Genetic toolkits of the red alga Pyropia tenera against the three most common diseases in Pyropia farms

Disease outbreaks devastate Pyropia aquaculture farms every year. The three most common and serious diseases are Olpidiopsis‐blight and red‐rot disease caused by oomycete pathogens and green‐spot disease caused by the PyroV1 virus. We hypothesized that a basic genetic profile of molecular defenses will be revealed by comparing and analyzing the genetic response of Pyropia tenera against the above three pathogens. RNAs isolated from infected thalli were hybridized onto an oligochip containing 15,115 primers designed from P. tenera expressed sequence tags (EST)s. Microarray profiles of the three diseases were compared and interpreted together with histochemical observation. Massive amounts of reactive oxygen species accumulated in P. tenera cells exposed to oomycete pathogens. Heat shock genes and serine proteases were the most highly up‐regulated genes in all infection experiments. Genes involved in RNA metabolism, ribosomal proteins and antioxidant metabolism were also highly up‐regulated. Genetic profiles of P. tenera in response to pathogens were most similar between the two biotrophic pathogens, Olpidiopsis pyropiae and PyroV1 virus. A group of plant resistance genes were specifically regulated against each pathogen. Our results suggested that disease response in P. tenera consists of a general constitutive defense and a genetic toolkit against specific pathogens.     

Green fluorescent protein (GFP) as gene expression reporter and vital marker for studying development and microbe-plant interaction in the tobacco pathogen Phythphthora parasitica var. nicotianae

PEG-mediated transformation of protoplasts in the presence of lipofectin was achieved in Phytophthora parasitica var. nicotianae, an oomycete pathogen of tobacco. Using oomycete promoter and terminator sequences, a plant-adapted green fluorescent protein (GFP) was introduced into the microorganism. The data show for the first time that this eukaryotic gene reporter can be used in an oomycete, both as a quantitative reporter of gene induction and as a vital marker allowing the study of development of Phytophthora in vitro and in the host plant.     

Alpha-proteobacteria cultivated from marine sponges display branching rod morphology

PCR amplification of two CHS gene fragments of the obligate biotroph Plasmopara viticola, the causal agent of downy mildew of grapevine, is described. While one fragment shows homology to fungal class IV chitin synthases, the other fragment groups with other oomycete chitin synthases to form a novel class of chitin synthases most closely related to class I-III. RT-PCR experiments indicate that PvCHS1 is constitutively expressed, whereas PvCHS2 is specifically transcribed in sporangiophores and sporangia. Analyses of wheat germ agglutinin labeling patterns by confocal laser scanning microscopy show that chitin is present on the surface of hyphal cell walls during in planta growth, and of sporangiophores and sporangia.     

An enzymatic fluorescent assay for the quantification of phosphite in a microtiter plate format

A sensitive fluorometric assay for the quantification of phosphite has been developed. The assay uses the enzymatic oxidation of phosphite to phosphate by a recombinant phosphite dehydrogenase with NAD⁺ as cosubstrate to produce the highly fluorescent reaction product resorufin. The optimized assay can be carried out in a 96-well microtiter plate format for high-throughput screening purposes and has a detection limit of 0.25 nmol phosphite. We used the method to quantify phosphite levels in plant tissue extracts and to determine phosphite dehydrogenase activity in transgenic plants. The assay is suitable for other biological or environmental samples. Because phosphite is a widely used fungicide to protect plants from pathogenic oomycetes, the assay provides a cost-effective and easy-to-use method to monitor the fate of phosphite following application.     

Current practices and emerging possibilities for reducing the spread of oomycete pathogens in terrestrial and aquatic production systems in the European Union

Diseases caused by oomycete pathogens are a global threat to forestry, agriculture and aquaculture. Because of their complex life cycles, characterised by dormant resting structures that enable their survival for years under hostile environmental conditions, reducing the spread of oomycetes is a challenging task. In this review, we present an overview of this challenge, starting from the need to understand the natural and anthropogenic dispersal pathways of these pathogens. Focusing on the European Union, we explore current legislation that forms a backbone for biosecurity protocols against the spread of oomycetes through trade and transport. We discuss the options for prevention, containment and long-term management of oomycetes in different production settings, emphasising the importance of prevention as the most cost-efficient strategy to reduce the spread of these pathogens. Finally, we highlight some of the new and emerging technologies and strategies as potential tools in the integrated pest management of animal and plant diseases caused by oomycetes. We emphasise the urgency of actions to halt the global spread of these pathogens.     

