Detection of the fumonisin-producing Fusarium fujikuroi species complex (FFSC) associated with wild grasses in Iran

INTRODUCTION

Poaceae is a large family of monocotyledonous flowering plants known as grasses. The Poaceae are among the most economically important plant families owing to their usages in food production, industry, and lawns (Inch & Gilbert 2003). Since the 1950s, grasses have been considered as the main source of food for domesticated animals in Iran. Northern and western parts of Iran are the main growth areas of rangeland plants in Iran (Parsa 1950).

Mycotoxigenic fungi which are natural contaminants of cereals worldwide (Goswami & Kistler 2004, 2005) have become also threatening for grass plants through several genera including Fusarium spp., Ustilago spp., and Aspergillus spp. (Inch & Gilbert 2003; Postic et al. 2012).

Fusarium ear rots are the most commonly and widely studied diseases of the Poaceae(Akinsanmi et al. 2003; Boutigny et al. 2011; Postic et al. 2012). At least 18 species of Fusarium Linkhave been assigned to Fusarium ear rots (Bottalico and Perrone, 2002). Among these, members of the Fusarium fujikuroi species complex (formerly the Gibberella fujikuroi species complex) are the most important and prevalent species, producing different mycotoxins includingmoniliformin (MON), fumonisins (FUMB), and beauvericin (BEA) (Logrieco et al. 1995, 2002).

The Fusarium mycotoxin contamination of grasses is a potential health hazard for animals grazing these plants (Desjardins & Proctor 2011; Postic et al. 2012). The mycotoxin–production ability of Fusarium spp. is diverse and particular strains may produce different mycotoxins (Goswami & Kistler 2005). Fumonisins are one of the most important carcinogenic mycotoxins that are mainly produced by F. proliferatum (Matsush.) Nirenberg ex Gerlach & Nirenberg and F. verticillioides (Sacc.) Nirenberg (Logrieco et al. 1995, 2002). More than 25 structurally related fumonisin analogues have been identified, with fumonisin B1 (FB1) as the most prevalent one (Logrieco et al. 1995; Desjardins 2006). So, rapid and accurate identificationof Fusarium spp, as well as detection of their mycotoxigenic properties is vital to reduce their harmful effects (Eskola et al. 2001). The aims of this study were to (i) identify the members of the FFSC associated with grass plants in Iran using morphological and molecular approaches; (ii) determine the genetic potential of FFSC isolates to produce fumonisin.

MATERIALS AND METHODS

Sample collection, isolation, and identification of Fusarium isolates

A survey was carried out in five agro–ecological zones of Kermanshah province of Iran, including Sarpol–e Zahab, Mahidasht, Eslamabad–e Gharb, Bisotun, and Kermanshah districts. In each agro–ecological zone, 12 farms were randomly selected and the infected grassy plants containing root, spike, and crown rot symptoms were collected. To identify the members of Poaceae, plants were transferred to the herbarium of Razi University. To isolate Fusarium isolates, the infected roots, crown, and head were cut into small pieces, surface sterilized with 96% ethanol for 30 sec followed with a fine rinse and subsequently plated on water agar (WA) (1.5%). The resulting single–spore Fusarium colonies were transferred to fresh potato dextrose agar (PDA) plates and maintained at 4^°C for further studies. To study the growth rates and pigment production of Fusarium spp. all the isolates were transferred onto PDA plates and incubated at ambient temperature. For microscopic observations, all the isolates of Fusarium were transferred to carnation leaf agar (CLA) (Fisher et al. 1982) media. Fusarium species were identified based on morphological characters according to the Fusarium laboratory manual (Leslie & Summerell 2006).

