Journal of General Plant Pathology

, Volume 75, Issue 2, pp 125–130

Genetic diversity and pathogenicity of Fusarium oxysporum isolated from wilted Welsh onion in Japan

Authors

  • Maha Laksha Mudiyanselage Chandrika Dissanayake
    • The United Graduate School of Agricultural SciencesTottori University
  • Rumi Kashima
    • Department of Biological and Environmental Sciences, Faculty of AgricultureYamaguchi University
  • Shuhei Tanaka
    • Department of Biological and Environmental Sciences, Faculty of AgricultureYamaguchi University
    • Department of Biological and Environmental Sciences, Faculty of AgricultureYamaguchi University
Fungal Diseases

DOI: 10.1007/s10327-009-0152-6

Cite this article as:
Dissanayake, M.L.M.C., Kashima, R., Tanaka, S. et al. J Gen Plant Pathol (2009) 75: 125. doi:10.1007/s10327-009-0152-6

Abstract

Thirty isolates of Fusarium oxysporum from wilted Welsh onion plants were examined for their diversity in nucleotide sequences of the ribosomal DNA (rDNA) intergenic spacer (IGS) region and for pathogenicity with regard to five Welsh onion cultivars. Phylogenetic analysis based on the IGS sequences revealed polyphyletic origins of the isolates and a relationship between phylogeny and pathogenicity; low virulence isolates differed genetically from those with high and moderate virulence. Mating type analysis revealed that all F. oxysporum isolates were MAT1-1 idiomorphs, suggesting that the pathogens may be clonal in the fields examined.

Keywords

Allium fistulosumFusarium wiltMating typePhylogeny

Fusarium wilt (also known as Fusarium basal rot) of Welsh onion (Japanese bunching onion, Allium fistulosum) has been reported to be caused by Fusarium oxysporum f. sp. cepae (FOC) (Kodama 1977; Takakuwa et al. 1977) and has been sporadically observed in Japan (Takeuchi et al. 1983). Compared with bulb onion (Allium cepa), Welsh onion has been known to be tolerant to FOC (Galván et al. 2008). During the summers of 2006 and 2007, however, wilted Welsh onion plants were frequently found in a large number of fields in some major Welsh onion-growing areas in Japan. Fusarium oxysporum, F. solani, and F. verticillioides were isolated from wilted Welsh onion plants in these fields (Kashima et al. 2007; Dissanayake et al. 2009). Among the isolates, F. oxysporum isolates were the major pathogen responsible for Fusarium wilt of Welsh onion. Pathogenicity assays showed differences in disease severity among cultivars of Welsh onion (Dissanayake et al. 2009), suggesting the presence of populations of F. oxysporum that differed in virulence. Swift et al. (2002) described populational variability in F. oxsporum f. sp. cepae (FOC) isolated from onions in Colorado, USA, by identifying five vegetative compatibility groups (VCGs 0421, 0422, 0423, and two VCGs with no code number). Their results suggest the presence of genetic diversity among the FOC populations. No study on genetic diversity has been reported on FOC in Japan, except for that of Yoo et al. (1993) on FOC isolates from Hokkaido Island belonging to a VCG with no code number. Therefore, FOC may be genetically homogenous in Japan, but they examined only two isolates of FOC in their study.

The intergenic spacer (IGS) region, which separates rDNA repeat units, is particularly suitable for studying relationships among isolates of F. oxysporum (Appel and Gordon 1996). Recent studies showed that phylogenetic analysis of the IGS region sequence was useful for population analysis of the pathogen (Kawabe et al. 2005, 2007; Enya et al. 2008). In addition, the mating type locus (MAT1), which regulates sexual reproduction in ascomycete fungi, was cloned from F. oxysporum, and molecular techniques have been developed to demonstrate the presence of the two mating-type idiomorphs, MAT1-1 and MAT1-2 (Arie et al. 2000). Each F. oxysporum isolate possesses one of the two idiomorphs. Therefore, the MAT1 idiomorph can be used as a molecular marker to predict the clonality of a F. oxysporum population (Abo et al. 2005; Elmer et al. 2007).

The objective of this study was to characterize F. oxysporum isolates from wilted Welsh onion in Japan according to sequence similarities within the IGS region, pathogenicity, and MAT1 idiomorphs of the isolates.

