Fusarium solani species complex (FSSC) Clade 2 encompasses at least eight phylogenetic species including F. phaseoli (bean root rot pathogen), Fusarium virguliforme and related species that have been reported from all major plantation regions in South America (Aoki et al. 2003). Sudden death syndrome of soybean, caused by Fusarium virguliforme, is one of the most destructive diseases affecting soybean production in North and South America (Aoki et al. 2003). Aoki et al. (2003) extensively investigated the bean root rot pathogen and SDS pathogenic strains from North and South America based on comprehensive morphological comparisons and molecular phylogenetic analyses of multilocus DNA sequences Table 1. According to this study, they described three morphologically and phylogenetically distinct species within Clade 2. Fusarium virguliforme (formerly known as F. solani f. sp. glycine) and F. phaseoli (formerly known as F. solani f. sp. phaseoli) were described for the United States strains and F. tucumaniae was illustrated for Argentinian strains (Roy 1997; Rupe et al. 2001). Aoki et al. (2005, 2012), based on detailed morphological comparisons, phenotypic and molecular phylogenetic analyses of multiple loci of DNA sequences, described F. brasiliense, F. cuneirostrum and F. crassistipitatum as novel SDS pathogens. Until now, no attempt has been made to classify members of the FSSC in Malaysia. Therefore, the objectives of the present study were to re-identify strains of FSSC stored in the Fusarium Culture Collection Unit of School of Biological Sciences, Universiti Sains Malaysia by using morphological and molecular markers. In this survey, 140 strains were investigated based on morphological characteristics as shown in Table 2. All strains were purified by the single-spore isolation technique. Single germinated macroconidia were transferred onto PDA plates and colony appearance was used to select isolates for further study. Fusarium strains were grown on PDA and carnation leaf agar (CLA) (Fisher et al. 1982) in 9 cm plastic Petri dishes. Cultures were incubated under 12 h alternating light (black/white) at 25 ± 2 °C for 1 week. Colony morphology and colour were based primarily on cultures grown on PDA. Cited colours are given according to Kornerup and Wanscher (1978). For comparison of mycelial growth rates, agar blocks ca 5 × 5 mm were cut from the margins of 1 week old cultures on CLA and transferred onto PDA and incubated at 25 °C for 1 week in the dark. Thirty randomly selected conidia of each septation class (macroconidia and microconidia), sporodochial phialides, chlamydospores and conidiophores were measured and analysed by the 2-Sample T-Test using MINITAB® 15 (Table 2). For species determination, the descriptions by Aoki et al. (2003, 2005, 2012) were adopted.

Table 1 Strains of Fusarium solani species complex recovered from various hosts and substrates in Malaysia
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Table 2 Comparison of morphological characteristics of representative strains within the Fusarium solani species complex
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Representative FSSC strains were grown on PDA with sterile dialysis membranes (Lui et al. 2000) for 5 days (Table 3). The mycelium was harvested and ground in a sterile mortar with liquid nitrogen to a fine powder and then DNA was extracted using a DNeasy® Plant Mini Kit (Qiagen) according to the manufacturer’s instruction. Amplification of the translation elongation factor-1α (tef1) gene, and internal transcribed spacer (ITS) regions was conducted utilising the primer pair ef1 and ef2 for tef1 (O’Donnell et al. 1998), and ITS1 and ITS4 for ITS region (White et al. 1990). PCRs were performed in a Peltier Thermal Cycler, PTC-100® (MJ Research, Inc. USA) in a total volume of 25 μl. The PCR mixture contained 4 μl 5× buffer (Promega, Madison, WI, USA), 4 mM MgCl2, 0.2 mM deoxynucleotide triphosphate (dNTP) (Promega), 0.8 μM of each primer, 0.75 units of Taq DNA polymerase (Promega®, USA), and 6 ng of template DNA. To prevent evaporation, the reactions were overlaid with 25 μl of sterilised mineral oil. PCR products were purified using Qiagen columns according to the manufacturer’s instructions and stored at −20 °C. The purified PCR products were sent to First BASE Laboratories Sdn. Bhd. for sequencing of tef1 gene and ITS regions in both directions (forward and reverse) using ABI 3730x1 model of sequencer. Forward and reversed sequences of tef1 gene and ITS regions were edited and aligned using BioEdit version 7.0.5 (Hall 1999). Consensus sequences were used as query to search for similarities using two sources: I. BLAST network services at the National Centre for Biotechnology Information (NCBI); II. FUSARIUM-ID v.1.0 database (http://fusarium.cbio.psu.edu) (Geiser et al. 2004).

