First report of Fusarium keratoplasticum causing strawberry root rot in Sinaloa, Mexico.

Strawberry (Fragaria × ananassa) is a fruit of economic importance for Mexico, occupying the third place in world production, with an approximate production of 861, 337 t (SIAP, 2021). In January 2021, in Culiacan, Sinaloa, Mexico (24°46'46″N; 107°27'04″ W), wilting symptoms (stunted growth, leaf yellowing and wilting, necrosis in vascular bundles, root rot and wilting) were observed on commercial strawberry crops, with an incidence of 5 to 10 %. Tissue samples from symptomatic roots were cut and disinfected with alcohol, sodium hypochlorite and sterile water, to later be plated on potato dextrose agar (PDA). Fifteen monosporic isolates were obtained by single-spore culturing (Hansen and Smith, 1932). Typical Fusarium spp. colonies were obtained from all root samples. On PDA the colonies were abundant with white aerial mycelium, hyphae were branched and septate, and light-yellow pigmentation was observed in the center of old cultures (Leslie and Summerell 2006). From 10-day-old cultures grown on carnation leaf agar medium, macroconidia were slightly curved, showing three marked septa, broad central cells, slightly tapered apices, foot-shaped basal cells and measured 59.6 - 73.4 (x̄ = 68.7) x 10.4 - 14.9 µm (x̄ = 13.6) (n = 40). The microconidia (n = 40) were thin-walled, hyaline, ovoid unicellular that measured 19.7 - 32.2 (x̄ = 26.6) x 8.8 - 11.8 µm (x̄ = 10.2). The translation elongation factor 1 alpha (EF1-α) gene (O'Donnell et al. 1998) was amplified by polymerase chain reaction and sequenced. Maximum likelihood analysis was carried out using the EF1-α sequence from the isolate FKTFRESCULSIN (GenBank accession no. OK491929) and other Neocosmospora and Fusarium species. Phylogenetic analysis revealed the isolate was Fusarium keratoplasticum (currently named Neocosmospora keratoplastica) belonging to the Fusarium solani species complex (FSSC). Pathogenicity tests were performed on strawberry plants (cultivar Camarosa) grown on autoclaved sandy loam soil mix. Twenty plants were inoculated by drenching with 20 ml of a conidial suspension (1 × 105 CFU/ml) in an isotonic saline solution of FKTFRESCULSIN grown on PDA. Ten uninoculated plants served as controls. Plants were maintained for 60 days under greenhouse conditions (25 to 30°C). The assay was conducted twice. Root and stem rot similar to that observed on the infected plants in the field was observed. No symptoms were observed on uninoculated control plants after 60 days. The pathogen was reisolated from necrotic tissue from all inoculated plants and identified as F. keratoplasticum by sequencing the partial EF1-α gene and based on its morphological characteristics, thus fulfilling Koch's postulates. To our knowledge, this is the first report of root rot and wilt of strawberry caused by F. keratoplasticum in Mexico; it also contributes knowledge to the scientific community, since there is little information about this pathogen causing damage to plants in the world. Strawberry is an important crop in Mexico, and the occurrence of this disease could threaten strawberry production.

PDR Transporter ABC1 Is Involved in the Innate Azole Resistance of the Human Fungal Pathogen Fusarium keratoplasticum.

Fusarium keratoplasticum is arguably the most common Fusarium solani species complex (FSSC) species associated with human infections. Invasive fusariosis is a life-threatening fungal infection that is difficult to treat with conventional azole antifungals. Azole drug resistance is often caused by the increased expression of pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters of the ABCG sub-family. Most investigations of Fusarium ABC transporters associated with azole antifungal drug resistance are limited to plant pathogens. Through the manual curation of the entire ABCG protein family of four FSSC species including the fully annotated genome of the plant pathogen Nectria haematococca we identified PDR transporters ABC1 and ABC2 as the efflux pump candidates most likely to be associated with the innate azole resistance phenotype of Fusarium keratoplasticum. An initial investigation of the transcriptional response of logarithmic phase F. keratoplasticum cells to 16 mg/L voriconazole confirmed strong upregulation (372-fold) of ABC1 while ABC2 mRNA levels were unaffected by voriconazole exposure over a 4 h time-period. Overexpression of F. keratoplasticum ABC1 and ABC2 in the genetically modified Saccharomyces cerevisiae host ADΔΔ caused up to ∼1,024-fold increased resistance to a number of xenobiotics, including azole antifungals. Although ABC1 and ABC2 were only moderately (20% and 10%, respectively) expressed compared to the Candida albicans multidrug efflux pump CDR1, overexpression of F. keratoplasticum ABC1 caused even higher resistance levels to certain xenobiotics (e.g., rhodamine 6G and nigericin) than CDR1. Our investigations suggest an important role for ABC1 orthologues in the innate azole resistance phenotype of FSSC species.

