Indian Journal of Experimental Biology Vol. 56, January 2018, pp. 29-38

Physicochemical mechanisms of resistance in to partellus (Swinhoe)

Mukesh K Dhillon1* & DP Chaudhary2 1Division of Entomology, ICAR-Indian Agricultural Research Institute, New Delhi 110 012, 2Biochemistry Laboratory, ICAR-Indian Institute of Research, Pusa Campus, New Delhi 110 012, India Received 03 May 2017; revised 20 August 2017

Variation in nutritional components and amounts of secondary metabolites not only affects the growth, development and survival of herbivores but also indirectly influences expression of host plant resistance to . In this study, we examined the role of different biochemical and morphological factors in sorghum as host plant defense against the spotted stem borer, Chilo partellus (Swinhoe). The genotypes IS 2205 and IS 2123 suffered lower deadheart incidence, and exhibited deleterious effects on development and survival of C. partellus, followed by ICSV 700, ICSV 708 and ICSV 25066 than the susceptible check, Swarna. The anthocyanin pigmentation in sorghum seedlings and C. partellus deadhearts were found significantly and negatively correlated with the larval period (r = −0.60** to −0.88**), while positively correlated with the larval and pupal weights, and larval survival and adult emergence (r = 0.58* to 0.95**). Conversely, the numbers of C. partellus exit holes in the stalk, larvae recovered, number of tunnels and stem tunneling length were significantly and positively correlated (*, ** = P ≤0.05 and 0.01, respectively) with the larval period (r = 0.72** to 0.89**), but significantly and negatively correlated with larval and pupal weights, larval survival and adult emergence (r = −0.54* to −0.84**). Although there was significant variation in morphological traits and biochemical composition of the sorghum genotypes, there was no direct link to expression of resistance to this insect, but for a few cases. The significant and negative association of total carotenoid, p-coumaric acid, zinc and iron contents with growth, development and survival of C. partellus, and damage parameters (r = −0.48* to −0.72**), indicated their role in expression of resistance to C. partellus in sorghum. However, the interaction among different biochemical compounds and the morphological traits, rather than a particular biochemical constituent played a greater role in host plant defense against C. partellus.

Keywords: Anthocyanin pigmentation, Antibiosis, Deadheart incidence, Host plant resistance, Mechanisms of resistance, , Spotted stem borer

Sorghum bicolor (L.) Moench is one of the most lesions on the leaves. The older larvae leave the important cereal crops in the semi-arid tropics, and whorl, bore into the stem where it cuts the growing insect pests are one of the major yield-reducing point resulting in “deadheart” symptom. In older factors. A number of stem borer species have been plants, the larva feeds inside the stem causing reported as serious pests of sorghum in Asia and extensive tunneling. Feeding and stem tunneling by Africa, of which spotted stem borer, Chilo partellus C. partellus larvae cause huge crop losses due to (Swinhoe) is the predominant species in Indian interference with translocation of metabolites and subcontinent, and South and eastern Africa, causing nutrients, thus resulting in poor development of serious damage to sorghum, maize and pearl millet1,2. grains, stem breakage, lodging, direct damage to It causes 18-25% yield loss in sorghum and maize3. panicles and loss in grain yield. Spotted stem borer attack in sorghum starts from two Several control strategies such as crop rotation, weeks old seedlings, affects all plant parts except the field sanitation, introduction of parasitoids and use of roots and persists up to crop harvest. The neonate synthetic pesticides have been employed for the larvae scrap the leaf chlorophyll, and the early instar control of C. partellus but their deployment have not larvae while feeding in the whorl cause irregular given satisfactory control particularly when the larvae shaped pinholes which later convert to elongated are feeding inside the stalks4. In such a scenario, host plant resistance could be exploited as one of the most ————— effective mean of minimizing losses due to insect *Correspondence: Telefax: +91 11 25842482 pests and sustainable sorghum production. Several E-mail: [email protected] sorghum germplasm accessions have been screened, 30 INDIAN J EXP BIOL, JANUARY 2018

and a number of C. partellus resistance sources have stem length tunneled, number of exit holes, and been identified5. However, resistance reaction to number of C. partellus larvae recovered from the test C. partellus has been noticed highly variable across plants. Data on numbers of plants with C. partellus climatic conditions which could be due to variability in deadhearts were recorded at 45 days after seedling feeding potential or varying insect pressure at different emergence (DAE), and expressed as percentage of the locations. Thus, the effect of stem borer damage on total number of plants. Observations on leaf grain yield may reflect in multiple traits, such as non- glossiness15 and anthocyanin pigmentation16 were preference for oviposition, reduced feeding by the first recorded at 7 DAE on a scale of 1 to 5. Five randomly instars on young leaves, low deadheart formation, selected plants were cut at the base before harvest, reduced tunneling, and tolerance to leaf damage and and exit holes were recorded per plant after removing stem tunneling6-8. A number of biochemical factors, the sheath leaves. The stalks of five randomly selected such as low sugar content, amino acids, total sugars, plants were split open to determine the number of tannins, total phenols, neutral detergent fiber, acid C. partellus larvae and the number of tunnels per detergent fiber and lignins9-12 have also been reported plant. The length of the stem tunneled (cm) by to be associated with resistance to stem borer in C. partellus and the total peduncle length (cm) were sorghum. However, Dhillon and Chaudhary13 reported measured from five randomly selected plants, and the that the plant defense against C. partellus in maize is data were presented as percent length tunneled per due to complex interactions among different plant in relation to total peduncle length. biochemical constituents rather than the concentration of particular biochemical constituent. Thus, selection Sorghum plant samples for biochemical analysis and developmental biology of C. partellus for resistance to stem borer based on individual The seedlings of test sorghum genotypes were parameter is difficult as sorghum genotype identified raised in the plastic pots (12L capacity) under resistant to leaf feeding damage and/or deadhearts may 14 glasshouse conditions on the potting mixture be susceptible to stem tunneling and vice versa . consisted of red soil and farm yard manure (2: 1). Therefore, in the present study we explored Before sowing, diammonium phosphate was applied biochemical interactions in diverse sorghum genotypes @ 50 g per pot. Ten seeds were sown in each pot and with biological and damage parameters to understand there were 10 pots for each sorghum genotype. The their role in plant defense against C. partellus. plants were watered as per need. The 21-days old

