Transcriptomic dataset of Phaseolus vulgaris leaves in response to the inoculation of pathogenic Xanthomonas citri pv. fuscans and its type III secretion system-defective mutant hrcV.

Xanthomonas citri pv. fuscans (Xcf) and Xanthomonas phaseoli pv. phaseoli (Xpp) are responsible for the Common Bacterial Blight (CBB), a major common bean (Phaseolus vulgaris) disease. The pathogenicity of Xcf and Xpp is known to be dependent upon a functional Type III Secretion System (T3SS) allowing the injection of numerous bacterial Type III Effectors (T3Es) into plant cells. T3Es have been described as able to disrupt plant defence and manipulate plant metabolism. In this work we described the transcriptomic response of one susceptible (Flavert) and one resistant (Vezer) cultivars of P. vulgaris to the inoculation of the virulent strain Xcf CFBP4885 or its avirulent T3SS-defective hrcV mutant (CFBP13802). Leaves of both bean cultivars were infiltrated with water or bacterial suspensions. Inoculated leaves were sampled at 24 or 48 h post inoculation (hpi). The experiment was independently repeated three times for total RNA extraction and sequencing analysis. Library construction and total RNA sequencing were performed with BGISEQ-500 at Beijing Genomics Institute (BGI, Hong-Kong), generating an average of 24M of paired-end reads of 100bp per sample. FastQC was used to check reads quality. Mapping analyses were made using a quasi-mapping alignment from Salmon (version 1.2.1) against the Phaseolus vulgaris reference genome (version 2.1), revealing the expression profiles of 36,978 transcripts in leaf tissues. Fastq raw data and count files from 36 samples are available in the Gene Expression Omnibus (GEO) repository of the National Center for Biotechnology Information (NCBI) under the accession number GSE271236. This dataset is a valuable resource to investigate the role of T3Es in subverting the cellular functions of bean.

A stage-dependent seed defense response to explain efficient seed transmission of Xanthomonas citri pv. fuscans to common bean.

Although seed represents an important means of plant pathogen dispersion, the seed-pathogen dialogue remains largely unexplored. A multiomic approach was performed at different seed developmental stages of common bean (Phaseolus vulgaris L.) during asymptomatic colonization by Xanthomonas citri pv. fuscans (Xcf), At the early seed developmental stages, we observed high transcriptional changes both in seeds with bacterial recognition and defense signal transduction genes, and in bacteria with up-regulation of the bacterial type 3 secretion system. This high transcriptional activity of defense genes in Xcf-colonized seeds during maturation refutes the widely diffused assumption considering seeds as passive carriers of microbes. At later seed maturation stages, few transcriptome changes indicated a less intense molecular dialogue between the host and the pathogen, but marked by changes in DNA methylation of plant defense genes, in response to Xcf colonization. We showed examples of pathogen-specific DNA methylations in colonized seeds acting as plant defense silencing to repress plant immune response during the germination process. Finally, we propose a novel plant-pathogen interaction model, specific to the seed tissues, highlighting the existence of distinct phases during seed-pathogen interaction with seeds being actively interacting with colonizing pathogens, then both belligerents switching to more passive mode at later stages.

Emergence and spread of Mycobacterium ulcerans at different geographic scales.

The classical lineage of Mycobacterium ulcerans is the most prevalent clonal group associated with Buruli ulcer in humans. Its reservoir is strongly associated with the environment. We analyzed together 1,045 isolates collected from 13 countries on two continents to define the evolutionary history and population dynamics of this lineage. We confirm that this lineage spread over 7,000 years from Australia to Africa with the emergence of outbreaks in distinct waves in the 18th and 19th centuries. In sharp contrast with its global spread over the last century, transmission chains are now mostly local, with little or no dissemination between endemic areas. This study provides new insights into the phylogeography and population dynamics of M. ulcerans, highlighting the importance of comparative genomic analyses to improve our understanding of pathogen transmission. IMPORTANCE: Mycobacterium ulcerans is an environmental mycobacterial pathogen that can cause Buruli ulcer, a severe cutaneous infection, mostly spread in Africa and Australia. We conducted a large genomic study of M. ulcerans, combining genomic and evolutionary approaches to decipher its evolutionary history and pattern of spread at different geographic scales. At the scale of villages in an endemic area of Benin, the circulating genotypes have been introduced in recent decades and are not randomly distributed along the river. On a global scale, M. ulcerans has been spreading for much longer, resulting in distinct and compartmentalized endemic foci across Africa and Australia.

