1. S. Savocchia, R. Billones-Baaijens, A. Cimmino, A. Evidente
Isolation and characterisation of phytotoxins produced by Botryosphaeriaceae and their role in grapevine trunk diseases
PIERLUIGI REVEGLIA
PhD Candidate
Supervisors:
S. Savocchia, R. Billones-Baaijens, A. Cimmino, A. Evidente

2. Botryosphaeria dieback (BD)
INTRODUCTION
Botryosphaeria dieback (BD)
A serious problem of grapevines worldwide
Ranked in the top five priority diseases of the Australian wine grape industry
Caused by species of fungi – Botryosphaeriaceae
11 species reported in Australian vineyards
Causes cankers, dieback and eventually death of vines
Photo: W. Pitt

3. Photo: L. Mugnai
In Europe, the disease also produces foliar symptoms associated with phytotoxins produced by BD pathogens

4. No foliar symptoms have been observed in Australian vineyards to date
Investigate the role of phytotoxins in the pathogenicity and symptom expression of Botryosphaeria dieback pathogens in Australian vineyards

5. RESEARCH QUESTIONS
Do Australian Botryosphaeriaceae spp. produce phytotoxins in vitro and in planta?
Are these phytotoxins the same/different from those reported in other countries?
Are the phytotoxins involved in pathogenicity and virulence of these pathogens?
What is their role in the symptomatology observed in vineyards?
What gene families are involved in phytotoxin production?

6. OBJECTIVE 1: Identify and biologically and chemically characterise the phytotoxic metabolites produced by Botryosphaeriaceae spp.
Botryosphaeria spp.
Botryosphaeria dothidea
Diplodia mutila
Diplodia seriata
Dothiorella vidmadera
Dothiorella sp.
Neofusicoccum australe
Neofusicoccum luteum
Neofusicoccum parvum
Spencermartinsia viticola
Liquid cultures
Liquid extraction
TLC analysis
Grapevine leaf assays

7. Production of phytotoxic metabolites
PURIFICATION
CHARACTERISATION
Silica gel column
Preparative TLC
1D and 2D NMR spectroscopy
ESI/LC MS
Reveglia et al., Natural Product Research, (accepted)

8. Phytotoxins from Spencermartinsia viticola
Reveglia et al., Arab. J. Chem., 2018 doi.org/ /j.arabjc

9. Phytotoxins from Dothiorella vidmadera
Reveglia et al., J. Agr. Food Chem., 2018, 66,

10. FIRST CONCLUSIONS: ….WHAT’S NEXT?
The most virulent and widespread Australian Botryosphaeriaceae spp. are capable to produce phytotoxins in vitro.
They can produce already known and novel phytotoxins.
….WHAT’S NEXT?
Are the phytotoxins produced in planta ? Are they involved in pathogenicity and virulence of these pathogens?
What is their role in the symptomatology observed in vineyards?
What gene families are involved in phytotoxin production?

11. Botryosphaeriaceae spp.: D. seriata (H142a) S. viticola (L19)
OBJECTIVE 2: Production and translocation of PMs in artificially-inoculated and naturally-infected plants
Botryosphaeriaceae spp.:
D. seriata (H142a)
S. viticola (L19)
Do. vidmadera (L5)
Varieties:
Chardonnay
Cabernet Sauvignon
inoculate with mycelia plugs
12 plants per variety-treatment
sub-samples will be collected at 6, 9 and 12 months.
Naturally infected plants: Tumbaramba and Harden

12. EXPERIMENTS IN PROGRESS:
Isolation
Phytotoxins
extraction
and LC-QqQ analysis
DNA extraction
and qPCR

13. OBJECTIVE 3: Investigate the Genetic mechanisms involved in the production of phytotoxins by Botryosphaeriaceae spp.
Amplification of (R)-mellein gene, SN477 (Chooi et al. 2015).
Designed 7 different primers: NP 1 – NP 7.
DNA extraction 
(R)-mellein gene detection 
Only NP 1 was amplified for Neofusicoccum parvum, no amplification for Diplodia seriata. 
….WHAT’S NEXT?
Purify PCR product and sequence.
Design novel primers for D. seriata

14. ACKNOWLEDGEMENTS