Incidence of potentially toxigenic fungi in maize (Zea mays L.) grain used as food and animal feed

Abstract

Recently, new maize hybrids have been introduced in northern Mexico to reduce importations of grain, which is mainly directed for the production of flour and animal feed. This represents a potential risk to both human and animal health because of the development of fungal pathogens under the local weather conditions; thus, the identification of tolerant hybrids has become a priority. In this way, the objective of this study was to observe the mycoflora development in maize. There were 12 yellow and 10 white seed maize hybrids randomly planted. At harvest time, a grain sample of each plot was collected, surface-sterilized, plated in culture media, and incubated. After 7 days, colonies were observed and analyzed. The major fungi encountered were Fusarium spp., Penicillium spp., and Aspergillus spp. When compared with the yellow hybrids, white hybrids had 34, 52 and 22% less infection by F. verticillioides, Aspergillus flavus, and Aspergillus niger, respectively, and almost the double infection with Penicillium spp. White hybrids presented 51% more healthy grains than the yellow hybrids.

Recientemente nuevos híbridos de maíz han sido introducidos al norte de México para reducir las importaciones de grano que es usado principalmente para producir harinas y alimento para animales. Esto representa un riesgo potencial para la salud humana y animal debido al desarrollo de hongos bajo las condiciones climáticas locales, por lo tanto, la identificación de híbridos tolerantes es una prioridad. En este contexto, el objetivo de este estudio fue el observar el desarrollo de micobiota en maíz. Doce híbridos de grano amarillo y diez de grano blanco fueron sembrados aleatoriamente. A la cosecha, una muestra de granos de cada parcela fue colectada, desinfectada, puesta en medio de cultivo e incubada. Después de siete días, las colonias fueron observadas y analizadas. Los hongos que principalmente se encontraron fueron Fusarium spp., Penicillium spp., y Aspergillus spp. Comparados con los híbridos amarillos, los híbridos blancos tuvieron 34, 52, y 22% menos infección por F. verticillioides, Aspergillus flavus, y Aspergillus niger, respectivamente, y casi el doble de infección por Penicillium spp. Los híbridos blancos presentaron 51% más grano sano que los híbridos amarillos.

Keywords:

• Zea mays
• mycoflora
• hybrids
• Fusarium
• Aspergillus

Palabras clave:

