Pantothenate Auxotrophy in a Naturally Occurring Biocontrol Yeast.

The genus Hanseniaspora is characterized by some of the smallest genomes among budding yeasts. These fungi are primarily found on plant surfaces and in fermented products and represent promising biocontrol agents against notorious fungal plant pathogens. In this work, we identify pantothenate auxotrophy of a Hanseniaspora meyeri isolate that shows strong antagonism against the plant pathogen Fusarium oxysporum. Furthermore, strong biocontrol activity in vitro required both pantothenate and biotin in the growth medium. We show that the H. meyeri isolate APC 12.1 can obtain the vitamin from plants and other fungi. The underlying reason for the auxotrophy is the lack of two key pantothenate biosynthesis genes, but six genes encoding putative pantothenate transporters are present in the genome. By constructing and using a Saccharomyces cerevisiae reporter strain, we identified one Hanseniaspora transporter that conferred pantothenate uptake activity to S. cerevisiae. Pantothenate auxotrophy is rare and has been described in only a few bacteria and in S. cerevisiae strains that were isolated from sake. Such auxotrophic strains may seem an unexpected and unlikely choice as potential biocontrol agents, but they may be particularly competitive in their ecological niche and their specific growth requirements are an inherent biocontainment strategy preventing uncontrolled growth in the environment. Auxotrophic strains, such as the H. meyeri isolate APC 12.1, may thus represent a promising strategy for developing biocontrol agents that will be easier to register than prototrophic strains, which are normally used for such applications. IMPORTANCE As a precursor of the essential coenzyme A (CoA), pantothenate is present in all organisms. Plants, bacteria, and fungi are known to synthesize this vitamin, while animals must obtain it through their diet. Pantothenate auxotrophy has not been described in naturally occurring, environmental fungi and is an unexpected property for an antagonistic yeast. Here, we report that yeasts from the genus Hanseniaspora lack key enzymes for pantothenate biosynthesis and identify a transporter responsible for the acquisition of pantothenate from the environment. Hanseniaspora isolates are strong antagonists of fungal plant pathogens. Their pantothenate auxotrophy is a natural biocontainment feature that could make such isolates interesting candidates for new biocontrol approaches and allow easier registration as plant protection agents than prototrophic strains.

Clinical Aureobasidium Isolates Are More Fungicide Sensitive than Many Agricultural Isolates.

Fungicide applications in agriculture and medicine can promote the evolution of resistant, pathogenic fungi, which is a growing problem for disease management in both settings. Nonpathogenic mycobiota are also exposed to fungicides, may become tolerant, and could turn into agricultural or medical problems, for example, due to climate change or in immunocompromised individuals. However, quantitative data about fungicide sensitivity of environmental fungi is mostly lacking. Aureobasidium species are widely distributed and frequently isolated yeast-like fungi. One species, A. pullulans, is used as a biocontrol agent, but is also encountered in clinical samples, regularly. Here, we compared 16 clinical and 30 agricultural Aureobasidium isolates based on whole-genome data and by sensitivity testing with the 3 fungicides captan, cyprodinil, and difenoconazole. Our phylogenetic analyses determined that 7 of the 16 clinical isolates did not belong to the species A. pullulans. These isolates clustered with other Aureobasidium species, including A. melanogenum, a recently separated species that expresses virulence traits that are mostly lacking in A. pullulans. Interestingly, the clinical Aureobasidium isolates were significantly more fungicide sensitive than many isolates from agricultural samples, which implies selection for fungicide tolerance of non-target fungi in agricultural ecosystems. IMPORTANCE Environmental microbiota are regularly found in clinical samples and can cause disease, in particular, in immunocompromised individuals. Organisms of the genus Aureobasidium belonging to this group are highly abundant, and some species are even described as pathogens. Many A. pullulans isolates from agricultural samples are tolerant to different fungicides, and it seems inevitable that such strains will eventually appear in the clinics. Selection for fungicide tolerance would be particularly worrisome for species A. melanogenum, which is also found in the environment and exhibits virulence traits. Based on our observation and the strains tested here, clinical Aureobasidium isolates are still fungicide sensitive. We, therefore, suggest monitoring fungicide sensitivity in species, such as A. pullulans and A. melanogenum, and to consider the development of fungicide tolerance in the evaluation process of fungicides.

