Long-term monitoring of the hypogeal Etruscan Tomba degli Scudi, Tarquinia, Italy. Early detection of black spots, investigation of fungal community, and evaluation of their biodeterioration potential.

AIMS: Hypogeal environments with cultural heritage interest pose a real challenge for their preservation and conservation. The ancient Etruscan Necropolis of Tarquinia, Italy, consists of 200 tombs decorated with extraordinary mural paintings, of great artistic and historical value. Since the beginning of the restoration campaign in 2016, a regular microbiological survey has been performed in the Tomba degli Scudi. The aim of this study was to investigate the nature of an expansion of black spots on the pictorial layers recently observed. METHODS AND RESULTS: To determine the origin of the black spots in the atrium chamber of the Tomba degli Scudi, the fungal community was sampled using various techniques: cellulose discs, swabs, and nylon membranes and investigated by a multi-analytical approach. The obtained results suggest that the identified fungal strains (e.g. Gliomastix murorum and Pseudogymnoascus pannorum) are common to many subterranean environments around the world, such as Lascaux cave. CONCLUSIONS: The continuous and long-term monitoring made it possible to detect alterations at an early stage and assess the harmfulness of different fungal strains. This work is a demonstration of the effectiveness of prevention and monitoring actions within these fragile and valuable environments.

The Zymoseptoria tritici Avirulence Factor AvrStb6 Accumulates in Hyphae Close to Stomata and Triggers a Wheat Defense Response Hindering Fungal Penetration.

Zymoseptoria tritici, the causal agent of Septoria tritici blotch, is one of Europe's most damaging wheat pathogens, causing significant economic losses. Genetic resistance is a common strategy to control the disease, Stb6 being a resistance gene used for more than 100 years in Europe. This study investigates the molecular mechanisms underlying Stb6-mediated resistance. Utilizing confocal microscopy imaging, we determined that Z. tritici epiphytic hyphae mainly accumulate the corresponding avirulence factor AvrStb6 in close proximity to stomata. Consequently, the progression of AvrStb6-expressing avirulent strains is hampered during penetration. The fungal growth inhibition co-occurs with a transcriptional reprogramming in wheat characterized by an induction of immune responses, genes involved in stomatal regulation, and cell wall-related genes. Overall, we shed light on the gene-for-gene resistance mechanisms in the wheat-Z. tritici pathosystem at the cytological and transcriptomic level, and our results highlight that stomatal penetration is a critical process for pathogenicity and resistance. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Analysis of Italian isolates of Pantoea stewartii subsp. stewartii and development of a real-time PCR-based diagnostic method.

Pantoea stewartii subsp. stewartii (Pss) causes Stewart's vascular wilt of maize, and it is responsible for serious crop losses. Pss is indigenous to North America and spreads with maize seeds. The presence of Pss has been notified in Italy since 2015. The risk assessment of the entry of Pss in the EU from the United States through seed trade is in the order of magnitude of hundred introductions per year. Several molecular or serological tests were developed for the detection of Pss and used as official analysis for the certification of commercial seeds. However, some of these tests lack adequate specificity, not allowing to correctly discriminate Pss from P. stewartii subsp. indologenes (Psi). Psi is occasionally present in maize seeds and is avirulent for maize. In this study, several Italian isolates of Pss recovered in 2015 and 2018 have been characterized by molecular, biochemical, and pathogenicity tests; moreover, their genomes have been assembled through MinION and Illumina-sequencing procedures. Genomic analysis reveals multiple introgression events. Exploiting these results, a new primer combination has been defined and verified by real-time PCR, allowing the development of a specific molecular test able to detect the presence of Pss down to the concentration of 103 CFU/ml in spiked samples of maize seed extracts. Due to the high analytical sensitivity and specificity achieved with this test, the detection of Pss has been improved disentangling the inconclusive results in Pss maize seed diagnosis, overcoming its misidentification in place of Psi. Altogether, this test addresses the critical issue associated with maize seeds imported from regions where Stewart's disease is endemic.

