Journal of General Plant Pathology

, Volume 75, Issue 1, pp 37–45 | Cite as

Pathogenic variation and molecular characterization of Fusarium species isolated from wilted Welsh onion in Japan

  • Maha Laksha Mudiyanselage Chandrika Dissanayake
  • Rumi Kashima
  • Shuhei Tanaka
  • Shin-ichi Ito
Fungal Diseases

Abstract

Thirty-two isolates of Fusarium species were obtained from wilted Welsh onion (Allium fistulosum) grown on nine farms from six regions in Japan and identified as F. oxysporum (18 isolates), F. verticillioides (7 isolates), and F. solani (7 isolates). The pathogenicity of 32 isolates was tested on five commercial cultivars of Welsh onion and two cultivars of bulb onion in a seedling assay in a greenhouse. The Fusarium isolates varied in the degree of disease severity on the cultivars. Five F. oxysporum isolates (08, 15, 17, 22, and 30) had a higher virulence on the cultivars than the other isolates. The host range of these five isolates was limited to Allium species. Molecular characterization of Fusarium isolates was performed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the internal transcribed spacer (ITS) regions of ribosomal DNA. The 32 isolates were grouped into eight types (four types for F. oxysporum, one for F. verticillioides, and three for F. solani). Restriction patterns of the ITS region were not related to pathogenicity. However, the haplotypes obtained with five enzymes (RsaI, HinfI, HaeIII, ScrFI, and MspI) and the phylogenetic analysis permitted the discernment of the three Fusarium species. The PCR-RFLP analysis should provide a rapid, simple method for differentiating Fusaruim species isolated from wilted Welsh onion in Japan.

Keywords

Allium fistulosum Fusarium spp. Pathogenicity Fusarium wilt 

Introduction

Welsh onion, also known as Japanese bunching onion (Allium fistulosum), is one of the commonly cultivated vegetables in East Asia, including Japan, China, and Korea (Inden and Asahira 1990; Yakuwa 2006). In Japan, young seedlings as well as adult plants of Welsh onion are high-value products used for hors d’oeuvre, soup, and sushi. Therefore, Welsh onion, cultivated continuously in fields throughout the year to fill market demand, surpasses bulb onion (Allium cepa) in economic importance in Japan, ranking sixth among vegetable crops in annual output (Yakuwa 2006).

Fusarium wilt, also known as Fusarium basal rot, of Welsh onion was first reported in Japan in 1977, and the causal pathogen was identified as Fusarium oxysporum f. sp. cepae (Kodama 1977; Takakuwa et al. 1977). Although Fusarium wilt of Welsh onion has been sporadically observed (Takeuchi et al. 1983), the disease has been rarely observed in most Welsh onion-growing areas in Japan. Therefore, Fusarium wilt of Welsh onion has not been considered an important disease in the country. However during the summer of 2006, wilted Welsh onion plants were frequently found in large number of fields in the major Welsh onion-growing areas of Japan. In the fields, initial symptoms were yellowing of the lower leaves, which eventually progressed to entire leaf necrosis and plant death. Infected basal plates of Welsh onion had a brown discolouration. The symptoms clearly differed from those of Fusarium root rot of Welsh onion caused by F. oxysporum, which include leaf blight, stunting, root rot, without any browning of the basal plates (Shinmura et al. 1998). Some fields in infested areas lost more than 50% of the Welsh onion plants in the summer. Thus, we needed a method to identify the pathogens that cause Fusarium wilt-like symptoms in Welsh onion.

In the present study, attempts were made to identify Fusarium species isolated from wilted Welsh onion and to determine their pathogenicity on commercial cultivars of Welsh onion. We also report here the utility of a DNA-based method, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the internal transcribed spacer (ITS) region, for differentiating Fusarium species isolated from wilted Welsh onion.

Materials and methods

Isolation, morphology and identification of the causal fungi

Wilted Welsh onion plants were received from nine farms within six regions; Saitama (one field), Kyoto (three), Kagoshima (two), Okayama (one), Miyazaki (one), and Hyogo (one) in the summer 2006 (Table 1). The plants were washed under running tap water. Sections of diseased tissues were surface sterilized in 70% ethanol for 30 s followed by 30 s in 0.5% (v/v) NaOCl, then rinsed in sterile distilled water and air-dried on sterile filter paper. The disinfected pieces were cut into 3–5 mm pieces, placed on F. oxysporum selective agar medium (Komada 1975) and incubated for 8–14 days at 25°C. After 2 or 3 days, Fusarium-like colonies were observed, and hyphal tips of the colonies were transferred to potato dextrose agar (PDA). The pure cultures of the isolates were obtained using a single-spore culture technique (Leslie and Summerell 2006). Representative isolates were maintained on PDA slants. Macroscopic and microscopic characteristics of the pure cultures were studied on PDA and synthetic nutrient agar (SNA; Leslie and Summerell 2006) cultures, and the species were identified using illustrated keys (Nelson et al. 1983; Leslie and Summerell 2006).
Table 1

