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Until now, molecular and biochemical methods have only been used to show whether or not Plasmodiophora brassicae is present in plant or soil samples but not to what extent. Here, in planta quantification of P. brassicae by whole-cell fatty acid (WCFA) measurements and real-time polymerase chain reaction (PCR) was evaluated. Arachidonic acid (ARA, 20:4) was the most abundant fatty acid in resting spores and was only found in infected roots, which indicates a potential of ARA as a biomarker for P. brassicae. A real-time PCR assay was developed using primers designed from the internal transcribed spacer region of the ribosomal DNA. Using these primers, it was possible to detect P. brassicae in infected roots 10 days after germination of plants sown in infested soil. A bioassay showed that the amounts of ARA found by WCFA analysis and the DNA found by real-time PCR in infected plants were well correlated. These measurements also correlated with the soil spore content and the assessed disease incidence and disease severity scores. Therefore, we conclude that WCFA analysis and real-time PCR are good tools for P. brassicae quantification that can be applied to basic studies of the pathogen and in resistance screens.

Mansikan mustalaikun aiheuttaja Colletotrichum acutatum on Keski-Euroopassa mansikan lisäksi tärkeä hedelmien, kuten omenan ja kirsikan pilaaja. Mansikan marjoihin kehittyvät tummat laikut pilaavat nopeasti satoa kosteassa ja lämpimässä. Tauti leviää helposti taimissa ja se kulkeutui tuontitaimien mukana mansikkaviljelmille Suomeenkin 2000-luvun alussa. Pohjoismainen yhteistyö mustalaikun tutkimuksessa alkoi vuonna 2008 Suomessa, Norjassa ja Tanskassa erilaisissa ilmasto-oloissa. Mustalaikun aiheuttajan säilymistä seurattiin keinollisesti tartutetussa mansikan lehti-, rönsy- ja juurakkomassassa kolmen vuoden ajan suomalaisilla ja kahden vuoden ajan muilla koepaikoilla. Suomessa kokeet olivat MTT:n tutkimuspaikoilla Jokioisilla, Mikkelissä ja Sotkamossa, Norjassa Oslon lähellä Åsissa ja länsirannikolla Ullensvangissa sekä Tanskassa Kööpenhaminassa. Taudinaiheuttajan tartutuskyky testattiin kokeiden aikana vuosittain keväällä ja syksyllä. Koealueilta otetuilla näytteillä tartutettiin pyydyskasveja, joista määritettiin C. acutatum - tartunta PCR-menetelmällä käyttäen lajispesifisiä alukkeita. Colletotrichum acutatum voi säilyä Pohjoismaiden oloissa maassa kasvinjätteessä kaksi vuotta, jopa pitempään. Vain Tanskassa sieni säilyi tartutuskykyisenä heikommin, yhden talven yli. Maan kosteus ja lämpöolot vaikuttavat sienen säilymiseen, mikä näkyi Åsin ja Mikkelin tuloksissa. Sen sijaan Jokioisilla, Sotkamossa ja Ullensvangissa C. acutatum säilyi vähintään kaksi vuotta maassa tai maan pinnalla. Aikaisempien havaintojen mukaan taudinaiheuttaja säilyi rikkakasvien jätteessä vuoden verran. Tämä aines hajoaa nopeammin ja säilyminen on siinä vähäisempää kuin mansikan jätteessä. Pohjoisessa alhaiset lämpötilat lisäävät taudin säilymisaikaa ja maan jäätyminen hidastaa sen tuhoutumista kasvinjätteestä. Kahden vuoden aikana koealueille kasvoi vesiheinää, rönsyleinikkiä, piharatamoa ja kurjenpolvea, joita testattaessa havaittiin piilevää C. acutatum -tartuntaa. Aikaisemmin on todettu valkoapilan ja eräiden muiden mansikkamailla kasvavien kasvien säilyttävän mustalaikkua vuodesta toiseen. Onkin mahdollista, että tartunta voi jäädä viljelylohkolle mansikan jälkeen leviämällä rikkakasveihin elävistä kasveista tai kasvinjätteestä ja jatkamalla niissä säilymistä edelleen. Mustalaikkua ei ole tavattu suomalaisessa taimiaineistossa, mutta se tulee varmasti edelleen mansikkaviljelmille tuontitaimien mukana, jos taimituotannossa ei huolehdita tautivapaudesta. Viljelykierto ja tehokas rikkakasvien torjunta auttavat torjumaan tautia. Norjassa C. acutatum on tärkein kirsikkasadon pilaaja ja sitä esiintyy myös omenalla. Tautia on havaittu omenalla myös Suomessa, samoin sitä esiintyy pihlajan ja pensasmustikan marjoissa.

