​Screening of Pigeonpea Varieties through Nylon Bag No-choice Bioassay for Host Plant Resistance to Helicoverpa armigera

Pigeonpea [Cajanus cajan (L.) Millsp.] is a multipurpose, hardy pulse legume grown in the tropics and sub-tropics. Insect pests cause an average of 30% loss in pulses, valued at US$ 815 million throughout the world. However, the legume pod borer, Helicoverpa armigera (Hübner) (Sharma, 2016) is the most important yield reducing factor, resulting in a loss of over US$2 billion million annually in the semi-arid tropics, despite the use of insecticides costing more than $500 million annually. Since H. armigera has developed high levels of resistance to insecticides, it has become difficult to manage this pest with conventional insecticides. In this context, host plant resistance is an important component for managing H. armigera in different crops and cropping systems. Screening of more than 14,000 accessions of pigeonpea for resistance to H. armigera showed low to moderate levelsof resistance in the cultivated genotypes (Jat et al., 2021; Reed and Lateef, 1990), but a few accessions of the wild relatives found resistant to H. armigera (Green et al., 2006).
       
Various morphological traits have been reported to be associated with resistance to H. armigera (Jat et al., 2021). Besides these traits, chemical compounds in the trichome exudates also influence host plant selection and colonization by H. armigera (Green et al., 2003; Hartlieb and Rembold, 1996).
               
Due to variations in the flowering times, the infestation of H. armigera varies over space and time, results in variations in the infestation levels across seasons and locations. According to Smith et al., (1994), it is important to screen the test genotypes for resistance to the target insects under the optimum and uniform level of insect infestation at the most susceptible stage of the crop. A technique that results in more than 80% infestation/ pod or leaf damage in the susceptible check or maximum differences in leaf/pod damage between resistant and susceptible checks can be used to screen for resistance to insect-pests. Sharma et al., (2005) standardized a detached leaf assay in chickpea for rapid screening of germplasm for resistance to H. armigera in a short span of time, with minimal cost, and under uniform insect infestation, which also provides useful information on antibiosis component of resistance to the target insect pest. Therefore, we used a nylon bag no-choice bioassay technique to screen for host plant resistance to H. armigera in pigeonpea under field conditions.

The experiments were conducted during the 2013 and 2014 rainy season at Pulses Farm, Department of Genetics and Plant Breeding, CCS Haryana Agricultural University, Hisar.
 
Number of Treatments      : 24 (6 varieties and 4 dates of sowing).
a) Varieties                        : Manak, Paras, Pusa-992, AL-201, PAU-881 and H03-41.
b) Date of sowing              : D1 (3^rd week of June), D2 (1^st week of July).
                                            D3 (2^nd week of July) and D4 (3^rd week of July).
Plants
 
The experiment was replicated thrice in a factorial random complete block design. Plot size was 1.8 × 4 m (4 rows × 4 m long) with 45 cm × 15 cm spacing. Varieties were sown on four different planting dates by following recommended agronomic practices. At flowering, 10 plants were randomly covered with a nylon bag (60 × 35 cm).
 
Insects
 
The culture was maintained on artificial diet in the laboratory for infestation of the pigeonpea cultivars tested.
 
Infestation of varieties with the larvae
 
Plants were infested with 24 h old 1, 2, 3, 4 and 5 larvae per plant inside the nylon bag by removing naturally presence of eggs and larvae. Uninfested plants served as a control. Larva were allowed to feed for 10 days, and data were recorded on pod damge and larval weight and the larvae were shifted to artificial diet until pupation. The plant were again covered with the nylon bag for further studies. Adult emergence and fecundity data were also recorded. At maturity, the data were recorded on percentage pod damage.
 
Morpho-physiological interactions between pod borer and varieties
 
25 days old fresh pods of pigeonpea were picked randomly from each genotype. Trichome density on the pods (top, middle and bottom canopy) were recorded (Sass, 1964). Pod wall thickness, pod length, and seed length and width (in mm) were measured by using Vernier Calipers.
       
To study the biochemical constituents of the seeds as well as the pod wall, the sufficient number of 15 day old pods were plucked from each replication. The pods were kept in paper bags in an airtight plastic container, and stored at 4°C until chemical analysis. One set of pods kept in a paper bag, oven dried at 60°C for 3 days, powdered them, and again oven dried at 50°C for 1 day to ensure complete drying of pods. Biochemical constituents were estimated by adopting following methods viz crude protein AOAC (1985), moisture (Mehta and Lodha, 1979), total soluble sugars (Dubios et al., 1956), fat (AOAC 1975), total phenols (Bray and Thorpe, 1954), tannins (AOAC 1965) and chlorophyll content (Hiscox and Israelstam, 1979).
 
