Identification and characterization of a fungal cutinase-like enzyme CpCut1 from Cladosporium sp. P7 for polyurethane degradation.

Plastic degradation by biological systems emerges as a prospective avenue for addressing the pressing global concern of plastic waste accumulation. The intricate chemical compositions and diverse structural facets inherent to polyurethanes (PU) substantially increase the complexity associated with PU waste management. Despite the extensive research endeavors spanning over decades, most known enzymes exhibit a propensity for hydrolyzing waterborne PU dispersion (i.e., the commercial Impranil DLN-SD), with only a limited capacity for the degradation of bulky PU materials. Here, we report a novel cutinase (CpCut1) derived from Cladosporium sp. P7, which demonstrates remarkable efficiency in the degrading of various polyester-PU materials. After 12-h incubation at 55°C, CpCut1 was capable of degrading 40.5% and 20.6% of thermoplastic PU film and post-consumer foam, respectively, while achieving complete depolymerization of Impranil DLN-SD. Further analysis of the degradation intermediates suggested that the activity of CpCut1 primarily targeted the ester bonds within the PU soft segments. The versatile performance of CpCut1 against a spectrum of polyester-PU materials positions it as a promising candidate for the bio-recycling of waste plastics.IMPORTANCEPolyurethane (PU) has a complex chemical composition that frequently incorporates a variety of additives, which poses significant obstacles to biodegradability and recyclability. Recent advances have unveiled microbial degradation and enzymatic depolymerization as promising waste PU disposal strategies. In this study, we identified a gene encoding a cutinase from the PU-degrading fungus Cladosporium sp. P7, which allowed the expression, purification, and characterization of the recombinant enzyme CpCut1. Furthermore, this study identified the products derived from the CpCut1 catalyzed PU degradation and proposed its underlying mechanism. These findings highlight the potential of this newly discovered fungal cutinase as a remarkably efficient tool in the degradation of PU materials.

Sequential breakdown of the Cf-9 leaf mould resistance locus in tomato by Fulvia fulva.

Leaf mould, caused by Fulvia fulva, is a devastating disease of tomato plants. In many commercial tomato cultivars, resistance to this disease is governed by the Cf-9 locus, which encodes five paralogous receptor-like proteins. Two of these proteins confer resistance: Cf-9C recognises the previously identified F. fulva effector Avr9 and provides resistance during all plant growth stages, while Cf-9B recognises the yet-unidentified F. fulva effector Avr9B and provides mature plant resistance only. In recent years, F. fulva strains have emerged that can overcome the Cf-9 locus, with Cf-9C circumvented through Avr9 deletion. To understand how Cf-9B is circumvented, we set out to identify Avr9B. Comparative genomics, transient expression assays and gene complementation experiments were used to identify Avr9B, while gene sequencing was used to assess Avr9B allelic variation across a world-wide strain collection. A strict correlation between Avr9 deletion and resistance-breaking mutations in Avr9B was observed in strains recently collected from Cf-9 cultivars, whereas Avr9 deletion but no mutations in Avr9B were observed in older strains. This research showcases how F. fulva has evolved to sequentially break down the Cf-9 locus and stresses the urgent need for commercial tomato cultivars that carry novel, stacked resistance genes active against this pathogen.

Photoinactivation by UVA radiation and visible light of Candida auris compared to other fungi.

In addition to the rising number of patients affected by viruses and bacteria, the number of fungal infections has also been rising over the years. Due to the increase in resistance to various antimycotics, investigations into further disinfection options are important. In this study, two yeasts (Candida auris and Saccharomyces cerevisiae) and a mold (Cladosporium cladosporioides) were irradiated at 365, 400, and 450 nm individually. The resulting log 1 reduction doses were determined and compared with other studies. Furthermore, fluorescence measurements of C. auris were performed to detect possible involved photosensitizers. A roughly exponential photoinactivation was observed for all three fungi and all irradiation wavelengths with higher D90 doses for longer wavelengths. The determined log 1 reduction doses of C. auris and S. cerevisiae converged with increasing wavelength. However, S. cerevisiae was more photosensitive than C. auris for all irradiation wavelengths and is therefore not a suitable C. auris surrogate for photoinactivation experiments. For the mold C. cladosporioides, much higher D90 doses were determined than for both yeasts. Concerning potential photosensitizers, flavins and various porphyrins were detected by fluorescence measurements. By excitation at 365 nm, another, so far unreported fluorophore and potential photosensitizer was also observed. Based on its fluorescence spectrum, we assume it to be thiamine.Graphic abstract.

Gypsum Cave Biofilm Communities are Strongly Influenced by Bat- And Arthropod-Related Fungi.

The Gypsum Karst of Sorbas, Almeria, southeast Spain, includes a few caves whose entrances are open and allow the entry and roosting of numerous bats. Caves are characterized by their diversity of gypsum speleothems, such as stalactites, coralloids, gypsum crusts, etc. Colored biofilms can be observed on the walls of most caves, among which the Covadura and C3 caves were studied. The objective was to determine the influence that bat mycobiomes may have on the fungal communities of biofilms. The results indicate that the fungi retrieved from white and yellow biofilms in Covadura Cave (Ascomycota, Mortierellomycota, Basidiomycota) showed a wide diversity, depending on their location, and were highly influenced by the bat population, the guano and the arthropods that thrive in the guano, while C3 Cave was more strongly influenced by soil- and arthropod-related fungi (Ascomycota, Mortierellomycota), due to the absence of roosting bats.

