Maize Genetics Cooperation Newsletter vol 81 2007
CRA - Istituto Sperimentale per la Cerealicoltura
The
development of plants able to overcome damage 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 fed 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).
Some
limiting factors in breeding for aflatoxin resistance
are the spatial and temporal variations in aflatoxin
accumulation that require 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 large genotype variability for this trait has been
found. Studies in this field
allowed identification and development of 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
which precludes their direct use in commercial hybrids. Current efforts are to map and
characterize the genetic factors involved in 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 are a valuable tool in
breeding programs to increase crop protection 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) using the non-wounding Silk Channel
Inoculation Assay (SCIA method, Zummo and Scott, Plant
Disease 73:313-316, 1989). The
silks of each primary ear were inoculated with 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 this 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% 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 no 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 plot 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. Environ. Microbiol. 37: 959-964, 1979).
Seven days after plating, percentage of kernels showing visible Aspergillus
mycelium was calculated. Variability
among inoculated hybrids was also observed for this parameter, with the 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
level of AFB1 in ground grain samples of the 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 �g/kg (average: 27 � 4.8), while in the controls AFB1 was
present in trace amounts or absent (SIB: 2.0 � 2.8; water-inoculated control
2.0 � 5.0). In this case,
variability also occurred among hybrids under investigation.
Studies
of the correlations between visual ear rot ratings, internal kernel infection
evaluation, aflatoxin content, silk channel length at
pollination, husk cover ratings, are in progress.
*This work was developed within the framework
of the Research Program AFLARID, Italian Ministry of Agriculture, Rome, Italy.
Please Note: Notes submitted to the Maize Genetics Cooperation
Newsletter may be cited only with consent of authors.