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Structure of the Australian chickpea Phoma rabiei population and risk to
resistant cultivars through fungal adaptation
Rebecca Ford, Prabhakaran Sambasivam, Yasir Mehmood,
Sukjhiwan Kaur, Jenny Davidson, Audrey Leo, Kristy Hobson, Kevin Moore
GRDC project UM00052
Phoma (syn. Ascochyta) rabiei
• Necrotrophic ascomycete fungus
• Largest biotic cause of loss of yield and grower confidence in Australia
• Quantitative resistance and relatively few widely adopted R/MR cultivars
• Unlikely that sexual recombination is occurring frequently in Australia (2nd MAT?)
Approach to ensuring stability of cultivar resistance
Depends on selection and incorporation of resistance genes that are effective against the entire fungal population
Therefore specific requirements are to:
1. Survey and monitor for changes within the P. rabiei population
– Assess pathogenic structure - to identify those most highly pathogenic
– Assess molecular structure – to identify important clonal or unique haplotypes
– Identify frequencies of highly pathogenic isolates in widely adapted haplotypes
2. Choose and apply isolates that represent the highest risk for selective resistance breeding, these are:
– Highly pathogenic on most broadly grown cultivars
– Members of a widely adapted haplotype (fit across growing regions)
– Supplied to the National chickpea breeding program
Region1 Region1
Region 2 Region 2
Region 3 Region 3
Region4
Region4
Region 5
Region 5
Region 6
Region 6
Australian chickpea growing regions
Isolate collection 1998-2015
• >1500 isolates
• Location and host genotype noted
• Single spored and validated through morphology and PCR
Overall pathogenic structure of the population
• No significant structure detected based on the host set used
• Continuum of pathogenic diversity
Elliott et al. Australasian Plant Pathology (2011)
AUDPC where Dim-1 = 72.48% of the variation, Dim-2 9.81%, and Dim-3 7.12%.
In 2010…
• Relatively few isolates were ranked highly pathogenic on resistant sources
• No isolate was found highly pathogenic on Genesis090
• Few were highly pathogenic on PBA HatTrick
Resistant sources
Susceptible check
However, a significant proportion of the 2013-2015 annual population was highly pathogenic
Based on ability to cause leaf and stem symptoms on a refined host set under controlled conditions
Genotype Rating Reference
ICC3996 Resistant Nasir et al. (2000)
Genesis090 Resistant Pulse Australia (2009)
PBA HatTrick Moderately Resistant Pulse Breeding Australia (2013)
Kyabra Susceptible DPI NSW (2014)
Plants scored at 21 days post inoculation:
Leaf lesions
1. No leaf symptoms, 3. Pin prick lesions, 5. Small lesions without pycnidia, 7. Individual
lesions with darker margins and some pycnidia and 9. Lesions with dark margin
coalesced with pycnidia
Stem lesions
1. No stem symptoms, 3. Pin prick lesions, 5. Small lesions without pycnidia, 7. Individual
lesions with darker margins and some pycnidia, no stem breakage and 9. Lesions with
dark margin coalesced with pycnidia and possible stem breakage
Isolates were rated as highly pathogenic
When:
• Isolates had a leaf score of 7 or 9 greater than 80% of the time on PBA HatTrick, Genesis 090 and ICC3996
And/or
• Isolates had a stem score of 7 or 9 greater than 10% of the time on PBA HatTrick and Genesis 090
• We haven’t yet found an isolate highly pathogenic based on stem lesions on ICC3996 in our controlled environment trials
Genotypes Pathotype Groups
1 2 3 4
Kyabra
HatTrick
Genesi090
ICC3996
Highly pathogenic isolates were put into pathotype groups
Highly Pathogenic isolates collected in 2013 (n=105 ~17% HP)
Isolate
Location of Collection
Host Cultivar
ICC3996
Genesis090
HatTrick
Rating
Group
FT 13092-1 Kingsford, SA Genesis090 Moderate High High High 4
FT 13092-2 Kingsford, SA Genesis090 Moderate High High High 4
FT 13092-4 Kingsford, SA Genesis090 Moderate Moderate High High 3
FT 13092-6 Kingsford, SA Genesis090 Low Moderate High High 2
TR5919 Tooraweenah, NSW HatTrick Low Low High High 1
TR5920 Tooraweenah, NSW HatTrick Low Low High High 1
TR5928 Tooraweenah, NSW HatTrick Low Low High High 1
TR5964 Garah, NSW HatTrick Low Low High High 1
TR6034 Curban, NSW HatTrick Low Low High High 1
TR6041 Curban, NSW HatTrick Low Low High High 1
TR6042 Curban, NSW HatTrick Low Low High High 1
FT 13092-3 Kingsford, SA Genesis090 Low Low High High 1
FT 13092-5 Kingsford, SA Genesis090 Low Low High High 1
FT 13093-1 Kingsford, SA Genesis090 Low Low High High 1
