Transcriptomic investigation of the interaction between a biocontrol yeast, Papiliotrema terrestris strain PT22AV, and the postharvest fungal pathogen Penicillium expansum on apple.

Biocontrol strategies offer a promising alternative to control plant pathogens achieving food safety and security. In this study we apply a RNAseq analysis during interaction between the biocontrol agent (BCA) Papiliotrema terrestris, the pathogen Penicillium expansum, and the host Malus domestica. Analysis of the BCA finds overall 802 upregulated DEGs (differentially expressed genes) when grown in apple tissue, with the majority being involved in nutrients uptake and oxidative stress response. This suggests that these processes are crucial for the BCA to colonize the fruit wounds and outcompete the pathogen. As to P. expansum analysis, 1017 DEGs are upregulated when grown in apple tissue, with the most represented GO categories being transcription, oxidation reduction process, and transmembrane transport. Analysis of the host M. domestica finds a higher number of DEGs in response to the pathogen compared to the BCA, with overexpression of genes involved in host defense signaling pathways in the presence of both of them, and a prevalence of pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) only during interaction with P. expansum. This analysis contributes to advance the knowledge on the molecular mechanisms that underlie biocontrol activity and the tritrophic interaction of the BCA with the pathogen and the host.

First Report of Diplodia quercivora and Neofusicoccum vitifusiforme Associated with Cankers and Necrosis of Holm Oak (Quercus ilex) in Declining Stands in Southern Italy.

The emergence of new plant diseases is an increasingly important concern. Climate change is likely to be among the factors causing most of the emerging diseases endangering forest and tree heritage around the world. Such diseases may be caused by latent pathogens or microorganisms cryptically associated with plants. The shift from a non-pathogenic to a pathogenic stage may depend on physiological alterations of the host, environmental changes, and/or stress factors. In some woods of the Salento Peninsula (Apulia Region, Italy), sudden declines of holm oak plants (Quercus ilex L.) have been observed since 2016. The morphological and molecular characterization of representative fungal isolates associated with cankers and necrosis in declining plants indicated that these isolates belong to the Botryosphaeriaceae family, and the most frequent species were Diplodia corticola and Diplodia quercivora, followed by Neofusicoccum vitifusiforme. In artificially inoculated young holm oak plants, both D. corticola and D. quercivora species produced intense and severe subcortical and leaf margin necrosis. N. vitifusiforme, although less aggressive, induced the same symptoms. Our research, in addition to confirming the involvement of D. corticola in olm oak decline, represents the first report of D. quercivora as a new pathogen of Q. ilex in Italy. Furthermore, to the best of our knowledge, we also found N. vitifusiforme as a new pathogen of Q. ilex.

Alkaline pH, Low Iron Availability, Poor Nitrogen Sources and CWI MAPK Signaling Are Associated with Increased Fusaric Acid Production in Fusarium oxysporum.

Fusaric acid (FA) is one of the first secondary metabolites isolated from phytopathogenic fungi belonging to the genus Fusarium. This molecule exerts a toxic effect on plants, rhizobacteria, fungi and animals, and it plays a crucial role in both plant and animal pathogenesis. In plants, metal chelation by FA is considered one of the possible mechanisms of action. Here, we evaluated the effect of different nitrogen sources, iron content, extracellular pH and cellular signalling pathways on the production of FA siderophores by the pathogen Fusarium oxysporum (Fol). Our results show that the nitrogen source affects iron chelating activity and FA production. Moreover, alkaline pH and iron limitation boost FA production, while acidic pH and iron sufficiency repress it independent of the nitrogen source. FA production is also positively regulated by the cell wall integrity (CWI) mitogen-activated protein kinase (MAPK) pathway and inhibited by the iron homeostasis transcriptional regulator HapX. Collectively, this study demonstrates that factors promoting virulence (i.e., alkaline pH, low iron availability, poor nitrogen sources and CWI MAPK signalling) are also associated with increased FA production in Fol. The obtained new insights on FA biosynthesis regulation can be used to prevent both Fol infection potential and toxin contamination.

Exopolysaccharide from the yeast Papiliotrema terrestris PT22AV for skin wound healing.

