CHAPTER 4: PHAEOSPHAERIA LEAF SPOT RESISTANCE IN SOUTHERN
4.2 Materials and Methods
Inbred lines used in the study comprised a sample from the following heterotic groups and their derivatives: a) CIMMYT (International Maize and Wheat Improvement Centre):
A and B; and b) Southern African: P, K64R, SC, N3, M and I group (Table 4.1). Gevers and Whythe (1987) and Mickelson et al. (2001) presented detailed descriptions of these groups. The 27 inbred lines used consisted of six GLS-resistant and six drought-tolerant ones from CIMMYT, and 15 conventional lines obtained from Seed Co in Zimbabwe.
These inbred lines are adapted to tropical east and Southern Africa. Inbred lines were divided into eight sets of three each; hence three inbred lines in one set were used as females and crossed with three inbred lines from another set used as males (Appendix 1), according to a North Carolina Design II Mating Scheme (Comstock and Robinson, 1948; 1952; Hallauer and Miranda, 1988). Each inbred line was used once as a female parent in one set and once as a male parent in another set, except the inbreds K64R, B11 and B12, which were used as females only, and CML489, A26 and CIM24, which were used as male parents only (Appendix 1, in sets 8 and 4, respectively). The inbreds CML489, CIM24 and A26 replaced K64R, B11 and B12 as male in set 4, which had failed to provide adequate pollen due to severe attack by maize streak virus.
Consequently, eight sets of hybrids comprising nine hybrids each were formed among the 27 inbreds. The 27 inbred lines, their 72 crosses and 24 standard hybrids that were used as controls effectively constituted the reference population for this study. Among the 24 standard hybrids SC627 and SC513 were used as GLS resistant check hybrids, while SC403, SC633, PAN6777, ZS255, ZS257, R201 and R215 were widely grown hybrids throughout Southern Africa. The PLS resistance of the standard hybrids was not known at the beginning of the study.
Table 4.1: Parent Inbred Lines used in a Design II Mating Scheme
Inbred Designation Heterotic Group Principal Selection Criteria
1 CML442 A Drought Tolerance
2 CML312 A Drought Tolerance
3 CML445 AB Drought Tolerance
4 CML395 B Drought Tolerance
5 CML444 B Drought Tolerance
6 CML488 B Drought Tolerance
7 A7 M Conventional
8 A8 M Conventional
9 A9 P Conventional
10 K64R K Conventional
11 B11 K Conventional
12 B12 K Conventional
13 A13 A GLS Resistance
14 A14 A GLS Resistance
15 A15 N GLS Resistance
16 B16 I GLS Resistance
17 B17 B GLS Resistance
18 B18 B GLS Resistance
19 B19 K Conventional
20 B20 KB Conventional
21 B21 K Conventional
22 B22 S Conventional
23 B23 S Conventional
24 B24 S Conventional
25 CML489 AB Conventional
26 A26 I Conventional
27 CIM24 A Drought Tolerance
4.2.2 Experimental Design
Hybrids were evaluated at Cedara (CED; 1076 m altitude) in South Africa and Rattray Arnold Research Station (RARS; 1350 m altitude) in Zimbabwe. Due to inadequate seed for some crosses, only 64 hybrids comprising 57 experimental and seven standard hybrids were evaluated in 2003/4 and the experiments were laid out as 8 X 8 simple lattice design. Ninety-six hybrids comprising 72 experimental and 24 hybrid checks were screened in 2004/5 and the experiments were laid as 12 X 8 alpha lattice block designs with two replications. Inbred parent lines were evaluated in 6 X 5 α-lattice designs at RARS in 2004/5. Established plant populations (plants/ha) were 44000 at Cedara and 53000 at RARS, while fertiliser (kg/ha) was applied as follows: 120 N: 33 P: 44 K at Cedara and 208 N: 35 P: 21 K at RARS. Total precipitation was as follows: 853 mm at Cedara (2003/4); 826 mm at RARS (2004/5) and 885 mm at Cedara (2004/5). Standard cultural practices, including hand planting, hand weeding and application of herbicides was followed, and fields were left to natural disease inoculation. Disease development was monitored every fortnight beginning from tassel emergence. Phaeosphaeria leaf spot severity was assessed at 50% silk emergence (PLS1) and at hard dough stages (PLS2) based on the visual assessment of the whole plot by estimating (i) percentage
leaf area diseased or necrotic (% LAD) and (ii) a rating scale of 1 to 9. Using this scale, 1 = no disease, 3 = lesions on lower leaves, 5 = disease on most leaves and lower leaves dead, 7 = lower leaves dead and many lesions on all leaves above the ear and 9
= almost all leaf tissue is dead or necrotic (Munkvold et al., 2001). At harvest grain yield was measured on a whole plot basis following standard practice used at CIMMYT (CIMMYT, 1985) and was adjusted to 12.5% moisture using the formula:
Grain Yield (t/ha) = [Grain Weight (kg/plot) x 10 x (100-MC)/ (100-12.5)/(Plot Area)], where MC = Grain Moisture Content.