Molecular methods

Genomic DNA of the FFSCisolates was extracted from 10 mg of freeze–dried mycelia using DNeasy® Plant Mini Kit (Qiagen) according to the manufacturer's protocol. Polymerase chain reaction (PCR) was performed according to Mulé et al. (2004). VER1/VER2, PRO1/PRO2, and SUB1/SUB2 (Mulé et al. 2004) were the primers specifically used to identify F. verticillioides, F. proliferatum, and F. subglutinans isolates (Wollenw. & Reinking) P.E. Nelson, Toussoun & Marasas, respectively (Table 1). Amplification reactions were performed according to Mulé et al. (2004). The polymerase chain reaction was performed at 95 ^°C (5 min) for hot start, followed by 35 cycles of 94 ^°C for 50 sec, 56 ^°C (50 sec.), 56 ^°C (50 sec.) and a final extension of 72 ^°C (1 min). FUM1 F/FUM1 R set of primers (Bluhm et al.2004, (Table 1) was also used to determine the fumonisin–producing isolates of FFSC, examined in this study. The PCR program for amplification of FUM1 F/FUM1 R was as described by Bluhm et al. (2004). The PCR products were visualized in 1% agarose gel electrophoresis.

Chemical detection of mycotoxins

From selected samples, potential mycotoxigenic Fusarium isolates were chemically analysed to determine the concentration of B1 and B2 fumonisins. Detection of mycotoxins was carried out using high performance liquid chromatography (HPLC) assay, according to Chehri et al. (2010).

RESULTS

Seventy–eight samples with mostly spike and crown rot symptoms were collected from different sites of Kermanshah province. Nine species from nine tribes of the Poaceae, including Avenawiestii, Bromussericeus, Dactylisglumerata, Aegilopscylindrical, Stipabarbata, Loliumperenne, MelicaJacquemontii, Agropyronrepens, and Ermopoapersica were identified based on pollen morphology using light microscopy (Table 2).

Based on morphological features, 22 isolates belonging to FFSC were recovered from infected tissues of grassy plants. These isolates were mainly identified as F. proliferatum (10), F. verticillioides (7)and F. subglutinans (5) (Table 2).

Table 1. Primers used for detection of the fumonisin–producing Fusarium fujikuroi species complex members.

* SUB: For Fusarium subglutinans; VER: For F. verticilioides; PRO: For F. prolofiratum; FUM: For Fumonisin

Table 2. Sampled regions in association with the FFSC isolates recovered from roots, crown, and inflorescences of wild grasses in Kermanshah province, Iran.

a = F. verticillioides, b = F. subglutinans, c = F. proliferatum

The macroscopic and microscopic characteristics were applied to identify Fusarium species. Ten isolates of F. proliferatum were characterized by the production of abundant aerial mycelium. All isolates produced slightly straight macroconidia with 3–5–septate. The conidiogenous cells producer false head and chain microconidia were monophialides and polyphialides.

Five isolates of F. subglutinans were characterized by the production of purple to dark purple cottony. All isolates produced slender and falcate macroconidia with 3–septate. The conidiogenous cells producer false head microconidia were monophialides and polyphialides. Also, based on morphological features, seven isolates were belonging to F. verticillioides. The colony of all isolates produced white, light purple to dark violet pigmentations. The macroconidia were commonly slender and almost straight with 3–5 septate, mostly 3 septate, with curved apical cell and foot shaped basal cell. The conidiogenous cells producer with false head and chain microconidia were monophialides.

All isolates of F. verticillioides and F. subglutinans were obtained from infected spikes, whereas five strains of F. proliferatum were isolated from infected spikes and the seven from infected root and crown. Members of FFSC were also molecularly distinguished using specific primers. The primers PRO1/2, VER1/2, and SUB1/2 produced DNA fragments of 585, 578, and 631 bp in all isolates of F. proliferatum, F. verticillioides, and F. subglutinans, respectively (Fig. 1–3).

PCR assays were also used to identify the chemotypes of all FFSC isolates. The expected PCR product for FUM1 (183 bp) was amplified in all PCR reactions. Eleven isolates belonging to F. proliferatum (seven isolates), F. verticillioides (three isolates), and F. subglutinans (one isolates), isolated from infected spike samples were identified as the potential fumonisin producers (Fig. 4). The highest amount of fumonisin producing isolates was observed in Sarpol–e Zahab (25%) and Bisotun (25%) regions of Kermanshah province (Table 2). Frequencies of the potential mycotoxigenic isolates are given in Table 2. The HPLC analysis of infected spike samples gave no positive results and fumonisin B1 and B2 were not detected.