INTRODUCTION: Exopolysaccharides (EPSs) are high-value functional biomaterials mainly produced by bacteria and fungi, with nutraceutical, therapeutic and industrial potentials. OBJECTIVES: This study sought to characterize and assess the biological properties of the EPS produced by the yeast Papiliotrema terrestris PT22AV. METHODS: After extracting the yeast's DNA and its molecular identification, the EPS from P. terrestris PT22AV strain was extracted and its physicochemical properties (structural, morphological, monosaccharide composition and molecular weight) were characterized. The EPS's in vitro biological activities and in vivo wound healing potential were also evaluated. RESULTS: The obtained EPS was water-soluble and revealed an average molecular weight (Mw) of 202 kDa. Mannose and glucose with 97% and 3% molar percentages, respectively, constituted the EPS. In vitro antibacterial activity analysis of the extracted EPS exhibited antibacterial activity (>80%) against Escherichia coli, Staphylococcus aureus, and Staphylococcus epidermidis at a concentration of 2 mg/mL. The EPS showed cytocompatibility against the human fibroblast and macrophage cell lines and the animal studies showed a dose-dependent wound healing capacity of the EPS with higher wound closure at 10 mg/mL compared to negative and positive control after 14 days. CONCLUSION: The EPS from P. terrestris PT22AV could serve as a promising source of biocompatible macromolecules with potential for skin wound healing.

Modulation of extracellular Penicillium expansum-driven acidification by Papiliotrema terrestris affects biosynthesis of patulin and has a possible role in biocontrol activity.

The active regulation of extracellular pH is critical for the virulence of fungal pathogens. Penicillium expansum is the causal agent of green-blue mold on stored pome fruits and during its infection process acidifies the host tissues by secreting organic acids. P. expansum is also the main producer of patulin (PAT), a mycotoxin found in pome fruit-based products and that represents a serious health hazard for its potential carcinogenicity. While it is known that PAT biosynthesis in P. expansum is regulated by nutritional factors such as carbon and nitrogen and by the pH, the mechanistic effects of biocontrol on PAT production by P. expansum are not known. In this work, we assessed how optimal and suboptimal concentrations of the biocontrol agent (BCA) Papiliotrema terrestris LS28 affect both extracellular pH and PAT biosynthesis in P. expansum. In wounded apples, the optimal and suboptimal concentrations of the BCA provided almost complete and partial protection from P. expansum infection, respectively, and reduced PAT contamination in both cases. However, the suboptimal concentration of the BCA increased the specific mycotoxigenic activity by P. expansum. In vitro, the rate of PAT biosynthesis was strictly related to the extracellular pH, with the highest amount of PAT detected in the pH range 4-7, whereas only traces were detectable at pH 3. Moreover, both in vitro and in apple wounds the BCA counteracted the extracellular P. expansum-driven acidification maintaining extracellular pH around 4, which is within the pH range that is optimal for PAT biosynthesis. Conversely, in the absence of LS28 the pathogen-driven acidification led to rapidly achieving acidic pH values (<3) that lie outside of the optimal pH range for PAT biosynthesis. Taken together, these results suggest that pH modulation by LS28 is important to counteract the host tissue acidification and, therefore, the virulence of P. expansum. On the other hand, the buffering of P. expansum-driven acidification provided by the BCA increases the specific rate of PAT biosynthesis through the extension of the time interval at which the pH value lies within the optimal range for PAT biosynthesis. Nevertheless, the antagonistic effect provided by the BCA greatly reduced the total amount of PAT.

Biodeterioration of carbographic ribbon: Isolation, identification of causal agents and forensic implications.