Diffuse Associations Between Drosophila suzukii and Filamentous Fungal Microbes May Alter Caneberry Disease Dynamics.

Interactions between microorganisms and frugivorous insects can modulate fruit rot disease epidemiology. Insect feeding and/or oviposition wounds may create opportunities for fungal infection. Passive and active dispersal of fungal inoculums by adult insects also increases disease incidence. In fall-bearing raspberries and blackberries, such vectoring interactions could increase crop damage from the invasive pestiferous vinegar fly Drosophila suzukii (spotted-wing drosophila). Periods of peak D. suzukii activity are known to overlap with several species of primary fruit rot pathogen, particularly Botrytis cinerea and Cladosporium cladosporioides, and previous work indicates that larvae co-occur with and feed on various filamentous fungi at low rates. To further our understanding of the epidemiological consequences that may emerge from these associations, we surveyed the filamentous fungal community associated with adult D. suzukii, isolating and molecularly identifying fungi externally and internally (indicating feeding) from field-collected adults over 3 years. We isolated and identified 37 unique genera of fungi in total, including known raspberry pathogens. Most fungi were detected infrequently, and flies acquired and carried fungi externally at higher richness, frequency, and density relative to internally. In a worst-case scenario laboratory vectoring assay, D. suzukii adults were able to transfer B. cinerea and C. cladosporioides to sterile media at 0, 24, 48, and 72 h after exposure to sporulating cultures in Petri dishes. These results collectively suggest an adventitious vectoring association between D. suzukii and fruit rot fungi that has the potential to alter caneberry disease dynamics.

First Report of Green Mould on Leaf of Phaseolus vulgaris Caused by Cladosporium tenuissimum in Liaoning, China.

Phaseolus vulgaris Linn. is a widely cultivated vegetable throughout the world. From spring 2019 to 2022, green mould symptoms were observed on leaves of P. vulgaris in the greenhouse in Liaoning, China, with disease incidence of 8-75% (plants) and 6-23% (leaves). Symptoms appeared as chlorotic lesions covered with dark green mould. The infections started at the apex or margin of the leaves and then spread inward with a characteristic "V" shape. Lesions exhibited curly morphology. 15 leaf samples with typical symptoms were collected from 5 different greenhouses. A total of 75 (5 replicates of each sample) leaf tissues (0.5 cm × 0.5 cm) were selected from the boundary between diseased and healthy parts. These samples were surface sterilized in 0.5% NaClO formin, rinsed 3 times in sterile distilled water and subsequently incubated at 28℃ on potato dextrose agar (PDA) supplemented with streptomycin (50 µg/ml). Numerous morphologically uniform colonies had been purified, with no other fungi observed. Afterwards, the strains were subcultured on malt extract agar (MEA). Colonies on MEA reached 70 to 80 mm diam after 14 days, smoke-grey to pale olivaceous-grey, woolly, sometimes radially wrinkled. The mycelia were pale olivaceous-grey, with hyphae measuring 1-5 µm wide (n = 20). The conidiophores were solitary or in groups of 2 to 5, and measured 50-280(-350) × 2.5-4 µm (n = 20), with 2-7 septa. The conidiogenous cells exhibited a cylindrical-oblong morphology and measured 10-44 × 5 µm (n = 20), with 0-2 septa, and the loci frequently thickened. The conidia were catenate in densely branched chains, ellipsoid to obovoid, smooth, and measured 2.5-5 × 2-3 µm (n = 50), with 0-4 septa. The morphological characteristics were similar to Cladosporium tenuissimum (Zhang 2003). The representative isolate KZ-19 was selected for molecular identification. The rDNA-ITS, translation elongation factor 1-α and actin genes were amplified, sequenced, and the resulting sequence data were submitted to GenBank (ITS: OQ931048; EF-1α: OQ954495; ACT: OQ954496). The BLAST results exhibited a 99 to 100% similarity with the sequences of C. tenuissimum type strain CBS 125995(ITS: HM148197; EF-1α: HM148442; ACT: HM148687). Furthermore, a multi-locus phylogenetic tree was constructed using the PhyloSuite (v 1. 2. 2) software, which revealed that the strains were most closely related to C. tenuissimum (Zhang et al. 2020). Based on both morphological and molecular characteristics, KZ-19 was finally identified as C. tenuissimum (Bensch 2012). Pathogenicity testing was performed on healthy 1-month-old P. vulgaris plants by inoculating the spore suspension (1×106 conidia/ml) of KZ-19 onto leaf surfaces, while control plants were simulated inoculated with sterile water, and five pots were used for each treatment. The test was performed under field conditions of 16-28°C (temperature) and 24-56% (relative humidity). Chlorotic lesions became evident within 2 days of inoculation, followed by the appearance of green mold on leaves after 7 days. No symptoms were observed in the control group. To fulfill Koch's postulates, the pathogen was re-isolated from three inoculated leaves. The morphological identification of re-isolated pathogens was similar to that of originally isolated pathogens. No infection was observed in non-inoculated control. To the best of our knowledge, this is the first report of C. tenuissimum causing green mould on P. vulgaris. As a ubiquitous saprobic hyphomycete, C. tenuissimum has been implicated in leaf mold in Punica granatum and Trifolium repens, larch bud blight, and strawberry blossom blight in previous years (He et al. 1987; Zhang et al. 2003; Zheng et al. 2010; Nam et al. 2015), presenting a potential threat to numerous crops. Therefore, an investigation of its distribution and pathogenic potential is essential in addition to the development of effective disease management strategies.