seedlings of each genotype were harvested from the Materials and Methods base in separate polythene bags and immediately Field evaluation of sorghum genotypes for C. partellus damage and morphological traits transferred in the ice-box. After completion of The four selected sorghum genotypes IS 2123, harvesting, the seedlings of each test sorghum ICSV 700, ICSV 708 and ICSV 25066 along with genotype were stored at −20°C. The refrigerated resistant (IS 2205) and susceptible (Swarna) checks seedling samples were then freeze dried in a were sown in 2 row plots of 2 m row length with row- lyophilizer at −50°C to avoid changes in chemical row spacing of 60 cm in the research fields of ICAR- composition of the seedlings. Dried sorghum Indian Agricultural Research Institute, New Delhi, seedlings were fine powdered (<80 mesh size) in a India, during 2011-2013 Kharif (July-October) mixer-grinder and stored in zip-lock plastic bags at seasons. These test sorghum genotypes have earlier −20°C in the refrigerator for biological and been reported to show resistance to C. partellus1,5. biochemical studies.

The seeds were sown with dibbling method in three Evaluation of different sorghum genotypes for antibiosis against replications in a randomized complete block design C. partellus (RCBD). One week after seedling emergence, The biological studies of C. partellus were carried thinning was carried out to maintain plant-plant on artificial diet17, impregnated with lyophilized spacing of 10 cm. The crop was grown and seedling powder of test sorghum genotypes under maintained following standard agronomic practices, laboratory conditions maintained at 28 ± 1°C, 60 ± except insecticide application. The observation were 10% RH, and 12 h photoperiod. The sorghum leaf recorded on plants with stem borer deadhearts, leaf powder of susceptible sorghum genotype glossiness, anthocyanin pigmentation, number of (a constituent of standard C. partellus artificial diet) tunnels caused by C. partellus per stem, percentage of was replaced with lyophilized seedling powder of DHILLON & CHAUDHARY: CHILO PARTELLUS RESISTANCE MECHANISMS IN SORGHUM 31

