220

http://journals.tubitak.gov.tr/biology/

Turkish Journal of Biology Turk J Biol(2017) 41: 220-230© TÜBİTAKdoi:10.3906/biy-1606-25

Characterization and pathogenicity of Fusarium proliferatum andFusarium verticillioides, causal agents of Fusarium ear rot of corn

Nur Ain Izzati MOHD ZAINUDIN*, Farah Aqila HAMZAH, Nor Azizah KUSAI, Nur Syuhada ZAMBRI, Suhaida SALLEHDepartment of Biology, Faculty of Science, Universiti Putra Malaysia, Serdang, Selangor, Malaysia

* Correspondence: ainizzati@upm.edu.my

1. IntroductionPathogenic fungi including Fusarium species are commonly found associated with corn. Fusarium ear rot is the most common fungal disease of corn ears. This type of disease was detected in cornfields around Malaysia by a series of samplings conducted from October 2006 to September 2013. Fusarium ear rot symptoms in the infected corns are generally detected on the ear tips or dispersed throughout the ear on a single kernel or clusters of kernels with white to light pink or salmon-colored mold (Munkvold, 2003). This symptom develops with white streaks radiating out from the stylar canal (Duncan and Howard, 2010). As the disease develops, the infected kernels will gradually obtain tan or brown color, or white streaks. This disease does not usually affect the entire ear. Nevertheless, the pathogens are able to colonize the entire corn starting from seed to kernels without the plant showing visible symptoms (Parsons and Munkvold, 2012).

Previous reports have demonstrated that Fusarium species are able to cause diseases, including F. verticillioides, F. proliferatum, and F. subglutinans (Bottalico, 1998; Srobarova et al., 2002; Logrieco et al., 2007; Suhaida and Nur Ain Izzati, 2013). These pathogens can infect all parts of the corn plant including the kernels, roots, and stalk.

Additionally, the infections they give to corn plants vary depending on environmental conditions, geographical regions, and the phase of maturity of the corn (Srobarova et al., 2002), as this disease can reduce yields and quality by up to 50% (Ding et al., 2008). This disease is often related to warm and dry years with a decay that usually begins with insect-damaged kernels. The infection of Fusarium isolates in the corn kernel can occur through kernel wounds, silk exposure, or systemically through roots (Logrieco et al., 2007). The infected corn ears contain fungi that live in the debris or on the soil surface. Insects are one of the vectors spreading the Fusarium spores into the kernel wounds of corn. Airborne spores can germinate and grow down the silk channel once it lies on the kernel wound.

Etiological study of Fusarium species associated with the disease is limited in tropical areas. Thus, this study has focused on the etiology of fungal isolates from Fusarium ear rot-infected corns planted in Malaysia. In order to identify the isolated fungi to species level, biological species was studied by sexual crossing and sequencing of translation elongation factor (tef) 1-α. In this type of study, the tef1-α gene region is considered a marker for single-locus identification of Fusarium species (Geiser et al., 2004). This gene encodes the part of protein

Abstract: Fusarium ear rot is a significant disease of corn caused by several toxigenic Fusarium species including Fusarium proliferatum and Fusarium verticillioides. Forty-one Fusarium isolates were recovered from corn with Fusarium ear rot disease symptoms collected from Peninsular Malaysia. Isolates were classified into three described species known as F. proliferatum, F. verticillioides, and F. solani. Based on sexual compatibility test, four isolates from F. proliferatum (MATD-1) were crossed-fertile with tester isolate F. proliferatum D024853 (MATD-2), producing perithecia in the presence of ascospores. Meanwhile, the isolates from F. verticillioides (MATA-2) were crossed with tester isolate F. verticillioides A00149 (MATA-1), but were found producing 11 isolates with barren perithecia and three infertile isolates, whereas in 11 isolates of F. verticillioides (MATA-1), seven isolates produced barren perithecia with four nonfertile isolates. In the pathogenicity test, all isolates were found pathogenic and displayed disease symptoms with variation in severity. The highest disease severity index value was observed in F. proliferatum B68c at 4.67, which was obtained in an updated report on the mating type of F. verticillioides and F. proliferatum isolated from Fusarium ear rot disease.