Fusarium isolates were obtained from sections of basal stem plates of wilted Welsh onion plants from 11 different areas of Japan during the summer of 2006 and 2007 (Table 1), with the use of a selective agar medium (Komada 1975). A total of 30 isolates were identified as F. oxysporum on the basis of illustrated keys (Nelson et al. 1983; Leslie and Summerell 2006). The pathogenicity of 18 isolates to A. fistulosum and A. cepa were confirmed in a previous study (Dissanayake et al. 2009).
Table 1

The list of fungal isolates used for phylogenetic analysis

Species, forma specialis/race

Isolate

Year

Geographic origin (field)

Source

GenBank accessionc

Mating type

Disease severity indexd

Y7

Y13

Y17

Y27

Y55

Mean

Fusarium oxysporum

01a

2006

Saitama (Koshigaya), Japan

This study

AB450459

MAT1-1

1.9

1.5

2.1

1.8

1.6

1.8x

07a

2006

Saitama (Koshigaya), Japan

This study

AB450460

MAT1-1

1.9

1.4

1.2

1.7

1.2

1.49x

08a

2006

Saitama (Koshigaya), Japan

This study

AB450461

MAT1-1

2.9

2.8

3.8

3.2

2.5

3.03z

13a

2006

Kyoto (Fushimi-SA), Japan

This study

AB450462

MAT1-1

2.1

2.4

2.7

2.9

3.0

2.61y

15a

2006

Kyoto (Fushimi-SA), Japan

This study

AB450463

MAT1-1

2.9

3.5

3.0

3.6

2.7

3.13z

17a

2006

Kagoshima (Osaki-1), Japan

This study

AB450464

MAT1-1

3.5

3.4

3.5

3.4

3.6

3.47z

18a

2006

Kagoshima (Osaki-2), Japan

This study

AB450465

MAT1-1

2.9

1.6

2.4

3.0

2.1

2.40y

19a

2006

Kagoshima (Osaki-4), Japan

This study

AB450466

MAT1-1

2.2

1.2

2.4

2.8

1.8

2.07y

20a

2006

Kagoshima (Osaki-5), Japan

This study

AB450467

MAT1-1

2.0

1.5

2.1

2.9

2.1

2.11y

22a

2006

Kagoshima (Osaki-6), Japan

This study

AB450468

MAT1-1

3.7

3.8

3.7

3.4

3.8

3.68z

25a

2006

Okayma (Okyama), Japan

This study

AB450469

MAT1-1

2.7

1.5

2.5

2.6

2.6

2.37y

26a

2006

Okayma (Okyama), Japan

This study

AB450470

MAT1-1

2.0

1.6

2.2

2.5

2.3

2.16y

27a

2006

Okayma (Okyama), Japan

This study

AB450471

MAT1-1

1.8

2.9

1.7

1.7

2.2

2.06y

28a

2006

Miyazaki (Miyazaki), Japan

This study

AB450472

MAT1-1

2.9

1.8

2.7

2.5

2.4

2.45y

30a

2006

Hyogo (Nishinomiya), Japan

This study

AB450473

MAT1-1

3.2

2.9

3.1

3.2

3.3

3.13z

31a

2006

Hyogo (Nishinomiya), Japan

This study

AB450474

MAT1-1

2.5

2.1

2.5

2.1

2.5

2.34y

32a

2006

Hyogo (Nishinomiya), Japan

This study

AB450475

MAT1-1

0.6

1.4

2.3

1.5

2.8

1.72x

37

2007

Shizuoka (Hamamatsu- 1), Japan

This study

AB450927

MAT1-1

4.0

3.7

3.7

3.3

3.6

3.66z

39

2007

Shizuoka (Hamamatsu-2), Japan

This study

AB450928

MAT1-1

1.8

1.6

1.3

1.3

1.0

1.41x

40

2007

Shizuoka (Hamamatsu-3), Japan

This study

AB450929

MAT1-1

0.8

0.8

1.0

1.3

1.0

0.98x

41

2007

Shizuoka (Hamamatsu-4), Japan

This study

AB450930

MAT1-1

3.4

3.8

2.8

3.2

3.0

3.24z

45

2007

Hokkaido (Ashikawa), Japan

This study

AB450932

MAT1-1

3.2

3.6

4.0

3.2

4.0

3.60z

46

2007

Nagano (Matsumoto), Japan

This study

AB450933

MAT1-1

2.2

2.8

2.4

2.4

2.0

2.36y

48

2007

Tokushima (Anan-1), Japan

This study

AB450934

MAT1-1

2.0

2.0

2.0

2.4

2.0

2.08y

50

2007

Tokushima (Anan-2), Japan

This study

AB450935

MAT1-1

0.7

1.0

0.0

1.3

0.7

0.73x

52

2007

Tokushima (Anan-3), Japan

This study

AB450936

MAT1-1

2.0

2.5

2.8

2.6

2.0

2.38y

54

2007

Nagano, Japan

This study