respective test sorghum genotype (as described separation gradient used was 90% B to 10% A in 2 min, above) keeping the quantity of other dietary 90% B to 60% B in 15 min, 60% B to 10% B in constituents as per standard artificial diet. The diet 20 min and 90% B to 10% B in 25min. Total run time thus prepared was poured in 250 mL capacity plastic was 25 min. The phenolic acids were detected using cups having lids fitted with wire-mesh. Each cup was PDA at 254 nm with the column condition set at poured with 50 mL diet, and allowed to settle for 4 h. 30ºC. The ferulic acid and p-coumaric acid peaks Fifteen neonate C. partellus larvae were released in were acquired using Empower Pro Software® by each cup, and there were five replications in a Waters Corporation (2005-08), and quantification was completely randomized design. Observations were done based on calibration standards (98% pure) recorded on larval weight, larval survival, larval obtained from Merck Frankfurter Str. Darmstadt, period, pupal period, pupal weight and adult Germany, run at five concentrations viz., 10, 25, 50, emergence for each test sorghum genotype. At 30 days 75, and 100 mg/mL. after infestation, the weight of each larva were recorded on electronic balance, and expressed as Statistical analysis mg/larva. Each larva was observed daily for the The normality test showed that the seasonal effects formation of pupa. The day of larval release to day of were non-significant, thus the field data of three pupation was considered as larval period, and from seasons on insect damage and plant morphological the day of pupation to adult emergence was parameters were pooled for analysis of variance considered as pupal period. Pupal weight was (ANOVA). The significance of differences between recorded on electronic balance for each pupa the genotypes for different C. partellus biological, and separately after 24 h of pupation, and data were plant damage, morphological and biochemical expressed as mg/pupa. Percentage larval survival and parameters were tested by F-test, while the adult emergence were calculated based on the total significance of differences between the genotype number of larvae released in each cup. means were judged by least significant differences (LSD) at P = 0.05 using statistical software SAS Estimation of phenolic acids and important nutritional version 9.2©. The data were also subjected to Pearson biochemicals in sorghum genotypes correlation analysis to understand the association of The lyophilized samples of different sorghum sorghum seedling biochemical parameters with genotypes mentioned above were used for various biological parameters of C. partellus. quantification of phenolic acids (ferulic acid and p-coumaric acid) and other important nutritional Results biochemical parameters. The quantification of Chilo partellus damage parameters in sorghum nutritional biochemical constituents viz., protein and There was significant variability among the test oil content18, starch19, total sugars20, chlorophyll21, sorghum genotypes for deadhearts by C. partellus 22 23 total carotenoids , and Iron and Zinc were done (F5,10= 2543.63; P <0.001), number of tunnels/stem using standard methods described by Dhillon and (F5,10= 104.28; P <0.001), percent stem area tunneled 13 Choudhary . The ferulic acid and p-coumaric acid (F5,10= 148.78; P <0.001), exit holes/stem from sorghum samples were extracted by alkaline (F5,10= 65.54; P <0.001), and number of stem borer 24 hydrolysis method , test samples were prepared as larvae recovered/stem (F5,10= 61.04; P <0.001). The per the procedure described by Dhillon and deadhearts were significantly lower in IS 2123 and IS Chaudhary13, and stored at −80ºC for HPLC analysis. 2205 as compared to other test genotypes. Genotypes All the samples were prepared and analyzed in three ICSV 700, ICSV 708 and ICSV 25066 also had replications in completely randomized design. The significantly lower deadhearts as compared to ferulic acid and p-coumaric acid were separated on a susceptible check, Swarna (Table 1). Conversely, the Waters 2707 Module HPLC System attached to a number of tunnels made by stem borer larvae, percent photodiode array detector (Model PDA 2998). A 10 µL stem area tunneled, exit holes, and number of larvae sample was injected into a reversed phase C18 recovered were significantly higher in IS 2123 and IS column (250 × 4.6 mm) (waters, Milford, MA) using 2205 as compared to other test genotypes including auto sampler (Waters 2707). The glacial acetic acid (2%) susceptible check, Swarna. However, the number of eluent A and Acetonitrile (100%) eluent B were used in a tunnels, exit holes and larvae recovered in ICSV 700 separation gradient with a flow rate of 1.0 mL/min. The were significantly on par, while lower in ICSV 708 as 32 INDIAN J EXP BIOL, JANUARY 2018

compared to susceptible check, Swarna (Table 1). genotypes including susceptible check, Swarna. Furthermore, the percent tunneling area was However, the pupal period was significantly longer on significantly lower in ICSV 700, ICSV 708 and ICSV ICSV 700 as compared to other test genotypes 25066 as compared to susceptible check, Swarna. including resistant (IS 2205) and susceptible (Swarna) checks (Table 2). The C. partellus larvae fed on IS Sorghum morphological traits 2123, IS 2205, ICSV 700, ICSV 708 and ICSV 25066 There were significant differences among the test had significantly lower larval and pupal weights, sorghum genotypes for leaf glossiness (F5,10= 87.42; larval survival and adult emergence than those reared P <0.001) and anthocyanin pigmentation (F5,10= on susceptible check, Swarna (Table 2). Furthermore, 12.76; P <0.001). The leaves of sorghum genotypes the lower larval and pupal weights, larval survival and IS 2205, ICSV 705, ICSV 708 and ICSV 25066 were adult emergence in C. partellus reared on ICSV 700, glossy, while IS 2123 and Swarna were non-glossy ICSV 708 and ICSV 25066 than the susceptible check (Table 1). The anthocyanin pigmentation in seedlings (Swarna) and higher than resistant check (IS 2205) of sorghum genotypes IS 2123, IS 2205, ICSV 700 indicates significant variability in levels of antibiosis and ICSV 708 was intermediate, however ICSV among test genotypes against spotted stem borer.

25066 and Swarna showed no anthocyanin Amount of various biochemical constituents in different pigmentation. sorghum genotypes

Phenolic acids Antibiosis in different sorghum genotypes against Chilo partellus There was significant variability in amounts of The biological parameters of C. partellus viz., ferulic acid (F5,10= 2428.88; P <0.001) and p-coumaric larval weight (F5,20= 1533.85; P <0.001), larval acid (F5,10= 2595.05; P <0.001) in the seedlings of survival (F5,20= 28.48; P <0.001), larval period (F5,20= test sorghum genotypes (Fig. 1). The ferulic and 145.82; P <0.001), pupal weight (F5,20= 45.07; p-coumaric acid contents were significantly higher in P <0.001), pupal period (F5,20= 34.40; P <0.001), and resistant (IS 2205) and susceptible (Swarna) checks as adult emergence (F5,20= 33.20; P <0.001) significantly compared to other test sorghum genotypes (Table 3). varied across test sorghum genotypes. The larval Furthermore, significantly lower amount of ferulic period was significantly longer on IS 2123 and the and p-coumaric acids in ICSV 708 and ICSV 700 as resistant check IS 2205 as compared to other test compared to other test sorghum genotypes reveals that