FT 13093-2 Kingsford, SA Genesis090 Low Low High High 1
FT 13093-3 Kingsford, SA Genesis090 Low Low High High 1
FT 13093-5 Kingsford, SA Genesis090 Low Low High High 1
13DON002 Donald, Vic Genesis090 N/A High N/A High -
13MUR002 Murtoa, Vic Genesis090 N/A High N/A High -
In general
• If an isolate is low pathogenic on Genesis090 it is highly pathogenic on PBA HatTrick
• If an isolate is moderately pathogenic on ICC3996 it is moderate to highly pathogenic on Genesis090
Isolate Location of Collection
Host Cultivar ICC3996 Genesis090
HatTrick
Rating Group
TR6415 Yallaroi, NSW HatTrick Moderate High High High 4
TR6417 Yallaroi, NSW HatTrick Moderate High High High 4
TR6400 Yallaroi, NSW HatTrick Low Low High High 1
TR6403 Yallaroi, NSW HatTrick Low Low High High 1
TR6405 Yallaroi, NSW HatTrick Low Low High High 1
TR6406 Yallaroi, NSW HatTrick Low Low High High 1
TR6421 Yallaroi, NSW HatTrick Low Low High High 1
TR6422 Yallaroi, NSW HatTrick Low Low High High 1
14HOR 017 Horsham, VIC Genesis 425 Low Low High High 1
14DON003 Donald, VIC Slasher Low Low High High 1
14DON004 Donald, VIC Slasher Low Low High High 1
FT14041 Salter Springs, SA Monarch Low Low High High 1
Highly Pathogenic isolates collected in 2014 (n=100 ~12% HP)
• Some Yallaroi, NSW isolates from PBA HatTrick are highly pathogenic on Genesis 090
Isolate Location of Collection Host Cultivar ICC3996 Genesis090 HatTrick Rating Group
15CUR002 Curyo, VIC Genesis090 Moderate High High High 4
15CUR005 Curyo, VIC Genesis090 Moderate High High High 4
FT15024 Moonta, SA Genesis090 Moderate High High High 4
FT15026 Moonta, SA Genesis090 Moderate High High High 4
FT15030 Moonta, SA Genesis090 Moderate High High High 4
15DON008 Donald, VIC Slasher Moderate High High High 4
15DON009 Donald, VIC Slasher Moderate High High High 4
15DON010 Donald, VIC Slasher Moderate High High High 4
FT15023 Moonta, SA Genesis090 Moderate Moderate High High 3
FT15025 Moonta, SA Genesis090 Moderate Moderate High High 3
FT15027 Moonta, SA Genesis090 Moderate Moderate High High 3
FT15028 Weetulta, SA Genesis090 Moderate Moderate High High 3
FT15029 Weetulta, SA Genesis090 Moderate Moderate High High 3
15DON007 Donald, VIC Slasher Moderate Moderate High High 3
FT15001 Weetulta, SA Genesis090 Low Low High High 1
FT15003 Weetulta, SA Genesis090 Low Low High High 1
FT15004 Weetulta, SA Genesis090 Low Low High High 1
FT15017 Crystal Brook, SA Genesis090 Low Low High High 1
TR7135 North Star, NSW HatTrick Low Low High High 1
TR7137 North Star, NSW HatTrick Low Low High High 1
FT15019 Crystal Brook, SA Slasher Low Low High High 1
FT15020 Crystal Brook, SA Slasher Low Low High High 1
FT15021 Crystal Brook, SA Slasher Low Low High High 1
FT15022 Crystal Brook, SA Slasher Low Low High High 1
Highly Pathogenic isolates collected in 2015 (n=60 ~40% HP)
154 Km
• Increased frequency of Group 4 highly pathogenic isolates from Genesis 090 and PBA Slasher?
• A growing population of Group 3 highly pathogenic isolates?
Tooraweenah
Kingsford
Garah
Curban
Donald Murtoa
Distribution of Highly Pathogenic isolates collected in 2013
Donald Horsham
Salter Springs
Yallori
Distribution of Highly Pathogenic isolates collected in 2014
Curyo
Donald
Weetulta
North Star
Moonta
Crystal Brook
Distribution of Highly Pathogenic isolates collected in 2015
Host Year No isolates
phenotyped
Frequency (%)
highly pathogenic
Genesis090
2013 20 40
2014 9 0
2015 28 50
PBA HatTrick
2013 77 10
2014 70 12
2015 15 13
PBA Slasher 2015 16 38
Higher frequency of HP isolates was collected on Genesis090 and more recently on PBA Slasher than on PBA HatTrick
Genesis090 47%
HatTrick 29%
Slasher 20%
Genesis425 2%
Monarch 2%
Total frequency 2013-2015
But of course, this is dependant on where the epidemic occurs and what cultivars are sown
Percentage of highly pathogenic isolates collected from host cultivars
Genesis090 59%
HatTrick 41%
In 2013
HatTrick 60% Genesis
425 10%
Monarch 10%
Slasher 20%
In 2014
Genesis090 50%
HatTrick 8%
Slasher 32%
In 2015
What we learn from molecular population structure analyses
Based on differences in allele frequencies at characterised and unique P. rabiei microsatellite loci we can determine:
• How similar isolates are to each other across the whole population and among target regions and if the population is becoming more or less diverse
• Given that the pathogen is propagating asexually, if there are particular clones (haplotypes) that are more frequent and hence potentially better adapted
In 2010…..