AB450937

MAT1-1

1.3

1.3

2.6

1.0

2.0

1.65x

56

2007

Nagano, Japan

This study

AB450938

MAT1-1

2.6

2.0

2.0

2.8

2.0

2.28y

60

2007

Kochi (Konan), Japan

This study

AB450939

MAT1-1

1.0

0.7

0.0

1.0

1.0

0.73x

62

2007

Kochi (Konan), Japan

This study

AB450941

MAT1-1

1.3

2.0

1.0

1.6

1.0

1.39x

63a, b

 

Hokkaido, Japan

K. Tsutsui

AB450942

MAT1-1

2.0

3.0

2.0

3.6

2.5

2.62y

7866

 

Chiba, Japan

J. Enya

AB306845

 

 

 

 

 

 

NRRL22538

 

USA

G.C. Mbofung

DQ831891

      

f.sp. batatas

0-17

 

Ibaraki, Japan

K. Watanabe

AB106050

MAT1-2

      

f.sp. radis-lycopersici

103044

 

Gifu, Japan

MAFF

AB106058

MAT1-1

      

f.sp. lycopersici race 1

26034

 

Italy

NRRL

AB106025

MAT1-1

      

6531

 

Kyushu, Japan

NBRC

AB106018

MAT1-1

      

f.sp. lycopersici race 2

12575

 

Tochigi, Japan

JCM

AB106027

MAT1-1

      

103038

 

Ibaraki, Japan

MAFF

AB106031

MAT1-1

      

4287

 

Spain

A. Di Pietro

AB 120973

MAT1-1

      

f.sp. lycopersici race 3

DA1/7

 

Florida, USA

H.C. Kistler

AB106047

MAT1-2

      

f.sp. lactucae

SB1-1

 

Nagano, Japan

J. Enya

AB306846

MAT1-2

      

f.sp. spinaciae

100027

 

Chiba, Japan

J. Enya

AB306844

MAT1-2

      

AK13

 

Akita, Japan

A. Sayama

AB211845

MAT1-2

      

f.sp. lilii

PFOL002

 

Nantou, Taiwan

W. Chung

AB383690

      

FOL067

 

Tainan, Taiwan

W.Chung

AB383684

      

f.sp. cubense

26024

 

Honduras

NRRL

AY527732

      

Gibberella fujikuroi

7610

 

USA

FGSC

AB106061

MAT1-2

      

Endash indicates no information available in the database

MAFF Microorganism Section of the GenBank in the Ministry of Agriculture, Forestry and Fisheries of Japanese Government (Tsukuba, Ibaraki, Japan); NRRL Agriculture Research Service culture collection on United State Department of Agriculture (Peoria, IL, USA); NBRC National Institute of Technology and Evaluation Biological Resource Centre (Kisarazu, Chiba, Japan); JCM Japan Collection of Micoorganisms; RIKEN The Institute of Physical and Chemical Research (Wako, Saitama, Japan); FGSC Fungal Genetics Stock Center (University of Kansas Medical Center, Kansas City, KS, USA)

aPathogenicity to Allium cepa was confirmed by Dissanayake et al. (2009)

bFusarium oxysporum strain isolated from bulb onion and provided by K. Tsutsui (Shippo seed, Toyonaka, Kagawa, Japan)

cGenBank/DDBJ accessions of the rDNA IGS sequences determined in this study (AB450459–AB450475 and AB450927–AB450942) and those reported from other formae speciales used for phylogenetic analysis

dDisease severity index was measured for each of five Welsh onion lines, Y7, Y13, Y17, Y27and Y50 using a five-rank scale, where 0 = no symptoms, 1 = plant height and root length less than three-fourth of those of the controls, 2 = plant height and root length half of those of the controls, 3 = plant height and root length less than one-fourth of those of the controls, 4 = death due to pre- and post-emergence damping-off. Disease severity index (DSI) was calculated as: DSI = ∑ (disease severity scale × number of plants at each scale)/total number of seeds sown. The mean disease severity index (MDSI) is mean of the disease severity indexes for the five cultivars. Within columns, means followed by the same letters (x, y, and z) do not different significantly according to Dennett’s t-test