• Zea mays
• micobiota
• híbridos
• Fusarium
• Aspergillus

Introduction

Maize is the third most important crop in the world after wheat and rice. In Mexico, the area cultivated with maize is around 8 million hectares, in which there are 18.3 million tons produced every year (http://www.siap.gob.mx). Out of those, around 12 million tons are used for human consumption and 5.9 million tons for animal feed. The requirements of the poultry, cattle, milk, egg, and pork industries are around 16 million tons per year, therefore, each year there is the need to import sorghum grain (5 million tons), whole yellow maize grain (3.2 million tons), and cores of yellow maize grain (2 million tons). To reduce the importations of these grains, the government initiated a program in which some areas needed to convert to yellow maize grain production. Since 2001, there has been an increase in the number of yellow grain hybrids, which the seed companies have brought into the northeast region of Mexico. These hybrids have different degrees of adaptation to the climatic conditions of the area and this situation may trigger problems with diseases, particularly on the maize ear, because grain quality will be at risk because of the exposure of maize to unpredictable climatic conditions that deteriorate the grain (Agrios, 1988). Maize grain is susceptible to a number of ear and kernel rots that cause damage in humid areas, especially when rainfall is above normal from silking to harvest (Shurtleff, 1977). Infection of maize kernels by toxigenic fungi remains a challenging problem despite the research progress (Munkvold, 2003b). The most common signs of infection are the presence of grain-borne molds (Williams & Rao, 1981) and symptoms like loss of color and viability, and premature germination before harvest time (Castor & Frederiksen, 1980). Grain molds produce toxins that are generated inside the maize ear and are referred as mycotoxins and are characterized as small molecular weight fungal metabolites which pose a health risk to humans and animals that (most often) ingest contaminated food or feed, or (less often) are exposed via inhalation of toxin-bearing spores (Bennett & Klich, 2003). Some of the mycotoxins can have carcinogenic, mutagenic, or teratogenic properties (Abramson, Mills, & Boycott, 1983; Bauduret, 1990). Ingestion of mycotoxins reduces livestock productivity and lowers the health quality of derived products. Recent reviews have determined that almost 25% of grain harvests worldwide have mycotoxin contamination (Fink-Gremmels, 1999). In addition, mycotoxins cause large economic losses on a global scale for many commercial sectors (Jestoi, Riteni, & Rizzo, 2004; Miller, 1999), such as crop producers, food and animal feed processors as well as for animal breeders. In northern temperate regions, Fusarium molds are probably the most important mycotoxin producing fungi (Chelkowski, 1989; Munkvold, 2003a). Grain infection by Fusarium verticillioides Sacc. (Nierenberg) (synonym F. moniliforme Sheld) [teleomorph: Giberella fijikuroi (Sawada)] resulted in systemic infection of plants and kernels (Munkvold, 1997) and symptomless endophytic infections were common and existed throughout the maize plant (Munkvold & Desjardins, 1997). However, local infection (via silks) was a more important pathway to kernels than was systemic infection. Maize grown in the southern United States is frequently contaminated with moldy fungi, including F. verticillioides, which was isolated from 91% of the maize samples from Georgia (Jurjevic et al., 2005). This pathogen has been the most prevalent in maize grain in lowland Africa (Cardwell, Kling, Mazita-Dixon, & Bosque-Perez, 2000), West Africa (Fandohan, Gnonlonfin, Hell, Marasas, & Wingfield, 2005), Argentina (Gonzalez, Resnik, & Pacin, 2003; Mego, Lori, Botta, & Presello, 2005; Presello, Iglesias, Botta, & Eyherabide, 2007), Australia (Blaney, Ramsey, & Tyler, 1986), Brasil (Almeida, Correa, Mallozzi, Sawazaki, & Soares, 2000; Ono et al., 1999; Orsi et al., 2000), Benin (Schulthess, Cardwell, & Gounou, 2002), Bostwana (Allotey, Simpanya, & Mpuchane, 2001), Costa Rica (Danielsen, Meyer, & Funk, 1998), Finland (Jestoi, 2005), France (Bauduret, 1990), Honduras (Julian et al., 1995), Mexico (Desjardins, Plattner, & Nelson, 1994; Figueroa-Gomez, Reynoso, Castro-Zambranno, Reyes-Velazquez, 2006; Rosales & Perez, 1981; Sanchez-Rangel, SanJuan-Badillo, & Plasencia, 2005), Pakistan (Fakhrum, 1998), Spain (Jimenez, Sanchis, Santamarina, & Hernandez, 1985; Marin, Ramos, Cuevas, & Sanchis, 2006), and Venezuela (Antolini & Garcia, 1994: http://www.ceniap.gob.ve/pbd/Congresos/jornadas%20de%20maiz/2%20jornadas/antolinij.htm). There are other fungi like Aspergillus flavus Link that also develops and produces mycotoxins in grain and was related to the negative effect of drought stress (Halfon-Meiri & Barkai-Golan, 1990; Rodriguez-del-Bosque, 1996), or when grain moisture levels were above 17.5% and temperatures above 24 °C (Trenk & Hartman, 1970). Also, maize left in the field for more than 3 weeks after physiological maturity increased the mould incidence, insect damage, and aflatoxin contamination (Kaaya et al., 2005). Recently, yellow maize hybrids from temperate US regions has been introduced to northern Mexico and is being cultivated in more than 118,000 hectares with the objective to reduce importations of grain that is mainly directed to produce animal feed. The introduction of new yellow hybrids represents a risk to animal and human health (Abramson et al., 1983; Bauduret, 1990), which observed that yellow maize samples were the most frequently contaminated with toxigenic strains of A. flavus. Also, they observed that white maize seemed to present a better microbiological quality than yellow maize. Similarly, Antolini and Garcia in 1994 (http://www.ceniap.gob.ve/pbd/Congresos/jornadas%20de%20maiz/2%20jornadas/antolinij.htm) observed that imported yellow maize was affected by Fusarium spp., A. flavus, and Penicillium spp., meanwhile white maize was affected more often by F. moniliforme. The information on grain fungi in both white and yellow maize hybrids in northern Tamaulipas will give a better idea of the future situation of maize in the region and the possible implications to maize producers.

