Food legumes are important component of modern agriculture in meeting nutritional requirement of human beings. They are also a critical component of large and small animal feeds. Pigeonpea [Cajanus cajan (L.) Millsp.] is a major grain legume crop of the semi-arid tropics, which is cultivated on 2.65 million tones, with an annual production of over 4.33 million tones contributing to about 5% of the total world production of pulses (FAOSTAT 2012; http://faostat. fao.org). Nearly 90% of the global pigeonpea cultivation is confined to India and Nepal, the remainder is in Africa (6%), the Caribbean (2%) and other Southeast Asian countries (N. Maxted and S. Kell., 2012). In India, the major pigeonpea growing states are Maharashtra, Uttar Pradesh, Karnataka, Madhya Pradesh, Andhra Pradesh and Gujarat.

It is a major source of protein to about 20% of the world population (Wanjari et al., 2003) and is an abundant source of minerals and vitamins (Saxena et al., 2002). It is an ideal supplement to cereal and tuber-based diets of resource constrained farmers. It comes well in marginal lands and it is popular among small and marginal farmers in the Indian subcontinent. The productivity of pigeonpea crop is very low in India with 799 kg/ha as compared to its productivity potential of 2000 kg/ha. Factors such as lack of improved cultivars, poor crop husbandry, pests and diseases are responsible for this. Wild relatives have now been reported to possess many agronomically important traits such as resistance to pests and diseases (Reddy et al., 1995; Sharma et al., 1984), salinity tolerance (Subbarao et al., 1991) and high protein content (Saxena et al., 1996), all of which would be useful in cultivated pigeonpea. Over an hundred fungal, bacterial and viral pathogens are known to attack pigeonpea crop in the world (Nene et al., 1989). Major diseases include Fusarium wilt caused by Fusarium udum Butler, sterility mosaic disease (SMD) caused by pigeonpea sterility mosaic virus (PPSMV) and Phytophthora blight by Phytophthora drechsleri and pests such as pod borer (Helicoverpa armigera), maruca (Maruca vitrata), pod fly (Melanagromyza obtusa), plume moth (Exelastis atomosa) cause substantial reduction to pigeonpea production (Kannaiyan et al., 1984). In SMD incidence, virtually no seed setting is seen in pigeonpea crop. The pigeonpea sterility mosaic virus (PSMV) is transmitted and perpetuated through a mite, Aceria cajani (Nene et al., 1977).

Sterility Mosaic disease (SMD) occurs with regularity under suitable conditions, spreads rapidly, leading to epidemics where in the yield losses depend on the growth stage at which infection occurs (Ganapathy et al., 2011). The affected plants look are green with excessive vegetative growth without any flowers and pod, hence, it is also referred to as green-plague: under congenial conditions it is  known to spread rapidly like a plague, leading to severe epidemics. Generally, an early infection results in a 95%~100% loss in yield (Reddy et al., 1990; Kulkarni et al., 2003), on the other hand, losses from late infection depend on the level of infection ranging from 26%~97% (Kannaiyan et al., 1984). Maximum virus incidence and yield losses occur in ratooned and perennial pigeonpea.

Srinivas et al. (1997) reported the inheritance of resistance and allelic relationship in three resistant pigeonpea sources for strain 2 of sterility mosaic pathogen. The inheritance of resistance has been determined by crossing resistant genotypes ICP 7035, ICP 7349 and ICP 8850 with susceptible genotypes BDN 1 and LRG 30and used to obtain information on their allelic relationship. Parents, F₁ and F₂ gene- rations screened using “infector hedge row technique” and indicated the dominance of resistance in certain crosses and the dominance of susceptibility in others. Amala Balu and Rathnasamy (2003) studied the pattern of inheritance of the sterility mosaic resistance in pigeonpea, where the two susceptible parents, Prabath and Co 5, two resistant parents, ICPL 83024 and ICPL 83027 and their F₁ hybrids and F₂ progenies were screened for sterility mosaic disease. The study reported the moderately resistant to SMD in F₁s while, in F₂ the rating scale for SMD ranged from 3 to 9. The F₂ generation of the four combinations fitted well with the segregating ratio of 13:3 for susceptibility and resistance indicating non-allelic interaction of two factors.

1 Results
1.1 Development of F₂ and F_2:3
Crossing of the parental genotypes and raising of mapping population were carried out under nylon net to avoid cross pollination through insect pollinators. Gullyal white, a highly susceptible cultivar to SMD was crossed with a resistant parent, BSMR736 and the resultant F_1s was raised. The F₂ seeds collected from a single F₁ plant which was conformed for true hybridity using DNA markers were used to plant over 300 F₂ plants. All the 300 F₂ plants were selfed to obtain the F_2:3 families that were used for phenotyping the incidence of SMD at Agricultural Research Station (ARS), Bidar. ARS, Bidar is known for its endemism for the occurrence of PSMD, hence called ‘hot spot’ of SMD. The hedge row coupled with artificial inoculation of diseased leaves together with the mites was successfully used in manifesting the PSMD. Sowing of ICP 8863, a susceptible variety, was done about 4 weeks prior to the sowing of actual experimental material. Over 10-15 days old hedge border rows and infector rows infected with PPSMV through the stapling of diseased leaves could successfully manifest the disease symptoms in more than 80 per cent of the plants (Figure 1).

Figure 1 Manifestation of Sterility Mosaic Disease F2:3 Families in SMD sick garden at ARS Bidar

In leaf stapling techniques, one diseased leaflet per primary leaf was stapled (Figure 2). As the stapled leaflets from the infected plants get dried, mites from the infected leaves migrate to healthy leaf and inoculates the virus. The PPSMV susceptible genotype
ICP 8863, used to developed infector hedge rows on the border of the experimental field was found ideal genotype in bring up the disease and the pathogen and mites multiplication in sufficient threshold on the hedge and successfully served as source of PPSMV inoculums. Generally, the mites are carried through wind onto the test rows in the field, which was evident by the observed symptoms on all susceptible progenies in the field.
 