First Reports of Cladosporium tenuissimum Causing Leaf spots on Hydrangea macrophylla in Anhui province in China.

Forever Summer Hydrangea (Hydrangea macrophylla) is a common flowering plant in the Yangtze River Valley area of China, and it is widely cultivated globally (Chen et al. 2015). In July 2023, H. macrophylla leaves exhibiting visible diseased lesions were reported in a nursery in Wuhu, Anhui Province, China. The incidence reached 40% in a 0.2 ha area. The primary disease symptom was multiple irregular necrotic spots (0.5 to 1 mm in diameter) appearing on the leaves. These spots on the leaves were faded yellow around the perimeter and grayish brown in the center.). 15 leaf samples were sterilized with 75% alcohol and rinsed three times in sterile distilled water, then transferred to antibiotic-added potato dextrose agar (PDA) for incubation at 27°C. The colonies were fluffy, flocculent, or hairy, dark green, gray-green to gray-brown in color, and spreading or protruding punctate with a colorless halo on PDA. The conidiophores were brown to dark brown, smooth or rough surface, mostly unbranched, clearly differentiated, erect or curved. The conidia displayed a light brown to brown hue, lemon shape, fusiform, elongated ellipsoid or others with obvious spore markings and spore umbilicus. Genomic DNA was extracted from fungal colonies on infected leaves of three collections separately (Braun et al. 2003) and the internal transcribed spacer regions (ITS), actin (ACT) genes and partial translation elongation factor-l-alpha (EF) were amplified and sequenced using the primers ITS1/4 (Yin et al. 2012), ACT-512F/ACT-783R and EF 1-728F/986R (Carbone and Kohn 1999), respectively. DNA sequences of isolates were identical and deposited in GenBank (accession no. OR362754 for ITS, OR611929 for ACT and PP209106 for EF). The consensus sequences from ITS, EF and ACT showed 100%, 98.98% and 100% identical to Cladosporium strains (accession no. OQ186140.1, MT154169.1 and OL322092.1), respectively. To confirm the pathogenicity of the isolates, hydrangeas were planted in 15-cm pots containing commercial potting mix (one plant/pot). Three healthy plants were inoculated at the five to eight leaf stage by spraying 50 µL of the isolate conidial suspension (4 × 106 spores/mL) on healthy leaves. Three plants treated with sterile distilled water were used as controls. After inoculation, all plants were placed in a humidity chamber (>95% relative humidity, 26°C) for 48 h and then transferred to a greenhouse at 22/27°C. All inoculated leaves exhibited symptoms similar to those observed in the nursery 10 days after inoculation, while no symptoms were observed for control leaves. The fungus was re-isolated and confirmed to be C. tenuissimum. Based on the above morphological characterization and molecular identification, the causal agent for this leaf spot disease was identified as C. tenuissimum. Although C. tenuissimum has been reported to cause disease on H. paniculata in northern China (Li et al.2021), this is the first time that C. tenuissimum has been found on H. macrophylla in southern China. This new disease of H. macrophylla caused by C. tenuissimum is a threat to urban greening and is worth further investigation.

Biocontrol Potential of Cladosporium sphaerospermum Against the Wheat Powdery Mildew Fungus Blumeria graminis f. sp. tritici.

Cladosporium spp. are known to be mycoparasites and inhibit phytopathogenic fungi. However, so far, little information is available on the impact of Cladosporium spp. on powdery mildews. Based on the morphological characteristics and molecular analysis, C. sphaerospermum was identified as a mycoparasite on the wheat powdery mildew fungus Blumeria graminis f. sp. tritici (Bgt), recently named B. graminis s. str. C. sphaerospermum was capable of preventing colony formation and conidial distribution of Bgt. The biomasses of Bgt notably decreased by 1.3, 2.2, 3.6, and 3.8 times at 2, 4, 6, and 8 days postinoculation (dpi), respectively. In addition, biomasses of C. sphaerospermum at 2, 4, 6, and 8 dpi significantly increased to 5.6, 13.9, 18.2, and 67.3 times, respectively. In vitro, C. sphaerospermum exudates significantly impaired appressorial formation of Bgt. Thus, C. sphaerospermum acts as a potential biological control agent by suppressing the formation, distribution, and development of Bgt conidia and is a viable alternative for managing the wheat powdery mildew. These results suggest that C. sphaerospermum is an antagonistic parasite of the wheat powdery mildew fungus and, hence, provide new knowledge about the biological control of phytopathogenic fungi.

