Tetramic acid-motif natural products from a marine fungus Tolypocladium cylindrosporum FB06 and their anti-Parkinson activities.

Tetramic acid-containing natural products are attracting significantly increasing attention from biologists and chemists due to their intriguing structures and biological activities. In the present study, two new tetramic acid alkaloids tolypyridone I (1) and tolypyridone J (2), together with five known ones (3-7), were isolated from cultures of a marine fungus Tolypocladium cylindrosporum FB06 isolate obtained from a marine sediment in Beaufort sea of North Alaska. Their structures were elucidated using 1D, 2D NMR, and HRESIMS. Their configurations were established on the basis of 1H coupling constants, ROESY correlations and DP4 calculations. Compound 2 was isolated as mixtures of rotational isomers with C-3 to C-7 axis between 4-hydroxy-2-pyridone and 1-ethyl-3,5-dimethylcyclohexane, hindering rotation. In our unbiased screening to discover neuroprotective compounds in an in vitro Parkinson's disease (PD) model, SH-SY5Y dopaminergic cells were treated with isolated compounds followed by treatment with 1-methyl-4-phenylpyridinium (MPP+), a parkinsonian neurotoxin. Among tested compounds, F-14329 (7) significantly protected cells from MPP+-induced cytotoxicity. MPP+-mediated cell death is known to be related to the regulation of Bcl-2 family proteins, specifically the down-regulation of anti-apoptotic Bcl-2 and the up-regulation of pro-apoptotic Bax levels. Treatment with 2 mmol/L of MPP+ for 24 h significantly reduced Bcl-2 levels compared to control treated with vehicle. However, treatment with F-14329 (7) attenuated such reduction. This study demonstrates that tetramic acid-motif compounds could be potential lead compounds for treating PD. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-023-00198-7.

Identification, Characterization, and Evaluation of Nematophagous Fungal Species of Arthrobotrys and Tolypocladium for the Management of Meloidogyne incognita.

Root-knot nematodes belonging to the genus Meloidogyne are agriculturally important pests, and biocontrol strategies offer safer alternatives for their management. In the present study, two fungal species from Indian soils were identified as Arthrobotrys thaumasia and Tolypocladium cylindrosporum based on morphological characteristics and further confirmed using molecular markers. In vitro evaluation of A. thaumasia against M. incognita and Caenorhabditis elegans showed 82 and 73% parasitism, respectively, whereas T. cylindrosporum gave 65.2 and 57.7% parasitism, respectively. Similarly, culture filtrates of A. thaumasia caused 57.7 and 53.7% mortality of M. incognita and C. elegans, respectively, whereas T. cylindrosporum caused higher mortality of 87.3 and 64%, respectively. Besides, greenhouse evaluation of both fungi against M. incognita infecting tomato significantly reduced nematode disease burden reflecting parasitic success measured as the total number of galls, egg masses, eggs per egg mass, and derived nematode multiplication factor. Application of A. thaumasia and T. cylindrosporum reduced nematode multiplication factor by 80 and 95%, respectively, compared with control. General metabolite profiling of tested fungi using gas chromatography-mass spectrometry and ultra-performance liquid chromatography-quadrupole/time of flight mass spectrometry reported for the first time here showed presence of various volatile and non-volatile compounds with nematicidal activity, viz., trimethyl-heptadiene, methyl-hexadecanol, dodecadienal, decane, terpendole E, dodecane, acetamido-6-anthraquinone, and hexadecanol. Also, other compounds such as undecane, dibutyl-disulfide, octadecenal, paganin, talathermophilin, dactylarin, tolypyridone A, tolypyridone B, pyridoxatin, and destruxin were identified, reported in the literature to possess antibacterial, antifungal, and insecticidal properties. This is the first report of the occurrence of both fungi from India and pioneer demonstration of T. cylindrosporum for root-knot nematode management.

Complete mitogenome of the entomopathogenic fungus Tolypocladium cylindrosporum.

In this study, the complete mitogenome of an entomopathogenic fungus Tolypocladium cylindrosporum is assembled and annotated. This circular mitogenome is 34,698 bp in length and consists of two rRNA genes (rnl and rns), 26 tRNA genes, 14 standard protein-coding genes of the oxidative phosphorylation system, and four intergenic free-standing ORFs. A total of six introns (all group I) were identified in atp9, cob, cox1, and rnl, and they may encode ribosomal protein S3, LAGLIDADG or GIY-YIG endonucleases. Phylogenetic analysis based on concatenated mitochondrial protein sequences confirms T. cylindrosporum in Ophiocordycipitaceae.

Characterization of Tolypocladium cylindrosporum (Hypocreales: Ophiocordycipitaceae) and Its Impact Against Aedes aegypti and Aedes albopictus Eggs at Low Temperature.

We examined the growth characteristics of Tolypocladium cylindrosporum IBT 41712 and its potential to infect eggs of Aedes aegypti and Ae. albopictus at a low temperature (15°C). When grown on Sabouraud dextrose agar supplemented with yeast extract, the IBT 41712 formed white colonies turning to a slightly darker, off-white color when mature. The mycelia bore swollen conidiophores producing smooth-walled, oblong to cylindrical conidia with varying sizes, ranging from 1.5 to 3.5 µm long. To determine the optimum temperature for the fungus, we cultured the fungus at eight temperatures (4°C, 12°C, 15°C, 21°C, 28°C, 33°C, 37°C, and 40°C) and measured the diametric growth. The optimum temperature for growth was 28°C since it had the highest diametric growth rate (2.1 ± 0.05 mm/day) and the fastest sporulation period (within 8-10 days of incubation). There was no fungal growth at the 3 highest temperatures (33°C, 37°C, and 40°C) but plates incubated at 33°C, when shifted to optimal temperature (28°C), showed visible growth indicating that following incubation at 33°C, the fungus remained viable. The IBT 41712 successfully infected mosquito eggs at 15°C. Fungal treatment induced egg hatch on moist seed-germination paper and this effect was more pronounced in Ae. aegypti compared to Ae. albopictus. When treated eggs were immersed in dH2O 21 days posttreatment, larval hatch of both Ae. aegypti (control = 91%, 1 × 107 conidia/ml, fungal treatment = 0%) and Ae. albopictus (control = 85%, fungal treatment = 28%) was significantly lower in fungal treatment compared to the controls. The ability of the strain to grow in a wide temperature range, and effectively infect mosquito eggs and induce egg hatch at a low temperature warrants further investigation for its potential as a mosquito control agent targeting eggs that overwinter or undergo long diapause.

