Investigation on Various Aboveground Traits to Identify Drought Tolerance in Cowpea Seedlings

Impacts of drought stress on crop production can significantly impair farmer’s revenue, hence adversely impacting the gross national product growth. For cowpea [Vigna unguiculata (L.) Walp.], which is a legume of economic importance, effects of drought at early vegetative growth could lead to substantial yield losses. However, little has been done with respect to breeding for cowpea cultivars withstanding drought at early vegetative growth. In addition, previous investigations have focused on how plantmorphology and root architecture can confer drought tolerance in cowpea, which is not sufficient in efforts to unravel unknown drought tolerance–related genetic mechanisms, potentially of great importance in breeding, and not pertaining to either plantmorphology or root architecture. Therefore, the objective of this study was to evaluate aboveground drought-related traits of cowpea genotypes at seedling stage. A total of 30 cowpea genotypes were greenhouse grown within boxes and the experimental design was completely randomized with three replicates. Drought stress was imposed for 28 days. Data on a total of 17 aboveground-related traits were collected. Results showed the following: 1) a large variation in these traits was found among the genotypes; 2) more trifoliate wilt/chlorosis tolerance but more unifoliate wilt/chlorosis susceptible were observed; 3) delayed senescence was related to the ability of maintaining a balanced chlorophyll content in both unifoliate and trifoliate leaves; and 4) the genotypes PI293469, PI349674, and PI293568 were found to be slow wilting and drought tolerant. These results could contribute to advancing breeding programs for drought tolerance in cowpea

Investigation on Various Aboveground Traits to Identify Drought Tolerance in Cowpea Seedlings

Impacts of drought stress on crop production can significantly impair farmer’s revenue, hence adversely impacting the gross national product growth. For cowpea [Vigna unguiculata (L.) Walp.], which is a legume of economic importance, effects of drought at early vegetative growth could lead to substantial yield losses. However, little has been done with respect to breeding for cowpea cultivars withstanding drought at early vegetative growth. In addition, previous investigations have focused on how plantmorphology and root architecture can confer drought tolerance in cowpea, which is not sufficient in efforts to unravel unknown drought tolerance–related genetic mechanisms, potentially of great importance in breeding, and not pertaining to either plantmorphology or root architecture. Therefore, the objective of this study was to evaluate aboveground drought-related traits of cowpea genotypes at seedling stage. A total of 30 cowpea genotypes were greenhouse grown within boxes and the experimental design was completely randomized with three replicates. Drought stress was imposed for 28 days. Data on a total of 17 aboveground-related traits were collected. Results showed the following: 1) a large variation in these traits was found among the genotypes; 2) more trifoliate wilt/chlorosis tolerance but more unifoliate wilt/chlorosis susceptible were observed; 3) delayed senescence was related to the ability of maintaining a balanced chlorophyll content in both unifoliate and trifoliate leaves; and 4) the genotypes PI293469, PI349674, and PI293568 were found to be slow wilting and drought tolerant. These results could contribute to advancing breeding programs for drought tolerance in cowpea

Genetic Resistance to the Downy Mildew Pathogen and Breeding towards Durable Disease Management in Spinach

Spinach (Spinacia oleracea) is a self-pollinated, dioecious winter crop. Prevalent challenges to the production of spinach include disease pressure imposed by downy mildew, which is caused by Peronospora effusa (=P. farinosa f. sp. spinaciae [Pfs]). A total of 19 new races of P. effusa have emerged, imposing serious challenges to the disease management in spinach production. Accordingly, this study was designed to explore the genetic components for establishing the basis of durable disease resistance development against the downy mildew pathogen (P. effusa 13) in spinach, through the use of various genome engineering approaches. Our results have led (Chapter 2) to the identification of potential stage specific resistance genes on the basis of the differential gene expression analysis between the downy mildew susceptible cultivar Viroflay and its near isogenic line NIL1 carrying downy mildew resistance gene RPF1. These genes included several novel genes encoding the NBS-LRR, RLK, PR- proteins along with the hormone associated genes, located on chromosomes 1, 3, 4, 5 and 6. Furthermore, the outcomes of this study propose a potential model for plant-pathogen interaction for the downy mildew infection in spinach by determining the stage specific resistance and susceptibility responses. The IsoSeq studies identified the potential genes encoding the RLK, RLP, PR- protein and additional WRKY and NAC domain proteins localized on chromosomes 1, 3, 4, 5 and 6 specific to the downy mildew infection in Viroflay and NIL1 (Chapter 3). Further, relatively new genes were identified for the disease infection at the initial or control (0 hours post infection) and late infection or stress stages (48 hours post infection). Additionally, the full-length transcripts of the resistant and susceptible genotypes were analyzed by using two distinct spinach genome assemblies in order to elucidate the potential differences between the genomes of the two spinach genotypes, respectively. Furthermore, a broader approach of gene pyramiding was used to stack the three Resistance to Peronospora farinosa (RPF) loci namely, RPF1, RPF2, and RPF3 resulting in stacking of three loci in a single spinach line and two loci in fifteen spinach lines. These findings will thus be useful in combatting multiple races of P. effusa, simultaneously (Chapter 4). Overall, our study has facilitated the broadening of the genetic basis for plant pathogen interaction specific to the P. effusa infection, which causes downy mildew in spinach. It provides remarkable value to the scientific community by: 1) increased understanding of the stage specific dynamics of downy mildew infection by determining resistance or susceptibility loci in spinach genome, 2) clarifying the disparities of using two different genome assemblies in spinach, 3) identifying potentially new genetic elements, thereby broadening the knowledge of existing chromosomal location of disease resistance loci in spinach, and 4) establishing durable resistance by assembling three resistance loci into a single spinach line. Furthermore, the newly identified genetic elements associated with resistance to the downy mildew pathogen during this study can be used to further study and breed disease resistance cultivars of spinach

