phoma rabiei population and risk to resistant cultivars ......tr6403 yallaroi, nsw hattrick low low...

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



TR5964 Garah, NSW HatTrick Low Low High High 1

TR6034 Curban, NSW HatTrick Low Low High High 1



FT 13092-3 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






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


• 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



15DON008 Donald, VIC Slasher Moderate High High High 4



FT15023 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



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





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


Curyo

Donald

Weetulta

North Star

Moonta

Crystal Brook


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







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 = 37 ARH01=75%

No haplotypes = 37 ARH01=75%



No haplotypes = 19

ARH01=59.57%

No haplotypes = 19

ARH01=59.57%





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

phoma rabiei population and risk to resistant cultivars ......tr6403 yallaroi, nsw hattrick low low...

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