Potential of popcorn germplasm as a source of resistance to ear rot

Because of its multi-purpose nature, popcorn has sparked the interest of the World Trade Organization as regards fungal contamination by mycotoxins. However, no investigations have been conducted on popcorn for resistance of genotypes to ear rot. The aim of this study was to evaluate the potential of popcorn genotypes as to resistance to ear rot and rotten kernels, as an initial step for the implementation of a breeding program with the popcorn crop in Northern Rio de Janeiro State, Brazil. Thirty-seven accessions from different ecogeographic regions of Latin America were evaluated in 2 cultivation periods, in a randomized block design with 4 replications. We evaluated the incidence of rotten ears, incidence of rotten ears caused by Fusarium spp., severity of ears with Fusarium spp. rot, and PLANT BREEDING Article Potential of popcorn germplasm as a source of resistance to ear rot Railan do Nascimento Ferreira Kurosawa1, Antonio Teixeira do Amaral Junior1*, Janieli Maganha Silva Vivas2, Amanda Gonçalves Guimarães1, Samila Barbosa Miranda2, Vicente Mussi Dias2, Carlos Alberto Scapim3 1. Universidade Estadual do Norte Fluminense “Darcy Ribeiro” Centro de Ciências e Tecnologias Agropecuárias Laboratório de Melhoramento Genético Vegetal Campos dos Goytacazes (RJ), Brazil. 2. Universidade Estadual do Norte Fluminense “Darcy Ribeiro” Laboratório de Entomologia e Fitopatologia Campos dos Goytacazes (RJ), Brazil. 3. Universidade Estadual de Maringá Programa de Pós-Graduação em Agronomia Maringá (PR), Brazil. *Corresponding author: amaraljr@pq.cnpq.br Received: May 11, 2016 – Accepted: Nov. 11, 2016 incidence of rotten kernels. The results were subjected to analysis of variance, and means were compared by the Scott-Knott clustering test (p < 0.05). A significant effect was observed for all evaluated variables, characterizing them as efficient in the discrimination of genotypic variability for reaction to fungal injuries in popcorn. The gene pool of the tropical and temperate Germplasm Collection evaluated here has the potential to generate superior segregants and provide hybrid combinations with alleles of resistance to diseases affecting ears and stored kernels. Based on the different variables and times, the experiment was conducted, and genotypes L65, L80, and IAC 125 showed the highest levels of resistance.


INTRODUCTION
Mycotoxins are products of the secondary metabolism of fungi that can affect one's health when ingested, inhaled, or in simple contact with the skin.Mycotoxins can appear in food primarily by fungal infection of crops from agricultural productions, being directly absorbed by men, or indirectly, when contaminated diets are supplied to animals in whom mycotoxins can accumulate in different organs or tissues and enter the human diet through consumption of meat, milk, and eggs (Marin et al. 2013).In the Italian market, for instance, the presence of mycotoxins was detected in baby foods in at least 31% of cereal-based nutrients, and 27% of contaminated samples showed simultaneous occurrence of different mycotoxins.The mycotoxin levels found reveal a situation that puts at risk the health of vulnerable populations, as is the case of the infant group (Juan et al. 2014).A study in Northern Nigeria investigated the occurrence of mycotoxins in urine samples of 120 farmers of the region, most of whom depended on corn, sorghum, peanut, and millet growing.Of the studied samples, 50.8% showed contamination by mycotoxins (Ezekiel et al. 2014).
In general, questions about food safety are complex, requiring more than 1 control measure to effectively overcome the risk posed to health, which includes actions involving the different steps of the food production chain (Pitt et al. 2013).In the agricultural production scenario, the occurrence of damaged, namely rotten kernels, is a consequence of ear rots in corn, which is always related to the presence of mycotoxins, caused by fungi of the genus Fusarium spp. in batches of grains stored either for human consumption or to be used as a component in the formulation of animal diets (Juliatti et al. 2007).Besides the hazard of contamination by mycotoxins, the effects of fungal development markedly reduce grain yield and the quality of the end product (Mendes et al. 2012).Moreover, fungi of the genus Fusarium spp.have caused serious damage to cereal production, resulting in reductions of 10 to 40% (Bottalico and Perrone 2002;Linkmeyer et al. 2016).Nevertheless, the quantification of economic losses in popcorn production remains unknown.
Only in the last decade, studies about the control of diseases in popcorn have become a concern to breeders (Arnhold 2008;Vieira et al. 2011;Sanches et al. 2011;Vieira et al. 2012;Noor et al. 2015;Vieira et al. 2016).To complicate matters, no research has been developed on resistance to ear rot in popcorn.In this regard, the evaluation of germplasm banks is of great importance during pre-breeding, mainly to support breeding programs in the identification of sources of resistance genes (Sanches et al. 2011;Resh et al. 2015;Vivas et al. 2015).
Corn has a great genetic importance worldwide held in genetic reservoirs, such that there is no shortage of favorable alleles in germplasm banks capable of contributing, among other aspects, with resistance to diseases, tolerance to abiotic stresses, greater productivity, and better nutritional quality (Prasanna 2012).The institution of development of this study has a popcorn germplasm bank with accessions from different eco-geographic regions that have been made available for research focused on resistance to diseases.This collection contains germplasm from Brazil and abroad -Latin America, Mexico, and USAobtained from grants and exchanges.
Given the impacts of mycotoxins, along with the scarcity of investigations on resistance to ear rot in popcorn, the present study was developed to evaluate the performance of genotypes from the Germplasm Collection concerning ear rot by quantifying the incidence and severity of symptoms and the percentage of rotten kernels resulting from susceptibility to infection by Fusarium spp.

