diversity, ecology and evolution of soilborne fungal...

María del Mar Jiménez-Gasco

Department of Plant Pathology & Environmental Microbiology The Pennsylvania State University

Diversity, ecology and evolution of soilborne fungal plant pathogens

Outline

Plant disease bias: What are we missing?

Fusarium oxysporum

Host-adapted endophytes Evolution of plant pathogenicity and virulence

Verticillium dahliae

Endophytic associations with “non-hosts”

Reproductive biology

Conclusions and implications

The world of endophytes

Plant disease bias: What are we missing?

Endophytes:

“Organisms that live inside plant hosts without causing disease”

Best studied: Clavicipitaceous endophytes Neotyphodium/Epichloë associated with cool season grasses

Increasing knowledge of the diversity of fungal endophytes found in tropical and other regions

Kuldau & Bacon (2008) Biocontrol 46: 57-71 Rodriguez, White, Arnold & Redman (2009) New Phytologist 182: 314–330

Higgins, Coley, Kursar & Arnold (2011) Mycologia 103: 247-260

Fusarium wilts

Fusarium oxysporum Fusarium wilts Anamorphic Ascomycete No teleomorph found — considered to be asexual Well known plant pathogen: causes disease to hundreds

of plants Inhabitant of cultivated and non-cultivated soils worldwide

Fusarium oxysporum Fusarium wilts Non pathogenic, saprotrophic populations Pathogenic populations (formae speciales) host specific (over 150 ff. spp. described) Pathogenic F. oxysporum invade the root cortex and

colonize the xylematic vessels Symptoms are mainly the result of water stress

Fusarium wilts

Morphologically undistinguishable

The primitive basis of a F. oxysporum forma specialis was a nonpathogenic population that evolved towards acquisition of pathogenicity on a particular host

For a long time it was assumed that formae speciales were natural subdivisions within the species

The evolutionary interpretation :

Evolutionary Hypotheses

Isolates with shared host range should be closely related

O’Donnell et al. (1998) PNAS 95: 2044-2049

Molecular phylogenetics proved this assumption wrong:

many formae speciales are polyphyletic

Convergent evolution? Cryptic sexual

recombination? Horizontal gene transfer?


Transference of 1 or 2 small Chromosomes

(conditionally dispensable or supernumerary chromosomes)

Ma et al. (2010) Nature 464:367-373

pathogenic

nonpathogenic

Isolate with transferred chromosomes

Recently, comparative genomics identified lineage-specific chromosomes in F. oxysporum f. sp. lycopersici (tomato)


F. oxysporum f. sp. ciceris pathogenic of Cicer spp.

Fusarium wilt of chickpeas

High pathogenic variability limits the use of host resistance

Pathotypes

Pathogenic races


Y W

R0

R5

Pathotypes induce different disease syndromes: Yellowing and wilting

Pathogenic races based on differential disease reactions on a set of cultivars Races 0, 1B/C, 2, 3, 4, 5, and 6


Jiménez-Gasco et al. (2002) Plant Pathol. 51:72-77 Demers et al. (2014) EJPP: 139:303-318

1 change

F.o.ciceris 9094 JG62

F.o.ciceris 1992R2N F.o.ciceris 7802 F.o.ciceris 7989 F.o.ciceris 9035 F.o.ciceris 1992R3N F.o.ciceris 1992R4N F.o.ciceris 1987T F.o.ciceris USA-W17 F.o.ciceris 9601 F.o.ciceris 9602 F.o.ciceris USA3-1JG62 F.o.ciceris 8605 F.o.ciceris 8606 F.o.ciceris 8607

F.o.ciceris 9093 PV1 F.o.ciceris 9168

1 change

57

F.o.melonis 26046 F.o.lycopersici 26200

F.o.lycopersici 26034 F.o.tulipae 28974

F.o.lycopersici 26383 F.o.lycopersici 26203

F.o.asparagi 28404 F.o.radicis-lycopersici 26379

F.oxysporum 9169

F.o. cubense 26022 F.o.spinaciae 26876

F.oxysporum 25356 F.o.melonis 26178 F.oxysporum 90101 F.oxysporum 90105

F.o.cubense 25609 F.o.lini 29094

F.oxysporum 28366

F.o.dianthi 28401 F.o.gladioli 28918

F.o.cubense 25607

F.oxysporum 25357 F.o.cubense 25605

F.o.canariensis 26035 F.o.cubense 26029

F.oxysporum 28371 Fusarium sp. 22903

62

76

57 86

61 58

83 65

74 58

88

100 65

F. o. ciceris

F. oxysporum NP to chickpea

TEF1α MP

F. o. ciceris is monophyletic

All F. o. ciceris races: • Identical sequences of genes: actin, β-tub, cal, EF-1α, histone