Characterization of Gene Function in Tomato Using CRISPR/CAS9 System

Nuclease based genome editing, specifically the CRISPR/CAS9 system, is being hailed as a novel tool to improve quality of crop plants. To test potential of CRISPR/CAS9 system and to characterize gene functions contributing to improve yield and quality of horticultural crops, we have developed knockout mutants in tomato (Solanum lycopersicum), a widely-consumed vegetables crop throughout world. A CRISPR/CAS9 construct was made containing the guide RNA of auxin-responsive protein/ IAA9 (SlAUX/IAA9) gene and MET1 gene. SlAUX/IAA9 encodes a short-lived transcription factor that affects a number of plant growth and developmental processes including leaf architecture. MET1 gene is responsible for maintenance of DNA methylation in plants leading to epigenetic changes effecting plant growth and developmental processes. We show that CRISPR/CAS9 system effectively knocked out SlAUX/IAA9 and generated mutations in MET1 gene sequence in tomato. The resulting AUX/IAA9 knockouts exhibited multiple phenotypes including change of leaf architecture from compound leaves to simple leaves. The knockout plants showed reduced plant height; early fruit set and reduced seed number in fruit. The SlAUX/IAA9 knockout did not altered patterns of ethylene production and lycopene accumulation in ripening fruits but significantly reduced ethylene production during fruit ripening. The changes associated with the knock out mutations were stable and inherited in the successive generation for the SlAUX/IAA9 knockout. However, some other genes contributed to variation in leaf architecture in the simple SlAUX/IAA9 leaf for the second generation of mutants. The MET1 Mutants showed a delayed flowering, fruiting and ripening suggesting a role of MET1 gene in maintaining DNA methylation essential for normal fruiting processes. The MET1 mutants showed that the epigenetic changes associated with MET1 mutants were reversed. Taken together, these results show that CRISPR/CAS9 system provides a method of choice to develop desirable mutants

Disinfectants Useful to Manage the Emerging Tomato Brown Rugose Fruit Virus in Greenhouse Tomato Production

Tomato brown rugose fruit virus (ToBRFV) is an emerging tobamovirus infecting tomato and pepper crops. First identified in 2014 in the Middle East, ToBRFV has spread rapidly around the world. Being seed-borne, resistance breaking and easy mechanical transmission, ToBRFV can spread quickly in a greenhouse through plant handling. Thus, selecting an effective disinfectant that is capable of deactivating virus infectivity is important. We aimed to identify these effective disinfectants for ToBRFV management in greenhouse tomato production, particularly for total cleaning. A useful disinfectant should be effective against ToBRFV infectivity without major phytotoxic effect on the test plants. In this study, we evaluated 11 disinfectants at various concentrations and assessed their efficacy in ToBRFV treatment on tomato plants that were pretreated with or without SP2700, a known antiviral plant activator of Ningnanmycin. SP2700 treated-plants generated systemic acquired resistance with a delay in symptom expression for 2–3 weeks in comparison to the mock control. Overall, 1% Virocid, 2% Virkon S, 0.25% sodium hypochlorite (5% Clorox bleach), and 2.5% trisodium phosphate (TSP) achieved complete deactivation of ToBRFV with 15 min exposure. However, TSP presented serious phytotoxicity. Our results offer practical solutions to manage this emerging disease affecting tomato production in greenhouses

