The objectives of the research described in this thesis were to determine whether either of the fungal genera Phoma Sacc. or Phomopsis (Sacc.) Bubak was associated with stem lesions of sunflower growing in Australia and, more specifically, whether any isolates found could be identified as belonging to either of the quarantine species Phomopsis helianthi Munt-Cvet et al. and Phoma macdonaldii Boerema through comparison to the respective Holotypes. If isolates from Australia were found not to be of these species, then the aim was to determine their identity by comparing the isolates against an outgroup of known species of Phomopsis and Phoma. Additionally, conducting pathogenicity tests assessed the potential threat of these isolates to sunflower production in Australia.
Isolates of both
Phomopsis and Phoma were successfully isolated from sunflower in Australia. The holotype of Phomopsis helianthi (CBS59.81) as well as herbarium and field isolates identified as P helianthi, were obtained from Yugoslavia, Italy, Hungary, Bulgaria, Russia, France, North America and South America. Isolates identified as belonging to other species of Phomopsis were obtained from herbaria. Isolates of Phoma species and Phoma macdonaldii, including the holotype (CBS381.67), were obtained from North America, South America, South Africa, and Yugoslavia.
Morphological features such as culture colour and texture, and spore-type and dimensions were observed and measured. The observations led to two main groupings of Phomopsis isolates that separated isolates of P helianthi having fine mycelium from
isolates from Australia having coarse or ropey mycelium. Spore analysis indicated that while conidial dimensions were similar in both groups, isolates of P helianthi mostly produced only β-conidia, while isolates from Australia produced mostly α-conidia. The main Australian group also included P longicolla Hobbs and P.leptostromiformis (Keuhn) Bubak, as well as an isolate from Italy (CBS187.87) identified as P helianthi and one from North America and those from Argentina. All Phoma isolates collected were putatively identified to be Phoma macdonaldii since colony colour, texture and spore dimensions of the holotype and the collected isolates were similar.
The entire genome was targeted in a RAPD (random amplified polymorphic DNA) analysis, while the β-tubulin gene and ITS (internally transcribed
spacer) region were subject to RFLP (restriction fragment length polymorphism) analysis. The ITSl and ITS2 regions of several isolates were sequenced and compared against sequences on GenBank. SCARs (sequence characterised amplified regions) were generated and used in a comparative analysis, and primers generated from cDNA sequences were also used to compare similarity of isolates.
The RFLP analyses of both the β-tubulin and the ITS provided less discrimination between isolates than did fingerprinting using RAPDs. Development of several RAPD polymorphisms present in Phomopsis helianthi into SCARs was not successful as amplicons were produced in other species. Primers amplifying part of a putative glutathione-S-transferase gene from P helianthi were found to be specific to P helianthi and could be used as a diagnostic marker.
RAPD analysis was the
most informative analysis as it clustered most of the Australian isolates together and all of the P helianthi together in a fashion similar to the morphological analyses. There was not a complete match of the RAPD groups to the morphological groupings however, with the two species of the outgroup - P. longicolla and P.leptostromiformis, as well as an isolate from Italy and one from North America and those from Argentina not clustering with the main Australian group nor with other species. Another five isolates from Australia did not cluster with any other isolate. Additionally an isolate from France, the isolate from Italy and two from North America that had previously been identified as P. helianthi were found by RAPD analysis not to belong to this group. Additionally isolates from Argentina were not P. helianthi.
None of the
Australian isolates clustered with any of the species from the outgroup. RAPD analysis also showed sub-clusters within the Australian cluster that correlated to locality of collection indicating little movement between populations. The P. helianthi grouping did not cluster according to country, indicating that there has been transfer of populations throughout Europe and America. In ITS sequencing, comparison of the P. helianthi holotype to sequences from Genbank indicates that the P helianthi identified in Italy (CBS 187.87) may not be P helianthi, however those from France are. In the ITS sequencing it appears there is a connection of the main Australian group to P longicolla, though this link may suggest that P. longicolla is the nearest relative it does not indicate that the isolates from Australia are P
longicolla. Other analyses do not show any link between local isolates taken from sunflower and P longicolla.
RAPD and ITS-RFLP analyses confirmed the morphological analyses suggesting that Phoma isolates taken from sunflower from the different countries belong to Phoma macdonaldii. ITS-RFLP analysis placed all the Phoma isolates from sunflower into one cluster of 100% similarity but distinct from species in the outgroup. RAPD analysis revealed genotypes according to country of origin and in the case of Ausfralian isolates to locality of collection. Populations from other countries were more diverse, and a number of smaller clusters were observed in the South African grouping that may pertain to locality collections.
Pathogenicity tests were carried out on isolates from Australia using potted sunflower in controlled conditions with
virulent isolates being further tested in field trials. Pathogenicity of isolates from Australia was compared to overseas isolates by in vitro assays using sunflower petioles. Pathogenicity studies show there are isolates of Phomopsis highly virulent to sunflower in Australia. Field tests revealed a high virulence of some isolates with significant sunflower lodging, grain yield loss and reduced oil content. Some of these isolates are not restricted to sunflower and may infect soybean and Noogoora burr. The virulence of the most virulent Australian isolates were comparable in in vitro studies to P helianthi isolates from Europe and North America, though the most virulent isolate of Phomopsis was that from Yugoslavia.
Phoma macdonaldii is confirmed to be present in Australian sunflower fields. While there are isolates of Phomopsis highly pathogenic
on sunflower in Australia and not restricted to a single host, they form little resemblance to P helianthi or species of the outgroup and remain unclassified.