Table 1 — Evaluation of different sorghum genotypes for Chilo partellus damage parameters and associated morphological traits Genotypes Stem borer Stem borer Stem Stem borer exit Stem borer Leaf glossiness Seedling deadhearts (%) tunnels/ stem tunneled (%) holes/stem larvae/stem score pigmentation score IS 2123 14.2 7.7 41.5 9.1 1.7 3.9 3.4 IS 2205 13.4 8.3 51.6 9.5 2.1 1.1 3.6 ICSV 700 22.3 2.9 9.6 3.6 0.9 1.7 3.4 ICSV 708 27.1 1.5 14.5 1.5 0.7 1.9 3.8 ICSV 25066 28.4 3.4 13.5 3.9 1.3 2.2 5 Swarna 41.7 3.5 34.5 4.2 1.1 5 4.9 F-probability <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 LSD (0.05) 3.65 0.85 4.49 1.27 0.21 0.5 0.64

Table 2 — Expression of antibiosis mechanism of resistance to Chilo partellus in different sorghum genotypes Larval weight at 30 Larval Larval period Pupal weight Pupal period Adult emergence Genotypes days (mg/larva) survival (%) (days) (mg/pupa) (days) (%) IS 2123 28.4 46.0 48.7 72.9 9.9 43.0 IS 2205 35.6 42.0 51.0 95.6 9.6 40.0 ICSV 700 114.4 67.0 40.3 100.5 12.8 62.0 ICSV 708 127.8 67.0 36.4 95.9 10.2 63.0 ICSV 25066 130.0 72.0 36.2 95.5 10.7 67.0 Swarna 135.3 81.0 35.2 110.1 10.0 77.0 F-probability <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 LSD (0.05) 3.73 8.44 1.68 5.38 0.59 7.35

DHILLON & CHAUDHARY: CHILO PARTELLUS RESISTANCE MECHANISMS IN SORGHUM 33

Fig. 1—Chromatograms of ferulic and p-coumaric acids separated from different sorghum genotypes using HPLC-PDA.

Table 3 — Amount of selected phenolic acids in 14 days old seedlings Nutritional factors of sorghum genotypes with variables levels of resistance to C. partellus Total protein (F5,10= 1780.21; P <0.001), oil (F5,10= Phenolic acids (mg/g) 30.80; P <0.001), total sugars (F5,10= 2250.72; Genotypes Ferulic acid p-Coumaric acid P <0.001), starch (F5,10= 84586.13; P <0.001), IS 2123 7.46 7.91 chlorophyll-a (F5,10= 829.47; P <0.001), chlorophyll-b IS 2205 13.40 14.05 (F5,10= 8974.69; P <0.001), total chlorophyll (F5,10= ICSV 700 5.25 5.08 5004.45; P <0.001), total carotenoids (F = 769.15; ICSV 708 3.73 3.76 5,10 ICSV 25066 5.87 7.64 P <0.001), iron (F5,10= 88203.68; P <0.001), and zinc Swarna 11.49 9.36 (F5,10= 3203.50; P <0.001) contents varied significantly F-probability <0.001 <0.001 in the seedlings of test sorghum genotypes. The LSD (0.05) 0.02 0.22 seedlings of moderately resistant sorghum genotype this variation is not in accordance to their levels of ICSV 708 and susceptible check, Swarna were found antibiosis, suggesting genotype-specific biochemical with significantly higher protein content as compared interactions of these phenolic acids in sorghum other sorghum genotypes including resistant check, IS defense against C. partellus. 2205 (Table 4). Conversely, the sugar content was 34 INDIAN J EXP BIOL, JANUARY 2018

significantly lower in ICSV 708 and susceptible check, higher in the seedlings of resistant check, IS 2205 and Swarna as compared to other test sorghum genotypes. It lower in susceptible check, Swarna as compared to other was found significantly higher in the seedlings of ICSV test sorghum genotypes, except in a few cases. 700 and ICSV 25066 as compared to resistant and Association of sorghum morphological and stem borer susceptible checks. The oil content was significantly damage parameters with various biological attributes of lower in the seedlings of ICSV 25066 and ICSV 708, C. partellus while rest of the test genotypes including resistant and Anthocyanin pigmentation was found significantly susceptible checks were on par with each other negatively correlated with larval period (r = −0.60), (Table 4). Starch content was significantly higher in all while significantly positively correlated (r = 0.58 to the test sorghum genotypes as compared to susceptible 0.65) with larval and pupal weights, larval survival and check, Swarna, being highest in ICSV 708. The content emergence of C. partellus adults (Table 5). The larval of chlorophyll-a was significantly lower in resistant period of C. partellus was found significantly check, IS 2205 being higher in IS 25066, while rest of negatively (r = −0.88), while larval and pupal weights, the test genotypes were on par with susceptible check, larval survival and adult emergence significantly Swarna. Conversely, chlorophyll-b content was positively correlated (r = 0.79 to 0.95) with deadhearts significantly lower in IS 25066 as compared to all the caused by this pest. The numbers of exit holes, larvae test genotypes including resistant and susceptible checks recovered, number of tunnels and stem tunneling (Table 4). Total chlorophyll content was significantly length were found significantly positively correlated higher in susceptible check, Swarna, being lowest in IS with larval period (r = 0.72 to 0.89), however, 2123 as compared to other test genotypes. The total these damage parameters were significantly and carotenoid content was significantly higher in the negatively correlated (r = −0.54 to −0.84) with larval seedlings of IS 2123 and lower in susceptible check, and pupal (except stem tunnel length) weights, larval Swarna than in other test sorghum genotypes (Table 4). survival and adult emergence of C. partellus adults However, iron and zinc contents were significantly (Table 5).