This study is part of a comprehensive investigation that was performed in regard to a case of alterations on a carbographic ribbon used in a typewriter that was found and seized by inner security operations of the Arma dei Carabinieri, Italy. Thirty-six coded scripts possessing potentially and criminally liable content were present on the tape; however, only the 6th and 7th scripts exhibited alterations of an uncertain nature. The study included sampling that was performed under sterile conditions of a large surface area of carbographic ribbons. A protocol based on physico-chemical, microbiological, and biomolecular tools was established. Preliminary results revealed the presence of fungal contamination that was primarily located on the inner surface of the 6th and 7th scripts on the black carbographic ribbon. One fungal strain was isolated and identified by universal ITS-PCR primer and rDNA sequencing as Alternaria infectoria strain NIS4. Fungal growth was monitored for 3 weeks in the laboratory under different environmental conditions (temperature, open-closed system, and substrate). The A. infectoria NIS4 strain exhibited the best growth at 28°C under a closed system with RH near saturation. We also noted that the fungal growth was abundant at 15°C. Moreover, this fungus (a potential human pathogen) possessed the ability to colonize the surface of the new carbographic ribbon even when using mineral medium; however, this only occurred in a closed system environment and not in open systems due to rapid desiccation. Under our experimental conditions, the A. infectoria NIS4 strain could degrade gelatin as an organic matter present in trace amounts that are often used as a binder in a carbographic ribbon emulsions. The results revealed that the isolated microorganism was the major biological candidate capable of altering the investigated carbographic ribbon; however, these alterations could only occur under favourable environmental conditions. AIMS: Identify the cause of microbial alterations on a carbographic ribbon in a typewriter used in a hypogean Italian criminal house named "covo." METHODS AND RESULTS: The isolation and identification of biodeteriogens (Alternaria infectoria NIS4) were performed using both culture-dependent and-independent methods, including ITS regions-primed PCR and rDNA techniques. Environmental scanning electron microscopy (ESEM) and optical observations were also performed. Growth tests and biodeterioration simulation tests on carbographic ribbons at the lab scale were performed under different environmental conditions. The A. infectoria NIS4 strain exhibited biodeterioration activity on carbographic ribbons under environmental conditions that were extremely favourable for growth. A high ability to colonize carbographic ribbon surfaces with fast and abundant growth at both 15°C and 28°C under lab-scale conditions at RH near saturation was observed. CONCLUSIONS: In this forensic case study, the ability of the isolated micromycetes A. infectoria NIS4 strain to colonize and induce alterations and degradation in a carbographic ribbon stored under indoor environmental conditions was examined. When favourable conditions change over time, the risk of microbial colonization and the damage produced by the fungal biodeterioration processes on the synthetic material objects has been confirmed. SIGNIFICANCE AND IMPACT OF STUDY: The current study contributes to the knowledge of biodeterioration processes in carbographic ribbon and the responsible agents, and our study provides an example of how environmental microbiology can also aid in forensic studies.

Mycorrhized Wheat Plants and Nitrogen Assimilation in Coexistence and Antagonism with Spontaneous Colonization of Pathogenic and Saprophytic Fungi in a Soil of Low Fertility.

The aim of the work was to study the biological interference of the spontaneous colonization of pathogenic and saprophytic endophytes on the nitrogen assimilation of mycorrhized wheat plants cultivated in soils deficient in N and P. The nitrogen assimilation efficiency of mycorrhized plants was determined by measuring the activities of nitrate reductase assimilatory and glutamine synthetase enzymes and free amino acid patterns. Mycorrhizal plants at two different sites showed an assimilative activity of nitrate and ammonium approximately 30% greater than control plants. This activity was associated with significant increases in the amino acids Arg, Glu Gln and Orn in the roots where those amino acids are part of the inorganic nitrogen assimilation of mycorrhizal fungi. The nutrient supply of mycorrhizal fungi at the root guaranteed the increased growth of the plant that was about 40% greater in fresh weight and 25% greater in productive yield than the controls. To better understand the biological interaction between plant and fungus, microbiological screening was carried out to identify colonies of radicular endophytic fungi. Fourteen fungal strains belonging to nine different species were classified. Among pathogenic fungi, the genus Fusarium was present in all the examined roots with different frequencies, depending on the site and the fungal population present in the roots, providing useful clues regarding the principle of spatial conflict and fungal spread within the root system.

Complete genome sequence of the biocontrol yeast Papiliotrema terrestris strain LS28.

Papiliotrema terrestris strain LS28 is a biocontrol agent selected for its antagonistic activity against several plant pathogens both in the field and postharvest. The availability of a genome sequencing sets the foundation for the identification of the genetic mechanisms of its antagonistic activity. The genome size is 21.29 Mbp with a G+C content of 58.65%, and genome annotation predicts 8,626 protein-encoding genes. Phylogenetic analysis based on whole-genome data confirms that P. terrestris is a Tremellomycetes more closely related to Papiliotrema flavescens than Papiliotrema laurentii.

