Daniela Bulgari

Can waste serve as a source of known or novel molecules for plant disease management? This review explores this question, which is closely linked to the necessity of mitigating the environmental impact of agriculture, specifically in terms of waste generation and greenhouse gas emissions. Furthermore, it addresses the increasing demand for safer, environmentally friendly alternatives to ensure sustainable plant production and protection. To satisfy these needs, research efforts over the past few decades have primarily focused on biocontrol strategies, with the valorization of waste being addressed only recently. Different approaches have been applied for extracting bioactive molecules from waste, ranging from green chemistry to microbial fermentation. We discuss secondary metabolites, elicitors, and biosurfactants obtained chemically and from fermentation. Microorganisms growing on waste can produce biomass or spores [bioactive ingredients of plant-protection products (PPPs)] and facilitate the extraction of active compounds. Although the production of spores on waste material is now well-established on an industrial scale, the extraction of biologicals from waste and/or their production through fermentation are still at the experimental stage. The growing knowledge on the bioactivity of agri-food waste–derived molecules against phytopathogens is paving the way for the development of new PPPs suitable for integrated pest management. The waste-based PPPs pipeline described here shows strong potential to generate both established and novel, regulation-compliant compounds without sacrificing disease-control efficacy.

Background: Solid state fermentation (SSF) is a biotechnological process in which microorganisms grow in the absence or near-absence of flow-free water. Recently, this process has re-gained attention due to the possibility to use raw materials and by-products as SSF substrates to produce value-added products. In this work, the volatilome produced by Streptomyces sp. DEF603 during solid-state fermentation (fVOCs) on defatted wheat bran has been characterised to identify molecules active against fungal phytopathogens. Results: Untargeted HS–SPME–GC–MS, combined with statistical analysis, highlighted a clear separation ( i ) between inoculated and non-inoculated samples and ( ii ) among inoculated samples collected at different time points. Variable Importance in Projection (VIP) statistics identified 21 potential volatile markers that significantly contributed to distinguishing different variables. Interestingly, the relative amount of 12 fVOCs increased during fermentation, including known antimicrobial compounds such as 2-pentanone, dimethyl trisulfide, and 2-methyl-1-butanol. Accordingly, the antifungal activity of fVOCs against Botrytis cinerea , Fusarium culmorum, and Alternaria alternata increased over time, reaching 98.5% inhibition of mycelium growth and complete inhibition of spore germination in Alternaria alternata . Conclusion: The results support the use of defatted wheat bran, a common agri-food waste, as a substrate for producing VOCs to be applied as novel, environmentally friendly biofumigants. Graphical Created in BioRender. Bulgari, D. (2026) https://BioRender.com/yk3z5s3

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.

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.

The continuous increase in biogas production poses the need for innovative applications for its by-products. Solid-state fermentation (SSF) has regained attention in the development of several products because of the possibility to use low-cost and easily available substrates, such as organic wastes. SSF represents a valuable process for agricultural digestate valorization in terms of enzyme production. In the present study, cellulase and esterase were produced by Trichoderma asperellum R on a digestate-based substrate in SSF, with esterase as the highest obtained activity. After assessing the effect of light on it, the esterase production in SSF was optimized using response surface methodology. The influence of substrate composition, temperature and humidity on the enzyme production was evaluated on two sets of data generated based on digestate concentration (50% and 70% w/w). The statistical analyses revealed that these parameters affected esterase production only when Trichoderma asperellum grew on substrate containing 50% w/w of digestate. The best esterase activity (264.6 mU/mg total protein) was achieved with the following optimized SSF parameters: 50% digestate, 50% fruits, 10% sawdust, 30 °C. The current finding of esterase production on digestate-based substrates makes the SSF method presented here a sustainable and completely circular technology.

SUMMARY: The Trichoderma genus includes soil-inhabiting fungi that provide important ecological services in their interaction with plants and other fungi. They are exploited for biocontrol. A collection of Trichoderma isolates from the Sardinia island (a biodiversity hotspot) had been previously characterized. Here we started a characterization of the viral components associated to 113 selected Trichoderma isolates, representatives of the collection. We carried out NGS sequencing of ribosome depleted total RNA following a bioinformatic pipeline that detects virus RNA-dependent RNA polymerases (RdRP) and other conserved virus protein sequences. This pipeline detected 17 viral RdRPs. Two of them correspond to viruses already detected in other regions of the world. The remaining 15 represent isolates of new virus species: surprisingly, eight of them are from new negative stranded RNA viruses, which for the first time are reported in the genus Trichoderma . Among them is a cogu-like virus, very closely related to plant-infecting viruses. Regarding the positive strand viruses, it is noticeable the presence of an ormycovirus belonging to a recently characterized group of bi-segmented ssRNA genome viruses with still uncertain phylogenetic assignment. Finally, for the first time we report a bipartite mononegavirales-infecting fungi: the proteins encoded by the second genomic RNA were used to re-evaluate a number of viruses in the Penicillimonavirus and Plasmopamonavirus genera, here shown to be bipartite and to encode a conserved polypeptide having structural conservation with the nucleocapsid (NC) domain of members of the Rabhdoviridae. IMPORTANCE Trichoderma is a genus of fungi of great biotechnological impact in multiple industrial fields. The possibility to investigate a diverse collection of Trichoderma isolates allowed us to characterize both double-stranded and single-stranded virus genomes belonging to three of the major phyla that constitute the RNA viral kingdom, thus further increasing the taxa of viruses infecting this genus. To our knowledge here we report for the first time negative-stranded RNA viruses infecting Trichoderma spp. and through in silico structural analysis a new conserved domain of nucleocapsids common among some mymonavirids. Obtaining such a library of mycoviruses could be the basis for further development of targeted virus-induced gene silencing or gene editing (VIGS/VIGE) tools; in addition, the many biotechnological applications of this fungus, will require to assess the qualitative (commercial) stability of strains, linked to positive or negative effects caused by mycovirus infections.

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.