Mogens Nicolaisen

Towards on-site pathogen detection using antibody-based sensors

In this paper, the recent progress within biosensors for plant pathogen detection will be reviewed. Bio-recognition layers on sensors can be designed in various ways, however the most popular approach is to immobilise antibodies for specific capture of analytes. Focus will be put on antibody surface-immobilisation strategies as well as the use of antibodies in the widely used sensors, quartz crystal microbalance, surface plasmon resonance and cantilevers. We will describe the available data on antibody-based plant pathogen detection and furthermore use examples from detection of the pathogens Salmonella, Listeria monocytogenes, Streptococcus mutans, Bacillus cereus, Bacillus anthracis, Campylobacter and Escherichia coli. We will touch upon optimal assay design and further discuss the strengths and limitations of current sensor technologies for detection of viruses, bacteria and fungi.

Real-time PCR for quantification of eleven individual Fusarium species in cereals

Contamination of cereals with Fusarium species is one of the major sources of mycotoxins in food and feed. Quantification of biomass of Fusarium species is essential to understand the interactions of individual species in disease development. In this study quantitative real-time PCR assays based on the elongation factor 1 alpha (EF1alpha) gene for the 11 Fusarium species F. graminearum, F. culmorum, F. poae, F. langsethiae, F. sporotrichioides, F. equiseti, F. tricinctum, F. avenaceum, F. verticillioides, F. subglutinans and F. proliferatum were developed and tested on 24 wheat and 24 maize field samples. The assays were found to be specific and sensitive. Generally, the results from the quantitative real-time PCR assays corresponded well with mycotoxin data of the field samples.

Distribution of phytoplasmas in infected plants as revealed by real-time PCR and bioimaging.

Phytoplasmas are cell wall-less bacteria inhabiting the phloem and utilizing it for their spread. Infected plants often show changes in growth pattern and a reduced crop yield. A quantitative real-time polymerase chain reaction (Q-PCR) assay and a bioimaging method were developed to quantify and localize phytoplasmas in situ. According to the Q-PCR assay, phytoplasmas accumulated disproportionately in source leaves of Euphorbia pulcherrima and, to a lesser extent, in petioles of source leaves and in stems. However, phytoplasma accumulation was small or nondetectable in sink organs (roots and sink leaves). For bioimaging, infected plant tissue was stained with vital fluorescence dyes and examined using confocal laser scanning microscopy. With a DNA-sensitive dye, the pathogens were detected exclusively in the phloem, where they formed dense masses in sieve tubes of Catharanthus roseus. Sieve tubes were identified by counterstaining with aniline blue for callose and multiphoton excitation. With a potentiometric dye, not all DNA-positive material was stained, suggesting that the dye stained metabolically active phytoplasmas only. Some highly infected sieve tubes contained phytoplasmas that were either inactive or dead upon staining.

Phytoplasma effector SAP54 induces indeterminate leaf-like flower development in Arabidopsis plants

Phytoplasmas are insect-transmitted bacterial plant pathogens that cause considerable damage to a diverse range of agricultural crops globally. Symptoms induced in infected plants suggest that these phytopathogens may modulate developmental processes within the plant host. We report herein that Aster Yellows phytoplasma strain Witches’ Broom (AY-WB) readily infects the model plant Arabidopsis (Arabidopsis thaliana) ecotype Columbia, inducing symptoms that are characteristic of phytoplasma infection, such as the production of green leaf-like flowers (virescence and phyllody) and increased formation of stems and branches (witches’ broom). We found that the majority of genes encoding secreted AY-WB proteins (SAPs), which are candidate effector proteins, are expressed in Arabidopsis and the AY-WB insect vector Macrosteles quadrilineatus (Hemiptera; Cicadellidae). To identify which of these effector proteins induce symptoms of phyllody and virescence, we individually expressed the effector genes in Arabidopsis. From this screen, we have identified a novel AY-WB effector protein, SAP54, that alters floral development, resulting in the production of leaf-like flowers that are similar to those produced by plants infected with this phytoplasma. This study offers novel insight into the effector profile of an insect-transmitted plant pathogen and reports to our knowledge the first example of a microbial pathogen effector protein that targets flower development in a host.

