Lifestyles of Colletotrichumand implications for

anthracnose integrated disease management

Paul Taylor

Professor of Plant Pathology

University of Melbourne

paulwjt@unimelb.edu.au

Outline

• Introduction to anthracnose caused by Colletotrichum spp and problems with control

• Lifestyles of Colletotrichum spp

• Current taxonomy of species

• Colletotrichum spp infecting chili in SE Asia

• Pathogenicity of species infecting chili

Anthracnose• Important disease affecting wide

range of plant species• Crop, orchard and post harvest

disease and maybe seed transmitted

• Biosecurity risk to importers and producers

C. siamense

C. scovillei

C. truncatum

C. gloeosporioidesC. acutatum, C. fruticola

Control of anthracnose caused by Colletotrichum spp.

• Difficult disease to control because:

– Pathogen may infect a wide host range

– Difficult to identify species

– Variable lifestyles of species during infection

– Lack of progress in identifying resistant genes

– Development of pathotypes within species

– Fungicide resistance

Life styles of Colletotrichum

• Important to understand the mechanism of infection and colonisation by Colletotrichumspecies

– major implications in the global transmission of the pathogens (biosecurity risk)

– the implementation of integrated disease control methods

General life cycle of Colletotrichumspecies

De Silva et al. (2017). Fungal Biology Reviews

Life styles of Colletotrichum

• Necrotrophic – infect and kill host tissue and extract nutrients from the dead host cells

• Hemibiotrophic – colonize living plant tissue and obtain nutrients from living host cells before moving to a necrotrophic life style

• Endophytic - lives within a plant for at least part of its life without causing apparent disease

• Latent or Quiescent –an inactive or dormant phase within an infected cell

C. truncatum in chili fruit –hemibiotrophic/necrotrophic/latent

Fungal transformation

using the green

fluorescent protein (GFP)

Auyong et al. (2012). Journal of Basic Microbiology

Intramural colonization

Hemibiotrophic colonisation - hyphae infect tissue by colonisingcell walls then changes to infect and kill cells (necrotrophic)

cu

epcu

ep

Necrotrophic infection

Latent infection on chili leaves

Leaves symptomless until senescence 5 weeks after inoculation at 103 spores/mL

10 μm

C

AP

GT

(A)

10 μm

IH

AP

(B)

Spore germination and appressoria12 h

Infection of leaves –intramural hyphaebeneath cuticle48 h

Ranathunge et al. (2012) Australasian Plant Pathology

Disease cycle of Colletotrichum truncatum

Sexual

Reproduction

?Crop

debris

C. truncatum infected fruits

Acervuli

formation

Seed infection

Water splash

dispersal of conidia

Infection of young

plants and seedlings

Pre- and post

emergent

infectionWater

splash

dispersal

of conidia

Leaf infection and quiescence

Colletotrichum lentis in lentil –hemibiotrophic/necrotrophic

http://www.agriculture.gov.sk.ca/

• Formation of multiseptate, multilobed vesicle in the epidermal cell

• Typical of the destructivum complex

Colletotrichum coccodes in potatoes - endophytic/necrotrophic

• Black dot –microsclerotia on tuber peel

• Infects by appressoria, then colonises entire plant via vascular system.

• After 12 weeks the roots and stolon tissue colonised

Appressoria formation and leaf infection

Is C. coccodes a plant pathogen?

Necrotic leaf lesion after inoculation with 106

spores/mL

Necrotic lesions on chili fruit 9 days after inoculation of wounded chili fruit

Problems with Colletotrichum taxonomy

• Lack of reliable morphological features

• Pathogenic variation

• Many older Colletotrichum names lack

type specimens and living strains for

molecular analysis

Type specimen of C. capsici 1912, now called C. truncatum

Molecular taxonomy of fungi

Fungal gene sequences

– ITS rDNA

– partial b-tubulin

– translation elongation factor 1-α

– glutamine synthetase

– glyceraldehyde 3-phosphate dehydrogenase

– actin

– histone

– mating type genes (ApMAT)

