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 softrot 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

Single-molecule real-time sequencing combined with optical mapping yields completely finished fungal genome

Next-generation sequencing (NGS) technologies have increased the scalability, speed, and resolution of genomic sequencing and, thus, have revolutionized genomic studies. However, eukaryotic genome sequencing initiatives typically yield considerably fragmented genome assemblies. Here, we assessed various state-of-the-art sequencing and assembly strategies in order to produce a contiguous and complete eukaryotic genome assembly, focusing on the filamentous fungus Verticillium dahliae. Compared with Illumina-based assemblies of the V. dahliae genome, hybrid assemblies that also include PacBio- generated long reads establish superior contiguity. Intriguingly, provided that sufficient sequence depth is reached, assemblies solely based on PacBio reads outperform hybrid assemblies and even result in fully assembled chromosomes. Furthermore, the addition of optical map data allowed us to produce a gapless and complete V. dahliae genome assembly of the expected eight chromosomes from telomere to telomere. Consequently, we can now study genomic regions that were previously not assembled or poorly assembled, including regions that are populated by repetitive sequences, such as transposons, allowing us to fully appreciate an organism’s biological complexity. Our data show that a combination of PacBio-generated long reads and optical mapping can be used to generate complete and gapless assemblies of fungal genomes. IMPORTANCE Studying whole-genome sequences has become an important aspect of biological research. The advent of nextgeneration sequencing (NGS) technologies has nowadays brought genomic science within reach of most research laboratories, including those that study nonmodel organisms. However, most genome sequencing initiatives typically yield (highly) fragmented genome assemblies. Nevertheless, considerable relevant information related to genome structure and evolution is likely hidden in those nonassembled regions. Here, we investigated a diverse set of strategies to obtain gapless genome assemblies, using the genome of a typical ascomycete fungus as the template. Eventually, we were able to show that a combination of PacBiogenerated long reads and optical mapping yields a gapless telomere-to-telomere genome assembly, allowing in-depth genome sanalyses to facilitate functional studies into an organism’s biology

Чинники та фактори зростання продуктивності праці на підприємстві

In this study, we functionally analyzed the gene family encoding necrosis- and ethylene-inducing-like proteins (NLPs) of the vascular wilt pathogen Verticillium dahliae. We show that the composition of the NLP gene family varies little among V. dahliae isolates. The cytotoxic activity of NLP family members of a tomato pathogenic V. dahliae strain was determined, demonstrating that only two of the seven NLPs induced plant cell death. The genes encoding these cytotoxic NLPs were found to be induced in V. dahliae upon colonization of tomato. Interestingly, targeted deletion of either of the two genes in V. dahliae significantly compromised virulence on tomato as well as on Arabidopsis plants, whereas deletion of only one of the two genes affected virulence on N. benthamiana. This could be attributed to differential induction of the two NLP genes in V. dahliae upon N. benthamiana colonization, revealing that the in planta induction of NLP genes varies between plant hosts. Intriguingly, one of the NLP genes appears to also affect vegetative growth and conidiospore production, as the corresponding deletion strain produced significantly less conidiospores and developed extensive aerial mycelium. In conclusion, we demonstrate that the expanded V. dahliae NLP family shows functional diversification, not only revealing differential cytotoxicity between family members, but also that the cytotoxic NLPs play a role in vegetative growth and asexual reproduction in addition to their contribution to virulence