Table 4 — Biochemical and nutritional composition of 14 days old seedlings of sorghum genotypes with variable levels of resistance to Chilo partellus Chlorophyll (mg/g) Genotypes Protein (%) Oil (%) Sugar (%) Starch (%) Total carotenoid Fe (mg/g) Zn (mg/g) a b Total (mg/g) IS 2123 20.33 2.61 8.62 29.79 2.62 2.39 5.00 12.67 26.01 4.59 IS 2205 21.32 2.68 8.33 32.59 2.49 3.11 5.59 12.52 35.61 5.03 ICSV 700 21.09 2.67 11.20 32.98 2.64 3.48 6.11 12.41 33.97 4.19 ICSV 708 22.91 2.29 7.37 35.38 2.58 3.58 6.14 12.45 29.78 4.71 ICSV 25066 15.43 2.18 11.13 33.02 3.21 1.93 5.16 12.64 29.63 3.27 Swarna 22.81 2.66 8.51 28.71 2.63 3.59 6.21 12.34 26.05 3.92 F-probability <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 LSD (0.05) 0.21 0.13 0.11 0.03 0.03 0.02 0.02 0.02 0.04 0.04

Table 5 — Association of seedling morphological and stem borer damage traits with various biological parameters of Chilo partellus fed on different sorghum genotypes Sorghum morphological/ Correlation coefficients (r) of Chilo partellus damage with biological parameters Chilo damage parameters Larval period Larval survival Larval weight Pupal period Pupal weight Adult emergence Sorghum morphological traits Leaf glossiness −0.19 0.35 0.16 −0.39 0.19 0.36 Pigmentation −0.60** 0.63** 0.58* −0.22 0.58* 0.65** Chilo partellus damage parameters Chilo deadhearts −0.88** 0.95** 0.79** −0.10 0.80** 0.95** Chilo exit holes 0.88** −0.82** −0.78** −0.36 −0.59** −0.82** Chilo larvae recovered 0.79** −0.78** −0.76** −0.46 −0.54* −0.77** No. of Chilo stem tunnels 0.89** −0.84** −0.78** −0.40 −0.60** −0.83** Stem tunnel length 0.72** −0.61** −0.57* −0.66** −0.31 −0.61** [*, ** = Correlation coefficients significant at P = 0.05, and 0.01, respectively] DHILLON & CHAUDHARY: CHILO PARTELLUS RESISTANCE MECHANISMS IN SORGHUM 35

Association among different biochemical constituents in carotenoid contents were also significantly and sorghum seedlings and with different biological parameters of negatively correlated with larval and pupal weights, C. partellus respectively (Table 6). Stem borer deadhearts were significantly positively correlated with chlorophyll-a (r = 0.55) and Total carotenoid content in sorghum seedlings significantly negatively correlated with total showed significant and negative correlation with carotenoid, iron and zinc contents (r = −0.48 to −0.61) chlorophyll-b, total chlorophyll and protein content in the seedlings of test sorghum genotypes (Table 6). (−0.69 to −0.97); chlorophyll-a with chlorophyll-b, oil, Oil, total carotenoid, zinc and p-coumaric acid contents protein and zinc (−0.66 to −0.87); starch with ferulic showed significantly positive (r = 0.49 to 0.69), while acid and oil; and protein with sugar and zinc (−0.67 to chlorophyll-a and -b contents negative correlation −0.70). However, the correlation coefficients between (r = −0.47 to −0.51) with larval period of C. partellus. chlorophyll-b, total chlorophyll and protein (0.72 to Total sugar content was significantly positively 0.90), ferulic and p-coumeric acids (0.94), starch and (r = 0.68), and ferulic acid and p-coumeric acid iron (0.58), and zinc and protein (0.67) were contents negatively correlated (r = −0.54 to −0.55) with significant and positive (Table 7). pupal period of C. partellus. Chlorophyll-a showed significantly positive correlation with larval survival, Discussion larval and pupal weights, and C. partellus adult Holistic plant nutrition determines suitability of a 25 emergence on different sorghum genotypes (r = 0.48 to host , while variation in host plant quality in terms of 0.66). Total carotenoid and zinc contents were found allelochemical composition is detrimental to growth, significantly negatively correlated with C. partellus development and reproduction of the insect26. Plant larval survival and adult emergence on different resistance to C. partellus is a complex phenomena sorghum genotypes (r = −0.58 to −0.69). Zinc and total involving many traits, which finally determines the