Nuclear small rDNA (18S) ITS5.8S

rDNA ITS Nuclear large rDNA (25S)

ITS4

ITS5 ITS1 ITS3

ITS2

Determine phylogenetic relationship between

species – based on sequence similarity

Taxonomy of Colletotrichum 2012

Jayawardena et al (2016). Mycosphere 7: 1192-1260

2012 - 119 Colletotrichum species in 9 species complexes

2016 - 190 Colletotrichum species in 11 species complexes

Phylogenetic tree based on combination of 4 fungal genes

Taxonomy of Colletotrichum2016

Redefined taxonomy of chili anthracnose De Silva et al.2016, Diao et al. 2016, Liu et al. 2016

• C. aenigma• C. conoides• C. fructicola• C. viniferum• C. hymenocallidis• C. endophytica• C. gloeosporioides• C. grossum• C. queenslandicum• C. siamense• C. asianum

gloeosporioides complex

• C. fioriniae• C. simmondsii• C. cairnsense sp nov• C. scovillei• C. nymphaeae• C. acutatum• C. brisbanense

acutatum complex

• C. coccodes

Not grouped

• C. cliviae• C. liaoningense• C. brevisporum

• C. sichuanensis

truncatum complex

• C. truncatum

boninense complex

• C. karstii

spaethianum complex

• C. incanum

New cliviae complex

• Australia, 3 species were known – now 6

• SE Asia and China, 4 species were known – now 25

(12 new reports from China)

Chili anthracnose in Australia & SE Asia

UOM 26- INDO

UOM 27- INDO

UOM 25- INDO

UOM 15- INDO

UOM 14- INDO

UOM 13- INDO

UOM 16- INDO

UOM 38- INDO

CPC30233- INDO

CPC30234- INDO

CPC30235- INDO

CPC30236- INDO

CPC30237- INDO

CPC30238- INDO

F7-4A- THAI

UOM 3- SRI

UOM 4- SRI

UOM 5- SRI

F4-1C- THAI

UOM 21- AUS

UOM 1- AUS

UOM 2- AUS

UOM 3- AUS

C_siamense_(syn_C_hymenocallidis)CBS_125378

C_siamense_(syn_C_jasmini_sambac)CBS_130420

C_siamense_CBS_130417

F1-3A- THAI

F7-3B-THAI

-F1-3CTHAI

CPC 28609- INDO

F5-4A-THAI

F5-1A-THAI

F7-1B-THAI

C_salsolae_ICMP_19051

COLL1298-TWN

COLL1290-TWN

CPC 28555-INDO

CPC 28612-INDO

CPC 28611-INDO

C_queenslandicum_ICMP_1778

UOM 20- AUS

UOM19-AUS

UOM 6-AUS

UOM 7-AUS

UOM 4- AUS

UOM 22- AUS

C_asianum_ICMP_18580

C_tropicale_ICMP_18653

CPC 28608- INDO

CPC 28607- INDO

UOM 42- INDO

UOM 43- INDO

C_aeschynomenes_ICMP_17673_ATCC_201874

F5-2D- THAI

C_musae_ICMP_19119

C_aenigma_ICMP_18608

C_nupharicola_ICMP_18658

C_fructicola_ICMP_18581

COLL1318-TWN

COLL 853-TWN

CPC 28644-INDO

CPC 28645-INDO

C_gloeosporioides_CBS_112999

C_alatae_ICMP_17919

C_xanthorrhoeae_CBS_127831

C_horii_ICMP_10492

C_henanenseLC3030_CGMCC_3_17354_LF238

C_ti_ICMP_4832

C_aotearoaICMP_18537

C._cordylinicola_ICMP_18579

C_psidii_CBS_145_29_ICMP_19120 N

C_clidemiae

C_kahawae_ICMP_17816

C_camelliaeCGMCC_3_14925_LC1364

C_theobromicola_CBS_124945

C._grevilleae_CBS_132879_CPC_15481

9993

87

88

73

99

99

54

88

94

99

60

32

99

99

100

81

99

100

99

99

99

100

80

100

42

53

100

55

99

95

9594

100

99

73

98

62

63

58

64

0.02

C. siamense

NEW

C. queenslandicum

NEW

C. fructicola

NEW

NEW

Gloeosporioides complex based on maximum likelihood analysis of ITS, TUB2, ApMat and GS genes