Fine mapping of two major QTLs conferring resistance to powdery mildew in tomato

Tomato (Solanum lycopersicum) is the most cultivated crop in the Solanaceae family and is a host for Oidium neolycopersici, the cause agent of powdery mildew disease. In wild species of tomato, genes (Ol-1–Ol-6) for monogenic resistance have been identified. Moreover, three quantitative resistance loci (QRLs), namely Ol-qtl1, Ol-qtl2 and Ol-qtl3, have been mapped in Solanum neorickii G1.1601. In this work, we developed several advanced backcross populations in order to fine-map these Ol-qtls. Resistant lines harboring individual Ol-qtl were produced and used in recombinant screening. Ten recombinants were identified in chromosomal regions carrying Ol-qtl1s. The recombinant individuals were used to produce recombinant families (RFs). By screening these RFs with molecular markers and testing them with O. neolycopersici, we could localize Ol-qtl1 in a region of about 2.3 Mbp on the long arm of chromosome 6 and Ol-qtl2 in a region of 2.5 Mbp on the short arm of chromosome 12. On the other hand, the presence of Ol-qtl3 locus was not confirmed in this study. The fine-mapping results further demonstrated the co-localization between Ol-qtls and genes for monogenic resistance; the Ol-qtl1 interval contains the Ol-1 gene and the Ol-qtl2 interval harbors the Lv gene that confers monogenic resistance to Leveillula taurica, another species of tomato powdery mildew

Force transmissibility and vibration power flow behaviour of inerter-based vibration isolators

This paper investigates the dynamics and performance of inerter-based vibration isolators. Force / displacement transmissibility and vibration power flow are obtained to evaluate the isolation performance. Both force and motion excitations are considered. It is demonstrated that the use of inerters can enhance vibration isolation performance by enlarging the frequency band of effective vibration isolation. It is found that adding inerters can introduce anti-resonances in the frequency-response curves and in the curves of the force and displacement transmissibility such that vibration transmission can be suppressed at interested excitation frequencies. It is found that the introduction of inerters enhances inertial coupling and thus have a large influence on the dynamic behaviour at high frequencies. It is shown that force and displacement transmissibility increases with the excitation frequency and tends to an asymptotic value as the excitation frequency increases. In the high-frequency range, it was shown that adding inerters can result in a lower level of input power. These findings provide a better understanding of the effects of introducing inerters to vibration isolation and demonstrate the performance benefits of inerter-based vibration isolators

Genetics and molecular mechanisms of resistance to powdery mildews in tomato (Solanum lycopersicum) and its wild relatives

Powdery mildews (PMs) cause disease in a wide range of plant species including important crops. Taking tomato as an example, here we review findings on the genetic basis and mechanisms of plant resistance to PMs. First, we present a summary of our research on tomato resistance to two PM species, with the focus on Oidium neolycopersici. We discuss the genetics of resistance to this pathogen in tomato. Then, we compare different forms of resistance mediated by different resistance genes based on molecular and cytological data. Also, we provide a comparison between these resistance genes in tomato with those in barley, Arabidopsis and wheat, in order to present a model for the genetic basis of resistance to PMs in plants. We try to accommodate these resistance mechanisms in the current model of plant innate immunity. At the end we discuss possibilities to translate these findings to practical approaches in breeding for resistance to PMs in crops

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

PRGdb: a bioinformatics platform for plant resistance gene analysis

PRGdb is a web accessible open-source (http://www.prgdb.org) database that represents the first bioinformatic resource providing a comprehensive overview of resistance genes (R-genes) in plants. PRGdb holds more than 16 000 known and putative R-genes belonging to 192 plant species challenged by 115 different pathogens and linked with useful biological information. The complete database includes a set of 73 manually curated reference R-genes, 6308 putative R-genes collected from NCBI and 10463 computationally predicted putative R-genes. Thanks to a user-friendly interface, data can be examined using different query tools. A home-made prediction pipeline called Disease Resistance Analysis and Gene Orthology (DRAGO), based on reference R-gene sequence data, was developed to search for plant resistance genes in public datasets such as Unigene and Genbank. New putative R-gene classes containing unknown domain combinations were discovered and characterized. The development of the PRG platform represents an important starting point to conduct various experimental tasks. The inferred cross-link between genomic and phenotypic information allows access to a large body of information to find answers to several biological questions. The database structure also permits easy integration with other data types and opens up prospects for future implementations

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