Thesis (Ph. D.)--University of Adelaide, Dept. of Applied and Molecular Ecology, 1999. / Includes bibliographical references (leaves 182-217).

Ascochyta blight, caused by Ascochyta rabiei (Pass.) Labrousse, is one of the most destructive diseases of chickpea (Cicer arietinum L.) worldwide. Despite the existence of highly resistant uncultivated genotypes, attempts to develop cultivars with a high level of durable resistance have been unsuccessful. This study investigated the chickpea defence response to A. rabiei using a functional genomics approach, which has the capacity to improve the overall understanding of the coordinated defence response at a molecular level. An existing cDNA library was used to generate a resource of Expressed Sequence Tags (ESTs) that, after clustering, comprised 516 unigenes. The unigenes were functionally annotated resulting in the identification of 20 specific defence-related unigenes, as well as numerous transcripts with possible involvement in the coordination of defence responses. To explore the expression patterns of the defence-related unigenes in an A. rabiei resistant and susceptible genotype, the unigenes were employed as probes in microarrays. Resulting expression data was analysed to identify differentially expressed unigenes over a time-course after infection. Comparison of the expression profiles from the resistant and susceptible genotype identified three putative genes that were exclusively up-regulated in the resistant genotype, thus may be involved in an effective defence response. Considering that a defence response can involve hundreds of genes, the entire set of chickpea unigenes were used to construct large-scale microarrays. To supplement the chickpea probes, 156 putative defence-related grasspea (Lathyrus sativus L.) ESTs and 41 lentil (Lens culinaris Med.) Resistance Gene Analogs (RGAs) were also included. Expression profiles for three chickpeas and one wild relative were generated over a time course. 97 differentially expressed ESTs were identified using a robust experimental system that included confirmation by quantitative RT-PCR. The results indicated that genes involved in the active defence response were similar to those governed by R-gene mediated resistance, including the production of reactive oxygen species and the hypersensitive response, down-regulation of 'housekeeping' gene expression, and expression of pathogenesis-related proteins. The comparison between resistant and susceptible genotypes identified certain gene expression 'signatures' that may be predictiv e of resistance. To further characterise the regulation of potential defence-related genes, the microarray was used to study expression profiles of the three chickpea genotypes (excluding the wild relative) after treatment with the defence signalling compounds, ethylene (E), salicylic acid (SA), and jasmonate (JA). 425 ESTs were differentially expressed, and comparison between genotypes revealed the presence of a wider range of inducible defence responses in resistant genotypes. Linking the results with the previous microarray results indicated the presence of other pathogen-specific signalling mechanisms in addition to E, SA and JA. The lower arsenal of defence-related gene expression observed in the susceptible genotype may be a result of 'breaks' in the pathways of defence-related gene activation. To draw together the findings of all experiments, a model was constructed for a hypothetical mechanism of chickpea resistance to A. rabiei. The model was synthesised based on the evidence gathered in this study and previously documented defence mechanisms in chickpea, and identified signal transduction as a key to resistance.

Field pea (Pisum sativum L.) is highly susceptible to ascochyta blight, primarily caused by the pathogen Mycosphaerella pinodes (Berk. & Blox.) Vestergr. Grasspea (Lathyrus sativus L.) has been reported to possess a moderate level of resistance to ascochyta blight caused by M. pinodes. The work reported in this thesis aimed to develop the various techniques that would be required to transfer ascochyta blight resistance from grasspea into field pea via somatic hybridisation. This thesis also assesses the feasibility of achieving this goal. Field pea shoot cultures were established on hormone-free MS medium, and a protoplast isolation protocol developed for both species. Grasspea shoot cultures were established on both RL and SSB8 medium. Friable grasspea callus was achieved on media supplemented with 2,4-D in the range 4.523 µM, whereas kinetin tested at any concentration did not appear to influence callus growth. A suspension culture of grasspea was developed for the first time, in B5 medium supplemented with 4.5 µM 2,4-D and 0.5 µM kinetin. Grasspea protoplasts were isolated from both in vitro seedlings and suspension cultures. Protocols for hybrid shoot culture on KM8p medium were developed via organogenesis and somatic embryogenesis.

Field pea (Pisum sativum L.) is highly susceptible to ascochyta blight, primarily caused by the pathogen Mycosphaerella pinodes (Berk. & Blox.) Vestergr. Grasspea (Lathyrus sativus L.) has been reported to possess a moderate level of resistance to ascochyta blight caused by M. pinodes. The work reported in this thesis aimed to develop the various techniques that would be required to transfer ascochyta blight resistance from grasspea into field pea via somatic hybridisation. This thesis also assesses the feasibility of achieving this goal. Field pea shoot cultures were established on hormone-free MS medium, and a protoplast isolation protocol developed for both species. Grasspea shoot cultures were established on both RL and SSB8 medium. Friable grasspea callus was achieved on media supplemented with 2,4-D in the range 4.523 µM, whereas kinetin tested at any concentration did not appear to influence callus growth. A suspension culture of grasspea was developed for the first time, in B5 medium supplemented with 4.5 µM 2,4-D and 0.5 µM kinetin. Grasspea protoplasts were isolated from both in vitro seedlings and suspension cultures. Protocols for hybrid shoot culture on KM8p medium were developed via organogenesis and somatic embryogenesis.

