Pieter van West

Saprolegniosis in salmonids, a disease caused by the oomycete Saprolegnia parasitica , poses a serious global threat to wild salmon and to aquaculture. To be able to functionally characterise genes in S. parasitica , it is essential to develop a stable transformation method for S. parasitica . We describe for the first time a method that can generate stable transgenic S. parasitica strains. Transformants were generated following the uptake and integration of a mutated gene from Achlya hypogyna conferring imidazole resistance, CYP51 using S. parasitica protoplasts in the presence of polyethylene glycol (PEG) and lipofectamine. This leads to production of CYP51 protein which catalyses a crucial demethylation step in the biosynthesis of ergosterol. As a result, there is no disruption of ergosterol synthesis and the transformants, but not the wild type S. parasitica , can grow in the presence of imidazole. Putative transformants growing in the presence of up to 10 mM imidazole were confirmed by PCR.

Saprolegniosis is a major destructive disease in freshwater aquaculture. The destructive economic impact of saprolegniosis on freshwater aquaculture necessitates further study on the range of Saprolegnia species within Atlantic salmon fish farms. This study undertook a thorough analysis of a total of 412 oomycete and fungal isolates that were successfully cultured and sequenced from 14 aquaculture sites in Scotland across a two-year sampling period. An ITS phylogenetic analysis of all isolates was performed according to whether they were isolated from fish or water samples and during enzootic or epizootic periods. Several genera of oomycetes were isolated from sampling sites, including Achlya, Leptolegnia, Phytophthora, and Pythium, but by far the most prevalent was Saprolegnia, accounting for 66% of all oomycetes isolated. An analysis of the ITS region of Saprolegnia parasitica showed five distinct phylotypes (S2–S6); S1 was not isolated from any site. Phylotype S2 was the most common and most widely distributed phylotype, being found at 12 of the 14 sampling sites. S2 was overwhelmingly sampled from fish (93.5%) and made up 91.1% of all S. parasitica phylotypes sampled during epizootics, as well as 67.2% of all Saprolegnia. This study indicates that a single phylotype may be responsible for Saprolegnia outbreaks in Atlantic salmon fish farms, and that water sampling and spore counts alone may be insufficient to predict Saprolegnia outbreaks in freshwater aquaculture.

The oomycete genus Phytophthora includes many plant pathogens important in agricultural and environmental systems. Natural interspecific hybridization has been reported several times in Phytophthora, and although the fundamental processes of interspecific hybridization and the consequences of subsequent ecological distribution are poorly understood, reports suggest some hybrids can infect a broader host range and display enhanced virulence compared to the putative parental species. During a survey carried out at the University of Aberdeen in 2014–2015, of oomycetes present in ornamental plants purchased via the internet, a batch of oomycete isolates remained unidentified, showing, in some isolates, features generally related to hybridization. The aim of this study was to determine whether hybridization events had occurred between endemic and introduced oomycetes, probably/possibly facilitated through the international plant trade. The list of isolates examined included a putative hybrid closely related to Phytophthora cryptogea. The putative hybrid isolate was further characterized, and pathogenicity were tests carried out on Eucalyptus globulus, using an isolate of P. cryptogea as a positive control. Cloning of ITS, COXI and β-tubulin genes resulted in different sequence versions of the putative hybrid isolate; after mapping and a polymorphism position comparison, it was concluded that the studied isolate contained genetic information from P. cryptogea, P. erythroseptica, P. kelmanii, P. sansomeana and Phytopythium chamaehyphon. A PCR-RFLP assay, a NEBcutter analysis and flow cytometry analysis (genomes ranged between 0.168 to 0.269 pg/2C) added further evidence of the hybrid nature of this isolate. The putative hybrid presented complex growing patterns ranging from rosaceous to chrysanthemum-like and had an optimum growth temperature of 25 °C. Although the putative hybrid produced visible symptoms of disease on E. globulus seedlings, assessment of the relative susceptibility of E. globulus to P. cryptogea and the putative hybrid indicated that P. cryptogea was significantly more virulent than the putative hybrid, based on mortality, disease severity and foliar symptoms.

The international plant trade is considered to be the main pathway causing the spread of oomycetes internationally, especially when live plants are accompanied by soil or potting substrates. Modern, rapid shipping technologies, together with inadequate management practices in nurseries, increase the probability of survival of plant pathogens and the subsequent chances of disease outbreaks in new locations. The survival of two oomycete soil-borne pathogens, Phytophthora cryptogea and Phytophthora cactorum, was studied in two different commercial potting substrates (peat-based and peat-free) in the absence of a plant host under simulated nursery conditions in a glasshouse for 21 months. Colony forming units (CFUs) of both pathogens were recovered 21 months after substrate inoculation, with a decrease in CFUs of between 92 and 99%, depending on the pathogen and substrate. Eucalyptus globulus plants were then planted into these inoculated pots. After 21 months, P. cryptogea and P. cactorum remained capable of producing diseases in E. globulus plants, resulting in up to 30% mortality and an up to 5-fold greater disease severity. These results highlight the ability of these pathogens to survive in the absence of a suitable host plant in potting substrates over time and to then have the capacity to infect a plant. This research adds to the body of essential evidence that is required to develop meaningful management practices and potting substrates at the nursery level to minimize the risk of the spread of oomycetes through the international plant trade.

The contribution of fungi to the degradation of plant litter and transformation of dissolved organic matter (humic substances, in particular) in freshwater ecosystems has received increasing attention recently. However, the role of Saprolegniales as one of the most common eukaryotic organisms is rarely studied. In this study, we isolated and phylogenetically placed 51 fungal and 62 Saprolegniales strains from 12 German lakes. We studied the cellulo-, lignino-, and chitinolytic activity of the strains using plate assays. Furthermore, we determined the capacity of 10 selected strains to utilize 95 different labile compounds, using Biolog FF MicroPlates™. Finally, the ability of three selected strains to utilize maltose and degrade/produce humic substances was measured. Cladosporium and Penicillium were amongst the most prevalent fungal strains, while Saprolegnia, Achlya, and Leptolegnia were the most frequent Saprolegniales strains. Although the isolated strains assigned to genera were phylogenetically similar, their enzymatic activity and physiological profiling were quite diverse. Our results indicate that Saprolegniales, in contrast to fungi, lack ligninolytic activity and are not involved in the production/transformation of humic substances. We hypothesize that Saprolegniales and fungi might have complementary roles in interacting with dissolved organic matter, which has ecological implications for carbon cycling in freshwater ecosystems.