Standardized agronomic practices for mechanical harvesting of the single-harvest garden pea in India.

The field investigations were conducted at Vegetable Research Farm, Department of vegetable Science, Punjab Agricultural University, Ludhiana with the objective to standarize the agronomic practices for mechical harvesting of garden pea. Therefore, effect of different agronomic traits including time of sowing, spacing and sowing method on yield and its attributing traits were studied on single-harvest garden pea. The experiment was laid out in split-plot design with 30 treatments comprising five dates of sowing in main plots and 2 different planting methods (flat and bed) sown at three different spacing in subplots and replicated three times. The bed size was standardized according to front-loading width of the pea combine. Significant interactions were observed between the date of sowing and spacing for a number of pods per plant, green seeds per pod, green pod yield per plant, green pod yield per plot; date of sowing and sowing method for pod length; spacing and sowing method for plant height. However, for all the traits, there was non-significant 3-way interaction. The late sown (20th December) crop resulted in the least number of pods per plant, green seeds per pod, green pod yield per plant and green pod yield per plot which was due to high temperature and low relative humidity conditions at pod development and filling stage. It is concluded that the garden pea cultivar Punjab-89 sown on 5th November at the spacing of 20 × 7.5 cm on beds (bed width of 1.0 m) resulted in significantly highest green pod yield of 12.75 kg/9 m2 in the single harvest. Therefore, the 3 beds of 1 m width can be harvested together with pea combine (because combine has working front width of 3.1 m) results into maximum yield when pea crops is sown on 5th November @ 20 × 7.5 cm spacing.

Biochemical probing of phloem sap defensive traits in Brassica juncea-B. fruticulosa introgression lines following Lipaphis erysimi infestation.

The lack of resistance to Lipaphis erysimi in cultivated Brassicas makes caused this pest highly devastating resulting in significant loss of rapeseed-mustard productivity in India. B. fruticulosa, a wild crucifer is known as an excellent source of resistance to L. erysimi. Therefore, we planned to assess defense associated biochemical alterations and molecular components of B. juncea-B. fruticulosa ILs to mustard aphid. Phenotypic assessment of ILs on the basis of aphid population per plant (APP) categorized genotypes into resistant (7.15-18.50 APP), moderately susceptible (42.29-53.33 APP) and susceptible (70.00-77.07 APP) genotypes. Mustard aphid infested minimally B. fruticulosa (0.80 APP) among tested genotypes. The maximum increase in catalase (CAT) activity was determined in B. fruticulosa and resistant ILs after 48 h (2.03 and 1.76-fold, respectively) and one week (2.98 and 1.79-fold, respectively) of mustard aphid infestation. The strong induction of CAT2 transcripts (19.25-fold) and CAT activity (5.88-fold) along with low aphid count in resistant IL, Ad4-64 (13.85 APP) suggested the pivotal role of CAT in resistance to mustard aphid. Guaiacol peroxidase (GPX) was significantly decreased following pest infestation at both infestation stages. The ascorbate content was highest in resistant IL, ADV-6RD (2.14-fold) after one week of aphid infestation. H2O2 content rapidly increased in B. juncea-B. fruticulosa derived lines after 48 h of aphid infestation. The negative and significant association between APP and CAT (- 0.56** and - 0.48*, respectively), glutathione (- 0.43* and - 0.40*, respectively), H2O2 (- 0.57** and - 0.43*, respectively) at both 48 h and one week infestation stages signified their role in deterring mustard aphid infestation. The positive and significant association between total sugars (0.33* at 7 DPI), reducing sugars (0.33* at 7 DPI), sucrose (0.36** at 48 h) and APP indicated that higher the sugars content, higher will be mustard aphid infestation in B. juncea derived ILs. The information being generated and key candidates (CAT2, ascorbate and H2O2) being identified may help in effective deployment of B. fruticulosa resistance in mustard breeding.

Genome wide association analyses to understand genetic basis of flowering and plant height under three levels of nitrogen application in Brassica juncea (L.) Czern & Coss.