Table 6 — Association of nutritional and anti-nutritional factors in sorghum seedlings with biological parameters of Chilo partellus Damage/ Correlation coefficients (r) of biochemical factors in sorghum seedlings with biological parameters of C. partellus biological Total Total Chlorophyll Total Ferulic p-Coumaric Starch Oil Fe Zn parameters protein sugars a b Total carotenoid acid acid Chilo deadhearts 0.12 −0.23 0.02 −0.20 0.55* 0.27 0.32 −0.60** −0.48* −0.61** −0.04 −0.26 Larval period 0.09 −0.18 −0.24 0.50* −0.51* −0.47* −0.21 0.49* 0.26 0.69** 0.40 0.52* Larval survival 0.04 −0.02 0.20 −0.31 0.58* 0.36 0.31 −0.60** −0.34 −0.67** −0.27 −0.46 Larval weight 0.01 0.09 0.15 −0.34 0.48* 0.35 0.24 −0.46 −0.25 −0.55* −0.29 −0.42 Pupal period −0.10 0.32 0.68** 0.10 0.24 0.10 0.15 −0.19 0.40 −0.19 −0.55* −0.54* Pupal weight 0.21 −0.11 0.12 0.04 0.66** 0.12 0.47* −0.72** −0.06 −0.42 0.11 −0.09 Adult emergence 0.01 −0.03 0.22 −0.32 0.56* 0.39 0.29 −0.58* −0.35 −0.69** −0.27 −0.45 [*, ** = Correlation coefficients significant at P = 0.05, and 0.01, respectively]

Table 7—Association among different nutritional and anti-nutritional factors in sorghum seedlings with variable levels of resistance to Chilo partellus Correlation coefficients (r) among different biochemical factors in sorghum seedlings Biochemical factors Total carotenoid A B C D E F G H I J A. Chlorophyll-a 0.44 B. Chlorophyll-b −0.91** −0.73** C. Total chlorophyll −0.97** −0.45 0.94** D. Ferulic acid −0.15 −0.36 0.11 −0.03 E. Iron −0.14 −0.18 0.21 0.18 0.13 F. Oil −0.34 −0.69** 0.43 0.22 0.58* 0.17 G. p-Coumeric acid 0.13 −0.18 −0.16 −0.29 0.94** 0.26 0.42 H. Protein −0.69** −0.91** 0.90** 0.72** 0.22 −0.01 0.54* −0.04 I. Starch 0.03 0.12 0.09 0.18 −0.57* 0.58* −0.55* −0.42 −0.10 J. Sugar 0.15 0.66** −0.41 −0.21 −0.29 0.25 −0.12 −0.17 −0.70** 0.04 Zinc −0.06 −0.87** 0.43 0.13 0.26 0.35 0.45 0.25 0.67** 0.17 −0.69** [*, ** = Correlation coefficients significant at P = 0.05, and 0.01, respectively] 36 INDIAN J EXP BIOL, JANUARY 2018

mechanism, expression and level of resistance27. release of phenolics from cereal bran in digestion is Present study shows that the C. partellus reared on never quantitative, thus their impact in antibiosis from germplasm genotypes IS 2205 and IS 2123 this route is minimal38. Present study revealed that the significantly reduced larval and pupal weights, amounts of ferulic and p-coumaric acids were although prolonged larval period, larval survival and adult highly variable in the test sorghum genotypes, these emergence, and resulted in lower deadhearts followed variations were not found in accordance to their levels by varieties ICSV 700, ICSV 708 and ICSV 25066 in of antibiosis, indicating genotype-specific role of these comparison to susceptible check, Swarna, indicating phenolic acids in sorghum defense against C. partellus. variable levels of antibiosis in these genotypes against No adverse effects of even higher concentrations of spotted stem borer. Earlier studies have also reported p-coumaric acid and ferulic acid have been reported on these genotypes as sources of resistance5, having the development of Sesamia nonagrioides (Lefebvre)39. antibiosis as the predominant mechanism of resistance Earlier, Dhillon and Chaudhary13 also demonstrated to C. partellus28,29. that the defense of maize plants against C. partellus is A number of plant phenological and insect damage not because of per se ferulic and p-coumaric acid parameters have been reported to be associated with concentrations, but due to their interaction with other stem borer deadhearts1,8, however, leaf feeding, biochemical constituents. deadhearts and stem tunneling by C. partellus larvae Present study revealed significant variability in are considered as primary yield reducing factors1,30. amount of protein, oil, total sugars, starch, chlorophyll, Present study revealed significant variability in carotenoids, iron, and zinc in the sorghum seedlings deadhearts, numbers of tunnels, percent stem area across test genotypes. Biochemical factors such as tunneled, number of exit holes and number of stem nitrogen, phosphorus, potash, silica, iron, sugars, amino borer larvae recovered per stem across test sorghum acids, carotenoid, protein content, surface wax, genotypes. The numbers of C. partellus exit holes, chlorophyll, etc. have also been reported to be larvae recovered, number of tunnels and stem tunneling associated with resistance/susceptibility to insect pests length were found significantly and positively in sorghum and maize13,15,40-43. The significant and correlated with larval period and negatively correlated negative association of total carotenoid, p-coumaric with larval and pupal (except stem tunnel length) acid, zinc and iron contents with different growth, weights, larval survival and adult emergence, while development, survival and damage parameters of reverse was the case with deadhearts, suggesting that C. partellus suggest their role in resistance to this pest these plant damage and insect biological parameters are in sorghum. Furthermore, the significant and positive important resistance determining factors in sorghum association between ferulic acid, p-coumaric acid and against spotted stem borer. There was significant oil; chlorophyll-a and total sugars; chlorophyll-b, total variation in test sorghum genotypes for leaf glossiness chlorophyll and protein; iron and starch; and protein and anthocyanin pigmentation, and the leaf and zinc; while significant and negative correlation of anthocyanin pigmentation showed significant and chlorophyll-a with chlorophyll-b, oil, protein and zinc; negative association with development and survival of ferulic acid with starch; oil with starch; protein with C. partellus. Leaf glossiness and anthocyanin total sugars; and total sugars with zinc, during present pigmentation in sorghum have also been reported studies indicate complex biochemical interactions earlier to be associated with resistance to C. partellus1. among the biochemical factors playing role in Insects compensate nutritional requirement by resistance/susceptibility to C. partellus. Inter- and intra- increasing the rate and quantity of food intake, thus specific interactions and synthesis of desired compounds leading to accumulation of desired amount of proteins, during insect growth also result in quantitative and amino acids, lipophilic metabolites and other qualitative variations in biochemical constituents in plant nutritional compounds required for growth, species/genotypes and insects29. Thus, variations for development and life processes29,31-33. The phenolics these biochemical constituents in the test sorghum are known to provide structural support, pigmentation, genotypes irrespective of their levels of resistance signalling, and defense against biotic and abiotic suggest that the interaction among different biochemical stresses in plants34-36. However, the origin of compounds and the morphological traits, rather than a hydrolysed ferulic and p-coumaric acids is mainly the particular biochemical constituent play a greater role in phenolic acid-carbohydrate complex of cell wall37, and host plant defense against C. partellus. DHILLON & CHAUDHARY: CHILO PARTELLUS RESISTANCE MECHANISMS IN SORGHUM 37