Secondary metabolites from the fungus Cladosporium xylophilum.

A new cladosporol derivative xylophilum A (1), together with 10 known compounds (2-11), were isolated from the rice fermentation of the fungus Cladosporium xylophilum. Their structures were established by extensive spectroscopic methods and comparison of their NMR data with literatures. The antimicrobial activity of compound 1 against 11 kinds of pathogenic microbial was evaluated, but no significant activity was found (MIC >100 µg/ml).

Changes in Electron Paramagnetic Resonance Parameters Caused by Addition of Amphotericin B to Cladosporium cladosporioides Melanin and DOPA-Melanin-Free Radical Studies.

Cladosporium cladosporioides are the pigmented soil fungi containing melanin. The aim of this work was to determine the influence of amphotericin B on free radicals in the natural melanin isolated from pigmented fungi Cladosporium cladosporioides and to compare it with the effect in synthetic DOPA-melanin. Electron paramagnetic resonance (EPR) spectra were measured at X-band (9.3 GHz) with microwave power in the range of 2.2-70 mW. Amplitudes, integral intensities, linewidths of the EPR spectra, and g factors, were analyzed. The concentrations of free radicals in the tested melanin samples were determined. Microwave saturation of EPR lines indicates the presence of pheomelanin in addition to eumelanin in Cladosporium cladosporioides. o-Semiquinone free radicals in concentrations ~1020 [spin/g] exist in the tested melanin samples and in their complexes with amphotericin B. Changes in concentrations of free radicals in the examined synthetic and natural melanin point out their participation in the formation of amphotericin B binding to melanin. A different influence of amphotericin B on free radical concentration in Cladosporium cladosporioides melanin and in DOPA-melanin may be caused by the occurrence of pheomelanin in addition to eumelanin in Cladosporium cladosporioides. The advanced spectral analysis in the wide range of microwave powers made it possible to compare changes in the free radical systems of different melanin polymers. This study is important for knowledge about the role of free radicals in the interactions of melanin with drugs.

The Genus Cladosporium: A Prospective Producer of Natural Products.

Cladosporium, a genus of ascomycete fungi in the Dematiaceae family, is primarily recognized as a widespread environmental saprotrophic fungus or plant endophyte. Further research has shown that the genus is distributed in various environments, particularly in marine ecosystems, such as coral reefs, mangroves and the polar region. Cladosporium, especially the marine-derived Cladosporium, is a highly resourceful group of fungi whose natural products have garnered attention due to their diverse chemical structures and biological activities, as well as their potential as sources of novel leads to compounds for drug production. This review covers the sources, distribution, bioactivities, biosynthesis and structural characteristics of compounds isolated from Cladosporium in the period between January 2000 and December 2022, and conducts a comparative analysis of the Cladosporium isolated compounds derived from marine and terrestrial sources. Our results reveal that 34% of Cladosporium-derived natural products are reported for the first time. And 71.79% of the first reported compounds were isolated from marine-derived Cladosporium. Cladosporium-derived compounds exhibit diverse skeletal chemical structures, concentrating in the categories of polyketides (48.47%), alkaloids (19.21%), steroids and terpenoids (17.03%). Over half of the natural products isolated from Cladosporium have been found to have various biological activities, including cytotoxic, antibacterial, antiviral, antifungal and enzyme-inhibitory activities. These findings testify to the tremendous potential of Cladosporium, especially the marine-derived Cladosporium, to yield novel bioactive natural products, providing a structural foundation for the development of new drugs.

New viruses of Cladosporium sp. expand considerably the taxonomic structure of Gammapartitivirus genus.

Despite the fact that Cladosporium sp. are ubiquitous fungi, their viromes have been little studied. By analysing a collection of Cladosporium fungi, two new partitiviruses named Cladosporium cladosporioides partitivirus 1 (CcPV1) and Cladosporium cladosporioides partitivirus 2 (CcPV2) co-infecting a strain of Cladosporium cladosporioides were identified. Their complete genome consists of two monocistronic dsRNA segments (RNA1 and RNA2) with a high percentage of pairwise identity on 5' and 3' end. The RNA directed RNA polymerase (RdRp) of both viruses and the capsid protein (CP) of CcPV1 display the classic characteristics required for their assignment to the Gammapartitivirus genus. In contrast, CcPV2 RNA2 encodes for a 41 KDa CP that is unusually smaller when aligned to CPs of other viruses classified in this genus. The structural role of this protein is confirmed by electrophoresis on acrylamide gel of purified viral particles. Despite the low percentage of identity between the capsid proteins of CcPV1 and CcPV2, their three-dimensional structures predicted by AlphaFold2 show strong similarities and confirm functional proximity. Fifteen similar viral sequences of unknown function were annotated using the CcPV2 CP sequence. The phylogeny of the CP was highly consistent with the phylogeny of their corresponding RdRp, supporting the organization of Gammapartitiviruses into three distinct clades despite stretching the current demarcation criteria. It is proposed that a new subgenus be created within the genus Gammapartitivirus for this new group.

