Pineapple (Ananas comosus) is one of the important commercial fruit crops cultivated in Malaysia (Malaysian Pineapple Industry Board 2015). During a disease survey from December 2010 to November 2011 in pineapple farms in the states of Kedah, Penang, Selangor and Johor, typical fusariosis symptoms were observed on Moris, Josapine and Gandul varieties. Common symptoms were lesion and brown discoloration of the fruitlet, rotten or sunken fruit skin and stem, gum exudation on some fruits, dry rot on leaf, stem bending, chlorosis, increasing number of leaves per spiral and natural cracks on the fruit (Fig. 1a-d), which is similar to pineapple fusariosis described by Rohrbach and Schmitt (1998) and Ploetz (2006). Wilt symptoms were generally observed on the leaves with yellowing or brown discoloration.

Fig. 1
figure 1

Fusariosis symptoms in the field a Dry rot on top of the fruit, b brown lesion on one side of the fruit, c rot at leaf base, d necrotic leaf spot

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Fungi were isolated from the infected fruits and leaves. The tissues between the margin of infected and apparently healthy fruit tissues were sampled. Isolates from infected leaves were obtained from the rotted leaf base and necrotic leaf tissues. The infected areas of the fruits and leaves (cut into 5 × 5 mm pieces) were surface sterilized by soaking in 1 % sodium hypochlorite for 3 min, rinsed with sterile distilled water, and then plated onto peptone pentachloronitrobenzene agar. All of the plates were incubated at 27 ± 1 °C under 12 h alternate light and dark. Mycelial growths from the tissues were then subcultured onto potato dextrose agar (PDA) and single-spored.

Morphological identification was based on procedures in the Fusarium Laboratory Manual (Leslie and Summerell 2006). Eight isolates of Fusarium produced white to dark purple colonies and white to dark violet pigmentation. Aerial mycelia were floccose and sometimes cottony in appearance (Fig. 2a, b). Macroconidia (3.3–3.6 μm x 37.8–43.7 μm) were slender to relatively straight, 2–3-septa (Fig. 2c). Microconidia (3.4–3.7 μm x 12.2 to 12.9 μm) were obovoid with a truncate base (Fig. 2d). Conidiophores produced mono- and polyphialides, and microconidia formed in short (4 conidia) to medium chains (20 conidia) (Fig. 2e, f). The characteristics observed best matched those of F. fujikuroi. The eight isolates that were recovered from the three pineapple varieties from were deposited in culture collection of Plant Pathology Lab, School of Biological Sciences, Universiti Sains Malaysia, Penang, Malaysia.

Fig. 2
figure 2

Morphological characteristics of F. fujikuroi a floccose and cottony colony, b dark violet pigmentation on lower surface of PDA, c slender to relatively straight 3–4 septate macroconidia, d obovoid microconidia e mono- and polyphialides, f V-shaped microconidial in chains

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Genomic DNA of the isolates was extracted using DNeasy® Plant Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. PCR was performed in a MyCycler® Thermal Cycler (Bio-Rad, Hercules, CA, USA). Molecular identification was done using a partial TEF (O’Donnell et al. 1998) and β-tubulin (Glass and Donaldson 1995; O’Donnell and Cigelnik 1997) sequences. PCR cycles for amplification of TEF was set at initial denaturation at 94 °C for 1 min, followed by 35 cycles of denaturation at 95 °C for 35 s, annealing at 59 °C for 55 s, extension at 72 °C for 1.5 min and final extension at 72 °C for 1 min. For β-tubulin, the PCR cycles were initial denaturation at 94 °C for 1 min followed by 39 cycles of denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 1 min and final extension at 72 °C for 5 min. Based on BLAST results against GenBank and Fusarium-ID databases, the isolates showed 98–100 % similarity to F. fujikuroi NRRL43610. All of the sequences were deposited in GenBank (Accession No. from KC584844 to KC584851 for TEF and from KC584866 to KC584873 for β-tubulin).

