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Characterization and molecular identification of Phytophthora spp.
associated with Phytophthorablight of vegetables in Texas.
R.D. French-Monar1, Z. G. Abad2, A. F. Patton III1 , and T. Isakeit3
1Dept. of Plant Pathology, AgriLife Extension-Texas A&M, Amarillo, TX, USA. 2USDA-APHIS-PPQ-PSPI Molecular Diagnostics Lab., Beltsville, MD, USA.3Dept. of Plant Pathology , Texas A&M University, College Station, TX, USA.
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Texas High Plains Plant Diagnostic Laboratory
Texas AgriLife Research and Extension Center6500 Amarillo Blvd. WAmarillo, Texas 79106
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TEXAS RAINFALL: GO EAST, GET WET
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Texas = 2.25X Size of Italy
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126
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Phytophthora blightυ Caused by Phytophthora capsici (oomycete)
υ Devastating on pepper, cucurbits, tomato, and eggplant
υ Spread by surface water, wind and splashing rains.
υ Present in Texas for over 60 years.
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Symptoms
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P. capsici in Texas High Plainsυ 2006: First known report on pumpkin and
winter squash (Isakeit, 2007): 36 acres – 2% field affected August; 78% lost next month– Yoakum County
υ 2007:10-12 acres of chile pepper in a 60 acre field affected by this pathogen.– Gaines County
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Morphological
υ These isolates had semi-papillate to papillatesporangia, caducous sporangia with pedicels that exceeded 35 μm in length.
υ Sporangia 37 to 40 X 21 to 23 μmυ Exhibited heterothallism; no chlamydospores. υ When paired with an opposite mating type,
oospores produced were mostly aplerotic.υ Oogonia were amphigynous in their antheridial
attachment.υ Oospores 25 to 27 μm in diameter.
French-Monar et al. 2006. Plant Dis. 90: 345-350
Isakeit, T. 2007. Plant Dis. 91: 633
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Phytophthora capsici from Capsicum annuum
Photos by Gloria Abad, Ph.D USDA/APHIS/MDL 8.18.08
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2007 Field (Yoakum County): No Phytophthora blight
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2007 Field (Gaines County)
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Phytophthora blight on peppers Ronald French
(Texas A&M-TCE)
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Phytophthora blight on bell pepper
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Phytophthora blight in Texas
Chile pepper
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Phytophthora sp. on watermelon
Texas
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Compatibility type determination
υ For compatibility type determination:– a 6-mm mycelial plug of a known A1 or A2 mating type
is added to one side of a clarified V8 juice agar plate.– on the opposite side, the isolate of unknown mating type
is added to the agar plate.– After 7 days, the center of the plate is observed under
the microscope for the oospore production.
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Isolates of Phytophthora
A1Ph 216 (Watermelon-S. Texas)
A1Ph 204 (Chile pepper-S. Texas)
A2Ph 205 (Winter squash-N. Texas)
A2Ph 206 (Pumpkin-N. Texas)
--Ph 207 (Chile pepper-Mexico)
A2Ph 214 (Chile pepper-N. Texas)A2Ph 213 (Chile pepper-N. Texas)A1Ph 212 (Chile Pepper-N. Texas)A1Ph 211 (Chile pepper-N. Texas)
Compatibility TypeIsolate
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Methodology (cont.)υ For mefenoxam sensitivity studies:
– 10% clarified V8 juice agar was amended with 0, 5, and 100 µg/ml of mefenoxam (Ridomil Gold EC).
– A plug of mycelium is set at the center of each plate.– After three days, colony diameters are measured at each
concentration.– Growth of >40% of control at 100 µg/ml= Resistant– Growth of >40% of control at 5 µg/ml = Intermediate– Growth of <40% of control= Sensitive.