Incidence of acute bee paralysis virus, black queen cell virus, chronic bee paralysis virus, deformed wing virus, Kashmir bee virus and sacbrood virus in honey bees (Apis mellifera) in Denmark

Samples of adult honey bees from apiaries with unusually high winter mortality and brood from hives with symptoms of disease were tested for presence of acute bee paralysis virus (ABPV), black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), deformed wing virus (DWV), Kashmir bee virus (KBV) and sacbrood virus (SBV) by RT-PCR. All six viruses were detected, but the frequencies varied significantly: SBV was detected in 78 apiaries, DWV in 55, ABPV in 11, CBPV in 4, BQCV in 1 and KBV in 1. This is the first record of KBV in Denmark. A large majority of the bee samples were infected with one or more viruses. Single, dual and triple infections were observed. Nucleotide sequences of the PCR products from each virus were determined and found to be 98–99% identical to GenBank accessions except CBPV, which was only 88–90% identical to known CBPV sequences.ZusammenfassungNeuere Untersuchungen zur Verbreitung von Bienenviren unter Verwendung molekulargenetischer Diagnosemethoden wurden in Europa lediglich in Frankreich und Deutschland durchgeführt. Um auch für Dänemark einen detaillierten Überblick zu erhalten, wurden adulte Honigbienen von Bienenständen mit außergewöhnlich hoher Wintermortalität sowie Brutwaben von Bienenvölkern mit Krankheitssymptomen mittels RT-PCR auf folgende Viruserkrankungen hin untersucht: Akute-Bienenparalyse-Virus (ABPV), Schwarze-Königinnenzellen-Virus (BQCV), Kaschmir-Bienen-Virus (KBV), Chronische-Bienenparalyse-Virus (CBPV), Verkrüppelte-Flügel-Virus (DWV) und Sackbrut-Virus (SBV). Die verwendeten Primer und RT-PCR-Verfahren wurden der Literatur entnommen. Alle Proben wurden auf das Vorkommen aller sechs Bienenviren hin analysiert. Um die PCR-Produkte eindeutig zu identifizieren, wurden repräsentative Proben sequenziert und die Sequenzen gegen die GenBank-Datenbank geblasted.Insgesamt wurden 96 Bienenstände auf das Vorkommen der sechs Bienenviren hin untersucht. Alle sechs Viren wurden nachgewiesen, allerdings in sehr unterschiedlicher Häufigkeit. SBV wurde in 78, DWV in 55, ABPV in 11, CBPV in 4 und BQCV sowie KBV in einem Bienenstand festgestellt. Die Mehrzahl der Bienenproben war von mehr als einem Bienenvirus befallen, wobei Einzel-, Doppel- und Tripelbefall vorkamen. Die Nukleotidsequenzen der PCR-Produkte aller Viren waren zu 98–99% identisch mit den GenBank-Sequenzen mit Ausnahme von CBPV, der lediglich eine Übereinstimmung von 88–90% zu den bekannten CBPV-Sequenzen aufwies. Die Häufigkeit der sechs Bienenviren in Dänemark unterscheidet sich erheblich von den Ergebnissen aus anderen europäischen Ländern. Im Gegensatz zu diesen Ländern war SBV am meisten verbreitet, gefolgt von DWV. Außerdem war die Häufigkeit von ABPV, BQCV und CBPV deutlich geringer als sonst beschrieben. KBV wurde erstmals in Dänemark nachgewiesen und erweitert die bisherigen Funde von KBV aus fünf anderen europäischen Ländern.

Linking fungal communities in roots, rhizosphere, and soil to the health status of Pisum sativum.

Changes in fungal communities associated with healthy and diseased pea roots were investigated using deep amplicon pyrosequencing in three spatial compartments: roots, rhizosphere, and surrounding soil. Thirty root systems were collected from three fields, half of which showing clear symptoms of root rot. In total, 500,461 internal transcribed spacer-1 sequences were obtained that were clustered into 123 (roots), 271 (rhizosphere), and 440 (bulk soil) nonsingleton operational taxonomic units (OTUs). Species richness was highest in bulk soils and lowest in roots; however, no notable differences in richness were observed between samples associated with diseased and healthy roots. Health status and field both had significant effects on fungal community structures in roots, whereas only field had significant effects on communities in rhizosphere and bulk soils. Indicator species analysis across the three fields identified a number of OTUs that were more abundant in healthy roots. Pathogens such as Fusarium oxysporum were abundant in diseased roots in some fields. Patterns of disease and causal agents of root rot were different among the three fields, which were also reflected in fungal communities. In conclusion, health status of roots was only vaguely reflected in rhizosphere and bulk soil fungal communities, whereas health status was more important for shaping root communities.