Seedling microbiota engineering using bacterial synthetic community inoculation on seeds.

Synthetic Communities (SynComs) are being developed and tested to manipulate plant microbiota and improve plant health. To date, only few studies proposed the use of SynCom on seed despite its potential for plant microbiota engineering. We developed and presented a simple and effective seedling microbiota engineering method using SynCom inoculation on seeds. The method was successful using a wide diversity of SynCom compositions and bacterial strains that are representative of the common bean seed microbiota. First, this method enables the modulation of seed microbiota composition and community size. Then, SynComs strongly outcompeted native seed and potting soil microbiota and contributed on average to 80% of the seedling microbiota. We showed that strain abundance on seed was a main driver of an effective seedling microbiota colonization. Also, selection was partly involved in seed and seedling colonization capacities since strains affiliated to Enterobacteriaceae and Erwiniaceae were good colonizers while Bacillaceae and Microbacteriaceae were poor colonizers. Additionally, the engineered seed microbiota modified the recruitment and assembly of seedling and rhizosphere microbiota through priority effects. This study shows that SynCom inoculation on seeds represents a promising approach to study plant microbiota assembly and its consequence on plant fitness.

The type VI secretion system of Stenotrophomonas rhizophila CFBP13503 limits the transmission of Xanthomonas campestris pv. campestris 8004 from radish seeds to seedlings.

Stenotrophomonas rhizophila CFBP13503 is a seedborne commensal bacterial strain, which is efficiently transmitted to seedlings and can outcompete the phytopathogenic bacterium Xanthomonas campestris pv. campestris (Xcc8004). The type VI secretion system (T6SS), an interference contact-dependent mechanism, is a critical component of interbacterial competition. The involvement of the T6SS of S. rhizophila CFBP13503 in the inhibition of Xcc8004 growth and seed-to-seedling transmission was assessed. The T6SS cluster of S. rhizophila CFBP13503 and nine putative effectors were identified. Deletion of two T6SS structural genes, hcp and tssB, abolished the competitive advantage of S. rhizophila against Xcc8004 in vitro. The population sizes of these two bacterial species were monitored in seedlings after inoculation of radish seeds with mixtures of Xcc8004 and either S. rhizophila wild-type (wt) strain or isogenic hcp mutant. A significant decrease in the population size of Xcc8004 was observed during confrontation with the S. rhizophila wt in comparison with T6SS-deletion mutants in germinated seeds and seedlings. We found that the T6SS distribution among 835 genomes of the Stenotrophomonas genus is scarce. In contrast, in all available S. rhizophila genomes, T6SS clusters are widespread and mainly belong to the T6SS group i4. In conclusion, the T6SS of S. rhizophila CFBP13503 is involved in the antibiosis against Xcc8004 and reduces seedling transmission of Xcc8004 in radish. The distribution of this T6SS cluster in the S. rhizophila complex could make it possible to exploit these strains as biocontrol agents against X. campestris pv. campestris.

First report of Xanthomonas campestris pv. campestris causing black rot on oilseed rape (Brassica napus L.) in France.