Next generation biosecurity: Towards genome based identification to prevent spread of agronomic pests and pathogens using nanopore sequencing.

The unintentional movement of agronomic pests and pathogens is steadily increasing due to the intensification of global trade. Being able to identify accurately and rapidly early stages of an invasion is critical for developing successful eradication or management strategies. For most invasive organisms, molecular diagnostics is today the method of choice for species identification. However, the currently implemented tools are often developed for certain taxa and need to be adapted for new species, making them ill-suited to cope with the current constant increase in new invasive species. To alleviate this impediment, we developed a fast and accurate sequencing tool allowing to modularly obtain genetic information at different taxonomical levels. Using whole genome amplification (WGA) followed by Oxford nanopore MinION sequencing, our workflow does not require any a priori knowledge on the investigated species and its classification. While mainly focusing on harmful plant pathogenic insects, we also demonstrate the suitability of our workflow for the molecular identification of bacteria (Erwinia amylovora and Escherichia coli), fungi (Cladosporium herbarum, Colletotrichum salicis, Neofabraea alba) and nematodes (Globodera rostochiensis). On average, the pairwise identity between the generated consensus sequences and best GenBank BLAST matches was 99.6 ± 0.6%. Additionally, assessing the generated insect genomic dataset, the potential power of the workflow to detect pesticide resistance genes, as well as arthropod-infecting viruses and endosymbiotic bacteria is demonstrated.

Dynamics of the Apple Fruit Microbiome after Harvest and Implications for Fruit Quality.

The contribution of the apple microbiome to the production chain of apple was so far largely unknown. Here, we describe the apple fruit microbiome and influences on its composition by parameters such as storage season, storage duration, storage technology, apple variety, and plant protection schemes. A combined culturing and metabarcoding approach revealed significant differences in the abundance, composition, and diversity of the apple fruit microbiome. We showed that relatively few genera contribute a large portion of the microbiome on fruit and that the fruit microbiome changes during the storage season depending on the storage conditions. In addition, we show that the plant protection regime has an influence on the diversity of the fruit microbiome and on the dynamics of pathogenic fungal genera during the storage season. For the genus Neofabraea, the quantitative results from the metabarcoding approach were validated with real-time PCR. In conclusion, we identified key parameters determining the composition and temporal changes of the apple fruit microbiome, and the main abiotic driving factors of microbiome diversity on apple fruit were characterized.

Impact of drying methods on the changes of fruit microstructure unveiled by X-ray micro-computed tomography.

A good understanding of the microstructural changes due to dehydration is critical to optimize fruit drying processes. By using X-ray micro-computed tomography, we quantified the changes in porosity, pore diameter, cell sphericity, cell diameter and cell elongation of apple parenchyma tissue during multiple convective drying scenarios: natural convection (air speed = 0.05 m s-1), forced convection (air speed = 0.5 m s-1) and coupled irradiation-convective drying (air speed = 0.05 m s-1 with radiation heating). Drying conditions affected the microstructure noticeably, in particular the formation of an elevated-porosity layer (tissue region where the porosity was higher than the initial porosity) and a deformed-cell layer (tissue region where the sphericity of the cells was lower than 0.75) near the sample surface. Using the combination of Eulerian and Lagrangian approaches, we linked the formation of the aforementioned layers to bulk shrinkage and deformation of individual cells. Forced convective drying resulted in a more porous structure and a higher degree of cell deformation compared to the other drying cases. Meanwhile, the coupled irradiation-convective drying induced the largest bulk shrinkage. The latter was caused by a large reduction in pore volume and the formation of large cell clusters in the deformed-cell layer.

Dispersal of harmful fruit fly pests by international trade and a loop-mediated isothermal amplification assay to prevent their introduction.