H3, rpb2, ITS, IGS… and over 15 microsatellite markers

• 1 VCG • 1 mtDNA RFLP pattern

What is the true ecological role of F. oxysporum?

Are all soil isolates able to establish asymptomatic infections in any plant?

Are there true saprobes?

Are nonpathogenic populations the ancestors of pathogenic ones?

Questions

25-PV60-1 - Spain 45-PV1-8 - Spain Fo506 - Italy Fo442 - Italy Focc14J - Israel Fsp7V - Algeria heliotropii 26412 IC100 - Syria IC51 - Syria IC64 - Syria IC77 - Syria IC94 - Syria JG62-21 - Spain JG62-24-A - Spain JG62-24-B - Spain JG62-25-I - Spain Fo8250 - Spain Sy96-18-2 - Syria

allium 38293 Fo550B - Italy Fo803 - Italy Fo813 - Italy gladioli 28911

asparagi 28973 batatas 22535 batatas 26409 bouvardiae 26219 carnation nonpathogen 26994 chrysanthemi 22539 dianthus 39464 erythroxyli 26574 Fo816 - Italy Fo817 - Italy Fo818 - Italy Fo91114 - Spain

passiflorae 22549 radicis lycopersici 26381 Fo802 - Italy

Fo807 - Italy lycopersici 26203 lycopersici 26383

tuberosi 22555 opuntiarum 28940

opuntiarum 28279 7.96 - Tunisia gladioli 28918

pini 38290 9-JG62-6 - Spain ET11A - Ethiopia ET1A - Ethiopia Fo420 - Italy Fo448 - Italy Fo9081 - Spain Fo511 - Italy Fo623A - Italy Fsp9 - Algeria hibiscus 38270 JG62-8 - Spain melonis 26178 Fo90101 - Spain Fo90105 - Spain Fo9169 - Morocco cc44J - Israel

cc61c - Israel conglutinans 38272 coreopsii Krane2 cubense 25367 ET10A - Ethiopia ET13B - Ethiopia ET14A - Ethiopia ET19A - Ethiopia ET23A - Ethiopia ET8A - Ethiopia ET9A - Ethiopia F4-29 - Syria raphani FRC1115 vasinfectum 25420

cyamopsis 38305 ET12A - Ethiopia ET15A - Ethiopia ET25A - Ethiopia ET7A - Ethiopia Fo804 - Italy ailanthus 38361

apii 38295 medicaginis 38285 embothrium 38303

litchi 38286 boxwood 38300 cyclaminis 36266 cc42K - Israel

Fo819 - Italy IC74 - Syria ET16B - Ethiopia

ET2A - Ethiopia ET4A - Ethiopia Fo812 - Italy IC67 - Syria IC70 - Syria elaeidis 38313

glycines 25598 lens 38271 prunus 38354

cubense 26022 pterocarpus 38317 agave 38336 cc3K - Israel melonis 26406 MX4 - Mexico MX446 - Mexico MX463 - Mexico MX472 - Mexico MX6 - Mexico MX719 - Mexico lycopersici 4287

ciceris 82108 race 0 ciceris 7989 race 1A ciceris USA 3-1 JG62 race 1BC ciceris 8605 race 2 ciceris 1992 R3N race 3 ciceris 8607 race 4 ciceris 8012 race 5 ciceris 9093 PV1 race 6

