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Studies on new plant phytoplasma and viruses infections and molecular dissection of virus resistance using Medicago truncatula

Saqib, Muhammad (2008) Studies on new plant phytoplasma and viruses infections and molecular dissection of virus resistance using Medicago truncatula. PhD thesis, Murdoch University.

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The work presented in this thesis is in two areas - study of novel pathogens resulting from new encounters between crop and native species and 'mining' for plant virus resistance genes in the model legume Medicago truncatula.

The history of agriculture in Western Australia (WA) is less than 150 years old. All major broadacre and horticultural crops grown in WA have been introduced from overseas. These introduced horticultural and field crops potentially carry pathogens which may be transferred to infect native vegetation. Conversely, cultivated plants are vulnerable to infection by pathogens present in indigenous plants. This potential for new disease encounters is compounded by expansion of agriculture to crop new land and by predicted climate changes. These changes may provide selective advantage to a particular pest or disease, enabling infection to increase and so increase crop losses or damage native species. Global trade in agricultural produce also increases the potential for introduction of exotic pathogens. The focus of the first part of the research was to look for new pathogens of crops and native plants in WA.

A series of field trips to study diseases in horticultural crops and native vegetation were made in the agricultural regions of Carnarvon, Broome, Kununurra, Perth and the surrounding metropolitan area. Although the initial focus was on virus diseases, the work expanded to study phytoplasma-associated diseases, because of their widespread occurrence and clear symptoms.

In the agricultural region around Kununurra the potyvirus Bean common mosaic virus (BCMV) was found infecting Phaseolus vulgaris crops. Sequencing of isolates collected provided the first reliable molecular confirmation of the presence of BCMV in Australia.

In joint work with K. Bayliss three commercial Paulownia tree plantations near Perth were found exhibiting symptoms of Witches'-Broom disease. The Paulownia trees were found to be associated with 'Candidatus Phytoplasma australiense' 16SrXII group. Chickpeas in the Kununurra region were found with symptoms of stunting, little leaf and proliferating branches and tested positive for phytoplasma. Sequencing confirmed the presence of a phytoplasma with high similarity to the 16SrII group 'Ca Phytoplasma aurantifolia' (peanut witches broom group). This is the first molecular evidence for a phytoplasma-associated disease in chickpea. Red clover (Trifolium pratense), several other pasture legumes and paddy melon (Cucumis myriocarpus) with symptoms of diminished leaf size, pallor, rugosity, leaf deformation, shoot proliferation and stunting were observed amongst pasture plots in south-western Australia. All species with these symptoms were positive for a phytoplasma resembling 'Ca Phytoplasma australiense, 16SrXII group. This association was confirmed for red clover and paddy melon by subsequent nested PCR and sequence analysis. This is the first time that 'Ca. Phytoplasma australiense, 16SrXII group, has been reported infecting these hosts in southern WA. Snakebean (Vigna unguiculata var. sesquipedalis) and tomato (Lycopersicon esculentum) plants with phytoplasma-like symptoms were found in the horticultural region at Broome. The symptoms on snakebean were typical of phytoplasma disease. Sequence analysis identified that the agent associated with the symptoms as a strain of sweet potato little leaf strain V4 (SPLL-V4) phytoplasma (16SrXII group, strain of 'Ca Phytoplasma australiense'). SPLL phytoplasma has not been reported in snakebean or tomato in this isolated agricultural region. In a survey in the Gascoyne region phytoplasma-like symptoms were found in tomato, eggplant and papaya. Previously in this region plants had been found to be associated with peanut witches broom phytoplasma 16SrII group 'Ca Phytoplasma aurantifolia'. Phytoplasma-like symptoms which included bunchy growth, witches' broom and 'little leaf' were observed in Allocasuarina fraseriana (Western Sheoak, Casuarina) and Acacia saligna (Acacia, Orange Wattle) trees in Kings Park and Botanic Garden Perth WA. Phytoplasma-associated disease was confirmed for the first time in native Australian casuarina and acacia trees in WA. Based on the identification of these phytoplasma associated diseases in WA, phytoplasma-associated diseases can be divided into two zones, because phytoplasma 16SrII group was found mostly in the north west of WA and the 16SrXII group in the south west of WA. This work has added to knowledge of the extent and distribution of phytoplasma disease in WA: it is concluded that crop-associated phytoplasma disease originated from native vegetation.