Materials and methods

Experiments were carried out during the spring and fall seasons of 2005 at INIFAP Rio Bravo Experimental Station located at 25° 58′N, 98° 00′W. Each experiment was conducted under irrigated conditions. There were 12 yellow grain hybrids: “DK 697”, “DK 1060”, “Garst 8222IT”, “Garst 8285”, “Garst 8288”, “Asgrow 7573Y”, “TechAg N83-N5”, “Golden Acres 8112”, “Golden Acres 8311”, “Golden Acres 8460”, “Pioneer 31R88”, and “Pioneer 31G98”; and 10 white grain hybrids: “Asgrow Tigre”, “Asgrow Puma”, “H-436”, “H-437”, “H-439”, “Asgrow 7573W”, “DK 2010”, “Pioneer 3025W”, “UAP-1790W”, and “UAP-1851W”. These genotypes were planted under a complete randomized block design with four replications. Each replication consisted of two rows. Population density was 55 thousand plants per hectare. Each experiment was irrigated three times (at 40, 70 and 90 days after planting) and was fertilized with a 140-40-00 (NPK) rate 1 month before planting. Weeds were controlled using Atrazine 0.75 kg a.i. ha⁻¹ plus Prowl® at 1.5 L ha⁻¹ before the first irrigation. At harvest time, a sample of 100 g of each plot was collected and kept under cool temperature for further analysis. One week later, 400 grains of each plot were surface-sterilized for 3 min with a 10% sodium hiphochlorite solution. After that, they were rinse three times with distilled water and put them to dry out in a ventilated chamber. Then, they were transferred to 0.5 PDA plates (Shurtleff and Averre, 1999) (seven seeds plate⁻¹) and left them for 7 days for incubation at 24 °C ± 1 °C. After this time, colonies developed in each grain were examined visually and by transferring mycelium and forming conidia onto microscope slides, they were examined under the microscope. Fungi identification was made using the morphological description of spores, mycelium, and with colony appearance, color and shape (Alexopoulos et al., 1996; Barnett and Hunter, 1998). Before analysis, mycoflora percentage was arcsine square root transformed to satisfy normality assumptions, but back-transformed results are shown in the tables. Data were subjected to ANOVA (Ott and Longnecker, 2001) to identify significant main effects of genotypes, grain color, and the interaction of treatments. Analysis of variance was conducted on all grain-borne fungi as well in healthy seeds, using the analysis of variance GLM procedure of SAS (SAS, Institute, Cary, NC. Version 9.0). Treatment means were separated with Tukey at P < 0.05. (SAS, Institute, Cary, NC. Version 9.0).

Results and discussion

Among all maize hybrids, the major fungal genera encountered were Fusarium spp., Penicillium spp., and Aspergillus spp. Out of those, Fusarium verticillioides was the Fusarium species mostly found in all genotypes (32.64%), followed by Aspergillus spp. (20.15%) and Penicillium spp. (14.77%). Nevertheless, there was a highly significant effect of maize grain color in the grain-borne fungi development ( Table 1). Yellow maize was attacked in greater proportion by Fusarium verticillioides, Aspergillus flavus, and Aspergillus niger. Meanwhile, white maize was preferred by Penicillium spp. and other Fusarium spp. When compared with yellow hybrids, white hybrids had 13.3%, 7%, and 2.6% less infection by F. verticillioides, A. flavus, and A. niger, respectively, and almost the double infection with Penicillium spp (9.3%). White hybrids presented 12.8% more healthy seeds than yellow hybrids ( Figure 1).

Figure 1. Development of mycoflora in white maize grains cultivated in Rio Bravo, Tamaulipas, Mexico.

Figura 1. Desarrollo de micobiota en granos de maíz blanco cultivado en Río Bravo, Tamaulipas, México.

Table 1. Percentage of mycoflora isolated from maize grain samples collected at Rio Bravo, Tamaulipas, Mexico.
Tabla 1. Porcentaje de micobiota aislada de muestras de grano de maíz colectadas en Río Bravo, Tamaulipas, México.

Grain color	Fusarium verticillioides	Penicillium spp.	Other Fusarium	Aspergillus flavus	Aspergillus niger	Healthy grain
Yellow	39.29 a*	10.09 a	0.31 a	13.49 a	11.46 a	25.30 b
White	25.98 b	19.42 b	1.16 a	6.45 b	8.90 a	38.10 a
*Maize grain colors with same letters in each column (fungi) are statistically similar according to Tukey's at P < 0.05.
Percentages represent the average infection observed in 400 data grain sample for each genotype in each grain color.

In both types of hybrids, genotype, grain color, and the interaction showed a highly significant effect on the incidence of F. verticillioides, Penicillium spp., A. flavus, and A. niger. There was a non-significant effect of the main factors on the presence of other Fusarium spp. Almost all yellow maize genotypes showed greater susceptibility to F. verticillioides, nevertheless, Asgrow 7573Y showed the least infection that was 48.3% less of the highest infection observed in DK697 ( Table 2; Figure 2). As we mentioned before, yellow maize was infected also by A. flavus and A. niger. There were no differences in the susceptibility of this type of hybrids with respect to A. flavus. Meanwhile, there were some differences for A. niger. Yellow hybrids Garst 8222IT and Garst 8285 showed the lowest infection (less than 1%) by this pathogen. On the other hand, the most susceptible one was Garst 8288 with 13.12%. Yellow hybrids, Asgrow 7573Y and DK 1060 significantly showed the highest healthy grain percentage (>50%). Meanwhile, Pioneer 31R88 was more vulnerable to almost all pathogens.