cubense FRC1564 ET5A - Ethiopia ET6A - Ethiopia

Fo526 - Italy lini 28922 canariensis 26035

cubense 25603 glycines 38328 perniciosum 22550

soil 25357 lisianthus 38353

F. commune 38348

batatas 25594 passiflorae 38273 coffee 38355

63

61

66

99

65

64

88 64

66

55

64

54 64

75

63

65

84

95

86 100

90

99 52

25-PV60-1 - Spain Fo442 - Italy Focc14J - Israel Fsp7V - Algeria IC94 - Syria IC77 - Syria JG62-24-A - Spain heliotropii 26412 IC64 - Syria Sy96-18-2 - Syria 45-PV1-8 - Spain JG62-25-I - Spain Fo8250 - Spain JG62-21 - Spain IC51 - Syria Fo506 - Italy

asparagi 28973 batatas 22535 batatas 26409 carnation nonpathogen 26994 Fo816 - Italy Fo817 - Italy Fo818 - Italy

erythroxyli 26574 dianthus 39464

passiflorae 22549 cc3K - Israel melonis 26406 lycopersici 4287

MX4 - Mexico MX446 - Mexico MX6 - Mexico MX719 - Mexico MX463 - Mexico MX472 - Mexico cubense FRC1564

passiflorae 38273 7.96 - Tunisia chrysanthemi 22539 radicis lycopersici 26381

agave 38336 allium 38293 Fo91114 - Spain

coffee 38355 Fo550B - Italy Fo802 - Italy Fo803 - Italy Fo807 - Italy Fo813 - Italy gladioli 28911 lycopersici 26203 tuberosi 22555 IC100 - Syria

lycopersici 26383 9-JG62-6 - Spain Fo420 - Italy Fo448- Italy Fo9081 - Spain Fo511 - Italy Fsp9 - Algeria JG62-8 - Spain Fo90101 - Spain Fo90105 - Spain Fo9169 - Morocco cyamopsis 38305 ET10A - Ethiopia

IC67 - Syria IC70 - Syria coreopsii Krane2 cubense 26022

pterocarpus 38317 ET15A - Ethiopia Fo812 - Italy ET5A - Ethiopia ET6A - Ethiopia ET8A - Ethiopia ET9A - Ethiopia

Fo804 - Italy ET7A - Ethiopia F4-29 - Syria hibiscus 38270

raphani FRC1115 vasinfectum 25420

lens 38271 apii 38295 ET25A - Ethiopia

boxwood 38300 embothrium 38303 medicaginis 38285

cyclaminis 36266 lisianthus 38353

canariensis 26035 ciceris 82108 race 0 ciceris 7989 race 1A ciceris USA 3-1 JG62 race 1BC ciceris 1992 R3N race 3 ciceris 8607 race 4 ciceris 8012 race 5 ciceris 9093 PV1 race 6

ciceris 8605 race 2 cubense 25603

perniciosum 22550 glycines 38328

soil 25357 Fo526 - Italy lini 28922 cc42K - Israel ET14A - Ethiopia Fo819 - Italy

IC74 - Syria melonis 26178 ET4A - Ethiopia

elaeidis 38313 ET19A - Ethiopia ET11A - Ethiopia JG62-24-B - Spain

conglutinans 38272 ET12A - Ethiopia Fo623A - Italy glycines 25598

litchi 38286 prunus 38354 ET16B - Ethiopia ET2A - Ethiopia

batatas 25594 bouvardiae 26219

opuntiarum 28940 opuntiarum 28279

ailanthus 38361 gladioli 28918

pini 38290 cc44J - Israel cc61c - Israel cubense 25367 ET13B - Ethiopia

ET1A - Ethiopia ET23A - Ethiopia F. commune 38348

99

58 97

81 88

100

100 74

86

64

93 70

99

96

60 100 60

63 99

64

98

52

64 100

64 100

96

86 90

94

100

98 59

100

100 98 63

100

100

99

100

55

100

100

71

54

100

gladioli 28918 pini 38290

glycines 25598 lens 38271

litchi 38286 medicaginis 38285


prunus 38354 pterocarpus 38317

canariensis 26035 cubense 25603 soil 25357 glycines 38328

perniciosum 22550 Fo526 - Italy lini 28922

lisianthus 38353 9-JG62-6 - Spain cc42K - Israel cc44J - Israel cc61c - Israel conglutinans 38272 coreopsii Krane2