Differences in fungal communities in the fur of two- and three-toed sloths revealed by ITS metabarcoding.

Sloths have dense fur on which insects, algae, bacteria and fungi coexist. Previous studies using cultivation-dependent methods and 18S rRNA sequencing revealed that the fungal communities in their furs comprise members of the phyla Ascomycota and Basidiomycota. In this note, we increase the resolution and knowledge of the mycobiome inhabiting the fur of the two- (Choloepus hoffmanni) and three-toed (Bradypus variegatus) sloths. Targeted amplicon metagenomic analysis of ITS2 nrDNA sequences obtained from 10 individuals of each species inhabiting the same site revealed significant differences in the structure of their fungal communities and also in the alpha-diversity estimators. The results suggest a specialization by host species and that the host effect is stronger than that of sex, age and animal weight. Capnodiales were the dominant order in sloths' fur and Cladosporium and Neodevriesia were the most abundant genera in Bradypus and Choloepus, respectively. The fungal communities suggest that the green algae that inhabit the fur of sloths possibly live lichenized with Ascomycota fungal species. The data shown in this note offer a more detailed view of the fungal content in the fur of these extraordinary animals and could help explain other mutualistic relationships in this complex ecosystem.

Bioremoval of heavy metals from aqueous solution using dead biomass of indigenous fungi derived from fertilizer industry effluents: isotherm models evaluation and batch optimization.

The present work investigated the utilization of dead biomass of the highly multi-heavy metals tolerant indigenous fungal strain NRCA8 isolated from the mycobiome of fertilizer industry effluents that containing multiple heavy metal ions at high levels to remove Pb2+, Ni2+, Zn2+, and Mn2+ as multiple solutes from multi-metals aqueous solutions for the first time. Based on morphotype, lipotype and genotype characteristics, NRCA8 was identified as Cladosporium sp. NRCA8. The optimal conditions for the bioremoval procedure in the batch system were pH 5.5 for maximum removal (91.30%, 43.25%, and 41.50%) of Pb2+, Zn2+ and Mn2+ but pH 6.0 supported the maximum bioremoval and uptake of Ni2+ (51.60% and 2.42 mg/g) by NRCA8 dead biomass from the multi-metals aqueous solution, respectively. The 30 min run time supported the highest removal efficiency and uptake capacity of all heavy metals under study. Moreover, the equilibrium between the sorbent NRCA8 fungal biomass and sorbates Ni2+, Pb2+ and Zn2+ was attained after increasing the dead biomass dose to 5.0 g/L. Dead NRCA8 biomass was described by scanning electron microscopy, energy-dispersive X-ray spectroscopy and Fourier transform infrared spectrometer before and after biosorption of Pb2+, Ni2+, Zn2+ and Mn2+ under multiple metals system. The Langmuir, Freundlich and Dubinin-Kaganer-Radushkevich isotherms were applied to characterize the adsorption equilibrium between Pb2+, Ni2+, Mn2+ and Zn2+ and the adsorbent NRCA8. By comparing the obtained coefficient of regression (R2) by Freundlich (0.997, 0.723, 0.999, and 0.917), Langmiur (0.974, 0.999, 0.974, and 0.911) and Dubinin-Radushkevich (0.9995, 0.756, 0.9996 and 0.900) isotherms values for Pb2+, Zn2+, Ni2+ and Mn2+ adsorption, respectively, it was found that the isotherms are proper in their own merits in characterization the possible of NRCA8 for removal of Pb2+, Zn2+, Ni2+ and Mn2+. DKR isotherm is the best for Pb2+ and Ni2+ (0.9995 and 0.9996) while Langmiur isotherm giving a good fit to the Zn2+ sorption (0.9990) as well as Freundlich isotherm giving a good fit to the Mn2+ sorption (0.9170). The efficiencies of Cladosporium sp. NRCA8 dead biomass for bioremoval of heavy metals from real wastewater under the optimized conditions were Pb2+, Ag+, Mn2+, Zn2+ and Al3+ ˃ Ni2+ ˃ Cr6+ ˃ Co2+ ˃ Fe3+ ˃ Cu2+ ˃ Cd2+. Dead NRCA8 biomass showed efficient ability to adsorb and reduce harmful components in the industrial effluents to a level acceptable for discharge into the environment.

On predatory fungi feeding on free-living amoebae harbouring yeast-like endoparasites.

Amoebae of the genus Vannella isolated from an ornamental fish aquarium were found to be infected with fungi. Upon plate culture, amoeba-trapping hyphal filaments were developed, and the amoeba trophozoites were found to harbour yeast-like parasites in their cytoplasm. Transfection of hyphae to a laboratory strain of Vannella resulted in the formation of conidia indicating the possible presence of zygomycetes of the genus Acaulopage, while efforts to culture the endoparasite remained unsuccessful. Biomolecular analysis based on rDNA revealed the presence of two distinct types of fungi, confirming the filamentous form as Acaulopage sp. (Zoopagomycota, Zoopagales) and identifying the yeast-like endoparasite as Cladosporium sp. (Ascomycota, Cladosporiales). To our knowledge, this is the first report of amoebae infected with Cladosporium.