Plants are often attacked by pathogens and insects. Their combined impact on plant performance and fitness depends on complicated three-way interactions and the plant’s ability to compensate for resource losses. Here, we evaluate the response of Barbarea vulgaris, a wild crucifer, to combined attack by an oomycete Albugo sp., a plant pathogen causing white rust, and a flea beetle, Phyllotreta nemorum. Plants from two B. vulgaris types that differ in resistance to P. nemorum were exposed to Albugo and P. nemorum alone and in combination and then monitored for pathogen infection, herbivore damage, defence compounds, nutritional quality, biomass and seed production. Albugo developed infections in the insect-resistant plants, whereas insect-susceptible plants were scarcely infected. Concentrations of Albugo DNA were higher in plants also exposed to herbivory; similarly, flea beetle larvae caused more damage on Albugo-infected plants. Concentrations of saponins and glucosinolates strongly increased when the plants were exposed to P. nemorum and when the insect-susceptible plants were exposed to Albugo, and some of these compounds increased even more in the combined treatment. The biomass of young insect-susceptible plants was lower following exposure to flea beetles, and the number of leaves of both plant types was negatively affected by combined exposure. After flowering, however, adult plants produced similar numbers of viable seeds, irrespective of treatment. Our findings support the concept that pathogens and herbivores can affect each other’s performance on a host plant and that the plant reacts by inducing specific and general defences. However, plants may be able to compensate for biomass loss from single and combined attacks over time.

Plasmodiophora brassicae is an obligate, biotrophic pathogen causing the club-root disease of crucifers. Despite its importance as a plant pathogen, little is known about P. brassicae at the molecular level as most of its life cycle takes place inside the plant host, and axenic culturing is impossible. Discovery of genes expressed during infection and gene organization are the first steps toward a better understanding of the pathogen-host interaction. Here, suppression subtractive hybridization was used to search for the P. brassicae genes expressed during plant infection. One-hundred and forty ESTs were found of which 49% proved to be P. brassicae genes. Ten novel P. brassicae genes were identified, and the genomic sequences surrounding four of the ESTs were acquired using genome walking. Alignment of the ESTs and the genomic DNA sequences confirmed that P. brassicae genes are intron rich and that the introns are small. These results show that it is possible to discover new P. brassicae genes from a mixed pool of both plant and pathogen cDNA. The results also revealed that some of the P. brassicae genes expressed in Chinese cabbage (Brassica rapa subsp. pekinensis) were identical to the genes expressed in the infection of Arabidopsis plants, indicating that these genes play an important role in P. brassicae infection.

Initially, molecular techniques were used to detect and distinguish Plasmodiophora pathotypes in soil. Meanwhile, chromosomes from 2.2 Mb to 680 kb are characterized and the total genome size is estimated to be approximately 20 Mb. Furthermore, the genomic gene structure and the cDNA structure of several genes have been revealed, and the expression of those genes has been linked to development of clubroot to some extent. In addition, the sequence data have reinforced the inclusion of the plasmodiophorids within the Cercozoa. The recent successes in molecular biology have produced new approaches for clubroot research.

Colletotrichum acutatum is a major fungal pathogen of fruit crops, which causes severe yield losses in strawberry production. A potential key factor in plant–pathogen interactions is fungal sesquiterpenoids which have mycotoxic and phytotoxic activities. The first committed step in sesquiterpenoid biosynthesis is performed by sesquiterpene synthases (TPS). Only a few TPSs have been functionally characterized from filamentous fungi and none from the genus Colletotrichum. Despite being an important fungal pathogen to agriculture, it is poorly understood at the molecular and chemical levels. The terpenoid biochemistry in Coll. acutatum strain SA 0-1 was studied and one Coll. acutatum TPS (CaTPS) was successfully cloned and characterized in yeast. CaTPS catalyses the biosynthesis of multiple sesquiterpenoids. The two major products are β-caryophyllene and an unidentified sesquiterpenoid along with α-humulene as one of the minor sesquiterpenoid products. These products were also secreted by the fungus in strawberry fruit medium along with several other sesquiterpenoids indicating other TPSs are active during in vitro growth. β-Caryophyllene and α-humulene are known cytotoxic products important for ecological interactions and are produced by SA 0-1. Interestingly, a gene expression analysis using quantitative real-time PCR revealed a significant increase in expression of CaTPS during strawberry fruit infection, thus indicating that it could be involved in fruit infection. This is, we believe, the first characterization of TPS in Colletotrichum spp. and terpenoid profiles of Coll. acutatum, which could facilitate studies on the role of terpenoids in the ecology of Coll. acutatum.