Figure 2. Development of mycoflora in yellow maize grains cultivated in Rio Bravo, Tamaulipas, Mexico.

Figura 2. Desarrollo de micobiota en granos de maíz amarillo cultivado en Río Bravo, Tamaulipas, México.

Table 2. Percentage of mycoflora isolated from yellow maize grain hybrids that were evaluated at Rio Bravo, Tamaulipas, Mexico.
Tabla 2. Porcentaje de micobiota aislada de híbridos de maíz de grano amarillo que fueron evaluados en Río Bravo, Tamaulipas, México.

Genotype	Fusarium Verticillioides	Penicillium spp.	Other Fusarium	Aspergillus flavus	Aspergillus niger
DK 697	77.29 a*	0 b	0 b	4.61 a	1.5 bc
DK 1060	34.71 cd	5.89 ab	0 b	4.76 a	4.38 abc
GARST 8222IT	52.63 bc	9.65 a	0 b	14.66 a	0.75 c
GARST 8285	53.48 bc	5.49 ab	0 b	11.58 a	0.88 c
GARST 8288	49.25 bd	4.51 ab	0 b	8.28 a	13.12 a
ASGROW 7573Y	28.95 d	9.9 a	0 b	6.23 a	1.76 abc
N83-N5	54.36 bc	3.01 ab	0 b	6.52 a	4.26 abc
G ACRES 8112	57.09 bc	3.86 ab	2.38 ab	4.74 a	3.01 abc
G ACRES 8311	47.07 bd	6.94 ab	0 b	2.26 a	7.64 abc
G ACRES 8460	53.38 bc	3.51 ab	0.75 ab	3.38 a	2.63 abc
PIONEER 31R88	60.16 ab	14.06 a	0 b	3.26 a	12.63 ab
PIONEER 31G98	47.86 bd	1.76 ab	3.01 a	12.16 a	2.51 abc
*Genotypes with the same letter in each column (fungi) have statistically similar fungal infection according to Tukey's P < 0.05.
Percentages are the average of 400 data grain sample in each genotype.

In general, white maize hybrids showed less susceptibility to pathogens, thus, they had more healthy grain percentage. The healthiest hybrid was Asgrow Tigre, with 72.53% of healthy grain, whereas Asgrow Puma, Pioneer 3025, UAP 1790W, and UAP 1851W were the most susceptible to grain fungi infection ( Table 3). F. verticillioides was the pathogen that infected significantly more grain among genotypes; nevertheless, Asgrow Tigre showed the least amount of infected grain followed by INIFAP H-439 and INIFAP H-437.

Table 3. Percentage of mycoflora isolated from white maize grain hybrids that were evaluated at Rio Bravo, Tamaulipas, Mexico.
Tabla 3. Porcentaje de micobiota aislada de híbridos de maíz de grano blanco que fueron evaluados en Río Bravo, Tamaulipas, México.

Genotype	Fusarium verticillioides	Penicillium spp.	Other Fusarium	Aspergillus flavus	Aspergillus niger
ASGROW TIGRE	22.56 b*	0.88 cd	0 a	1.05 ab	2.63 ab
ASGROW PUMA	37.19 ab	10.38 ab	6.77 a	2.38 ab	4.56 ab
INIFAP H-439	24.56 b	0 d	4.51 a	7.52 a	11.16 a
INIFAP H-437	32.46 ab	9.64 bc	0 a	2.5 ab	2.63 ab
ASGROW 7573W	36.46 ab	0.88 bc	5.26 a	4.76 ab	2.51 ab
DK 2010	33.08 ab	8.77 bc	0.75 a	5.14 ab	2.98 ab
INIFAP H-436	36.96 ab	10.26 ab	0.75 a	0 b	0 b
PIONEER 3025	34.21 ab	14.54 a	0.75 a	6.51 a	5.39 ab
UAP 1790W	55.64 a	5.49 bcd	0 a	1.05 ab	0 b
UAP 1851W	39.22 ab	12.28 ab	0.75 a	0 b	1.75 ab
*Genotypes with the same letter in each column (fungi) have statistically similar fungal infection according to Tukey's P < 0.05.
Percentages are the average of 400 data grain sample in each genotype.