Peroxisome Proliferator Activated Receptor-γ Agonistic Compounds from the Jellyfish-Derived Fungus Cladosporium oxysporum.

In our search for peroxisome proliferator-activated receptor (PPAR) agonists, five undescribed compounds, namely two acyclic diterpenes (1 and 2; cladopsol A and cladopsol B), two sesquiterpenes (3 and 4; cladopsol C and cladopsol D), and one C21-ecdysteroid (5; cladopsol E), and 15 known compounds were isolated from the jellyfish-derived fungus - Cladosporium oxysporum. The structures of the undescribed compounds were defined using UV, NMR, HR-ESI-MS, and electronic circular dichroism (ECD) spectroscopy and a modified Mosher's method. Luciferase reporter assay and docking analysis suggested that cladopsol B may function as a PPAR-γ partial agonist with a potential antidiabetic lead which may evade the side effects of full agonists. Moreover, cladopsol B stimulated glucose uptake in HepG2 cells with an efficacy comparable to that of rosiglitazone, but with less side effect induced by lipid accumulation in 3T3-L1 cells. Therefore, cladopsol B could serve as a molecular skeleton in a study of advanced antidiabetic lead with less side effect.

First Report of Cladosporium tenuissimum causing spot diseases on leaves and fruits of cucurbits in Brazil.

Cucurbitaceae crops are widely cultivated in the Northeast region of Brazil, which is the biggest producer of melon and watermelon in the country (Oliveira, 2020). Between November and December 2020 leaves of pumpkins (Cucurbita maxima L.) and watermelon (Citrullus lanatus L.), and leaves and fruits of melon plants (Cucumis melo L.) were collected with moderate to severe necrotic, irregular, and brown lesions from farms in the state of Rio Grande do Norte, Brazil. Fragments of diseased tissues were cut into small pieces and surface disinfested in 70% ethanol for 30 seconds, then in 2% sodium hypochlorite for 1 minute, and washed in sterile distilled water. Disinfested pieces of tissue were plated on potato dextrose agar (PDA) and incubated for seven days in the dark at 28 ± 2 °C. A total of 12 fungal isolates (four from pumpkins, one from watermelon, and seven from melons) were isolated from leaves and symptomatic fruits. All isolates in this study shared similar morphological characteristics. The colonies were dark gray to olive green in color with a velvety texture and surrounded by gray-white hyphae. The conidiophores were erect, tall, dark, and irregularly branched at the apex containing dark conidia, with 0 to 3 septa, variable in shape and size, forming chains that were often branched, globose, or subglobose with 3 to 4.5 µm in diameter. DNA from each isolate was extracted using the SDS method (Smith et al., 2001) and submitted to PCR amplification of the ITS and TEF1α regions with the primers ITS1/ITS4 (White et al. 1990) and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. The amplicons were sequenced and deposited in GenBank: ITS (OP493545-OP493556) and TEF1α (OP536836-OP536847). Blastn analysis of the ITS and TEF1α partial sequences revealed that all 12 isolates belong to the species Cladosporium tenuissimum, with 100% nucleotide similarity with sequences of many C. tenuissimum isolates deposited in GenBank. A phylogenetic tree was constructed using the Maximum Parsimony Analysis, with the concatenated sequences (ITS-TEF1α) on MEGAX software (version 11.0.8) (Tamura et al, 2018). All 12 isolates clustered in the same clade and were closely related to isolates A2PP5, A3I1, and XCHK2 with the respective accession numbers KU605789.1, KU605790.1, and MG873071.1 from GenBank, with 99% bootstrap support. The pathogenicity of the 12 isolates was evaluated in pumpkin and melon plants in a greenhouse. Spore suspensions (10 6 conidia/ml -1) were sprayed on the leaves of healthy seedlings until runoff, only water was sprayed on control plants as the mock, and five seedlings of each crop (melon and pumpkin) were inoculated in each treatment. All plants were covered with plastic bags for two days. Spots, similar to those observed on diseased plants in the field, developed on the inoculated leaves (after seven days from the inoculation day, no symptoms were observed on plants from the mock treatment) and the fungal morphology was identical to that observed on the originally diseased leaves, fulfilling Koch's postulate. The pathogenicity test was repeated and yielded the same results. The fact that all 12 isolates were pathogenic on pumpkin and melon leaves, indicates that many Cucurbits are susceptible to C. tenuissimum infection. Many growers in the region are reporting similar symptoms in their melon plantations and it appears that the disease incidence is getting more severe year after year, based on growers's reports. Therefore, more research needs to be conducted to determine the epidemiology and the extension of the economic impact caused by this pathogen to Cucurbits to develop strategies for disease control. To the best of our knowledge, this is the first report of C. tenuissimum causing disease in Cucurbits in Brazil.