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Sensitivity to mefenoxam
1316A2Ph 214 (Chile pepper-N. Texas)
4842--Ph 207 (Chile pepper-Mexico)
2622A1Ph 204 (Chile pepper-S. Texas)
3121A2Ph 216 (Watermelon-S. Texas)
2519A2Ph 213 (Chile pepper-N. Texas)2418A1 Ph 212 (Chile Pepper-N. Texas)2415A1Ph 211 (Chile pepper-N. Texas)
2430A2 Ph 206 (Pumpkin-N. Texas)2733A2Ph 205 (Winter squash-N. Texas)
100 ppm5 ppm
Percent growthIsolate (compatibility type)
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0 ppm 5 ppm 100 ppm
Sensitive fungal isolate
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Fully resistant isolate
0 ppm 5 ppm 100 ppm
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Growth rate at 25° C
0
10
20
30
Average daily growth in
millimeters
Ph207P
epST
Ph205S
quNT
Ph206P
umNT
Ph211P
epNT
Ph212P
epNT
Ph213P
epNT
Ph214P
epNT
Ph216W
mST
Isolate Average Exp 1 and 2
aab ab ab bc
c bc c
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Growth rate at 30° C
0
10
20
30
Average daily growth in
millimeters
Ph207P
epST
Ph205S
quNT
Ph206P
umNT
Ph211P
epNT
Ph212P
epNT
Ph213P
epNT
Ph214P
epNT
Ph216W
mST
Isolate Average Exp 1 and 2
a
ab ab ab bcc bc c
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ITS Phylogenyυ Isolates from diverse backgrounds were grown in
clarified V8 juice broth.υ Isolates were lyophilized.υ DNA extraction using Qiagen DNeasy Kit.υ PCR amplification
– ITS1, 5.8 S, ITS 2 region (Nuclear)= 752 bpϖ Primers ITS 4 and ITS 5ϖ Temperature cycle parameters: 93°, 53°, 72°
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ITS Phylogenyυ Neighbor joining phylogenetic tree with 1000 bp
based on the ITS1-5.8S-ITS2 complete region of Phytophthora Clade 2 (Cooke et al 2000).
υ Tree shows the position of samples from vegetables in Texas to representative sequences of P. capsici and other known taxa in the clade.
υ Pythium vexans is outgroup. υ Scale bar unit: number of nucleotide substitutions
per site.
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PHYLIP_1
0.1
Ph204(chile ST)P.capsici.EF660564.Chile
Phyt..sp. carolinana Ph268.99.6102.Abad (Strawberry)P.capsici.A2.P223.1.DEL.CIDP.capsici.DQ464031.224.CapsicumPh216(watermelon ST)Ph205 (winter squash NT)Ph204 (chile pepper ST)P.sp.glovera.AF279125P.sp.glovera.AF279126P.sp.glovera.AF279124Ph206 (pumpkin NT)
Ph211 (chile pepper NT)Ph213 (chile pepper NT)Ph212 (chile pepper NT)Ph207 (chile pepper Mexico)P.mexicana.WPC0646A10
P.colocasiae.AF266786P.meadii.AY251649P.botryosa.AF266784P.citrophthora.AF266785
P.siskiyouensis.EF523387.1P.tropicalis.AY946257.P4742P.citricola.AF266788P.inflata.AF266789
P.multivesiculata.AF266790P.cactorum.AF266772
P.heveae.AF266770P.ilicis.AY423297
P.megakarya.AF266782P.megasperma.AF541897
P.fragariae.v.fragariae.AF2667P.lateralis.AF266804
P.kernoviae.AY940661P.insolita.AF271222
Pythium.vexans.AY598713.CBS.11
Phytophthora capsici
Phytophthora sp 1
Phytophthora sp 2
Phytophthora mexicana
ITS
Neighbor joining phylogenetic tree with 1000 bs based on the ITS1-5.8S-ITS2 complete region of Phytophthora Clade 2 (Cooke et al 2000) showing the position of samples from vegetables in Texas to representative sequences of P. capsici and other known taxa in the clade. Pythium vexans is outgroup. Scale bar unit: number of nucleotide substitutions per site.
CL2
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Phy. sp. caroliniana Ph268.99.6102 from strawberry NC- Homothallica
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υ How can the pathogen survive and persist in the Texas High Plains?
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Common Purslane (Portulaca oleracea)
Andy Bennett
French-Monar et al. 2006. Plant Dis. 90:345-350.
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Carolina Wild Geranium (Geranium carolinianum)
Daniela Bell
French-Monar et al. 2006. Plant Dis. 90:345-350.
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Nightshades (Solanum spp.)
http://www.agf.gov.bc.ca French-Monar et al. 2006. Plant Dis. 90:345-350.
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Conclusionsυ Phytophthora blight devastation in Texas may not
be solely the responsibility of P. capsiciυ Up to 4 species of Phytophthora might be involved
in Phytophthora blight of vegetables.υ Isolates from watermelon (2008, S. Texas), winter
squash (2006, N. Texas) and chile pepper (collection, S. Texas) had a perfect alignment for P. capsici.
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Conclusionsυ Isolates from chile pepper in N. Texas perfectly
aligned with P. mexicana.υ The pumpkin isolate (N. Texas) did not group with
any Phytophthora sp. and could represent a new species.
υ Chile pepper isolate from S. Texas did not grouped with another isolate from chile pepper (Mexico) and closely related to a species to be reported by G. Abad on strawberry.
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Thank You
H. D. Thurston