Host

The formation of base maize populations with white or yellow endosperms began (C₀) in the Spring-Summer cycle 2007, by recombining several sources of desirable germplasm in a nursery established in the ejido of Cinta Larga, Hgo. (20° 13´ 47” N, 99° 12´ 52” O), selected due to its high incidence of head smut under natural conditions. In the Autumn-Winter cycle 2007/08, in a nursery established in a farmer’s plot in Valle de Bravo, Mex. (19° 11´ 42” N, 100° 07´ 52” O), the C₀ seeds were planted, and best plants were self-pollinated obtaining the C₀-S₁ seed. The nursery in Valle de Bravo, Mex. was established due to the presence of head smut and to advance a planting season under subtropical conditions without frost.

From this cycle onwards, a S₁ population improvement program was followed, recombining S₁ families selected for agronomic characters and resistance to head smut in the Spring-Summer cycle in Hidalgo and generating new S₁ lines in the Autumn-Winter cycle in Valle de Bravo, Mex.

In 2009, two breeding cycles were completed, resulting in C₂-S₁ seed with a total of 258 lines of white endosperm, and 71 lines of yellow endosperm. In these lines, the response to infection by S. reilianum f. sp. zeae was evaluated by artificial inoculation of seeds and crops in greenhouse conditions.

In 2010, in the state of Hidalgo, C₂-S₁ lines were planted and were simultaneaously recombined and self-pollinated, resulting in C₃ and C₂-S₂ seeds. To obtain the C₂-S₂ seed, six or seven plants of the best artificially inoculated C₂-S₁ lines were selected based on agronomic traits with a pressure of selection to head smut with 0 to 7.7% of incidence in the lines of white endosperm and 0 to 12.5% in the lines of yellow endosperm. After harvest, seeds from 123 and 114 C₂-S₂ ears with white or yellow endosperm, respectively, were inoculated, planted in the greenhouse, and evaluated for resistance to the pathogen.

Pathogen

The study used S. reilianum f. sp. zeae teliospores obtained from tassels and ears of maize plants infected in natural conditions in the state of Hidalgo. The teliospores were disinfested in a 1% CuSO₄ solution for 24 h, washed with three changes of sterile distilled water and recovered in filter paper for drying.

To determine the viability of the inoculum, a suspension of teliospores was prepared with a concentration of 50 000 teliospores mL^-1 in sterile distilled water and 0.5 mL of this suspension was spread in Petri dishes with a PDA medium. The dishes were incubated at 25 °C in darkness. The percentage of germination was determined after 96 h counting 4 compound microscope fields (40X), and the percentage of germinated teliospores was recorded.

Inoculation of the host

A suspension of teliospores at a concentration of 1.7x10⁷ spores mL^-1 was prepared in sterile distilled water with sodium carboxymethylcellulose as a sticker (NaCMC; Droguería Metropolitana, Mexico, D. F.). Twenty two seeds of each maize line, were submerged for 1 min in this suspension of teliospores and dried at 22 °C during 48 h. As a control and source of comparison, seed of the susceptible hybrid AS-910 was used.

The inoculated seeds were planted in pots with sterilized soils at a depth of 2 cm. The soil had a pH of 8.0, electric conductivity of 5.08 dSm^-1, 3.23% of organic matter and a sandy-loam texture (sand 75.6%, lime 14.5%, and clay 9.9%). The pots (40 cm wide x 40 cm high) were placed in a greenhouse and distributed in a completely randomized. The treatments were 123 lines of white endosperm and 114 of yellow endosperm. The experimental unit was a pot with 22 plants and 2 repetitions per treatment. After planting, the pots were watered at field capacity. Temperature and moisture in the greenhouse and soil were taken with a data recorder (WatchDog Micro Station, Spectrum Technologies Inc.).

Evaluation of the incidence of the disease

In C₂-S₁ and C₂-S₂ lines, resistance and susceptibilty were recorded during flowering by visual observation of the symptoms of smut, both in the ear and tassel of the plants of each line. A plant was considered as susceptible when presented sori in the ear, tassel, or both.

The percentage of incidence was determined as the quotient between plants with symptoms and the total of plants in the experimental unit, multiplied by 100; later, these values were transformed by arcsine to homogenize the variances. According to the incidence, the lines were classified according to the following scale: 0%=highly resistant, 11-25%= moderately resistant, 26-50%= moderately susceptible, 51-75%= susceptible, and 76-100%= highly susceptible. The data were anlyzed using the SAS analysis system and a mean comparison.

Evaluation of incidence in lines C₂-S₁

In the lines with white endosperm, only one was highly susceptible to the disease, with an incidence of 92.3%. In the yellow endosperm lines, the highest incidence values were moderately susceptible with 28.6, 30, 33.3, and 40%. The frequency of the disease in the 258 C₂-S₁ lines with white endosperm and 71 C₂-S₁ lines with yellow endosperm is shown in Figures 1 and 2, and the analysis of variance included in Table 1, showing that only one line with white endosperm presented an average incidence value of 92.3%, higher than the control, with incidence of 77.8% of the disease.

Figure 2 Frequency distribution of incidence of head smut in C₂-S₁ lines with white endosperm. 

Table 1 Response of S₁ lines with white or yellow endosperm to the infection of head smut (Sporisorium reilianum f. sp. zeae) with artificial seed inoculation. 

In the greenhouse, average temperatures for soil and environment were 19.4 and 22.1°C, respectively, and a relative humidty of 70%, adequate conditions for the development of the disease.