Herbaria collections containing plants with disease symptoms are highly valuable, and they are often the only way to investigate outbreaks and epidemics from the past as the number of viable isolates in culture collections is often limited. Species belonging to the Colletotrichum acutatum complex infect a range of important crops. As members of the C. acutatum complex are easily confused with other Colletotrichum species, molecular methods are central for the correct identification. We performed molecular analyses on 21 herbaria specimens, displaying anthracnose symptoms, collected in Norway and Denmark before the first confirmed findings of C. acutatum complex members in this region. Sequencing parts of the fungal ITS regions showed that members of the species complex were present in 13 of the 21 specimens collected in different parts of Norway and Denmark between 1948 and 1991, representing seven plant hosts (three cherry species, apple, raspberry and rhododendron). This is the first time herbarium specimens have been used to study these pathogens under Nordic conditions. Differences in the ITS sequences suggest the presence of different genotypes within the complex, indicating a well-established population.

Strawberry, Fragaria × ananassa, and leatherleaf fern, Rumohra adiantiformis, are two important crops in Costa Rica. One of the most severe diseases affecting these crops is anthracnose, caused by members of the fungal genus, Colletotrichum (teleomorph; Glomerella). Eighty single-spore isolates from strawberry and leatherleaf fern were identified as Colletotrichum acutatum by species-specific PCR, and were further characterised by Universally Primed PCR (UP-PCR) fingerprinting analysis, and sequence analysis of the ribosomal internal transcribed spacer (ITS) region. Morphological differences, genotypic variation revealed by UP-PCR fingerprinting analysis, and a single sequence polymorphism within the ITS2 region were found between the isolates from strawberry and leatherleaf fern, respectively. The UPGMA cluster analysis of the fingerprints clearly separated the isolates derived from strawberry and leatherleaf fern into two different clusters. Pathogenicity assays on detached strawberry fruits confirmed the apparent difference between the two groups of isolates. It is therefore suggested that the pathogens responsible for strawberry anthracnose fruit rot and leatherleaf fern anthracnose in Costa Rica, belong to two distinct subpopulations of C. acutatum.

The oomycete Albugo candida has long been considered a broad spectrum generalist pathogen, but recent studies suggest that it is diverged into several more specialized species in addition to the generalist Albugo candida sensu stricto . Whereas these species cause the disease white blister rust in many crucifer plants, asymptomatic endophytic infections may be important in the epidemiology of others. One of the plant species attacked by Albugo sp. is the wild crucifer Barbarea vulgaris ssp. arcuata , which is diverged into two phytochemically and genetically different types with different geographical distributions in Europe. These were previously shown to differ strongly in propensity to develop white rust upon controlled infections in the greenhouse. Here, we analyse the phylogenetic relatedness of this local Albugo sp. field isolate to other species and lines of Albugo spp., including others collected on B. vulgaris . We further ask whether the difference in incidence of white rust between the two types of B. vulgaris are also expressed in natural populations. Phylogenetically, the local Albugo sp. field isolate clustered tightly together with previously analysed samples from B. vulgaris , supporting that the Albugo sp. infecting B. vulgaris may indeed be an independent specialized species. White blister rust and Albugo DNA was only detected in two populations of the plant type that frequently develops symptoms upon controlled inoculations. The lack of white rust and Albugo sp. DNA in the other plant type may be due to either resistance, preventing infection, or asymptomatic infection of other tissues than leaves, which we analysed.

Plants are sessile organisms that are under constant attack from microbes. They rely on both preformed defenses, and their innate immune system to ward of the microbial pathogens. Preformed defences include for example the cell wall and cuticle, which act as physical barriers to microbial colonization. The plant immune system is composed of surveillance systems that perceive several general microbe elicitors, which allow plants to switch from growth and development into a defense mode, rejecting most potentially harmful microbes. The elicitors are essential structures for pathogen survival and are conserved among pathogens. The conserved microbe-specific molecules, referred to as microbe- or pathogen-associated molecular patterns (MAMPs or PAMPs), are recognized by the plant innate immune systems pattern recognition receptors (PRRs). General elicitors like flagellin (Flg), elongation factor Tu (EF-Tu), peptidoglycan (PGN), lipopolysaccharides (LPS), Ax21 (Activator of XA21-mediated immunity in rice), fungal chitin, and β-glucans from oomycetes are recognized by plant surface localized PRRs. Several of the MAMPs and their corresponding PRRs have, in recent years, been identified. This review focuses on the current knowledge regarding important MAMPs from bacteria, fungi, and oomycetes, their structure, the plant PRRs that recognizes them, and how they induce MAMP-triggered immunity (MTI) in plants.

A protocol involving PCR, shot-gun cloning and sequencing was developed as a pre-diagnostic screening tool working directly on disease symptoms. The method was used to show the presence of biotrophic and non-biotrophic eukaryotic plant pathogens in leaves and fruits.

