Natural Occurrence of Fusarium species in Maize Kernels at Gholestan Province in Northern Iran

MATERIALS AND METHODS

Maize sampling: A total of 41 (5-10 kg) samples, related to pre harvest time, harvest time, enter to silo and exit from silo were collected in the areas of Gholestan province from North of Iran which is located in the endemic esophageal cancer (OC) area in 2004, mostly intended for animal and some for human consumption without indicating noticeable signs of mold contamination.

Isolation of Fusarium: All grain from each sample were outside disinfected for 1 min with a 5% NaOCl solution, washed two times in sterile distilled water and dried in a laminar flow cabinet. Then all samples were grounded using a Romer analytical mill (Union, MO, USA) and one-third selected for isolation Fusarium species. Isolate were made in two ways as described: (1) grounded maize were plated onto Peptone PCNB (Pentachloronitro benzene) agar or Nash-Snyder agar in three plates (Nash and Snyder, 1962) modified by Nelson et al. (1983) and (2) 25 g of grounded samples was added to 200 mL deionoised water while shook at 140 rpm for 30 min then 1 mL of each was placed into the same medium in three plates. This medium contained 1000 mg L^-1 of streptomycin sulfate, to suppress the growth of bacteria. Pentachloronitrobenzene (PCNB) cause reduction in growth of fast growing fungi including Zygomycetes such as Mucor and Rhizopus and prevent their extra ordinary generation and their colony mixing (Singleton et al., 1992). The plates were incubated for 5-7 days at 25-27°C in the dark. With observing the colonies, prepared a single spore of them.

Single spore isolation: This method was used to gain unmixed cultures, divide species in mixed cultures and to permit for uniform and consistent production of conidia (Singleton et al., 1992). Therefore because some colonies with close vicinity may mixed cultures, so isolates were purified by preparing single-spore cultures as follows: by scraping conidia from PDA (Phtato dextrose agar) a suspension of conidia in 10 mL sterile distilled water was prepared to obtain a concentration of 1-10 conidia in a drop viewed under the low power objective (10x) of a microscope. A droplet of suspension was splashed on the PDA plates and incubated at 25-27°C for 24 h. After verification of plates with stereomicroscope and observing the single germinated conidia, it was removed along with small piece of agar and transferred to PDA slants.

Identification of Fusarium species: For identification of Fusarium species, subcultures were transferred to PDA (potato dextrose agar) and SNA (Spezieller Nahrstoffarmer Agar) respectively for macroscopic and microscopic identification and incubated for 5-7 days at 25-27°C. In case along time passes from Fusarium culture or, Fusarium are several times subcultured, the mutates may occurs. To identify the species macroscopic specifications such as color, type of growth of mycelium; and microscopic specifications such as type of phialides (monophialides or polyphialides), presence or absence of microconidia chain, false heads, presence or absence and shape of macroconidia, presence or absence of chlamydospores was investigated. The identification of Fusarium species was made upon Nelson et al. (1983).

The isolation Frequencies (Fq) and Relative density (Rd) of species isolated were calculated as follows:

