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.

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.

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.

Enhancing phytoextraction of Cd by combining poplar (clone "I-214") with Pseudomonas fluorescens and microbial consortia.

The plant-microorganism combinations may contribute to the success of phytoextraction of heavy metal-polluted soil. The purpose of this study was to investigate the effects of cadmium (Cd) soil concentration on selected physiological parameters of the poplar clone "I-214" inoculated at root level with a strain (BT4) of Pseudomonas fluorescens and a commercial product based on microbial consortia (Micosat F Fito®). Plants were subjected to Cd treatment of 40 mg kg(-1) in greenhouse. The effects of plant-microbe interactions, plant growth, leaf physiology, and microbial activity were periodically monitored. Metal concentration and translocation factors in plant tissues proved enhanced Cd uptake in roots of plants inoculated with P. fluorescens and transfer to shoots in plants inoculated with Micosat F Fito®, suggesting a promising strategy for using microbes in support of Cd uptake. Plant-microbe integration increased total removal of Cd, without interfering with plant growth, while improving the photosynthetic capacity. Two major mechanisms of metal phytoextraction inducted by microbial inoculation may be suggested: improved Cd accumulation in roots inoculated with P. fluorescens, implying phytostabilization prospective and high Cd transfer to shoots of inoculated plants, outlining enhanced metal translocation.

Antifungal cyclic lipopeptides from Bacillus amyloliquefaciens strain BO5A.

A bioassay-guided fractionation of Bacillus amyloliquefaciens strain BO5A afforded the isolation of two new cyclic lipopeptides (1 and 2) as the major lipid constituents (>60%) of the CHCl3-MeOH (2:1) extract. The chemical structures of the isolated metabolites were elucidated by spectroscopic methods, including 1D and 2D NMR spectroscopy, mass spectrometry (MS), secondary ion mass spectrometry (MS1, MS2), and chemical degradation. The compounds are members of the surfactins family and are based on a heptapeptide chain composed by Glu-Val-Leu-Val-Asp-Leu-Leu. Its N-terminal end is N-acylated by an (R)-3-hydroxy fatty acid with linear alkyl chains of 16:0 and 15:0 (1 and 2, respectively). The 3-hydroxyl group closes a 25-membered lactone ring with the carboxylic group of the C-terminal amino acid. The isolated compounds were tested for their inhibitory activity against the four pathogenic fungi Fusarium oxysporum, Aspergillus niger, Botrytis cinerea, and Penicillium italicum and the biocontrol fungus Trichoderma harzianum. Compound 2 displayed activity against all tested pathogens.

Antifungal lipopeptides from Bacillus amyloliquefaciens strain BO7.

Three new lipopeptides (1-3) were isolated from the organic extract of Bacillus amyloliquefaciens strain (BO7). These compounds represented the major constituents (>60%) of the total cell lipids extractable with CHCl(3)/MeOH (2:1). Elucidation of their chemical structure was carried out by spectroscopic analyses, including 1D and 2D NMR spectroscopy, mass spectrometry (MS), and secondary ion mass spectrometry (MS/MS), along with chemical degradation. The compounds are members of the surfactins family and are based on the heptapeptide Glu-Leu-Leu-Ala-Asp-Leu-Leu, N-acylated to the N-terminal by an (R)-3-hydroxy fatty acid with linear alkyl chains from 16:0 to 18:0 (1-3, respectively). An ester bond between the 3-hydroxyl group of the fatty acid and the carboxylic group of the C-terminal amino acid closes a 13-membered lactone ring. The bacterial lipopeptides, particularly compound 3, displayed strong and dose-dependent antifungal activity against the plant pathogenic fungus Fusarium oxysporum.

Depression and recovery of daily life autonomy in patients with stroke receiving home rehabilitative treatment.

BACKGROUND: The object of this study was to evaluate the prevalence of post stroke depression and its possible role as a predictive negative factor in patients receiving home rehabilitation treatment. METHODS: We analyzed 103 patients with stroke by correlating comorbidities, clinical and blood test parameters and characteristics of the lesion with depression class identified according to the Hamilton scale and the outcome of the rehabilitation program. RESULTS: A significant association between hypertension and post-stroke severe depression emerged in the female patients. CONCLUSIONS: Since the literature offers conflicting data, our results may contribute a stimulus for further studies. KEYWORDS: Home care; Rehabilitation; Stroke; Depression; Hypertension.

Occurrence of mycotoxin in Farro samples from southern Italy.

The occurrence of nine mycotoxins and of contamination by pre- and postharvest fungal pathogens of cereals was investigated in samples of stored Triticum monococcum L., Triticum dicoccon Schrank (emmer), and Triticum spelta L. (spelt). In Italy, all three species are collectively referred to as farro. The samples examined were harvested in summer 2000 from eight different sites in southern Italy. Conventional fluorimetric and diode array-based high-performance liquid chromatography (HPLC) analyses and HPLC-mass spectrometry analyses were used to identify fumonisin B1 in five samples (up to 70.00 microg/ kg), ochratoxin A in seven samples (up to 4.07 microg/kg), and beauvericin in three samples (up to 4.44 mg/kg). Enniatin B was detected in one sample (30.00 microg/kg), but no zearalenone or fusaproliferin was found. Deoxynivalenol and aflatoxins were not evaluated. The potentially mycotoxigenic fungal species detected were Alternaria alternata, Fusarium proliferatum, Fusarium tricinctum, Penicillium verrucosum, and Penicillium chrysogenum. This is the first report of the natural occurrence of mycotoxins in farro samples.