In October 2022, v-shaped necrotic lesions were observed on the leaf margins of field-grown winter oilseed rape (WOSR), Brassica napus L., in western France (Ille-et-Vilaine (35) and Maine-et-Loire (49) departments). Disease incidence on volunteers and cultivated WOSR was generally low (5-10 %) but occasionally up to 80% on some fields. Leaf sections sampled from the margin of necrotic leaf tissue were dilacerated in sterile deionized water and the extract was spread onto tryptone soya agar (TSA) with cycloheximide (100 mg.L-1) and Polyflor (Syngenta, France) (2ml.L-1, containing 5 mg.L-1 propiconazole) then incubated at 28°C for 2 days. Colonies were yellow-pigmented, mucoid, and convex, which are morphological characteristics of Xanthomonas spp. colonies. The partial fyuA and gyrB gene sequences were amplified for eight isolated strains (CFBP 9155, CFBP 9156, CFBP 9157, CFBP 9158, CFBP 9159, CFBP 9161, CFBP 9162, and CFBP 9163) using primers of Fargier et al. (2011), and sequenced (Genoscreen, France). The sequences were deposited under numbers OR232891 to OR232898 for fyuA and OR634932 to OR634939 for gyrB. BLASTN analysis of the sequenced fyuA amplicon showed 100% identity and query coverage with the fyuA fragment of Xanthomonas campestris pv. campestris (Xcc) CFBP 6865R (Bellenot et al., 2022). BLASTN analysis of the sequenced gyrB amplicon showed two allelic forms: one showed 100% identity and query coverage with the gyrB fragment of Xcc strain CFBP 6865R (Bellenot et al., 2022), the other one showed 100% identity and query coverage with the type strain Xcc CFBP 5241 (ATCC33913) (Vorhölter et al., 2003). Moreover, two qPCR tools were used to identify the strains successfully as Xcc (Köhl et al., 2011; Rezki et al., 2016) which target the same gene encoding a hypothetical protein and whose primers overlap. The pathogenicity of the eight isolated strains was validated using a bacterial suspension (108 CFU.ml-1) for i) leaf spraying until runoff onto the leaf surfaces of WOSR plants previously maintained at saturated humidity for 48 hours, ii) wound-leaf inoculation of the two youngest true leaves with scissors that had been dipped into the bacterial suspension. Both tests were performed on 3-week-old WOSR plants of the Aviso (INRAE) genotype. Deionized water was used as negative control. Strains CFBP 5241 and the strain CFBP 4954 (Fargier et al., 2007) were used as positive controls for disease expression. Tested plants (seven for spray inoculation and four for wound-leaf inoculation per strain and control condition) were incubated in a greenhouse at 20°C/24°C (night/day). Isolated strains and the strain CFBP 4954 caused yellow lesions with both inoculation methods that necrotized starting about 10 days post inoculation (dpi). The spots coalesced within 14 dpi to form necrotic areas. The type strain CFBP 5241 caused mild symptoms, with only yellow lesions that did not coalesce. Plants inoculated with water remained symptomless. To complete Koch's postulate, re-isolations were achieved. Re-isolated strains on TSA showed the same colony morphology as described above. All re-isolated strains were identified as Xcc based on partial gyrB sequencing and Xcc specific qPCR test (Rezki et al., 2016). This first report in France and the recent identification in Serbia (Popovic et al., 2013) may illustrate the emergence of the disease on this crop in Europe. The prevalence and consequences of this disease should be evaluated over a wider geographic area.

Whole Genome Sequences of Nine Xanthomonas Strains Responsible for Common Bacterial Blight of Bean.

We report the complete and circularized genome sequences of nine strains of Xanthomonas phaseoli pv. phaseoli and Xanthomonas citri pv. fuscans, which cause common bacterial blight of bean. These assemblies provide high-quality material for functional and evolutionary studies of these legume pathogens.

Suspicions of two bridgehead invasions of Xylella fastidiosa subsp. multiplex in France.

Of American origin, a wide diversity of Xylella fastidiosa strains belonging to different subspecies have been reported in Europe since 2013 and its discovery in Italian olive groves. Strains from the subspecies multiplex (ST6 and ST7) were first identified in France in 2015 in urban and natural areas. To trace back the most probable scenario of introduction in France, the molecular evolution rate of this subspecies was estimated at 3.2165 × 10-7 substitutions per site per year, based on heterochronous genome sequences collected worldwide. This rate allowed the dating of the divergence between French and American strains in 1987 for ST6 and in 1971 for ST7. The development of a new VNTR-13 scheme allowed tracing the spread of the bacterium in France, hypothesizing an American origin. Our results suggest that both sequence types were initially introduced and spread in Provence-Alpes-Côte d'Azur (PACA); then they were introduced in Corsica in two waves from the PACA bridgehead populations.

Single Seed Microbiota: Assembly and Transmission from Parent Plant to Seedling.

