Assessment of Fusariotoxins and Toxigenic Potential of Fusarium Spp. in Maize Grains Collected from Pakistan

A total of 65 maize grain samples ( Zea Mays L. ) collected from cold and hot climatic zones of Pakistan lead to isolation of 10 Fusarium supposing deep freezing method. Occurrence of fusarium spp. was high (75%) in cold climatic zones as compared to hot zones (25%). Fumonisins were recorded in both climatic zones, though their incidence was higher in hot climatic zone (range 4,379-9,990 µg/kg). Incidence of A and B trichothecenes was higher in cool climatic zone. Zearalenone (ZON) with mean level 500 µg/kg was positive in only 10% samples of hot climatic zone. Furthermore, co-contamination of fusariotoxins was also observed for fumonisins with ZON, A and B trichothecenes. Among 33 recovered isolates, F. proliferatum, F. tricinctum, F. subglutinans, F. poae, F. nivale and F. acuminatum were found toxigenic.


INTRODUCTION
Maize (Zea mays L.) is the third largest cereal crop of Pakistan, after wheat and rice. It is grown in irrigated as well as rain-fed areas. Its production has increased from 3,593 to 4, 631 MT between 2008(Economic Survey, 2014. Punjab and Khyber Pakhtunkhwa (KPK) provinces are the major maize growing areas. Maize is a very versatile cereal, providing food, feed, adhesives, oil, syrups, flakes, alcoholic and non-alcoholic drinks, starch, ethanol and pop-corn, etc. (El-Imam et al., 2012). Many feed mills utilize maize as the principal raw material. This important food source can be infested by different parasites, pests/ insects and fungi in field as well as during storage (Fandhon et al., 2003;Matny, 2014). Among these, fungi rank second in deteriorating the maize quality (Saleemi et al., 2012). Maize is considered as an ideal substrate for fungal growth because of its high carbohydrate contents (Trung et al., 2008). Spores of fungi are ubiquitously present in air and soil. Whenever, they find optimum conditions, they geminate and produce 'Mycotoxins' (Niaz and Dawar, 2009) chemically diverse group of toxic secondary metabolites of certain pathogenic fungi (Sultana et al., 2013). The major toxigenic genera commonly recorded on maize in tropical regions, including Pakistan, are Aspergillus, Penicillium and Fusarium (Orisi et al., 2000;Saleemi et al., 2012). Aspergillus and Penicillium are mainly responsible for aflatoxins and ochratoxin A production. Fusarium spp. produces a wide range of mycotoxins, viz., fusariotoxins (Tahira et al., 2015).
Fusarium is a major genus of the filamentous fungi widely distributed in soil in association with plants, particularly cereal Hanif et al.,: Fusariotoxins And Toxigenic Potential of Fusarium Spp. J. Bioresource Manage. (2015) 2(2): 63-72. grains. They may colonize in maize at all stages of their development, from the early hours of kernel germination to harvest time and even postharvest decay of grains (Fandhon et al., 2003).
In Pakistan, comprehensive research work has been carried out on seed borne mycoflora of different maize varieties (Niaz and Dawar, 2009;Saleemi et al., 2012). Data concerning mycotoxins in maize grains of Pakistan were well reported (Khatoon et al., 2012). However, toxigenic potential of Fusarium spp. and scenario of fusariotoxins, particularly fumonisins, are missing. In view of foregoing, present study was planned to investigate the Fusarium mycoflora, fusariotoxigenic species and fusariotoxins in maize collected from different climatic zones.

Sampling
Randomly 65 maize grains samples (1.0kg) were collected from major maize growing areas.
On the basis of meteorological data, sampling areas were divided into cold (Swat, Murree) and hot (Peshawar, Faisalabad, Sahiwal and Multan) climatic zones. This study was carried out at the premises of Romer Labs, Pakistan and plant pathology department (Arid Agriculture University Rawalpindi). The maize samples were preserved at -20ºC till analysis to prevent further fungal contamination and mycotoxin production.

