Physiological And Biochemical Mechanism Of Brassinosteroids Mediated Tolerance To Verticillium Wilt In Cotton

Upland cotton (Gossypium hirsutum L.), the main cultivated cotton around the world, is highly susceptible to Verticillium wilt, a disease caused by a soil born necrotrophic fungal pathogen Verticillium dahliae. Commonly used cultural practices are futile to control this disease due to the absence of host specificity and extreme variability of pathogenicity. In this scenario, breeding for resistant cotton varieties would be the solitary approach to manage the disease and to reduce the economic losses. Pursuing such a purpose requires a comprehensive understanding of the mechanisms underlying pathogen infection and the corresponding host defense responses. Therefore, the present study was carried out to elucidate the physiological and biochemical mechanisms during stress and tolerance of cotton to Verticillium wilt. Considering the protective role of brassinosteroids (BRs) for abiotic stresses and inducing disease resistance, a biologoically active BR 24-epibrassinolide (EBR) was used to mitigate Verticillium wilt in cotton. The main results were summarized as follow.1. Effect of Verticillium dahliae toxin (Vd toxin) on cell structure, plant morphology and biochemical characteristics of upland cottonExposure of the cotton plant roots to V. dahliae toxin (Vd toxin) caused substantial changes at cellular/organ level and affected the physiological and biochemical responses of five different genotypes of G. hirsutum. For instance, Vd toxin decreased the uptake of basic nutrition solution (BNS) up to 75% in NIAB-846,70% in Zheda B,63% in Zheda R,56% in NIAB-111 and 34% in NIAB-999 than non treated control after 24 h of stress application. Similarly, Vd toxin triggered the activity of superoxide dismutase (SOD), decreased the contents of reduced ascorbate (ASA) and glutathione (GSH) with least decrease in NIAB-999 thus accounted for relatively low accumulation of malondialdehyde (MDA) when compared with other genotypes. In addition, the toxic effects of Vd toxin were persistent even after two weeks of its removal and resulted in suppression of the plant growth, particularly root growth parameters i.e root surface area, root length, root diameter and root volume which were significantly correlated with BNS uptake. This could be further associated with smashing of root tips, closing of stomata, damage of cellular organelles in root and leaf cells of Vd toxin treated plants. On the basis of integrated score, cotton genotypes were categorized for relative tolerance to Verticillium wilt in an order of NIAB-999>NIAB-111>Zheda R>Zheda B>NIAB-846. Our findings indicated that Vd toxin can be used as a pathogen free approach to induce stress at cellular level and to monitor the mechanism of Verticillium wilt.2. Role of brassinosteroids to alleviate negative effects of Vd toxin on cotton callus growthTreatment of cotton callus (G. hirsutum) with EBR induced Vd toxin stress tolerance and improved the callus growth. The effects were highly ameliorative when callus was treated with EBR prior to its exposure to Vd toxin (pre-EBR treatment) than EBR applied along with Vd toxin simultaneously (co-EBR treatment). Pre-EBR treated callus remained green; while 65% and 90% callus browning was observed in co-EBR and Vd toxin alone treated callus, respectively. Likewise, the fresh weight of the pre-EBR treated callus after three days was 52% higher than that of Vd toxin alone treatment whereas, this increase was only 23% in co-EBR treated callus. Meanwhile, EBR treatment of the cotton callus increased the contents of Chi a, Chi b, carotenoids, polyphenol oxidase (PPO), phenylalanine dehydrogenase (PAL), cinnamyl alcohol dehydrogenase (CAD), shikimate dehydrogenase (SKDH), total phenols, flavonoids, soluble sugars and proteins over Vd toxin alone treatment with higher increments being observed in pre-EBR treated callus. Furthermore, both pre- and co-EBR mimicked the DNA damage and tail moments of comet were less frequent in contrast to gigantic DNA damage in Vd toxin alone treated callus. The number/structure of mitochondria, granum and stroma thylakoids were also improved in EBR treated callus. In addition, the attachment of ribosomes with the endoplasmic reticulum was more profound in EBR treated callus. Taken together, application of EBR induced resistance in cotton callus against Vd toxin mainly by protecting photosynthetic apparatus, enhancing photosynthetic pigments and regulating secondary metabolism.3. Brassinosteroids attenuated Verticillium wilt in upland cotton by modulating the carbohydrates metabolism, plasma membrane ATPases and intracellular osmolytesWe extended our studies to whole cotton plant to monitor the mechanism of Verticillium wilt and protective role of root and shoot pre-EBR treatment in G. hirsutum using different levels of root (5,10,15 nM) and shoot (50,100,200 nM) applied EBR. Results revealed that in absence of EBR, Vd toxin caused 83% plant wilting and accumulation of glycine betaine and proline which were 33% and 61% higher than non treated control, respectively. However, the accumulation of these osmolytes was decreased in EBR treated plants with minimum values at 5 nM and 200 nM. Furthermore, the results depicted a remarkable decline in soluble sugars, photosynthesis (Pn), transpiration (Tr), chlorophyll content and chlorophyll fluorescence (Fv/Fm) in Vd toxin alone treated plants than Vd toxin+EBR. Moreover, the activities of sucrose synthase (SuSy), sucrose phosphate synthase (SPS) and acid invertase (AI) were high in Vd toxin+EBR treated plants and accompanied with increased root and shoot biomass than Vd toxin alone treated plants. Moreover, EBR elevated the activities of total ATPase, Na+K+-ATPase while contents of Ca+2 Mg+2-ATPase and