Timely transition to flowering, maturity and plant height are important for agronomic adaptation and productivity of Indian mustard (B. juncea), which is a major edible oilseed crop of low input ecologies in Indian subcontinent. Breeding manipulation for these traits is difficult because of the involvement of multiple interacting genetic and environmental factors. Here, we report a genetic analysis of these traits using a population comprising 92 diverse genotypes of mustard. These genotypes were evaluated under deficient (N75), normal (N100) or excess (N125) conditions of nitrogen (N) application. Lower N availability induced early flowering and maturity in most genotypes, while high N conditions delayed both. A genotyping-by-sequencing approach helped to identify 406,888 SNP markers and undertake genome wide association studies (GWAS). 282 significant marker-trait associations (MTA's) were identified. We detected strong interactions between GWAS loci and nitrogen levels. Though some trait associated SNPs were detected repeatedly across fertility gradients, majority were identified under deficient or normal levels of N applications. Annotation of the genomic region (s) within ± 50 kb of the peak SNPs facilitated prediction of 30 candidate genes belonging to light perception, circadian, floral meristem identity, flowering regulation, gibberellic acid pathways and plant development. These included over one copy each of AGL24, AP1, FVE, FRI, GID1A and GNC. FLC and CO were predicted on chromosomes A02 and B08 respectively. CDF1, CO, FLC, AGL24, GNC and FAF2 appeared to influence the variation for plant height. Our findings may help in improving phenotypic plasticity of mustard across fertility gradients through marker-assisted breeding strategies.

Association genetics of the parameters related to nitrogen use efficiency in Brassica juncea L.

KEY MESSAGE: Genome wide association studies allowed prediction of 17 candidate genes for association with nitrogen use efficiency. Novel information obtained may provide better understanding of genomic controls underlying germplasm variations for this trait in Indian mustard. Nitrogen use efficiency (NUE) of Indian mustard (Brassica juncea (L.) Czern & Coss.) is low and most breeding efforts to combine NUE with crop performance have not succeeded. Underlying genetics also remain unexplored. We tested 92 SNP-genotyped inbred lines for yield component traits, N uptake efficiency (NUPEFF), nitrogen utilization efficiency (NUTEFF), nitrogen harvest index (NHI) and NUE for two years at two nitrogen doses (No without added N and N100 added @100 kg/ha). Genotypes IC-2489-88, M-633, MCP-632, HUJM 1080, GR-325 and DJ-65 recorded high NUE at low N. These also showed improved crop performance under high N. One determinate mustard genotype DJ-113 DT-3 revealed maximum NUTEFF. Genome wide association studies (GWAS) facilitated recognition of 17 quantitative trait loci (QTLs). Environment specificity was high. B-genome chromosomes (B02, B03, B05, B07 and B08) harbored many useful loci. We also used regional association mapping (RAM) to supplement results from GWAS. Annotation of the genomic regions around peak SNPs helped to predict several gene candidates for root architecture, N uptake, assimilation and remobilization. CAT9 (At1g05940) was consistently envisaged for both NUE and NUPEFF. Major N transporter genes, NRT1.8 and NRT3.1 were predicted for explaining variation for NUTEFF and NUPEFF, respectively. Most significant amino acid transporter gene, AAP1 appeared associated with NUE under limited N conditions. All these candidates were predicted in the regions of high linkage disequilibrium. Sequence information of the predicted candidate genes will permit development of molecular markers to aid breeding for high NUE.

Genetics of days to flowering, maturity and plant height in natural and derived forms of Brassica rapa L.