Key words: Fusarium ear rot, Fusarium proliferatum, Fusarium verticillioides, mating type, translation elongation factor 1-α

Received: 09.06.2016 Accepted/Published Online: 22.09.2016 Final Version: 20.02.2017

Research Article

MOHD ZAINUDIN et al. / Turk J Biol

221

translation machinery and appears consistently single-copy in Fusarium species as well as presenting a high level of sequence polymorphism among closely related species (Geiser et al., 2004). Based on biological species, F. verticillioides and F. proliferatum belonged to mating population (MPs) A and D, respectively.

Hence, the objectives of the present study are to identify the Fusarium species associated with Fusarium ear rot in Peninsular Malaysia based on phenotypic, biological, and molecular characterization and to examine the pathogenicity phenotypes of fungal isolates. We hypothesized that several Fusarium species within the Gibberella fujikuroi species complex would be present and that F. verticillioides or F. proliferatum would be the pathogenic species. Our results will provide additional information on the biodiversity of Fusarium species associated with Fusarium ear rot and aid in developing regulatory policies for corn. The isolates of the pathogens obtained can also be utilized in controlling disease.

2. Materials and methods2.1. Fungal isolationFusarium ear rot samples were obtained from seven different geographical areas, namely Seri Medan (Johor), Senggarang (Johor), Serdang (Selangor), Semenyih (Selangor), Jerantut (Pahang), Cameron Highlands (Pahang), and Seberang Perai (Pulau Pinang). All infected samples were surface-sterilized with 10% Clorox and rinsed using sterile distilled water for 2 min each. Samples were then blotted and transferred onto peptone-pentachloronitrobenzene agar. This process was then followed by subsequent incubation at room temperature for 7 days. Single spore isolation was carried out by streaking the Fusarium conidia on potato dextrose agar (PDA). The single-spored colony was transferred onto PDA and incubated at room temperature under fluorescence and UV light for 7 days. The isolates were then preserved as aqueous spore suspensions made using 25% glycerol complete medium with xylose. Reference isolates used in sexual development were A-00149 (MATA-1), A-00999 (MATA-2), D-04854 (MATD-1), and D-04853 (MATD-2), which were obtained from the Department of Plant Pathology, Kansas State University, Manhattan, Kansas, USA.2.2. Phenotypic characterizationFor morphological characteristics and descriptions the system proposed by Leslie and Summerell (2006) was adopted. Pure cultures of the Fusarium isolates were subcultured onto carnation leaf agar and incubated under near-UV light for 5 days. The microscopic characteristics were observed such as shapes and sizes of macroconidia and microconidia, number of septa, shapes of the apical

and basal cells of the macroconidia, length of microconidia in chains, conidiogenous cells, and chlamydospores. These microscopic characteristics of Fusarium cultures were recorded using light microscope (Zeiss, model Axio Camera) under 400× magnification. Furthermore, macroscopic characteristics such as growth rate, colony features, growth rings, and pigmentation on PDA were observed.2.3. Molecular characterization based on translation elongation factor geneIsolates were cultured on PDA and incubated for 7 days at 28 ± 1 °C with a 12-h photoperiod under a combination of cool white florescent and black light. DNA extraction was completed using the UltraClean Microbial DNA Isolation Kit following the instructions of the manufacturer, unless otherwise mentioned. The quality of DNA was evaluated following separation in 1% agarose gel. Amplification of genes was done using a thermal cycler (Biometra, TProfessional).

Amplification for tef-1α was conducted under the conditions of 94 °C for 1 min, followed by 34 cycles of 94 °C for 30 s, 62 °C for 45 s, and 72 °C for 1 min and finally 4 °C until further analysis. The primer sequences for tef-1α are EF1 (5′-AACATGCGTGAGATTGTAAGT-3′) and EF2 (5′-TAGTGACCCTTGGCCCAGTTG-3′) (O’Donnell and Cigelnik, 1997). The appearance of fragments was 700 bp in sized for the tef1-α gene (Nitschke et al., 2009).