RESULTS AND DISCUSSION

In the present study the Fusarium species in 41 maize samples that related to pre-harvest time, harvest time, enter to silo and exit from silo, which collected in 2004 from North of Iran at Gholestan province were investigated. Fusarium species were identified according to the Nelson et al. (1983). From the total samples, 1008 Fusarium species were isolated. F. verticillioides (with 60.41% of total isolates) was the most predominant species, followed by F. proliferatum (13.39%), F. oxysporum (7.44%), F. culmorum (5.64%), F. solani (4.17%), F. equiseti (1.48%), F. poae (1.19%) and other species with lower incidence (5.96%) (Table 1). Occurrence of F. verticillioides in harvest stage with frequency of 41.46% and relative density of 30.35% was the highest amount, compared to other stages. F. proliferatum was also the highest amount in harvest stage weighed against other stages with frequency of 21.95% and relative density of 6.55%. Occurrence of F. oxysporum, F. culmorum and F. solani had also the highest statistics during harvest stage in comparison to other stages with frequency of 14.63, 12.19, 7.31% and relative density of 3.57, 2.38 and 1.78%, respectively. But F. equiseti and F. poae were the most prevalent in exit from silo stage with frequency and relative density of 4.87 and 0.69% (for F. equiseti) and 4.87 and 87% (for F. poae), respectively in compare with other stages (Table 2). Frequency of F. verticillioides in enter to silo stage comparing to the same stage was 100% that indicate this fungus has been existed in all the samples in this stage (Table 3). The result of this study showed that, 60.41% of the species was F. verticillioides, which demonstrated the high frequency of this seed-born species in maize. Similar studies also found that F. verticillioides has the same prevalence in other countries such as Brazil (Rodriguez and Sabino, 2002) USA (Jurjevic et al., 2004), Nigeria (Bankole et al., 2003; Bankole and Mabekoje, 2004; Timothy et al., 2007) Colombia (Acuna et al., 2005) and Spain (Jurado et al., 2006). F. proliferatum is the second most predominant Fusarium species with frequency and relative density of 46.34 and 13.39%, respectively in this study. These results are in accordance with other results in Iran and other countries (Zare and Ershad, 1997; Srobarova1 et al., 2002; Ghiasian et al., 2004; Acuna et al., 2005). Presence of F. verticillioides and F. proliferatum were found in earlier studies in northern Iran as the most common seed-borne fungi on farm rice, maize and wheat kernels (Zare and Ershad, 1997). Concerning locality, it was demonstrated that, incidence of F. verticillioides was much higher in Mazandaran province in compare with other areas in Iran including Khuzestan and Kermanshah provinces, from field-testing results gained on the fungal mycoflora research made in 2000, regarding to maize kernels in the main maize producing areas (Ghiasian et al., 2004).

Table 1:	Frequency, relative density of Fusarium species in maize from Gholestan provinces in northern Iran

Another study about maize has shown that F. verticillioides and F. proliferatum were the most major species isolated from maize, after Aspergillus species, in the northern parts of Iran (Boujari and Ershad, 1995). Also F. verticillioides with 92% and F. proliferatum with 8% incidence was isolated from maize in North of Iran and Karaj town in Iran (Zamani and Alizadeh, 2000). In spite of Norred (1993) study, which indicated the incidence of F. verticillioides in OC (Oesophaseal cancer area surrounding North of Iran, is low, the other studies, which mentioned above and the present work indicate the most incidences of F. verticillioides in these areas. The occurrence of Fusarium species in the maize samples that recovered in our study agreed with surveys carried out worldwide where F. verticillioides, F. proliferatum and F. graminearum are the most frequently isolated species in maize and F. equiseti, F. poae, F. sporotrichioides and F. culmorum are considered less frequent (Logrieco et al., 2002). The difference between their study and the present study is that in our study no F. graminearum and F. sporotrichioides isolated instead we isolate F. solani and F. oxysporum. In 2007 in Mexico in maize through morphological and phylogenetic analysis, seven species of Fusarium identified as F. chlamydosporum, F. napiforme, F. poae, F. pseudonygamai, F. solani, F. subglutinans and F. verticillioides, were found to be associated with ear rot disease and F. chlamydosporum, F. poae, F. pseudonygamai, F. subglutinans and F. verticillioides were found within asymptomatic kernels as well (Morales-Rodriguez et al., 2007). Not only high incidence of F. verticillioides demonstrates its importance as a seed-borne fungus, often causing symptomless infections in maize grains, but also it is because of its potential toxigenicity. F. verticillioides and F. proliferatum, the main species isolated in the present study, are the most common fumonisin producing fungi associated with corn (Marasas et al., 2001). The spread of this species has harmful influence on human and animal health because of its capability to generate fumonisins which is a group of toxic and carcinogenic metabolites (Ghiasian et al., 2005).