Global trade of plant products represents one of the major driving forces for the spread of invasive insect pests. This visualization illustrates the problem of unintended dispersal of economically harmful fruit fly pests using geospatial maps based on interception data from the Swiss import control process. Furthermore, it reports the development of a molecular diagnostic assay for rapid identification of these pests at points of entry such as sea- and airports as a prevention measure. The assay reliably differentiates between target and non-target species within one hour and has been successfully evaluated for on-site use at a Swiss point of entry.

A Loop-mediated Isothermal Amplification (LAMP) Assay for Rapid Identification of Bemisia tabaci.

The whitefly Bemisia tabaci (Gennadius) is an invasive pest of considerable importance, affecting the production of vegetable and ornamental crops in many countries around the world. Severe yield losses are caused by direct feeding, and even more importantly, also by the transmission of more than 100 harmful plant pathogenic viruses. As for other invasive pests, increased international trade facilitates the dispersal of B. tabaci to areas beyond its native range. Inspections of plant import products at points of entry such as seaports and airports are, therefore, seen as an important prevention measure. However, this last line of defense against pest invasions is only effective if rapid identification methods for suspicious insect specimens are readily available. Because the morphological differentiation between the regulated B. tabaci and close relatives without quarantine status is difficult for non-taxonomists, a rapid molecular identification assay based on the loop-mediated isothermal amplification (LAMP) technology has been developed. This publication reports the detailed protocol of the novel assay describing rapid DNA extraction, set-up of the LAMP reaction, as well as interpretation of its read-out, which allows identifying B. tabaci specimens within one hour. Compared to existing protocols for the detection of specific B. tabaci biotypes, the developed method targets the whole B. tabaci species complex in one assay. Moreover the assay is designed to be applied on-site by plant health inspectors with minimal laboratory training directly at points of entry. Thorough validation performed under laboratory and on-site conditions demonstrates that the reported LAMP assay is a rapid and reliable identification tool, improving the management of B. tabaci.

Seasonal Dynamics of Glyphosate and AMPA in Lake Greifensee: Rapid Microbial Degradation in the Epilimnion During Summer.

Occurrence and fate of glyphosate, a widely used herbicide, and its main metabolite AMPA was investigated in Lake Greifensee, Switzerland. Monthly vertical concentration profiles in the lake showed an increase of glyphosate concentrations in the epilimnion from 15 ng/L in March to 145 ng/L in July, followed by a sharp decline to <5 ng/L in August. A similar pattern was observed for AMPA. Concentrations of glyphosate and AMPA in the two main tributaries generally were much higher than in the lake. Simulations using a numerical lake model indicated that a substantial amount of glyphosate and AMPA dissipated in the epilimnion, mainly in July and August, with half-lives of only ≈2-4 days which is ≫100 times faster than in the preceding months. Fast dissipation coincided with high water temperatures and phytoplankton densities, and low phosphate concentrations. This indicates that glyphosate might have been used as an alternative phosphorus source by bacterio- and phytoplankton. Metagenomic analysis of lake water revealed the presence of organisms known to be capable of degrading glyphosate and AMPA.

From laboratory to point of entry: development and implementation of a loop-mediated isothermal amplification (LAMP)-based genetic identification system to prevent introduction of quarantine insect species.

BACKGROUND: Rapid genetic on-site identification methods at points of entry, such as seaports and airports, have the potential to become important tools to prevent the introduction and spread of economically harmful pest species that are unintentionally transported by the global trade of plant commodities. This paper reports the development and evaluation of a loop-mediated isothermal amplification (LAMP)-based identification system to prevent introduction of the three most frequently encountered regulated quarantine insect species groups at Swiss borders, Bemisia tabaci, Thrips palmi and several regulated fruit flies of the genera Bactrocera and Zeugodacus. RESULTS: The LAMP primers were designed to target a fragment of the mitochondrial cytochrome c oxidase subunit I gene and were generated based on publicly available DNA sequences. Laboratory evaluations analysing 282 insect specimens suspected to be quarantine organisms revealed an overall test efficiency of 99%. Additional on-site evaluation at a point of entry using 37 specimens performed by plant health inspectors with minimal laboratory training resulted in an overall test efficiency of 95%. During both evaluation rounds, there were no false-positives and the observed false-negatives were attributable to human-induced manipulation errors. To overcome the possibility of accidental introduction of pests as a result of rare false-negative results, samples yielding negative results in the LAMP method were also subjected to DNA barcoding. CONCLUSION: Our LAMP assays reliably differentiated between the tested regulated and non-regulated insect species within <1 h. Hence, LAMP assays represent suitable tools for rapid on-site identification of harmful pests, which might facilitate an accelerated import control process for plant commodities. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Development and evaluation of a bioinformatics approach for designing molecular assays for viral detection.