Amplification products of the tef1-α gene were cleaned using the QIAquick Gel Extraction Kit according to the manufacturer’s instructions. The tef1-α gene fragment was sequenced using an ABI 3730xl sequencer by MyTACG Bioscience Company (Selangor, Malaysia). Sequences were analyzed and BLAST searched against GenBank (http://blast.ncbi.nlm.nih.gov/) and FUSARIUM-ID (http://isolate.fusariumdb.org/) (Geiser et al., 2004). Nucleotide BLAST was used to find maximum identity and the number of bases of the aligned sequence. Sequence alignments and analyses were performed using MEGA version 6 (Tamura et al., 2007). A maximum-likelihood phylogenetic tree was constructed using bootstrapping (1000 replicates).2.4. Mating type and crosses analysesAll isolates were subjected to the identification of their mating type gene (MAT) allele. Specific primers were used to amplify the MAT region of Fusarium species: a forward primer FMAT1-1a (5’-GTTCATCAAAGGGCAAGCG-3’) was used for MAT1-1 and the reverse primer was FMAT1-1b (5’-TAAGCGCCCTCTTAACGCCTTC-3), while a forward primer FMAT1-2c (5’-AGCGTCATTATTCGATCAAG-3’) and a reverse primer of FMAT1-2d (5’-CTACGTTGAGAGCTGTACAG-3’) were utilized for MAT1-2 (Steenkamp et al., 2000). The program of the thermal cycler was set as follows: 1 min at 94 °C, followed by 35 cycles of 30 s at 92 °C, 30 s at 67 °C, and 30 s at 72 °C

MOHD ZAINUDIN et al. / Turk J Biol

222

and then 4 °C until further analysis. PCR products were electrophoresed, stained, and visualized under UV light. The amplified amplicon indicates the mating type for the isolates.

All isolates were crossed with standard, female-fertile tester isolates (A-00149, A-00999, D-04854, and D-04853). Sexual crosses were made on carrot agar as previously described by Leslie and Summerell (2006). Meanwhile, fertility was determined by the presence of numerous perithecia with a cirrhus of ascospores oozing from the perithecia 28 days after fertilization. Isolates producing eight mature ascospores and presenting germinated ascospores were indicated as fertile isolates. Positive and negative crosses were carried out in triplicate. Field isolates were tested as both male and female parents in crosses with standard testers after the MAT allele in the field isolate was determined. Male fertility was scored in crosses where the field isolate was found male while the standard tester isolate was considered as the female parent. Female fertility was examined in crosses where the field isolate was identified as the female parent while the standard tester isolate was the male. The perithecia produced from each cross plate were individually squeezed open, which demonstrated the presence of ascospores observed using a compound microscope (Carl Zeiss, Germany). 2.5. Pathogenicity testAll corn varieties of Thailand Supersweet were used in the pathogenicity test. Conidial suspension of Fusarium was prepared following the procedure of Siti Nordahliawate et al. (2008). First, 20 mL of distilled water was poured onto 7-day-old Fusarium cultures and the conidia were harvested using a sterile hockey stick. The concentration was modified to 2 × 106 conidia/mL using a hemocytometer. After that, 1 mL of conidia suspension was injected to the ear region using a sterile syringe. Two controls were set up, one inoculated with sterile distilled water (C1) and one not inoculated (C2).

After 7 days of inoculation, the inoculated cobs were manually dehusked and scored for the discoloration of kernels around the inoculation area. Evaluation was done based on a disease scale from 0 to 5 (0- no visible symptoms, 1- moccasin, 2- tan1, 3- tan2, 4- tan3, 5- tan4 with/without decay) following the scoring system proposed by Lori et al. (2008) with slight modification for corn on symptom color (Glynn, 2005). Disease severity index (DSI) was then calculated by the formula DSI = Σ (A × n) /ΣB, where A = disease scale (0, 1, 2, 3, 4, 5), n = number of maize samples in the specific scale, and B = total number of maize samples, as modified by Elmer (2002), in which the disease scale is based on the color of infection. Other visible symptoms including mycelial growth and rot were also recorded. All data from the DSI were analyzed using SPSS 19.0 based on the Friedman test (P < 0.05).

To ascertain the pathogenicity phenotypes of F. proliferatum and F. verticillioides, all inoculated ears showing Fusarium ear rot symptom were reisolated, single-spored, and reidentified based on their morphological appearance as proposed by Leslie and Summerell (2006). Treatments and controls were completed in four replicates.