Saprolegnia parasitica, an oomycete pathogen with a fishy appetite: new challenges for an old problem

Water moulds (oomycetes) of the order Saprolegniales, such as Saprolegnia and Aphanomyces species, are responsible for devastating infections on fish in aquaculture, fish farms and hobby fish tanks. Members of the genus Saprolegnia cause Saprolegniosis, a disease that is characterised by visible white or grey patches of filamentous mycelium on the body or fins of freshwater fish. Up till 2002, Saprolegnia infections in aquaculture were kept under control with malachite green, an organic dye that is very efficient at killing the pathogen. However, the use of malachite green has been banned worldwide due to its carcinogenic and toxicological effects and this has resulted in a dramatic re-emergence of Saprolegnia infections in aquaculture. As a consequence Saprolegnia parasitica is now, economically, a very important fish pathogen, especially on catfish, salmon and trout species, and warrants further investigation to develop new alternative control strategies.     

Control of damping-off in tomato seedlings exerted by Serratia spp. strains and identification of inhibitory bacterial volatiles in vitro

Serratia marcescens can be a plant growth promoting bacteria (PGPB) and an opportunistic human and plant pathogen. We have identified and characterized strains of related species of Serratia and evaluated their biological control of damping-off of tomato seeds caused by Pythium cryptoirregulare. Serratia ureilytica, S. bockelmannii and S. nevei were identified by phylogenetic analysis of partial gyrB gene sequence and average nucleotide identity (ANI). Tomato seeds inoculated with S. ureilytica ILBB 145 showed higher germination percentage and reduced damping-off in greenhouse experiment resembling a commercial operation, and volatiles produced by this strain caused the nearly complete inhibition in vitro of P. cryptoirregulare. Analysis of volatile organic compounds (VOCs) showed that ILBB 145 produced dimethyl disulfide (DMDS), which can partially account for this inhibition. Serratia bockelmannii ILBB 162 performance against damping-off was intermediate and the inhibition of P. cryptoirregulare in vitro was lower and explained by volatile and diffusible metabolites. Both strains augmented DMDS production in the presence of P. cryptoirregulare, suggesting this compound may play a role in the context of interspecific competition. Serratia nevei ILBB 219 showed the lowest inhibition of P. cryptoirregulare in vitro, no DMDS production, and no biocontrol in planta. Draft genomes of the three strains were annotated and individual genes and biosynthesis gene clusters were identified in relation with the observed phenotypes. We report S. ureilytica – a low risk species- with activity as a biological control agent and DMDS produced by this bacterial species putatively involved in seed and seedling protection against P. cryptoirregulare.     

Genetic structure of Italian population of the grapevine downy mildew agent,Plasmopara viticola

Downy mildew, caused by the Oomycete Plasmopara viticola, is one of the most important diseases affecting the Eurasian grapevine, Vitis vinifera. The pathogen originated in Northern America and its presence was signalled for the first time into Europe in 1878. In this study, the genetic variability and structure of Italian P. viticola population was investigated by genotyping 106 P. viticola strains belonging to 12 different regions, at 31 microsatellite markers. As a result of the high percentage of missing data, 96 strains and 19 loci were retained for the data analysis. The overall Italian population presents absence of clones, evidence of sexual and asexual reproduction and a low genetic diversity, as expected for an introduced pathogen, but a slightly higher genetic diversity than that found in other European populations, based on allelic diversity at the investigated microsatellite loci. Out of 19 loci, half shows deviation from Hardy–Weinberg equilibrium and, indeed, structure analysis indicates the presence of two separate genetic clusters, with little but significantly different distribution according to geography (west–east gradient) and climatic conditions. Overall, the analysis of the P. viticola population, 140 years after its appearance in Italy, provides indication on the pathogen adaptability. This should be taken into consideration in the future for preserving the durability of disease resistant varieties in open field. In this view, all the disease control methods available should be integrated in order to reduce the selection of pathogen strains able to overcome plant resistance.