The Neolithic site "La Marmotta": DNA metabarcoding to identify the microbial deterioration of waterlogged archeological wood.

Introduction: The evaluation of biological degradation of waterlogged archeological wood is crucial to choose the conservative and protective treatments to be applied to the wooden material. The waterlogged environmental conditions are characterized by oxygen scarcity, only allowing the growth of adapted microbes capable to degrade the organic wooden material, mainly erosion bacteria and soft-rot fungi. In this work, we characterized and evaluated the biodegradation state and the microbial communities of wooden fragments preserved in storage tanks. These were preserved by waterlogging within the Neolithic village "La Marmotta," currently found under the Bracciano Lake (Lazio, Italy). Methods: The waterlogged wood samples were first identified taxonomically with an optical microscope, also allowing an evaluation of their preservation state. The microbial community was then evaluated through the sequencing of Internal Transcribed Spacer sequences for fungi and 16S for bacteria with the Oxford Nanopore Technologies (ONT) MinION platform. Results: The identified microbial community appears to be consistent with the waterlogged samples, as many bacteria attributable to the erosion of wood and ligninolytic fungi have been sequenced. Discussion: The reported results highlight the first use of targeted metabarcoding by ONT applied to study the biodeterioration of waterlogged archeological wood.

A highly polymorphic effector protein promotes fungal virulence through suppression of plant-associated Actinobacteria.

Plant pathogens secrete effector proteins to support host colonization through a wide range of molecular mechanisms, while plant immune systems evolved receptors to recognize effectors or their activities to mount immune responses to halt pathogens. Importantly, plants do not act as single organisms, but rather as holobionts that actively shape their microbiota as a determinant of health. The soil-borne fungal pathogen Verticillium dahliae was recently demonstrated to exploit the VdAve1 effector to manipulate the host microbiota to promote vascular wilt disease in the absence of the corresponding immune receptor Ve1. We identify a multiallelic V. dahliae gene displaying c. 65% sequence similarity to VdAve1, named VdAve1-like (VdAve1L), which shows extreme sequence variation, including alleles that encode dysfunctional proteins, indicative of selection pressure to overcome host recognition. We show that the orphan cell surface receptor Ve2, encoded at the Ve locus, does not recognize VdAve1L. Additionally, we demonstrate that the full-length variant VdAve1L2 possesses antimicrobial activity, like VdAve1, yet with a divergent activity spectrum, that is exploited by V. dahliae to mediate tomato colonization through the direct suppression of antagonistic Actinobacteria in the host microbiota. Our findings open up strategies for more targeted biocontrol against microbial plant pathogens.

Nanopore Hybrid Assembly of Biscogniauxia mediterranea Isolated from Quercus cerris Affected by Charcoal Disease in an Endangered Coastal Wood.

Biscogniauxia mediterranea is the causal agent of charcoal disease, affecting oak decline under the trigger of various biotic and abiotic factors, including climate change. Here, we report the genome assembly of an Italian B. mediterranea strain obtained using hybrid sequencing technologies combining long and short reads.

Evolutionary Trajectories toward Ceftazidime-Avibactam Resistance in Klebsiella pneumoniae Clinical Isolates.