Bibliography: leaves 182-217. This study was conducted to determine the etiology of a blight disease of chickpea in south-eastern Australia and the factors affecting disease development. The disease had previously been identified as phoma blight. Pathogenicity testing revealed two isolates subsequently identified as Asochyta rabiei, the first conclusive identification in the southern hemisphere. Greenhouse screening of chickpea varieties identified types resistant to ascochyta blight. The effects of plant age and environmental conditions on disease development were investigated under controlled conditions in growth rooms. Seedlings were more susceptible than older plants. The optimum conditions for ascochyta blight were 20° C and a 48-96 h period of leaf wetness. Through field trials it was found that disease intensity increased over time, especially in cv. Desavic. The means of penetration of the chickpea host was established in histological studies. This study provided advance warning of this disease for the expanding chickpea industry, and has allowed the implementation of appropriate disease management strategies. It is recommended that cv. Desavic should not be grown where ascochyta blight is likely to be a problem.

Successful chickpea production in western Canada typically requires multiple applications of fungicides to minimize the severity of Ascochyta blight (AB) caused by <i>Ascochyta rabiei</i>. Although planting resistant cultivars could be economical and environmentally safer than fungicide usage, varieties with a high level of resistance are not available. The objectives of this research were i) to determine the effect of different seeding arrangement treatments on ascochyta blight severity and seed yield of two cultivars (moderately resistant and susceptible) of kabuli chickpea; ii) to compare one and four fungicide applications at recommended and reduced rates and their impact on disease severity and cost; and iii) to assess organ-specific reaction to AB in chickpea in leaves, stems and pods of 12 desi and 12 kabuli varieties that are of economic significance to western Canada. <p>Treatments significantly influenced AB severity on both moderately resistant and susceptible cultivars in a season with a severe epidemic. Seed yield was significantly influenced by treatments for both varieties in both years. Contrast analyses revealed that four fungicide applications significantly reduced the AB severity for both varieties in a season with a severe epidemic and for the susceptible variety in a season with a moderate epidemic. Seed yield of both varieties was significantly higher under four fungicide applications compared to a single application. Solid seeding and paired row arrangements did not differ in their effect on seed yield and AB severity for both varieties in both years, except that the susceptible variety benefited from paired row planting with respect to seed yield and reduced AB severity in the season with a severe epidemic. Reducing fungicide rates and seeding rate could reduce the cost of cultivation without significantly affecting disease control and yield. Economic assessment revealed that in a severe epidemic season, the gross returns were high for the moderately resistant variety under four fungicide applications than one fungicide application. Gross returns for the susceptible variety were higher under four fungicide applications in both years.<p>There were differences among varieties for AB severity on leaves, stems and pods, seed yield and 1000 seed weight at all site-years tested. The variation was greater in kabuli varieties than desi varieties. AB severity on leaves, stems and pods was lower under high fungicide regimes, with few exceptions. Varieties with a fern leaf type had lower AB severity than those with unifoliate leaves. There was a positive correlation among AB severity on leaves, stems and pods. No differences in organ-specific reaction were observed.