Phenothiazinium Photosensitizers Associated with Silver Nanoparticles in Enhancement of Antimicrobial Photodynamic Therapy.

Antimicrobial photodynamic therapy (APDT) and silver nanoparticles (AgNPs) are known as promising alternatives for the control of microorganisms. This study aims to evaluate the antifungal activity of APDT, particularly by using the association of low concentrations of phenothiazinium photosensitizers (PS) methylene blue (MB), new methylene blue N (NMBN), and new methylene blue N Zinc (NMBN-Zn) in association with biosynthesized AgNPs. The AgNPs were characterized by UV-Vis spectrophotometry, transmission electron microscopy, and the dynamic light scattering method. The minimum inhibitory concentration of compounds in APDT against Candida albicans and Fusarium keratoplasticum was obtained and the Fractional Inhibitory Concentration Index determined the antifungal effect. The toxicity of compounds and associations in APDT were evaluated in Galleria mellonella. The AgNPs presented a surface plasmon band peak at 420 nm, hydrodynamic diameter of 86.72 nm, and zeta potential of -28.6 mV. AgNPs-PS showed a wider and displaced plasmon band peak due to PS ligands on the surface and decreased zeta potential. AgNPs-NMBN and AgNPs-NMBN-Zn associations presented synergistic effect in APDT with 15 J cm-2 against both fungi and did not show toxicity to G. mellonella. Hence, the enhancement of antifungal activity with low concentrations of compounds and absence of toxicity makes APDT with AgNPs-PS a promising therapeutic alternative for fungal infections.

Antimicrobial photodynamic therapy with phenothiazinium photosensitizers in non-vertebrate model Galleria mellonella infected with Fusarium keratoplasticum and Fusarium moniliforme.

Fusarium keratoplasticum and Fusarium moniliforme are filamentous fungi common in the environment and cause mycosis in both animals and plants. Human infections include mycetoma, keratitis and onychomycosis, while deeper mycosis occurs in immunocompromised patients. Most of the Fusarium spp. are frequently resistant to treatment with currently used antifungals. The frequent occurrence of antifungal resistance has motivated the study of antimicrobial photodynamic therapy as an alternative treatment for fungal infections. Many studies have investigated the in vitro use of antimicrobial photodynamic therapy to kill fungi, but rarely in animal models of infection. Thus, here we employed the invertebrate wax moth Galleria mellonella to study the in vivo effects of antimicrobial photodynamic therapy with three different phenothiazinium photosensitizers, methylene blue, new methylene blue N and the pentacyclic S137 against infection with microconidia of Fusarium keratoplasticum and Fusarium moniliforme. The effect of antimicrobial photodynamic therapy using these photosensitizers and light-emitting diodes with an emission peak at 635 nm and an integrated irradiance from 570 to 670 nm of 9.8 mW cm-2 was investigated regarding the toxicity, fungal burden, larval survival and cellular immune response. The results from this model indicate that antimicrobial photodynamic therapy with methylene blue, new methylene blue N and S137 is efficient for the treatment of infection with F. keratoplasticum and F. moniliforme. The efficiency can be attributed to the fungal cell damage caused by antimicrobial photodynamic therapy which facilitates the action of the host immune response.

Biofilm Formation and Resistance to Fungicides in Clinically Relevant Members of the Fungal Genus Fusarium.

Clinically relevant members of the fungal genus, Fusarium, exhibit an extraordinary genetic diversity and cause a wide spectrum of infections in both healthy individuals and immunocompromised patients. Generally, Fusarium species are intrinsically resistant to all systemic antifungals. We investigated whether the presence or absence of the ability to produce biofilms across and within Fusarium species complexes is linked to higher resistance against antifungals. A collection of 41 Fusarium strains, obtained from 38 patients with superficial and systemic infections, and three infected crops, were tested, including 25 species within the Fusarium fujikuroi species complex, 14 from the Fusarium solani species complex (FSSC), one Fusarium dimerum species complex, and one Fusarium oxysporum species complex isolate. Of all isolates tested, only seven strains from two species of FSSC, five F. petroliphilum and two F. keratoplasticum strains, recovered from blood, nail scrapings, and nasal biopsy samples, could produce biofilms under the tested conditions. In the liquid culture tested, sessile biofilm-forming Fusarium strains exhibited elevated minimum inhibitory concentrations (MICs) for amphotericin B, voriconazole, and posaconazole, compared to their planktonic counterparts, indicating that the ability to form biofilm may significantly increase resistance. Collectively, this suggests that once a surface adherent biofilm has been established, therapies designed to kill planktonic cells of Fusarium are ineffective.

