Abstract: Fusarium is one of the most important pathogenic and toxigenic fungi widely distributed all over the world, including Iran. Fusarium species are found frequently in stored agriculture products especially wheat. The objective of this study was to identify Fusarium species associated with stored wheat seeds and their pathogenicity on root and head of wheat in Kermanshah, the leading province in wheat production in Iran. In this survey 75 seed samples of stored wheat were collected from 10 different regions during 2006-2008 and tested for the presence of Fusarium. Fusarium spp. were found in 51 (68 %) of 75 samples. A total of 580 Fusarium strains were isolated, identified and preserved. All these strains belong to 20 Fusarium spp. according to morphological characters. Each conidial suspension of selected strains representing all species was evaluated for their pathogenicity on roots and spikes of healthy wheat var. Fallat in the greenhouse. F. graminearum, F. crookwellense, F. trichothecioides, F. culmorum and F. verticillioides were the most pathogenic to wheat’s head. Foot rot assessment revealed that F. pseudograminearum and F. culmorum were the most damaging species. Of the Fusarium isolates, F. graminearum was the most prevalent followed by F. verticillioides and F. proliferatum. This is the first comprehensive report on identity and distribution of Fusarium spp. from stored wheat seeds in Iran while F. nelsonii was reported for the first time from wheat seeds in Iran.
INTRODUCTION
Wheat is the world’s most widely grown cereal crop and is a food staple for most of the World. Wheat provides approximately 20% of food calorie in the world (Kachuei et al., 2009) and is the most important daily food in Iran like many countries in the world and is the main cereal for human consumption in Iran (Farshadfar et al., 2008). Fusarium represents one of the major fungal genera, which are ubiquitous in distribution and found frequently in stored agriculture commodities such as cereals (Kachuei et al., 2009; Yli-Mattila, 2010). Fusarium species is responsible for many economical important plant diseases such as root rot, crown rot, vascular wilts, canker and head blight on cereals (Leslie and Summerell, 2006). Also, they can cause root, stem and ear rot, resulting in severe reductions in small-grain cereals yield, often estimated at between 10 -40% (Bottalico and Perrone, 2002). Fusarium spp. can also produce fumonisins and trichothecenes in cereals which are detrimental to animal and human health (Gamanya and Sibanda, 2001). Fumonisins are a group of structurally related toxic compounds produced by Fusarium verticillioides (formerly known as F. moniliforme) and related species. Additionally, F. proliferatum and F. nygamai have all been reported to produce fumonisins (Yli-Mattila, 2010). Fumonisin B1 is known to be produced in wheat and hence occurs in human foodstuffs and animal feedstuffs (Shephard et al., 2005). There is more information concerning Fusarium species associated with wheat on the field and stored wheat both on the international and national levels (Bottalico and Perrone 2002; Torp and Nirenberg, 2004; Darvishnia et al., 2006; Kachuei et al. 2009; Yli-Mattila, 2010) but there are few reports concerning Fusarium species associated with wheat in store compared with those in the field. The occurrence Fusarium species and the production of fumonisins in crops and their products are partly determined by environmental factors in the field, during transportation and storage. Kermanshah Province with having different climatic regions is capable in keeping the most of Fusarium species in their different fields, so, it is necessary to isolate and identify the Fusarium spp. colonizing the stored wheat seeds. The current study has been performed to pursue the following goals: (1) to determine the contaminating Fusarium species in stored wheat located in ten main wheat producing and storage regions in Kermanshah province and (2) to check the pathogenicity of Fusarium species on root and head of wheat in Kermanshah province of Iran.
MATERIALS AND METHODS
Collection of seed samples and isolation of Fusarium spp.: Seventy five stored wheat seed samples were collected from 10 different climatic regions in Kermanshah, Iran during 2006-2008 (Table 1, 2). The isolation frequency (Fr) and the relative density (RD) Fusarium species were calculated according to Gonzalez et al. (2008). The seed samples were surface sterilized with 1% sodium hypochlorite solution for 3 min, rinsed with several changes of sterilized distilled water and dried on sterile filter papers. From each sample, 400 grains were placed on pentachloronitrobenzene agar (PPA) (Nash and Snyder, 1962) and Water Agar (WA) plates and incubated under the standard incubation conditions (Salleh and Sulaiman, 1984) for 48 h. The resulting single-spored Fusarium colonies were kept in 15% glycerol and stored at -80°C for further studies.
