Introduction

Sоmе major hazards in the production of lentil (Lens culinaris Medik) include susceptibility to various fungal diseases such as Ascochyta blight, rust, wilt, collar rot, botrytis and stem rot which affect the production of lentil in Pakistan (^{Chaudhry et al., 2008}). Vascular wilt caused by Fusarium oxysporium is the most important disease of lentil in many lentil growing areas of Pakistan and worldwide. This wilt pathogen survives unfavorable conditions in the soil either as a dormant form (chlamydospores) or saprophytically that can remain viable for several years without a suitable host (^{Bayaa et al., 1997}; ^{Erskine et al., 2011}; ^{Pouralibaba et al., 2015}a). Wilt may cause 5-10 % yield losses but, sometimes, severe damage may cause a whole crop failure under favorable conditions for the disease development (^{Chaudhary and Amarjit, 2002}; ^{Sarwar et al., 2014}), especially in a warm spring and a dry and hot summer (^{Agarwal et al., 1993}; Sarwar et al., 2014). The damage also depends upon the crop stage being affected and the severity of the pathogen strain (^{Taylor et al., 2007}). The disease occurs both at seedling and flowering stages. In early sown crops wilting may occur in November but the disease ceases during the cool months of December and January In February it starts to reappear and the maximum incidence of the disease occurs in March (^{Bashir and Malik, 1988}). Under favorable conditions susceptible genotypes showed 100 % yield losses in Pakistan (^{Chaudhry et al., 2008}).

Breeding for host resistance is the most effective, efficient and environment friendly method (^{Sarwar et al., 2014}). Most of the commercial varieties in the field are susceptible to Fusarium wilt in Pakistan and work is in progress to screen resistant/tolerant genotypes (^{Chaudhry et al., 2008}). Therefore, the objective of this study was to evaluate the responses of different lentil populations to biotic stress (Fusarium wilt disease) in a wilt sick plot.

Materials and Methods

Three distinct sets comprising eight variable populations (segregating generations, their parents and standard varieties) were evaluated at the Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad, in a wilt sick plot developed with locally available F. oxysporum f. sp. lentis isolate. Each set comprised eight populations, viz. four segregating generations (recombinant, mutant and recombinant mutant), their two parents and two standard varieties (Table 1).

Table 1 Lentil populations (each one identified along with some details of the genetic nature of their patents), their segregating generations and standard varieties in each one of eight-population sets used in a plot infected by Fusarium in Faisalabad, Pakistan. 

P₁: Parent-1; P₂: Parent-2; F₃: Recombinant segregating generation; M₃(P₁): Mutant segregating generation of P₁; М₃(Р₂): Mutant segregating generation of P₂; F₃M₂: Recombinant mutant segregating generation; SV₁: Standard variety-1; SV₂: Standard variety-2

The experimental design was nested (hierarchical) and it was same for all sets (^{Akhtar et al, 2015}), in each population of true breeding genotypes (parents and standard varieties) and, similarly, in each population of segregating generations (recombinant, mutant and recombinant mutants), two rows of each genotype were taken as progenies and twenty plants of each progeny were sown in field. These plants were equally divided into two rows to obtain two observations (replicates) of a trait in ten plants of a progeny. These progenies were considered as treatments in the experiment. Each experimental unit was 1 m long keeping plant-to-plant and row-to-row distances of 10 and 30 cm, respectively. Sowing was done by dibbling two seeds per hill at uniform depth. All other agronomic practices were kept uniform.

Fusarium wilt of lentil may occur from the seedling stage to the reproductive stage. However, seedling wilt can cause more yield losses. Thus, visual observations of disease symptoms were started after two weeks of seed germination and total number of wilt infected plants was noted till crop approached near physiological maturity in each plant progeny row for each population to calculate disease incidence by using the formula:

The level of resistance/susceptibility of each plant progeny row was determined by using the rating scale of ^{Baya et al. (1997)} and ^{Stoilova and chavdarov (2006}), with some modifications (Table 2). Plants bearing damage due to Fusarium wilt in all progeny rows were uprooted and sent to laboratory to be analyzed, whereas the pathogens were isolated on gram meal agar medium to confirm that the disease was caused by Fusarium oxysporium f. sp. lentis.