Figure 2 Pigeonpea plants are stapling with SMD infected leaf samples

Table 1 Segregation pattern of PSMD incidence deduduced in F2 individuals based on the reaction of F2:3 families of the cross Gullyal white×BSMR 736 to PSMD field evaluated in a SMD sick garden at ARS Bidar

Table 2 Distribution of F2:3 families of the cross Gullyal white×BSMR 736 in the PSMD score ranges of resistant, moderately resistant and susceptible based on disease incidence in SMD sick garden at ARS Bidar

It was suggestive from the results that the PPSMV resistance was governed by two genes designated as SV₁ and SV₂ (Table3). Singh et al. (1983) the dominant allele of one gene (SV1) has inhibitory action on the character (resistance) govern by other (SV2) gene. Based on these assumption, the presence of dominant allele of SV1 gene in one locus suppresses the action of dominant allele of SV2 (resistance) gene present on another locus resulting in susceptible phenotype. Only those plants with recessive allele of SV1 gene and dominant allele of SV2 gene might have shown resistant phenotype. On the basis of this hypothesis we propose that the following types of genotypes might be possible for resistance and susceptible phenotypes in plants.

Table 3 Types of genotypes might be possible for resistance and susceptible phenotypes in plants

3 Materials and Methods
The PPSMV resistant variety-BSMR 736 and susceptible variety-Gullyal white were obtained from Agricultural Research Station (ARS), Gulbarga and Karnataka to develop suitable segregating generations and recombinant inbred line population. BSMR736 variety released from Agricultural Research Station, Badnapur, Maharashtra, is known to be consistently and stably resistant to PPSMV (Saxena et al., 2008) Gullyal white, a highly preferred variety for its dhal quality is also highly susceptible for PPSMV infection. These lines were maintained by continuous selfing for several generations in the pigeonpea breeding programme at Agricultural Research Station, Gulbarga.

The individual flowers of the female parent, Gullyal white, were hand emasculated and pollinated with the pollen dust from the male parent, BSMR736, in the cool hours of the day to get sufficient F₁ seeds. The F₁s plants along with their parents were grown and true F₁ plants were selfed by covering nylon net to prevent out crossing through honey bees and other insect pollinators. The F₁ plants were raised and the mature seeds were collected and advanced to F₂ generation. A set of 300 individual F₂ plants from Gullyal white×BSMR 736 cross were grown in the field under the nylon net at Main Agricultural Research Station (MARS), Dharwad. The plot chosen for this experiment was positioned to have sugarcane field on one side and rice field on the other side, both known to favors the manifestation of SMD in pigeonpea.

Before actual sowing of F_2:3 families, ‘infector-hedge - rows’ of susceptible variety ICP8863 were established. Briefly, an infector-hedge consisting of two widely spaced rows of the susceptible cultivar, ICP8863 was sown one month prior to actual planting experiment. The susceptible check was sown in regular intervals of test sample, which would serve as spreader of disease on the field. The F_2:3 families were sown in two replications, where at least 30~40 plants per replication was maintained. A spacing of 60 cm between the rows and 30 cm between plants within a row was maintained with recommended package of practices without any control measures for SMD.

3.1 Artificial inoculation
The PPSMV artificial inoculation in the field was done according to “leaf stapling” and “infector-hedge” techniques (Nene and Reddy, 1976). The PPSMV infected pigeonpea leaves along with mites were collected from farmers’ fields of Afzalpur village near Bidar and used for artificial inoculation. One diseased leaflet per primary leaf was generally used. The diseased leaflet was folded on the primary leaf in such a way that its lower surface came into contact with the primary leaf of the test seedlings. As the stapled leaflets from the infected plants get dried, mites from the infected leaves migrate to healthy leaf and inoculates the virus.

3.2 Ratooning
The ratooned and perennial pigeonpea is known to have highest SMD incidence. In case of late infection of PPSMV, symptoms may not occur even if the genotype is susceptible to PPSMV, however, when plants are ratooned symptoms appear prominently on the new growth (Kumar et al., 2002; Jones et al., 2004). Ratooning is known to favor the manifestation of SMD in pigeonpea. The experimental F_2:3 families of present study were cut from first branch position from ground and irrigated to encourage the sprouting. After 30 days of ratooning, the disease incidence was scored. The ratios of F_2:3 to SMD was grouped the following the standard scale (Singh et al., 2003) as given below: 0~10% of plants infected: Resistant; 10.1%~30% of plants infected: Moderately resistant; 30.1%~100% of plants infected: Susceptible.

Authors’ Contributions
Shivarudrappa B Bhairappanavar conducted the study. B Fakrudin analyzed and interpreted the data. Shivarudrappa B Bhairappanavar wrote the manuscript. B Fakrudin designed the study by assistance. K.R.S.Sambasiva Rao reviewed the manuscript.

Acknowledgments
This research work was supported by Indian Council of Agricultural Research (ICAR) for the financial support (AKI-PGI) and Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India (DBT Programme Support Project). We thank the Project Monitoring and Mentoring Committee (PMMC) members, Dr. V. P. Gupta, Dr. N. Seetharama; Dr. P. Balasubramaniam, Dr. M. B. Chetti, Dr P.U. Krishanraj and Dr. Y.D.Narayana for their helpful suggestions from time to time. We acknowledge the timely help of Dr.C.R.Konda, in facilitating disease studies at Bidar.

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