First Report of black spot of Shine Muscat fruit caused by Cladosporium allicinum in China.

Shine muscat is a Vitis vinifera hybrid (Akitsu-21 × Hakunan), that has emerged as a popular table grape cultivar in China. In recent years, shine muscat has been widely cultivated with 66,667 ha being cultivated in 2021. In November 2021, symptoms of fruit spot were observed on shine muscat during the storage at 0~3℃ and 85%~90% RH, while stored at National Agricultural Product Preservation Engineering Technology Research Center, in Tianjin (N 116°20', E 39°09'), China. The incidence of this disease was about 35%. Affected grape berries initially had small brown spots. The spots on the fruit expanded to an ellipse or circular sunken area with a black center. The central peel of the diseased spots were ruptured and collapsed. The diseased fruits eventually fell off the vine. To isolate the pathogen, grape peels with typical symptoms were cut into small pieces, sterilized with 75% ethanol for 45-sec, rinsed with sterilized distilled water three times, and then transferred onto potato dextrose agar (PDA) medium.The plates were incubated at 25°C in the dark. After 10 days, 26 single spore isolates with similar morphology were obtained from 30 symptomatic grape berries. Fungal colonies were grayish brown, with abundant conidia on the obverse-side on PDA. Conidiophores were cylindrical, straight with unbranched, solitary or clustered, elongation at the tip and ranged in size from 3.2-6.8 × 35.6-150.9 µm (n=50). Conidia were grew in chains, ovoid, aseptate, and 2.2-6.0 × 8.3-16.8 µm (n=50). The morphological characteristics were consistent with Cladosporium allicinum (Bensch et al. 2012). Molecular data were also used to support the microscopic identification by extracting genomic DNA from 26 isolates using a Plant Genomic DNA kit (Tiangen, China). Amplicons were generated for the internal transcribed spacer (ITS), translation elongation factor 1-alpha(tef1-α), and actin (act) using the following primers ITS1/ITS4, EF1-728F/ EF1-986R and ACT-512F/ ACT-783R, respectively (Bensch et al. 2012). Blast analysis showed that three amplified fragments of 26 isolates were highly similar to C. allicinum, with 98.96~100% sequence identity with Cladosporium allicinum accessions in GenBank (ITS, OK661041; tef1-α, MF473332; act, LN834537). Three amplified fragments of representative isolate YG03 were deposited in GenBank with accession nos. OP799670 for ITS, OP888001 for tef1-α and OP887999 for act, respectively. Neighbor-joining trees based on concatenated sequences of three genes were constructed using MEGA5.2. The results showed that the strain YG03 from shine muscat was closely related to C. allicinum. Pathogenicity tests of 26 isolates were performed on healthy shine muscat berries using pin pricks and a humidor. In each wound, 5 µL of conidial suspension (1×106 conidia/mL) and sterile distilled water were inoculated on 30 berries, and maintained in a dark incubator at 25°C, 90% relative humidity. Each treatment was repeated twice. After 10 days, the wounded berries inoculated with the spore suspension showed dark brown spots, similar to the original diseased fruits, while no symptoms were observed on the control treament. Pathogen re-isolated from inoculated fruits were identical to the original strains on colony and microscopic morphology, and identified to Cladosporium allicinum based on act gene by molecular method, thereby fulfilling Koch's postulates. C.allicinum has been reported causing leaf spot on 11 host plants around the world (Bensch et al. 2012, 2015; Quaedvlieg et al. 2014; Jurisoo et al. 2019). To our knowledge, this is the first report of C. allicinum causing black spot on fruit of Vitis vinifera worldwide. The identification of this disease could establish a foundation for developing management strategies to reduce losses in storage period.

First Report of Cladosporium tenuissimum causing Postharvest rot of Elaeagnus conferta Fruit in Kunming, China.

