Xuanli Ma

Co-occurrence of an Aphanomyces sp. and Phytopththora clandestina in subterranean clover pastures in the high rainfall areas of the lower south-west of Western Australia

A survey was undertaken, firstly, to study the distribution of Phytophthora clandestina in the high rainfall, highly leached soils in the cool climate region of the lower south-west of Western Australia and, secondly, to determine the incidence of an undescribed Aphanomyces sp. in these same subterranean clover-based (Trifolium subterraneum) pastures as the occurrence of Aphanomyces in Western Australia has never been studied. P. clandestina and an Aphanomyces sp. were both frequently detected in this survey. P. clandestina was detected from 16.1% and the Aphanomyces sp. was detected from 11.4% of the individual plants sampled. Of the 44 locations sampled, P. clandestina was detected from 71 root samples from across 22 locations, while the Aphanomyces sp. was detected from 50 samples taken from across 23 locations. There were 31 locations from where at least one of the pathogens was detected, of which 14 yielded both pathogens. It is noteworthy that there were eight samples across six locations from where both pathogens were detected in the roots of the same individual plant. Although P. clandestina and the Aphanomyces sp. were detected in a similar number of samples, their distribution patterns were different. P. clandestina was distributed evenly across the area surveyed. In contrast, the Aphanomyces sp. was not detected across the Torbay, Albany and Napier districts.

Infection Processes and Involvement of Defense-Related Genes in the Expression of Resistance in Cultivars of Subterranean Clover (Trifolium subterraneum) to Phytophthora clandestina

Studies on infection processes and gene expression were done to determine differential responses of cultivars of Trifolium subterraneum resistant and susceptible to infection by races of Phytophthora clandestina. In the infection process study, one race was inoculated onto the roots of T. subterraneum cvs. Woogenellup and Junee (compatible or incompatible interactions, respectively). There were no differences in relation to the processes of cyst attachment, germination, and hyphal penetration. There were, however, major differences in infection progression observed post-penetration between compatible and incompatible interactions. In susceptible cv. Woogenellup, hyphae grew into the vascular bundles and produced intercellular antheridia and oogonia in the cortex and stele by 4 days postinoculation (dpi), oospores in the cortex and stele by 8 dpi, when sporangia were evident on the surface of the root. Infected taproots were discolored. Early destruction of taproots prevented emergence of lateral roots. Roots of resistant cv. Junee showed no oospores or sporangia and no disease at 8 dpi. In the gene expression studies, two races of P. clandestina were inoculated onto three cultivars of T. subterraneum. Results showed that three genes known to be associated with plant defense against plant pathogens were differentially expressed in the roots during compatible and incompatible interactions. Phenylalanine ammonia lyase and chalcone synthase genes were activated 4 h postinoculation (hpi) and cytochrome P450 trans-cinnamic acid 4-monooxygenase gene was activated 8 hpi in the incompatible interactions in cvs. Denmark and Junee following inoculation with Race 177. In contrast, in compatible interactions in cv. Woogenellup, there were no significant changes in the activation of these three genes following inoculation, indicating that these three genes were associated with the expression of resistance to Race 177 of the pathogen by the host. To confirm this result, in the second test, cv. Woogenellup was challenged by Race 000 of P. clandestina. In this incompatible interaction, cv. Woogenellup was resistant and expressed highly all three genes in the manner similar to the incompatible interactions observed in the first test.

Taxonomic and pathogenic characteristics of a new species Aphanomyces trifolii causing root rot of subterranean clover (Trifolium subterraneum) in Western Australia

