Search

Apple proliferation (AP), caused by 'Candidatus Phytoplasma mali', is one of the most important phytoplasma diseases in Europe. Because of its economic importance, efforts are focused on the study of 'Ca. Phytoplasma mali' characterization, molecular mechanisms of disease induction, transmission by insect vectors, and plant defense responses. However, very few studies have investigated the effect of 'Ca. Phytoplasma mali' on microbial diversity. In this work, we describe for the first time the endophytic bacterial community associated with healthy and AP phytoplasma-infected apple roots, suggesting the hypothesis that 'Ca. Phytoplasma mali' influences microbial diversity in apple plants. En- dophytic bacterial diversity associated with healthy and 'Candidatus Phytoplasma mali'-infected apple (Malus domestica, Borkh.) trees was analyzed by cultivation-dependent and -independent methods. Sequence analysis of 16S rRNA gene showed the presence of Proteobacteria, Acidobacteria, Bacteroidetes, Actinobacteria, Chlamydiae, and Firmicutes. Bacterial group distribution differed in healthy and diseased plants. Library analyses underscored 24 and 17 operational taxonomic units (OTUs) in healthy and infected roots, respectively, with a dominance of the Betaproteobacteria group. Pseudomonas fluorescens was prevalent in healthy roots, while Burkholderia sp. in the infected ones. The combination of culturing methods and cloning analysis allowed an accurate description of the endophytic community in apple trees, revealing that bacterial richness in healthy plants is higher than that observed in those infected by 'Ca. Phytoplasma mali'. Evidences from the present study reinforce the idea that uncultivable bacterial pathogens can modify the composition of endophytic bacterial communities associated with infected plants. [ABSTRACT FROM AUTHOR]

An abstract on the study relating to the characterization of the benefits of endophytic bacteria from a healthy but Candidatus phytoplasma-infected mali plant is presented.

Agro-industrial residues have transitions from being an environmental problem to being a cost-effective source of biopolymers and value-added chemicals. However, the efficient extraction of the desired products from these residues requires pretreatments. Fungal biorefinery is a fascinating approach for the biotransformation of raw materials into multiple products in a single batch. In this study, the ability of Trichoderma asperellum R to convert fruit scrap and green waste into value-added chemicals was tested in solid-state and in nonsterile conditions. A solid-state fermentation protocol for a tray bioreactor was developed using spawn as the inoculum for nonsterile substrates. T. asperellum R drove the fermentation of both substrates, shaping the metabolites that were enriched in the secondary plant metabolites. Strain R showed cellulase activity only when inoculated on fruit scraps, resulting in increased amounts of polysaccharides in the crude extract. This extract was also enriched in vanillic acid and limonoid, which are intriguing compounds due to the increasing interest in their potential as biological nitrification inhibitors or food additives. Finally, trimethoxybenzaldehyde, an interesting chemical building block, was identified in the extracts of the Trichoderma -guided fermentation. The overall results showed that the application of T. asperellum R has potential as a driver to facilitate the extraction of bioactive substances from nonsterile recalcitrant substrates.

Fusarium verticillioides is an important plant pathogen in maize and other cereals that is seldom detected as the cause of human fusariosis. Here, we provide the analysis of the available diversity of F. verticillioides sequenced worldwide and report the first two genome assemblies and annotations (including mitochondrial DNA) of Fusarium verticillioides from clinical settings. Fusarium verticillioides 05-0160 (IUM05-0160) and Fusarium verticillioides 09-1037 (IUM09-1037) strains were obtained from the bone marrow and blood of two immunocompromised patients, respectively. The phylogenomic analysis confirmed the species identity of our two strains. Comparative genomic analyses among the reannotated F. verticillioides genomes ( n = 46) did not lead to the identification of unique genes specific to the clinical samples. Two subgroups in the F. verticillioides clade were also identified and confirmed by a mitochondrial diversity study. Clinical strains ( n = 4) were positioned in the multigene phylogenetic tree without any correlation between the host and the tree branches, grouping with plant-derived strains. To investigate the existence of a potential fitness advantage of our two clinical strains, we compared demethylase inhibitor fungicides susceptibility against the reference Fusarium verticillioides 7600, showing, on average, lower susceptibility to agricultural and medical-used antifungals. A significant reduction in susceptibility was observed for itraconazole and tetraconazole, which might be explained by structural changes in CYP51A and CYP51C sequences. By providing the first two annotated genomes of F. verticillioides from clinical settings comprehensive of their mitogenomes, this study can serve as a base for exploring the fitness and adaptation capacities of Fusarium verticillioides infecting different kingdoms.