Fungal isolates of Fusarium species collected from wilted Welsh onion (Allium fistulosum)

Isolatea

Geographical origin (field)

Species

Disease severity indexb

RFLP typec

Y7

Y13

Y17

Y27

Y50

Mean

01

Saitama (Koshigaya)

F. oxysporum

1.9

1.5

2.1

1.8

1.6

1.78x

I

02

Saitama (Koshigaya)

F. solani

1.7

0.9

1.4

1.6

1.3

1.40x

V

03

Saitama (Koshigaya)

F. verticillioides

1.2

0.9

1.6

1.6

1.7

1.40x

VIII

04

Saitama (Koshigaya)

F. oxysporum

0.9

1.0

1.3

1.9

1.0

1.23x

I

05

Saitama (Koshigaya)

F. solani

1.3

0.7

1.3

1.6

1.3

1.24x

VII

06

Saitama (Koshigaya)

F. verticillioides

0.7

1.3

2.2

1.6

0.7

1.30x

VIII

07

Saitama (Koshigaya)

F. oxysporum

1.9

1.4

1.2

1.7

1.2

1.49x

IV

08

Saitama (Koshigaya)

F. oxysporum

2.9

2.8

3.8

3.2

2.5

3.03z

I

09

Kyoto (Fushimi-OF)

F. verticillioides

1.6

1.7

2.0

2.0

1.8

1.83x

VIII

10

Kyoto (Fushimi-OF)

F. verticillioides

1.1

1.0

1.5

1.8

1.5

1.39x

VIII

11

Kyoto (Fushimi-IY)

F. verticillioides

1.8

1.2

1.0

1.6

1.7

1.46x

VIII

12

Kyoto (Fushimi-SA)

F. verticillioides

2.0

0.7

2.0

2.3

2.2

1.83x

VIII

13

Kyoto (Fushimi-SA)

F. oxysporum

2.1

2.4

2.7

2.9

3.0

2.61y

I

14

Kyoto (Fushimi-SA)

F. verticillioides

2.2

0.8

1.6

3.0

1.5

1.81x

VIII

15

Kyoto (Fushimi-SA)

F. oxysporum

2.9

3.5

3.0

3.6

2.7

3.13z

IV

16

Kagoshima(Osaki-YO)

F. solani

1.0

1.3

1.5

1.7

1.5

1.40x

V

17

Kagoshima (Osaki-YO)

F. oxysporum

3.5

3.4

3.5

3.4

3.6

3.47z

I

18

Kagoshima (Osaki-YO)

F. oxysporum

2.9

1.6

2.4

3.0

2.1

2.40y

IV

19

Kagoshima (Osaki-YO)

F. oxysporum

2.2

1.2

2.4

2.8

1.8

2.07y

I

20

Kagoshima (Osaki-NA)

F. oxysporum

2.0

1.5

2.1

2.9

2.1

2.11y

I

21

Kagoshima (Osaki-NA)

F. solani

1.4

0.8

1.2

2.6

0.4

1.29x

VI

22

Kagoshima (Osaki-NA)

F. oxysporum

3.7

3.8

3.7

3.4

3.8

3.68z

I

23

Kagoshima (Osaki-NA)

F. solani

0.8

0.9

1.0

1.6

1.0

1.07x

V

24

Okayama (Okayama)

F. solani

1.6

1.0

1.3

1.5

1.8

1.44x

VI

25

Okayama (Okayama)

F. oxysporum

2.7

1.5

2.5

2.6

2.6

2.37y

IV

26

Okayama (Okayama)

F. oxysporum

2.0

0.6

2.2

2.5

2.3

1.92x

I

27

Okayama (Okayama)

F. oxysporum

1.3

2.9

1.1

1.2

1.2

1.54x

I

28

Miyazaki (Miyazaki)

F. oxysporum

2.9

1.8

2.7

2.5

2.4

2.45y

I

29

Miyazaki (Miyazaki)

F. solani

1.4

0.9

1.3

1.3

1.7

1.32x

VI

30

Hyogo (Nishinomiya)

F. oxysporum

3.2

2.9

3.1

3.2

3.3

3.13z

I

31

Hyogo (Nishinomiya)

F. oxysporum

2.5

2.1

2.5

2.1

2.5

2.34y

III

32

Hyogo (Nishinomiya)