KEY MESSAGE: Genome wide association studies enabled prediction of many candidate genes for flowering, maturity and plant height under differing day-length conditions. Some genes were envisaged only from derived B. rapa. Flowering and plant height are the key life history traits. These are crucial for adaptation and productivity. Current investigations aimed to examine genotypic differences governing days to flowering, maturity and plant height under contrasting day-length conditions; and identify genomic regions governing the observed phenotypic variations. An association panel comprising 195 inbred lines, representing natural (NR) and derived (DR) forms of Brassica rapa (AA; 2n = 20), was evaluated at two sowing dates and two locations, representing different day-length regimes. Derived B. rapa is a unique pre-breeding material extracted from B. juncea (AABB; 2n = 36). Population structure analysis, using DArT genotypes established derived B. rapa as a genetic resource distinct from natural B. rapa. Genome wide association studies facilitated detection of many trait associated SNPs. Chromosomes A03, A05 and A09 harboured majority of these. Functional annotation of the associated SNPs and surrounding genome space(s) helped to predict 43 candidate genes. Many of these were predicted under specific day-length conditions. Important among these were the genes encoding floral meristem identity (SPL3, SPL15, AP3, BAM2), photoperiodic responses (COL2, AGL18, SPT, NF-YC4), gibberellic acid biosynthesis (GA1) and regulation of flowering (EBS). Some of the predicted genes were detected for DR subpanel alone. Genes controlling hormones, auxins and gibberellins appeared important for the regulation of plant height. Many of the significant SNPs were located on chromosomes harbouring previously reported QTLs and candidate genes. The identified loci may be used for marker-assisted selection after due validation.

Molecular and genetic analysis of defensive responses of Brassica juncea - B. fruticulosa introgression lines to Sclerotinia infection.

Sclerotinia stem rot caused by Sclerotinia sclerotiorum is a major disease of crop brassicas, with inadequate variation for resistance in primary gene pools. We utilized a wild Brassicaceae species with excellent resistance against stem rot to develop a set of B. juncea - B. fruticulosa introgression lines (ILs). These were assessed for resistance using a highly reproducible stem inoculation technique against a virulent pathogen isolate. Over 40% of ILs showed higher levels of resistance. IL-43, IL-175, IL-215, IL-223 and IL-277 were most resistant ILs over three crop seasons. Sequence reads (21x) from the three most diverse ILs were then used to create B. juncea pseudomolecules, by replacing SNPs of reference B. juncea with those of re-sequenced ILs. Genotyping by sequencing (GBS) was also carried out for 88 ILs. Resultant sequence tags were then mapped on to the B. juncea pseudomolecules, and SNP genotypes prepared for each IL. Genome wide association studies helped to map resistance responses to stem rot. A total of 13 significant loci were identified on seven B. juncea chromosomes (A01, A03, A04, A05, A08, A09 and B05). Annotation of the genomic region around identified SNPs allowed identification of 20 candidate genes belonging to major disease resistance protein families, including TIR-NBS-LRR class, Chitinase, Malectin/receptor-like protein kinase, defensin-like (DEFL), desulfoglucosinolate sulfotransferase protein and lipoxygenase. A majority of the significant SNPs could be validated using whole genome sequences (21x) from five advanced generation lines being bred for Sclerotinia resistance as compared to three susceptible B. juncea germplasm lines. Our findings not only provide critical new understanding of the defensive pathway of B. fruticulosa resistance, but will also enable development of marker candidates for assisted transfer of introgressed resistant loci in to agronomically superior cultivars of crop Brassica.

Detection of First Marker Trait Associations for Resistance Against Sclerotinia sclerotiorum in Brassica juncea-Erucastrum cardaminoides Introgression Lines.

A set of 96 Brassica juncea-Erucastrum cardaminoides introgression lines (ILs) were developed with genomic regions associated with Sclerotinia stem rot (Sclerotinia sclerotiorum) resistance from a wild Brassicaceous species E. cardaminoides. ILs were assessed for their resistance responses to stem inoculation with S. sclerotiorum, over three crop seasons (season I, 2011/2012; II, 2014/2015; III, 2016-2017). Initially, ILs were genotyped with transferable SSR markers and subsequently through genotyping by sequencing. SSR based association mapping identified six marker loci associated to resistance in both A and B genomes. Subsequent genome-wide association analysis (GWAS) of 84 ILs recognized a large number of SNPs associated to resistance, in chromosomes A03, A06, and B03. Chromosomes A03 and A06 harbored the maximum number of resistance related SNPs. Annotation of linked genomic regions highlighted an array of resistance mechanisms in terms of signal transduction pathways, hypersensitive responses and production of anti-fungal proteins and metabolites. Of major importance was the clustering of SNPs, encoding multiple resistance genes on small regions spanning approximately 885 kb region on chromosome A03 and 74 kb on B03. Five SNPs on chromosome A03 (6,390,210-381) were associated with LRR-RLK (receptor like kinases) genes that encode LRR-protein kinase family proteins. Genetic factors associated with pathogen-associated molecular patterns (PAMPs) and effector-triggered immunity (ETI) were predicted on chromosome A03, exhibiting 11 SNPs (6,274,763-994). These belonged to three R-Genes encoding TIR-NBS-LRR proteins. Marker trait associations (MTAs) identified will facilitate marker assisted introgression of these critical resistances, into new cultivars of B. juncea initially and, subsequently, into other crop Brassica species.

