Unravelling the taxonomy of the Colletotrichum species causing

anthracnose in chili in Australia and SE Asia

Dilani de Silva

Prof. Paul Taylor, Prof. Pedro Crous, Prof. Peter Ades

Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Parkville, Victoria 3010,

Australia

Chili

• Chili (Capsicum annuum-Family- Solanaceae) is one of the most important vegetable crops grown throughout the world

• The highest amount of chili cultivation occurs in Queensland (73 %) mainly in Bowen-Burdekin and Bundaberg regions (ABS 2014)

Anthracnose disease

•Anthracnose disease in fruits is a post harvest disease •Symptoms of anthracnose disease are water soaked, black sunken lesions with the presence of concentric rings of acervuli •Caused by a complex of Colletotrichum species.

(Mahasuk et al. 2009)

Colletotrichum spp. Kingdom -Fungi Division -Ascomycota Class -Sordariomycetes Order -Glomerellales Family -Glomerellaceae Genus - Colletotrichum

The genus was recently voted the eighth most important group of plant

pathogenic fungi in the world (Dean et al.

2012).

C. gloeosporioides species complex

C. acutatum species complex

Damm et al. 2012

C. truncatum

Anthracnose disease of chili (Capsicum sp)

Redefined taxonomy of chili anthracnose 2014

• C. scovillei • C. nymphaeae • C. acutatum • C. brisbanense • C. siamense • C. fructicola • C. asianum • C. truncatum • C. coccodes

C. gloeosporioides complex

C. acutatum complex

• Australian Capsicum industry at risk to infection by introduction of exotic pathogens from other countries

Objectives

• Confirm the taxonomy of Colletotrichum isolates from chili using morphological characters and multi gene phylogeny

• Assess pathogenicity of the major Colletotrichum species using fruit bioassays

Confirmation of the taxonomy of Colletotrichum isolates

• Identification of Colletotrichum sp infecting chili in Australia and SE Asian

countries including Thailand, Sri Lanka, Indonesia, Taiwan, Bhutan and Malaysia.

• Characterisation of Colletotrichum sp determined using:

morphological characters

multi gene phylogenetic analyses

Microscopic features of C. siamense isolate one week old culture on PDA (a) production of conidiomata on chili peduncle (b) appressoria (c) asexual conidia (d) setae (e) conidia attached to conidophores (f)

d e

a b c

f

Taxonomy and multigene phylogeney

DNA extraction PCR amplification ITS, CHS, ACT, GAPDH, ApMat, GS and TUB2

Sequence alignment and phylogenetic analysis

Sequencing (AGRF)

Phylogenetic analysis of Colletotrichum acutatum complex based on Maximum likelihood analysis of the ITS, TUB2, GAPDH, and ACT regions.

A-Australia T-Thailand M-Malaysia

C. scovillei

C. simmondsii

T6

T8

T9

T7

T5

T10

T14

T11

T12

T13

T1

T3

M1

T2

T4

M6

M3

M4

M5

C scovillei CBS 126529

C guajavae CPC 18893

AUS 13

AUS 18

AUS 17

AUS 12

AUS 11

AUS 16

C chrysanthemi CBS 126518

C nymphaeae CBS 515 78

C brisbanense CBS 292.67

C indonesiense CBS 127551

C simmondsii BRIP 28519

AUS 14

AUS 15

AUS8

AUS9

AUS10

C lupini CBS 109225

C acutatum CBS 127539

C acutatum CBS 112996

C godetiae CBS 133 44

C dematium CBS 125 25

C coccodes CBS 369 75

C spaethianum CBS 167 49

C xanthorrhoeae CBS 127831

C theobromicola CBS 124945

C brasiliense CBS 128501

C boninense CBS 123755

C karstii CBS 132134

100

92

100

100

99

67

100

100

9759

72

57

100

58

96

99

99

21

17

62

23

10

18

97

99

67

70

84

59

42

55

68

76

99

82

29

58

0.02

Colletotrichum ?

T1

T2

T7

T6

T4

T5

C_siamense_CBS_130417_Thai_Coffee_

C_siamense_CBS_125378_ICMP 18642

T3

A3

A1

A2

C_salsolae_ICMP_19051

T9

S5

S3

S4

C_queenslandicum_ICMP_1778

A4

A6

A7

C_asianum_ICMP_18580

C_alienum_ICMP_12071

C_tropicale_ICMP_18653

T8

C_musae_ICMP 19119

C_fructicola_ICMP_18581

C_nupharicola_ICMP_18658

C_gloeosporioides_CBS_112999

C_alatae_ICMP_17919

C_xanthorrhoeae_CBS_127831

C_horii_ICMP 10492

C. cordylinicola_ICMP_18579

C_psidii_ICMP_19120

C_kahawae_ICMP_17816

C_clidemiae

C_theobromicola_CBS_124945

98

82

100

100

68

37

100

100

100

15

100

100

80

100

45

69

100

91

79

100

69

91

97

90

76

56

57

21

28

20

100

21

0.01

Phylogenetic analysis of Colletotrichum gloeosporioides complex based on Bayesian analysis, ITS, TUB2, Ap Mat, and GS regions.

C. queenslandicum ?

C. siamense

Colletotrichum sp ?