From January 2019 to April 2020, 32 KPC-producing, ceftazidime-avibactam (CZA)-resistant Klebsiella pneumoniae strains were isolated in a university hospital in Rome, Italy. These strains belonged to the sequence type 512 (ST512), ST101, and ST307 high-risk clones. Nine different CZA-resistant KPC-3 protein variants were identified, five of them never previously reported (KPC-66 to KPC-70). Among the nine, KPC-31, KPC-39, KPC-49, KPC-66, KP-68, KPC-69, and KPC-70 showed amino acid substitutions, insertions, and deletions in the Ω loop of the protein. KPC-29 has a duplication, while the novel KPC-67 has a triplication, of the KDD triplet in the 270-loop, a secondary loop of the KPC-3 protein. Genomics performed on contemporary resistant and susceptible clones underlined that these novel mutations emerged in blaKPC-3 genes located on conserved plasmids: in ST512, all blaKPC-3 mutant genes were located in pKpQIL plasmids, while the three novel blaKPC-3 mutants identified in ST101 were on FIIk-FIA(HI1)-R plasmids. Selection also promoted multiplication of the carbapenemase gene copy number by transposition, recombination, and fusion of resident plasmids. When expressed in Escherichia coli recipient cells cloned in the high-copy-number pTOPO vector, the Ω loop mutated variants showed the CZA-resistant phenotype associated with susceptibility to carbapenems, while KPC variants with insertions in the 270-loop showed residual activity on carbapenems. The investigation of CZA resistance mechanisms offered the unique opportunity to study vertical, horizontal, and oblique evolutionary trajectories of K. pneumoniae high-risk clones.

Draft Genome Sequence of a New Fusarium Isolate Belonging to Fusarium tricinctum Species Complex Collected From Hazelnut in Central Italy.

In summer 2019, during a survey on the health status of a hazelnut orchard located in the Tuscia area (the province of Viterbo, Latium, Italy), nuts showing symptoms, such as brown-grayish spots at the bottom of the nuts progressing upward to the apex, and necrotic patches on the bracts and, sometimes, on the petioles, were found and collected for further studies. This syndrome is associated with the nut gray necrosis (NGN), whose main causal agent is Fusarium lateritium. Aiming to increase knowledge about this fungal pathogen, the whole-genome sequencing of a strain isolated from symptomatic hazelnut was performed using long Nanopore reads technology in combination with the higher precision of the Illumina reads, generating a high-quality genome assembly. The following phylogenetic and comparative genomics analysis suggested that this isolate is caused by the F. tricinctum species complex rather than F. lateritium one, as initially hypothesized. Thus, this study demonstrates that different Fusarium species can infect Corylus avellana producing the same symptomatology. In addition, it sheds light onto the genetic features of the pathogen in subject, clarifying facets about its biology, epidemiology, infection mechanisms, and host spectrum, with the future objective to develop specific and efficient control strategies.

Repetitive Elements Contribute to the Diversity and Evolution of Centromeres in the Fungal Genus Verticillium.

Centromeres are chromosomal regions that are crucial for chromosome segregation during mitosis and meiosis, and failed centromere formation can contribute to chromosomal anomalies. Despite this conserved function, centromeres differ significantly between and even within species. Thus far, systematic studies into the organization and evolution of fungal centromeres remain scarce. In this study, we identified the centromeres in each of the 10 species of the fungal genus Verticillium and characterized their organization and evolution. Chromatin immunoprecipitation of the centromere-specific histone CenH3 (ChIP-seq) and chromatin conformation capture (Hi-C) followed by high-throughput sequencing identified eight conserved, large (∼150-kb), AT-, and repeat-rich regional centromeres that are embedded in heterochromatin in the plant pathogen Verticillium dahliae Using Hi-C, we similarly identified repeat-rich centromeres in the other Verticillium species. Strikingly, a single degenerated long terminal repeat (LTR) retrotransposon is strongly associated with centromeric regions in some but not all Verticillium species. Extensive chromosomal rearrangements occurred during Verticillium evolution, of which some could be linked to centromeres, suggesting that centromeres contributed to chromosomal evolution. The size and organization of centromeres differ considerably between species, and centromere size was found to correlate with the genome-wide repeat content. Overall, our study highlights the contribution of repetitive elements to the diversity and rapid evolution of centromeres within the fungal genus VerticilliumIMPORTANCE The genus Verticillium contains 10 species of plant-associated fungi, some of which are notorious pathogens. Verticillium species evolved by frequent chromosomal rearrangements that contribute to genome plasticity. Centromeres are instrumental for separation of chromosomes during mitosis and meiosis, and failed centromere functionality can lead to chromosomal anomalies. Here, we used a combination of experimental techniques to identify and characterize centromeres in each of the Verticillium species. Intriguingly, we could strongly associate a single repetitive element to the centromeres of some of the Verticillium species. The presence of this element in the centromeres coincides with increased centromere sizes and genome-wide repeat expansions. Collectively, our findings signify a role of repetitive elements in the function, organization, and rapid evolution of centromeres in a set of closely related fungal species.