Elaeagnus conferta Roxb. is a perennial evergreen climbing shrub and is mainly native to India, Vietnam, Malaysia, and South China (Gupta & Singh, 2021). Various parts of this plant are used to treat multiple diseases(Gupta et al., 2021). Between during the months of March and April of 2021, in Kunming city of grower fields, Yunnan Province (N 25°02'; E 102°42'), southwest China. Some postharvest E. conferta fruits showed brown spots of decay with a greyish mycelium, which symptom only appears on fruit, and did not find it on this plant. The incidence of this disease in postharvest E. conferta fruits ranges from 45 % to 65 % in natural conditions. This pathogen is harmful and causes many plant diseases. Such as rice, oriental persimmon, pear, panicles of mango, and so on (Cho & Shin, 2004; Guillén-Sánchez et al., 2007; Lee et al., 2009). The infected fruit samples surface was disinfected with 75 % ethanol and 0.3 % NaClO for 30 s and 2 min respectively, then aseptic water washing three times. The fruit tissue is rich in carbohydrates and water content, which aid the growth of fungal species. Putting these diseased tissues on a potato dextrose agar (PDA) medium, cultured at 25 ± 1 ℃ for 7 days. The colonies grow on the PDA medium, then separated and puried again. Three pure cultures (YNGH01, YNGH03, YNGH05) were obtained, which were stored in 15 % glycerol at -80 ℃ refrigerator in the State Key Laboratory for Conservation and Utilization of Bio-Resources, Yunnan Agricultural University. After 7 days of cultivation, the colonies were round and the diameter attained up to 38 mm, the surface of the colony showed tiled, fluffy, with a velvet-like texture, greyish-green to smoke-gray, slightly raised in the middle, the edges were radial hollow and wrinkle (Fig. 1A). Conidiophores were solitary, erect, unbranched or rarely branched, slightly flexuous at the apex, septate, dark brown, 254 to 680 µm long, 3.6 to 4.5 µm wide, top of the conidiophores or the rostral were slightly swollen (Fig. 1B). Conidia were light gray or grey, solitary or bispora, irregular in shape and size (Fig. 1C), nearly circular (3.21 × 3.31 µm), oval to lemon-shaped (6.59 × 3.21 µm) or elliptical (8.35 × 4.65 µm). The CTAB method extracts 3 isolates (YNGH01, YNGH03, YNGH05) genomic DNA (Aboul-Maaty & Oraby, 2019). To confirm identity with molecular identification, performed by three different genomic DNA regions, fragments of internal transcribed spacer (ITS), partial translation elongation factor-1 alpha (TEF-1α), and actin (ACT) genomic regions. These genomic DNA were amplified with primers ITS1/4, EF1-728F/986R, and ACT-512F/783R, respectively (Carbone & Kohn, 1999). The sequences of these isolates were uploaded to GenBank (YNGH01: ON753810, ON868696, ON912090 YNGH03: ON753812, ON868698, ON912092, and YNGH05: ON753814, ON868700, ON912094). NCBI's BLASTn search of those ITS sequences showed 99.81% similar to C. tenuissimum (MG873077.1), and sequences TEF-1α and ACT were 100% identical to several isolates of C. tenuissimum (OM256526.1 and MT154171.1). Combined the ITS region, TEF-1α, and actin (ACT) genomic regions of isolates YNGH01, YNGH03 and YNGH05 to construct a phylogenetic tree with MEGA11. Maximum likelihood phylogenetic analyses further confirmed the results (Fig. 2)(Santos et al., 2020). Healthy and mature E. conferta fruits were used for pathogenicity test. Pathogens were washed with sterilized water at a final concentration of 2× 106 spores/mL (Jo et al., 2018). The test was divided into A and B groups (A: The surface of fruits was pierced with a sterilized needle that carried pathogenic fungus of final concentration at 2×106 spores/mL B: Sprayed at the concentration of 2×106 spores/mL on fruits). The control fruits were treated with sterilized water and stored at 25 ± 1 ℃ with a relative humidity of 80 %, average group with 10 fruits in this test, which was repeated three times. After 7 days, the fruits of group A were initially sesame seed size of the disease spots, nearly round, irregular, with grayish-brown spots, and slightly depressed. Later, the lesion gradually turns dark brown (Fig. 1D). And group B began with small patches of brown fungal growth on the pericarp, with the development of the disease, the necrotic spots enlarged and developed irregular and coalesced, the color of spots became gray or black gradually (Fig. 1E). The symptoms were similar to previously observed and the pathogen was reisolated and identified as C. tenuissimum. Control fruits were healthy (Fig. 1F). The pathogens test fulfilled Koch's postulates. According to morphology (Bensch et al., 2012), rDNA-ITS, TEF-1α, and ACT sequence analysis, phylogenetic analysis, and pathogenicity test, the pathogen was identified as C. tenuissimum. To our knowledge, this is the first report of C. tenuissimum occurring on E. conferta fruits in China.

Identification and Pathogenicity of Cladosporium, Fusarium, and Diaporthe spp. Associated with Late-Season Bunch Rots of Grape.

Fungal pathogens continue to pose a significant threat to grape production. Previous studies of pathogens associated with late-season bunch rots in Mid-Atlantic vineyards had elucidated the primary causal agents of these diseases, but the significance and identity of the less commonly isolated genera was unclear. Therefore, to more fully understand the identity and pathogenicity of Cladosporium, Fusarium, and Diaporthe spp. associated with late-season bunch rots of wine grapes in the Mid-Atlantic, phylogenic analyses and pathogenicity assays were conducted. Isolates were characterized to the species level by sequencing the TEF1 and Actin, TEF1 and TUB2, and TEF1 genes for 10, 7, and 9 isolates of Cladosporium, Diaporthe, and Fusarium, respectively. Four Cladosporium, three Fusarium, and three Diaporthe species were identified, and C. allicinum, C. perangustum, C. pseudocladosporioides, F. graminearum, and D. guangxiensis had not yet been isolated from grape in North America. The pathogenicity of each species was evaluated on detached table and wine grapes, and D. eres, D. ampelina, D. guangxiensis, and F. fujikuroi were found to be the most aggressive on both table and wine grapes. Further investigations through more extensive isolate collection and of myotoxicity testing may be warranted due to the prevalence and pathogenicity of D. eres and F. fujikuroi.