Statistical analysis
 
Data were subjected to analysis of variance Steel and Torrie (1980) by factorial analysis. Significance of differences between the genotypes was judged by F-test, and the genotypic means were compared by the least significant difference (LSD) at P 0.05.

Insect density × pod damage relationships
 
There were significant differences between varieties and larvae released per plant (F8,32 = 0.516; p£0.05) and sowing dates×varieties×larvae released per plant (F8,32 = 1.032; p≤0.05) (Table 1). There were significant differences in larval weight, pupation and adult emergence across sowing dates and varieties (F8,32 = 0.256; p≤0.05), (F8,32 = 0.313; p£0.05), (F8,32 = 0.626; p≤0.05) (Table 2), varieties (F8,32 = 6.42; p≤0.05) and sowings×varieties (F8,32 = 12.85; p≤0.05) and sowing dates (F8,32 = 5.63; p≤0.05), varieties (F8,32 = 6.90; p≤0.05) and sowings×varieties (F8,32 = 19.50; p≤0.05).
       

 

 
Pod damage is the most common parameter for assessing genotypic resistance or susceptibility to H. armigera. Maximum chickpea pod damage was observed when six third-instar larvae per three plants released in the greenhouse and eight larvae per plant under field conditions (Sharma et al., 2005). Under detached leaf assay, significantly lower larval weight gain and lowest pod damage was in chickpea cultivars ICCV 097105 and ICCV 92944.
       
Susceptibility of a test genotype in the field conditions and under detached leaf assay is also influenced by non-preference for oviposition and feeding, tolerance and antibiosis. As these factors are important component of resistance, nylon bag no-choice bioassay technique can be used to evaluate germplasm and breeding lines under uniform insect pressure and environmental conditions.
 
Association of morphological traits with expression of resistance to H. armigera
 
Trichome density of pods of top canopy
 
In pooled over years results, type A and B trichomes were significantly and negatively correlated with pod damage (r = -0.730*, r = -0.768*, r­ = -0.531*, r = -0.729*) and (r = -0.864*, r = -0.734*, r = -0.662*, r = -0.776*, respectively) (Table 3) in D₁, D₂, D₃ and D₄ sown crop.
 

 
Trichome density of pods of middle canopy
 
In pooled results, type A and B trichomes were significantly and negatively correlated with pod damage (r = -0.751*, r = -0.766*) in D₁ and D₂ and (r = -0.729*, r = -0.730*, r = -0.742*, respectively) in D₁, D₃ and D₄ sown crop.
 
Trichome density of pods of lower canopy
 
In pooled over years results, type A and B trichomes were significantly and negatively correlated with pod damage (r = -0.725*) in D₂ sown crop.
       
But, C type of trichomes were positively correlated (r = 0.794*, r = 0.760*), (r = 0.646*, r = 0.803*) and (r = 0.964**, r = 0.639*, r ­= 0.510*, r = 0.832*) with pod damage in D₁, D₂, D₃ and D₄ sowings (Table 3).
       
Trichomes type A and B of top and middle pod canopy (slope = -0.50; -1.11, -0.25; -0.32) and (slope = -0.37; -0.50; -0.46; -0.62) were negatively correlated with pod damage, with a negative slope in D₁, D₂, D₃ and D₄ sowings (Fig 1 and 2). Trichomes type B of top and middle pod canopy (slope = -2.23; -3.55; -1.89; -0.72) and (slope = -3.18; -2.20; -3.03; -2.52) were negatively correlated with pupation, with a negative slope.
 

 

       
Trichomes type A of middle canopy in D₁ and D₂ sowings (slope = -0.24; -0.30) were negatively correlated with fecundity, with a negative slope (Fig 5).
       
However, trichomes of type C of top and middle canopy  in D₁, D₂, D₃ and D₄ sowings (slope = 0.45; 0.50; 0.57, 0.50) and (slope = 0.1.53; 0.93; 1.74; 1.31) (Fig 3 and 4), (slope = 2.04; 1.53; 1.82; 1.69), (slope = 1.48; 1.83; 1.63; 2.21) were positively associated with pod damage, pupation and fecundity with a positive slope.
 

