Sonja S. Klemsdal

Genetic variation between Phytophthora cactorum isolates differing in their ability to cause crown rot in strawberry

Analysis of 44 isolates of Phytophthora cactorum, isolated from strawberry and other hosts, by AFLP showed that the crown rot pathotype is different from leather rot isolates and from P. cactorum isolated from other hosts. 16 of 23 crown rot isolates, including isolates from Europe, Japan, Australia, and New Zealand, were identical in an analysis based on 96 polymorphic bands from seven primer combinations. Leather rot isolates of strawberry could not be distinguished from isolates from other hosts. The pathogenicity test of all 44 isolates on strawberry plants mostly gave unambiguous results, except for three American isolates, which seemed to have reduced aggressiveness compared to the crown rot isolates. These isolates also differed in the AFLP analysis. Comparing information on the origin of the isolates with results from the pathogenicity test, showed that isolates from strawberry fruits or petioles could be either leather rot or crown rot pathotypes. None of the isolates from hosts other than strawberry caused crown rot symptoms in strawberry.

AFLP analysis of Russian Alternaria tenuissima populations from wheat kernels and other hosts

Alternaria tenuissima is a common pathogen on a number of plants described in several geographic regions of the world. Genetic variation within and between Russian Far East, North West and Caucasus populations of A. tenuissima from wheat was examined. In addition, genetic differences between isolates from various hosts were estimated. In total, 101 isolates of A. tenuissima were studied using amplified fragment length polymorphism (AFLP) with four primer combinations. Wright’s fixation index (Fst), gene flow (Nm) and gene diversity (Hs) were calculated. AFLP banding patterns indicated significant genetic distance and at the same time a low level of gene flow between the Far East and the two other groups of isolates originating from the European part of country. The degree of similarity between the North West and Caucasus populations was very high, as was the migration rate. Isolates analysed by UPGMA-based cluster analysis were grouped according to location of origin but irrespective of plant host. Based on the Fst value, the group of isolates originating from wheat and barley were not found to differ significantly from each other.

Identification of Pythium aphanidermatum using the RAPD technique.

The suitability of random amplified polymorphic DNA for identification of Pythium aphanidermatum was investigated. Three oligoprimers were selected after testing three isolates of P. aphanidermatum and one isolate of P. ultimum with a total of 40 primers. The selected 10-mer primers were used with 20 isolates of P. aphanidermatum , four isolates of P. deliense , two isolates of P. ultimum , two isolates of P. irregulars and one isolate of P. paroecandrum . Most of the P. aphanidermatum isolates (13 of 20), were obtained from samples of Cucumis sativus , or water from cucumber greenhouses. The three selected primers gave identical fingerprints for 18 of the 20 P. aphanidermatum isolates, including all the isolates from cucumbers. Two of the primers gave fingerprints that could be used to differentiate between isolates of the Pythium species studied. The banding pattern of P. aphanidermatum given by the third primer could not easily be distinguished from the fingerprint of P. deliense . However, when used in conjunction with the other two primers, the third primer can be used to verify the identity of P. aphanidermatum .

Fusarium langsethiae (Torp and Nirenberg), investigation of alternative infection routes in oats

Fusarium langsethiae is a recently characterized fungus within the genus Fusarium. It is found as a grain contaminant of small grain cereals such as oats and barley, and to a lesser extent wheat. Fusarium langsethiae is particularly widespread in the Nordic countries and the UK where it poses a serious problem as the main producer of T-2 and HT-2 mycotoxins. The biology of F. langsethiae and its interaction with the plant remains poorly understood, partly hampered by difficulties reproducing a natural level of infection under controlled conditions. The reported study was designed as a series of glasshouse experiments to advance our understanding of F. langsethiae biology by investigating alternative infection routes and its proliferation in oats, Avena sativa. Various methods of seed, soil, and seedling inoculation, boot injection and spray inoculation, were tested. The results clearly show a strong preference of F. langsethiae for the panicle, ruling out alternative infection routes. At relatively low temperatures spray infection, accompanied by prolonged humidity, ensured a thorough establishment of the fungus both at flowering and at early dough stage. Boot injection proved to be a reliable working tool for production of an even and predictable grain infection. Apart from in the panicle, considerable fungal proliferation was only detected in flag leaf nodes, and was a direct consequence of the boot injection method. Fungal presence in the node tissue also correlated with significant stunting of infected shoots. In light of the results the pathogenic and endophytic abilities of F. langsethiae are discussed.