Identification of Fusarium spp.: To study the growth rates and pigment production of Fusarium spp., all strains were ransferred onto Potato Dextrose Agar (PDA) plates and incubated as above. For microscopic observations, all the strains of Fusarium were transferred to Carnation Leaf Agar (CLA) (Fisher et al., 1982), Spezieller Nahrstoffarmer Agar (SNA) (Nirenberg, 1976) and Potassium Chloride Agar (KClA) (Fisher et al., 1983) plates. The species were identified on the basis of macroscopic characteristics such as pigmentations and growth rates on PDA plates, as well as their microscopic features including size of macroconidia, presence of microconidia and its production in chains or false heads, type of conidiogenous cells (monophialidic and polyplialidic conidiophores) and also absence or presence of chlamydospores (Gerlach and Nirenberg, 1982). Identification of species was based on species description of Gerlach and Nirenberg (1982), Nelson et al. (1983), Marasas et al. (1998) and Leslie and Summerell (2006).
Pathogenicity assay on root and foot of wheat: All of the identified Fusarium species were tested for their pathogenicity on apparently healthy wheat seedlings variety Fallat. The inoculation was carried out on root. Prior to inoculation, a maximum of 10 seedlings were washed before inoculation. For inoculation, six isolates of each identified Fusarium species were grown on PDA plates and conidial suspension of each individual strain was prepared by scrapping the mycelium with sterile distilled water onto 7 day-old cultures, shaken thoroughly and the concentration was adjusted to 2×106 conidia mL-1 using a haemocytometer.
| Table 1: | Climatic data for each sampling region of the Kermanshah province |
| Table 2: | Fusarium spp. identified on wheat samples |
F.an=F. anthophillum, F.av=F. avenaceum, F.ch=F. chlamydosporum,
F.co=F. compactum, F.cu=F. culmorum, F.eq=F. equiseti, F.gr=F. graminearum,
F.ny=F. nygamai, F.ps=F. pseudograminearum, F.pr=F. proliferatum, F.ne=F.
nelsonii, F.sa=F. sambucinum, F.sc=F. scripi, F.so=F. solani, F.sp=F. sporotrichioides,
F.se=F. semitectum, F.tr=F. trichothecioides, F.ca=F. camptoceras, F.ve=F.
verticillioides, F. crookwellense=F.cr |
|
Fourteen days after planting, 20 mL of the spore suspension of six isolates of each Fusarium species was sprayed on the root of the plants. After the inoculum, plants were transferred to the greenhouse bench. The control plants were sprayed with 20 mL of sterile distilled water. Three replications were maintained for each strain and the experiment was repeated twice. Disease percentages of infected roots from all plants in each pot were averaged and the means per pot of percent infected roots were used in the statistical analysis. The inoculated fungi were re-isolated from the infected plants to prove the Koch’s postulates.
Pathogenicity assay on wheat spikes: For Pathogenicity assay was used of Xue et al. (2006). All of the identified Fusarium species were tested for their pathogenicity on apparently healthy wheat (Fallat variety) and individual plants were used for inoculation tests on head. The healthy wheat’s (Fallat variety) were inoculated 10-14 days after heading. Prior to inoculation, a maximum of 10 spikes per pot were randomly selected, while the remainder and those from lateral tillers were removed. For inoculation, six isolates of each identified Fusarium species were grown on PDA plates and conidial suspension of each individual strain was prepared by scrapping the mycelium with sterile distilled water onto 7 day-old cultures, shaken thoroughly and the concentration was adjusted to 1×106 conidia mL-1 using a haemocytometer. Plants were sprayed with the spore suspension at 0.2 mL per spike. After the inoculum, plants were transferred to humidity chamber in a growth chamber for 48 h. The growth chamber was operated at 25°C with a 12 h photoperiod and the humidity chamber was maintained at or near 100% RH by the continuous operation of two ultrasonic humidifiers. After incubation, plants were returned to the greenhouse bench. The negative control, plants were sprayed with 0.2 mL sterile distilled water per spike and for positive control, plants were sprayed with 0.2 mL of aggressive F. graminearum isolate. Symptoms of Fusarium head blight (FHB) were rated as disease severity at 4, 7, 14, 21 and 28 days after inoculation and as percentage of Infected Spikelet’s (IS) after 25 days, when plants were at the soft dough stage (Xue et al., 2006). Disease severity was estimated visually in situ for each inoculated spike on a 0 (no visible FHB symptoms) to 9 (severely diseased, spike dead) scale described by Xue et al. (2004). Disease severities and percentages of IS from all plants in each pot were averaged and the means per pot of percent IS were used in the statistical analysis.