Acknowledgement 16 Dhillon MK, Sharma HC, Singh R & Naresh JS, Influence of cytoplasmic male-sterility on expression of physico-chemical The authors gratefully acknowledge funding by traits associated with resistance to sorghum shoot fly, ICAR-Indian Agricultural Research Institute, New Atherigona soccata. SABRAO J Breed Genet, 38 (2006) 105. Delhi. We also wish to thank Dr. H.C. Sharma, 17 Sharma HC, Taneja SL, Leuschner K & Nwanze KF, ICRISAT, India for providing seeds of test sorghum Techniques to screen sorghum for resistance to insect pests. genotypes. Information Bulletin No. 32. International Crops Research Institute for the Semi-Arid Tropics, Patancheru 502 324,

Andhra Pradesh, India, 1992. References 18 AOAC, Official Methods of Analysts. In: Association of 1 Sharma HC, Dhillon MK, Pampapathy G & Reddy BVS, Official Analytical Chemicals, Edn 10, (Washington DC, Inheritance of resistance to spotted stem borer, Chilo partellus USA), 1970, 744. in sorghum, Sorghum bicolor. Euphytica, 156 (2007) 117. 2 Dhillon MK, Kalia VK & Gujar GT, Insect pests and their 19 Clegg KM, The application of anthrone reagent to the management: Current status and future need of research in estimation of starch in cereals. J Sci Food Agri 7 (1956) 40. quality maize. In: Maize: Nutrition dynamics and novel uses, 20 Nelson N, A photometric adoption of the Somogyi's method (Ed. Choudhary DP, Sapna & Kumar S, Springer, New York, for determination of glucose. J Biol Chem 153 (1944) 375. USA), 2014a, 95. 21 Hiscox JD & Israelstam GF, Different methods of chlorophyll 3 Dhaliwal GS, Jindal V & Mohindru B, Crop losses due to extraction. Can J Bot, 57 (1979) 1332. insect pests: global and Indian scenario. Indian J Entomol, 77 22 Rodriguez DB & Kimura M, Harvest Plus Handbook for (2015) 165. Carotenoid Analysis, Edn I (IFPRI, Washington, DC), 2004. 4 Kfir R, Overholt WA, Khan ZR & Polaszek A, Biology and 23 Prasad R, Shivay YS, Kumar D & Sharma SN, Learning by management of economically important lepidopteran cereal doing exercises in soil fertility: A practical manual for soil stem borers in Africa. Annu Rev Entomol, 47 (2002) 701. fertility. Division of Agronomy, Indian Agricultural Research 5 Sharma HC, Reddy BVS, Dhillon MK, Venkateswaran K, Institute, New Delhi, India, 2006. Singh BU, Pampapathy G, Folkertsma R, Hash CT & Sharma 24 Hung PV, Hatcher DW & Baker W, Phenolic acid KK, Host plant resistance to insects in Sorghum: Present status composition of sprouted wheats by ultra-performance liquid and need for future research. SAT eJournal, 1 (2005) 1. chromatography (UPLC) and their antioxidant activities. Food 6 Chapman RF, Woodhead S & Bernays EA, Survival and Chem, 126 (2011) 1896. dispersal of young larvae of Chilo partellus (Swinhoe) 25 Beck SD, Nutrition, adaptation and environment. In: Insect (: Pyralidae) in two cultivars of sorghum. Bull and Mite Nutrition: Significance and Implications in Ecology Entomol Res, 73 (1983) 65. and Pest Management, (Ed. Rodriguez JG, North-Holland 7 Dabrowski ZT & Kidiavai EL, Resistance of some sorghum Publications, Amsterdam, The Netherlands), 1972, 1. lines to the spotted stalk borer, Chilo partellus, under western 26 Baldwin IT, Halitschke R, Kessler A & Schittko U, Merging Kenyan conditions. Insect Sci Appl, 4 (1983) 119. molecular and ecological approaches in plant–insect 8 Woodhead S & Taneja SL, The importance of the behaviour of interactions. Curr Opin Plant Biol, 4 (2001) 351. young larvae in sorghum resistance to Chilo partellus. 27 Huang Y, Sharma HC & Dhillon MK, Bridging conventional Entomol Exp Applic, 45 (1987) 47. and molecular genetics of sorghum insect resistance. In: Plant 9 Khurana AD & Verma AN, Amino acid contents in sorghum Genetics and Genomics: Crops and Models Vol. 11: Genomics plants, resistant/ susceptible to stem borer and shoot fly. Indian of the Saccharinae, (Ed. Paterson AK, Springer, New York, J Entomol 44 (1982) 184. USA), 2013, 367. 10 Khurana AD & Verma AN, Some biochemical plant 28 Kumar KV, Sharma HC & Reddy DK, Antibiosis mechanism characters in relation to susceptibility of sorghum to stem borer of resistance to spotted stem borer, Chilo partellus in sorghum, and shoot fly. Indian J Entomol 45 (1983) 29. Sorghum bicolor. Crop Prot, 25 (2006) 66. 11 Khurana AD & Verma AN, Some physical plant characters in 29 Dhillon MK & Kumar S, Amino acid profiling of different relation to stem borer and shoot fly resistance in sorghum. sorghum genotypes and Chilo partellus (Swinhoe) larvae for Indian J Entomol 47 (1985) 14. their contribution in resistance. Arthro Plant Interact, 11 12 Torto B, Hassanali A & Saxena KN, Chemical aspects of (2017) 537. Chilo partellus feeding on certain sorghum cultivars. Insect Sci 30 Alghali AM, Effect of cultivar, time, and amount of Chilo Appl, 11 (1990) 649. partellus Swinhoe (Lepidoptera: Pyralidae) infestation on 13 Dhillon MK & Chaudhary DP, Biochemical interactions for sorghum yield components in Kenya. Trop Pest Manage, 32 antibiosis mechanism of resistance to Chilo partellus (1986) 126. (Swinhoe) in different maize types. Arthro Plant Interact, 9 31 Chapman RF, The Insects: Structure and Function, 4th edition, (2015) 373. (Cambridge University Press, UK), 1998. 14 Ajala SO & Saxena KN, Interrelationship among Chilo 32 Dhillon MK, Kumar S & Gujar GT, A common HPLC-PDA partellus (Swinhoe) damage parameters and their contribution method for amino acid analysis in insects and plants. Indian J to grain yield reduction in maize (Zea mays L.). Appl Entomol Exp Biol, 52 (2014b) 73. Zool, 29 (1994) 469. 33 Kumar S & Dhillon MK, Lipophilic metabolite profiling of 15 Dhillon MK, Sharma HC, Singh R & Naresh JS, Mechanisms maize and sorghum seeds and seedlings, and their pest spotted of resistance to shoot fly, Atherigona soccata in sorghum. stem borer larvae: A standardized GC-MS based approach. Euphytica, 144 (2005) 301. Indian J Exp Biol, 53 (2015) 170. 38 INDIAN J EXP BIOL, JANUARY 2018