Real-Time Quantitative Expression Studies of the Zearalenone Biosynthetic Gene Cluster in Fusarium graminearum

The estrogenic mycotoxin zearalenone (ZON) produced by some Fusarium spp. causes reproductive problems and hyperestrogenic syndromes in mammals. In an effort to elucidate the molecular pathways of ZON production, we present a comparative real-time quantitative polymerase chain reaction expression study of seven contiguous genes in the ZON biosynthetic cluster on sterile rice and during wheat and oat infection. Under ZON production on rice, the polyketide synthase (PKS) genes PKS4 and PKS13, alcohol oxidase FG12056 gene, and transcriptional regulator FG02398 gene showed similarly upregulated patterns, whereas the nonribosomal peptide synthetase (NPS) FG02394, the K(+) channel beta subunit FG12015, and the protein kinase FG02399 displayed a variant pattern. During the same time period under wheat infection when no ZON was produced, the PKS genes and the NPS were downregulated relative to rice whereas the K(+) channel beta subunit gene FG12015 was markedly upregulated, suggesting that it may play a role in the infection process. This is the first expression study of ZON biosynthetic genes in planta. The results give insight into the regulation and activities of the ZON gene cluster under different experimental systems and suggest a connection between ZON and a K(+) channel that could reveal a novel function for ZON in Fusarium spp.

Pythium polare, a new heterothallic oomycete causing brown discolouration of Sanionia uncinata in the Arctic and Antarctic

Pythium polare sp. nov. is a new heterothallic oomycete species isolated from fresh water and moss from various locations in both the Arctic and Antarctic. This water mould is able to infect stems and leaves of Sanionia moss (Sanionia uncinata). Pythium polare causes brown discolouration in in vitro inoculation tests at 5 °C after 5 weeks of inoculation. It is characterized by globose sporangia with various lengths of discharge tubes releasing zoospores and aplerotic oospores with usually one to five antheridia. The sexual structures are only produced in a dual culture of antheridial and oogonial isolates. Phylogenetic analysis, based on ITS sequencing, places all isolated strains of P. polare in a unique new clade, hence it is considered a novel species. Pythium canariense and Pythium violae are the most closely related species of P. polare based both on morphology and the phylogenetic analysis.

Identification of up-regulated genes during zearalenone biosynthesis in Fusarium

The Fusarium genus includes devastating plant pathogenic fungi that cause diseases in cereals around the world. They produce several mycotoxins, including the estrogenic compound zearalenone. To better understand the molecular mechanisms determining zearalenone production, we performed differential display RT-PCR under conditions where Fusarium graminearum and F. culmorum produced high amounts of zearalenone. We found 133 expressed sequence tags (ESTs) and 54 of these were considered to be up-regulated during high zearalenone production. Several of the ESTs were confirmed to be up-regulated by real-time qPCR, but none showed any significant down-regulation in the zearalenone negative mutant ΔPKS4-T9, or were similar to typical gene expression patterns of previously described zearalenone-related genes. Some of the up-regulated ESTs were similar to genes involved in secondary metabolite production, lipid metabolism, transcriptional activation, provision of precursors, signal transduction, transport or detoxification. Several of the ESTs were also located adjacent to one another in the genome and therefore might represent genes involved in the same biosynthetic pathway. Members of six such putative pathways could be found. All sequences were compared to the MIPS F. graminearum Genome Database to verify autocalled gene predictions experimentally and to introduce new exons and gene structures.

Occurrence of Pythium ultimum var. ultimum in a greenhouse on Spitsbergen Island, Svalbard.

Pythium ultimum var. ultimum was isolated from carrot seedlings with damping off and from soil used for growing the plant in a greenhouse on Spitsbergen Island, Svalbard. The fungus caused severe damping off of carrot, cucumber and tomato seedlings after artificial inoculation. The rDNA internal transcribed spacer sequences of the Svalbard isolate were identical to those of Canadian and Japanese isolates of P. ultimum var. ultimum. The results suggest that the pathogen in the greenhouse on Svalbard was probably introduced from temperate regions through contaminated plants and/or soil imported to the island. This is the first record of P. ultimum var. ultimum within the Arctic zone.