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?
Evolutionary Hypotheses
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)
Evolutionary Hypotheses
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
Fusarium wilt of chickpeas
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
Fusarium wilt of chickpeas
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
opuntiarum 28940 opuntiarum 28279
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
opuntiarum 28279 opuntiarum 28940
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
opuntiarum 28279 opuntiarum 28940
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
F. oxysporum field study
Demers et al. (2015) Appl. Environ. Microbiol. 81: 81-90
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
F. oxysporum field study
Demers et al. (2015) Appl. Environ. Microbiol. 81: 81-90
Plan
ts
Soil
Endophytic and soil F. oxysporum from field-grown tomatoes
Demers et al. (2015) Appl. Environ. Microbiol. 81: 81-90
F. oxysporum field study
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: