diversity of Cylindrocarpon species - Australasian Plant Pathology ...

Species composition and genetic
diversity of Cylindrocarpon species
found commonly infecting New Zealand
grapevines
Blessy Pathrose
Supervisors: Dr Hayley Ridgway, Dr Eirian Jones and
Dr Marlene Jaspers
23/06/2011 1

Black foot disease of grapevines
An important problem in vineyards worldwide,
including:
South Africa
North America
Portugal
Australia
France
Italy
Sicily
New Zealand
It occurs in young vines, mature vines and mother
vines
Disease development is slow, usually in vines < 5 year
old and sometimes in first year of planting

Symptoms of Black foot disease
Chlorosis
Weak shoot growth
Delayed/absent bud break
Abnormal root
development

Photo by Francois Halleen
Black foot ‐ decline of plants

Photo by Francois Halleen
Vascular streaking

Cylindrocarpon species
Causal agents are Cylindrocarpon spp.
In all major viticulture regions throughout the world
In New Zealand, 3 species are associated with black
foot:
C. destructans C. liriodendri C.macrodidymum

Objective 1: To determine the population
composition of Cylindrocarpon species in NZ
The composition is currently unknown
A 2005 survey of NZ vineyards recovered 174 isolates of
Cylindrocarpon species from symptomatic vines
Isolates were obtained from the
8 main grape growing regions:
Auckland (14)
Gisborne/Hawkes bay (34)
Martinborough/Nelson/
Marlborough (98)
Waipara, North Canterbury (10)
Otago (18)

Identification of Cylindrocarpon species
infecting New Zealand grapevines
Identification by morphological features was insufficient
to differentiate the species
Process of molecular identification
Genomic DNA was extracted from
mycelium

MW C. macrodidymum C. liriodendri C. destructans ‐ve
Amplification with species specific primers CymaF1 & CymaR1
Species Specific Primers:
• Lizel Mostert, Stellenbosch University
C. macrodidymum – CymaF1 & CymaR1 = 300 bp
C. liriodendri – CyliF1 & CyliR1 = 200 bp
• Hayley Ridgway, Lincoln University
C. destructans – CydeF1 & CydeR1 = 200 bp
PCR products from the tubulin gene sequenced:
-10% of isolates from sp-sp PCR
- 14 unidentified isolates

Objective 2: Characterising genetic diversity of
the three predominant Cylindrocarpon spp.
Selection of UP-PCR Primers:
• 5 out of 11 chosen
• 3 test isolates
• Chosen for # polymophisms & band
distribution
UP-PCR using AA2M2 primer for
C. liriodendri
DNA fingerprints were generated for
151 isolates using UP-PCR:
• C. liriodendri (57) , C. macrodidymum
(41) , C. destructans (53)
Neighbour Joining Tree:
• ~ 70 data points per isolate
• Generated using PAUP software
• Identified main genetic groups

Objective 1:
Results
– 41 isolates of C. macrodidymum, 57 isolates of C. liriodendri,
53 isolates of C. destructans were identified
– DNA sequencing confirmed the identification as 100%
accurate for C. destructans, C. liriodendri and C.
macrodidymum
– Unidentified Cylindrocarpon spp. were identified as
C. pauciseptatum (9 isolates) Cylindrocladiella parva (1) and a
novel Cylindrocarpon species (4 isolates)
– 9 isolates remain unidentified

Distribution of Cylindrocarpon species in major
grape growing regions of New Zealand
100%
90%
North Island
South Island
Cylindrocarpon novel spp.
80%
70%
60%
Cylindrocarpon pauciseptatum
Cylindrocarpon macrodidymum
50%
40%
Cylindrocarpon liriodendri
30%
Cylindrocarpon destructans
20%
10%
0%
n= 14 13 21 3 3 95 11 18
Pearson Chi Squared
P= 0.000

Objective 2:C. macrodidymum
• NJ tree produced two
main branches (1
change)
• No clustering by
region, genetically
dissimilar individuals
found at all sites
• Inter – and intra –
vineyard variability
observed
• Clonal isolates were
identified

C. destructans
• NJ tree produced
three main branches
(1 change)
• No clustering by
region, genetically
dissimilar individuals
found at all sites
• Inter – and intra –
vineyard variability
observed
• Clonal isolates were
identified

C. liriodendri
• NJ tree produced six
main branches (1
change)
• No clustering by
region, genetically
dissimilar individuals
found at all sites
• Inter – and intra –
vineyard variability
observed
• Clonal isolates were
identified
• Isolate Ack1b
genetically distinct

Conclusions
• Five species of Cylindrocarpon identified
• Similar frequencies of C. macrodidymum (41), C. liriodendri (57) &
C. destructans (53)
• Four isolates of a novel Cylindrocarpon sp. ,1 isolate of
Cylindrocladiella parva and 9 isolates of C. pauciseptatum identified
• Differences in the distribution of the 3 main species:
– Higher proportion of C. destructans in the South Island
– Higher proportion of C. macrodidymum in the North Island
– Proportion of C .liriodendri, C. pauciseptatum, novel
Cylindrocarpon spp. isolates similar in both Islands
• Populations of the three main Cylindrocarpon species were
genetically diverse with low numbers of clonal isolates
• Inter – and intra – vineyard genetic variability observed

Acknowledgements
• Carolyn Bleach – Collecting isolates
• Winegrowers New Zealand –for research
funding
• Lincoln University –for the PhD scholarship
• Australasian Plant Pathology Society –
Sponsorship
• Lizel Mostert, Stellenbosch University
• ICPP –Travel Grant

Chlorosis
Thank You
23/06/2011 18