Mycological Analysis
For the Fusarium spp. isolation, deep freezing method was used as described by Mostafa et al. (2011). Surface of maize grains was disinfected by 1% Sodiumhypochlorite for one minute and rinsed twice in sterile distilled water for 30 seconds. Samples were plated @ 12 seeds/sample /plate on three layered sterilized moistened filter paper in 9 cm glass Petri dishes and incubated at 22±0.5 ºC for 24 hours. Plates were then placed at -20ºC for next 24 hours followed by incubation at 22 ± 0.5ºC for five days. After incubation, slides were prepared using Lactophenol cotton blue stain (LCBS) as mounting reagent and examined under low (20x) and high (100x) magnification for the presence of mycelia and conidial structures and identified following Ninreberg et al. (1982).

Fusariotoxins Analysis
Total fumonisins (FB1, FB2 and FB3) were analyzed using direct competitive ELISA. Samples were prepared following manufacturer's protocol provided along with AgraQuant ® ELISA kit (Romer Labs Inc, USA).

Mycotoxigenic Potential
To assess the mycotoxigenic ability, 20 gm of rice was taken in 500 ml Erlenmeyer flask and 100 ml distilled water and autoclaved at 121ºC for 30 minutes. Each flask was inoculated and incubated at 15 ºC with daily shaking, for the first 3-4 days for even distribution of inoculums (Khatoon et al., 2008). The detection and quantification of fusariotoxins in fungal cultures was carried out by using ELISA for fumonisins and HPTLC for ZON, A and Btrichothecenes respectively.

Fusarium Incidence
Maize is considered as the best substrate for germination of fungal spores because of its high carbohydrates contents (Fandhon et al., 2005). Spores may spread in several ways, viz., birds, insects and wind, etc. Fungal spores may enter maize via silk hairs of comb and may create wounds in grains and grow (Matny, 2013 and2014). In present study a higher incidence of Fusarium spp. infestation in cold zone (75%) was observed. It might be due to low temperatures (15-25°C) and     (Sultana et al., 2013). ** Analysis by ELISA. higher relative humidity levels which provides favorable environment for Fusarium spp. growth (Mangan et al., 2010). Furthermore, water activity of maize samples also play key role for the colonization of fungi. Generally, water activity (aw >0.7) leads to fungal growth and <0.7 considered as a measure to prevent fungal growth (Mostafa et al., 2011). Time of sampling may also be an important contributing factor for this variation (Backhouse and Burguess, 2002). Present samples from hot zone (Faisalabad Sahiwal and Multan) were collected in July when temperature and relative humidity were >40 ºC and ≤55%, respectively. However, in cold zone, harvesting of spring maize starts at the end of September (having temperature ≤30ºC and ~65% relative humidity). The higher temperature and lower relative humidity could be the reason for low (25%) incidence of Fusarium spp. infestation in hot zone. This is the first report from Pakistan suggesting fumonisins as the most frequent fusariotoxin. A total of 28 fumonisins analogues have been identified and characterized so far, but FB1, FB2 and FB3 are abundantly found in naturally contaminated food (Pietri et al., 2004). Fumonisins are more abundant in maize as compare to sorghum, rice and spices (Fandhon et al., 2005;Saleemi et al., 2012). The occurrence of fumonisins is associated with warmer and dry climate (Marsasa et al., 2001;Pietri et al., 2004;Galvano et al., 2005;Fandhon et al. 2005;Binder et al., 2007), which explains their higher incidence in Sahiwal and Faisalabad having hot climate. Detected levels of fumonisins in present samples were found far beyond (4.3 to 9.99 times) the allowable limit of EC, i.e., 1000 µg/kg (EC, 2007). These are strongly associated with esophageal cancer in human, leukoencephalomalacia (ELEM) in horses, pulmonary edema syndrome (PES) in pigs and liver cancer in livestock and poultry (Shephard et al., 2000;Gelderblom et al., 2001;Matny, 2014). The international agency for research on cancer (IARC), has classified fumonisins as potential human carcinogen (type 2B carcinogen;IARC, 2002).
A and B trichothecenes were detected in maize grain samples from both cold and hot zones. Among these, European commission has defined regulatory limits for DON i.e. 1250 µg/kg (EC, 2007). In present study, DON with mean level of 913µg/kg was observed and found below the EC regulatory limits.
Trichothecenes are associated with various acute and chronic symptoms in human and animals. In human, trichothecenes are mainly responsible for impaired fertility, protein metabolism disturbance, lung diseases and cancer (Khatoon et al., 2012;Sultana et al., 2013;Kos et al., 2014). Maize unfit for human consumption is usually consumed in animal feed as an energy source. Contaminated maize can cause vomiting, diarrhea, feed refusal, skin inflammation, hemorrhagic syndrome in internal organs, cellular damage to bone marrow, thymus and spleen (immunosuppressant), and disturbance of nervous system in animal (CAST, 2003;Galvano et al., 2005).
Zearalenone, an important fusariotoxin, has been well recognized as maize contaminant. ZON induces hyperestrogenism in animal and human (CAST, 2003;Khatoon et al., 2012). It is also declared as hepatotoxic in human (Zindine et al., 2007). Due to its toxicity, European Commission (EC) has defined its permissible level in maize (75 µg/kg; EC, 2007). In present study only 10% samples collected from Sahiwal (hot zone) were positive for ZON. Although, the incidence of ZON in present study was 10% but positive samples were contaminated with 6.66 folds higher level than the EC legislation (EC, 2007).
Co-contamination of mycotoxins is another aspect that has been highlighted because of mycotoxin-mycotoxin interactions. Various research studies revealed that combination of mycotoxins exerts synergistic, additive or antagonistic effects in consumers (Streit et al., 2012;Sultana et al., 2013). Fusariotoxins data of present study suggested that fumonisins were co-contaminated with trichothecenes. This co-contamination may prove more lethal even at lower levels (CAST, 2003;Striet et al., 2013). Similar findings were reported from Vietnam (Wang et al., 2000) and South East Asia (Yamashita, et al., 1995).
The multiple mycotoxin contamination in kernels may be due multispecies infestation of Fusarium. Furthermore, Fusarium does not grow alone, rather it forms complex either with other Fusarium or non-Fusarium species (Diaz and Borenman, 1994;Matny, 2014).