Symphyotrichum novi-belgii (L.) G.L. Nesom (synonym Aster novi-belgii L.) is an autumn flowering perennial used in gardens and as a cut flower. During the last 20 years, it has been developed as a potted plant, thereby increasing its economic importance. In Denmark, 7 to 8 million S. novi-belgii plants are produced annually, making it one of the 10 most popular potted plant crops ( http://floradania.dk/index.php?id=165 ). In general, S. novi-belgii is a healthy plant, but it can be severely attacked by powdery mildew both in greenhouse production and outdoors, and diseased plants have been observed in most parts of the country. Infected plants show typical symptoms: leaf surfaces become covered with white mycelium and as the disease progresses infected leaves turn yellow and die. Powdery mildew is regarded the main disease problem in S. novi-belgii and it causes problems year round in greenhouse production. Normally, the disease is controlled by fungicides, but once out of the production system, symptom development in the retail trade will reduce the plant's appeal to customers to a degree that prevents sales. The powdery mildew identified in this study was collected in a small research field at Aarslev, Denmark in September 2004. Since collection, the pathogen has been maintained in a greenhouse on S. novi-belgii and it has been used for disease resistance screening. However, lack of proper identification of the causal agent has hindered the development of powdery mildew resistant cultivars. To identify the pathogen, the internal transcribed spacer region (ITS) of the rDNA was amplified using primers ITS1 and ITS4 (2) and sequenced. The resulting sequence was deposited in GenBank (Accession No. HM769725). BLASTn analysis of the 598-bp fragment showed 99% identity to Golovinomyces cichoracearum (DC.) V.P. Heluta from Rudbeckia laciniata L. (Accession No. AB077622). The powdery mildew colonies were slightly pink with barrel-shaped, hyaline conidia borne in chains of three to four. The length of the conidia was 30 ± 4 μm and the width was 13 ± 1 μm (n = 105). Foot cells of the conidiophores were 101 ± 16 μm long and 12 ± 5 μm wide (n = 50) with a slight constriction at the base. Chasmothecia were not observed. These morphological characteristics confirmed the identification as G. cichoracearum (1). To fulfill Koch's postulates, 10 healthy S. novi-belgii ‘Victoria Fanny’ plants were inoculated in an inoculation tower by shaking infected S. novi-belgii plants over the tower, resulting in a spore density of 47 spores/mm2 on the leaf surface. The infected plants were placed in a growth chamber with 16 h of light (200 μmol·m–2·s–1) and day and night temperatures of 20 and 15°C, respectively. Symptoms developed on all plants after 11 days. Colony morphology on the leaves and the morphological characteristics were as described above. Conidia were washed off the leaves, DNA extracted, and the ITS was amplified by PCR. The resulting PCR product was sequenced and was identical to HM769725. To our knowledge, this is the first report of G. cichoracearum on S. novi-belgii in Denmark. References: (1) U. Braun. The Powdery Mildews (Erysiphales) of Europe. Gustav Fischer Verlag, Jena Germany, 1995. (2) T. J. White et al. Page 315 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis et al., eds. Academic Press, New York, 1990.

Strawberry (Fragaria × ananassa) is the most important small fruit crop in Denmark. The quarantine pathogen Colletotrichum acutatum was detected for the first time in June 2000 in Denmark in a production field on the island of Falster. Strawberry plants of cv. Kimberly showed typical symptoms of anthracnose fruit rot. On mature fruits, brown-to-black lesions with spore masses that were orange to salmon in color were observed. Mummified berries were also observed. The fungus was isolated and identified on the basis of morphological characteristics, and identification was confirmed using enzyme-linked immunosorbent assay at the Central Science Laboratory, York, U.K. Species-specific polymerase chain reaction with the C. acutatum-specific primer pairs acut1/col2 (1) and CaInt2/ITS4 (3) also supported the identification. Additionally, the internal transcribed spacer regions, ITS1 and ITS2, of the ribosomal DNA were sequenced in both directions (GenBank Accession No. AY818361). Homology searches with this sequence using BLAST also confirmed the identity. Colonies grown on potato dextrose agar developed white-to-grey aerial mycelium with salmon-colored spore masses, and were beige to black on the reverse side. Conidia were 11.3 (7.3 to 16.6) μm × 3.9 (2.5 to 5.2) μm, hyaline, cylindrical with at least one pointed end, and aseptate. Mycelial growth rate was 8.4 mm per day at 25°C which is similar to earlier reports (2). Spray-inoculated (106 conidia per ml) strawberry fruits cv. Elsanta developed brown, sunken, irregular lesions with salmon-colored acervuli after 2 to 5 days at 25°C. Koch's postulates were fulfilled since the reisolated fungus from these lesions developed the same morphological characteristics as described above. To our knowledge, this is the first report of C. acutatum in Denmark. References: (1) P. V. Martinez-Culebras et al. J. Phytopathol. 151:135, 2003. (2) B. J. Smith et al. Plant Dis. 74:69, 1990. (3) S. Sreenivasaprasad et al. Plant Pathol. 45:650, 1996.