1. Characterization of Fusarium oxysporum f. sp
Characterization of Fusarium oxysporum f. sp. vasinfectum isolates from cottonseed imported from Australia into California for dairy feed
J. Liu, A. A. Bell, M. H. Wheeler, and R. D. Stipanovic
USDA-ARS
Cotton Pathology Research Unit
College Station, TX
2008 Beltwide Cotton Conferences
Nashville, Tennessee, January 8 –11, 2008

2. Background to The Problem
Fusarium oxysporum f. sp. vasinfectum (Fov) is a continuing threat to cotton production in the United States.
Newly recognized (2000) Fov race 4 isolates in California caused serous damages to cotton production.
A new Fov biotype was identified in 1993 in Australia.

3. Characters of Australian Fov Biotype
The Australian Fov biotype is distinct from American Fov isolates in that they cause wilt in root dip assays, but not in stem puncture inoculations.
The Australian Fov isolates are favored by heavy clay soils and do not require nematodes for severe disease, a character shared with race 3 isolates.
This pathogen has now spread to 6 of 10 cotton production areas in Australia in spite stringent control measures.
They are a threat to cotton grown on heavy alkaline soil in the U.S. (i.e. Texas to California)

4. Bell’s Australian Fov Isolates from Cottonseed
Around 2001 and 2002, several shiploads of cottonseed were imported into California from Australia for dairy feed.
In 2003, Bell screened several samples of these seeds and isolated 17 Fusarium oxysporum isolates at a 0.1% rate.
The pathogenicity of one of Bell’s isolates is similar to Kochman’s Australian isolates.

5. Objective of This Study
Determine the genetic relatedness of Bell’s Australian cottonseed isolates to U.S. isolates and to isolates of Australian biotype through characterization of partial sequences of the translation elongation factor 1-a (EF-1a), phosphate permase (PHO), b-tubulin, and mating type genes (Mat1-1 and Mat1-2).

6. Isolates Whose Sequences Were Obtained from NCBI Database for Comparison*
ATCC (R1)
36198 (R6)
16611 (R2)
31665 (R8)
16613 (R4) CA-14
16612 (R3) CA-3, CA-11
Aust-16 and Aust-19
F. oxysporum 1502
F. oxysporum a-1 and SUF959
F. oxysporum BW-1517, BW-7108 and BW-1537
* Kim et al., 2005, Plant Disease;
Yoshida et al., 1998, Ann. Phytopathol. Soc. Jpn.
Brubaker and Wang, 2006, NCBI

7. Isolates Investigated in This Study
Bell’s Australian seed isolates:
AustSeed 14
AustSeed 5, 6, 7, 8, 9, 12, 18, 19, 20, 21 (10 total)
AustSeed 17
AustSeed 1, 2, 3, 4 (4 total)
AustSeed 23
Australian Fov isolates:
Aust K-1, K-2, K-3, K-4 (4 total, Kochman’s Australian isolate)
U.S. Fov isolates (race 1 lineage):
Fov 11 (ATCC from California)
Fov 218 (from Lawrenburg, North Carolina)

8. EF-1a
PHO
b-Tubulin
Race 1 lineage
Race 8 lineage
Race 4 lineage
Race 3 lineage
Australian biotype ?

9. EF-1a
PHO
b-Tubulin
Race 1 lineage
Race 8 lineage
Race 4 lineage
Race 3 lineage
Australian biotype ?

10. EF-1a
PHO
b-Tubulin
Race 1 lineage
Race 8 lineage
Race 4 lineage
Race 3 lineage
Australian biotype ?

11. EF-1a
PHO
b-Tubulin
Race 1 lineage
Race 8 lineage
Race 4 lineage
Race 3 lineage
Australian biotype ?

12. EF-1a
PHO
b-Tubulin
Race 1 lineage
Race 8 lineage
Race 4 lineage
Race 3 lineage
Australian biotype ?

13. EF-1a ?

14. Mat1-1 California race 3 isolates, CA-11 and CA-3, were kindly
provided by Dr. Michael Davis

15. Mat1-2

16. Indel Analysis
Insertion and deletion information are not accurately represented in the phylogenetic analysis.
Some of the indel information in the present study are congruent with phylogenetic information.
Intels shared by at least two groups of isolates are discussed bellow.

17. Indel Analysis Informative indels in EF-1a: Insertion at position 295:
AustSeed (race 3 lineage)
CA (race 3 lineage)
ATCC (race 3 lineage)
Insertion at position 295 and 296:
AustSeed 1(4) (race ?)
Insertion at position 332:
Aust-16, Aust (Australian biotype)
Aust K-1 group (4) (Australian biotype)
AustSeed 1 group (4) (race ?)

18. Deletion at position 356-58: Aust-16, Aust-19 (Australian biotype)
Aust K-1 group (4) (Australian biotype)
Deletion at position :
AustSeed 14 (race 3 lineage)
Deletion at position :
AustSeed 1 group (4) (race ?)

19. MAT1-1 MAT1-2 VCG 3 AustSeed 1 group (4) Belong to no known lineage
Aust K-1 group (4)
Australian biotype
VCG 2
VCG 1
AustSeed 5 group (10)
AustSeed 17
Race 3 lineage
AustSeed 14
Race 3 lineage
CA-11
ATCC R3
CA-3
VCG 5
VCG 4
AustSeed 23
Race 1 lineage
Fov 11 group (2)
Race 1 lineage
(VCG data from Bell, 2003)

20. Conclusion
Both mating types are present in Bell’s seed isolates including those belonging to the same race lineages.
The most aggressive of Bell’s seed isolates (i.e., AustSeed 14) belong to race 3 lineage based on EF-1a, PHO, and b-tubulin partial sequences.
Indel analysis of the EF-1a gene reveals a close evolutionary relationship between AustSeed 14 (race 3) and Australian biotype.
AustSeed 14 belongs to the same VCG as the Kochman’s Australian isolates; it displays similar pathogenicity although it belongs to a different mating type.
Race 3 isolates favors alkaline clay soil and does not require nematodes to cause severe disease; this is also true for Australian biotypes.
Australian seed isolates may present a threat to the U.S. cotton industry.