Relationship between Components of Resistance to Late Leaf Spot in Groundnut Botanical Genotypes

| Late leaf spot (LLS) is the most destructive, widespread

Valencia and Spanish types, has an upright-growth habit (bunch type) with a growth period of three to four months and seeds without dormancy. Seeds of running types are usually used for direct consumption and confectionary purposes, whereas those of the Valencia and Spanish types are generally grown for oil extraction. (De Waele and Swanevelder, 2001). Pixley et al. (1990) compared LLS epidemic rates and leaf area dynamics on the susceptible cv Florunner and three other partially resistant lines. Percent necrotic area in three leaf canopy layers (estimated by using a modified Horsfall-Barratt diagram), defoliation of the main stem (determined by counting missing leaflets) and leaf area index were recorded at seven to 10 day intervals. The leaf area index (LAI) was calculated as: LAI = specific leaf area x fraction leaf x biomass. This technique assumes that specific leaf area and the ratio of leaf weight to total aboveground plant weight (fraction leaf ) are similar for neighboring plants of the same age and genotype. The specific leaf area is the ratio of leaf area to leaf mass. Leaf spot induced defoliation of Florunner progressed more rapidly on the other three partially resistant lines. Maintenance of higher LAI by the partially resistant lines was associated with sustained leaf production until maturity.
Objective of improvements in varieties of groundnut have included resistance to diseases especially leaf spot and other agronomic desirable characteristics. In last 60 years of groundnut production in Pakistan about 12 lines have been released for commercial cultivation. The groundnut varieties like B-4, Accession No. 45 and Accession No. 334 released before 1980 were developed under irrigated ecological conditions. These varieties were released for cultivation in all over the Pakistan but their responses to diseases had not been studied (Hussain and Ahmed, 1984). Later on variety BARI-11 (Naeem et al., 2012), was developed at Barani Agricultural Research Institute Chakwal which is drought tolerant, has more shelling percentage and has resistance against leaf spot and root rot diseases.
The aim of present study was to evaluate groundnut germplasm to sort out sources of resistance against LLS disease under field and lab conditions in semiarid tropics of Pakistan.

Materials and Methods
Screening under field conditions In total, 153 groundnut genotypes were collected from different sources to investigate the sources of multivariate resistance against LLS under field conditions and detached leaf assay (Table 7). Among these 103 genotypes are Virginia, 45-Spanish and 5-Valencia botanical type. The experiment primarily was laid out in completely randomized design. All the genotypes were sown in plots of size measuring 1m × 0.3m (single row of 1m length) across the fertility and slop gradient. The sowing was done in already infested fields (Coffelt and Porter, 1986) and additionally LLS diseased leaves were collected at harvesting of previous year's crop and kept under room conditions in craft paper bags were added in soil at time of sowing (Kishor et al., 2005). LLS naturally infected leaves were collected from farmer fields and LLS spots were excised and kept under a moist chamber lined with aluminum foil for 48 hours. These sporulated excised spots were blended in Molinex to obtain spore suspension for artificial inoculation. LLS conidial suspension (2 × 10 4 mL -1 ) was maintained under heamocytometer and sprayed inoculum on onset of summer rainfall.

Assessment of spots per leaf (SL) and defoliation (Def )
Severity of leaf spot on groundnut entries was evaluated. Numbers of spots per leaf (SL) were counted. Defoliation (Def ) was assessed on 1 to 10 scale where 1 is no defoliation and 10 is 100% leaves defoliated. Degree of sporulation (S) was determined on ten leaflets with infected leaves collected from 3 rd and 4 th node of randomly selected 5 plants from each plot. Collected leaf lets were washed with tap water and swabbed with cotton thereafter incubated    (Pande and Rao, 2001). All lesions of sample were examined under stereoscope (Swift SM80HF, Made in Japan). Disease data were recorded and sporulation assessed on 0-5 scale modified from (Melouk and Banks, 1984) described in detail where, 0; No sporulation (Immune), 1: Spars sporulation, one to two stromata sporulating (Highly Resistant) 2; More than two stromata sporulating but less than half of total stromata on a spot (Resistant) 3; Sporulation moderate, half of the total stromata sporulating (Moderately susceptible) 4; Whole of the spot sporulating, (Susceptible) and 5; Heavy sporulation. Spores long, arose looking like mycelium and sometimes stromata sporulate on both sides of leaf (Highly susceptible).
In earlier studies assessments were based on sporulated stromata out of total stromata after counting of total stromata. Whereas, present studies emphasis is on number of sporulated stromata and intensity of sporulation rather on non-sporulated stromata. Data were recorded under stereoscopic microscope at 2X and 4X magnifications according to ease of counting.
Statistical analysis ANOVA-1 was used for one-way comparison of means and significant variate was correlated to study their dependency on each other.