Table 3 Strain number, geographical origin, and GenBank accession numbers of representative strains within the Fusarium solani species complex
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All FSSC strains were successfully investigated based on macroscopic and microscopic characteristics. One-hundred and thirty seven strains (97.9 %) were identified as F. solani and three strains (2.1 %) belonged to F. virguliforme (Fig. 1). All three strains produced pink to bluish-gray mycelium. Aerial conidiophores, unbranched or sparsely branched, up to 250 μm long and 2.5–7.0 μm wide at base, abundantly formed on CLA at 25 °C (Fig. 1a). Aerial phialides were simple, cylindrical or subcylindrical. Sporodochial conidiophores were subcylindrical or ampulliform with distinctive collarette at the tip, 20–28 μm long, 2.5–3.0 μm wide at base (Fig. 1b). Macroconidia arising from sporodochia were falcate and sometimes widest at middle, with 3-5-septa and mostly 3-septate, with a tapered and curved apical cell and distinctly notched basal cell (Fig. 1c–d). Microconidia were comma-shaped, elongated oval to sometimes short-clavate and ellipsoidal shaped with a swollen apex often rounded and mostly 0-2-septate (Fig. 1f). The size of conidia; 0–1(−2)-septate = (11–) 15.6–26 (−33.1) × (3.8–) 5–6.4 (−7.4) μm; 3-septate= (30–) 47–56 (−65) × (4.2–) 4.6–5.4 (−6) μm; 4-septate (45–) 53–58 (−65) × (4.7–) 5.2–5.4 (−6) μm; 5-septate 56–65 × 5.3–6.1 μm. Two types of chlamydospores formed, relatively abundant in mycelium, smooth and rough-walled, 5–14 μm in diameter, may occasionally be found within macroconidia. Chlamydospores formed mostly singly, and in pairs, rarely in chains, mostly subglobose, terminal or intercalar (Fig. 1e).

Fig. 1
figure 1

Fusarium virguliforme grown on CLA, 2 weeks, 25 °C, cultured in the dark. a Slender, unbranched aerial conidiophores. b Branched sporodochial conidiophores forming falcate to curved cylindrical conidia. c–d Falcate and comma-shaped macroconidia produced in sporodochial. e Terminal and intercalary chlamydospores, and produced chlamydospores in conidia. f Comma-shaped microconidia were formed on conidiophores in hyphae. Scale bars: a= 50 μm, b–f = 20 μm

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A list of species names and culture collection numbers, geographical origins, original substrates, and GenBank accession numbers of the individual strains used in this study are in Table 3. Single bands of DNA fragments approximately 550-bp and 700-bp were successfully amplified for ITS region and tef1 gene from all three strains, respectively. From similarities searched at NCBI and FUSARIUM-ID database, all three strains were similar to F. virguliforme with the percentage of maximum identity (100 %). Based on morphological features combined with molecular analysis using tef1 gene and ITS regions sequences, F. virguliforme was reported for the first time in Malaysia.

Fusarium virguliforme is one of the most important causal agents of sudden death syndrome (SDS) in soybean and has been reported from all major growing regions in North America (Aoki et al. 2003, 2005, 2012). Kolander et al. (2012) showed that the diversity and number of hosts for F. virguliforme are greater than previously reported by Aoki et al. (2005, 2012) and indicated that agricultural crops other than soybean can be damaged by F. virguliforme and increase pathogen inoculum in soil. Therefore, further information regarding this well-known plant pathogen within the region is needed. To the best of our knowledge this is the first report on occurrence of this species in agricultural soil in Malaysia.