AUS- Australia

INDO- Indonesia

SRI- Sri Lanka

THAI-Thailand

TWN- Taiwan

Gloeosporioides complex

• C. siamense widespread throughput SE Asia and Aust

• C. siamense and C. queenslandicum associated with other hosts, as is C. fructicola

• New species in Indonesia (Sulawesi), Taiwan and Thailand (Kanchanburi)

C_scovillei_CBS:120708

C_scovillei_CBS_126529

C_guajavae_CPC_18893

C_chrysanthemi_CBS_126518

AUS_13

AUS_18

AUS_16

AUS_11

AUS_12

AUS_17

C_walleri_CBS:125472

C_cosmi_CBS:853.73

C_nymphaeae_CBS_515_78

INDO_3

C_brisbanense_CBS_292.67

C_laticiphilum_CBS:112989

C_indonesiense_CBS_127551

C_sloanei_IMI:364297

C_simmondsii_BRIP_28519

AUS_14

AUS8

AUS9

AUS10

AUS_15

C_lupini_CBS_109225

C_fioriniae_CBS:128517

C_acutatum_CBS_127539

C_acutatum_CBS_112996

C_godetiae_CBS_133_44

C_boninense_CBS_123755

100

60

100

97

80

63

86

98

94

58

58

99

99

69

91

77

88

90

94

99

75

95

0.02

T211-THAI

COLL153-- TWN

MJ8- THAI

COLL329-TWN

MJ3-THAI

COLL-524- TWN

MJ7-THAI

MJ8-THAI

316-THAI

COLL683- TWN

MJ5- THAI

COLL1300-TWN

GA1-THAI

GA3- THAI

GA2- THAI

GA5- THAI

UOM- 16- INDO

UOM- 11- INDO

UOM 12- INDO

UOM M5- MLS

UOM M4- MLS

UOM M3-MLS

UOM M1-MLS

UOM 8- INDO

313- THAI

UOM 7- INDO

UOM M6-MLS

COLL 1301- TWN

211- THAI

UOM6- INDO

314- THAI

UOM- 9- INDO

311- THAI

UOM 4- INDO

C. cairnsense

C. simmondsii

C. scovillei

C_scovillei_CBS:120708

UOM 6- INDO

UOM 5- INDO

314- THAI

UOM 1- INDO

UOM 2- INDO

UOM 3- INDO

UOM 14- INDO

UOM 13- INDO

UOM 15- INDO

C. brisbanense

GA4- THAI

COLL-883- TWN

UOM- 10- INDO

UOM- 10- INDO

99

95

Acutatum complex based on maximum likelihood analysis of ITS, TUB2, GAPDH, ACT, HIS and CHS genes

AUS- Australia

INDO- Indonesia

SRI- Sri Lanka

THAI-Thailand

MLS- Malaysia

TWN- Taiwan

Acutatum complex

• C. scovillei widespread throughout SE Asia but not in Aust.

• C. cairnsense only in Australia

• Limited diversity of acutatum complex species outside of Aust

Colletotrichum cairnsense sp. nov.