F. oxysporum

0.6

1.4

2.3

1.5

2.8

1.72x

II

I

33

Hokkaido

F.oxysporum f.sp.cepae A

2.0

3.0

2.0

3.6

2.5

2.62y

34

Tottori

F.oxysporum f.sp.cepae B

2.5

3.0

1.8

2.9

2.5

2.54y

I

a01–32, field isolates collected during the summer 2006; 33–34, reference stains of Fusarium oxysporum f. sp. cepae

bDisease severity was assessed for each of five Welsh onion cultivars (Y7, Y13, Y17, Y27, and Y50) using a 0–4 scale, where 0 = no symptoms; 1 = plant height and root length were less than three fourth of those of the controls; 2 = plant height and root length were half as those of the controls; 3 = plant height and root length were less than one fourth of those of the controls; 4 = death of seedling due to pre- and postemergence damping off. Disease severity index was calculated as: Disease severity index = ∑ (disease severity scale × number of plants at each severity)/total number of seeds sown. Mean disease severity index is the average of disease severity indexes against five cultivars. Within columns, means followed by the same letters do not different significantly according to Dennett’s t-test

cRELPs observed among the 34 Fusarium isolates digested with five restriction enzymes (RsaI, ScrFI, HinfI, HaeIII, and MspI) (Figs. 3, 4)

Pathogenicity tests

Thirty-two isolates of Fusarium spp. obtained from wilted Welsh onion plants and two of reference strains (F. oxysporum f. sp. cepae A and B) (Table 1) were examined for their pathogenicity to five commercial cultivars of Welsh onion (Y7, Y13, Y17, Y27, and Y50) from Nissan seed, Nishinomiya, Japan) and two cultivars of bulb onion (Kinkyu from Kaneko seed, Maebashi, Japan; and Tsuri from Matsunaga seed, Konan, Japan). Each isolate was grown in an Erlenmeyer flask containing 100 ml of potato dextrose broth (PDB; Difco, Detroit, MI, USA) and shaken at 120 rpm at 25°C under constant light. After 7 days, fungal cultures were aseptically filtered through four layers of sterile gauze cloth. The spore concentration was estimated with a haemocytometer and adjusted to ca. 5 × 105 conidia/ml. Welsh onion seeds were surface-disinfested with 0.05% (v/v) NaOCl for 10 min, rinsed twice in sterile distilled water and allowed to dry in a laminar flow hood. Seeds were immersed in a conidial suspension of a fungal isolate for 1 h. Seeds immersed in sterile distilled water served as the control. After treatment, seeds were sown in cell-type plastic growing trays (cell size: 3 × 3 × 4.5 cm) filled with steam-sterilized soil mixture (5:1 coconut fibre to sand, pH 5.8). Trays were divided into groups inoculated with only one strain to avoid contamination and maintained in greenhouse at 28–32°C under natural light. Seedlings were watered as required to maintain normal growth. Symptoms such as seedling damping-off, stunted growth and reduced root length of seedling were recorded at 3-day intervals for 21 days of emergence of the seedlings. Disease severity was measured using a 0–4 scale, where 0 = no symptoms; 1 = plant height and root length were less than three fourths of those of the controls; 2 = plant height and root length were half as those of the controls; 3 = plant height and root length were less than one fourth of those of the controls; 4 = death due to pre- and postemergence damping-off. Disease severity index was calculated as: Disease severity index = ∑(disease severity scale × number of plants at each severity scale)/total number of seeds sown. Mean disease severity index is the average of disease severity indexes against five cultivars. For each fungal isolates, eight seeds of each cultivar were used as the replicates. Pathogenicity tests were repeated three times for each fungal isolates.

Host range test

The host range of the five isolates of F. oxysporum (isolates 08, 15, 17, 22, and 30) with high virulence in the pathogenicity tests and the two reference strains of F. oxysporum f. sp. cepae was tested against 17 plant species: maize (Zea mays), rice (Oryza sativa), Japanese radish (Raphanus sativus), Chinese cabbage (Brassica campestris, pekinensis group), soybean (Glycine max), carrot (Daucus carota var. sativa), cucumber (Cucumis sativus), pumpkin (Cucurbitapepo), pea (Pisum sativum), tomato (Lycopersicon esculentum), bell pepper (Capsicum annuum), eggplant (Solanum melongena), broccoli (Brassica oleracea, italica group), lettuce (Lactuca sativa), cowpea (Vigna unguiculata), bulb onion (A. cepa), and Welsh onion (A. fistulosum). Seeds of all plant species were surface-sterilized and immersed in conidial suspension as described. Seeds immersed in sterile distilled water served as controls. One to eight seeds per each fungal isolates were sown in cells of plastic growing trays (cell size: 4 × 4 × 5.5 cm) and maintained in a greenhouse at 28–32°C under natural light. Seedlings were watered as required to maintain normal growth. Four weeks after seeding, disease severity was measured as described in the pathogenicity test. For each fungal isolate, 25 seeds of each plant species were used as the replicates. Pathogenicity tests were repeated three times for each fungal isolate.