RESULTS
Seventy five wheat samples were collected and analyzed for the occurrence of Fusarium spp. Fifty one samples out of 75 were found positive for Fusarium species. All samples were collected from Gilan Gharb positive for Fusarium incidence (Table 2). A total of 580 isolates of Fusarium species were isolated and identified through morphological characters. According to morphological characters all these strains are belonging to 20 Fusarium spp. including: F. crookwellense, F. culmorum, F. trichothecioides, F. graminearum, F. sambucinum, F. scirpi, F. equiseti, F. compactum, F. avenaceum, F. chlamydosporum, F. sporotrichioides, F. semitectum, F. nygamai, F. solani, F. camptoceras, F. nelsonii, F. anthophilum, F. proliferatum, F. pseudograminearum and F. verticillioides (Table 2, 3 and Fig. 1, 2) that were preserved in the Fusarium Culture Collection Unit, Universiti Sains Malaysia (USM). In this survey F. nelsonii is reported for the first time from Iran. All of the F. nelsonii isolates were identified according with Marasas et al. (1998) description. F. nelsonii is similar to F. semitectum and F. camptoceras (Section Arthrosporiella) but F. nelsonii produce straight or curved and falcate macroconidia with usually 3-septate that can be distinguished from F. camptoceras and F. semitectum on the basis of its carmine red pigmentation in PDA and short macroconidia (Table 3, Fig. 1, 2). Of the Fusarium isolates collected in Kermanshah province, Iran, F. graminearum was the most prevalent with relative density of 20%, followed by F. verticillioides and F. proliferatum with relative density of 12 and 10%, respectively (Table 4).
Ten days after inoculation, disease symptoms were observed on the roots. Initially, the roots showed brownish discoloration and eventually turned to dark color which indicates rotting of the root tissues. The results in pathogenicity tests indicated that F. pseudograminearum had the greatest infected roots (75%), followed by F. culmorum (50%) F. verticillioides (12%), F. graminearum (12%) and F. proliferatum (12%) are the most pathogenic on the roots. Fusarium avenaceum, F. crookwellense and F. trichothecioides resulted in 6% infected roots, which were significantly lower than those of the five highly pathogenic species and therefore were moderately pathogenic. The remaining species did not cause any symptoms (Table 4).
The eight Fusarium spp. were different in the rate of FHB symptom appeared on tested plant four days after inoculation with isolates of F. graminearum, F. crookwellense, F. trichothecioides, F. culmorum, F. chlamydosporum, F. culmorum, F. avenaceum, F. verticillioides and F. sporotrichioides and 10-21 days after inoculation with isolates of the remaining species.
| Fig. 1: | Coniphore shapes of all Fusarium spp. (a) F. crookwellense,
(b) F. culmorum, (c) F. trichothecioides, (d) F. graminearum,
(e) F. sambucinum, (f-g) F. scirpi, (h) F. equiseti,
(i) F. compactum, (j) F. avenaceum, (k) F. chlamydosporum,
(l) F. sporotrichioides, (m) F. semitectum, (n)
F. nygamai, (o) F. solani, (p) F. nelsonii, (q)
F. camptoceras, (r) F. pseudograminearum, (s) F. anthophilum,
(t) F. proliferatum and (u-v) F. verticillioides |
| Fig. 2: | Macroconidial shapes and sizes of Fusarium spp. (a)
F. crookwellense, (b) F. culmorum, (c) F. trichothecioides,
(d) F. graminearum, (e) F. sambucinum, (f) F. scirpi,
(g) F. equiseti, (h) F. compactum, (i) F. avenaceum,
(j) F. chlamydosporum, (k) F. sporotrichioides, (l)
F. pseudograminearum, (m) F. semitectum, (n) F. nygamai,
(o) F. solani, (p) F. nelsonii, (q) F. camptoceras,
(r) r=F. anthophilum, (s) F. proliferatum and (t) F.