Results and Discussion
Screening of groundnut germplasm under field conditions against LLS during consecutive three years First year: First observation on disease severity was recorded at flowering stage and none of genotypes showed diseased symptoms. Five genotypes exhibited minimum disease severity and showed moderately resistant response. Whereas, 31 genotypes were moderately susceptible, 100 were susceptible and 17 were found highly susceptible with more than 51 % diseased leaf area. Second observation was done at pod development stage, where only one genotype i.e. Chakori showed moderately susceptible response, while others 120 genotypes were susceptible and 32 genotypes showed highly susceptible response to LLS under field conditions (Table 1).

Leaf Spot Reaction Index (LSRI)
Non-consistent grouping of groundnut germplasm under field plot screening necessitated to study more than one variate in addition to diseased area per leaf.
One-way analysis of variance of multiple variables for LLS expression on botanical groundnut genotypes showed that spots per leaf (SL), sporulation (S), SL × S, SL × Diseased area (DA), and Defoliation (Def ) × SL variables and interactions were significant to measure the disease expression (     (Table 4).
Correlation between SL and DA values showed increase in number of infections resulted in increase in diseased area per leaf in Virginia, Valencia and pooled analysis of genotypes but in case of Spanish type plants number of infection lesion did not cause increase in diseased area per leaf significantly. It may be concluded that lesion size was smaller in Spanish type plants. Correlation between SL and S has same trend in all plant types. There was a negative correlation between Def and SL. Correlation between interaction of SL × S and S are significant and positive (Table 6). Previous studies showed that amount of SL, Def, S and reaction between amount of SL and S showed a significant difference in wild and cultivated groundnut entries. Groundnut genotypes were grouped on bases of more or less sporulation (Melouk and Banks, 1984;. To obtain maximum sporulation necrotic area of LLS on groundnut leaves was removed. It is thought that more necrotic area results in more sporulation of fungus under optimum humidity and temperature conditions (Creen and Wynne, 1986;Nova et al., 1989). In general number of conidia per lesion was significantly higher in susceptible genotypes than in resistant genotypes (Rao et al., 1995).
Selection of genotypes with low sporulation levels could be expected to identify genotypes with desirable levels of other resistance components. A high level of resistance to LLS was identified in groundnut lines derived from interspecific crosses with A. durenensis. These homozygous lines were used as parents to incorporate resistance into high yielding breeding lines and to produce a segregating population for molecular marker studies (Anderson et al., 2000).
In Late groundnut host pathogen system (Nova et al., 1989) obtained sporulation by incubating necrotic area of groundnut leaves under high humidity moist chamber conditions. Resistance to LLS could be associated with low partitioning, late maturity and undesirable pod and seed characteristics (Nigam and Dwivedi, 2000). Luo et al. (2005) identified genes for resistance to LLS using micro array and real-time polymerase chain reaction (PCR). They detected 56 genes in several functional categories which could be used for marker-assisted selection in breeding programs.
In present study number of spots (necrotic areas) x sporulation was significant rather than diseased area (necrotic area + yellow hallo). The susceptibility of plant organ was also affected by the age of leaves; older leaves were more susceptible than younger ones. More resistant varieties, which were affected on their younger leaves, can suffer severe damage on their older leaves (Raymond et al., 1985). High defoliation scores after monsoon season left only few leaves on upper nodes on some genotypes so fluctuated results are observed due to younger leaves on upper nodes.

Conclusions and Recommendations
The graduation in the susceptibility of genotypes to toxin producing pathogens were purely quantitative. Absolute resistance i.e. incompatibility, cannot be found in such quantitative resistance responses. In the absence of quantitative resistance two main strategies are advocated to keep LLS of peanut under some limits. The first is to reduce the level of inoculum during the intercrop period, which will reduce the amount of inoculums available to start an epidemic after the peanut crop emerges and second strategy is to reduce the rate of increase during the cropping period.

Author's Contribution
M. Ijaz conceived the idea of this study, designed experimental layout, did data analysis, and provided technical inputs at every step of this study. S.R.A. Shah wrote manuscript and formatted according to journal keeping in view authors guidelines. A. Afzal worked on data recording, and reviewed the manuscript. M. I. Haq critically reviewed the manuscript and contributed in formatting.