Table 2:	Frequency and relative density of Fusarium species in maize derived from four collected stages in compare with whole sampling stages in Gholestan province

Table 3:	Frequency and relative density of Fusarium species in maize derived from each of four collected stages comparing to the same stage in Gholestan province

International Agency for Research on Cancer (IARC) has also labeled fumonisins as Group 2B carcinogens, possibly carcinogenic to humans (IARC, 1993). F. oxysporum produces toxins such as Diacetoxyscirpenol and T-2 toxin. F. culmorum generates B trichothecene, especially deoxynivalenol which is harm for human in case exceeding 1000 ng g^-1. F. solani causes kratitis and is known as an opportunistic pathogen in human and animals. Also it produces toxins like HT-2 toxin and Neosolaniol. F. equiseti produces toxins such as Equisetin, Diacetoxyscirpenol and nivalenol and at last F. poae generates type A of trichothecene such as T-2 toxin and enniatins and beauvericin (Marasas et al., 1984; Morrison et al., 2002; Logrieco et al., 2002). F. verticillioides (called before F. moniliforme) is one of the most prevalent fungi which are related to main human and animal nutritional samples such as maize. Due to early definition of this fungus, it was found suspicious of being involved in human and animal diseases. F. moniliforme is in the section Liseola along with F. proliferatum, F. subglutinans and F. anthophilum. F. moniliforme is the reason for ear rot and stalk rot of maize and it is widely infecting the maize kernels. Maize kernels may get infected through the silks, through holes and fissures in the pericarp or at points where the pericarp scratched by the emerging seedling and as a result of systemic infection of the maize plant by F. moniliforme (Nelson, 1992). Temperature and osmotic stress are the most important factors that influence on host susceptibility to fungal disease (Conrath et al., 2002). It is possible that Fusarium spp. populations vary within and between harvesting seasons (Bateman and Murray, 2001). Among fungal genera that contaminate maize, F. verticillioides and F. proliferatum are dominant over a wide range of temperature and a_w conditions (Doohan et al., 2003). Values of Topt and Tmax for F. verticilloides were set at 31 and 35°C, respectively corresponding with evidence that indicated F. verticillioides grow faster at higher temperatures (Reid et al., 1999). Production of F. verticillioides occurs during higher temperatures and drier years compared to warm and dry conditions of the subtropics and dry land maize (Vigier et al., 1997). F. verticillioides, the causal agent of Fusarium ear rot, produces a whitish-colored mold growth that tends to be scattered on the ear (Reid et al., 1999). Fusarium species contaminating maize kernels are competing with other fungi including the other Fusarium species and competition between these fungi can have clear influence on final concentration of mycotoxins. F. verticillioides suppresses the growth of other maize ear fungi including F. graminearum and Aspergillus flavus. Evidences indicate that F. verticillioides has the higher ability of colonization rather than other Fusarium species in maize (Stewart et al., 2002). In this study among all samples, occurrence of F. verticillioides was the most prevalent with frequency and relative density of 87.8 and 60.41% followed by F. proliferatum with frequency and relative density of 46.34 and 13.39%, respectively. After these two species F. oxysporum, F. solani and F. culmorum graded highest occurrence sequenced with frequencies of 29.26, 24.39 and 21.95% followed by F. equiseti and F. poae with frequency of 9.75% for each case (Table 1). Eventually, the results of this study indicate the high incidence of F. verticillioides followed by F. proliferatum resulting from the temperature, that is dominant in North of Iran like Gholestan province.

CONCLUSION

This study expresses high occurrence of F. verticillioides with frequency and relative density of 87.8 and 60.41% followed by F. proliferatum with frequency and relative density of 46.34 and 13.39%, respectively which indicate prevalence of these two species in Iranian maize kernels especially in wet area.