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).
Anthracnose disease of chili (Capsicum sp) C. acutatum species complex C. gloeosporioides species complex C. truncatum Damm et al. 2012
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. acutatum complex C. gloeosporioides 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
a b c d e f 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)
Taxonomy and multigene phylogeney DNA extraction PCR amplification Sequencing (AGRF) ITS, CHS, ACT, GAPDH, ApMat, GS and TUB2 Sequence alignment and phylogenetic analysis
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 67 99 18 10 23 70 42 T6 T8 T9 55 T7 T5 76 59 T10 T14 T11 68 84 T12 T13 82 T1 29 T3 M1 99 97 67 58 T2 T4 M6 M3 99 M4 M5 C scovillei CBS 126529 C guajavae CPC 18893 96 AUS 13 AUS 18 AUS 17 99 AUS 12 58 AUS 11 AUS 16 C chrysanthemi CBS 126518 62 C nymphaeae CBS 515 78 17 C brisbanense CBS 292.67 C indonesiense CBS 127551 C simmondsii BRIP 28519 21 AUS 14 99 AUS 15 57 AUS8 72 97 AUS9 59 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 92 C karstii CBS 132134 C. scovillei Colletotrichum? C. simmondsii 0.02
Phylogenetic analysis of Colletotrichum gloeosporioides complex based on Bayesian analysis, ITS, TUB2, Ap Mat, and GS regions. A-Australia T-Thailand S-Sri Lanka 37 68 80 45 90 T1 76 T2 56 T7 T6 57 T4 97 91 T5 28 C_siamense_CBS_130417_Thai_Coffee_ 20 C_siamense_CBS_125378_ICMP 18642 T3 A3 21 69 A1 A2 C_salsolae_ICMP_19051 T9 S5 79 21 S3 S4 C_queenslandicum_ICMP_1778 91 15 69 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 82 98 C_clidemiae C_theobromicola_CBS_124945 C. siamense Colletotrichum sp? C. queenslandicum? 0.01
1.00 1.00 /0.95 1.00 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
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 33 38 98 83 99 67 A2 91 A3 98 A1 97 T3 Colletotrichum siamense Thai Coffe ICMP 18578 S1 S2 S3 T5 99 T6 T4 T7 T1 99 T2 68 T9 A4 A6 A7 T8 Colletotrichum salsolae ICMP 19051 Colletotrichum queenslandicum ICMP 1778 Colletotrichum asianum ICMP 18580 Colletotrichum alienum ICMP 12071 Colletotrichum tropicale ICMP 18653 Colletotrichum musae ICMP 19119 Colletotrichum fructicola ICMP 18581 Colletotrichum nupharicola ICMP 18187 Colletotrichum gloeosporioides ICMP 17821 Colletotrichum xanthorrhoeae ICMP17903 99 89 82 92 91 70 99 59 73 9965 Colletotrichum alatae ICMP 17919 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 0.02
Summary 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.
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
Determination of pathogenicity Disease severity = (DCS) lesion length Fruit length X Disease severity scale developed by (Montri et al. 2009)
Identification of pathotypes Different pathotypes can be identified within the same species based on qualitative differences in disease severity
Disease severity of different 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 Colletotrichum species 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
Future studies 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
Acknowledgment Principle supervisor Prof. Paul Taylor Co-supervisors Prof. Pedro Crous, Dr. Peter Ades Collaborative researchers from Australia and other countries
Thank you..