Genomic DNA extraction

Each Fusarium isolate was grown in PDB for 3 days on an orbital shaker (120 rpm) at 25°C. Then, the culture was filtered through a millipore filter (0.8 μm), and the harvested mycelium was lyophilized. Genomic DNA was isolated from the lyophilized mycelium (ca. 0.1 g) in the presence of lysis buffer (100 mM Tris–HCl [pH 8.0], 50 mM ethylene diamine tetraacetic acid [EDTA],100 mM NaCl, 10 mM 2-mercaptoethanol, 1% (w/v) sodium dodecyl sulphate [SDS]), then extracted with phenol/chloroform/isoamylalcohol (25:24:1) and chloroform/isoamylalcohol (24:1). DNA was then precipitated by adding two volumes of absolute ethanol and pelleted by centrifugation for 15 min at 15,000×g. The pellet was washed with 70% ethanol, air dried and resuspended in TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH 8.0). RNA was degraded by treatment with RNase A (50 μg/ml) for 30 min at 37°C. DNA concentration and purity was spectrophotometrically measured using an RNA/DNA calculator (GeneQuant II; Pharmacia Biotech, Freiburg, Germany).

Diagnostic PCR analysis using species-specific primers

Identity of F. verticillioides isolates was confirmed by PCR using species-specific primers, VERT-1 (5′-GTCAGAATCCATGCCAGAACG-3′) and VERT-2 (5′-CACCCGCAGCAATCCATCAG-3′) for F. verticillioides under the conditions described by Patiño et al. (2004), and PRO1 (5′-CTTTCCGCCAAGTTTCTTC-3′) and PRO2 (5′-TGTCAGTAACTCGACGTTGTTG-3′) for F. proliferatum under the conditions described by Mule et al. (2004). PCR was repeated three times and the presence of a single amplicon was confirmed by 1.5% agarose gel electrophoresis followed by ethidium bromide staining.

PCR-RFLP of ITS region

ITS region of ribosomal DNA (rDNA) was amplified by PCR. The primers used for the PCR were 5′-TCCGTAGGTGAACCTGCGG-3′ (ITS1) from the 18S and 5′-TCCTCCGCTTATTGATATGC-3′ (ITS4) from the 28S rDNA sequences (White et al. 1990). PCR was performed in 20 μl of reaction mixture containing 20 pmol of both primers, 0.2 U ExTaq DNA polymerase (Takara, Otsu, Japan), 200 μM of each dNTP, 2 μl of 10× PCR buffers and 0.2 μg of template DNA with a thermal cycler (PC-800, Astec, Fukuoka, Japan). An initial denaturation step for 3 min at 95°C was followed by 30 cycles of denaturation for 40 s at 94°C, annealing for 40 s at 58°C, extension for 40 s at 72°C and final extension step for 5 min at 72°C. Negative controls (no template DNA) were included in every assay. Amplicons were separated by electrophoresis on 1.5% agarose gels and visualized by staining with ethidium bromide. Amplification tests were repeated four times and confirmed the presence of a single product by electrophoresis. Amplified PCR products were purified using Min Elute DNA cleanup system (Qiagen, Hilden, Germany) according to manufacturer’s instructions. Samples (1 μg) of purified DNA were digested for 4 h at 37°C with five restriction enzymes: RsaI, ScrFI, HinfI, HaeIII, (New England Biolabs, Beverly, MA, USA) and MspI (Nippon Gene, Toyama, Japan). The digested DNA fragments were separated on 1.5% agarose gels. Gels were stained with ethidium bromide and photographed under a UV transilluminator.

Data analysis

All pathogenicity and host range tests were conducted in a completely randomized design. All data from each of the repeated experiments were analyzed separately and subjected to analysis of variance (ANOVA). Mean disease severity index were compared with control mean using Dennett’s t-test (JMP statistical discovery software; SAS Institute, Cary, NC, USA). To record RFLP patterns, we prepared a binary data matrix for each isolate, on which the DNA bands obtained for each individual were scored based on their presence (1) or absence (0). The genetic similarities between individuals were estimated using Nei and Li (1979) coefficient. The similarity matrix generated was then used to produce a dendrogram by unweighted paired-group method with arithmetic (UPGMA) using the REALOctan 2.0 program, which was distributed personally from Dr. Mitsuru Okuda, National Agricultural Research Center for Kyushu Okinawa region (KONARC, Japan).

Results

Field observation and symptom development

Wilted Welsh onion plants were observed on nine individual farms located in six regions in Japan; Saitama (one field), Kyoto (three), Kagoshima (two), Okayama (one), Miyazaki (one), and Hyogo (one). The disease incidence reached up to 50% or more in the fields in Kyoto. The disease incidence in the other locations ranged from 10–30%. In the fields, the lower leaves yellowed, and eventually the entire leaf necrosed, causing plant death (Fig. 1a). Infected basal plates of Welsh onion were discoloured brown (Fig. 1b).
Fig. 1

Symptoms on wilted Welsh onion from a field. a Clorosis and necrosis of wilted leaves of diseased plants, b Infected, browned vascular system of wilted Welsh onion

Isolation, morphology and identification of the causal fungi

Thirty-two strains of Fusarium spp. were isolated from wilted Welsh onion plants in the nine farms described using Komada’s Fusarium-selective medium. All the isolates used in the present study were identified as either F. oxysporum (18 isolates), F. verticillioides (7), or F. solani (7) based on macro- and microscopic characteristics (Table 1).