Mid parent heterosis (MPH) for PLS was calculated for each hybrid (Falconer, 1989) using the formula:
MPH (%) = (F1-MP)/MP x 100,
Where F1 = mean of the F1 hybrid performance, MP = mean of two parents making the cross using the formula: (P1 + P2)/2, where P1 and P2 are the means of the inbred parents.
4.2.3 Statistical Analyses
General analyses of variance were performed for all hybrids and inbred lines, including control hybrids using IRRISTAT (2003) computer package for PLS and grain yield data for each site. Genetic analyses for PLS were performed in SAS (SAS Institute, 1997) as a fixed effects model for experimental hybrids, as described by Hallauer and Miranda (1988) for across environments using the following linear model:
Yijkpq = µ+Sp + gi(Sp) + gj(Sp) + hij(Sp) + Eq + rk(SE)pq + (ES)pq + (Eg)iq(Sp) +(Eg)jq(Sp) + (Eh)ijq(Sp) + eijkpq
Where i =1, 2, 3; j = 1, 2, 3; k = 1, 2; p = 1, 2, 3, 4, 5, 6, 7, 8; q = 1, 2 and Yijkpq denotes the value of the hybrid of a mating of the ith female line, the jth male line, in the kth block, within set p and in the qth environment. The terms are defined as follows:
µ = Grand mean
Sp = the average effect of the pth set
gi(Sp) = the GCA effect common to all hybrid of the ith female line nested within pth set, gj(Sp) = the GCA effect common to all hybrid of the jth male line nested within pth set,
hij(Sp) = the SCA effect specific to hybrid of the ith female and jth male line nested within pth set, Eq = average effect of qth environment
r(SE) = the effect of the kth replication nested within the pth set and qth environment
(ES)pq = the interaction between set effects and the environment
(Eg)iq(Sp) and +(Eg)jq(Sp) = the interaction between environment and GCA nested within sets (Eh)ijq(Sp) = the interaction between environment and SCA nested within sets
eijkpq = the random experimental error.
According to Hallauer and Miranda (1988), the main effects due to females (sets) and males (sets) are equivalent to the GCA (general combining ability), while male x female (sets) interaction effects represent the SCA (specific combining ability) effects in a diallel mating. General combining ability and SCA effect estimates for inbred lines and crosses, respectively, were determined by line x tester analysis in Agrobase (2005) computer package. Using the variance ratios in REML (GenStat, 2003), heritability estimates were calculated as suggested by Hallauer and Miranda (1988) for the fully inbred parents (F = 1) (where σ2m = σ2f = ½σ2A; and σ2mf = σ2D ) using the formulae:
a) h2 = 2σ2m/(σ2/r + σ2mf + 2σ2m) for one environment, and
b) h2 =2σ2m/( σ2/re + σ2fme/e + 2σ2me/e + σ2mf + 2σ2m) for across environments,
Where σ2m = male(set) variance, σ2 = random error variance; σ2mf = male x female (set) variance; σ2fme = environment x male x female (set) variance; σ2me = environment x male (set) variance; r = number of replications and e = number of environments. σ2f = fema (set) variance; σ2A = additive variance and σ2D = dominance variance. Heritability estimates were calculated using the male variance (σ2m) to avoid the upward bias of heritability due to maternal effects if the female variance (σ2f) was used.