Subterranean clover (Trifolium subterraneum) is grown extensively as a pasture legume in agronomic regions with Mediterranean-type climates in parts of Africa, Asia, Australia, Europe, North America and South America. Root diseases of subterranean clover, especially those caused by oomycete pathogens including Aphanomyces, Phytophthora and Pythium, greatly reduce productivity by significantly decreasing germination, seedling establishment, plant survival and seed set. For this reason, experiments were conducted to determine the species of Aphanomyces causing root disease on subterranean clover in the high-rainfall areas of south-west Western Australia. The effects of flooding, temperature and inoculum concentration on the development of root disease on subterranean clover caused by this Aphanomyces sp. were also investigated as was its host range. Morphological and molecular characteristics were used to identify the pathogen as a new species Aphanomyces trifolii sp. nov. (O’Rourke et al.), which forms a distinct clade with its nearest relative being A. cladogamus. A. trifolii caused significant lateral root pruning as well as hypocotyl collapse and tap root disease of subterranean clover. The level of disease was greater in treatments where soil was flooded for 24 h rather than for 6 h or in unflooded treatments. The pathogen caused more disease at 18/13oC than at lower (10/5oC) or higher (25/20oC) temperatures. The pathogen caused more disease at 1% inoculum than at 0.5 or 0.2% (% inoculum : dry weight of soil). In greenhouse trials, A. trifolii also caused root disease on annual medic (M. polymorpha and M. truncatula), dwarf beans (Phaseolus vulgaris) and tomatoes (Solanum lycopersicum). However, the pathogen did not cause disease on peas (Pisum sativum), chickpea (Cicer arietinum), wheat (Triticum aestivum), annual ryegrass (Lolium rigidium) or capsicum (Capsicum annuum). A. trifolii is a serious pathogen in the high-rainfall areas of south-west Western Australia and is likely a significant cause of root disease and subsequent decline in subterranean clover pastures across southern Australia.

Molecular variation among isolates belonging to eight races of Phytophthora clandestina

Phytophthora clandestina, a serious pathogen of subterranean clover (Trifolium subterraneum), has only been recorded in Australia. A rapid method was used to characterise genetic variation among 61 isolates of P. clandestina belonging to eight pathogenic races based on DNA sequencing and single-strand conformation polymorphism (SSCP) analyses of the β-tubulin gene of the pathogen. The β-tubulin gene among those tested displayed a high degree of variability among isolates. Cluster analysis of the SSCP profiles grouped the 61 isolates into two main clusters (A and B). ClusterAwith 3 subclusters viz; I (race 177), II (race 000) and III (races 001, 121, 101, 143 and 157) and B with race 173 alone. In addition, SSCP of β-tubulin also successfully differentiated between races 173 and 177, the two most prevalent and most virulent of the races studied. This is the first time that the β-tubulin gene has been used to study intra-species variation in this pathogen. In addition to showing relationship among the strains, it also provides a practical means for rapid monitoring of current and future differences in the distribution of P. clandestina strains, giving subterranean clover breeders and farmers a sound basis for the selection/breeding and deployment of appropriate cultivars to counter the predominant strain populations in specific localities.

Grevillea (Proteaceae) seed coats contain inhibitors for seed germination

The purpose of this research was to examine the effect of the seed coat on seed dormancy in Grevillea (Proteaceae) species, and to further investigate the existence of germination inhibitors in Grevillea seed coat extracts. Seed dormancy of 18 Grevillea accessions involving 17 species was investigated: results indicated that removal of seed coat increased seed germination from 0–6% (intact seeds) to 83–100% for the Grevillea accessions and removal of half seed coat resulted in no increase in seed germination. Grevillea seed coat extracts reduced germination of barley, canola, lupin and ryegrass seeds by 48, 57, 10 and 38% respectively. The extracts also reduced seedling growth of the above four species. Ryegrass seeds showed no germination on the 3rd day after imbibition in the presence of Grevillea seed coat extracts compared with 88% germination for the control. Thus, our results showed that seed coat is a major factor determining Grevillea seed dormancy and removal of seed coat dramatically increased seed germination. Grevillea seed coat extracts reduced seed germination and seedling growth of other species. We conclude that there is exogenous seed dormancy in Grevillea species and the chemical(s) in the seed coat is a major factor inhibiting seed germination.

Ploidy variation and karyotype analysis in Hemerocallis spp. (Xanthorrhoeaceae) and implications on daylily breeding

Daylily or Hemerocallis spp. (Xanthorrhoeaceae) is one of the most economically important flowering crops in the world. Ploidy level of 149 genotypes both local to China and introduced from the United States and New Zealand were assessed with an outcome of 79 diploids, 22 triploids and 48 tetraploids. A large proportion of introduced cultivars were tetraploid (42%), half were diploid and the rest, 8%, were triploid. Most Chinese cultivars were diploid; only one was tetraploid. Among the 29 wild genotypes collected from the Taihang Mountain range, 13 (45%) were triploid and 16 (55%) were diploid; no tetraploids were identified. For the 31 genotypes with known ploidy, 22 matched previous counts with the rest showing a lower ploidy. As different ploidy is common in daylily, we suggest that ploidy levels should be assessed in breeding programmes for specific breeding purposes. Karyotypes of three diploid, three triploid and two tetraploid genotypes were constructed and they were assigned to ‘3A’, ‘2B’ and ‘3B’ categories based on Stebbins’ karyotype classification.