verticillioides (Scale bars = 20 μ |
| Table 3: | Morphological characters of Fusarium spp. associated with wheat seeds |
| +: Presence, -: Absence, Ch: Chlamydospore, Poly: Polyphialidic, Mono: Monophialidic, Pdfs: Poorly developed foot shape, Nfs: Notch or foot shape, Fs: Foot shape | |
| Table 4: | Percentage of infected spikelets, roots and relative density of different Fusarium species isolated from stored wheat samples in Kermanshah province, Iran |
Infected spikelets first were appeared water-soaked, lose their chlorophyll, Pinkish-red mycelium and conidia developed abundantly in the infected spikelets and the infection spreads to adjacent spikelets or through the entire head. On average of the six isolates of the eight Fusarium spp., F. graminearum had the greatest IS (66%), followed by F. crookwellense (54%), F. trichothecioides (45%), F. chlamydosporum (45%), F. culmorum (45%), F. avenaceum (36%), F. verticillioides (24%) and F. sporotrichioides (24%). Therefore, these eight species were considered highly to moderately pathogenic. The remaining species had <12% IS and were weakly pathogenic and disease progressed slowly and lower severities were generally observed for F. proliferatum, F. compatum, F. equiseti and F. nygamai. Disease reached maximum severity 28 days after inoculation, when plants were at or near maturity.
DISCUSSION
Several studies have shown that Fusarium spp. can be readily isolated from cereal grains and according this subject, results obtained in this study were in agreement with the previous findings (Nicholson et al., 2003; Snijders, 1990; Dawson et al., 2004). Fusarium graminearum is one of the most frequently found Fusarium species on European cereals where it is more common in wet and warm climate of central and Southern Europe (European Commission, 2003). Common species in cooler regions are F. culmorum and F. avenaceum (Lemmensa et al., 1993; Wong et al., 1992; Wilcoxon et al., 1988, Yli-Mattila et al., 2004). In this study, we also identified F. graminearum isolates in all of the regions of Kermanshah Province especially in warm regions and F. culmorum and F. avenaceum isolates are only on moderate to cold regions of Kermanshah province (Sahneh, Bisotun, Sonqor and Kangavar) (Table 2). Also, F. camptoceras has been isolated from stored wheat samples in warm site (Qasr Shirin), so indicate an agreement with the previous literatures (Marasas et al., 1998).
In this survey we identified different species within Section Discolor including: F. crookwellense, F. culmorum, F. trichothecioides, F. graminearum, F. sambucinum and F. pseudograminearum. Results indicated that all of these species expect for F. sambucinum are responsible in root rot and FHB (Table 4). This subject has been confirmed by other researchers (Bottalico and Perrone, 2002). The occurrence of mycotoxins produced by species Section Discolor in small cereal grains, particularly in wheat, is of great concern worldwide, because their presence in processed feeds and foods seems unavoidable. Consequently, they have been associated with chronic or acute mycotoxicoses in livestock and, to a lesser extent, in humans (Bottalico and Perrone, 2002; IARC, 1993).
The fungal isolation assays made on wheat grain samples collected throughout the Kermanshah province clearly indicate that F. graminearum is the primary pathogen causing Fusarium head blight. From 2007 to 2008 F. graminearum comprised nearly 20% of all Fusarium species isolated from infected grains. Similar results were found by other researchers (Wilcoxon et al., 1988; Gilbert et al., 1995). In this survey the member of Fusarium Section Liseola especially F. proliferatum (10%) and F. verticillioides (12%) also comprised the most frequencies after F. graminearum especially in moderate to warm area in this survey (Table 2 and 4) that were very important in pathogenicity assay. F. verticillioides and F. proliferatum appeared FHB symptoms 4 and 14 days, respectively after inoculation that indicated these species are very important in FHB. Besides of FHB, these species are responsible for root rot disease and causing seedling blight. This results is agreement with other international studies in the USA, Canada, Argentina (Walker et al., 2001; Gonzalez et al., 2008) and Europe (Bottalico and Perrone, 2002; Torp and Nirenberg, 2004). Furthermore, ability of these two species in synthesize mycotoxins has been illustrated by Bottalico (1998).