Genomic DNA was extracted from the mycelia using Nucleon PhytoPure DNA Extraction Kit (GE Healthcare, Buckinghamshire, UK), according to the manufacturer’s instruction. The nucleotide sequences of partial IGS region was determined with amplification reactions using primer pair FIGS11 (5′-GTAAGCCGTCCTTCGCCTCG-3′) and FIGS12 (5′-GCAAAATTCAATAGTATGGC-3′) (Kawabe et al. 2005). PCR was performed in 20 μl of reaction mixture containing 1.0 μM of both primers, 0.2 units ExTaq DNA polymerase (Takara Bio, Otsu, Shiga, Japan), 200 μM of each dNTP, 2 μl of 10 × PCR buffers, and 20 ng of template DNA with a thermal cycler (PC-800; Astec, Fukuoka, Japan). An initial denaturation step for 2 min at 94°C was followed by 35 cycles of denaturation for 1 min at 94°C, annealing for 30 s at 58°C, and extension for 1 min at 72°C; with a final extension step for 7 min at 72°C. Negative controls (no template DNA) were included in every assay.

The amplified DNA fragments were purified by ethanol precipitation and then labelled using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). The DNA fragments were sequenced on an ABI PRISM 3100 genetic analyzer (Applied Biosystems). The sequence data determined in the present study were submitted to DDBJ (DNA Data Bank of Japan, Centre for Information Biology and DNA Data Bank of Japan, National Institute of Genetics, Mishima, Japan) (Table 1). The IGS sequences of F. oxysporum isolates from other plant species and Gibberella fujikuroi strains obtained from GenBank databases were used in phylogenetic analyses as reference sequences and the outgroup, respectively (Table 1). DNA sequences were aligned with the program ClustalX 1.81 (Thompson et al. 1997), and the resulting multiple-alignment file was edited with the program BioEdit (Hall 1999). The distance matrix for aligned sequences was calculated using Kimura’s two-parameter model (Kimura 1980) and analyzed with a neighbor-joining (NJ) algorithm (Saitou and Nei 1987). Clade stability was assessed using 1,000 bootstrap replications.

The partial IGS sequences of each F. oxysporum isolate and reference strains were analyzed phylogenetically, and the isolates clustered into four distinct clades (Fig. 1A–D). We included two IGS sequences for FOC from the GenBank database in the phylogenetic tree. Isolate 7866 from Chiba, Japan grouped into clade A, while isolate 22538 from the USA was included in clade C. These results suggest that F, oxysporum isolates from wilted Welsh onion are polyphyletic. Galván et al. (2008) used an amplified fragment length polymorphism marker (AFLP) with isolates of F. oxysporum from bulb onion (A. cepa) from the Netherlands and Uruguay, which clustered into two clades, suggesting polyphyly for F. oxysporum isolates pathogenic to bulb onion. Because F. oxysporum isolates from other plant species comprised three clades (Fig. 1A–C), phylogenetic relationships may exist among isolates belonging to different formae speciales including cepae, batatas, lycopersici, radicis-lycopersici, spinaciae, lilii, lactucae and cubense.
https://static-content.springer.com/image/art%3A10.1007%2Fs10327-009-0152-6/MediaObjects/10327_2009_152_Fig1_HTML.gif
Fig. 1

Phylogenetic tree derived using the neighbour-joining method from the partial sequences of the rDNA IGS region in Fusarium oxysporum isolates from wilted Welsh onion plants and other formae speciales. The distances were determined by Kimura’s two-parameter method. An approximately 600-bp fragment was amplified from genomic DNA with the primers FIGS11 and FIGHS12, and the determined nucleotide sequences were phylogenetically analyzed. Gibberella fujikuroi (FGSC 7610) is the outgroup. The four distinctive groups proposed are labeled A, B, C and D. The numbers beside branches represent the percentage of congruent clusters in 1,000 bootstrap trials with values greater than 80%. The bar indicates 2% sequence dissimilarity

The mating type of each isolate was determined with a MAT-specific PCR assay as previously reported (Arie et al. 2000). On the basis of the PCR amplification results, all the F. oxysporum isolates examined were considered to be MAT1-1 idiomorphs (Table 1), suggesting that the F. oxysporum isolates examined did not reproduce sexually by themselves.