cubense 25367 cyamopsis 38305 ET10A - Ethiopia ET11A - Ethiopia ET12A - Ethiopia ET13B - Ethiopia ET14A - Ethiopia ET16B - Ethiopia ET19A - Ethiopia ET1A - Ethiopia ET23A - Ethiopia ET25A - Ethiopia ET2A - Ethiopia ET4A - Ethiopia ET7A - Ethiopia ET8A - Ethiopia ET9A - Ethiopia F4-29 - Syria Fo420 - Italy Fo448-- Italy Fo9081 - Spain Fo511 - Italy Fo804 - Italy Fo812 - Italy Fo819 - Italy Fsp9 - Algeria hibiscus 38270 IC67 - Syria IC70 - Syria IC74 - Syria JG62-8 - Spain melonis 26178 Fo90101 - Spain Fo90105 - Spain Fo9169 - Morocco raphani FRC1115

vasinfectum 25420 elaeidis 38313

F. commune 38348

61

64

100

71

64

94

95

25-PV60-1 - Spain 45-PV1-8 - Spain Fo506 - Italy Fo442 - Italy Focc14J - Israel Fsp7V - Algeria heliotropii 26412 IC51 - Syria IC64 - Syria IC77 - Syria IC94 - Syria JG62-21 - Spain JG62-24-A - Spain JG62-24-B - Spain JG62-25-I - Spain Fo8250 - Spain Sy96-18-2 - Syria

allium 38293 asparagi 28973 batatas 22535 batatas 26409 bouvardiae 26219 carnation nonpathogen 26994 chrysanthemi 22539 dianthus 39464 erythroxyli 26574 Fo550B - Italy Fo802 - Italy Fo803 - Italy Fo807 - Italy Fo813 - Italy Fo816 - Italy Fo817 - Italy Fo818 - Italy Fo91114 - Spain gladioli 28911 IC100 - Syria lycopersici 26203 lycopersici 26383 passiflorae 22549 radicis lycopersici 26381 tuberosi 22555 7.96 - Tunisia

ciceris 82108 race 0 ciceris 7989 race 1A ciceris USA 3-1 JG62 race 1BC

ciceris 8605 race 2 ciceris 1992 R3N race 3 ciceris 8607 race 4 ciceris 8012 race 5 ciceris 9093 PV1 race 6 ET5A - Ethiopia ET6A - Ethiopia agave 38336

batatas 25594 coffee 38355 passiflorae 38273 cc3K - Israel

melonis 26406 MX446 - Mexico MX719 - Mexico lycopersici 4287

MX4 - Mexico MX463 - Mexico MX472 - Mexico MX6 - Mexico

ailanthus 38361 apii 38295 boxwood 38300

cubense FRC1564 cubense 26022 cyclaminis 36266 embothrium 38303

ET15A - Ethiopia Fo623A - Italy

65

85

62 62

65

89

63

66

Lineage I

Lineage I

Lineage I

Lineage II

Lineage II

Lineage II

Lineage III

Lineage III

Lineage III TEF, MP

IGS, MP

β-tubulin, MP

100

58

68

60 85 97

91

65 63

57

61

58

81

100

62

59

60

95 66

63 83

62

85

99 65

100 68

100 60

batatas 25594 passiflorae 38273

coffee 38355 batatas 26409

passiflorae 22549 radicis lycopersici 26381 Foc816 - Italy Foc817 - Italy Foc818 - Italy

Foc 91114 - Spain carnation nonpathogen 26994 batatas 22535 chrysanthemi 22539 bouvardiae 26219 erythroxyli 26574 asparagi 28973 dianthus 39464

lycopersici 26203 lycopersici 26383

tuberosi 22555 Foc550B - Italy Foc803 - Italy Foc813 - Italy gladioli 28911 allium 38293