The recoveries of Fusarium species from the 2008 samples were extremely low and high in 2006 and 2007 samples because of heavy rains. In 2008 there was little rain, whereas in 2006 and 2007, there was more raining. It seemed that, probably in wet weather, spores of the Fusarium spp. are abundant and are transferred to wheat heads by air currents. Of course in addition to primarily climatic conditions, distribution of Fusarium species is due to other several factors, including agronomic factors, such as soil cultivation, nitrogen fertilization, fungicides, crop rotation, rainfall determines and host genotype (Xue et al., 2006). Also, one possible explanation to these facts is that rainfall determines the availability of water for fungi, allow the spores to germinate and cause other activities. In Kermanshah province, the rainfall is irregular and subsequently causes moisture stress. On the other hand Fusarium spp. are capable of attacking weaker plants including those under water stress. Several authors have indicated that drought stress and higher temperatures contribute to an intensified infection rate by F. verticillioides and F. proliferatum while F. subglutinans and F. graminearum are common pathogens of maize ear rot in continental and humid climate (Lew et al., 1991; Miller, 2001; Logrieco et al., 2002; Munkvold, 2003).
Results from this study indicated that F. sambucinum, F. sporotrichioides and F. culmorum were collected throughout moderate to cold areas and F. nygamai, F. scirpi, F. proliferatum, F. anthophilum and F. chlamydosporum were isolated in stored wheat samples from moderate to warm areas. So, results of this study are in agreement with the previous literature (Desjardins, 1995; Leslie et al., 1990; Burgess and Summerell, 1992). In this study, we have isolated as F. equiseti in Iran with average frequency. This species was rarely isolated from wheat in Tehran, East Azarbayejan and Mazandaran provinces by Kachuei et al. (2009) while the latter case has been recently reported as Fusarium head blight by Moosawi-Jorf et al. (2007) that is in agreement with our results. Other Fusarium species that were present at low frequency levels were F. crookwellense, F. culmorum, F. trichothecioides, F. sambucinum, F. scirpi, F. compactum, F. avenaceum, F. sporotrichioides, F. solani, F. camptoceras, F. nelsonii, F. anthophilum and F. pseudograminearum (Table 4), which was in accordance with other studies (Kachuei et al., 2009; Moosawi-Jorf et al., 2007). In this survey F. nelsonii is reported for the first time from Iran. Production of toxin and related metabolites by Fusarium Section Arthrosporiella can be important as was previously shown (Mule et al., 1997; Jimenez et al., 1997). Studies on fungal species isolated from cereals in other provinces have demonstrated their ability to produce trichothecenes (Rezayat et al., 1996; Haratian et al., 2008), fumonisins (Ghiasian et al., 2005) and zearalenone (Zamani-Zadeh and Khorsandi, 1995). We believe that this study will serve as a basis for further studies on Fusarium species particularly their mycotoxin profiles in cereals from west of Iran and in developing proper management strategies to control Fusarium diseases and reduce the risks of mycotoxin contamination.
CONCLUSIONS
This study demonstrates that wheat samples collected from Kermanshah Province, Iran varied in their fungal distribution. Fusarium species in Section Discolor were the most important in pathogenicity assay. Also, the results of this study indicated some of the Fusarium species are capable of surviving and transferring by wheat seeds which are pathogenic on wheat and responsible for two forms of disease; foot rot affects roots and crowns and includes an early stage causing seedling blight and FHB. Off course, it is important to include the results of toxicology and risk assessment of toxic compounds. This is because Fusarium species are mostly saprophytes or have lost their pathogenic capacity but produce different mycotoxins.
ACKNOWLEDGMENTS
Khosrow Chehri acknowledges the Universiti Sains Malayisa, Penang, Malaysia for providing necessary facilities and Research Grants No. 300/PBIOLOGI/811009 to carry out this research.