Melia azedarach L. is a Meliaceae that has shown important insecticidal activities. However, few researchers have extensively studied the toxicology of aqueous extracts of M. azedarach (MAE). Therefore, the main objective of this study was to characterize the phyto-eco-toxicological profile of MAE. First, a botanical and phytochemical characterization of MAE was performed using a histological, and metabolomic multi-analytical approach. Second, the toxicological effects on pollinating insects (Apis mellifera ligustica) and soil collembola (Folsomia candida) were evaluated. In addition, acute toxicity was evaluated in zebrafish (Danio rerio) to assess effects on aquatic fauna, and toxicity was determined in human neuroblastoma (SH-SY5Y) and fibroblast (FB-21) cell models. Finally, phytotoxic effects on germination of Cucumis sativus L., Brassica rapa L. and Sorghum vulgare L. were considered. Metabolomic analyses revealed the presence of not only limonoids but also numerous alkaloids, flavonoids and terpenoids in MAE. Histological analyses allowed us to better localize the areas of leaf deposition of the identified secondary metabolites. Regarding the ecotoxicological data, no significant toxicity was observed in bees and collembola at all doses tested. In contrast, severe cardiac abnormalities were observed in zebrafish embryos at concentrations as low as 25 μg/mL. In addition, MAE showed toxicity at 1.6 μg/mL and 6.25 μg/mL in FB-21 and SH-SY5Y cells, respectively. Finally, MAE inhibited seed germination with inhibitory concentrations starting from 5.50 μg/mL in B. rapa, 20 μg/mL in S. vulgare, and 31 μg/mL in C. sativus. Although M. azedarach extracts are considered valuable natural insecticides, their ecological impact cannot be underestimated. Even the use of an environmentally friendly solvent (an aqueous solution), for the first time, is not without side effects. Therefore, the data collected in this study show the importance of evaluating the dosages, modes of administration and production methods of M. azedarach phytoextracts in agricultural settings., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Genetics in Drosophila have revealed the role of neuropeptides in development and behavior. However, determining when and where neuropeptides are released has been challenging. Furthermore, the cell biology underlying neuropeptide release has largely been unexplored. Thus, it has not been possible to determine whether changes in neuropeptide immunofluorescence reflect traffic and/or release, and in neurons where such changes are not detectable, conclusions about neuropeptide release have been formulated based on the assumption that electrical and Ca 2+ recordings are accurate and quantitative predictors of release. Recently, the advent of optical detection of neuropeptides tagged with fluorescent proteins and fluorogen-activating proteins (FAPs) has made it feasible to directly image vesicle traffic and exocytosis that mediates neuropeptide release in peripheral synapses and in the brain. In fact, these approaches have led to the discovery of unexpected insights concerning neuropeptide release. Here procedures are presented for optimizing fluorescence imaging of neuropeptides tagged with green fluorescent protein or a FAP., (© 2024 Cold Spring Harbor Laboratory Press.)

Electrophysiological studies of synaptic function do not robustly report release of neuropeptides and neurotrophins. These limitations have been overcome with the presynaptic expression of optical release reporters based on green fluorescent protein and fluorogen-activating protein. Here we describe how to image neuropeptide release in Drosophila at the neuromuscular junction and in the adult brain., (© 2024 Cold Spring Harbor Laboratory Press.)