• 241 isolates collected from 5 major growing regions were assessed for diversity at 20 loci
• Overall low diversity (H = 0.094; Ĝ = 22.91%)
• Low diversity among regions (φpt = 0.040, P > 0.05), suggests recent founder effect
• Highest diversity among isolates from Kingsford, SA
Leo et al. Mycological Research (2013); Leo et al. Biological Invasions (2014)
In 2010, a frequent haplotype was detected
Isolate Geographic Origin Host origin Year Pathogenicity Haplotype
09KAL09 Kalkee, western Victoria Genesis 090 2009 High ARH046
09KAN32 Kaniva, western Victoria CICA 0512 2009 High ARH091
09KIN06 Kingsford, SE South Australia Howzat 2009 High ARH069
09MEL03 Melton, Yorke Peninsula, South Australia Genesis 114 2009 High ARH073
09MEL35 Melton, Yorke Peninsula, South Australia Howzat 2009 High ARH088
10MEL004 Melton, Yorke Peninsula, South Australia Genesis 090 2010 High ARH01
10MEL005 Melton, Yorke Peninsula, South Australia Genesis 090 2010 High ARH01
10MEL009 Melton, Yorke Peninsula, South Australia Genesis 090 2010 High ARH01
10MEL017 Melton, Yorke Peninsula, South Australia Genesis 090 2010 High ARH01
10MEL018 Melton, Yorke Peninsula, South Australia Genesis 090 2010 High ARH01
10MEL027 Melton, Yorke Peninsula, South Australia Genesis 090 2010 High ARH01
T12437 Warra, southern Queensland HatTrick 2010 High ARH01
• ARH01 accounted for 47% of the surveyed population
• Detected in all chickpea growing regions
• Detected on all resistance sources
• Many of the ARH01 isolates were highly pathogenic
A more comprehensive survey revealed
Number of alleles
Locus Total Region1 Region2 Region3 Region4 Region5 Region6 Diversity
ArA03TA 8 5 3 4 2 3 4 0.178
ArH05TA 6 2 4 2 3 2 3 0.129
ArR12DA 3 2 1 2 2 2 2 0.036
Me14-1-56B 2 2 1 1 1 1 1 0.036
Me14-1-63B 3 1 2 1 3 2 1 0.032
Me14-1-83B 2 2 1 1 1 1 1 0.018
Me14-1-91B 2 2 1 2 1 1 1 0.020
Mean 0.064
• In the 2013 -2015 population, just 7 of the 20 loci were informative enough to provide structural information
• Regions contained 12 – 16 of the 26 informative alleles
• Region 1 (central Qld) had the highest number of
different alleles
AHuttel et al. Genome (1999) BLeo et al. Mycological Research (2013)
• 655 isolates
• 103 haplotypes
• Most frequently detected = ARH01 (~63%)
Haplotypes detected 2013-2015
Where: • Line distance = Relatedness • Circle size = Frequency
No haplotypes = 9 ARH01=55.55%
No haplotypes = 9 ARH01=55.55%
No haplotypes = 37 ARH01=75%
No haplotypes = 37 ARH01=75%
No haplotypes = 23 ARH01=69.62%
No haplotypes = 23 ARH01=69.62%
No haplotypes = 19
ARH01=59.57%
No haplotypes = 19
ARH01=59.57%
No haplotypes = 13 ARH01=55.53%
No haplotypes = 13 ARH01=55.53%
No haplotypes = 17 ARH01=55.84%
No haplotypes = 17 ARH01=55.84%
In 2013-2015, ARH01 was by far the most frequently detected haplotype across all regions
Year No Isolates No Haplotypes Frequency (%)
ARH01
2013 161 32 59
2014 254 31 64
2015 240 40 63
Overall, the ARH01 frequency was stable 2013-2015
• ~63% irrespective of sixe of population sampled or host cultivar
• However, peaks in ARH01 frequency were observed over time and within regions
Year Population
and No
isolates
Frequency
(%) ARH01
2013 1 (48) 67
2 (48) 84
2014 1 (73) 63
2 (73) 63
2015 1 (46) 63
2 (31) 62
Host (isolates collected) Frequency (%)
of ARH01 of all
isolates on host
Mean diversity
among all isolates
PBA HatTrick (383) 63 0.048
Genesis090 (61) 54 0.052
CICA1152 (19) 47 0.051
PBA Boundary (14) 47 0.038
PBA Slasher (17) 45 0.063
2013-2015 ARH01 was by far the most frequently detected haplotype on all host cultivars
In 2015, ARH01 was even more frequently detected than before on two of the most widely grown cultivars
Host (isolates collected) Frequency (%) of
ARH01
Mean diversity among
all isolates
PBA HatTrick (117) 70 0.052
Genesis090 (34) 73 0.046
ARH01 isolates were more frequently highly pathogenic than isolates of other haplotype groups 2013-2015
ARH01 75%
ARH125 5%
ARH08 4%
ARH129 4%
ARH07 2%
ARH77 2% ARH116
2%
ARH136 2%
ARH178 2%
ARH180 2%
Overall, 75% of 54 highly pathogenic isolates were ARH01 haplotype, irrespective of host or region
Selective adaptation towards highly pathogenic ARH01 isolates?