A Multispecies Cluster of VIM-1 Carbapenemase-Producing Enterobacterales Linked by a Novel, Highly Conjugative, and Broad-Host-Range IncA Plasmid Forebodes the Reemergence of VIM-1.

In this study, we investigated VIM-1-producing Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Citrobacter freundii, and Enterobacter cloacae strains, isolated in 2019 during a period of active surveillance of carbapenem-resistant Enterobacterales in a large university hospital in Italy. VIM-1-producing strains colonized the gut of patients, with up to three different VIM-1-positive bacterial species isolated from a single rectal swab, but also caused bloodstream infection in one colonized patient. In the multispecies cluster, blaVIM-1 was identified in a 5-gene cassette class 1 integron, associated with several genetic determinants, including the blaSHV-12, qnrS1, and mph(A) genes, located on a highly conjugative and broad-host-range IncA plasmid. The characteristics and origin of this IncA plasmid were studied.

Dynamic virulence-related regions of the plant pathogenic fungus Verticillium dahliae display enhanced sequence conservation.

Plant pathogens continuously evolve to evade host immune responses. During host colonization, many fungal pathogens secrete effectors to perturb such responses, but these in turn may become recognized by host immune receptors. To facilitate the evolution of effector repertoires, such as the elimination of recognized effectors, effector genes often reside in genomic regions that display increased plasticity, a phenomenon that is captured in the two-speed genome hypothesis. The genome of the vascular wilt fungus Verticillium dahliae displays regions with extensive presence/absence polymorphisms, so-called lineage-specific regions, that are enriched in in planta-induced putative effector genes. As expected, comparative genomics reveals differential degrees of sequence divergence between lineage-specific regions and the core genome. Unanticipated, lineage-specific regions display markedly higher sequence conservation in coding as well as noncoding regions than the core genome. We provide evidence that disqualifies horizontal transfer to explain the observed sequence conservation and conclude that sequence divergence occurs at a slower pace in lineage-specific regions of the V. dahliae genome. We hypothesize that differences in chromatin organisation may explain lower nucleotide substitution rates in the plastic, lineage-specific regions of V. dahliae.

Long-Read Annotation: Automated Eukaryotic Genome Annotation Based on Long-Read cDNA Sequencing.

Single-molecule full-length complementary DNA (cDNA) sequencing can aid genome annotation by revealing transcript structure and alternative splice forms, yet current annotation pipelines do not incorporate such information. Here we present long-read annotation (LoReAn) software, an automated annotation pipeline utilizing short- and long-read cDNA sequencing, protein evidence, and ab initio prediction to generate accurate genome annotations. Based on annotations of two fungal genomes (Verticillium dahliae and Plicaturopsis crispa) and two plant genomes (Arabidopsis [Arabidopsis thaliana] and Oryza sativa), we show that LoReAn outperforms popular annotation pipelines by integrating single-molecule cDNA-sequencing data generated from either the Pacific Biosciences or MinION sequencing platforms, correctly predicting gene structure, and capturing genes missed by other annotation pipelines.

Arabidopsis thaliana ambient temperature responsive lncRNAs.