Foc802 - Italy Foc807 Italy 45 PV1 8 - Spain JG62 24 Micro - Spain JG62 24 Macro - Spain JG62 21 - Spain JG62 25 1 - Spain 25 PV60 1 - Spain IC 51 - Syria IC 64 - Syria IC 77 - Syria IC 94 - Syria NP 8250 - Spain Foc442 - Italy Foc506 - Italy Foc cc14J - Israel Fsp7V - Algeria Sy96 18 2 - Syria heliotropii 26412 IC 100 - Syria


ciceris 82108 race 0 ciceris 7989 race 1A ciceris USA 3 1JG race 1BC ciceris 8605 race 2 ciceris 1992 R3N race 3 ciceris 8607 race 4 ciceris 8012 race 5 ciceris 9093 PV1 race 6 ET 5A - Ethiopia ET 6A - Ethiopia 7 96 - Tunisia

coreopsii Krane2 cubense 25367 JG62 8 - Spain 9 JG62 6 - Spain ET 11A - Ethiopia ET 1A - Ethiopia melonis 26178 NP 90101 - Spain NP 90105 - Spain NP 9169 - Morocco Fo420 - Italy Fo448 CM - Italy Fo9081 - Spain Foc511 - Italy Fsp9 - Algeria hibiscus 38270

ET 10A - Ethiopia ET 13B - Ethiopia ET 14A - Ethiopia ET 19A - Ethiopia ET 23A - Ethiopia ET 8A - Ethiopia ET 9A - Ethiopia

vasinfectum 25420 cc44J - Israel cc61c - Israel F4 29 - Syria raphani 1115 conglutinans 38272 ET 12A - Ethiopia ET 25A - Ethiopia ET 7A - Ethiopia Foc804 - Italy

cyamopsis 38305 ET 16B - Ethiopia ET 2A - Ethiopia ET 4A - Ethiopia IC 67 - Syria IC 70 - Syria IC 74 - Syria cc42K - Israel Foc819 - Italy Foc812 - Italy

elaeidis 38313 ET 15A - Ethiopia Foc623A - Italy

glycines 25598 medicaginis 38285 apii 38295

embothrium 38303 cyclaminis 36266 boxwood 38300

lens 38271 litchi 38286 prunus 38354 ailanthus 38361 cubense 26022 pterocarpus 38317

melonis 26406 MX 446 - Mexico MX 719 - Mexico cc3K - Israel MX 4 - Mexico MX 463 - Mexico MX 472 - Mexico MX 6 - Mexico agave 38336 Foc526 - Italy

lini 28922 cubense 1564 gladioli 28918

pini 38290 canariensis 26035

cubense 25603 soil 25357

perniciosum 22550 glycine 38328

lisianthus 38353 allium 38348

F. redolens 91117

1 change

25-PV60-1 - Spain 45-PV1-8 - Spain Fo506 - Italy

Fo442 - Italy

Focc14J - Israel

Fsp7V - Algeria heliotropii 26412 IC100 - Syria

IC51 - Syria

IC64 - Syria

IC77 - Syria IC94 - Syria JG62-21 - Spain JG62-24-A - Spain

JG62-24-B - Spain

JG62-25-I - Spain

Fo8250 - Spain

Sy96-18-2 - Syria

65

Chickpea isolates from different areas are phylogenetically related – Identical sequences

for TEF, β-tubulin, IGS, and 4 microsatellites

MP tree based on TEF and β-tubulin 75 chickpea endophytic isolates

100

58

68

60 85 97

91

65 63

57

61

58

81

100

62

59

60

95 66

63

F. redolens 91117

1 change

cubense 25367 JG62 8 - Spain 9 JG62 6 - Spain ET 11A - Ethiopia ET 1A - Ethiopia melonis 26178 NP 90101 - Spain NP 90105 - Spain NP 9169 - Morocco Fo420 - Italy

Fo448 CM - Italy

Fo9081 - Spain Foc511 - Italy Fsp9 - Algeria

hibiscus 38270 ET 10A - Ethiopia ET 13B - Ethiopia ET 14A - Ethiopia ET 19A - Ethiopia ET 23A - Ethiopia ET 8A - Ethiopia ET 9A - Ethiopia

vasinfectum 25420 cc44J - Israel cc61c - Israel F4 29 - Syria raphani 1115 conglutinans 38272

ET 12A - Ethiopia ET 25A - Ethiopia ET 7A - Ethiopia Foc804 - Italy cyamopsis 38305

ET 16B - Ethiopia ET 2A - Ethiopia ET 4A - Ethiopia IC 67 - Syria IC 70 - Syria

IC 74 - Syria cc42K - Israel Foc819 - Italy

Foc812 - Italy

83

62

85

99 65

100 68

100 60

batatas 25594 passiflorae 38273

coffee 38355 batatas 26409

passiflorae 22549 radicis lycopersici 26381 Foc816 - Italy Foc817 - Italy Foc818 - Italy