Fusariumoxysporum isolates produced white to pale violet colonies on PDA with aerial mycelia and had a cottony or somewhat ropey texture. The colour of the undersurface of the colonies among the isolates varied from pink or light to dark violet or dark magenta. Microconidia were formed in false heads (Fig. 2a) on short monophialides (Fig. 2b). Uni- or bicellular, and ovoid to ellipsoid microconidia were abundant (Fig. 2c). Canoe-shaped macroconidia with a long apical cell and a foot-shaped basal cell formed 3–5 septa (Fig. 2d). Chlamydospores were mostly single or rarely in short chains in two-week-old cultures. On some PDA cultures, macroconidia were produced from orange sporodochia.
Fig. 2

Macroconidia and microconidia produced by three Fusarium species isolated from wilted Welsh onion. F. oxysporium microconidia in situ on short monophialides (a, b), microconidia (c) and macroconidia (d), F. solani microconidia in situ on elongated monophialides (e, f) microconidia (g) and macroconidia (h). F. verticillioides microconidia in long chains in situ on nonbranched monophialides (i, j), microconidia (k) and macroconidia (l). Bar represents 50 μm in a, b, e, f, i and j, and 25 μm in c, d, g, h, k and l

Fusarium solani isolates on PDA formed cream- or white colonies, and in some isolates the undersurface was light violet. The conidia formed on false heads (Fig. 2e) on elongated phialides (Fig. 2f). The oval or elliptical microconidia were mono- or bicellular (Fig 2g). Although macroconidia were similar to those of F. oxysporum, they were wider than those of F. oxysporum and had a conspicuous wall. Their apical and basal cells were round or foot-shaped and had three or five septa (Fig. 2h).

Fusarium verticillioides colonies on PDA produced white mycelia initially and developed to dark violet pigment (almost black) with age. However, some isolates had violet or pink mycelia, similar to F. oxysporum. Microconidia were produced from unbranched monophialides (Fig. 2i) in long chains in the aerial mycelium (Fig 2j). All isolates of this species produced abundant microconidia that were monocellular and oval or elliptical in shape (Fig. 2). Macroconidia were very long, slender, bent in shape, and had three to five septa with a curved or tapered apical cell (Fig. 2l). Orange sporodochia were present, and chlamydospores were absent.

DNA from all the isolates was subjected to PCR amplification with species-specific primers, VERT-1/VERT2 and PRO1/PRO2. A single fragment of about 800 bp was amplified with VERT-1/VERT2 from only the seven isolates that were morphologically identified as F. verticillioides (data not shown). No PCR product was observed with F. proliferatum-specific primers, PRO1/PRO2, with any of the fungal DNA used.

Pathogenicity of Fusarium isolates

In pathogenicity tests, symptoms appeared as pre- and postemergence damping-off, and any surviving seedlings had stunted growth with short root systems. These symptoms agreed with those reported in previous studies (Kodama 1983; Srivastava and Qadri 1984). The control plants, raised from seeds immersed in distilled water, remained symptomless during experiments. On the basis of the disease severity index for the number of emerged, healthy and diseased seedlings, five (08, 15, 17, 22, and 30) isolates were strongly pathogenic on tested cultivars, causing the highest disease severity (more than 3) and greatest reduction in seed germination (50–65%) and highest seedling mortality (62.5–100%). With these five isolates, postemergence damping-off was evident within 2–3 days of emergence, and most of the seedlings inoculated were dead within two weeks. Seedlings that survived were stunted (less than half of control plants) with short roots. Although the seedlings inoculated with eight F. oxysporum isolates (13, 18, 19, 20, 25, 26, 28, and 31) and two F. oxysporum f. sp. cepae strains had more than 52.5% survival rates, the average plant height of surviving seedlings was between 50–60%, and root length was 42.5–50% of those of control plants, respectively. F. oxysporum isolates (13, 18, 19, 20, 25, 26, 28, and 31) and two F. oxysporum f. sp. cepae strains induced moderate disease severity (from 2 to 3). The other F. oxysporum isolates and F. verticillioides and F. solani were weakly pathogenic on tested cultivars, causing less stunted growth of seedlings and reduction in root length (less than 25%) and low disease severity (<2). Thus, the isolates analyzed in this study varied from 0 to 4 in disease severity on the five cultivars tested (Table 1). Welsh onion cultivars differed in their susceptibility to each of the 32 isolates. Bulb onion cultivars in general were highly sensitive to all F. oxysporum isolates tested.