BACKGROUND: Long non-coding RNAs (lncRNAs) have emerged as new class of regulatory molecules in animals where they regulate gene expression at transcriptional and post-transcriptional level. Recent studies also identified lncRNAs in plant genomes, revealing a new level of transcriptional complexity in plants. Thousands of lncRNAs have been predicted in the Arabidopsis thaliana genome, but only a few have been studied in depth. RESULTS: Here we report the identification of Arabidopsis lncRNAs that are expressed during the vegetative stage of development in either the shoot apical meristem or in leaves. We found that hundreds of lncRNAs are expressed in these tissues, of which 50 show differential expression upon an increase in ambient temperature. One of these lncRNAs, FLINC, is down-regulated at higher ambient temperature and affects ambient temperature-mediated flowering in Arabidopsis. CONCLUSION: A number of ambient temperature responsive lncRNAs were identified with potential roles in the regulation of temperature-dependent developmental changes, such as the transition from the vegetative to the reproductive (flowering) phase. The challenge for the future is to characterize the biological function and molecular mode of action of the large number of ambient temperature-regulated lncRNAs that have been identified in this study.

Gapless Genome Assembly of the Potato and Tomato Early Blight Pathogen Alternaria solani.

The Alternaria genus consists of saprophytic fungi as well as plant-pathogenic species that have significant economic impact. To date, the genomes of multiple Alternaria species have been sequenced. These studies have yielded valuable data for molecular studies on Alternaria fungi. However, most of the current Alternaria genome assemblies are highly fragmented, thereby hampering the identification of genes that are involved in causing disease. Here, we report a gapless genome assembly of A. solani, the causal agent of early blight in tomato and potato. The genome assembly is a significant step toward a better understanding of pathogenicity of A. solani.

Evolution within the fungal genus Verticillium is characterized by chromosomal rearrangement and gene loss.

The fungal genus Verticillium contains ten species, some of which are notorious plant pathogens causing vascular wilt diseases in host plants, while others are known as saprophytes and opportunistic plant pathogens. Whereas the genome of V. dahliae, the most notorious plant pathogen of the genus, has been well characterized, evolution and speciation of other members of the genus received little attention thus far. Here, we sequenced the genomes of the nine haploid Verticillium spp. to study evolutionary trajectories of their divergence from a last common ancestor. Frequent occurrence of chromosomal rearrangement and gene family loss was identified. In addition to ∼11 000 genes that are shared at least between two species, only 200-600 species-specific genes occur. Intriguingly, these species-specific genes show different features than the shared genes.

A gapless genome sequence of the fungus Botrytis cinerea.

Following earlier incomplete and fragmented versions of a genome sequence for the grey mould Botrytis cinerea, a gapless, near-finished genome sequence for B. cinerea strain B05.10 is reported. The assembly comprised 18 chromosomes and was confirmed by an optical map and a genetic map based on approximately 75 000 single nucleotide polymorphism (SNP) markers. All chromosomes contained fully assembled centromeric regions, and 10 chromosomes had telomeres on both ends. The genetic map consisted of 4153 cM and a comparison of the genetic distances with the physical distances identified 40 recombination hotspots. The linkage map also identified two mutations, located in the previously described genes Bos1 and BcsdhB, that conferred resistance to the fungicides boscalid and iprodione. The genome was predicted to encode 11 701 proteins. RNAseq data from >20 different samples were used to validate and improve gene models. Manual curation of chromosome 1 revealed interesting features, such as the occurrence of a dicistronic transcript and fully overlapping genes in opposite orientations, as well as many spliced antisense transcripts. Manual curation also revealed that the untranslated regions (UTRs) of genes can be complex and long, with many UTRs exceeding lengths of 1 kb and possessing multiple introns. Community annotation is in progress.

Comparative genomics of Beauveria bassiana: uncovering signatures of virulence against mosquitoes.