The seed acts as the primary inoculum source for the plant microbiota. Understanding the processes involved in its assembly and dynamics during germination and seedling emergence has the potential to allow for the improvement of crop establishment. Changes in the bacterial community structure were tracked in 1,000 individual seeds that were collected throughout seed developments of beans and radishes. Seeds were associated with a dominant bacterial taxon that represented more than 75% of all reads. The identity of this taxon was highly variable between the plants and within the seeds of the same plant. We identified selection as the main ecological process governing the succession of dominant taxa during seed filling and maturation. In a second step, we evaluated the seedling transmission of seed-borne taxa in 160 individual plants. While the initial bacterial abundance on seeds was not a good predictor of seedling transmission, the identities of the seed-borne taxa modified the phenotypes of seedlings. Overall, this work revealed that individual seeds are colonized by a few bacterial taxa of highly variable identity, which appears to be important for the early stages of plant development. IMPORTANCE Seeds are key components of plant fitness and are central to the sustainability of the agri-food system. Both the seed quality for food consumption and the seed vigor in agricultural settings can be influenced by the seed microbiota. Understanding the ecological processes involved in seed microbiota assembly will inform future practices for promoting the presence of important seed microorganisms for plant health and productivity. Our results highlighted that seeds were associated with one dominant bacterial taxon of variable taxonomic identity. This variety of dominant taxa was due to (i) spatial heterogeneity between and within plants and (ii) primary succession during seed development. According to neutral models, selection was the main driver of microbial community assembly for both plant species.

Analysis of the Diversity of Xylophilus ampelinus Strains Held in CIRM-CFBP Reveals a Strongly Homogenous Species.

Xylophilus ampelinus is the causal agent of blight and canker on grapevine. Only a few data are available on this species implying that the occurrence of this pathogen may be underestimated, and its actual ecological niche may not be understood. Moreover, its genetic diversity is not well known. To improve our knowledge of this species, an analysis of the complete genome sequences available in NCBI was performed. It appeared that several sequences are misidentified. The complete genome sequence of the type strain was obtained and primers designed in order to sequence gyrB and rpoD genes for the strains held in CIRM-CFBP. The genetic barcoding data were obtained for 93 strains, isolated over 35 years and from several geographical origins. The species revealed to be strongly homogenous, displaying nearly identical sequences for all strains. However, the oldest strains of this collection were isolated in 2001 therefore, a new isolation campaign and epidemiological surveys are necessary, along with the obtention of new complete genome sequences for this species.

Seed microbiota revealed by a large-scale meta-analysis including 50 plant species.

Seed microbiota constitutes a primary inoculum for plants that is gaining attention owing to its role for plant health and productivity. Here, we performed a meta-analysis on 63 seed microbiota studies covering 50 plant species to synthesize knowledge on the diversity of this habitat. Seed microbiota are diverse and extremely variable, with taxa richness varying from one to thousands of taxa. Hence, seed microbiota presents a variable (i.e. flexible) microbial fraction but we also identified a stable (i.e. core) fraction across samples. Around 30 bacterial and fungal taxa are present in most plant species and in samples from all over the world. Core taxa, such as Pantoea agglomerans, Pseudomonas viridiflava, P. fluorescens, Cladosporium perangustum and Alternaria sp., are dominant seed taxa. The characterization of the core and flexible seed microbiota provided here will help uncover seed microbiota roles for plant health and design effective microbiome engineering.

Improving Common Bacterial Blight Phenotyping by Using Rub Inoculation and Machine Learning: Cheaper, Better, Faster, Stronger.

Accurate assessment of plant symptoms plays a key role for measuring the impact of pathogens during plant-pathogen interaction. Common bacterial blight caused by Xanthomonas phaseoli pv. phaseoli and X. citri pv. fuscans is a major threat to common bean. The pathogenicity of these bacteria is variable among strains and depends mainly on a type III secretion system and associated type III effectors such as transcription activator-like effectors. Because the impact of a single gene is often small and difficult to detect, a discriminating methodology is required to distinguish the slight phenotype changes induced during the progression of the disease. Here, we compared two different inoculation and symptom assessment methods for their ability to distinguish two tal mutants from their corresponding wild-type strains. Interestingly, rub inoculation of the first leaves combined with symptom assessment by machine learning-based imaging allowed significant distinction between wild-type and mutant strains. By contrast, dip inoculation of first-trifoliate leaves combined with chlorophyll fluorescence imaging did not differentiate the strains. Furthermore, the new method developed here led to the miniaturization of pathogenicity tests and significant time savings.

Complete and Circularized Genome Sequences of Three Xanthomonas Strains Pathogenic on Soybean and Alfalfa.

