Studies On Physiological Mechanisms Of Rust Resistance Improved By Silicon And Its Molecular Marker In Asparagus Bean

Rust disease, caused by the pathogen Uromyces vignae, is one of the most important diseases of asparagus bean (Vigna unguiculata ssp. sesquipedlis (L.) Verd.) in China. To develop a new method to prevent rust and breeding varieties resistant to the disease is an important programme both in asparagus bean production and research.Silicon (Si) is not considered as one of the essential elements for plant growth, but the roles of Si on alleviating biotic and abiotic stresses in plants have drawn much attention worldwide. Many previous studies have reported that Si could improve diseases resistance of cucumber, gourd, rice, barley and wheat etc. Recently, most investigations of disease resistance enhanced by Si were concentrated on graminaceous plants, which contain higher level of silicon (1-10% of dry weight), and some cucurbit species. Little information is available in legume species, which contain relatively lower levels of silicon.The traditional breeding programme for disease resistance is mainly based on field inoculation and phenotype screening, which need a great deal of experience, special condition and time consuming. Molecular marker-assisted selection (MAS), where molecular markers linked to the gene facilitate the indirect selection of the linked gene in breeding populations, is a new breeding method combined modern biotechnology with traditional genetics. The application of MAS could dramatically decrease the time needed and increase selection efficiency. Development of molecular markers linked to the asparagus bean rust resistance gene is an essential step towards both MAS and map-based gene cloning.To explore the physiological mechanism of rust resistance enhanced by Si in asparagus bean, two asparagus bean cultivars differing in their resistance to rust disease, 'ZN016' (highly resistant) and 'Zhijiang 282' (susceptible), were grown on substrate to study the effects of root-applied Si (K₂SiO₃ 1.7 mM) on chlorophyll fluorescence, photosynthesis, reactive oxygen species (ROS) and antioxidants system. Bulked segregant analysis (BSA) and AFLP were applied to a F₂ population derived from these two cultivars to identify an AFLP marker linked to rust resistance gene, and then convert it to SCAR marker. The main results are summarized as follows:1. Exogenous Si remarkably prevent the decrease of the photosynthesis rate (Pn), the stomatal conductance (Gs) and the increase of intercellular CO₂ concentration (Ci) for the plants of susceptible cultivar infected by U. vignae. Si contributed those plants to keep higher total respiratory activity (V_t), alternative pathway activity (V_alt), and activity of the alternative respiratory pathway (ρV_alt). With the application of Si, the decrease of chlorophyll fluorescence parameters, such as Fv/Fm, Fv/Fo,Φ_PSII, NPQ, Fv'/Fm', Rid, and ETR, were greatly delayed. However, there were no obvious differences in those parameters between Si supplied and Si-deprived plants for the susceptible variety without inoculation, and for the highly resistant variety regardless of Si application or U. vignae infection. The SiO₂ content of Si-supplied plants is 36.42% higher than the control. Si significantly suppressed rust development of 'Zhijiang 282', the disease index being 31.7% lower in Si-supplied than in Si-deprived plants.2. Si promoted the activities of peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), phenylalanine ammonia-lyase (PAL), superoxide dismutase (SOD) and glutathione reductase (GR), decreased O₂^- generation rate, H₂O₂ and malondialdehyde (MDA) content, and increased the contents of phenolic compound, reduced glutathione (GSH) and ascorbic acid (AsA) when the plants were inoculated with U. vignae for both cultivars. And a higher effect of Si was found from susceptible cultivar than resistant cultivar. There were no consistent effects of Si on those parameters above when the plants were not infected by U. vignae. Exogenous Si failed to change the POD isoform pattern for both cultivars.3. Application of Si greatly increased the activities of POD, APX, CAT, SOD, and GR in three sub-cellular fractions for both inoculated cultivars. Exogenous Si also increased the content of AsA in three sub-cellular fractions and the content of phenolic compound in cytoplasm and chloroplast fraction. The content of MDA in cytoplasm and chloroplast of susceptible cultivar was significantly reduced by Si-application. The POD isoform patterns in cytoplasm and chloroplast for two cultivars were different from each other, whereas, there was no difference in mitochondria. Exogenous Si still failed to change the POD isoform pattern in any cell fractions. Application of Si could effectively prevent the decomposition of chlorophyll for inoculated susceptible cultivar; increase the membrane potential of decrease the swelling of mitochondria, and increase the respiration rate, the activities of cytochrome C oxidase (CCO) and ATPase for both of the cultivars. Therefore, Si might increase the ability of scavenging ROS in chloroplast and mitochondria, the activities of CCO and ATPase, the stability of membrane structure and function, which eventually led to the remarkable increase of respiration, photosynthesis and enhance its resistance to rust.4. Based on the results from field inoculation on different populations derived from two parents, 'ZN016' (highly resistance) and 'Zhijiang 282' (susceptible), rust resistance of asparagus bean was controlled by a single dominant gene. An AFLP analyses system special for asparagus bean was established. 64 primer combinations used to screen the parental DNA in AFLP analysis amplified 2019 discrete genomic fragments (on the average of 31.55 products per primer pair). With 52 primer pairs gave 124 polymorphic bands between the resistant and susceptible parental genotypes, on the average of 2.0 polymorphic bands per primer combination. Two primer combinations, E-AAG/M-CTG and E-ACC/M-CTG, produced a polymorphic band only in the resistant parent and resistant bulk, but absent in susceptible parent and susceptible bulk.5. These two polymorphic bands were retrieved, cloned and then sequenced. They were 150 bp and 321 bp, respectively. Based on the sequence of the cloned fragments, two pairs of oligonucleotide primers were designed with Primer Premier 5.0 software, respectively. The SCAR primers of E-AAG/M-CTG amplified a monomorphic band of 98 bp (named ABRS_AAG/CTG98 in resistant parent and resistant F₂ individuals, as expected from the sequence data, but it was absent in the cv. Zhijiang 282 (susceptible) and susceptible F₂ individuals. To estimate the genetic distance of ABRS_AAG/CTG98 marker from the resistance gene, linkage analysis was carried out with Mapmaker/Exp version 3.0 software. We estimated that the SCAR primer ABRS_AAG/CTG98 is 5.4 cM distant from the resistance gene. However, the SCAR primers designed for E-ACC/M-CTG were not converted successfully as expected.