Host Preferences and Pathogenomics of Pseudomonas fuscovaginae in Australia

The bacterial plant pathogen Pseudomonas fuscovaginae causes sheath brown rot disease on a broad range of hosts. Since the first detection from Japan in 1976, it has been reported from various agro-ecological regions around the world. In 2005, P. fuscovaginae infecting rice was first reported from Australia in the major rice-growing region of southern New South Wales (NSW). The pathogenicity and aggressiveness of five strains namely, DAR77318, DAR77320, DAR77795, DAR77797 and DAR77800, were identified and characterised. Although there are several reports on host preference and pathogenomics of strains of P. fuscovaginae, no such research has been conducted for the identified Australian strains. Nor is it known whether strains isolated from rice pose a threat to cereal crops including wheat.
In this thesis, the potential host range of the five strains of P. fuscovaginae from Australia were investigated utilising pathogenicity and virulence assays. Comparisons were made with four strains of P. fuscovaginae isolated from rice, originating from Japan (ICMP5940), Burundi (ICMP9995) and Madagascar (ICMP9996, ICMP9998), and strain ICMP10137 isolated from wheat in Brazil. Pathogenicity and virulence assays were conducted on ten cultivars of wheat, by both seed-soaking and pinprick inoculation methods. In addition, two cultivars each of durum wheat, triticale and barley were inoculated by the seed-soaking method. All five of the Australian strains of P. fuscovaginae demonstrated pathogenicity and virulence at heading on wheat cultivars, Chara, Ventura, Mace, Spitfire, EGA Gregory, Lincoln, Sunvale and Wyalkatchem, as indicated by percent lesion length on flag leaf sheaths. These and two other wheat cultivars namely, EGA Wedgetail and Rosella were susceptible to infection by P. fuscovaginae at seed germination, as indicated by reduced seedling height. Similar effects were observed for the triticale (cv. Hawkeye, cv. Tahara), durum-wheat (cv. EGA Bellaroi, cv. Tjilkuri) and barley (cv. Schooner, cv. Commander) at seed germination. This is the first report on the pathogenicity of rice-infecting strains of P. fuscovaginae from Australia on wheat.
Furthermore, ten cultivars of rice that are commonly grown in southern NSW were inoculated with the aforementioned strains of P. fuscovaginae by the pinprick method. Based on the percent lesion length on flag leaf sheaths at heading, all the rice cultivars investigated, Amaroo, Doongara, Illabong, Koshihikari, Kyeema, Langi, Opus, Quest, Reiziq, Sherpa, were susceptible to P. fuscovaginae. Strain ICMP10137, isolated from wheat, expressed pathogenicity and virulence on all the rice cultivars, demonstrating the cross-species pathogenicity of P. fuscovaginae. By providing host resistance information on a range of cultivars, this host preference study provides insights for variety selection for cultivation and breeding of resistant cultivars.
Four additional strains of P. fuscovaginae isolated from wheat, UPB0526, UPB0588 and UPB0589 originating from Mexico and UPB1013 from Nepal, and UPB0407 isolated from Leersia hexandra (Swamp rice grass) in Burundi, were able to cause significant (α = 0.05) pathogenicity on rice (cv. Amaroo), at the seed germination stage. Strain UPB0407 was also virulent on both rice (cv. Amaroo) and wheat (cv. Rosella) at seed germination. The results demonstrate the potential threat of strains infecting rice to infect wheat and other cereals crops. Furthermore, it indicates the possibility of the pathogen to be present in rice-wheat cropping systems.
In order to determine the presence of P. fuscovaginae in rice-wheat cropping systems in southern NSW, a survey of both rice and wheat crops was conducted, covering the Murrumbidgee and Coleambally irrigation areas, and the Murray valley. Disease symptoms associated with sheath brown rot were observed in three out of 16 rice crops and one out of 17 wheat crops. However, P. fuscovaginae could not be identified from the bacteria isolated from them.
Notably, there were significant (α = 0.05) strain to cultivar interactions observed in expressing disease symptoms in rice, at heading. However, the strain to cultivar interactions were not significant (α = 0.05) in wheat at heading. Similar observations were made with wheat, durum wheat, triticale, and barley, at seed germination. These results suggested that different host resistance mechanisms are engaged at different stages of plant growth in wheat. To explain these observations of host-pathogen interactions, further investigations were carried out on the molecular interactions between the hosts and strains of P. fuscovaginae.
Few molecular studies have been conducted on the pathogenicity and virulence factors of P. fuscovaginae and only a small number of pathogenomic studies supported by whole genome analyses have been conducted on rice-infecting strains. However, none of these included strains from Australia. In this thesis, a comparative genome analysis was conducted of P. fuscovaginae, with a perspective of host-preference. Assembled draft genomes of rice-infecting DAR77795, DAR77800, ICMP5940, SE-1 from the Philippines, CB98818 from China and UPB0736 from Madagascar were compared with the newly assembled genomes of wheat-infecting UPB0526, UPB0588 and UPB0589 from Mexico, UPB1013 from Nepal and UPB0497 isolated from swamp rice grass in Burundi. The results show a highly diverse core-genome of P. fuscovaginae. Some distinct differences between the strains could be observed. Strains of P. fuscovaginae isolated from rice had 119 unique genes encoding hypothetical proteins, non-ribosomal peptide synthetases, peptide synthetases, long chain fatty acid CoA ligases, and large exo-proteins involved in haem utilisation and adhesion functions. Sixty-five genes appeared to be more common to wheat-infecting P. fuscovaginae strains. The genome of UPB0407 appeared to have acquired a number of phage-related genes and hypothetical proteins that were not present in other strains.
Further analysis revealed that one of the genes coding for hypothetical proteins/non-ribosomal protein synthesis to be a homologue of phytotoxic syringopeptin synthetase A (sypA) of P. syringae pv. syringae. Therefore, a mutagenesis protocol was undertaken utilising the pKNOCK system to knock-out this gene in strains DAR77795 and DAR77800. The subsequent effect of this knock-out on pathogenicity and virulence on both rice and wheat seeds and rice seedlings was analysed. The inability to synthesize this syringopeptin homologue was found to affect the virulence of DAR77795 and DAR77800 mutants, as indicated by the root lengths and shoot lengths of inoculated seeds that were comparable to those of the control treatment of sterile distilled water, and inability of the mutants to cause necrotic lesions on the inoculated seedlings in comparison to the respective wildtypes. The syringopeptin homologue appears to be a non-host-specific toxin affecting both rice and wheat, contributing to the pathogenicity of both strains of P. fuscovaginae. Genome mining for the region targeted by the mutagenesis within the syringopeptin gene homologue showed that it is conserved in all 10 of the P. fuscovaginae genomes (DAR77795, DAR77800, SE-1, UPB0736, UPB0526, UPB0588 and UPB0589, UPB1013, ICMP5940, CB98818) assessed. Therefore, this phytotoxin could be an important factor responsible for non-host specific pathogenicity and virulence reported for P. fuscovaginae.
Another gene, which was present in only seven of the examined genomes and showed high variability among the strains of P. fuscovaginae, was also targeted for mutagenesis to determine its role in pathogenicity and virulence. Several techniques such as the deletion-insertion by pEX system, and gene knockout by pKNOCK system failed to generate stable mutants in both DAR77795 and DAR77800. Therefore, these large exo-proteins involved in haemaglutinin and adhesion functions could not be studied further. However, during the experimental process, the ability to produce competent cells from P. fuscovaginae was demonstrated. Thus, direct transformation with plasmids could be possible for future studies of the pathogen.