Ciborinia camelliae Kohn is the causal agent of camellia flower blight. The fungus infects only the flowers of camellias. C. camelliae isolates obtained from symptomatic samples, collected in 13 different localities worldwide, were characterized by Multi-Locus Sequence Typing (MLST) using the following: (i) a nuclear ribosomal DNA internal transcribed spacer; (ii) subunit 2 of β-tubulin (β-TUB II), (iii) elongation factor 1-α (EF1α); and (iv) glycerol-3-phosphate dehydrogenase (GPDH). The variability of the strains was assessed using a universally primed-polymerase chain reaction (UP-PCR) with six universal primers. Gene sequence comparison showed high similarity among all the European strains and highlighted the diversity of the New Zealand and Chinese representative strains. The profiles obtained by UP-PCR confirmed the significant diversity of extra-European strains and identified subgroups within the European population. The presence of shared genetic profiles obtained from strains isolated in different countries (New Zealand and France) suggests the movement of strains from one location to another, which is probably due to the exchange of infected plant material. Moreover, our study shows the overall high intraspecific variability of C. camelliae , which is likely due to the sexual reproduction of the fungus, suggesting the risk of emergence of new pathotypes adapting to novel camellia varieties.

Trichoderma genus includes soil-inhabiting fungi that provide important ecosystem services in their interaction with plants and other fungi, as well as biocontrol of fungal plant diseases. A collection of Trichoderma isolates from Sardinia has been previously characterized, but here we selected 113 isolates, representatives of the collection, and characterized their viral components. We carried out high-throughput sequencing of ribosome-depleted total RNA following a bioinformatics pipeline that detects virus-derived RNA-directed RNA polymerases (RdRps) and other conserved viral protein sequences. This pipeline detected seventeen viral RdRps with two of them corresponding to viruses already detected in other regions of the world and the remaining fifteen representing isolates of new putative virus species. Surprisingly, eight of them are from new negative-sense RNA viruses, a first in the genus Trichoderma . Among them is a cogu-like virus, closely related to plant-infecting viruses. Regarding the positive-sense viruses, we report the presence of an 'ormycovirus' belonging to a recently characterized group of bisegmented single-stranded RNA viruses with uncertain phylogenetic assignment. Finally, for the first time, we report a bisegmented member of Mononegavirales which infects fungi. The proteins encoded by the second genomic RNA of this virus were used to re-evaluate several viruses in the Penicillimonavirus and Plasmopamonavirus genera, here shown to be bisegmented and encoding a conserved polypeptide that has structural conservation with the nucleocapsid domain of rhabdoviruses., Competing Interests: None declared., (© The Author(s) 2023. Published by Oxford University Press.)

Neuronal dense-core vesicles (DCVs) contain neuropeptides and much larger proteins that affect synaptic growth and plasticity. Rather than using full collapse exocytosis that commonly mediates peptide hormone release by endocrine cells, DCVs at the Drosophila neuromuscular junction release their contents via fusion pores formed by kiss-and-run exocytosis. Here, we used fluorogen-activating protein (FAP) imaging to reveal the permeability range of synaptic DCV fusion pores and then show that this constraint is circumvented by cAMP-induced extra fusions with dilating pores that result in DCV emptying. These Ca2+-independent full fusions require PKA-R2, a PKA phosphorylation site on Complexin and the acute presynaptic function of Rugose, the homolog of mammalian neurobeachin, a PKA-R2 anchor implicated in learning and autism. Therefore, localized Ca2+-independent cAMP signaling opens dilating fusion pores to release large cargoes that cannot pass through the narrower fusion pores that mediate spontaneous and activity-dependent neuropeptide release. These results imply that the fusion pore is a variable filter that differentially sets the composition of proteins released at the synapse by independent exocytosis triggers responsible for routine peptidergic transmission (Ca2+) and synaptic development (cAMP)., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

In this study, the agricultural digestate from anaerobic biogas production mixed with food wastes was used as a substrate to grow Trichoderma reesei RUT-C30 and Trichoderma atroviride Ta13 in solid-state fermentation (SSF) and produce high-value bioproducts, such as bioactive molecules to be used as ingredients for biostimulants. The Trichoderma spp. reached their maximum growth after 6 and 3 SSF days, respectively. Both Trichoderma species were able to produce cellulase, esterase, and citric and malic acids, while T. atroviride also produced gibberellins and oxylipins as shown by ultraperformance liquid chromatography with quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) profiling. Experimental evaluation of germination parameters highlighted a significant promotion of tomato seed germination and root elongation induced by T. atroviride crude extracts from SSF. This study suggests an innovative sustainable use of the whole digestate mixed with agro-food waste as a valuable substrate in fungal biorefineries. Here, it has been applied to produce plant growth-promoting fungi and bioactive molecules for sustainable agriculture.