34 Dixon RA & Paiva NL, Stress-induced phenylpropanoid 39 Santiago R, Butrón A, Arnason JT, Reid LM, Souto XC & metabolism. Plant Cell, 7 (1995) 1085. Malvar RA, Putative role of pith cell wall phenylpropanoids in 35 Berbehenn RV, Martin MM & Hagerman AE, Reassessment Sesamia nonagrioides (Lepidoptera: Noctuidae) resistance. J of the roles of the peritrophic envelope and hydrolysis in Agri Food Chem, 54 (2006) 2274. protecting polyphagous grasshoppers from ingested 40 Hedin PA, Williams WP, Davis FM & Buckley PM, Role of hydrolyzable tannins. J Chem Ecol, 22 (1996) 1901. amino acids, protein and fiber in leaf feeding resistance of corn 36 Douglas CJ, Phenylpropanoid metabolism and lignin to the fall armyworm. J Chem Ecol, 16 (1990) 1977. biosynthesis: from weed to trees. Trends Plant Sci, 1 (1996) 41 Kumar H, Resistance in maize to Chilo partellus (Swinhoe) 171. (Lepidoptera: Pyralidae): An overview. Crop Prot, 16 (1997) 37 Fincher GB & Stone BH, Cell walls and their components in 243. cereal grain technology. Adv Cereal Sci Technol, 8 (1986) 207. 42 Rao CN & Panwar VPS, Biochemical plant factors affecting 38 Sen A, Role of conjugated phenolic amines in the resistance of resistance to Chilo partellus (Swinhoe) in maize. Ann Plant maize towards Sitophilus zeamais and Prostephanus truncates. Prot Sci, 10 (2002) 28. In: Proceeding of the National Symposium on Biochemical 43 Chamarthi SK, Sharma HC, Sahrawat KL, Narasu LM & Bases of Host Plant Resistance to Insects, (Ed. Dhillon MK, Physico-chemical basis of resistance to shoot fly, Ananthakrishnan T, National Academy of Agricultural Atherigona soccata in sorghum, Sorghum bicolor. J Appl Sciences, New Delhi-110012, India), 1996, 125. Entomol, 135 (2011) 446.