BACKGROUND: Viruses belonging to the Flaviviridae and Bunyaviridae families show considerable genetic diversity. However, this diversity is not necessarily taken into account when developing diagnostic assays, which are often based on the pairwise alignment of a limited number of sequences. Our objective was to develop and evaluate a bioinformatics workflow addressing two recurrent issues of molecular assay design: (i) the high intraspecies genetic diversity in viruses and (ii) the potential for cross-reactivity with close relatives. METHODOLOGY: The workflow developed herein was based on two consecutive BLASTn steps; the first was utilized to select highly conserved regions among the viral taxon of interest, and the second was employed to assess the degree of similarity of these highly-conserved regions to close relatives. Subsequently, the workflow was tested on a set of eight viral species, including various strains from the Flaviviridae and Bunyaviridae families. PRINCIPAL FINDINGS: The genetic diversity ranges from as low as 0.45% variable sites over the complete genome of the Japanese encephalitis virus to more than 16% of variable sites on segment L of the Crimean-Congo hemorrhagic fever virus. Our proposed bioinformatics workflow allowed the selection-based on computing scores-of the best target for a diagnostic molecular assay for the eight viral species investigated. CONCLUSIONS/SIGNIFICANCE: Our bioinformatics workflow allowed rapid selection of highly conserved and specific genomic fragments among the investigated viruses, while considering up to several hundred complete genomic sequences. The pertinence of this workflow will increase in parallel to the number of sequences made publicly available. We hypothesize that our workflow might be utilized to select diagnostic molecular markers for higher organisms with more complex genomes, provided the sequences are made available.

Evaluation of a real-time PCR and a loop-mediated isothermal amplification for detection of Xanthomonas arboricola pv. pruni in plant tissue samples.

Operational capacity of real-time PCR and loop-mediated isothermal amplification (LAMP) diagnostic assays for detection of Xanthomonas arboricola pv. pruni was established in a ring-test involving four laboratories. Symptomatic and healthy almond leaf samples with two methods of sample preparation were analyzed. Kappa coefficient, sensitivity, specificity, likelihood ratios and post-test probability of detection were estimated to manage the risk associated with the use of the two methods.

Phylogeography and population structure of the biologically invasive phytopathogen Erwinia amylovora inferred using minisatellites.

Erwinia amylovora causes a major disease of pome fruit trees worldwide, and is regulated as a quarantine organism in many countries. While some diversity of isolates has been observed, molecular epidemiology of this bacterium is hindered by a lack of simple molecular typing techniques with sufficiently high resolution. We report a molecular typing system of E. amylovora based on variable number of tandem repeats (VNTR) analysis. Repeats in the E. amylovora genome were identified with comparative genomic tools, and VNTR markers were developed and validated. A Multiple-Locus VNTR Analysis (MLVA) was applied to E. amylovora isolates from bacterial collections representing global and regional distribution of the pathogen. Based on six repeats, MLVA allowed the distinction of 227 haplotypes among a collection of 833 isolates of worldwide origin. Three geographically separated groups were recognized among global isolates using Bayesian clustering methods. Analysis of regional outbreaks confirmed presence of diverse haplotypes but also high representation of certain haplotypes during outbreaks. MLVA analysis is a practical method for epidemiological studies of E. amylovora, identifying previously unresolved population structure within outbreaks. Knowledge of such structure can increase our understanding on how plant diseases emerge and spread over a given geographical region.

Comparative genomics of 12 strains of Erwinia amylovora identifies a pan-genome with a large conserved core.