Host range test

All F. oxysporum isolates and two F. oxysporum f. sp. cepae stains tested for host range clearly demonstrated Allium-specific infection. F. oxysporum isolates reduced seed germination and increased seedling mortality due to pre- and postemergence damping-off only in Welsh onion and bulb onion seedlings. In contrast, no symptoms were visible on the other inoculated plant species (data not shown). The forma specialis concept in the F. oxysporum is restricted to those causing wilt of a specific host (Snyder and Toussoun 1965; Correll 1991). Thus, the five high virulent F. oxysporium isolates were considered as Fusarium oxysporum f. sp. cepae.

PCR-RFLP of the ITS region

A single amplification product of ca. 570 bp was obtained for DNA of all 32 isolates listed in Table 1 (Fig. 3a). All restriction endonucleases tested (RsaI, HinfI, HaeIII, ScrFI, and MspI) had restriction sites in the amplified ITS region for at least one Fusarium species except for one isolate (05) that had no restriction site for the restriction endonucleases tested in the region. According to restriction patterns generated by the five enzymes, eight RFLP groups were easily defined (Fig. 3a–f). The restriction enzyme RsaI digested DNA amplicons at one site only in F. verticillioides isolates (Fig. 3b). HinfI, MspI and HaeIII enzymes provided species-specific banding patterns (Fig. 3c–e). The ScrFI enzyme had two restriction sites in the ITS region of F. oxysporum isolates, but none in the ITS of isolate 32. Analysis of ScrFI restriction patterns indicated a single site in F. solani and F. verticillioides, giving rise to the same fragment size (Fig. 3f). No polymorphisms were observed within F. verticillioides isolates with these five restriction enzymes.
Fig. 3

Representative data for PCR-RFLP products for the rDNA ITS regions of Fusarium isolates obtained from Welsh onion. The ITS regions (ITS1, ITS4, and 5.8S) of the isolates were amplified, and the products (a) were digested with RsaI (b), HinfI (c), MspI (d), HaeIII (e), or ScrFI (f). Lanes 1 and 2, F. oxysporum; lanes 3 and 4, F. solani; lanes 5 and 6, F. verticillioides. M, 100-bp molecular size marker

The similarity of RFLP banding patterns among the 32 isolates ranged from 28.6 to 100%. In the dendrogram based on fragment size using the UPGMA method, we identified three groups that corresponded to species classification. Isolates of F. oxysporum were divided into two genetic groups with a similarity of 61.6%, one of which consisted of three subclusters. Isolates of F. verticillioides were clustered in a single group with coincident RFLP banding patterns (100% similarity). In contrast, isolates of F. solani clustered in two groups with only 40% similarity (Fig. 4). Genetic diversity was observed among isolates of each Fusarium species that were obtained, even from within the same field.
Fig. 4

UPGMA dendrogram constructed from PCR-RFLP patterns of the ITS region of the Fusarium isolates used in this study. The bar at the top denotes the percentage similarity between isolates, and the numbers to the left of the clusters numbers (I–VIII) correspond to the isolates in Table 1. The level of support for each node, evaluated as percentage of 1,000 bootstrap replicates, is given above the nodes (only values higher than 70% are shown)

Discussion

The recent occurrence of Fusarium wilt of Welsh onion in the major growing areas of Japan requires our attention, because the disease has been rare even in fields where Welsh onion has been cultivated every year. There have been few studies on Fusarium wilt of Welsh onion and the causal agent of the disease (Takakuwa et al. 1977; Kodama 1983; Takeuchi et al. 1983) in contrast to Fusarium wilt (also known as Fusarium basal rot) of bulb onion, which is a worldwide disease found wherever bulb onion cultivars are grown (Abawi and Lorbeer 1972; Sokhi et al. 1974; Ashour et al. 1980; Kodama 1983; Entwistle 1990; Galván et al. 2008). To the best of our knowledge, the present report is the first detailed study on the pathogenic variation and genetic diversity of Fusarium species causing wilt of Welsh onion in Japan.

Fusarium oxysporum, which has been assumed to be the causal agent of Fusarium wilt, indeed predominated in our collection. Our results showed a significant variation in virulence among F. oxysporum isolates from wilted Welsh onion from six different regions of Japan. These results agree with previous studies (Özer et al. 2004; Galván et al. 2008), which showed variation in virulence of F. oxysporum isolates on bulb onion.

We unexpectedly found that both F. verticillioides and F. solani species colonized Welsh onion plants and caused wilting, because these two species have not previously been known to be pathogens of Welsh onion in Japan (Phytopathological Society of Japan 2000). Fusarium verticillioides has not been recognized as a common plant pathogen in Japan, nor is there a record of F. verticillioides on Welsh onion or other Allium spp., although this pathogen can cause severe rotting and wilting in maize (Oren et al. 2003).