Foc 91114 - Spain carnation nonpathogen 26994 batatas 22535 chrysanthemi 22539 bouvardiae 26219 erythroxyli 26574 asparagi 28973 dianthus 39464

lycopersici 26203 lycopersici 26383

tuberosi 22555 Foc550B - Italy Foc803 - Italy Foc813 - Italy gladioli 28911 allium 38293

Foc802 - Italy Foc807 Italy 45 PV1 8 - Spain JG62 24 Micro - Spain JG62 24 Macro - Spain JG62 21 - Spain JG62 25 1 - Spain 25 PV60 1 - Spain IC 51 - Syria IC 64 - Syria IC 77 - Syria IC 94 - Syria NP 8250 - Spain Foc442 - Italy Foc506 - Italy Foc cc14J - Israel Fsp7V - Algeria Sy96 18 2 - Syria heliotropii 26412 IC 100 - Syria


ciceris 82108 race 0 ciceris 7989 race 1A ciceris USA 3 1JG race 1BC ciceris 8605 race 2 ciceris 1992 R3N race 3 ciceris 8607 race 4 ciceris 8012 race 5 ciceris 9093 PV1 race 6 ET 5A - Ethiopia ET 6A - Ethiopia 7 96 - Tunisia

coreopsii Krane2 cubense 25367 JG62 8 - Spain 9 JG62 6 - Spain ET 11A - Ethiopia ET 1A - Ethiopia melonis 26178 NP 90101 - Spain NP 90105 - Spain NP 9169 - Morocco Fo420 - Italy Fo448 CM - Italy Fo9081 - Spain Foc511 - Italy Fsp9 - Algeria hibiscus 38270

ET 10A - Ethiopia ET 13B - Ethiopia ET 14A - Ethiopia ET 19A - Ethiopia ET 23A - Ethiopia ET 8A - Ethiopia ET 9A - Ethiopia

vasinfectum 25420 cc44J - Israel cc61c - Israel F4 29 - Syria raphani 1115 conglutinans 38272 ET 12A - Ethiopia ET 25A - Ethiopia ET 7A - Ethiopia Foc804 - Italy

cyamopsis 38305 ET 16B - Ethiopia ET 2A - Ethiopia ET 4A - Ethiopia IC 67 - Syria IC 70 - Syria IC 74 - Syria cc42K - Israel Foc819 - Italy Foc812 - Italy

elaeidis 38313 ET 15A - Ethiopia Foc623A - Italy

glycines 25598 medicaginis 38285 apii 38295

embothrium 38303 cyclaminis 36266 boxwood 38300

lens 38271 litchi 38286 prunus 38354 ailanthus 38361 cubense 26022 pterocarpus 38317

melonis 26406 MX 446 - Mexico MX 719 - Mexico cc3K - Israel MX 4 - Mexico MX 463 - Mexico MX 472 - Mexico MX 6 - Mexico agave 38336 Foc526 - Italy

lini 28922 cubense 1564 gladioli 28918

pini 38290 canariensis 26035

cubense 25603 soil 25357

perniciosum 22550 glycine 38328

lisianthus 38353 allium 38348

Interpretation: Endophytic F. oxysporum are adapted to their host

plant

Interpretation: Endophytic F. oxysporum may be seed-transmitted

Chickpea isolates from different areas are phylogenetically related – Identical sequences

for TEF, β-tubulin, IGS, and 4 microsatellites

Endophytic and soil F. oxysporum from field-grown tomatoes

Demers et al. (2015) Appl. Environ. Microbiol. 81: 81-90

Hypothesis Populations of F. oxysporum endophytes will be different and less diverse than populations from surrounding soil

F. oxysporum field study

• 609 isolates studied, up to 7 different TEF haplotypes per plant

0.000 0.050 0.100 0.150 0.200 0.250 0.300 0.350 0.400 0.450 0.500

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Freq

uenc

y

Haplotype

Field 1 populations Field 2 populations

• Populations in each field had a different composition (HST, P < 0.001)

• Soil populations within a field had the same composition (pair-wise comparisons, HST, P> 0.05) HST= Haplotipe statistic



