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1 Sexual reproduction of Phyllosticta citricarpa, the causal agent of citrus black spot Nga Tran, Andrew Miles, Ralf Dietzgen, and André Drenth Centre for Plant Science, Queensland Alliance for Agriculture and Food Innovation, The University of Queensland

2 Citrus World production appx. 120M tons in 2011 (FAO, 2012) In Australia Production 390,000 A$500M ~ 28,000 ha in 2011, ~ 70% orange Queensland: ~ 3,600 ha (~13% of Australia s citrus) ~ 50% Australia s mandarin (Sette, 2011) Citrus production regions in Australia (Map provided by Citrus Australia Ltd.) 2

3 Citrus black spot (CBS) First reported in Australia in 1895 Reported in other citrus growing regions with summer rainfall Causes fruit blemishes Yield reduction (up to 50%) CBS infected areas in Australia Map adapted from by Miles, AK. Citrus black spot (Phyllosticta citricarpa) (photo by Shuey, T.) 3

4 Symptoms Hard spot Virulent spot Freckle spot Cracked spot Speckled blotch Hard spot lesions on leaf 4

5 Causal agent - Phyllosticta citricarpa Phyllosticta citricarpa McAlp (Pcit) Synonyms: Guignardia citricarpa Kiely, Letodothiorella sp. B C E Fruiting bodies and spore morphology of Phyllosticta citricarpa. A- Vertical section through Pycnidium, B- Conidia, C- Spermatia, D- Vertical section through Pseudothecium, E- Ascospore and conidia. Scale bars: A, D = 50 µm; B, C and E = 10 µm (A and D: Wikee et al., 2011) 5

6 Disease cycle Spermatia Miles, AK. 6

7 Disease cycle Pseudothecia Formed on leaf litter within days after leaf drop Alternate wetting and drying required Ascospores Spermatia Primary inoculum Released from pseudothecia Wetting of leaves required Dispersed by wind When and how ascospores are formed? Miles, AK. What is the role of spermatia in reproduction of P. citricarpa? 7

8 Sexual reproduction Sexual reproduction of other ascomycetes Homothallism vs heterothallism Mating type (MAT) genes involved in sexual reproduction In heterothallism, MAT genes found in isolates with complementary mating types (MAT1-1 and MAT1-2) MAT1-1 and MAT1-2 need outcross to proceed mating Spermatia involved in mating in some cases In P. citricarpa, MAT1-1 and MAT1-2 identified (Wang NY, in preparation) 8

9 Research question Question: What is the mating system of Phyllosticta citricarpa causing citrus black spot in Australia? Hypothesis: P. citricarpa is heterothallic and spermatia are involved in sexual reproduction of the fungus Ascospores of P. citricarpa 9

10 Methods P. citricarpa isolates A used in the mating assays Accession number B Mating type C MAT MAT MAT MAT MAT MAT MAT MAT1-2 A Isolates in bold have been characterised and identified by DNA sequence analysis (Miles et al., 2013) B Queensland Department of Primary Industries and Fisheries Plant Pathology Herbarium (BRIP) C Mating type identified by Wang NY. (in preparation) 10

11 Methods In vitro agar mating assay Mycelial strips of (i) MAT1-1 (ii) MAT1-2, or (iii) MAT1-1 + MAT1-2 paired parallel on ½ PDA, in triplicate Incubate under light/dark cycle of 12h/12h at 25 o C Assess vegetative compatibility Examine reproductive structures In vitro agar mating assay 11

12 Methods In vitro leaf disc mating assay Autoclaved lemon leaf discs of 1 cm dia. placed on moistened filter paper Mycelial plugs of (i) MAT1-1 (ii) MAT1-2, or (iii) MAT1-1 + MAT1-2 paired at leaf disc edges Plates exposed to 16 treatment regimes, e.g. fertilising with spermatia suspension, light, temperatures, wetting and drying Reproductive structures weekly examined under microscope Leaf disc mating assay (Mondal et al., 2004 with modifications) 12

13 Methods In planta mating assay Leaves inoculated with (i) MAT1-1 (ii) MAT1-2, or (iii) MAT1-1 + MAT1-2 Fertilised weekly with spermatia of opposite mating types Presence of reproductive structures examined weekly Sterilised blotting paper soaked in spore suspension Blotting folded to cover 2 sides of leaves Plants cover by plastic bag for 96 hours Leaves harvested, incubated, treated with wetting and drying in laboratory or kept intact 13

14 Methods Experimental mycelium and spermatia mating techniques P. citricarpa isolates of Mat1-1 and Mat1-2 were paired with all possible combinations via mycelium and spermatia Examine microscopically for reproductive structures Detail of methods pending publication 14

15 Results In vitro agar mating Incompatibility of P. citricarpa colonies observed Sexual reproduction was not induced 15

16 Results In vitro leaf disc mating Pycnidia with conidia, spermogonia with spermatia produced abundantly Some vegetatively incompatibility observed No sexual structure observed so far, after 6 months (Assessment is underway) In planta mating No sexual structure was induced 16

17 Results Mycelium and spermatia mating Combinations of opposite mating types readily and consistently produced fertile pseudothecia with ascospores Mat1-1 isolates and were not compatible with any Mat1-2 isolates Combinations of same mating type did not produce any reproductive structure Pseudothecia with asci and ascospores (inset) of P. citricarpa were successfully, 4 week after pairing 17

18 Conclusions P. citricarpa is vegetatively incompatible heterothallic Preliminary evidence for spermatia having a role Some isolates might carry incomplete or non-functional mating genes Question: What is the mating system of Phyllosticta citricarpa causing citrus black spot in Australia? P (partly) Hypothesis: P. citricarpa is heterothallic...and... P spermatia are involved in sexual reproduction of the fungus 18

19 Future works Confirm role of spermatia Investigate the possibility of non-functional mating gene isolates Study genetics of the offsprings Ascospores of P. citricarpa 19

20 Acknowledgment PhD Scholarship sponsors: UQI tuition fee awards Ernest Singer scholarships (UQ senate) Horticulture Innovation Australia Ltd project CT13021 Joint Florida and Australia citrus black spot research initiative and Citrus Research and Development Foundation, Florida. Citrus Teams (QAAFI and the University of Florida), staff and fellow students at 2CW (ESP) Research and Development for Primary Industries Pty Ltd 20

21 Thank you! 21

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