The aim of the second part of the research was to screen and map a virus resistance gene in the model legume M. truncatula to better understand host/pathogen interactions of legume-infecting viruses. Natural resistance genes found in M. truncatula could then be used to locate similar genes in grain legumes (e.g. chickpea and lupins) for practical applications. M. truncatula is a model legume which has a relatively small genome. International consortia have been established to develop genomic resources for M. truncatula. The M. truncatula core collection (from SARDI, South Australia) totalling 230 accessions was screened for resistance/susceptibility to four legume-infecting viruses: Alfalfa mosaic virus (AMV), Cucumber mosaic virus (CMV), Bean yellow mosaic virus (BYMV) and Subterranean clover mottle virus (SCMoV). Five plants from each of the 230 phenotypically distinct members of the M. truncatula core collection were challenged with one isolate of each virus using infectious sap together with five uninoculated control plants for each accession. The symptoms that developed were recorded and virus presence was confirmed by ELISA for inoculated and systemic leaves. Accessions that were potentially resistant were retested to check for escapes. The result from this screen was that 5 accessions were potentially resistant to AMV, 56 to BYMV, 21 to CMV and 42 to SCMoV. The remaining accessions were susceptible to all four viruses with symptoms which ranged from no apparent symptoms (symptomless systemic infection) to highly susceptible and plant death. In continuing work with DAFWA (Dr R. Jones) accessions potentially resistant to AMV, BYMV and CMV are being challenged with additional isolates to check for the presence of genes providing broader resistance.

The Sobemovirus SCMoV was chosen for further study because it is the most widespread viral pathogen of subterranean clover pastures in Australia. It is also a high titre, mechanically transmitted virus which gave the least escapes on infection. SCMoV has a linear, single-stranded positive-sense RNA genome of 4.25 Kb. Making use of natural resistance is an effective means to reduce pasture losses caused by SCMoV. From the screen of the core collection of M. truncatula, amongst the lines resistant to SCMoV a single dominant hypersensitive resistance was detected in line DZA-315. To accelerate mapping of the SCMoV resistance gene, an F8 RIL population of a cross between the resistant line (DZA-315) and a susceptible line (Jemalong-J6, A-17) was sourced and obtained from INRA Toulouse. A total of 166 RILs were phenotyped for resistance and susceptibility to SCMoV. Resistant and susceptible lines showed parental phenotypic symptoms with 84 being susceptible and 82 being resistant. This indicated the presence of a single resistance (R) gene. This phenotypic data was combined with genotypic data (76 polymorphic molecular markers) already available for this RIL population to provide a framework map. Mapmaker and Mapmanager mapping programs were used to locate the position of the resistance gene. This framework map indicated a position for the resistance gene on the long arm of chromosome 6.

Additional polymorphic SSR markers flanking the R gene locus on chromosome 6 were used to map the position of the R gene more closely. These SSR markers were developed from a parental cross of M. truncatula line A17 and A20 at UC Davis and from a parental cross between line A17 and DZA 315 developed at INRA Toulouse. Ten new polymorphic SSR markers were identified and located on the long arm of chromosome 6 after analysis of the F8 RIL population. When combined with the other phenotypic and genotypic data a more accurate map position for the SCMoV R gene was obtained. The results indicate that the R gene to SCMoV is located on the long arm of M. truncatula chromosome 6 between position 35 to 38 centimorgans (cM). The closest marker to the SCMoV R gene is marker mtic153 which is about 2.3 cM away. From existing maps of M. truncatula most of the R genes located in this region are of the TIR-NBS-LRR type and occur in R gene clusters. A series of BACs that span the region of interest have been identified in which SCMoV R gene should be present.

M. truncatula has been used as a model legume to study a number of symbiotic (e.g. rhizobium) and pathogenic interactions (e.g. fungal and nematode), but this is the only example of its use to study legume-virus interactions. The results obtained indicate the potential of using M. truncatula as a model to study resistance response to other legume viruses and provide a firm basis for identifying the hypersensitive R gene that confers resistance to SCMoV.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Biological Sciences and Biotechnology
Supervisor(s): Jones, Michael
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