 

 

 
Pod wall thickness
 
Pod wall thickness was significantly and negatively correlated with pod damage (r = -0.909**, -0.739*, -0.612*,
-0.801*) (Table 3) with a negative slope (slope = -2.43; -3.17; -4.54; -4.11) (Fig 6) in D₁, D₂, D₃ and D₄ sowings.
 

 
Association of biochemical traits with expression of resistance to H. armigera
 
Chlorophyll content (mg g^-1)
 
Chlorophyll content of seeds as well as pod wall was significantly and positively correlated with pod borer damage in D₃ and D₄ sowings (r = 0.655*, r = 0.753*) and in D₁ sowing (r = 0.626*) (Table 4).
 

 
Crude protein (%)
 
Crude protein content of seeds as well as pod wall was significantly and positively correlated with pod borer damage in D₂, D₃ and D₄ sowings (r = 0.639*, r = 0.810*, r = 0.711*) and in D₁ sowing (r = 0.740*) (Table 4). Path coefficients shows positive slope with larval weight (slope = 1.13; 0.93; 1.24; 0.21) (Fig 7), pupation (slope = 10.30; 5.13; 8.67; 9.72) (Fig 8), adult emergence (slope = 5.56; 2.08; 6.44; 12.95) (Fig 9) and fecundity (slope = 4.17; 3.79; 7.03; 5.52) (Fig 10), respectively in D₁, D₂, D₃ and D₄ sowings.
 

 

 

 

 
Total soluble sugar (%)
 
Total soluble sugars of seeds as well as pod wall were significantly and positively correlated with pod borer damage in D₁, D₂ and D₄ sowings (r = 0.738*, r = 0.793*) and (r = 0.698*, r = 0.898**, r = 0.819*), respectively (Table 4).
       
Total soluble sugar content (slope = 6.42; 2.88; 1.06; 1.0) was positively correlated with pod borer damage (Fig 11), larval weight (slope = 4.50; 1.43; 0.99; 0.62), pupation (slope = 40.16; 20.31; 30.97; 30.94) (Fig 12) and fecundity (slope = 20.47; 3.95; 2.90; 3.03) with a positive slope in D₁, D₂, D₃ and D₄ sowings, respectively.
 

 

 
Fat content (%)
 
Fat content of seeds as well as pod wall was significantly and negatively correlated with pod borer damage (r = -0.884**, r = -0.675*) and (r = -0.743*) in D₁ and D₂ sowings (Table 4).
 
Phenol content (mg g^-1)
 
Phenol content of seeds as well as pod wall was significantly and negatively correlated with pod borer damage (r = -0.900**, r = -0.625*) and (r = -0.656*, r = -0.697*) in D₁ and D₂ sowings (Table 4).
       
Phenol content was negatively correlated with pod borer damage (slope = -2.40; -2.50; -2.30; -3.75) (Fig 13), larval weight (slop = -1.73; -1.92; -0.72; -1.89) (Fig 14), pupation (slope = -16.70; -8.87; -13.08; -14.23) (Fig 15), adult emergence (slope = -8.96; -4.38; -6.67; -13.84), and fecundity (slope = -6.51; -9.20; -1.73; -11.50) (Fig 16) respectively, with a negative slope in D₁, D₂, D₃ and D₄ sowings.
 

 

 

 

 
Tannin content (µg g-1)
 
The tannin content of the seeds as well as pod wall was also significantly and negatively correlated with borer damage (r = -0.792*, r = -0.812*, r = -0.676*) and (r = -0.630*), respectively in D₁, D₂ and D₄ sowings.
       
Path coefficients of trichome density, pod wall thickness, phenol and tannins content exhibited direct effects and correlation in the same direction (-ve) suggesting the importance of these traits against H. armigera resistance and these traits can be used as a resistance source criteria. To understand the mechanisms of expression of resistance to H. armigera under field conditions is a long-term process. And hence, it is difficult to identify stable source of resistance under natural infestation in the field.
               
Trichome density, orientation and their types also influences the expression to insect pests in pigeonpea (Aruna et al., 2005; Jat et al., 2021; Sharma et al., 2009). Total phenolic content, phenols and flavonoids were negatively correlated, while sugar content and green pods were positively associated with susceptibility to insect pests in pigeonpea and cowpea (Jakhar et al., 2017; Tripathi and Purohit, 1983).