Lines with the greatest susceptibility to the disease had up to 92.3 and 40% of incidence in white or yellow endosperm maize, respectively. These results suggest that maize with white endosperm is more susceptible than those with yellow endosperm.

The frequency of the disease in the 258 inoculated lines with white endosperm was: 106 lines (41.1%) did not show the disease, 47 (18.2%) had an incidence of 1-10%, 58 (22.5%) with an incidence of 11-20%, 46 (17.8 %) with an incidence of 21-70%, only one line had an average incidence value of 92.3%, greater than in the control which had a value of 77.8% of disease incidence.

In the case of the 71 S₁ yellow endosperm lines, the distribution of the incidence was: 35 lines (49.3%) did not show the disease, 17 (23.9%) had an incidence of 1-10%, 13 (18.3%) had an incidence of 11-20%, and 6 (8.4%) had an incidence of 21-40%. In the greenhouse, the average temperature values were 19.4 and 22.1 °C for soil and environment, respectively, and a relative humidity of 70%. These values were favourable for disease development.

Evaluation of incidence in C₂-S₂ lines

The highest head smut incidence in the tassel in C₂-S₂ lines with white endosperm was 42.8% and up to 28.5% in yellow endosperm lines, whereas in the control the incidence was 86.1%.

Out of the 123 lines with white endosperm, 98 (79.7%) did not show the disease and in the 114 yellow endosperm lines, 84 (73.7%) did not show the disease (Table 2). The frequency of the disease in C₂-S₂ lines with white and yellow endosperm is shown in Figures 3 and 4. The analysis of variance is shown in Table 2.

Table 2 Response of C₂-S₂ lines with white or yellow endosperm to the infection of head smut (Sporisorium reilianum f. sp. zeae) with artificial seed inoculation. 

Figure 3 Frequency distribution of incidence of head smut in C₂-S₁ lines with yellow endosperm. 

Figure 4 Frequency distribution of incidence of head smut in C₂-S₁ lines with white endosperm. 

Figure 5 Frequency distribution of incidence of head smut in C₂-S₁ lines with yellow endosperm. 

In the greenhouse, during the development of this study, average temperatures of 18.47 °C and 21.61 °C were recorded in the soil and in the environment, respectively, and an average relative humidity of 69.76%.

The response of maize plants to infection by S. reilianum f. sp. zeae has been evaluated with the aim of identifying resistant germplasm. To do this, different seed inoculation methods have been used. ^{Njuguna and Odhianbo (1989)} reported incidences of up to 62.8% when placing teliospores on the seeds at planting time. ^{Stromberg et al. (1984)}, after inoculating the seeds with substrates contaminated with teliospores, obtained maximum incidences of 56% and 35.5% in maize lines and hybrids, respectively. In this study, seed inoculation was done with a 1% NaCMC solution and 1.7x10⁷ teliospores mL^-1 (^{Quezada et al., 2013}), producing up to 92.3% incidences in C₂-S₁ lines with white endosperm and up to 40% in those with yellow endosperm. Using seed inoculation, it is possible to simulate the natural conditions of the infection, ensuring that the pathogen is in direct contact with the host during the seed germination process (^{Prom et al., 2011}).

When advancing the C₂-S₁ lines to C₂-S₂, the proportion of lines with some degree of incidence decreased, and the proportion of lines with an incidence of 0% increased from 41.1 to 79.7% and from 49.3 to 73.7%, in maize with white or yellow endosperm, respectively. These results show that this method of inoculation leads to higher levels of infection and reliable selection in a program for the selection of germplasm resistant to head smut and helps in identifying lines with different response to infection. In this study, the presence or absence of sori in the ear or tassel was used as an indicator of the resistance or susceptibility. According to ^{Zhao et al. (2015)}, resistance is determined by the suppression of the invasion of tissues inside the plant and not with the suppression of pathogen penetration.

Not only is important to rely in an efficient inoculation method, but temperature and soil humidity are also important factors in the epidemic. In this study, an excellent germination of teliospores was determined resulting in an infection at 18.47 and 19.4 °C, which differs with ^{Matyac and Kommedahl (1985a)}, and ^{Baier and Krüger (1962)}, who report that temperatures ranging between 23 and 30 °C are ideal for the germination of teliospores and seedling infection. In this study, the results obtained can be explained by the lower temperatures in the Central Mexican Highlands, where the disease is present at seed germination and first stages of development of the seedlings, the crucial stages for the infection, or by the presence of different genetic backgrounds with different behavior depending on the weather conditions and the genotype of the host (^{Pecina et al., 2004}). The average soil humidity remained at 26.18 kPa, equivalent to 0.2618 bar, resulting in an incidence of up to 92.3% in the first cycle of evaluation. This level of humidity is different to reports by ^{Matyac and Kommedahl (1985a)}, who stated that a soil humidity of 1.5 bar leads to a greater number of infected plants, with incidences of 32%.

In most maize genetic improvement programs, evaluations and selection against diseases are done under field conditions. This study showed that it is possible to combine the use of a greenhouse (greater selection pressure in controlled conditions) while perform breeding work in the field only with germplasm that has previously been evaluated and selected in the greenhouse.

Finally, the lines selected in this study have been used in the formation of synthetic varieties with a good agronomic behavior and resistance to head smut, as well as being incorporated into breeding programs for resistance to head smut in the states of Mexico and Hidalgo.