Melanization of Fusarium keratoplasticum (F. solani Species Complex) During Disseminated Fusariosis in a Patient with Acute Leukemia.

Fusarium spp. are recognized as the second most frequently filamentous fungi causing opportunistic infections and particularly important due to the increasing number of immunocompromised patients. F. keratoplasticum (a member of F. solani species complex) is one of the Fusarium species commonly associated with human infection, and therefore, studies on the virulence of this fungus are needed. This study aimed to confirm the presence of melanin in F. keratoplasticum from a patient with systemic fusariosis. Immunofluorescence labeling with anti-melanin monoclonal antibody (MAb) was used to examine an expression of melanin in F. keratoplasticum in vitro and during infection. Electron spin resonance identified the particles extracted from F. keratoplasticum as stable free radical consistent with melanin. Lesional skin from the sites with fusariosis contained hyphal structures that could be labeled by melanin-binding MAb, while digestion of the tissue yielded dark particles that were reactive. These findings suggest that F. keratoplasticum hyphae and chlamydospores can produce melanin in vitro and that hyphae can synthesize pigment in vivo. Given the potential role of melanin in virulence of other fungi, this pigment in F. keratoplasticum may play a role in the pathogenesis of fusariosis.

Fatal Fusarium solani species complex infections in elasmobranchs: the first case report for black spotted stingray (Taeniura melanopsila) and a literature review.

Fusarium species are environmental saprophytic fungi. Among the many Fusarium species, members of the Fusarium solani species complex (FSSC) are the most prevalent and virulent in causing human and animal infections. In this study, we describe the first case of fatal FSSC infection in a black spotted stingray and three concomitant infections in scalloped hammerhead sharks. In the stingray, cutaneous lesions were characterised by ulcers and haemorrhage of the ventral pectoral fin, or 'ray', especially around the head; while cutaneous lesions in the sharks were characterised by ulcers, haemorrhage, as well as white and purulent exudates at the cephalic canals of the cephalofoil and lateral line. Histological sections of the cutaneous lesions revealed slender (1-4 µm in diameter), branching, septate fungal hyphae. Internal transcribed spacer region and 28S nrDNA sequencing of the fungal isolates from the fish showed two isolates were F. keratoplasticum (FSSC 2) and the other two were FSSC 12. Environmental investigation revealed the FSSC strains isolated from water and biofilms in tanks that housed the elasmobranchs were also F. keratoplasticum and FSSC 12. Fusarium is associated with major infections in elasmobranchs and FSSC 12 is an emerging cause of infections in marine animals. DNA sequencing is so far the most reliable method for accurate identification of Fusarium species.

Disseminated fusariosis and hematologic malignancies, a still devastating association. Report of three new cases.

BACKGROUND: Fungi of the genus Fusarium are primarily plant pathogens and saprobes that produce disseminated infections in immunologically deficient humans. After aspergillosis, disseminated fusariosis is the second most common cause of invasive infection by filamentous fungi in patients with hematologic malignancies or those undergoing transplants of hematopoietic progenitors. AIMS: Disseminated fusariosis (DF) is considered an extremely rare infection and has reached a stable incidence rate, but its high mortality rate and the lack of an optimal management protocol have raised increasing interest in this mycosis. METHODS: We present three cases of DF produced by Fusarium oxysporum species complex, Fusarium solani species complex and the highly unusual Fusarium dimerum in patients with advanced hematological malignancies diagnosed in our hospital between 2007 and 2011. The species level identification of the Fusarium isolates was established by sequencing their TEF1 gene. RESULTS: The isolates showed low susceptibility to most of the antifungal agents analyzed, except that observed for F. dimerum to amphotericin B (AmB) and terbinafine, and F. oxysporum species complex to AmB. Interestingly, the strain of F. solani species complex exhibited high MIC values for AmB and voriconazole, notwithstanding these drugs were used for treatment with good results. Other relevant aspects to be considered in the treatment of DF are surgically cleaning foci of infection, withdrawing presumably contaminated catheters and recovery from neutropenia. CONCLUSIONS: The prevention of infection in colonized patients, the maintenance of a high level of diagnostic suspicion for early diagnosis, and the combined, vigorous and prolonged use of L-AmB and voriconazole are essential to decrease the mortality rate of this devastating infection.