BACKGROUND: Entomopathogenic fungi such as Beauveria bassiana are promising biological agents for control of malaria mosquitoes. Indeed, infection with B. bassiana reduces the lifespan of mosquitoes in the laboratory and in the field. Natural isolates of B. bassiana show up to 10-fold differences in virulence between the most and the least virulent isolate. In this study, we sequenced the genomes of five isolates representing the extremes of low/high virulence and three RNA libraries, and applied a genome comparison approach to uncover genetic mechanisms underpinning virulence. RESULTS: A high-quality, near-complete genome assembly was achieved for the highly virulent isolate Bb8028, which was compared to the assemblies of the four other isolates. Whole genome analysis showed a high level of genetic diversity between the five isolates (2.85-16.8 SNPs/kb), which grouped into two distinct phylogenetic clusters. Mating type gene analysis revealed the presence of either the MAT1-1-1 or the MAT1-2-1 gene. Moreover, a putative new MAT gene (MAT1-2-8) was detected in the MAT1-2 locus. Comparative genome analysis revealed that Bb8028 contains 163 genes exclusive for this isolate. These unique genes have a tendency to cluster in the genome and to be often located near the telomeres. Among the genes unique to Bb8028 are a Non-Ribosomal Peptide Synthetase (NRPS) secondary metabolite gene cluster, a polyketide synthase (PKS) gene, and five genes with homology to bacterial toxins. A survey of candidate virulence genes for B. bassiana is presented. CONCLUSIONS: Our results indicate several genes and molecular processes that may underpin virulence towards mosquitoes. Thus, the genome sequences of five isolates of B. bassiana provide a better understanding of the natural variation in virulence and will offer a major resource for future research on this important biological control agent.

Transposons passively and actively contribute to evolution of the two-speed genome of a fungal pathogen.

Genomic plasticity enables adaptation to changing environments, which is especially relevant for pathogens that engage in "arms races" with their hosts. In many pathogens, genes mediating virulence cluster in highly variable, transposon-rich, physically distinct genomic compartments. However, understanding of the evolution of these compartments, and the role of transposons therein, remains limited. Here, we show that transposons are the major driving force for adaptive genome evolution in the fungal plant pathogen Verticillium dahliae We show that highly variable lineage-specific (LS) regions evolved by genomic rearrangements that are mediated by erroneous double-strand repair, often utilizing transposons. We furthermore show that recent genetic duplications are enhanced in LS regions, against an older episode of duplication events. Finally, LS regions are enriched in active transposons, which contribute to local genome plasticity. Thus, we provide evidence for genome shaping by transposons, both in an active and passive manner, which impacts the evolution of pathogen virulence.

RNA-sequencing of Cercospora beticola DMI-sensitive and -resistant isolates after treatment with tetraconazole identifies common and contrasting pathway induction.

Cercospora beticola causes Cercospora leaf spot of sugar beet. Cercospora leaf spot management measures often include application of the sterol demethylation inhibitor (DMI) class of fungicides. The reliance on DMIs and the consequent selection pressures imposed by their widespread use has led to the emergence of resistance in C. beticola populations. Insight into the molecular basis of tetraconazole resistance may lead to molecular tools to identify DMI-resistant strains for fungicide resistance management programs. Previous work has shown that expression of the gene encoding the DMI target enzyme (CYP51) is generally higher and inducible in DMI-resistant C. beticola field strains. In this study, we extended the molecular basis of DMI resistance in this pathosystem by profiling the transcriptional response of two C. beticola strains contrasting for resistance to tetraconazole. A majority of the genes in the ergosterol biosynthesis pathway were induced to similar levels in both strains with the exception of CbCyp51, which was induced several-fold higher in the DMI-resistant strain. In contrast, a secondary metabolite gene cluster was induced in the resistance strain, but repressed in the sensitive strain. Genes encoding proteins with various cell membrane fortification processes were induced in the resistance strain. Site-directed and ectopic mutants of candidate DMI-resistance genes all resulted in significantly higher EC50 values than the wild-type strain, suggesting that the cell wall and/or membrane modified as a result of the transformation process increased resistance to tetraconazole. Taken together, this study identifies important cell membrane components and provides insight into the molecular events underlying DMI resistance in C. beticola.