Screening of Maize Inbred Lines Under Artificial Epiphytotic Condition for their Reaction to Turcicum Leaf Blight (TLB) and Common Leaf Rust (CLR)

Maize is among the leading cereal crops selected to achieve food self-sufficiency in Ethiopia [2]. Although, improved cultivars have been largely included in the national extension package, the national average yield of maize is only 3.45 tons/ha [3], which is far below the world average of 5.5 tons/ha. The low yield is attributed to a number of factors such as Biotic (Diseases, insect pests, and weeds), abiotic (moisture, soil fertility, etc). Among biotic factors, foliar diseases such as turcicum leaf blight (Exserohilum trurcicum) and common rust (Puccinia sorghi Schw) are generally among the important constraints in tropical maize production [4]. In Ethiopia, the two diseases can cause yield loss in the range of 12 to 61.0 percent depending up on the genotype (Unpublished data). Previously the disease was limited to specific areas and varieties, but currently the disease become very important almost in all maize growing agro-ecologies due to climate change and pathogens virulent and/or avirulent shifts.

Bako (TLB only). Maize lines were evaluated against CLR and TLB diseases. The treatment was arranged following a randomized complete block design (RCBD) with three replications for two disease types, separately. The plots were ploughed with tractor and disc harrowed twice before planting. The distance between rows and plants were 75 cm and 25cm, respectively. All plots were planted by hand with two seeds per hole. Inorganic fertilizer (Dap & Urea) and all agronomic practices were applied based on the area recommendations.

Inoculation and disease assessment
Inoculation ways: The TLB pathogens were isolated by collecting diseased maize leaf lesions and placing in a moist chamber. After two-three days newly formed spores on the surface of the lesions was picked up with the help of fine flattened needle under a dissecting microscope placed in a droplet of sterile water and streak across the surface hardened, acidified water agar in petri-plates. After 6hrs the spores start to germinate, and it was cut out of the agar and transferred to hard, acidified PDA. After two weeks of incubation at 20-25 ˚C, this culture was transferred to fresh plates of acidified PDA for multiplication. When the fungus growth was covered the surface of petri-plate fully, the cultures were ready for use. The spore (TLB) suspension@ 60,000spores/ ml was applied in the whorl using atomizer hand sprayers. Inoculation was made twice a week for three weeks, when plants were 30-45cm high.
Inoculum (rust) was collected naturally infected leaves showing large number of pustules. Collection of rust uredospore was done by lightly tapping the leaves in to a cup or a suitable container. The spores were dried and kept in tightly sealed glass jars and stored at minus 20 ˚C. Maize plants under field conditions were inoculated first time at around 6-8 leaf stage and it repeated within 2 weeks. Rust spore suspension @ 60,000 spores/ml was prepared and applied in the whorl using hand atomizer. To avoid spores clump together on the upper surface of the water, the spore suspension was agitated (stirred) continuously and tween 20 was added in the solutions.

Disease severity estimation
Maize lines were pheno typed for TLB and rust severity when the diseases are appeared using standard 1-5 scale, 1 being complete resistant and 5 being the complete susceptible [6]. Based on this rating scale over two years, maize lines were categorized into four groups namely, resistant (R) genotypes with a score < 2.0; moderately resistant (MR) 2.1-3.0; moderately susceptible (MS) 3.1-3.5 and highly susceptible (S) > 3.5.
Area under disease progress curve (AUDPC) AUDPC (% day) was calculated from severity and it was recorded 6 times at ten-day interval starting from one set of disease for each year. Disease severity was recorded from 10 randomly selected and tagged plants in each plot for AUDPC calculation. AUDPC was calculated using the formula suggested by Wilcoxson et al. [7].
Where, X i is the cumulative disease severity expressed as a proportion at the i th observation; t i is the time (days after planting) at the i th observation and n is total number of observations.

Data analysis
Analysis of variance (ANOVA) was used for disease data as randomized block design (RCBD) and following the procedure described by Gomez & Gomez [8], using SAS computer software. Mean separation was done based on LSD at 5% probability level. Disease data was analyzed after checking for good fitness to ANOVA.     (Table 1). Among26 QPM resistance maize lines, 14 (53.8%) resistance maize lines were obtained both from Bako and Ambo in the same screening season, whereas 12 (46.2) resistance maize lines were got only from Ambo screening site., in 2015 (Table 1). Moderately, responses were recorded at Ambo and Bako on seven and eighteen maize lines in 2015, respectively. Only one QPM maize line showed susceptible response at Bako experimental site (Table 1).

S.no Maize Lines
Severity ( (Table  4). Moderately resistant (MR) responses were recorded on 11 QPM maize lines, and only 2 lines showed moderately susceptible (MS) responses for this disease. Relatively higher Area under disease curve (AUDPC) andseverity of Turcicum leaf blight was recorded on the normal maize lines than QPM maize lines at Ambo, in 2016 (Table 4). Among 20 normal maize lines, 9 lines showed resistant responses for Turcicum leaf blight (TLB) at Ambo, in 2016 (Table  4). Moderately resistant responses were recorded on four normal maize lines. Moderately susceptible responses were recorded on five normal maize lines, and only 2 normal maize lines (AMB15N-37LD-27 and AMB15N-21-5) showed susceptible responses for TLB at Ambo, in 2016 (Table 4).
Among 20 QPM maize lines, five lines were showed resistant (R) responses for Common leaf rust (CLR) at Ambo, in 2016 (Table 5). Moderately resistant (MR) and moderately susceptible (MS) responses were recorded on ten and four QPM maize lines, respectively. Only one QPM maize line (AMB15QTWP3-32) showed susceptible (S) response for CLR disease at Ambo, in 2016 ( Table 5).
Out of 20 normal maize lines, eleven lines showed resistant responses for CLR disease at Ambo, in 2016 (Table 5). Moderately resistant responses were recorded on nine normal maize lines. Relatively higher AUDPC and severity of CLR was recorded on QPM maize lines than normal maize lines at Ambo, in 2016 (Table  5).

Conclusion and Recommendation
Turcicum leaf blight (TLB) and Common leaf rust (CLR) is among the major foliar diseases of maize in Ethiopia. Screening was done at Ambo (TLB and CLR) and Bako (TLB only) for two consecutive years (2015 and 2016 growing seasons) in order to know the responses of maize lines (QPM and Normal maize lines) for two diseases. This was done on 73 maize lines (53 QPM and 20 Normal) for TLB, and on 105 maize lines (53 QPM and 52 normal) for CLR. In general, higher mean severity and mean AUDPC of CLR was recorded on QPM than non-QPM (normal) maize lines whereas higher mean severity and mean AUDPC of TLB was recorded on non-QPM than QPM maize lines. Therefore, attention should be given for both diseases during screening in order to develop resistant maize varieties for both maize types. Those selected resistance maize lines from this screening should be used in breeding program and finding of resistant maize lines for both diseases will be continued using conventional and molecular methods.