Susceptibility of Cabbage Maggot Larvae (Diptera: Anthomyiidae) to Hypocreales Entomopathogenic Fungi.

The pathogenicity of six Metarhizium spp., four Beauveria bassiana (Balsamo) Vuillemin, and four Tolypocladium cylindrosporum Gams (Ascomycota: Hypocreales) fungal pathogens exposed to third-instar Delia radicum L. was evaluated in laboratory bioassays. The presence of intra- and intergeneric variations concerning the pathogenicity of the isolates was investigated. Results show that all Metarhizium spp. and T. cylindrosporum isolates caused a noteworthy mortality to the third instar and consequently reduced adult eclosion. The well-known standard, F52 strain (identified as Metarhizium brunneum), resulted in up to 79% reduction in D. radicum eclosion. The other Metarhizium isolates including UAMH 9197 (Metarhizium anisopliae) and UAMH 2801 (M. brunneum), as well as T. cylindrosporum DAOM 167325 and DAOM 183952, produced a mean eclosion reduction of >50%. While the pathogenicity of Metarhizium spp. and T. cylindrosporum is similar, the B. bassiana isolates are undoubtedly less pathogenic. Based on the results obtained with the selected isolates, no intrageneric differences relative to the pathogenicity of the isolates appeared to be present. Globally, this study deepened the knowledge about D. radicum susceptibility toward Hypocreales entomopathogenic fungi, chiefly T. cylindrosporum. The implications of this study regarding the development of a biological control agent are discussed.

Pathogenic and enzyme activities of the entomopathogenic fungus Tolypocladium cylindrosporum (Ascomycota: Hypocreales) from Tierra del Fuego, Argentina

Tolypocladium cylindrosporum is an entomopathogenic fungi that has been studied as a biological control agent against insects of several orders. The fungus has been isolated from the soil as well as from insects of the orders Coleoptera, Lepidoptera, Diptera and Hymenoptera. In this study, we analyzed the ability of a strain of T. cylindrosporum, isolated from soil samples taken in Tierra del Fuego, Argentina, to produce hydrolytic enzymes, and to study the relationship of those activities to the fungus pathogenicity against pest aphids. We have made the traditional and molecular characterization of this strain of T. cylindrosporum. The expression of hydrolase activity in the fungal strain was estimated at three incubation temperatures (4ºC, 12ºC and 24ºC), on different agar media supplemented with the following specific substrates: chitin azure, Tween ® 20, casein, and urea for chitinase, lipase, protease, and urease activity, respectively. The hydrolytic-enzyme activity was estimated qualitatively according to the presence of a halo of clarification through hydrolase action, besides was expressed semi-quantitatively as the ratio between the hydrolytic-halo and colony diameters. The pathogenicity of the fungus was tested on adults of the aphid Rhopalosiphum padi at three temperatures of incubation (4ºC, 12ºC and 24ºC). The suspension was adjusted to a concentration of 1x10 7 conidia/ml. In pathogenicity assays at seven days post-inoculation, the fungus caused the mortality of adults of Ropalosiphum padi at different temperatures also showed a broad ability to grow on several agar-culture media, supplemented with different carbon sources at the three incubation temperatures tested. Although, the growth was greater with higher incubation temperatures (with maximum levels at 24°C), the fungus reached similar colony diameters after 15 days of incubation on the medium supplemented with Tween® 20 at the lower two incubation temperatures of 4°C or 12°C. In accordance with the results on colony diameters, the fungus revealed an ability to degrade casein, chitin derivatives, Tween® 20, and urea as evidenced by the appearance of a halo around the fungal colony. Because of its origin and temperature tolerance, this Argentine strain has great potential for use as a biocontrol agent for insect pest control in cold and temperate environments. Rev. Biol. Trop. 60 (2): 833-841. Epub 2012 June 01.

El hongo entomopatógeno Tolypocladium cylindrosporum ha sido estudiado como un agente de control biológico contra insectos de varios órdenes. Esta especie fue aislada del suelo, así como de insectos de los órdenes Coleoptera, Lepidoptera, Diptera e Hymenoptera. En el presente trabajo hemos analizado la capacidad de una cepa de T. cylindrosporum (LPSC Nº1065) aislada del suelo en Tierra del Fuego, Argentina, para producir enzimas hidrolíticas y determinar la relación de esta actividad con la patogenicidad del hongo para combatir la plaga de los áfidos en diferentes temperaturas (4º, 12º y 24ºC). En los ensayos de patogenicidad, siete días posteriores a la inoculación, se registró mortalidad en los adultos del áfido Ropalosiphum padi a diferentes temperaturas y también se demostró una amplia capacidad de crecer en varios medios de cultivos complementados con diferentes fuentes de carbono bajo las tres temperaturas de incubación ensayadas. Debido a su origen y a la tolerancia que tiene a bajas temperaturas esta cepa, presenta un gran potencial para su uso como agente de control biológico para las plagas de insectos de ambientes fríos y templados.