The plant pathogen Erwinia amylovora can be divided into two host-specific groupings; strains infecting a broad range of hosts within the Rosaceae subfamily Spiraeoideae (e.g., Malus, Pyrus, Crataegus, Sorbus) and strains infecting Rubus (raspberries and blackberries). Comparative genomic analysis of 12 strains representing distinct populations (e.g., geographic, temporal, host origin) of E. amylovora was used to describe the pan-genome of this major pathogen. The pan-genome contains 5751 coding sequences and is highly conserved relative to other phytopathogenic bacteria comprising on average 89% conserved, core genes. The chromosomes of Spiraeoideae-infecting strains were highly homogeneous, while greater genetic diversity was observed between Spiraeoideae- and Rubus-infecting strains (and among individual Rubus-infecting strains), the majority of which was attributed to variable genomic islands. Based on genomic distance scores and phylogenetic analysis, the Rubus-infecting strain ATCC BAA-2158 was genetically more closely related to the Spiraeoideae-infecting strains of E. amylovora than it was to the other Rubus-infecting strains. Analysis of the accessory genomes of Spiraeoideae- and Rubus-infecting strains has identified putative host-specific determinants including variation in the effector protein HopX1(Ea) and a putative secondary metabolite pathway only present in Rubus-infecting strains.

Erwinia amylovora loop-mediated isothermal amplification (LAMP) assay for rapid pathogen detection and on-site diagnosis of fire blight.

Several molecular methods have been developed for the detection of Erwinia amylovora, the causal agent of fire blight in pear and apple, but none are truly applicable for on-site use in the field. We developed a fast, reliable and field applicable detection method using a novel target on the E. amylovora chromosome that we identified by applying a comparative genomic pipeline. The target coding sequences (CDSs) are both uniquely specific for and all-inclusive of E. amylovora genotypes. This avoids potential false negatives that can occur with most commonly used methods based on amplification of plasmid gene targets, which can vary among strains. Loop-mediated isothermal AMPlification (LAMP) with OptiGene Genie II chemistry and instrumentation proved to be an exceptionally rapid (under 15 min) and robust method for detecting E. amylovora in orchards, as well as simple to use in the plant diagnostic laboratory. Comparative validation results using plant samples from inoculated greenhouse trials and from natural field infections (of regional and temporal diverse origin) showed that our LAMP had an equivalent or greater performance regarding sensitivity, specificity, speed and simplicity than real-time PCR (TaqMan), other LAMP assays, immunoassays and plating, demonstrating its utility for routine testing.

Comparative analysis of the Hrp pathogenicity island of Rubus- and Spiraeoideae-infecting Erwinia amylovora strains identifies the IT region as a remnant of an integrative conjugative element.

The Hrp pathogenicity island (hrpPAI) of Erwinia amylovora not only encodes a type III secretion system (T3SS) and other genes required for pathogenesis on host plants, but also includes the so-called island transfer (IT) region, a region that originates from an integrative conjugative element (ICE). Comparative genomic analysis of the IT regions of two Spiraeoideae- and three Rubus-infecting strains revealed that the regions in Spiraeoideae-infecting strains were syntenic and highly conserved in length and genetic information, but that the IT regions of the Rubus-infecting strains varied in gene content and length, showing a mosaic structure. None of the ICEs in E. amylovora strains were complete, as conserved ICE genes and the left border were missing, probably due to reductive genome evolution. Comparison of the hrpPAI region of E. amylovora strains to syntenic regions from other Erwinia spp. indicates that the hrpPAI and the IT regions are the result of several insertion and deletion events that have occurred within the ICE. It also suggests that the T3SS was present in a common ancestor of the pathoadapted Erwinia spp. and that insertion and deletion events in the IT region occurred during speciation.

Complete genome sequence of clinical isolate Pantoea ananatis LMG 5342.

The enterobacterium Pantoea ananatis is an ecologically versatile species. It has been found in the environment, as plant epiphyte and endophyte, as an emerging phytopathogen, and as a presumptive, opportunistic human pathogen. Here, we report the complete genome sequence of P. ananatis LMG 5342, isolated from a human wound.