H+K+-ATPase were lowered as compared with Vd toxin alone treated plants. The study amplified the understanding of Verticillium wilt and demonstrated the potential role of EBR in mediating tolerance to Vd toxin induced stress of V. dahliae in cotton.4. Understanding the physiological and biochemical basis of innate and brassinosteroid induced tolerance to Verticillium wilt in cotton leavesGreenhouse pot experiment was conducted to investigate the genotypic differences in the leaves of G. hirsutum (sensitive to Verticillium wilt), G. barbadense (resistant) and their Hybrid (tolerant) for the responses to Vd toxin stress and to monitor the protective role of shoot applied EBR. The results showed tolerance in G. barbadense and Hybrid which was evident from low accumulation of superoxides (O2-), increased osmotic potential (OP), high relative water content (RWC), low suppression in PAL, PPO, SKDH activities. Besides, the contents of soluble protein, callose and DPPH were high while increases in phenols and flavonoids were lower than G. hirsutum. Furthermore, the activities of ROS scavenging enzymes were also lower than G. hirsutum and suggest modest oxidative stress in these two genotypes. Shoot application of EBR induced tolerance by enhancing osmoregulation, antioxidant enzymes and detoxification signaling in cotton leaves. For instance, the activity of AI was high in Vd toxin+EBR treated plants of all the studied genotypes and might be a cause of increased OP that could maintain high values of RWC in Vd toxin treated plants. Besides, EBR application constitutively increased the activities of SOD, catalase (CAT), dehydroascorbate reductase (DHAR) and glutathione-s-transferase (GST) that could lower O2- contents and accelerate the process of detoxification in cotton leaves to have sustainable plant growth.5. Genotypic differences in carbohydrate, secondary metabolism, antioxidative capacities in cotton root and amino acid contents in cotton root exudatesVd toxin induced oxidative burst, decreased the activities of SuSy, SPS, AI, DPPH, dehydrogenase, increased the levels of glutathione peroxidases (GPX), glutathione reductase (GR), SOD, GSH, ASA, PAL, SKDH, flavonoids and lipid peroxidation in roots of all the studied genotypes. In contrast, shoot applied EBR increased the root dehydrogenase activity and lowered the levels of O2- and H2O2, MDA, flavonoids, DPPH and GR in all the genotypes studied. Furthermore, G. hirsutum depicted highest accumulation of O2-, H2O2, MDA, and decreased root dehydrogenase activity than G. barbadense and Hybrid. Likewise, the activities of AI, SuSy and SPS remained unaltered in Hybrid, but decreased in other two genotypes with greater reduction in G. hirsutum. PAL and SKDH activity were increased under Vd toxin stress in all genotypes; and EBR application further elevated their levels in G. barbadense and Hybrid only. Interestingly, CAD activity was much higher in G. barbadense and increment in callose content was greater than other two genotypes. Similarly, Vd toxin triggered the activities of GR, GPX and SOD, GST and DHAR in all genotypes with greater magnitude in G. barbadense while GSH and ASA increased largely in Hybrid. Likewise, inoculation of plant roots with V. dahliae increased the contents of methionine (Met), valine (Val), serine (Ser) in root exudates of all genotypes and EBR application further elevated their contents. Besides, G. barbadense and Hybrid have higher contents of flavonoids in root tissue and tyrosin (Tyr), arginine (Arg) and phenylalanine (Phe) in root exudates. These amino acids were found to significantly decrease the fungal growth in in vitro assays.6. Liquid chromatography mass spectroscopy (LC-MS) analysis reveal that phenolic compounds and endogenous plant hormones contribute to the brassinosteroid mediated disease resistanceMeasurement of enzyme dynamics and transcripts levels of PAL and CAD revealed an earlier and greater increase in their activity and gene expression in V. dahliae alone treated plants which declined with the passage of time with less reduction in V. dahliae+EBR. Liquid chromatography mass spectroscopy (LC-MS) of methanolic extracts depicted the presence of benzoic acid, cinnamic acid and m-hydroxybenzoic acid in both leaf tissue and Vd toxin, while gallic acid, o-pathalic acid, t-hydroxy cinnamic acid and vanillic acid were detected only from leaf tissue. Besides, V. dahliae inoculation and EBR application either alone or combined (V. dahliae+EBR) induced significant changes in the contents of phenolic compounds. The contents of benzoic acid, m-hydroxybenzoic acid and cinnamic acid were decreased while o-pathalic acid and t-hydroxy cinnamic acid accumulated during earlier stages of infection with greater effect seen in V. dahliae alone treated plants than V. dahliae+EBR. Furthermore, the contents of indole acetic acid (IAA), jasmonic acid (JA) and salicylic acid (SA) were initially decreased and then increased with higher values in V. dahliae alone treated plants. In contrast, the contents of abscissic acid (ABA) and gibberellic acid (GA3) continuously increased due to V. dahliae inoculation and were further increased with EBR application. Besides, the expression of NPR1, DREB2B, LOX1 involved in SA, ABA and JA synthesis respectively, were also increased during 24 hpi with greater increase in V. dahliae alone treated plants while in V. dahliae+EBR during following days of infection. The combined effects of these changes resulted in relatively low oxidative burst, electrolyte leakage, less number of diseased leaf discs, diseased plants, less growth inhibition and fungus colonization (%) in V. dahliae+EBR than V. dahliae alone treated plants. All these studies suggested that EBR could be utilized as a potent growth regulator to attain tolerance against Verticillium wilt in cotton.