Soil and endophyte population differentiation

• Diversity of endophyte populations (SHA = 2.729) was significantly higher than soil populations (SHA = 2.672) for both fields (P < 0.001)

• Total soil population was different from total endophyte population (Snn, HST, P < 0.05)

0.000 0.050 0.100 0.150 0.200 0.250 0.300 0.350 0.400 0.450

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

Freq

uenc

y

Haplotype

Endophyte populations

Soil populations

SHA= Shannon diversity index

HST= Haplotipe statistic; Snn= Nearest neighbor statistic



Plan

ts

Soil

Endophytic and soil F. oxysporum from field-grown tomatoes



Ecological scenario for the evolution of pathogenicity and host specificity in Fusarium oxysporum

Cortex-colonizing endophytic lineages symbiotic, non-pathogenic? mutualistis, commensals

Vascular-colonizing pathogenic lineages

parasitic, pathogenic forma specialis

HORIZONTAL TRANSFER?

Acquisition of pathogenicity

factors?

Diverse resident soil population

(remnants of past recombination events?) saprobes, symbionts, plant pathogenic?

HOST PLANT SOIL

Host specificity and pathogenicity may be evolving independently

Nit M VCG 1213

Nit M VCG 1213

Nit 1 VCG 1213

Nit 1 EC5-LR-GM1

Nit 3 EC5-LR-GM1

Nit M EC5-LR-GM1

An endophyte isolated from old asymptomatic ‘Gros Michel’ bananas: Positive for TR4, but not pathogenic to ‘Cavendish’

Sampling in Ecuador

Panama disease: F. oxysporum f. sp. cubense

Verticillium wilts

Verticillium dahliae Verticillium wilts

Over 400 dicotyledonous hosts Survives in soil as microsclerotia Causes vascular wilts Symptoms of wilting are most evident on warm, dry,

sunny days There is some evidence of host-adapted populations

Rotations with non-host plant species

Examples for potato crops in the US: North East: Potato/oat, barley, corn North West: Potato/winter wheat, barley Florida: Potato/sudangrass

Results regarding a reduction in Verticillium wilt in the subsequent potato crop have been inconsistent

The mechanism responsible for reducing disease has been assumed to be a reduction of inoculum buildup

Verticillium wilts and crop rotations: Verticillium wilts

POTATO OAT

Two potato cultivars: ‘Snowden’— highly susceptible ‘Reba’— moderately resistant

Oat Surface disinfested, NP-10/EPAA

Verticillium wilts: Case study in PA

Hay mix: Timothy (Phleum pretense) Alfalfa (Medicago sativa) Red clover (Trifolium pratense) Hairy chess (Bromus commutatus)

Verticillium dahliae is an endophyte of oat plants

Malcolm, Kuldau, Gugino & Jimenez-Gasco (2013) Phytopathology 103: 538-544

Potato Potato ‘Snowden’ ‘Reba’ Oats

Reaction to Verticillium Wilt Susceptible

Moderately resistant Asymptomatic

N 30 30 26 Number of genotypes 7 4 2

Shannon index 1.434 0.717 0.161

Microsatellite analysis of V. dahliae populations

Scientifically interesting: hidden biology of V. dahliae Reservoirs of inoculum and diversity What does this mean in terms of management? Can these endophytic interactions explain why the effect of

rotations with an asymptomatic host in the subsequent symptomatic host is so inconsistent?

Genotype shifts

Potential scenarios and key factors: Differences in virulence to potato in the different

genotypes Contribution of inoculum to the soil population as a result

of the endophytic colonization of an asymptomatic host Can we ultimately design ad hoc rotations that specifically target

the resident V. dahliae soil population?