We report the complete and circularized genome sequences of two strains of Xanthomonas citri pv. glycines causing bacterial pustule on soybean and one strain of Xanthomonas euvesicatoria pv. alfalfae causing bacterial leaf and stem spot on alfalfa. These assemblies provide high-quality material for functional and evolutionary studies of these legume pathogens.

Complete and Circularized Genome Sequences of 17 Xanthomonas Strains Responsible for Common Bacterial Blight of Bean.

We report the complete and circularized genome sequences of 17 strains of Xanthomonas citri pv. fuscans and Xanthomonas phaseoli pv. phaseoli, which cause common bacterial blight of bean. These new assemblies combining PacBio and short-read sequencing methods provide high-quality material for studying the evolution of these plant pathogens.

Common bacterial blight of bean: a model of seed transmission and pathological convergence.

BACKGROUND: Xanthomonas citri pv. fuscans (Xcf) and Xanthomonas phaseoli pv. phaseoli (Xpp) are the causal agents of common bacterial blight of bean (CBB), an important disease worldwide that remains difficult to control. These pathogens belong to distinct species within the Xanthomonas genus and have undergone a dynamic evolutionary history including the horizontal transfer of genes encoding factors probably involved in adaptation to and pathogenicity on common bean. Seed transmission is a key point of the CBB disease cycle, favouring both vertical transmission of the pathogen and worldwide distribution of the disease through global seed trade. TAXONOMY: Kingdom: Bacteria; phylum: Proteobacteria; class: Gammaproteobacteria; order: Lysobacterales (also known as Xanthomonadales); family: Lysobacteraceae (also known as Xanthomonadaceae); genus: Xanthomonas; species: X. citri pv. fuscans and X. phaseoli pv. phaseoli (Xcf-Xpp). HOST RANGE: The main host of Xcf-Xpp is the common bean (Phaseolus vulgaris). Lima bean (Phaseolus lunatus) and members of the Vigna genus (Vigna aconitifolia, Vigna angularis, Vigna mungo, Vigna radiata, and Vigna umbellata) are also natural hosts of Xcf-Xpp. Natural occurrence of Xcf-Xpp has been reported for a handful of other legumes such as Calopogonium sp., Pueraria sp., pea (Pisum sativum), Lablab purpureus, Macroptilium lathyroides, and Strophostyles helvola. There are conflicting reports concerning the natural occurrence of CBB agents on tepary bean (Phaseolus acutifolius) and cowpea (Vigna unguiculata subsp. unguiculata). SYMPTOMS: CBB symptoms occur on all aerial parts of beans, that is, seedlings, leaves, stems, pods, and seeds. Symptoms initially appear as water-soaked spots evolving into necrosis on leaves, pustules on pods, and cankers on twigs. In severe infections, defoliation and wilting may occur. DISTRIBUTION: CBB is distributed worldwide, meaning that it is frequently encountered in most places where bean is cultivated in the Americas, Asia, Africa, and Oceania, except for arid tropical areas. Xcf-Xpp are regulated nonquarantine pathogens in Europe and are listed in the A2 list by the European and Mediterranean Plant Protection Organization (EPPO). GENOME: The genome consists of a single circular chromosome plus one to four extrachromosomal plasmids of various sizes, for a total mean size of 5.27 Mb with 64.7% GC content and an average predicted number of 4,181 coding sequences. DISEASE CONTROL: Management of CBB is based on integrated approaches that comprise measures aimed at avoiding Xcf-Xpp introduction through infected seeds, cultural practices to limit Xcf-Xpp survival between host crops, whenever possible the use of tolerant or resistant bean genotypes, and chemical treatments, mainly restricted to copper compounds. The use of pathogen-free seeds is essential in an effective management strategy and requires appropriate sampling, detection, and identification methods. USEFUL WEBSITES: https://gd.eppo.int/taxon/XANTPH, https://gd.eppo.int/taxon/XANTFF, and http://www.cost.eu/COST_Actions/ca/CA16107.

Novel Virulent Bacteriophages Infecting Mediterranean Isolates of the Plant Pest Xylella fastidiosa and Xanthomonas albilineans.