The pathogenicity of the 30 F. oxysporum isolates was evaluated by inoculating five Welsh onion lines, Y7, Y13, Y17, Y27, and Y55 (Nissan Seed, Nishinomiya, Hyogo, Japan). Surface-disinfested seeds of each cultivar were dipped in a spore suspension (5 × 105 conidia/ml sterile water) for 1 h or in sterile water for the noninoculated control. After inoculation, seeds were sown in plastic cell-type growing trays (cell size, 3 × 3 × 4.5 cm) filled with a steam-sterilized soil mixture (coconut fibre:sand = 5:1 v/v, pH 5.8). The experiment was set up in a greenhouse at 28–32°C (Abawi and Lorbeer 1972; Kodama 1983) under natural light in a completely randomized design. For each fungal isolate, the pathogenicity assay was done three times using eight seeds of each cultivar. Each plant was evaluated for disease 21 days after inoculation using a five-rank scale, where 0 = no symptoms, 1 = plant height and root length less than three-fourths of those of the controls, 2 = plant height and root length half of those of the controls, 3 = plant height and root length less than one-fourth of those of the controls, and 4 = death due to pre- or post-emergence damping-off. A disease severity index (DSI) was calculated as DSI = ∑(disease severity scale × number of plants at each scale)/total number of seeds sown. Mean disease severity index (MDSI) was also calculated as the mean disease severity indices for five cultivars. All data from each repeated experiment were analyzed separately and subjected to analysis of variance (ANOVA). MDSI values were compared with the control mean using Dennett’s t-test (JMP statistical discovery software; SAS Institute, Cary, NC, USA).

The F. oxysporum isolates examined had differences in their DSI. Based on the MDSI, the virulence of each isolate was recorded as low (MDSI: <2), moderate (MDSI: 2–3) or high (MDSI: >3) (Table 1). These results agree with those of previous studies, which showed variation in the virulence of F. oxysporum isolates from bulb onion (Özer et al. 2004; Galván et al. 2008) and Welsh onion (Dissanayake et al. 2009).

Of eight high virulence isolates (08, 15, 17, 22, 30, 37, 41, and 45), five (22, 30, 37, 41, and 45) formed a distinct subgroup (C1), which was consistently supported with bootstrapping of over 87%, whereas the other three high virulence isolates (08, 15, and 17) grouped into clade A (Fig. 1). This result suggests that F. oxysporum isolates highly virulent to Welsh onion are not monophyletic in Japan. The low virulence isolates clearly separated into two groups (subgroup C2 and clade D) except for isolate 07, which grouped in clade A. All moderate virulence isolates were grouped into two separate clades (A and B) with high virulence isolates. Therefore, some of high and moderate virulence isolates may share the similar genetic background. These results also suggest that low virulence isolates may be phylogenetically differentiated from those of high and moderate virulence. It is interesting to note that clade C included both high and low virulence isolates (C1 and C2, respectively). These isolates with various virulences in clade C may be useful for studying the evolution of this pathogen. In addition, none of the F. oxysporum isolates examined in this study had any relationship between their genetic profile and geographic origin, suggesting that these F. oxysporum isolates may have originated independently at each locality.

The present study demonstrated, for the first time, the genetic diversity of F. oxysporum isolated from wilted Welsh onion in Japan. Of the 30 F. oxysporum isolates from wilted Welsh onion examined in this study, 18 isolates were also pathogenic to onion; thus they are likely to be F. oxysporum f. sp. cepae. Although the formae speciales of the other 12 isolates need to be determined, the findings of this study would provide basic information for breeding programs of Welsh onion resistant to Fusarium diseases.

Acknowledgments

We thank Mr. Masataka Funahashi, Nissan Seed, Nishinomiya, Japan, for providing wilted Welsh onion samples. This work was supported in part by Nissan Seed Foundation.

Copyright information

© The Phytopathological Society of Japan and Springer 2009