Molecular Tools for the Yeast Papiliotrema terrestris LS28 and Identification of Yap1 as a Transcription Factor Involved in Biocontrol Activity.

Fungal attacks on stored fruit and vegetables are responsible for losses of products. There is an active research field to develop alternative strategies for postharvest disease management, and the use of biocontrol agents represents a promising approach. Understanding the molecular bases of the biocontrol activity of these agents is crucial to potentiate their effectiveness. The yeast Papiliotrema terrestris is a biocontrol agent against postharvest pathogens. Phenotypic studies suggest that it exerts its antagonistic activity through competition for nutrients and space, which relies on its resistance to oxidative and other cellular stresses. In this study, we developed tools for genetic manipulation in P. terrestris to perform targeted gene replacement and functional complementation of the transcription factors Yap1 and Rim101. In vitro phenotypic analyses revealed a conserved role of Yap1 and Rim101 in broad resistance to oxidative stress and alkaline pH sensing, respectively. In vivo analyses revealed that P. terrestris yap1Δ and rim101Δ mutants display decreased ability to colonize wounded fruit compared to that of the parental wild-type (WT) strain; the yap1Δ mutant also displays reduced biocontrol activity against the postharvest pathogens Penicillium expansum and Monilinia fructigena, indicating an important role for resistance to oxidative stress in timely wound colonization and biocontrol activity of P. terrestris In conclusion, the availability of molecular tools developed in the present study provides a foundation to elucidate the genetic mechanisms underlying biocontrol activity of P. terrestris, with the goal of enhancing this activity for the practical use of P. terrestris in pest management programs based on biological and integrated control.IMPORTANCE The use of fungicides represents the most effective and widely used strategy for controlling postharvest diseases. However, their extensive use has raised several concerns, such as the emergence of plant pathogens' resistance as well as the health risks associated with the persistence of chemical residues in fruit, in vegetables, and in the environment. These factors have brought attention to alternative methods for controlling postharvest diseases, such as the utilization of biocontrol agents. In the present study, we developed genetic resources to investigate at the molecular level the mechanisms involved in the biocontrol activity of Papiliotrema terrestris, a basidiomycete yeast that is an effective biocontrol agent against widespread fungal pathogens, including Penicillium expansum, the etiological agent of blue mold disease of pome fruits. A deeper understanding of how postharvest biocontrol agents operate is the basic requirement to promote the utilization of biological (and integrated) control for the reduction of chemical fungicides.

A bacterial endophyte exploits chemotropism of a fungal pathogen for plant colonization.

Soil-inhabiting fungal pathogens use chemical signals released by roots to direct hyphal growth towards the host plant. Whether other soil microorganisms exploit this capacity for their own benefit is currently unknown. Here we show that the endophytic rhizobacterium Rahnella aquatilis locates hyphae of the root-infecting fungal pathogen Fusarium oxysporum through pH-mediated chemotaxis and uses them as highways to efficiently access and colonize plant roots. Secretion of gluconic acid (GlcA) by R. aquatilis in the rhizosphere leads to acidification and counteracts F. oxysporum-induced alkalinisation, a known virulence mechanism, thereby preventing fungal infection. Genetic abrogation or biochemical inhibition of GlcA-mediated acidification abolished biocontrol activity of R. aquatilis and restored fungal infection. These findings reveal a new way by which bacterial endophytes hijack hyphae of a fungal pathogen in the soil to gain preferential access to plant roots, thereby protecting the host from infection.

Complete Genome Sequence of the Biocontrol Agent Yeast Rhodotorula kratochvilovae Strain LS11.

Rhodotorula kratochvilovae strain LS11 is a biocontrol agent (BCA) selected for its antagonistic activity against several plant pathogens both in the field and postharvest. Genome assembly includes 62 contigs for a total of 22.56 Mbp and a G+C content of 66.6%. Genome annotation predicts 7,642 protein-encoding genes.