• Baseline frequency of ARH01 in the Aust pop is ~63%
• Baseline frequency of highly pathogenic isolates in the Australian population is 12-40%
• In 2015 40% of all isolates were highly pathogenic, 80% of these were of ARH01
2015 (n = 24)
ARH01 80%
ARH180 4%
ARH178 4%
ARH77 4%
ARH116 4%
ARH08 4%
ARH01 72%
ARH07 5%
ARH08 6%
ARH125 11%
ARH129 6%
2013 (n = 18)
ARHO1 73%
ARH125 9%
ARH129 18%
2014 (n = 12)
Group 1 62%
Group 2 2%
Group 3 13%
Group 4 23%
Genotypes Pathotype Groups
1 2 3 4
Kyabra
HatTrick
Genesi090
ICC3996
Almost a quarter of the ARH01 highly pathogenic isolates were of the most pathogenic group (4), able to cause significant disease
on broadly sown cultivars
Initial work towards dissecting functional differences among pathotype groups
• Differences in pathogen establishment and early invasion mechanisms will impact management strategies – different fungicides cause different disruptive effects
Isolate ICC3996 Genesis090 HatTrick Overall Group
FT 13092-1 Moderate High High High 4
FT 13092-4 Moderate Moderate High High 3
A initial comparison between isolates of different pathogenicity groups
Germination rate – isolate FT13092-1 (Group 4)
• Slower and lower on ICC3996 • At 24 hpi as much germination on Genesis090 and PBA HatTrick as on Kyabra
Germination rate – isolate FT13092-4 (Group 3)
• Slower and lower on ICC3996 but less than FT13092-1 (Group 4) • At 24 hpi less on Genesis090 than on PBA HatTrick
Germ tube length growth rate – isolate FT13092-1 (Group 4)
• Slower and lower on ICC3996 • At 24 hpi as much on Genesis090 and PBA HatTrick as on Kyabra
Germ tube length growth rate – isolate FT13092-4 (Group 3)
• No significant difference between Kyabra and PBA HatTrick
Summary of initial histopathology
• Isolates perform differently on different hosts and among each other
– The significant differences in germination percentage and germ tube length between the two isolates assessed matches with their pathogenicity group rating (4 and 3) - clear evidence of diversity within the population
• Based on the germination percentage and germ tube length of two highly aggressive isolates:
– both ICC3996 and Genesis090 are able to retard the growth of the fungus compared to the moderately susceptible PBA HatTrick and the susceptible Kyabra
• Is this due to:
– Production of antifungal compounds that are sent to attack the spore at a very early time point in the interaction (from 2 hpi)?
– An ability of ICC3996 and Genesis090 to recognise the spore faster?
In summary - reviewing the risk Although there is narrow whole genome diversity, a wide range of pathogenicity exists
within the Australian P. rabiei population
We are detecting a growing number of isolates that are highly pathogenic on our best resistant cultivars
Many highly pathogenic isolates are part of a clonal population, suggesting they are part of a founder population that is selecting for the fittest isolates adapted to all growing regions and our widely deployed cultivars
PBA HatTrick and Genesis090 are already compromised and we predict others such as PBA Slasher will too, very soon
Immediate recommendations • Deploy new resistant cultivars with different resistance genes to PBA HatTrick and
Genesis090
• Use the most recently detected highly pathogenic isolates (always one season behind the pathogen)
• Continue to monitor the spatially and temporally distribution and frequency of highly pathogenic isolates of large clonal haplotypes in the field to determine potential adaptation to current management practices