Mycotoxigenic Potential
In the past, researchers revealed Fusarium spp. as the main phytopathogenic fungi. However, since last two decades, interest for the investigation was turned over to its toxicity (Saleemi et al., 2012). Various studies conducted in different regions of the world revealed that maize is highly susceptible to the invasion of pathogenic fungi, particularly Fusarium and Aspergillus spp. (Marsasa et al., 2001;Verga et al., 2005;Hussain et al., 2013). During course of present study, 17 isolates of F. proliferatum recovered from maize grain sample collected from both climatic zones. It was due to its wider range of optimum conditions. Twelve (12) isolates of F. proliferatum produced fumonisins with mean of 116.64 mg/kg. Only one isolate of F. subglutinans (out of 3) produced fumonisins with mean level of 11.74 mg/kg, though a previous study suggested it as a non-fumonisins producing species (Acuna et al., 2005).This discrepancy may be due to differences in strains; some strains hold ability to produce toxins while others lack such an ability (Niaz And Dawar., 2009). Presently, F. nivale, F. tricinctum and F. poae were trichothecenes producers. F. acuminatum produced ZON and fumonisins, whereas F. Anthophilum produced NEOS and HT-2 toxins. These findings are in agreement with previously reports (Langseth et al., 2000;CAST, 2003). F. graminerum is considered as fusariotoxin producers, but in present study, it was a non-toxigenic, which may an inter-strain variation or culture media differences (Matny, 2014).

Conclusion
Fusarium spp. were toxigenic and can produce different mycotoxins. Cooccurrence of mycotoxins may exaggerate the situation. This is a matter of great concern because maize and its products/ byproducts have a significant place in human food and animal feed.
Mycotoxins, particularly fumonisins, may pose a serious health concern to human and animals via their exposure to contaminated maize. A continuous monitoring is needed in order to avoid negative impact on human and animal health. Moreover, a detailed investigation at biochemical and molecular levels is suggested to give a broader understanding of the biodiversity of toxigenic Fusarium species in Pakistan and mechanism of mycotoxins production needs to be elucidated. Good agricultural practices, i.e. irrigation/ water supply, timely harvesting activity, proper drying and storage, are important tools for minimizing mycotoxin contamination. An indigenous data based on the toxigenic nature of all commonly occurring toxigenic and non-toxigenic fungal species should also be generated.