Unravelling the taxonomy of the Colletotrichum species causing anthracnose in chili in Australia and SE Asia

Transcription

1 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

2 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)

3 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)

4 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).

5 Anthracnose disease of chili (Capsicum sp) C. acutatum species complex C. gloeosporioides species complex C. truncatum Damm et al. 2012

6 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

7 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

8 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

9 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)

10 Taxonomy and multigene phylogeney DNA extraction PCR amplification Sequencing (AGRF) ITS, CHS, ACT, GAPDH, ApMat, GS and TUB2 Sequence alignment and phylogenetic analysis

11 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 T6 T8 T9 55 T7 T T10 T14 T T12 T13 82 T1 29 T3 M T2 T4 M6 M3 99 M4 M5 C scovillei CBS C guajavae CPC AUS 13 AUS 18 AUS AUS AUS 11 AUS 16 C chrysanthemi CBS C nymphaeae CBS C brisbanense CBS C indonesiense CBS C simmondsii BRIP AUS AUS AUS AUS9 59 AUS10 C lupini CBS C acutatum CBS C acutatum CBS C godetiae CBS C dematium CBS C coccodes CBS C spaethianum CBS C xanthorrhoeae CBS C theobromicola CBS C brasiliense CBS C boninense CBS C karstii CBS C. scovillei Colletotrichum? C. simmondsii 0.02

12 Phylogenetic analysis of Colletotrichum gloeosporioides complex based on Bayesian analysis, ITS, TUB2, Ap Mat, and GS regions. A-Australia T-Thailand S-Sri Lanka T1 76 T2 56 T7 T6 57 T T5 28 C_siamense_CBS_130417_Thai_Coffee_ 20 C_siamense_CBS_125378_ICMP T3 A A1 A2 C_salsolae_ICMP_19051 T9 S S3 S4 C_queenslandicum_ICMP_ A4 A6 A7 C_asianum_ICMP_18580 C_alienum_ICMP_12071 C_tropicale_ICMP_18653 T8 C_musae_ICMP C_fructicola_ICMP_18581 C_nupharicola_ICMP_18658 C_gloeosporioides_CBS_ C_alatae_ICMP_17919 C_xanthorrhoeae_CBS_ C_horii_ICMP C. cordylinicola_icmp_18579 C_psidii_ICMP_19120 C_kahawae_ICMP_ C_clidemiae C_theobromicola_CBS_ C. siamense Colletotrichum sp? C. queenslandicum? 0.01

13

14 / T1 T2 T7 T6 T4 T5 C siamense CBS Thai Coffee T3 AUS 21 A1 A2 A3 T9 S5 S3 S4 C salsolae ICMP C queenslandicum ICMP 1778 AUS 19 AUS 20 A6 A7 A4 AUS 22 C asianum ICMP C alienum ICMP C tropicale ICMP T8 C musae ICMP C fructicola ICMP C nupharicola ICMP C gloeosporioides CBS C alatae ICMP C xanthorrhoeae CBS C horii ICMP C. cordylinicola ICMP C psidii ICMP C kahawae ICMP C clidemiae C theobromicola CBS

15 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 A2 91 A3 98 A1 97 T3 Colletotrichum siamense Thai Coffe ICMP S1 S2 S3 T5 99 T6 T4 T7 T1 99 T2 68 T9 A4 A6 A7 T8 Colletotrichum salsolae ICMP Colletotrichum queenslandicum ICMP 1778 Colletotrichum asianum ICMP Colletotrichum alienum ICMP Colletotrichum tropicale ICMP Colletotrichum musae ICMP Colletotrichum fructicola ICMP Colletotrichum nupharicola ICMP Colletotrichum gloeosporioides ICMP Colletotrichum xanthorrhoeae ICMP Colletotrichum alatae ICMP Colletotrichum horii ICMP Colletotrichum aotearoa ICMP Colletotrichum cordylinicola ICMP Colletotrichum psidii ICMP Colletotrichum kahawae ICMP Colletotrichum clidemiae ICMP Colletotrichum theobromicola ICMP

16 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.

17 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

18 Anthracnose symptoms on chili fruit 10 days after inoculation W NW Day 1 Day 10

19 Determination of pathogenicity Disease severity = (DCS) lesion length Fruit length X Disease severity scale developed by (Montri et al. 2009)

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

21 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

22

23 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

24 Acknowledgment Principle supervisor Prof. Paul Taylor Co-supervisors Prof. Pedro Crous, Dr. Peter Ades Collaborative researchers from Australia and other countries

25 Thank you..