Disinfectants Useful to Manage the Emerging Tomato Brown Rugose Fruit Virus in Greenhouse Tomato Production

Tomato brown rugose fruit virus (ToBRFV) is an emerging tobamovirus infecting tomato and pepper crops. First identified in 2014 in the Middle East, ToBRFV has spread rapidly around the world. Being seed-borne, resistance breaking and easy mechanical transmission, ToBRFV can spread quickly in a greenhouse through plant handling. Thus, selecting an effective disinfectant that is capable of deactivating virus infectivity is important. We aimed to identify these effective disinfectants for ToBRFV management in greenhouse tomato production, particularly for total cleaning. A useful disinfectant should be effective against ToBRFV infectivity without major phytotoxic effect on the test plants. In this study, we evaluated 11 disinfectants at various concentrations and assessed their efficacy in ToBRFV treatment on tomato plants that were pretreated with or without SP2700, a known antiviral plant activator of Ningnanmycin. SP2700 treated-plants generated systemic acquired resistance with a delay in symptom expression for 2–3 weeks in comparison to the mock control. Overall, 1% Virocid, 2% Virkon S, 0.25% sodium hypochlorite (5% Clorox bleach), and 2.5% trisodium phosphate (TSP) achieved complete deactivation of ToBRFV with 15 min exposure. However, TSP presented serious phytotoxicity. Our results offer practical solutions to manage this emerging disease affecting tomato production in greenhouses

Assessment of in vivo antiepileptic potential and phytochemical analysis of Cassia absus seed extracts

Cassia absus, a member of Fabaceae family, has been a part of traditional medicine for various ailments such as Hypertension, Diabetes, and Cancer. This family of plants has been utilized for Anticonvulsant and Anxiolytic effects. The ongoing investigation is aimed to seek the antiepileptic potential of C. absus seed extracts in pentylenetetrazole-induced kindling mice. The seeds of C. absus were subjected to a sequential extraction process for the preparation of n-hexane, chloroform, methanol, and aqueous extracts. The PTZ-induced kindling model was employed to assess the antiepileptic activity of each extract. Seizure activity and antioxidant biomarkers in the brain tissue such as levels of CAT, SOD, tGSH, and MDA were assessed. Mechanism of action was elucidated by Flumazenil. Through GC-MS analysis, the phytochemical components in the chloroform extract of C. absus were evaluated.The outcomes showed that C. absus extracts markedly reduced the seizure activity in kindling mice. The extracts exhibited significant Antioxidant properties by enhancing the levels of antioxidant biomarkers in the brain tissue such as CAT, SOD, and tGSH, and decreasing the MDA level. The results demonstrated that C. absus extracts showed antiepileptic effects may be via GABA pathway.According to the results of this investigation, C. absus has significant antiepileptic potential in PTZ-induced kindling mice via GABA pathway modulation and combating reactive oxygen species

Genome-Wide Association Study and Genomic Prediction for Bacterial Wilt Resistance in Common Bean (Phaseolus vulgaris) Core Collection

Common bean (Phaseolus vulgaris) is one of the major legume crops cultivated worldwide. Bacterial wilt (BW) of common bean (Curtobacterium flaccumfaciens pv. flaccumfaciens), being a seed-borne disease, has been a challenge in common bean producing regions. A genome-wide association study (GWAS) was conducted to identify SNP markers associated with BW resistance in the USDA common bean core collection. A total of 168 accessions were evaluated for resistance against three different isolates of BW. Our study identified a total of 14 single nucleotide polymorphism (SNP) markers associated with the resistance to BW isolates 528, 557, and 597 using mixed linear models (MLMs) in BLINK, FarmCPU, GAPIT, and TASSEL 5. These SNPs were located on chromosomes Phaseolus vulgaris [Pv]02, Pv04, Pv08, and Pv09 for isolate 528; Pv07, Pv10, and Pv11 for isolate 557; and Pv04, Pv08, and Pv10 for isolate 597. The genomic prediction accuracy was assessed by utilizing seven GP models with 1) all the 4,568 SNPs and 2) the 14 SNP markers. The overall prediction accuracy (PA) ranged from 0.30 to 0.56 for resistance against the three BW isolates. A total of 14 candidate genes were discovered for BW resistance located on chromosomes Pv02, Pv04, Pv07, Pv08, and Pv09. This study revealed vital information for developing genetic resistance against the BW pathogen in common bean. Accordingly, the identified SNP markers and candidate genes can be utilized in common bean molecular breeding programs to develop novel resistant cultivars