3. Results3.1. Morphological characterization of F. proliferatum and F. verticillioidesFusarium proliferatum was discovered producing thin-walled, slender, and rather straight macroconidia measuring around 29.97–86.88 µm × 2.53–3.55 µm (Figure 1a). The apical cell was curved and formed with 3–5 septa. Microconidia were noticed to be oval to obovoid in shape measuring around 3.76–37.59 µm × 1.12–4.58 µm (Figure 1b). These microconidia arose in a false head (Figure 1c) and/or moderate lengths of chains (Figure 1d). They derived from monophialide (Figure 1e) and/or also polyphialide (Figure 1f) conidiogenous cells. Chlamydospores were absent in the culture of F. proliferatum. On the PDA, F. proliferatum initially produced white aerial mycelia and became a cottony light orange pigment with age (Figures 1g and 1h). The growth rate of the colony was seen to be slow and measured at around 0.88–0.95 cm/day at room temperature (25 ± 2 °C). The colony covered the medium plates after 10 days of incubation.

Macroconidia of F. verticillioides were slender, slightly straight, thin-walled, and typically formed with three septa (Figure 1i). Their apical cells were blunted and barely notched on basal cells with the size of 20.51–48.26 µm × 3.07–5.08 µm. The microconidia were oval to obovoid-shaped and measured around 3.05–26.31 µm × 1.71–3.51 µm in size (Figure 1j). Compared to the cultures of F. proliferatum, F. verticillioides was found to produce longer chains of microconidia (Figure 1k), which also arose in false heads (Figure 1l). The culture of F. verticillioides also generated microconidia that only derived from monophialide (Figure 1m) conidiogenous cells. No polyphialide conidiogenous cells were produced by F. verticillioides. Fusarium verticillioides culture did not produce chlamydospores. However, they produced swollen cells (Figure 1n) that could be easily misidentified as chlamydospores. The culture of F. verticillioides grew moderately slowly on PDA at room temperature (25 ± 2 °C) with a growth rate ranging from 0.98 to 1.03 cm/day. The cultures were cottony flats, which initially grew as white mycelia and covered the medium plates after 9 days of incubation. The cultures produced orange pigmentation (Figures 1o and 1p).

MOHD ZAINUDIN et al. / Turk J Biol

223

3.2. Translation elongation factor 1-α (tef1-α) gene analysisFor amplification of the tef1-α gene, an amplicon of 600–700 bp was amplified for all isolates. Based on the BLAST

search, all sequences showed 99%–100% similarity with the deposited fungi. The sequences were deposited in GenBank with the accession numbers indicated in the Table. Phylogenetic relationships were analyzed using the

Figure 1. Morphological characteristics of F. proliferatum (a-h) and F. verticillioides (i-p): (a) 5-septate macroconidia, (b) pyriform-shaped microconidia, (c) microconidia forms in false heads, (d) microconidia forms in moderate length of chains, (e) monophialide conidiogenesis, (f) polyphialide conidiogenesis, (g, h) light orange pigmentation on PDA, (i) 3-septate macroconidia, (j) pyriform-shaped microconidia, (k) microconidia forms in long length of chains, (l) microconidia forms in false heads, (m) branched monophialides, (n) thick cell wall of swollen cells, (o, p) and orange pigmentation on PDA.

MOHD ZAINUDIN et al. / Turk J Biol

224

Table. Fusarium isolates from Fusarium ear rot samples in corn.

No. Isolate Species Geographic origin GenBank accession no.

