Combination of Pseudo-LC-NMR and HRMS/MS-Based Molecular Networking for the Rapid Identification of Antimicrobial Metabolites From Fusarium petroliphilum

An endophytic fungal strain isolated from a seagrass endemic to the Mediterranean Sea (Posidonia oceanica) was studied in order to identify its antimicrobial constituents and further characterize the composition of its metabolome. It was identified as Fusarium petroliphilum by in-depth phylogenetic analyses. The ethyl acetate extract of that strain exhibited antimicrobial activities and an ability to inhibit quorum sensing of Staphylococcus aureus. To perform this study with a few tens of mg of extract, an innovative one-step generic strategy was devised. On one side, the extract was analyzed by UHPLC-HRMS/MS molecular networking for dereplication. On the other side, semi-preparative HPLC using a similar gradient profile was used for a single-step high-resolution fractionation. All fractions were systematically profiled by 1H-NMR. The data were assembled into a 2D contour map, which we call “pseudo-LC-NMR,” and combined with those of UHPLC-HRMS/MS. This further highlighted the connection within structurally related compounds, facilitated data interpretation, and provided an unbiased quantitative profiling of the main extract constituents. This innovative strategy led to an unambiguous characterization of all major specialized metabolites of that extract and to the localization of its bioactive compounds. Altogether, this approach identified 22 compounds, 13 of them being new natural products and six being inhibitors of the quorum sensing mechanism of S. aureus and Pseudomonas aeruginosa. Minor analogues were also identified by annotation propagation through the corresponding HRMS/MS molecular network, which enabled a consistent annotation of 27 additional metabolites. This approach was designed to be generic and applicable to natural extracts of the same polarity range.

First report of Fusarium petroliphilum causing fruit rot of spaghetti squash in California

Spaghetti squash (Cucurbita pepo L. subsp. pepo) is a yellow-skinned squash that forms translucent spaghetti-like strands when cooked. California leads the nation in total squash production, the majority of which is grown in the San Joaquin Valley. In October of 2019, severe fruit rot of C. pepo L. subsp. Pepo (C. pepo) was observed in fruit harvested from seven cultivated fields in San Joaquin County, California. Infected fields incurred up to 30% postharvest losses. At harvest, fruit appeared healthy. After ten days in a shaded storage shed, scattered buff to tan ringed lesions extending into the flesh of infected fruits were observed. Lesions had visible sporodochia at the center that were variable in size and continued to expand in storage. Tissue (∼1 mm3) from the lesion margins of symptomatic fruit (n=8) was surface sterilized in 75 % ethanol for 1 min then 0.6% sodium hypochlorite for a minute, and aseptically transferred to half strength acidified potato dextrose agar (0.5 APDA) and incubated at 22–25 °C. Fungal colonies which grew from the pieces were light yellow, with mycelium that was flat and mucoid. Sporodochial conidia were falcate and robust with 3 to 5 septa and measured from 44.2 to 51.6 × 4.6 to 5.9 μm (average 46.3 × 5.2 μm). Aerial conidia were profuse, borne on short monophialides, ovoid to reniform, and measured 5.1 to 12.6 μm × 3.2 to 5.6 μm (average 4.2 × 6.1 μm). DNA extracted from two isolates, was amplified with primers ITS1/ITS4, and EF1-728F/EF1-986R using PCR, to obtain sequences from the internal transcribed spacer (ITS) (White 1990), and elongation factor 1α (EF1α) (Carbone et al. 1999) genetic regions. Sequences from both isolates were identical. Sequences from isolate MVAP50001827, GenBank nos. MZ081401 (ITS) and MZ102267 (EF-1α) matched 100% to sequences of representative isolates of Fusarium petroliphilum (Q.T. Chen & X.H. Fu; Short et al., 2013, MB 802539) from Cucurbita species, MF535516 (ITS) and MF580776 (EF-1α) respectively (González, V. et al. 2018). To fulfill Koch’s postulates, conidia were harvested from a culture of isolate MVAP50001827 and grown for 7 days on 0.5 APDA at room temperature (22–25 °C). A 3-cc syringe with a 25-gauge needle was used to wound and inject 200 μl of 1 × 106 conidia ml–1 into three equally spaced points 1 mm deep into the rind of C. pepo fruit (n=4). C. pepo fruit (n=4) serving as negative controls were treated similarly with 200 μl of sterile deionized water. Fruit was incubated in a growth chamber at 27 °C under 12-h diurnal cycle lighting conditions. Ten days post inoculation, lesions densely covered with white sporodochia had expanded to 7 cm diameter and 5 cm deep on average (average fruit size 31×11 cm). Twenty days post inoculation, severe fruit rot was observed. F. petroliphilum did not grow from the controls, and was successfully reisolated from the symptomatic inoculated fruits, completing Koch’s postulates. Seeds inside the inoculated fruits were completely colonized and covered in conidia. Twenty-five seeds from the source seed lot was tested for F. petroliphilum by surface sterilizing and plating onto 0.5 APDA. No F. petroliphilum grew from tested seed. Postharvest fungal diseases can affect profitability of winter squash, which is often held in storage, and sold when market prices are optimal. To our knowledge, this is the first report of Fusarium petroliphilum infecting spaghetti squash (Cucurbita pepo L. subsp. pepo) in California.