Alternative hosts for potato mop-top virus, genus Pomovirus and its vectorSpongospora subterranea f.sp. subterranea

SummarySeventeen weed species common in the potato fields in Denmark were grown in a hydroponic system infested with viruliferous zoospores ofSpongospora subterranea f.sp.subterranea carrying potato mop-top virus (PMTV). The plants were examined for infection with PMTV andS.s.s. using DAS-ELISA and based on visible symptoms. Only two weed species were found to be infected with PMTV,Chenopodium album andSolanum nigrum, whereas 13 became infected withS.s.s. C. album was infected with PMTV by mechanical inoculation were the infection became systemically and the leaves showed necrotic spots.S. nigrum became local infected with PMTV by mechanical inoculation. By inoculation with viruliferousS.s.s. the roots ofS. nigrum became infected but in three weeks PMTV had not spread into the top of the plants.Nicotiana benthamiana was used as a control susceptible toS.s.s. and PMTV.

DNA barcoding for identification of 'Candidatus Phytoplasmas' using a fragment of the elongation factor Tu gene.

Background Phytoplasmas are bacterial phytopathogens responsible for significant losses in agricultural production worldwide. Several molecular markers are available for identification of groups or strains of phytoplasmas. However, they often cannot be used for identification of phytoplasmas from different groups simultaneously or are too long for routine diagnostics. DNA barcoding recently emerged as a convenient tool for species identification. Here, the development of a universal DNA barcode based on the elongation factor Tu (tuf) gene for phytoplasma identification is reported. Methodology/Principal Findings We designed a new set of primers and amplified a 420–444 bp fragment of tuf from all 91 phytoplasmas strains tested (16S rRNA groups -I through -VII, -IX through -XII, -XV, and -XX). Comparison of NJ trees constructed from the tuf barcode and a 1.2 kbp fragment of the 16S ribosomal gene revealed that the tuf tree is highly congruent with the 16S rRNA tree and had higher inter- and intra- group sequence divergence. Mean K2P inter−/intra- group divergences of the tuf barcode did not overlap and had approximately one order of magnitude difference for most groups, suggesting the presence of a DNA barcoding gap. The use of the tuf barcode allowed separation of main ribosomal groups and most of their subgroups. Phytoplasma tuf barcodes were deposited in the NCBI GenBank and Q-bank databases. Conclusions/Significance This study demonstrates that DNA barcoding principles can be applied for identification of phytoplasmas. Our findings suggest that the tuf barcode performs as well or better than a 1.2 kbp fragment of the 16S rRNA gene and thus provides an easy procedure for phytoplasma identification. The obtained sequences were used to create a publicly available reference database that can be used by plant health services and researchers for online phytoplasma identification.

Succession of root-associated fungi in Pisum sativum during a plant growth cycle as examined by 454 pyrosequencing

PurposeRoots are inhabited by a broad range of fungi, including pathogens and mycorrhizal fungi, with functional traits related to plant health and nutrition. Management of these fungi in agroecosystems requires profound knowledge about their ecology. The main objective of this study was to examine succession patterns of root-associated fungi in pea during a full plant growth cycle.MethodsPlants were grown in pots with field soil in a growth chamber under controlled conditions. Fungal communities in pea roots were analyzed at different plant growth stages including the vegetative growth, flowering and senescence, using 454 pyrosequencing.ResultsOne hundred and twenty one non-singleton operational taxonomic units (OTUs) representing fungal species were detected. Pathogenic and arbuscular mycorrhizal fungi dominated during the vegetative growth stage, whereas saprotrophic fungi dominated during plant senescence.ConclusionsIn conclusion, the results from the present study demonstrated highly diverse fungal communities in pea roots with clear succession patterns related to fungal traits.

Molecular characterization of root-associated fungal communities in relation to health status of Pisum sativum using barcoded pyrosequencing

Background and AimsRoot-associated fungi are known to be important for plant health and nutrition, but only few studies have addressed their diversity in relation to plant health status.MethodsFungal diversity in roots of healthy and diseased Pisum sativum plants was examined in terms of barcoded pyrosequencing of the nuclear ribosomal internal transcribed spacer 1. The CLOTU program was used for filtering and clustering of sequences, and Chao 1 estimator was used to calculate fungal richness.ResultsPrincipal component analyses (PCA) showed that the structure of root-associated fungal communities differed between sites and a clear relationship between root-associated fungal communities and plant health status was found. For example, the arbuscular mycorrhizal (AM) fungus Glomus caledonium was prevalent in roots of healthy plants, whereas the pathogenic fungus Phoma sojicola was prevalent in roots of diseased plants.ConclusionsThe present study revealed clear differences in composition of root-associated fungi from four field sites, and correlations between abundance of several root-associated fungi and plant health status were found.