Microbial-based products are a promising alternative to agrochemicals in sustainable agriculture. However, little is known about their impact on human health even if some of them, i.e., Bacillus and Paenibacillus species, have been increasingly implicated in different human diseases. In this study, 18 bacteria were isolated from 2 commercial biostimulants, and they were genotypically and phenotypically characterized to highlight specific virulence properties. Some isolated bacteria were identified as belonging to the genus Bacillus by BLAST and RDP analyses, a genus in-depth studied for plant growth-promoting ability. Moreover, 16S rRNA phylogenetic analysis showed that seven isolates grouped with Bacillus species while two and four clustered, respectively, with Neobacillus and Peribacillus. Unusually, bacterial strains belonging to Franconibacter and Stenotrophomonas were isolated from biostimulants. Although Bacillus species are generally considered nonpathogenic, most of the species have shown to swim, swarm, and produced biofilms, that can be related to bacterial virulence. The evaluation of toxins encoding genes revealed that five isolates had the potential ability to produce the enterotoxin T. In conclusion, the pathogenic potential of microorganisms included in commercial products should be deeply verified, in our opinion. The approach proposed in this study could help in this crucial step., (© 2022. The Author(s).)

This study aimed at valorizing digestate through Trichoderma spp. solid-state fermentation (SSF) to produce a potentially ameliorated fertilizer combined with fungal biomass as a value-added bioproduct. Plant-growth-promoting Trichoderma atroviride Ta13, T. reesei RUT-C30, T. asperellum R, and T. harzianum T-22 were tested on different SSF substrates: whole digestate (WD), digestate dried up with wood sawdust (SSF1), and digestate enriched with food waste and dried up with wood sawdust (SSF2). The fungal biomass was quantified by using a qPCR assay. The growth of the four Trichoderma spp. was only observed on the SSF2 substrate. The highest quantity of mycelium was produced by T. reesei RUT-30 (689.80 ± 80.53 mg/g substrate), followed by T. atroviride Ta13, and T. asperellum R (584.24 ± 13.36 and 444.79 ± 91.02 mg/g substrate). The germination of Lepidium sativum seeds was evaluated in order to assess the phytoxicity of the Trichoderma -enriched substrate. The treatments with 7.5% SSF2-R, 3.75% SSF2-T-22, and 1.8% SSF2-Ta13 equally enhanced the root elongation in comparison to the non-fermented SSF-2. This study demonstrated that digestate, mixed with agro-food waste, was able to support the cultivation of Trichoderma spp., paving the way to the valorization of fermented digestate as a proper biofertilizer.

Quinoin is a type 1 ribosome-inactivating protein (RIP) we previously isolated from the seeds of pseudocereal quinoa ( Chenopodium quinoa ) and is known as a functional food for its beneficial effects on human health. As the presence of RIPs in edible plants could be potentially risky, here we further characterised biochemically the protein (complete amino acid sequence, homologies/differences with other RIPs and three-dimensional homology modeling) and explored its possible defensive role against pathogens. Quinoin consists of 254 amino acid residues, without cysteinyl residues. As demonstrated by similarities and homology modeling, quinoin preserves the amino acid residues of the active site (Tyr75, Tyr122, Glu177, Arg180, Phe181 and Trp206; quinoin numbering) and the RIP-fold characteristic of RIPs. The polypeptide chain of quinoin contains two N-glycosylation sites at Asn115 and Asp231, the second of which appears to be linked to sugars. Moreover, by comparative MALDI-TOF tryptic peptide mapping, two differently glycosylated forms of quinoin, named pre-quinoin-1 and pre-quinoin-2 (~0.11 mg/100 g and ~0.85 mg/100 g of seeds, respectively) were characterised. Finally, quinoin possesses: (i) strong antiviral activity, both in vitro and in vivo towards Tobacco Necrosis Virus (TNV); (ii) a growth inhibition effect on the bacterial pathogens of plants; and (iii) a slight antifungal effect against two Cryphonectria parasitica strains.