Genotypes
Thirty-seven accessions of popcorn from the Germplasm Bank were used to evaluate their reaction to Fusarium ear rot (Table 1).A randomized block design with 4 replications was adopted in the 2 harvests.Experimental units consisted of simple 3.0 m rows containing 16 plants per row.The experimental field was prepared mechanically by harrowing followed by furrowing.Seeding was performed manually, with 0.2 m spacing between plants and 0.9 m between rows.Cultivation practices included seedling thinning 15 days after emergence, control of weeds by hoeing, periodic irrigation with maintenance of soil at field capacity, and topdressing performed 30 and 45 days after emergence using a 20-0-20 NPK formulation with 300 and 200 kg•ha −1 urea, respectively.

Implementation of the experiment
The ears of each genotype were harvested by visually observing when the bracts were dry, at each cultivation period, to prevent early or late harvesting.

Evaluated traits
Incidence of rotten kernels (IRK): after the ears were threshed, two 100-kernel samples were counted and represented the portions of work.The incidence of rotten kernels was expressed in percentage, determined according to the criteria for classification of corn established by Ordinance no.11 from the Brazilian Ministry of Agriculture, Livestock and Food Supply, of April 12, 1996.The method consists of the visual segregation of symptomatic kernels showing discoloration in over ¼ of their total surface.
Incidence of rotten ears (IRE): determined adopting the total number of ears obtained from the harvest of each experimental unit.Ears infected at more than 50% by the action of ear-rot fungi were counted, and the incidence of rotten ears was the result of the division between the number of rotten ears and the total number of ears harvested per plot on the field, with values expressed in percentage.
Severity of ear rot caused by Fusarium spp.(SRF): the diagrammatic scale for ear rot caused by Fusarium spp.proposed by the International Maize and Wheat Improvement Center (CIMMYT 1985) was adopted, with values of 0, 10, 20, 30, and 40%, according to the rot severity level.
Incidence of ears damaged by Fusarium spp.(IDF): after the characteristic symptom of the disease was detected, all symptomatic ears infected by Fusarium spp.were counted.The incidence was obtained in percentage, as the division between the number of symptomatic ears and the total number of ears harvested per plot on the field, with values expressed in percentage.

Statistical analysis
Data were subjected to analysis of variance, and, when a significant difference was detected, the Scott-Knott means-clustering algorithm was used (p < 0.05).All analyses were performed using the computer resources of Genes software (Cruz 2013).

RESULTS AND DISCUSSION
There was a significant effect of the sources of variation genotype and time for the group of evaluated traits (data not shown), revealing the existence of genotypes with contrasting responses, among the evaluated accessions.This is of fundamental importance for applied breeding, from the perspective of potentiation of variability for the generation of superior segregants, or even for studies of heritability.These results also suggest that the 2 planting times were distinct enough to cause differences between the evaluated traits.The source of variation genotype versus time interaction revealed a significant effect on IDF, SRF, and IRK, whereby we concluded that the genotypes have different performances, determining the use of estimates of means based on each cultivation time.
The means-clustering test for IDF performed for the conditions of the first harvest formed 4 groups (Table 2).
Th e group of genotypes resistant to the occurrence of the disease was composed of 18.92% of the studied accessions, namely L80, L65, L70, from UENF; varieties PARA 172, URUG 298, Amarelo, and URUG 298 Roxo, from CIMMYT; and, lastly, hybrid IAC 125 (Table 2).Th ere were no resistant genotypes in this harvest originating from UEM.For the second harvest, the means-clustering test formed 3 groups.The group of most resistant genotypes corresponded to  Concerning SRF, for the first harvest, 2 statistically distinct groups were formed regarding severity of Fusarium ear rot.The group of resistant genotypes included 32.43% of the studied accessions, and variations between resistant and susceptible accessions reached 82.68% between genotypes BOYA 462 and L55, respectively (Table 2).For the second harvest, the means test generated statistically distinct groups, among which the group of most resistant genotypes comprised 37.84% of the studied accessions that responded positively to severity of Fusarium spp.The most significant variation between resistant and susceptible genotypes reached the magnitude of 71.19% (Table 2).