A-Australia T-Thailand S-Sri Lanka

T1

T2

T7

T6

T4

T5

C siamense CBS 130417 Thai Coffee

T3

AUS 21

A1

A2

A3

T9

S5

S3

S4

C salsolae ICMP 19051

C queenslandicum ICMP 1778

AUS 19

AUS 20

A6

A7

A4

AUS 22

C asianum ICMP 18580

C alienum ICMP 12071

C tropicale ICMP 18653

T8

C musae ICMP 19119

C fructicola ICMP 18581

C nupharicola ICMP 18658

C gloeosporioides CBS 112999

C alatae ICMP 17919

C xanthorrhoeae CBS 127831

C horii ICMP 10492

C. cordylinicola ICMP 18579

C psidii ICMP 19120

C kahawae ICMP 17816

C clidemiae

C theobromicola CBS 124945

0.01

1.00

1.00 /0.95

1.00

A2

A3

A1

T3

Colletotrichum siamense Thai Coffe ICMP 18578

S1

S2

S3

T5

T6

T4

T7

T1

T2

T9

Colletotrichum salsolae ICMP 19051

Colletotrichum queenslandicum ICMP 1778

A4

A6

A7

Colletotrichum asianum ICMP 18580

Colletotrichum alienum ICMP 12071

Colletotrichum tropicale ICMP 18653

T8

Colletotrichum musae ICMP 19119

Colletotrichum fructicola ICMP 18581

Colletotrichum nupharicola ICMP 18187

Colletotrichum gloeosporioides ICMP 17821

Colletotrichum alatae ICMP 17919

Colletotrichum xanthorrhoeae ICMP17903

Colletotrichum horii ICMP 10492

Colletotrichum aotearoa ICMP 18537

Colletotrichum cordylinicola ICMP 18579

Colletotrichum psidii ICMP 19120

Colletotrichum kahawae ICMP 17816

Colletotrichum clidemiae ICMP 18658

Colletotrichum theobromicola ICMP 18649

82

89

99

100

99

100

100

38

33

98

83

100

100

99

67

70

92

91

100

91

98

97

99

99

59

73

65

99

68

0.02

Concatenated use of ApMat and GS loci in phylogenetic analysis is able to resolve species in the C. gleosporioides species complex.

A- Australia

T- Thailand

S- Sri Lanka

• Phylogenetic analysis of C. acutatum complex identified major species belonging to C. scovillei, C. simmondsii and a potential new species in Australia

• C. gloeosporioides complex identified isolates close to C. siamense and C. queenslandicum, with potential new species in Australia and Sri Lanka.

• C. scovillei has not been identified in Australia.

• C. queenslandicum, C. simmondsii have never been reported as pathogens of chili in Australia.

Summary

Assessment of pathogenicity of the major Colletotrichum species

• Major Colletotrichum species in Australia and SE Asia were assessed for pathogenicity by inoculating chili fruit of C. annuum and C. chinense PBC932

• The fruit bioassay included both wounding and non-wounding inoculation of fruits

Anthracnose symptoms on chili fruit 10 days after inoculation

W

NW

Day 1 Day 10

Disease severity scale developed by (Montri et al. 2009)

Disease severity =

(DCS)

lesion length

Fruit length X 100

Determination of pathogenicity

Different pathotypes can be identified within the same species based on qualitative differences in disease severity

Identification of pathotypes

Disease severity of different Colletotrichum species infecting chili fruits

Species name Identified species DCS

W UW

Aus 1 C. siamense 3 0

Aus 2 C. siamense 5 1

Aus 3 C. siamense 7 0

S1 C. siamense 3 0

S2 C. siamense 3 1

S3 C. siamense 5 0

TG1 C. siamense 3 0

TG2 C. siamense 3 0

TG3 C. siamense 3 1

TG4 C. siamense 3 0

TG5 C. siamense 3 1

TG6 C. siamense 1 0

TG7 C. siamense 3 1

TG9 C. siamense 3 0

Aus 4 C. queenslandicum 7 0

Aus 5 C. queenslandicum 5 0

Aus 6 C. queenslandicum 5 0

Species name Identified species DCS

W UW

Aus 9 C. simmondsii 1 0

Aus 10 C. simmondsii 7 0

Aus 11 Colletotrichum ? 7 3

Aus 12 Colletotrichum? 3 1

Aus 13 Colletotrichum? 5 1

T1 C. scovillei 5 0

T2 C. scovillei 5 0

T3 C. scovillei 5 0

T4 C. scovillei 7 5

M1 C. scovillei 7 3

M2 C. scovillei 1 0

M3 C. scovillei 7 5

M4 C. scovillei 7 3

M5 C. scovillei 7 3

M6 C. scovillei 7 0

•More isolates will be collected from Australia and other SE Asian countries to analyse the impact of distribution of Colletotrichum species infecting chili. •Identification of potentially new Colletotrichum species and further characterization of the taxonomy has to be completed using multi-gene phylogeny •Further characterisation on identification of pathotypes by screening more isolates from Australia and other countries on different varieties of chili

Future studies

Acknowledgment

• Principle supervisor Prof. Paul Taylor

• Co-supervisors Prof. Pedro Crous,

Dr. Peter Ades

• Collaborative researchers from Australia and other countries

Thank you…..