MAT allele

aFertility bDSI

1 B102c F. verticillioides Serdang, Selangor KP340020 2 Barren 1.842 B103c F. verticillioides Serdang, Selangor KP340021 2 Barren 3.673 B106c F. verticillioides Serdang, Selangor KP340017 2 Barren 3.834 B1371c F. verticillioides Semenyih, Selangor KP340014 2 NF 2.845 B144c F. verticillioides Serdang, Selangor KP340023 2 NF 3.346 B146c F. verticillioides Serdang, Selangor KP340024 2 Barren 3.507 B52c F. proliferatum Serdang, Selangor KP340030 1 Barren 1.508 B55c F. proliferatum Serdang, Selangor KP340027 1 Barren 3.509 B56c F. proliferatum Serdang, Selangor KP340032 1 NF 4.5010 B57c F. proliferatum Serdang, Selangor KP340029 1 NF 3.1711 B60c F. verticillioides Serdang, Selangor KP340013 1 NF 2.8312 B66c F. proliferatum Serdang, Selangor KP340033 1 F 3.1713 B67c F. proliferatum Serdang, Selangor KP340034 1 NF 3.6714 B68c F. proliferatum Serdang, Selangor KP340035 1 F 4.6715 B75c F. proliferatum Serdang, Selangor KP340036 1 Barren 4.0016 B89c F. proliferatum Serdang, Selangor KP340019 1 NF 2.8417 B92c F. proliferatum Serdang, Selangor KP340037 1 F 4.1718 B99c F. proliferatum Serdang, Selangor KP340038 1 F 3.1719 C116c F. verticillioides Cameron Highlands, Pahang KP340022 2 Barren 4.5020 C121c F. verticillioides Cameron Highlands, Pahang KP340028 1 Barren 4.0021 C1372c F. proliferatum Jerantut, Pahang KP340041 2 NF 2.8422 J1361c F. verticillioides Seri Medan, Johor KP340011 1 NF 2.3423 J1362c F. verticillioides Seri Medan, Johor KP340010 1 NF 2.1724 J1363c F. verticillioides Senggarang, Johor KP340026 1 Barren 2.0025 J1364c F. verticillioides Senggarang, Johor KP340012 1 Barren 4.5026 J44c F. verticillioides Senggarang, Johor KP340009 1 NF 1.8427 P1366c F. proliferatum Seberang Prai, Pulau Pinang KP340039 1 Barren 4. 0028 P1367c F. verticillioides Seberang Prai, Pulau Pinang KP340006 1 Barren 2.3429 P1368c F. verticillioides Seberang Prai, Pulau Pinang KP340025 2 Barren 3.5030 P1369c F. verticillioides Seberang Prai, Pulau Pinang KP340018 2 Barren 3.1731 P1370c F. verticillioides Seberang Prai, Pulau Pinang KP340015 2 NF 1.8432 P175c F. verticillioides Seberang Prai, Pulau Pinang KP340004 1 Barren 3.6733 P179c F. verticillioides Seberang Prai, Pulau Pinang KP340008 2 Barren 3.3434 P180c F. verticillioides Seberang Prai, Pulau Pinang KP340007 2 Barren 1.5035 P183c F. verticillioides Seberang Prai, Pulau Pinang KP340040 1 Barren 1.6736 P191c F. proliferatum Seberang Prai, Pulau Pinang KP340031 1 Barren 1.6737 P197c F. verticillioides Seberang Prai, Pulau Pinang KP340005 2 Barren 4.0038 P201c F. verticillioides Seberang Prai, Pulau Pinang KP340016 2 Barren 2.8439 P202c F. proliferatum Seberang Prai, Pulau Pinang KP340043 1 Barren 3.5040 P204c F. verticillioides Seberang Prai, Pulau Pinang KP340042 1 Barren 4.50

aBarren: numerous perithecia, but no ascospore cirrhi; NF: Nonfertile. bDSI: All inoculated corn were significantly different from the control.

MOHD ZAINUDIN et al. / Turk J Biol

225

maximum-likelihood method as shown in Figure 2. The phylogenetic tree can be divided into two main clades, which are clade I and II. Clade I comprises subclades A

and B. Isolates of F. verticillioides were grouped together in subclade A while the isolates of F. proliferatum were grouped in subclade B. Isolate B1365 (F. solani) in this

Figure 2. Maximum-likelihood tree showing the relationship of 41 isolates of Fusarium species isolated from Fusarium ear rot of corn inferred from the sequence of tef-1α. The tree was generated using the Tamura–Nei method with 1000 replicates and bootstrap values of more than 60% are shown next to the branches. Trichoderma harzianum DUCC 7302 is the outgroup.

