CRA-Istituto Sperimentale per la Cerealicoltura, Sezione di Bergamo,

Via Stezzano 24, 24126 Bergamo (Italy)

Evaluation of maize hybrid genotypes for resistance to Aspergillus flavus*

Balconi, C, Berardo, M, Ferrari, F, Pisacane, V, Della Porta, G, Verderio, A, Motto, M

The development of plants able to overcome damages caused by fungal pathogens has been a significant challenge for maize breeders. Although selection eliminates genotypes particularly susceptible to diseases, cultivated hybrids frequently show serious fungal infection (Munkvold, Annu. Rev. Phytopathol., 41:99-116, 2003).

Aspergillus flavus and Aspergillus parasiticus are responsible for both pre- and post-harvest accumulation of aflatoxins (AF) in maize; concern about aflatoxin contamination is due to its potential carcinogenicity (Counc.Agric.Sci.Technol.Rep., CAST, Ames, IA, 2003). Aflatoxin B1 is the principal member of the family; it has an extremely high carcinogenic potential to some species of animals and a widespread occurrence in some food (Moreno et al., Plant Breed. 118:1-16, 1999).

In Italy, attention was focused on aflatoxins in 2003, when particularly favourable climatic conditions caused heavy A. flavus attack of maize. Milk produced by farm livestock feed with maize grains contaminated by A. flavus, showed an unusual presence of aflatoxin M₁ (AFM₁ milk toxin) (Piva and Pietri, Informatore Agrario, 14: 7-8, 2004).

A limiting factor in breeding for aflatoxin resistance is the spatial and temporal variation in aflatoxin accumulation that requires inoculation and a high number of plants, the lack of a reliable and inexpensive screening methodology, and the low metabolic activity of maize plants after physiological maturity (Payne. Crit. Rev. Plant Sci.; 10:423-440, 1992). In maize, resistance to aflatoxin is under genetic control and a large genotype variability for such character has been found. Studies on this field allowed to identify and to develop sources of genetic resistance, such as inbred lines ( Mp420, Mp313E, Mp715, Tex6, LB31, CI2) and populations (GT-MAS: gk) (Betran et al. Crop Sci., 42:1894-1901, 2002). However, the majority of these sources of resistance lack acceptable agronomic performance and adaptation that preclude their direct use in commercial hybrids. Current efforts are to map and characterize the genetic factors involved in the resistance and to transfer them through marker-assisted selection to more suitable elite genotypes (Rocheford and White, Proc. Aflatoxin/Fumonisin Workshop 2000, Yosemite, CA, http:www.nal.usda.gov/fsrio/ppd/ars06.pdf, 2002.

Beneficial secondary traits such as husk covering and tightness, physical properties of the pericarp, and drought or heat stress tolerance are factors contributing to aflatoxin resistance. In general, the hybrids with good husk cover show a greater resistance to insect damage and accumulate lower levels of aflatoxins (Betran et al. Crop Sci., 42:1894-1901, 2002). The incidence and severity of A.flavus infection and aflatoxin contamination are highly dependent on genotype, cultural practices, and environmental conditions (Brown et al., In K.K.Sinha and D Bhatnagar (eds), Mycotoxins in agriculture and food safety, Marcel Dekker, New York., 1998).

Reliable methods for screening and evaluation of maize genotypes for improving tolerance to Aspergillus attacks is a valuable tool in breeding programs to increase crop protections against fungal diseases. Accordingly, the aim of our research was to evaluate and compare 34 maize hybrids (FAO 300-400-500-600-700) for A. flavus resistance and for aflatoxin accumulation in field trials. The test included: i)self pollinated ( A.flavus) inoculated ears, ii) self-pollinated non-inoculated ears (SIB), iii) sterile water inoculated ears. The inoculation experiment was replicated at two different planting dates. Environmental conditions, such as temperature and rainfall, were recorded.

At pollination, silk channel (region within the husk between the tip of the cob and tip of the husk where the silks emerge) length was recorded for each hybrid; variability for this trait was observed among the genotypes, with values ranging from 3.1 cm to 10.6 cm (average: 7.0 ± 1,8). Ten hand pollinated plants per plot were inoculated with a fresh spore suspension (mixture of 5 A.flavus isolates from Northern Italy, supplied by Dr. Battilani-University of Piacenza), 7 days after pollination (DAP), by the non-wounding Silk Channel Inoculation Assay (SCIA method, Zummo and Scott . Plant Disease, 73:313-316, 1989) applied to each primary ear. The silks of each primary ear were inoculated spraying 1,5 ml of 10⁸ spore/ml fungal suspension; controls were non-inoculated and sterile water-inoculated plants.

At maturity, ears were manually harvested and husk cover was evaluated using a visual rating ranging from 1 (good tight long husks extending beyond the tip of the ear) to 5 (poor:loose short husks with exposed ear tips). Also at this stage, variability among hybrids was recorded for husk morphological trait; for this parameter 9 hybrids scored 1 (ear tip un-exposed), 21 scored between 1 and 2 (1-2 cm ear tip exposed), and 4 scored between 2 and 3 (2-4 cm ear tip exposed).

After hand de-husking; the severity of ear A. flavus attack was evaluated using rating scales (% of kernels with visible symptoms of infection, such as rot and mycelium growth; Disease Severity Rating, DSR, ranging from 1=0%-no infection, 2=1-3%, 3=4-10%, 4=11-25%, 5=26-50%, 6=51-75%, 7=76-100% of visibly infected kernels/ear, see Reid et al. Technical Bull, 1996-5E, Research Branch, Agriculture and Agri-Food Canada, 1996). Individual ear rating using a visual scale, as described above, allowed a discernible screening of the 34 hybrids tested for A.flavus resistance; variability in the hybrid response was observed (DSR: 2,45 ± 0,96). For all entries, non-inoculated (SIB) and sterile water-inoculated ears, as control, had absence or very low disease symptoms (DSR respectively, 1.02±0.06 for SIB and 1.01 ± 0.03 for water-inoculated). This result indicates that the non-wounding silk channel inoculation technique applied was effective in inducing A.flavus attack.

After visual inspection, ears of each plots were dried, shelled, and the kernels bulked. To evaluate internal kernel infection, 50 kernels, randomly chosen from each sample, were surface-disinfected and plated on DRBC agar (King et al., Appl. and Environ. Microbiol., 37: 959-964, 1979). Seven days after plating, percentage of kernels showing visible Aspergillus mycelium, was calculated. Also for this parameter variability among inoculated hybrids was observed , with value of contaminated kernels ranging from 0 to 88 % (average 16.4±1.5). In contrast, controls showed a percentage of internal contaminated kernels lower than that observed in the corresponding inoculated hybrids (SIB: 0.94 ± 1.81, water-inoculated control: 0.6 ± 1.03).

The levels of AFB₁ in ground grain samples of hybrids under study was evaluated using enzyme-immunoassay-ELISA kit (Kit Ridascreen-Aflatoxin B₁ 30/15-R-Biopharm-Art. Not: R1211). AFB₁ level for inoculated hybrids ranged from 0 to 80 µg/kg (average: 27± 4.8), while in the controls was present in traces or absent (SIB: 2.0 ± 2.8; water-inoculated control 2.0± 5.0). Also in this case variability occurred among hybrids under investigation.

Correlations between visual ear rot ratings, internal kernel infection evaluation, aflatoxin content, silk channel length at pollination, husk cover ratings, are in progress.

*The work was developed within the framework of the Research Program AFLARID, Italian Ministry of Agriculture, Rome, Italy