Fusarium proliferatum, a species that is easily misidentified as F. verticillioides because of its closely related morphology (Nelson et al. 1983; Leslie et al. 1996), has been isolated from Allium spp., including bulb onion and garlic in different countries (du Toit et al. 2003; Dugan et al. 2003; Stankovic et al. 2007). Therefore, in the present study we used species-specific primers to confirm the identity of F. verticillioides. Results of PCR analysis agreed with those of the morphological identification, confirming that these isolates were F. verticillioides. Colonization of Welsh onion plants by F. verticillioides may pose a serious health risk because the fungus is associated with the production of mycotoxins (fumonisins) (Marasas 1995; Bottalico and Perrone 2002). Therefore, these isolates should be tested for toxin production.

According to the results of PCR-RFLP, the 32 isolates examined were divided into eight RFLP types. Of the eight RFLP types, four types (I–IV) were represented by F. oxysporum isolates. However, there was no clear relationship between polymorphisms in the ITS region and disease severity. We also detected genetic diversity among F. solani isolates (types V–VII). The PCR-RFLP studies on the ITS region identified three enzymes (HinfI, MspI, and HaeIII) to differentiate the three species with very low interspecific polymorphism.

With regard to the origin of the pathogens that recently caused severe Fusarium wilt of Welsh onion in Japan, three possibilities should be considered. First, the pathogens might have been present in the field soil at low levels but were not noticed because of resistance in Welsh onion until inoculum levels exceeded threshold levels for significant epidemics after many years of continuous cropping of Welsh onion in the same field. Second, the pathogens may have spread through recent use of contaminated seeds or susceptible cultivars in the fields. However, this scenario is less likely because the disease was not associated with any particular seed lots or cultivars and was observed only in the fields where Welsh onion has been continuously cultivated for over 10 years. Third, the disease may be due to fungal populations that are becoming more virulent on Welsh onion cultivars. This may be the most probable because of the Allium species, A. fistulosum is thought to be one of the most resistant to F. oxysporum (Abawi and Lorbeer 1972; Sokhi et al. 1974; Galván et al. 2008).

In conclusion, the present study provides information about the causal agents of Fusarium wilt in Welsh onion in Japan and reveals for the first time pathogenic and genetic variability in these pathogens. This study also demonstrated that PCR-RFLP of amplified ITS regions may be useful for differentiating F. oxysporum, F. verticillioides, and F. solani, which were isolated from wilted Welsh onion plants.

Notes

Acknowledgments

We thank Mr. Masataka Funahashi, Nissan Seed, Nishinomiya, Japan, for wilted Welsh onion samples; Dr. Mitsuru Okuda, National Agricultural Research Center for Kyushu Okinawa region (KONARC, Japan), for the computer software to construct dendrograms. This work was supported in part by the Nissan Seed Foundation.