Genotype
For a reliable indication of promising genotypes in terms of resistance to ear rots, especially those caused by Fusarium spp, it is necessary to study the set of factors influencing the occurrence and severity of post-harvest diseases.Thus, considering the 3 variables simultaneously and the cultivation times, it was found that lines L65, L80 and triple hybrid IAC 125 possess favorable alleles for traits that provide resistance to ear rots, such as those caused by fungi of the genus Fusarium spp.Pacheco et al. (2005), evaluating cycles of selection of BRS Angela, observed that, up to selection cycle VI, variety BRS Angela showed higher levels of ear health when compared with controls IAC 112 and Zélia.This fact may indicate congregation of favorable alleles for resistance to ear diseases for lines extracted from the genetically improved cultivar BRS Angela, as is the case of line L65.Inferring about the potential of popcorn cultivars, Miranda et al. (2003) evaluated variety Viçosa for agronomic traits, and, for percentage of diseased ears, this cultivar showed an average resistance of 20% in relation to the others investigated in the range of 10% to 30%.Regarding triple commercial hybrid IAC 125, the estimates for ear health are possibly linked to the good performance per se of the genotype.
For the trait IRK, 3 groups were formed in the first harvest, whereas there was no statistical difference between the genotypes for the second cultivation period (Table 3).That being said, the factor cultivation time significantly changed the rates of contamination of in rotten kernels, making the season effect remarkable.The first harvest, in general, favored the occurrence of rotten kernels for all studied genotypes and, therefore, all accessions evaluated showed values above the maximum tolerated limits for commercial classification, according to Normative Instruction no.61 of the Brazilian Ministry of Agriculture, Livestock, and Supply (MAPA) of December 22, 2011.The second harvest significantly favored the health quality of the kernels, at a magnitude of 54.05% of the genotypes evaluated when compared with the first harvest.However, the values fitting the maximum commercial tolerance limits established by MAPA in 2011 do not necessarily apply to all genotypes that showed significant reductions of rotten kernels between the first and second harvests.For instance, lines L51, L52, and L75 showed significant reductions in the rotten-kernel incidence between periods; however, their lowest incidence values did not meet the minimum tolerance requirements established.However, at the second harvest, 54.05% of genotypes fitted type 01, with variations of 0% to 1.75% of rotten kernels; 21.62% fitted type 02, with variations of 2.12% to 2.87% of rotten kernels; and 16.21% of genotypes fitted group 03, with variations of 3% to 4.5% of rotten kernels (Table 3).It is known that the rotten kernels are a reflection of ear rots, caused mainly by fungi, still on the field.Initially, it was found that genotypes L65, L80, and IAC 125, which best stood out against ear rots, were also present in the group of genotypes most resistant to the incidence of kernel rot, and are thus promising to be used in breeding programs.

CONCLUSION
The adopted variables were efficient in detecting the existing variability for severity of Fusarium spp., allowing a reliable discrimination of superior accessions.
Genotypes L65, L80, and IAC 125 displayed the highest levels of resistance to incidence and severity of rot caused by Fusarim spp.and can thus be considered of interest for the introgression of resistance genes in popcorn breeding programs.

Table 1 .
Description of popcorn genotypes from the Germplasm Bank.

Table 2 .
Incidence of ears damaged by Fusarium spp.and severity of ear rot caused by Fusarium spp. in 37 popcorn genotypes evaluated in the fi rst (2013/2014) and second (2014) crops.
.*Means followed by common lett ers represent a statistically homogeneous group by the Scott-Knott algorithm.IDF = Incidence of ears damaged by

Table 3 .
Incidence of rotten kernels in in 37 popcorn genotypes evaluated in the first (2013/2014) and second (2014) harvests.
*Means followed by common letters represent a statistically homogeneous group by the Scott-Knott algorithm.