MOHD ZAINUDIN et al. / Turk J Biol

226

study was used as a Fusarium control as the species does not belong to Section Liseola and it formed a distinct clade (clade II) that was clearly separated from other isolates from GenBank (F. verticillioides isolate K311, F. proliferatum isolate 3069). All isolates of F. verticillioides and F. proliferatum formed separate subclades in the tree. In this study, sequence variations were not observed among isolates of F. verticillioides, whereas sequence variations were detected among the isolates of F. proliferatum B52c and B57c from other isolates and these isolates were grouped into separate branches. Trichoderma harzianum DUCC 7302, which was used as the outgroup isolate, was also grouped in a separate clade. 3.3. Mating type and crosses analysisAll 15 isolates of F. proliferatum and 25 isolates of F. verticillioides were assigned to different mating population groups known as MP-D and MP-A, respectively. The isolates of F. proliferatum were crossed with standard tester isolates D-04854 (MATD-1) and D-04853 (MATD-2), while F. verticillioides was crossed with the standard isolates A-00149 (MATA-1) and A-00999 (MATA-2) based on their MAT allele types. Among the 15 isolates from MP-D tested, only four isolates were crossed-fertile along with only six others producing barren perithecia without the presence of asci and ascospores (Table; Figure 3). The remaining five isolates were recorded as nonfertile when crossed with standard tester isolates. There were 25 isolates recorded for MP-A with 18 of them producing barren perithecia, while the remaining seven isolates were nonfertile (Table). The barren perithecia are a case when perithecia were produced without the existence of asci and ascospores.3.4. Pathogenicity testTo assure that the Fusarium isolates were pathogenic to corn and caused the ear rot disease, a pathogenicity test was conducted. All 40 isolates were identified as pathogenic and caused ear rot symptoms with a significantly different DSI from the control (P ≤ 0.05). Isolates F. proliferatum B68c and F. verticillioides C116c and J1364c and P204c demonstrated the highest DSI scores of 4.67 and 4.50, respectively, as shown in the Table. Some isolates of Fusarium exhibited low susceptibility to infection with low DSI values: F. proliferatum B52c and F. verticillioides P108c with DSI of 1.50. All treatments were found significantly different (P ≤ 0.05) from the control. Hence, they were confirmed as pathogenic to corn. After 7 days of artificial inoculation, no symptom was detected on control corn ears, neither on C1 nor C2. In contrast, the ears inoculated with all isolates of F. proliferatum and F. verticillioides showed typical symptom of ear rot disease. The ears inoculated with F. proliferatum and F. verticillioides showed tan4 color of infected kernels with decay (Figure 4). There were also visible mycelial growths observed at the infected

area. Since the conidial suspension was inoculated to the ear of corn, the symptoms of fungal colonization only emerged at the inoculated region and did not spread to the entire ear. The infected ears appeared in a group of kernels. However, the symptoms spread outward to the husk in some treatments, but were nonvisible on the other parts of corn plants. Mycelial growth was observed on all infected kernels. The growth of corn plants after inoculation of the pathogen was normal. There was no insect damage on the inoculated corn ears.

4. DiscussionFusarium verticillioides especially is known as a strong producer of mycotoxin fumonisins that can also be produced by F. proliferatum. These toxins are suspected to be carcinogenic for humans and are implicated in a number of animal diseases (Rahjoo et al., 2008). In fact, according to Leslie and Summerell (2006), fumonisins are phytotoxic, but their role in plant diseases caused by F. verticillioides has yet to be clearly defined. Corn and corn-related products are usually at the highest risk for contamination. Based on the work of Shim et al. (2003), toxin production is higher in damaged corn kernels than corn germ tissues. Fumonisin is sufficiently thermostable; thus, the compound cannot be destroyed during cooking or the processing of corn grain. F. verticillioides can also produce fusaric acid and various derivatives and trace levels of beauvericin. The isolates were also characterized based on the tef1-α gene sequence because morphological characteristics could easily result in misidentification. According to Rahjoo et al. (2008), it is difficult to differentiate species within the Gibberella fujikuroi species complex using morphological characteristics. Identification using morphological characters is the initial instrument crucial to sorting the species into smaller groups before applying other methods of identification.

There are limitations found when utilizing morphological characters for identifying the G. fujikuroi species complex as some species may share similar morphological characteristics, such as F. proliferatum and F. verticillioides. Based on morphological characteristics, F. proliferatum is most likely to be confused with F. verticillioides where both can be found in chains of varying length (Leslie and Summerell, 2006). Moreover, relying on the presence of polyphialides alone is not rigid evidence to differentiate F. proliferatum and F. verticillioides. Misidentification between these two species of Fusarium often occurs since they have similar pigmentation, shape of macroconidia and microconidia, and formation of false heads. Thus, the molecular method, which is used to differentiate DNA sequences of genes, has been used to support morphological identification of F. proliferatum and F. verticillioides as well as other Fusarium species.