Xylella fastidiosa (Xf) is a plant pathogen causing significant losses in agriculture worldwide. Originating from America, this bacterium caused recent epidemics in southern Europe and is thus considered an emerging pathogen. As the European regulations do not authorize antibiotic treatment in plants, alternative treatments are urgently needed to control the spread of the pathogen and eventually to cure infected crops. One such alternative is the use of phage therapy, developed more than 100 years ago to cure human dysentery and nowadays adapted to agriculture. The first step towards phage therapy is the isolation of the appropriate bacteriophages. With this goal, we searched for phages able to infect Xf strains that are endemic in the Mediterranean area. However, as Xf is truly a fastidious organism, we chose the phylogenetically closest and relatively fast-growing organism X. albineans as a surrogate host for the isolation step. Our results showed the isolation from various sources and preliminary characterization of several phages active on different Xf strains, namely, from the fastidiosa (Xff), multiplex (Xfm), and pauca (Xfp) subspecies, as well as on X. albilineans. We sequenced their genomes, described their genomic features, and provided a phylogeny analysis that allowed us to propose new taxonomic elements. Among the 14 genomes sequenced, we could identify two new phage species, belonging to two new genera of the Caudoviricetes order, namely, Usmevirus (Podoviridae family) and Subavirus (Siphoviridae family). Interestingly, no specific phages could be isolated from infected plant samples, whereas one was isolated from vector insects captured in a contaminated area, and several from surface and sewage waters from the Marseille area.

Cross-linkage between bacterial taxonomy and gene functions: a study of metagenome-assembled genomes of gut microbiota in adult non-alcoholic fatty liver disease.

BACKGROUND: The reconstruction of metagenome-assembled genomes (MAGs) has emerged as a powerful approach for combining the taxonomic and functional content of microbial populations. AIM: To use this new approach to highlight mechanisms linking gut microbiota to NAFLD severity METHODS: Stool samples were collected from 96 NAFLD patients on the day of liver biopsy. Shotgun DNA sequencing of the gut microbiota was performed on an Illumina HiSeq3000 system. Contigs were binned into MAGs according to their co-abundances and tetranucleotide frequencies using Metabat v.0.32.4. Predicted protein-coding genes were clustered in orthologous groups (OGs) with DIAMOND against the EggNOG v4.5 database. Liver biopsies were read in accordance with the NASH CRN classification. RESULTS: Fifty-four patients had NASH and 44 had significant fibrosis (F ≥ 2). Sequencing of DNA extracted from stools resulted in 13.8 + 3.2 million paired-end reads per sample. Of the 4,000 reconstructed MAGs, 220 in NASH patients, 192 in non-NASH patients, 203 in F ≥ 2 patients and 230 in F0-1 patients had > 70% completeness and < 5% contamination. Within these MAGs, 28 OGs were associated with NASH, 33 with significant fibrosis, and seven with both NASH and significant fibrosis. The study of MAGs showed associations between NAFLD severity and some gut bacteria with microbiota functions related to hydrogen sulfide production, citrate transport, hemicellulose degradation, aldehyde production and vitamin B12 synthesis. CONCLUSION: Using new metagenomics methods, our study unveils potential mechanisms by which certain bacteria from the gut microbiota could protect or contribute to the development of NASH and liver fibrosis in NAFLD.

Temporal dynamics of bacterial communities during seed development and maturation.

Seed microbiota acts as a starting point for the assembly of the plant microbiota and contributes to successful plant establishment. To date, the order and timing of microbial taxa immigration during seed development and maturation remained unknown. We investigated the temporal dynamics of seed bacterial communities in bean and radish. A high phylogenetic turnover was observed for both plant species with few taxa associated with all seed developmental stages. Greater heterogeneity in communities structure within each stage was observed for radish. While, about one-third of radish seed bacterial taxa were detected in buds, flowers and fruits, very few taxa seem to be transmitted by the floral route in bean. In the latter species, bacterial populations belonging to the P. fluorescens species complex were found either in buds, flowers and fruits or in seeds. The relative phylogenetic proximity of these bacterial populations combined with their habitat specificity led us to explore the genetic determinants involved in successful seed transmission in bean. Comparative genomic analyses of representatives bacterial strains revealed dozens of coding sequences specifically associated with seed-transmitted strains. This study provided a first glimpse on processes involved in seed microbiota assembly, which could be used for designing plant-beneficial microbial consortia.

Correction to: Common bean resistance to Xanthomonas is associated with upregulation of the salicylic acid pathway and downregulation of photosynthesis.

An amendment to this paper has been published and can be accessed via the original article.