Characterisation and inhibition studies of Helicoverpa armigera (Hübner) gut α-amylase.

BACKGROUND: The survival of a devastating pest, Helicoverpa armigera, is mainly dependent on the availability of α-amylase. Therefore, characterising H. armigera α-amylase and targeting it with effective inhibitors could aid in reducing its damaging effects. RESULTS: H. armigera gut possessed four isozymes of α-amylase. The molecular weight of the major purified isozyme ranged from 79 to 81 kDa. The purified enzyme was identified to be α-amylase on the basis of products formed from starch. The optimum pH and temperature were 10.0 and 50 °C respectively. The activation energy was 5.7 kcal mol(-1) . The enzyme showed high activity with starch and amylopectin, whereas dextrins were poor substrates. The Michaelis constant Km with starch, amylose and amylopectin was 0.45, 1.23 and 0.11 mg mL(-1) respectively. ZnSO4 , FeSO4 , CuSO4 , citric acid, oxalic acid and salicylic acid were potent inhibitors. ZnSO4 , salicylic acid and pigeonpea α-amylase inhibitor (∼21.0 kDa) acted primarily as competitive inhibitors, FeSO4 and citric acid displayed mainly anticompetitive behaviour, while CuSO4 and oxalic acid behaved mainly as non-competitive inhibitors. CONCLUSIONS: The identification of effective ecofriendly inhibitors could help in managing H. armigera infestation. © 2014 Society of Chemical Industry.

Induced resistance by oxidative shifts in pigeonpea (Cajanus cajan L.) following Helicoverpa armigera (Hübner) herbivory.

BACKGROUND: Oxidative responses in leaves, developing seeds and the pod wall of nine pigeonpea genotypes were investigated against Helicoverpa armigera feeding. Out of nine genotypes, four were moderately resistant, three were intermediate and two were moderately susceptible genotypes. RESULTS: A significant shift in the oxidative status of pigeonpea following herbivory was depicted by the upregulation of diamine oxidase (DAO), polyamine oxidase (PAO) and lipoxygenase 2 (LOX 2) activities. Polyphenol oxidase (PPO) activity was significantly higher in the infested pod wall and leaves of moderately resistant genotypes than in those of moderately susceptible genotypes. H. armigera infestation markedly enhanced phenylalanine ammonia lyase (PAL) and tyrosine ammonia lyase (TAL) activities in wounded tissues. The decline in ascorbate peroxidase (APX) activity and ascorbate content was lower in moderately resistant genotypes than in moderately susceptible genotypes. A significant decrease in LOX 3 activity was also observed in the infested pod wall of moderately resistant and intermediate genotypes. A lower malondialdehyde (MDA) content and higher proline content of the infested pod wall and developing seeds was observed. Higher activities of PPO, PAL and proline content in leaves of uninfested moderately resistant genotypes could either be an unrelated observation or alternatively could help in identifying H. armigera-resistant genotypes. CONCLUSION: The increase in activities of PPO, DAO, PAO, PAL and TAL and higher proline and lower MDA content upon herbivory suggested their integrated contribution in providing resistance to pigeonpea against H. armigera.

Structural features, substrate specificity, kinetic properties of insect α-amylase and specificity of plant α-amylase inhibitors.

