecological interactions and functional ... · summary •about 15% of identified...

Insects and fungi: ecological interactions and functional biodiversity

PhD candidate Antonino MALACRINÒ

Tutor

Prof. Leonardo SCHENA

Co-tutor Prof. Virgilio CALECA

Interactions

Introduction Aims & Scope Materials & Methods Results Discussion Conclusions

Fungi

Bacteria

Insects

Plants

Other Invertebrates and animals

Soil and organic debris

Viruses

Micro-organisms

Interactions with micro-organisms


Fungi

Bacteria

Insects

Micro-organisms

Bacteria: • Symbiosis

• Feeding • Reproduction • Protection • Others…

• Pathogens

Fungi: • Feeding activity (e.g. bark

beetles or harvester ants) • Insect pathogens

• Other interactions (?)


Aims

• Which is the fungal community associated to insect pests?

• Can international harbours be a point-of-entry of known and potential fungal pathogens carried by scolytid beetles?

• Can insects impact on soil microbial community? Is it possible to have a vice-versa effect?

What’s metabarcoding?

Sample DNA PCR Sequencing Bioinformatics


Sequencing Data handling OTU table Data analysis Insights



PART ONE

FUNGI ASSOCIATED TO BACTROCERA OLEAE


Aims and Scope

Bactrocera oleae (Rossi) damages:

o For table olives growers

o For oil producers:

- Increase of oil acidity

- Decrease of oil quality

- Decrease of income

• Study the fungal community of B. oleae

• Among these fungi, are there species that are causal agents of plant diseases?

Metabarcoding

DNA extraction

PCR ITS2 region


Amplicons Cloning Sanger sequencing

Amplicons with MID tags Pyrosequencing

454 FLX Titanium


Coordinate1(26.26%)

Coordinate2(16.37%

)

0

10

20

30

40

50

60

70

80

10 500 1000 1500 2000

NumberofobservedOTU

S

Numberofsequences

Males

Females

Results

0 2 4 6 8 10 12

Persistence(nr.ofsamplescontainingeachOTU)

log10ofm

axim

umabundance

0

0,02

0,04

0,06

0,08

0,1

0,12

0,14

0,16

0

1

2

3

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8

1 2 4 8 16 32 64 128 256 512 1024 2048 4096

FrequencyofO

TUs

foreachabundanceclass

AbundanceclassesofcoreOTUs

1

10

102

103

104 A) B)

0 2 4 6 8 10 12

Persistence(nr.ofsamplescontainingeachOTU)

log10ofm

axim

umabundance

0

0,02

0,04

0,06

0,08

0,1

0,12

0,14

0,16

0

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1 2 4 8 16 32 64 128 256 512 1024 2048 4096

FrequencyofO

TUs

foreachabundanceclass

AbundanceclassesofcoreOTUs

1

10

102

103

104 A) B)

Results


Cladosporium 55.60%

NPPF 21.58% Alternaria

12.60%

Auerobasidium 7.92%

Colletotrichum 0.89%

Pseudocercospora 0.78%

Pheomoniella 0.30%

Fusarium 0.25%

Neofisucoccum 0.05%

Phellinus 0.03%

Altra 2.30%


• NGS→ 57,964 sequences (after denoising) → 108 OTUs

Results

Cladosporium 55.6% Sooty moulds

Alternaria 12.60% Fruit – Sooty moulds

Aureobasidium 7.92% Sooty moulds

Colletotrichum 0.89% Fruit

Pseudocercospora 0.78% Fruit - Leaves

Phaeomoniella 0.30% Wood

Fusarium 0.25% Fruit

Neofusicoccum 0.05% Fruit

Phellinus 0.03% Wood


Comparison with data from olive leaves and fruits

Female flies Male flies Olive leaves Olive fruits


Comparison with data from olive leaves

Genus Bactrocera Olive leaves

Cladosporium 55.6% + 10.5%

Alternaria 12.6% + 1.5%

Aureobasidium 7.9% - 27.3%

Pseudocercospora 0.8% - 10.1%

Colletotrichum 0.9% - 18.6%

Summary

• About 15% of identified OTUs are fungi that cause diseases to olive trees or fruits • Colletotrichum acutatum and C. gleosporioides → causal agent of olive

anthracnosis

• Other plant pathogenic fungi (e.g. Alternaria spp.)

• New insights about the epidemiology of these fungi and the role of B. oleae, not only as direct source of damage, but also as potential carrier of plant pathogenic fungi



PART TWO

FUNGI ASSOCIATED TO SCOLITID BEETLES


Bark and Ambrosia beetles

Ver

tica

l tra

nsm

issi

on

Horizontal transmission


Seebens et al. 2013 Ecol. Lett.