Phylogenetic analysis was done using TEF and β-tubulin sequences and analyzed using Molecular Evolutionary Genetic Analysis (MEGA) version 5.1 (Tamura et al. 2011). Several F. fujikuroi isolates from rice from Malaysia, Taiwan, Thailand, Korea and the Philippines were included in the analysis (Table 1). Twenty-one members of Fusarium fujikuroi species complex representing Asian, American and African clades (O’Donnell et al. 1998), F. guttiforme, F. ananatum and F. proliferatum from pineapple were also included in the phylogenetic analysis (Table 1). The tree was rooted with F. oxysporum (O’Donnell et al. 1998).

Table 1 Host and geographic origin of Fusarium species used in phylogenetic analysis
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The Maximum likelihood tree generated (Fig. 3) showed that all of the pineapple fusariosis isolates clustered with F. fujikuroi from rice. Isolates of F. guttiforme and F. ananatum which have been reported to be associated with fusariosis or fruit rot of pineapple, as well as isolates of F. proliferatum formed separate clade. The results of the present study indicate that in addition to F. guttiforme and F. ananatum, F. fujikuroi is also associated with pineapple fusariosis.

Fig. 3
figure 3

Maximum likelihood tree inferred from combined TEF and β-tubulin sequences showing the placement of F. fujikuroi isolates from pineapple fusariosis. The bootstrap support values higher than 85 % from 1000 replicates are shown at the nodes. Sequences of F. oxysporum NRRL20433 and NRRL22902 were used to root the tree

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A pathogenicity test was conducted using four representative isolates (isolates 37, 59, 68 and 219). The test was conducted on Gandul, Josapine and Moris varieties using a pricking technique for pineapple fruits and an agar plug technique for pineapple leaves (Dianese et al. 1981). Detached matured fruits and leaves were surface sterilized using 1 % sodium hypochlorite for 3 min and then rinsed with sterile distilled water. For the inoculation test on fruits, the skin was wounded with a sterile tooth pick before a colonized tooth pick with mycelia was inserted into the wounded area. All of the tooth picks were left intact until the end of the experiment. On leaves, a fine sterile needle was used to wound the healthy leaf surface and a 5 mm mycelia plug (from 7-day-old culture) was placed in the wounded area. Control fruits were pricked with a sterile tooth pick without inoculum while control leaves were inoculated with a PDA plug without inoculum. All of the inoculated fruits were arranged in a container that contained moist sterile filter paper to maintain moisture and then covered with a plastic wrap (Cling Wrap). Inoculated leaves were also covered with plastic wrap for one week to maintain moisture content. Symptom development was observed one week after inoculation.

Two weeks after inoculation, the fruits were cut vertically and brown lesions around the inoculated areas were observed as well as mycelia growth in some of the inoculated areas (Fig. 4a). On leaves, a small necrotic spot appeared on the inoculated leaves which turned brown and grew into a dark brown lesion after eight weeks incubation (Fig. 4b). All inoculated fruits and leaves of Gandul, Josapine and Moris varieties showed fusariosis symptoms similar to those observed on infected pineapple plant in the field. The F. fujikuroi isolates were re-isolated from brown lesions on the infected fruit tissues as well as from black and brown necrotic spots on leaves, thus completing Koch’s postulates.

Fig. 4
figure 4

Pathogenicity test on pineapple fruit and leaf. a brown to black lesions on pineapple fruit. b necrotic lesions on pineapple leaf

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The present study revealed that F. fujikuroi isolates caused pineapple fusariosis in Peninsular Malaysia. Although morphologically, F. fujikuroi was similar with F. proliferatum, in the present study F. fujikuroi isolates produced colony with cottony appearance and formation of short chain microconidia which were minor morphological differences that can be used to differentiate both species (Gerlach and Nirenberg 1982). Moreover, in phylogenetic analysis isolates of F. fujikuroi and F. proliferatum were clearly grouped into separate clades. Fusarium fujikuroi is a well-known pathogen of bakanae disease of rice. Besides rice, F. fujikuroi has been reported as saprophytic or endophytic colonisers of vanilla (Pinaria et al. 2010) and was isolated from human skin (O’Donnell et al. 2010). According to Slippers et al. (2005), fungi are more likely to undergo plant host jumps as a result of human movement and weather changes. Movement of pineapple plants for trading and exchange of planting materials between plantations and farmers within Peninsular Malaysia may have introduced and spread the pathogen into new areas. To our knowledge, this is the first report of F. fujikuroi associated with pineapple fusariosis in Peninsular Malaysia.