MOHD ZAINUDIN et al. / Turk J Biol

227

Figure 3. Sexual development of F. proliferatum (MP-D). Isolate B66c (MATD-1) × D24853 (MATD-2) (a, c, e, g), and isolate B68c (MATD-1) × D24853 (MATD-2) (b, d, f, h). (a, b) cross plates; (c, d) perithecia on plate; (e, f) ascospores in asci; (g, h) ascospores.

a

c

e

g

b

d

f

h

MOHD ZAINUDIN et al. / Turk J Biol

228

In this study, the tef1-α gene is the marker used to describe Fusarium species isolated from Fusarium ear rot-infected corn samples, which were identified as F. verticillioides, F. proliferatum, and F. solani according to the closest matches

in BLAST searches and the FUSARIUM-ID server. Based on the phylogenetic tree obtained, isolates of F. verticillioides and F. proliferatum formed separate subclades in their tree but in the same main clade I, which further suggests that

Figure 4. Pathogenicity phenotypes of F. proliferatum and F. verticillioides isolates producing disease symptoms of tan lesion with fungal mass on the kernels after 7 days of inoculation (circle). (a) Control corn with no inoculation after 7 days of incubation has showed no symptoms of Fusarium ear rot; (b–d) low, medium, and high severity of F. proliferatum isolates P191c, P177c, and B56c; (e–g) low, medium, and high severity after inoculation with F. verticillioides isolates B44c, B55c, and C116c, respectively.

MOHD ZAINUDIN et al. / Turk J Biol

229

F. verticillioides and F. proliferatum are closely related. The bootstrap value was 68% between subclades A and B. In subclade A, the isolates gave the same gene sequence for each other and the control isolates of F. verticillioides K311 (Accession No. KF562131) taken from the NCBI. This gives an idea that all isolates belonging in subclade A and phylogenetically closely related to F. verticillioides K311 were isolated from corn that originated from Malaysia. Subclade A was well supported by a bootstrap value of 86% in this analysis. In subclade B, the isolates also gave the same gene sequence for each other and the referred F. proliferatum isolate 3069 (Accession No. KJ024979) from NCBI. Furthermore, subclade B was well supported by a bootstrap value of 99% in this analysis. The isolate of F. solani is in a distinct clade, indicating that F. solani is not related with F. verticillioides and F. proliferatum. The main clade II show 100% similarity between the F. solani isolates and the F. solani from GenBank (Accession No. DQ247444) as there may be F. solani isolates sharing the same control isolates from GenBank. Trichoderma harzianum DUCC 7302 was used as an outgroup since an outgroup is essential in determining which traits of the clade are derived from it or ancestral. Phylogenetic analysis based on the tef1-α gene clearly grouped the different species into separate groups with high bootstrap values. The grouping of F. proliferatum and F. verticillioides in a single cluster shows that they are closely related and are in accordance with the biogeographic hypothesis formulated by O’Donnell et al. (1998).

F. proliferatum and F. verticillioides were confirmed as pathogens of Fusarium ear rot disease and this finding is consistent with numerous previous reports. Although both mating types are found in this population, none of the F. proliferatum or F. verticillioides is female-fertile when crossed under laboratory conditions. This phenomenon was probably caused by some populations that reproduced asexually, as reported by Mohamed Nor (2014) for F. fujikuroi. Nonetheless, it could also occur to other Malaysian isolates of Fusarium species although the hosts are different.

In conclusion, the isolates of F. proliferatum and F. verticillioides from Fusarium ear rot in Malaysia have been discovered to be genetically similar regardless of their geographical origins. Both MAT1-1 and MAT1-2 were present in the field, while most of the isolates produced barren perithecia with low fertility. All isolates of F. proliferatum and F. verticillioides were noted to be pathogenic towards corn and the study of disease control is crucial since both pathogens are able to produce harmful mycotoxins.

AcknowledgmentsThis study was partially supported by a Fundamental Research Grant Scheme (grant project no. 01-11-08-661) to Nur Ain Izzati Mohd Zainudin. Nor Azizah Kusai and Suhaida Salleh were given a scholarship from the Graduate Research Fund of UPM.

References

Bottalico A (1998). Fusarium diseases of cereals: species complex and related mycotoxin profiles, in Europe. J Plant Pathol 80: 85-103.

Ding JQ, Wang XM, Chander S, Yan JB, Li JS (2008). QTL mapping of resistance to Fusarium ear rot using a RIL population in maize. Mol Breeding 22: 395-403.