Other complexes based on maximum likelihood analysis of ITS, TUB2, GAPDH, ACT and CHS genes

C. karstii

C. cliviae

INDO- Indonesia

THAI-Thailand

boninense complex

cliviae complex

C karstii CBS:125468

C karstii CAUOS1 Chili China

C karstii CAUOS7 Chili China

C karstii CBS:127595

C karstii CBS 132134

C karstii CBS:128545

28553 -INDO

28554 -INDO

28601 -INDO

28602-INDO

C phyllanthi CBS:175.67

C annellatum CBS:129826

C petchii CBS:378.94

C boninense CBS 123755

C beeveri CBS:128527

C brassicicola CBS:101059

C brasiliense CBS 128501

C constrictum CBS:128504

C.truncatum CBS151.35

C liaoningenses CAUOS2

C brevisporum L57/LC0600

C liaoningenses CAUOS3

CA2-THAI

CA3-THAI

CA1-THAI

CPC 28639-THAI

CPC 28638- THAI

C sichuanensis LJTJ3

C sichuanensis LJTJ16

C cliviae CBS:125375

C cliviae CSSS2

C cliviae CSSS1

C tropicicola L58/LC0598

96

100

99

81

73

60

88

100

100

100

10099

100100

99

98

100

5099

90

53

89

40

48

42

0.02

Cliviae complex

• Isolates from Thailand from leaves in Chiang Rai and fruit in Bangkok

• C. sichuanensis probably misidentified and is in fact C. cliviae

a

e

c

d

b

f

b

Isolates from cliviae complex

Colletotrichum spp causing anthracnose in Thailand

Location No. isolates

Species

Chiang Mai 10 C. scovillei

KU site 4 C. scovillei

Ratchaburi 2 C. siamense

Kanchana Buri 21

C. siamenseNew species

Nakhon Pathom 3 C. siamense

Chiang Rai 32

C. scovilleiC. cliviae

Bangkok 3 C. cliviae

Species name C. bangchang C. chinensis PBC:932

RED GREEN RED GREEN

W NW W NW W NW W NW

cliviae complex C. cliviae 1 0 0 0 0 0 0 0

boninense complex C. karstii 1 1 1 1 1 0 0 0

gloeosporioidescomplex

C. fructicola 1 0 1 1 1 0 0 0

new_4 (Thailand) 1 0 0 0 1 0 0 0

C. siamense 1 1 1 1 1 0 0 0

C. queenslandicum 1 1 1 1 1 0 1 0

new_1 (Taiwan) 1 0 1 0 1 0 0 0

new_2 (Indonesia) 1 0 1 1 1 0 0 0

new_3 (Indonesia) 1 0 1 0 0 0 0 0

acutatumcomplex

C. cairnsense 1 1 1 1 1 0 1 0

C. scovillei 1 1 1 1 1 1 1 1

C. simmondsii 1 1 1 1 1 0 0 0

C. brisbanense 1 1 1 1 1 1 0 0

Pathogenicity of Colletotrichum species (ability to cause infection on wounded & non-wounded fruit)

Pathogenicity of species• Results do not consider virulence or pathotype

differences between isolates

• C. scovillei most pathogenic – infect PBC, NW

• C. cairnsense & C. queenslandicum next most pathogenic but not able to infect PBC, NW

• Species were less likely to infect non wounded fruit – cuticle resistance?

• Wounded green fruit of PBC was generally more resistant then wounded red fruit, no difference for non-wounded fruit

To Wound or not to Wound fruit in a bioassay?

• Wounding

–Mongkolporn’s group at Kasetsart Uni identified pathotypes of Colletotrichum spp on red and green fruit; and resistance genes in Capsicumspecies

• Non wounding

– At Uni of Melb, a cutinase pathogenicity gene was cloned from C. truncatum and shown to be important in establishing initial infection of chili fruit.

Pathogenicity summary

• Cuticle of the fruit plays an important role as the primary mechanism of defence against infection by Colletotrichum spp. – especially in C. annuum.

• Cellular defence may be inducible resistance but only identified in Capsicum species – C. baccatum, C. chinense.

• Bioassays to screen Capsicum germplasm for resistance should be based on both wounding and non-wounding methods.

UM Plant Pathology PhD students

PhD student Dilani de Silva

Assoc Prof Orarat Mongkolporn, Kasetsart University, Thailand

Dr Marti Pottorff, AVRDC - World Vegetable Centre, Taiwan