Eye Infections Caused by Filamentous Fungi: Spectrum and Antifungal Susceptibility of the Prevailing Agents in Germany

Fungal eye infections can lead to loss of vision and blindness. The disease is most prevalent in the tropics, although case numbers in moderate climates are increasing as well. This study aimed to determine the dominating filamentous fungi causing eye infections in Germany and their antifungal susceptibility profiles in order to improve treatment, including cases with unidentified pathogenic fungi. As such, we studied all filamentous fungi isolated from the eye or associated materials that were sent to the NRZMyk between 2014 and 2020. All strains were molecularly identified and antifungal susceptibility testing according to the EUCAST protocol was performed for common species. In total, 242 strains of 66 species were received. Fusarium was the dominating genus, followed by Aspergillus, Purpureocillium, Alternaria, and Scedosporium. The most prevalent species in eye samples were Fusarium petroliphilum, F. keratoplasticum, and F. solani of the Fusarium solani species complex. The spectrum of species comprises less susceptible taxa for amphotericin B, natamycin, and azoles, including voriconazole. Natamycin is effective for most species but not for Aspergillus flavus or Purpureocillium spp. Some strains of F. solani show MICs higher than 16 mg/L. Our data underline the importance of species identification for correct treatment.

CRISPR/Cas9-Mediated Gene Replacement in the Fungal Keratitis Pathogen Fusarium solani var. petroliphilum

Fungal keratitis (FK) is a site-threatening infection of the cornea associated with ocular trauma and contact lens wear. Members of the Fusarium solani species complex (FSSC) are predominant agents of FK worldwide, but genes that support their corneal virulence are poorly understood. As a means to bolster genetic analysis in FSSC pathogens, we sought to employ a CRISPR/Cas9 system in an FK isolate identified as Fusarium petroliphilum. Briefly, this approach involves the introduction of two components into fungal protoplasts: (1) A purified Cas9 protein complexed with guide RNAs that will direct the ribonuclease to cut on either side of the gene of interest, and (2) a “repair template” comprised of a hygromycin resistance cassette flanked by 40 bp of homology outside of the Cas9 cuts. In this way, Cas9-induced double strand breaks should potentiate double homologous replacement of the repair template at the desired locus. We targeted a putative ura3 ortholog since its deletion would result in an easily discernable uracil auxotrophy. Indeed, 10% of hygromycin-resistant transformants displayed the auxotrophic phenotype, all of which harbored the expected ura3 gene deletion. By contrast, none of the transformants from the repair template control (i.e., no Cas9) displayed the auxotrophic phenotype, indicating that Cas9 cutting was indeed required to promote homologous integration. Taken together, these data demonstrate that the in vitro Cas9 system is an easy and efficient approach for reverse genetics in FSSC organisms, including clinical isolates, which should enhance virulence research in these important but understudied ocular pathogens.