This research has shown that maize grain is infected in northern Mexico by Fusarium spp., Penicillium spp., and Aspergillus spp., and from this three genera, F. verticillioides was the Fusarium species that prevails in grain obtained in northeast Mexico. Our results are similar to those observed by Antolini and Garcia in 1994 (http://www.ceniap.gob.ve/pbd/Congresos/jornadas%20de%20maiz/2%20jornadas/antolinij.htm); Almeida et al., 2000; Allotey et al., 2001; Bauduret, 1990; Blaney et al., 1986; Cardwell et al., 2000; Danielsen et al., 1998; Fandohan et al., 2005; Fakhrum, 1998; Figueroa-Gomez et al., 2006; Gonzalez et al., 2003; Jestoi, 2005; Jimenez et al., 1985; Julian et al., 1995; Jurjevic et al., 2005; Marin et al., 2006; Mego et al., 2005; Ono et al., 1999; Orsi et al., 2000; Presello, Iglesias, Botta, & Eyherabide, 2007; Sanchez-Rangel et al., 2005; and Schulthess et al., 2002. This is the first time that white and yellow maize hybrids have been compared in a mycoflora study in northern Mexico, and by our results, like in the ones by Abramson et al. (1983) and Bauduret (1990), white maize hybrids consistently exhibit reduced grain-borne fungi than yellow maize hybrids. This is an important issue because in the following years, yellow maize will substitute white maize in a large scale in northern Mexico due to the high demand of yellow grain for animal feed (http//: www.siap.gob.mx). Similarly to Antolini and Garcia (http://www.ceniap.gob.ve/pbd/Congresos/jornadas%20de%20maiz/2%20jornadas/antolinij.htm), our results showed that Fusarium spp. and Aspergillus spp. were present in mostly all yellow maize hybrids, therefore, this situation need to be considered in projects that will be established in the area. We observed differences among genotypes, nevertheless, all were susceptible to infection by Fusarium spp. and Aspergillus spp. Is very difficult to reduce the presence of these molds in the grain, especially if we consider that (a) F. verticillioides can be seed transmitted and may affect emergence of seedlings (Headrick & Pataky, 1989) and cause systemic infection of maize (Wilke, Bronson, Thomas, & Munkvold, 2007), (b) F. verticillioides in the stem predisposed kernel infection (Schulthess et al., 2002), (c) symptomless systemic infection can be initiated under a broad range of temperature conditions (Wilke et al., 2007), and (d) despite cultural practices (Rodriguez-del-Bosque et al., 1995; Rodriguez-del-Bosque, 1996), including crop rotation, conservation tillage, optimum planting date, and management of irrigation and fertilization, there is little effect on infection control and subsequent mycotoxin accumulation (Munkvold, 2003b). Because most mycotoxin problems develop in the field (Figueroa-Gomez et al., 2006), strategies are needed to prevent infection of growing plants by toxigenic fungi. Developing genetic resistance to A. flavus, and F. verticillioides in maize is a high priority (Munkvold, 2003b). Nevertheless, we need to consider that moldy fungi increase their presence when grain is stored under non-ventilated facilities (Fandohan et al., 2005), so providing storage with a well ventilated system will help to prevent development of these fungi. Also, avoiding to left maize in the field for more than 3 weeks after physiological maturity will reduce mould incidence, insect damage (Schulthess et al., 2002) and aflatoxin development (Kaaya, Warren, Kyamanywa, & Kyamuhangire, 2005), and the use of transgenic genotypes could result in better grain quality (lesser mold damage and decreased the potential for the development of fumonisins), as a result of lower disease and severity incidence (Munkvold, Hellmich, & Showers, 1997). Additionally, mexican northwest isolates of F. verticillioides have more fumonisin production than the ones from central Mexico (Sanchez-Rangel et al., 2005), thus, under all this circumstances, further research is needed to develop genotypes that presents resistance to grain-borne fungi (Presello et al., 2007) and to evaluate the effects of transgenic and non-transgenic genotypes on expression of fumonisin and aflatoxin concentrations under naturally occurring population pests that are one of the main problems during the grain filling period in northern Mexico.

Conclusion

This research has shown that maize grain is infected in northern Mexico by Fusarium spp., Penicillium spp., and Aspergillus spp., and from this three genera, F. verticillioides was the Fusarium species that prevails in grain. White maize hybrids consistently exhibit reduced grain-borne fungi than the yellow maize hybrids. This is an important issue because in the following years, yellow maize will substitute white maize in a large scale in northern Mexico because of high demand of yellow grain for animal feed, therefore, this situation need to be considered in projects that will be established in the area in the near future because grain could be contaminated with mycotoxin production due to the development of fungal pathogens in the grain.

Acknowledgement

The author thank Monsanto, Syngenta and UAP seed companies, and INIFAP for providing the genetic material to perform the study.