References

  1. Abawi GS, Lorbeer JW (1972) Several aspects of the ecology and pathology of Fusarium oxysporum f. sp. cepae. Phytopathology 62:870–876CrossRefGoogle Scholar
  2. Ashour WA, Elewa IS, Ali AA, Dabash T (1980) The role of some systemic and nonsystemic fungicides and fertilization on the enzyme activity and the control of Fusarium oxysporum f. sp. cepae, the cause of basal rot disease in onion. Agric Res Rev 58:145–161Google Scholar
  3. Bottalico A, Perrone G (2002) Toxigenic Fusarium species and mycotoxins associated with head blight in small grain cereals in Europe. Eur J Plant Pathol 108:611–624CrossRefGoogle Scholar
  4. Correll JC (1991) The relationship between formae speciales, races, and vegetative compatibility groups in Fusarium oxysporum. Phytopathology 81:1061–1064Google Scholar
  5. du Toit LJ, Inglis DA, Pelter GQ (2003) Fusarium proliferatum pathogenic on onion bulbs in Washington. Plant Dis 87:750CrossRefGoogle Scholar
  6. Dugan FM, Hellier BC, Lupien SL (2003) First report of Fusarium proliferatum causing rot of Garlic bulbs in North America. Plant Pathol 52:426CrossRefGoogle Scholar
  7. Entwistle AR (1990) Root diseases. In: Rabinowitch HD, Brewster JL (eds) Onions and allied crops, vol II. CRC Press, Boca Raton, FL, pp 103–154Google Scholar
  8. Galván GA, Koning-Boucoiran CFS, Koopman WJM, Burger-Meijer K, González PH, Waalwijk C, Kik C, Scholten OE (2008) Genetic variation among Fusarium isolates from onion, and resistance to Fusarium basal rot in related Allium species. Eur J Plant Pathol 121:499–512CrossRefGoogle Scholar
  9. Inden H, Asahira T (1990) Japanese bunching onion (Allium fistulosum L.). In: Rabinowitch HD, Brewster JL (eds) Onions and allied crops, vol III. CRC Press, Boca Raton, FL, pp 159–178Google Scholar
  10. Komada H (1975) Development of a selective medium for quantitative isolation of Fusarium oxysporum from natural soils. Rev Plant Prot Res 8:114–125Google Scholar
  11. Kodama F (1977) Fusarium wilt disease of Welsh onion caused by Fusarium oxysporum (Abstract in Japanese). Ann Phytopathol Soc Jpn 43:340Google Scholar
  12. Kodama F (1983) Studies on basal rot of onion caused by Fusarium oxysporum f. sp. cepae and its control. Rep Hokkaido Pref Agric Expt Stn 39:1–65Google Scholar
  13. Leslie JF, Summerell BA (2006) The Fusarium laboratory manual. Blackwell, AmesGoogle Scholar
  14. Leslie JF, Marasas WFO, Shephard GS, Sydenham EW, Stockenstrom S, Thiel PG (1996) Duckling toxicity and the production of fumonisin and moniliformin by isolates in the A and F mating populations of Gibberella fujikuroi (Fusarium moniliforme). Appl Environ Microbiol 62:1182–1187PubMedGoogle Scholar
  15. Marasas WFO (1995) Fumonisins: their implications for human and animal health. Nat Toxins 3:193–198PubMedCrossRefGoogle Scholar
  16. Mulè G, Susca A, Stea G, Moretti A (2004) A species-specific PCR assay based on the calmodulin partial gene for identification of Fusarium verticillioides, F. proliferatum and F. subglutinans. Eur J Plant Pathol 110:495–502CrossRefGoogle Scholar
  17. Nei M, Li WH (1979) Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci USA 76:5269–5273PubMedCrossRefGoogle Scholar
  18. Nelson PE, Toussoun TA, Marasas WFO (1983) Fusarium species: An illustrated manual for identification. Pennsylvania State University Press, University ParkGoogle Scholar
  19. Oren L, Ezrati S, Cohen D, Sharon A (2003) Early events in the Fusarium verticillioides—maize interaction characterize by using a green fluorescent protein expressing transgenic isolate. Appl Environ Microbiol 69:1695–1701PubMedCrossRefGoogle Scholar
  20. Özer N, Köycü ND, Chilosi G, Magro P (2004) Resistance to Fusarium basal rot of onion in greenhouse and field and associated expression of antifungal compounds. Phytoparasitica 32:388–394CrossRefGoogle Scholar
  21. Patiño B, Mirete S, González-Jaén MT, Mulé G, Rodríguez MT, Vázquez C (2004) PCR detection assay of fumonisin-producing Fusarium verticillioides strains. J Food Protect 67:1278–1283Google Scholar
  22. Phytopathological Society of Japan (ed) (2000) Common names of plant diseases in Japan. Plant Protection Association, Tokyo, JapanGoogle Scholar
  23. Shinmura A, Sakamoto N, Hayashi T, Hoshi H, Tani A (1998) Occurrence of Fusarium root rot of Welsh onion caused by F. oxysporum. Bull Hokkaido Pref Agric Exp Stn 74:35–41Google Scholar
  24. Snyder WC, Toussoun TA (1965) Current status of taxonomy in Fusarium species and their perfect stages. Phytopathology 55:833–837Google Scholar
  25. Sokhi SS, Singh DP, Joshi MC (1974) Sources of resistance to basal rot of onions caused by Fusarium oxysporum. Indian J Mycol Plant Pathol 4:214–215Google Scholar
  26. Srivastava KJ, Qadri SMH (1984) Some studies of damping-off disease of onion (Allium cepa L.). Indian Bot Rep 3:147–148Google Scholar
  27. Stankovic S, Levic J, Petrovic T, Logrieco A, Moretti A (2007) Pathogenicity and mycotoxin production by Fusarium proliferatum isolated from onion and garlic in Serbia. Eur J Plant Pathol 118:165–172CrossRefGoogle Scholar
  28. Takakuwa M, Ishizaka N, Kodama F, Saito I (1977) Host range of Fusarium oxysporum f. sp. cepae, causal fungus of Fusarium basal rot of onion. Ann Phytopathol Soc Jpn 43:479–481Google Scholar
  29. Takeuchi T, Sinohara S, Nagai Y (1983) Survey on occurrence of Fusarium wilt of Welsh onion and its control. Bull Chiba Agric Exp Stn 24:1–6Google Scholar
  30. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR Protocols: a guide to methods and applications. Academic Press, San Diego, CA, pp 315–322Google Scholar
  31. Yakuwa T (2006) Welsh onion or Japanese bunching onion. In: The Japanese Society for Horticultural Science Horticulture in Japan (ed). Shokabo publications, Tokyo, pp 165–166Google Scholar

Copyright information

© The Phytopathological Society of Japan and Springer 2008

Authors and Affiliations

  • Maha Laksha Mudiyanselage Chandrika Dissanayake
    • 1
  • Rumi Kashima
    • 2
  • Shuhei Tanaka
    • 2
  • Shin-ichi Ito
    • 2
  1. 1.The United Graduate School of Agricultural SciencesTottori UniversityTottoriJapan
  2. 2.Department of Biological and Environmental Sciences, Faculty of AgricultureYamaguchi UniversityYamaguchiJapan

Personalised recommendations