Implications in management

Potato Potato ‘Snowden’ ‘Reba’ Oats

VCG analysis of V. dahliae populations

VCG

2A & 4B 4A 4B 4A

2A & 4A 4B

VCGs are still used in V. dahliae research because they are correlated with biological information

MP tree IGS & Polymorphic sequences (SNPs)

Jimenez-Gasco et al. (2014) Phytopathology 104:650-659

Phylogenetics of VCGs

1. Digest genomic DNA with restriction enzyme

2. Ligate barcoded adaptors to ends of restriction fragments

3. Pool fragments from different isolates

4. PCR amplifies subset of fragments

5. Illumina sequences of ends of fragments aligned to reference genome

Davey et al. 2011, Nature Reviews Genetics

Genotyping-by-sequencing: Reduced Representation Libraries

Reduced Representation

Libraries

Sequencing reads are aligned to the V. dahliae reference genome (Vd.LS17) so SNPs can be identified

Strict criteria (e.g., number of reads with specific nucleotide) used to identify signals from sequencing errors, and for SNP calling

Genotyping-by-sequencing: Bioinformatics

141 isolates genotyped: host, geographical origin, host plant, VCG… 26,748 SNPs SNPs distributed in all 8 chromosomes

0

500

1000

1500

2000

2500

3000

0 500 1000 1500 2000 2500 3000

Contig size (kbp)

SNPs

per

con

tig

r = 0.97

SNPs evenly distributed throughout genome

Genotyping-by-sequencing in V. dahliae

4B

4A

6

2B824

2B334

2A

1A/1B

SNP Neighbor-joining tree

26,748 SNPs (>99% bootstrap)

2BR1 Recombinants between clades I and II?

Additional recombinants?

Clade I

Clade II

How can recombination hypotheses be tested?

Genotype-by-Sequencing

Phylogenetics of VCGs

C C G A C C T T Recombinant lineage

A C G T A C G T Parent lineage 1

C A T A C G T G Parent lineage 2

VCG1A

VCG2B

Sequence is a mix of nucleotides from VCG1A and VCG2B

Putative recombinant haplotype

Detecting recombination GBS, VCGs, and recombination

Milgroom , Jiménez-Gasco et al. (2014) PLoS ONE 9: e106740

443 unique recombination events detected Number of recombination events correlates with size of contigs

in the reference genome Recombination events are distributed in all 8 chromosomes

(30-82 recombination events/chromosome)

Recombination is evenly distributed throughout genome

r = 0.91

Contig size (kbp)

Reco

mbi

natio

n ev

ents

pe

r con

tig

0

5

10

15

20

25

30

35

40

45

50

0 500 1000 1500 2000 2500 3000

GBS, VCGs, and recombination

Recombination events by clades

2BR1

4B

395

VEMS281

(492)

V496 & V498

320

(V1287)

Total 443

2BR1 79

Clade II 338

Clade I 22

Clade II-2 90

Clade II-1 65

2A 22

1A/1B 2

4A

6

2B824

2B334

20

GBS, VCGs, and recombination

Clonality dominates populations of V. dahliae…but:

New lineages arose by recombination between clonal lineages: V. dahliae may not have been completely asexual

Recombinant isolates are associated to new variants of the pathogen or new epidemics of Verticillium wilt:

VCG 6: recombinant between 4A and 2B824 recently found in pepper in CA 2B334: recombinant between 1A/B and 2B824 recently found in artichoke in Spain

Current clonal population structure has been a consequence of agriculture and selection of lineages adapted to crops

Is recombination still occurring?

Recombination in V. dahliae

Plant Pathology 101: The Disease Triangle

Pathogen

Host Environment

Ecological niche Soil Plant

Diagnosis Pathogenic variability Reproductive systems

Conducive weather Resistance Interactions

In Plant Pathology we have largely ignored the ecology of well-known phytopathogenic fungi when not engaged in disease, but this may be key for:

Emergence of new pathogens or host expansion

Epidemiological studies (reservoir of inoculum and/or diversity)

Development of environmentally sustainable disease management strategies (e.g. biocontrol, rotations)

Comparative genomics and evolution (pathogenicity)

Studies on basic biology (reproductive modes)

Conclusions

My research group Jill Demers Glenna Malcolm Mónica Berbegal Laura del Sol Bautista Freddy Magdama Brian Aynardi Sarah Bardsley Jennifer Yanez Carla Garzon Esther Shin Sarah Colihan Jess Krocker Kristina Gans Jen Evans

Great collaborators Rafael M. Jiménez-Díaz (U. Cordoba,Spain) Beth K. Gugino (PSU) Michael Milgroom (Cornell U.)

Thanks to:

diversity, ecology and evolution of soilborne fungal...

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