Duncan KE, Howard RJ (2010). Biology of maize kernel infection by Fusarium verticillioides. Mol Plant Microbe In 23: 6-16.

Elmer WH (2002). Influence of inoculum density of Fusarium oxysporum f. sp. cyclaminis and sodium chloride on cyclamen and development of Fusarium wilt. Plant Dis. 86: 389-393.

Geiser DM, Jimenz Gasco MM, Kang S, Mkalowska I, Veeraraghavan N, Ward TJ, Zhang N, Kuldau GA (2004). FUSARIUM-ID v. 1.0: a DNA sequence database for identifying Fusarium. Eur J Plant Pathol 110: 473-479.

Glynn EF (2005). Color Chart. Kansas City, MO, USA: Stowers Institute for Medical Research.

Leslie JF, Summerell BA (2006). The Fusarium Laboratory Manual. Oxford, UK: Blackwell Publishing.

Logrieco A, Moretti A, Perrone G, Mulè G (2007). Biodiversity of complexes of mycotoxigenic fungal species associated with Fusarium ear rot of maize and Aspergillus rot of grape. Int J Food Microbial 1-2: 11-16.

Lori GA, Wolcan SM, Larran S (2008). Fusarium yellows of celery caused by Fusarium oxysporum f. sp. apii in Argentina. J Plant Pathol 90: 173-178.

Mohamed Nor NMI (2014). Genetics of the Southeast Asian populations and interspecific hybrids of Fusarium spp. PhD, Kansas State University, Manhattan, KS, USA.

Munkvold GP (2003). Epidemiology of Fusarium diseases and their mycotoxins in maize ears. Eur J Plant Pathol 109: 705-713.

Nitschke E, Nihlgard M, Varrelmann M (2009). Differentiation of eleven Fusarium spp. isolated from sugar beet, using restriction fragment analysis of a polymerase chain reaction–amplified translation elongation factor 1α gene fragment. Phytopathology 99: 921-929.

O’Donnell K, Cigelnik E (1997). Two divergent intragenomic rDNA ITS2 types within a monophyletic lineage of the fungus Fusarium are nonorthologous. Mol Phylogenet Evol 7: 103-116.

O’Donnell K, Cigelnik E, Nirenberg HI (1998). Molecular systematics and phylogeography of the Gibberella fujikuroi species complex. Mycologia 90: 465-493.

MOHD ZAINUDIN et al. / Turk J Biol

230

Parsons MW, Munkvold GP (2012). Effects of planting date and environmental factors on Fusarium ear rot symptoms and fumonisin B1 accumulation in maize grown in six North American locations. Plant Pathol 61: 1130-1142.

Rahjoo V, Zad J, Javan-Nikkhah M, Mirzadi Gohari A, Okhovvat SM, Bihamta MR (2008). Morphological and molecular identification of Fusarium isolated from maize ears in Iran. J Plant Pathol 90: 463-468.

Shim WB, Flaherty JE, Woloshuk CP (2003). Comparison of fumonisin B1 biosynthesis in maize germ and degermed kernels by Fusarium verticillioides. J Food Protect 66: 2116-2122.

Siti Nordahliawate MS, Nur Ain Izzati MZ, Azmi AR, Salleh B (2008). Distribution, morphological characterization and pathogenicity of Fusarium sacchari associated with Pokkah boeng disease of sugarcane in Peninsular Malaysia. Pertanika J Trop Agric Sci 31: 279-286.

Srobarova A, Moretti A, Ferracane R, Ritieni A, Logrieco A (2002). Toxigenic Fusarium species of Liseola section in pre-harvest ear rot, and associated mycotoxins in Slovakia. Eur J Plant Pathol 108: 299-306.

Steenkamp ET, Wingfield BD, Coutinho TA, Zeller KA, Wingfield MJ, Marasas WFO, Leslie JF (2000). PCR-based identification of MAT-1 and MAT-2 in the Gibberella fujikuroi species complex. Appl Environ Microbiol 66: 4378-4382.

Suhaida S, Nur Ain Izzati MZ (2013). The efficacy of Trichoderma harzianum T73s as a biocontrol agent of Fusarium ear rot disease of maize. Int J Agr Biol 15: 1175-1180.

Tamura K, Dudley J, Nei M, Kumar S (2007). MEGA6: Molecular evolutionary genetics analysis (MEGA) software. Mol Biol Evol 24: 1596-1599.