Aims and Scope

• Study the fungal community associated to both beetles, collected at international harbours, with a high-throughput culture-independent approach


Insects


Insects

1. Commonly trapped beetles in italian harbours

2. Native to Europe and introduced in several other Countries

3. Wide host and climatic range


Insects


Insects

Orthotomicus erosus


Insects

Xyleborinus saxesenii


Results • ≃60,000 sequences

• 294 OTUs (0.99 cut-off)


-2.0 -1.6 -1.2 -0.8 -0.4 0.0 0.4 0.8 1.2 1.6

C1 (35.75%)

-1.8

-1.5

-1.2

-0.9

-0.6

-0.3

0.0

0.3

0.6

C2 (

30.7

0%

)

PseudoF = 8.295; P = 0.01

0

15

30

45

1 100 200 300 400 500 600 700 800 900

Nu

mb

er

of O

TU

s

Number of sequences

X. saxesenii O. erosus

A) B)

0

30

60

90

1 100 200 300 400 500 600 700 800 900

Number of sequences

Ravenna Salerno Marghera

Ravenna Salerno Maghera

X. saxesenii

O. erosus

B)

0

15

30

45

1 100 200 300 400 500 600 700 800 900

Nu

mb

er

of O

TU

s

Number of sequences


A) B)

0

30

60

90

1 100 200 300 400 500 600 700 800 900

Number of sequences

Ravenna Salerno Marghera

Ravenna Salerno Maghera

X. saxesenii

O. erosus

B)

0

10

20

30

40

50

60

O. erosus X. saxesenii

Observed OTUs

**

Results


Aspergillus ruber

Uncultured

Devriesia xanthorrhoeae Geosmithia sp.

Pleospora herbarum

Fusarium equiseti

Ophiostoma ips

Aspergillus spelunceus

Alternaria tenuissima

Botrytis cinerea Fusarium incarnatum Boeremia hedericola

Altra

Core OTUs – Ortotomichus erosus


Core OTUs – Xyleborinus saxesenii


Uncultured

Botryotinia fuckeliana

Cladosporium cladosporioides Aspergillus candidus

Aureobasidium pullulans

Aspergillus tritici

Aspergillus ruber

Alternaria tenuissima

Yeasts

Altra

104 3052

O. erosus X. saxesenii

18

5525

7 20

27

34

Marghera

RavennaSalerno

A) B)


PC1 (35.75%)

PC

2 (

30

.7%

)

Results


Discussion

• First study that used NGS metabarcoding to assess the whole fungal community associated to bark and ambrosia beetles trapped at international harbors

• Main taxa • Ophiostoma

• Geosmithia

• Acremonium

• Fusarium

• Aspergillus

• Alternaria


Discussion

• New associations with fungi that could represent agents of plant diseases

• Food sources in non-native environments

• High number of unknown sequences

• In-out travelling populations – horizontal transfer

• Extend this approach to other pests

• Illumina SBS


Conclusions

• New scenario:

Bark and Ambrosia beetles not only direct source of damages, but also potential carriers of known/unknown plant pathogens

• Surveillance should be extended also to microorganisms associated to wood-associated insect



PART THREE

INTERACTIONS BETWEEN APHIDS AND SOIL MICROBIOTA

Above- belowground interactions

• Aboveground and belowground communities interact • Spatial scale

• Temporal scale

• Ecological effects

• Plant growth

• Microbial ecology



1. Can aphids and/or aphid’s endosymbionts

impact the soil microbial community?

(Top – Down effects)

2. Can soil microbial community impact the aphid

microbiota? (Bottom – Up effects)

Questions


Solanum tuberosum Solanum vernei

Plants Aphids Soil

Aphids Leaves

Roots Soil


Bacteria

Fungi

AM Fungi

16S

ITS2

18S



1. Can aphids or aphid’s endosymbionts impact the

soil microbial community?



microbiome? (Bottom – Up effects)

Questions

Soil microbial community



Aphids impact on soil bacterial community


1. Can aphids or aphid’s endosymbionts impact the

soil microbial community?



microbiome? (Bottom – Up effects)

Questions


Soil microbial community impact on aphids’ microbiome


1. Does plant species matter?

2. Can belowground microbial community shape

the plant microbiota?

Further questions

Plant species structures microbial community


Belowground microbial community shapes plant microbial assembly


16S

PLANT SPECIES APHID PRESENCE Aphid genotype

PLANT SPECIES APHID PRESENCE

-

LEA

VES

SOIL SOIL

PLANT SPECIES SOIL

PLANT SPECIES SOIL

SOIL PLANT SPECIES

SOIL PLANT SPECIES

SOIL PLANT SPECIES

RO

OTS

ITS 18S



Future work

• Identify the microbial actors of these interactions

• Identify the effectors that lead to these effects

• Extend the analysis to other plant species

• Disclose the role of H. defensa

• Networks

With aphids

Network analysis


Without aphids

Concluding remarks

• Fungal community associated to B. oleae

• Scolytid beetles trapped at harbors carry unknown fungi

• Aboveground-Belowground insect/fungi interactions

• Novel and unexpected answers to this ecological topic still poorly understood.


Thank you! Any question?

insects and fungi: ecological interactions and functional ... · summary •about 15% of identified...

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