Table 2 Disease scale for determining level of resistance/susceptibility of each plant, progeny from every population, against Fusarium wilt. 

Results and Discussion

The plants within all populations had the capacity to withstand Fusarium wilt disease. No plant in any population showed susceptibility to the disease (Table 3), which showed that the parents, standard varieties and plant progenies in segregating populations were developed through selection of single plants showing resistance against Fusarium wilt disease. However, ^{Stoilova and Chavdarov (2006}) reported different results after screening 32 genotypes of lentil, out of which three were susceptible.

Table 3 Response of different lentil populations to Fusarium wilt disease and percent disease infection. 

%DI: Percent of disease incidence

In Set-1, the mutant population of parent A i.e. M₃ (A_M) was found highly resistant to Fusarium wilt, since it showed least or even no effect of F oxysporium. Parental populations p J (A) and P₂ (B), segregating populations M₃ (B_M) and F3M2 (AB_M) and Standard varieties SV-1 (C) and SV-2 (D) showed resistance against Fusarium wilt, that is, the disease affected few plants. The plants within recombinant population F₃ (АВң) tolerated wilt disease, which means that a high number of plants wilted in this population (Table 3), as compared to the standard varieties.

In Set-2, the parents 'A' and 'B' and all segregating populations, i.e. АВң, A_M, B_M and AB_M, were highly resistant with no wilted plant found in these populations. Such populations may be used as a reliable source of resistance against F oxysporium in lentil. The standard variety 'D' showed resistant behavior, whereas standard variety 'C' was tolerant to wilt disease (Table 3). In Set-3, the parent 'B' and all segregating populations (АВң, A_M, B_M and AB_M) were highly resistant, whereas Parent 'A' along with both standard varieties had resistance to F oxysporium (Table 3).

Among all sets, the populations of Set-2 and Set-3 were a good source of variation for lentil resistance against Fusarium wilt disease (Table 4) as shown by the number of plants which survived. This may be evaluated and utilized for the selection of disease resistant plants as reported by ^{Baya et al. (1997)}, who found similar variability for resistance against Fusarium wilt disease.

Table 4 Progeny wise response expressed as the frequency that lentil populations were scored into the disease rating scale for Fusarium wilt in all sets. 

Our research revealed that in all sets, the parents, standard varieties and plant progenies in segregating populations showed capacity to withstand Fusarium wilt disease. All mutated populations from all sets (A_M, B_M and AB_M) showed resistant behavior as compared to recombinant population АВ_H (Table 3). Moreover, A_M population appeared to be highly resistant to Fusarium wilt disease in all sets. Such type of resistant breeding material can minimize the impact of this disease on lentil production. The results of our research also indicated the worth of induced mutation in developing resistant genotypes. This is similar to ^{Taylor et al. (2007}) and ^{Pouralibaba et al. (2015}a), who suggested that the most effective, economical and environment friendly method to control the disease is using resistant cultivars. Besides, ^{Pouralibaba et al. (2015a, b)} showed an interaction between pathogen and host genotypes, which should be considered for planning breeding and the pathotypes should be studied before setting up a breeding programs in order to select the resistant cultivars.

Conclusions

The breeding populations (parents, standard varieties and segregating generations) within Set 2 and Set-3 might be used as sources of resistance against Fusarium wilt of lentil in breeding programs. The mutated populations (A_M, B_M and AB_M) showed more resistance, as compared to recombinant populations, thus implying that the use of induced mutation is effective for developing resistance in lentil. Resistant plants of lentil may be the beginning of an integrated disease management program in lentil crop.