BACKGROUND: α-Amylase is an important digestive enzyme required for the optimal growth and development of insects. Several insect α-amylases had been purified and their physical and chemical properties were characterized. Insect α-amylases of different orders display variability in structure, properties and substrate specificity. Such diverse properties of amylases could be due to different feeding habits and gut environment of insects. KEY POINTS: In this review, structural features and properties of several insect α-amylases were compared. This could be helpful in exploring the diversity in characteristics of α-amylase between the members of the same class (insecta). Properties like pH optima are reflected in enzyme structural features. In plants, α-amylase inhibitors (α-AIs) occur as part of natural defense mechanisms against pests by interfering in their digestion process and thus could also provide access to new pest management strategies. AIs are quite specific in their action; therefore, these could be employed according to their effectiveness against target amylases. Potential of transgenics with α-AIs has also been discussed for insect resistance and controlling infestation. CONCLUSIONS: The differences in structural features of insect α-amylases provided reasons for their efficient functioning at different pH and the specificity towards various substrates. Various proteinaceous and non-proteinaceous inhibitors discussed could be helpful in controlling pest infestation. In depth detailed studies are required on proteinaceous α-AI-α-amylase interaction at different pH's as well as the insect proteinase action on these inhibitors before selecting the α-AI for making transgenics resistant to particular insect.

Nitrate reductase and nitrite as additional components of defense system in pigeonpea (Cajanus cajan L.) against Helicoverpa armigera herbivory.

Amylase inhibitors serve as attractive candidates of defense mechanisms against insect attack. Therefore, the impediment of Helicoverpa armigera digestion can be the effective way of controlling this pest population. Nitrite was found to be a potent mixed non-competitive competitive inhibitor of partially purified α-amylase of H. armigera gut. This observation impelled us to determine the response of nitrite and nitrate reductase (NR) towards H. armigera infestation in nine pigeonpea genotypes (four moderately resistant, three intermediate and two moderately susceptible). The significant upregulation of NR in moderately resistant genotypes after pod borer infestation suggested NR as one of the factors that determine their resistance status against insect attack. The pod borer attack caused greater reduction of nitrate and significant accumulation of nitrite in moderately resistant genotypes. The activity of nitrite reductase (NiR) was also enhanced more in moderately resistant genotypes than moderately susceptible genotypes on account of H. armigera herbivory. Expression of resistance to H. armigera was further revealed when significant negative association between NR, NiR, nitrite and percent pod damage was observed. This is the first report that suggests nitrite to be a potent inhibitor of H. armigera α-amylase and also the involvement of nitrite and NR in providing resistance against H. armigera herbivory.

Red pericarp introgression lines derived from interspecific crosses of rice: physicochemical characteristics, antioxidative properties and phenolic content.

BACKGROUND: Antioxidative properties and physicochemical characteristics of introgression lines (ILs) and their recurrent parents were analyzed. In addition, catalase (CAT) and superoxide dismutase (SOD) activities and free radical-scavenging capacity were evaluated, since these are important antioxidative properties for developing nutraceutical and functional foods. RESULTS: Comparative analysis of the brown and milled rice fractions of ILs with their respective recurrent parents revealed 2.26- and 1.22-fold increase in total phenolics, 1.95- and 2.09-fold increase in flavonoids, 8.38- and 6.80-fold increase in proanthocyanidins and 1.55- and 1.20-fold increase in tannins in brown and milled rice fractions respectively. Higher CAT (1.36- and 1.89-fold) and SOD (1.71- and 2.02-fold) activities and Trolox equivalent antioxidant capacity (TEAC, 7.13- and 1.98-fold) were observed in brown and milled rice fractions respectively of ILs compared with their respective recurrent parents. A high and positive correlation was obtained between TEAC and total phenols (0.73, P ≤ 0.01), flavonoids (0.66, P ≤ 0.05) and proanthocyanidins (0.69, P ≤ 0.05). The yield parameters and physicochemical characteristics of the grains, in general, were comparable in the ILs and their respective recurrent parents. CONCLUSION: The ILs of rice reported in the present study exhibited significant positive differences in the content of phenolic constituents and antioxidant properties with good grain quality characteristics over their recurrent parents, indicating their potential as a natural source of phytochemicals for nutraceutical and functional food development.