Random amplified polymorphic DNA (RAPD) markers were used in three genetic studies in lentil (<i>Lens culinaris</i> Medikus). The first study involved development of RAPD markers linked with genes for resistance to ascochyta blight, caused by <i>Ascochyta fabae</i> f. sp. lentis Gossen et al. Seventy F₂-derived F₃ (F_2:3) lines were field screened for ascochyta blight reaction in each of two hybrid populations, Indianhead x Eston and ILL 5588 x Eston, that were segregating for resistance to ascochyta blight. Resistance to ascochyta blight in ILL 5588 lentil was conferred by a single dominant gene (<i>Ral₁</i>), whereas resistance in Indianhead lentil was conferred by a single recessive gene (<i>ral₂</i>). An efficient DNA extraction procedure and a PCR protocol that yielded RAPD markers with high resolution were developed for lentil. Bulked segregant analysis was used to produce four bulks of DNA from resistant vs. susceptible F₂ plants in each of these two populations which were then screened for RAPD markers using 400 random oligonucleotide primers. One RAPD marker, UBC227₁₂₉₀, was linked to the recessive gene, <i>ral₂</i>, in Indianhead lentil in repulsion phase with a map distance of 14.1 ± 4.5 cM. No RAPD marker was linked with the <i>Ral₁</i> gene in ILL 5588 lentil. In the second study, RAPD markers were used to study phylogeny of the genus Lens. DNA, extracted from 23 accessions of all five taxa of the genus Lens (culinaris, orientalis, nigricans, odemensis and ervoides), was screened for RAPD polymorphisms, using 11 random oligonucleotide primers. Two accessions of the differentiated cytotype of L. nigricans were also included. One hundred and forty eight polymorphic RAPD markers were resolved. A dendrogram for these RAPD markers, using the unweighted pair group method, clearly separated all accessions into their supposedly related taxa. Lens orientalis was the undisputed progenitor of the cultivated lentil, <i>Lens culinaris</i>. A low level of RAPD polymorphism was observed in <i>Lens culinaris</i> and L. ervoides. The differentiated cytotype of L. nigricans was well separated from the normal cytotype of L. nigricans and was closely associated with the L. odemensis accessions, indicating its close genetic similarity to L. odemensis. Principal component analysis (PCA) also indicated a similar relationship among these accessions, but resulted in a better resolution of the groupings. In the third study, the genetic polymorphism of seven Canadian lentil cultivars were studied using RAPD markers. Four lentil cultivars, CDC Gold, CDC Matador, Eston and Indianhead each had a unique, cultivar-specific RAPD marker, allowing their identification.

Successful chickpea production in western Canada typically requires multiple applications of fungicides to minimize the severity of Ascochyta blight (AB) caused by <i>Ascochyta rabiei</i>. Although planting resistant cultivars could be economical and environmentally safer than fungicide usage, varieties with a high level of resistance are not available. The objectives of this research were i) to determine the effect of different seeding arrangement treatments on ascochyta blight severity and seed yield of two cultivars (moderately resistant and susceptible) of kabuli chickpea; ii) to compare one and four fungicide applications at recommended and reduced rates and their impact on disease severity and cost; and iii) to assess organ-specific reaction to AB in chickpea in leaves, stems and pods of 12 desi and 12 kabuli varieties that are of economic significance to western Canada. <p>Treatments significantly influenced AB severity on both moderately resistant and susceptible cultivars in a season with a severe epidemic. Seed yield was significantly influenced by treatments for both varieties in both years. Contrast analyses revealed that four fungicide applications significantly reduced the AB severity for both varieties in a season with a severe epidemic and for the susceptible variety in a season with a moderate epidemic. Seed yield of both varieties was significantly higher under four fungicide applications compared to a single application. Solid seeding and paired row arrangements did not differ in their effect on seed yield and AB severity for both varieties in both years, except that the susceptible variety benefited from paired row planting with respect to seed yield and reduced AB severity in the season with a severe epidemic. Reducing fungicide rates and seeding rate could reduce the cost of cultivation without significantly affecting disease control and yield. Economic assessment revealed that in a severe epidemic season, the gross returns were high for the moderately resistant variety under four fungicide applications than one fungicide application. Gross returns for the susceptible variety were higher under four fungicide applications in both years.<p>There were differences among varieties for AB severity on leaves, stems and pods, seed yield and 1000 seed weight at all site-years tested. The variation was greater in kabuli varieties than desi varieties. AB severity on leaves, stems and pods was lower under high fungicide regimes, with few exceptions. Varieties with a fern leaf type had lower AB severity than those with unifoliate leaves. There was a positive correlation among AB severity on leaves, stems and pods. No differences in organ-specific reaction were observed.

Bibliography: leaves 182-217. / xx, 217, [18] leaves, [17] leaves of plates : ill. (chiefly col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This study was conducted to determine the etiology of a blight disease of chickpea in south-eastern Australia and the factors affecting disease development. The disease had previously been identified as phoma blight. Pathogenicity testing revealed two isolates subsequently identified as Asochyta rabiei, the first conclusive identification in the southern hemisphere. Greenhouse screening of chickpea varieties identified types resistant to ascochyta blight. The effects of plant age and environmental conditions on disease development were investigated under controlled conditions in growth rooms. Seedlings were more susceptible than older plants. The optimum conditions for ascochyta blight were 20C and a 48-96 h period of leaf wetness. Through field trials it was found that disease intensity increased over time, especially in cv. Desavic. The means of penetration of the chickpea host was established in histological studies. This study provided advance warning of this disease for the expanding chickpea industry, and has allowed the implementation of appropriate disease management strategies. It is recommended that cv. Desavic should not be grown where ascochyta blight is likely to be a problem. / Thesis (Ph.D.)--University of Adelaide, Dept. of Applied and Molecular Ecology, 1999

Thesis (Ph.D.)--University of Western Australia, 2008.