Czapek-Dox Medium Research Articles

Seasonal and Geographical Impact on the Mycotoxigenicity of Aspergillus and Fusarium Species Isolated from Smallholder Dairy Cattle Feeds and Feedstuffs in Free State and Limpopo Provinces of South Africa.

This study evaluated the impact of seasonal and geographical variations on the toxigenicity of Aspergillus and Fusarium strains previously isolated from smallholder dairy cattle feeds and feedstuffs sampled during summer and winter in the Free State and Limpopo provinces of South Africa (SA). In total, 112 potential toxigenic fungal species were obtained and determined for their capability to produce mycotoxins on solid Czapek Yeast Extract Agar (CYA); followed by liquid chromatography-mass spectrometry (LC-MS/MS) analysis. Our result revealed that 41.96% of the fungal species produced their respective mycotoxins, including aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), and zearalenone (ZEN), with higher levels of AFB1 (0.22 to 1045.80 µg/kg) and AFB2 (0.11 to 3.44 µg/kg) produced by fungal species isolated from summer samples than those in winter [(0.69 to 14.44 µg/kg) and (0.21 to 2.26 µg/kg), respectively]. The same pattern was also observed for AFB1 and AFB2 in Limpopo (0.43 to 1045.80 µg/kg and 0.13 to 3.44 µg/kg) and Free State (0.22 to 576.14 µg/kg and 0.11 to 2.82 µg/kg), respectively. More so, ZEN concentrations in summer (7.75 to 97.18 µg/kg) were higher than in winter (5.20 to 15.90 µg/kg). A similar observation was also noted for ZEN in Limpopo (7.80 to 97.18 µg/kg) and Free State (5.20 to 15.90 µg/kg). These findings were confirmed via Welch and Brown-Forsythe tests with significantly (p ≤ 0.05) higher mycotoxin levels produced by fungal strains obtained in samples during summer than those in winter. In contrast, the concentrations of mycotoxins produced by the fungal species from both provinces were not significantly (p > 0.05) different.

Characterization and Fungicide Screening of a New Pathogen That Causes Leaf Spot on Rehmannia glutinosa

Outbreaks of leaf spot disease occurred in Rehmannia glutinosa fields in Henan Province, China, in 2019, with the incidence ranging from 20% to 40%. R. glutinosa plants with diseased leaves were collected, and 25 isolates were obtained. Pathogenicity tests, morphological observations, and phylogenetic analyses were conducted to identify the pathogens, and the biological characteristics and control agents of the pathogens were studied. Five isolates of pathogenic fungi were isolated. Three isolates were identified as Fusarium equiseti, which is a new pathogen causing R. glutinosa leaf disease; the other two isolates were identified as Fusarium acuminatum. The mycelia of F. equiseti grew fastest on Czapek medium, and the optimal temperature and pH were 25 °C and 10.0, respectively. The mycelia of F. equiseti grew from 5 °C t o 35 °C, and the lethal temperature was 55 °C. The optimal carbon and nitrogen sources were soluble starch and peptone, respectively. Eight fungicides had inhibitory effects on the mycelial growth of F. equiseti and F. acuminatum. Prochloraz had higher activities against F. equiseti and F. acuminatum, with EC50 values of 0.139 mg·L−1 and 0.123 mg·L−1, respectively. These results provide useful information that will aid the development of management strategies to control leaf diseases of R. glutinosa caused by F. equiseti and F. acuminatum.

Biotransformation of grease waste into fatty acid by Penicillium chrysogenum SNP5 through media engineering and artificial neural network.

Degradation of grease waste remains a challenging task. Current work deals with the biotransformation of grease waste into fatty acids under submerged fermentation using Penicillium chrysogenum SNP5 through media formulation and artificial neural network (ANN). Fermentation media was formulated to ameliorate the uptake of hydrocarbon by enhancing alkane hydroxylase (AlkB) activity, extracellular release of fatty acids and inhibiting beta-oxidation of fatty acid by regulating transketolase. Further, the process parameters of fermentation were optimized through Artificial Neural Network (ANN) using three critical variables viz; inoculum size (spores/ml), pH, and incubation time (days) while media engineering was done with the optimal supplementation of various medium components such as glucose, YPD, MnSO4, tetrahydrobiopterin (THB) and phloretin. The maximum conversion of 66.5% of grease waste into fatty acid was achieved at optimum conditions: inoculums size 3.36 × 107 spores/ml, incubation time 11.5days, pH 7.2 along with formulated media composed of 1% grease in czapek-dox medium supplemented with 55.5mM glucose, 0.5% YPD, 16.6mM hexadecane, 1mM MnSO4, 1mM THB, and 1mM phloretin. The presence of long-chain fatty acids in purified extracts such as oleic acid and octadecanoic acid as end products has valued the evolved process as another source of alternative fuel.

Amazonian soil fungi are efficient degraders of glyphosate herbicide; novel isolates of Penicillium, Aspergillus, and Trichoderma.

Pesticide residues that contaminate the environment circulate within the hydrological cycle can accumulate within the food chain and cause problems to both environmental and human health. Microbes, however, are well known for their metabolic versatility and the ability to degrade chemically stable substances, including recalcitrant xenobiotics. The current study focused on bio-prospecting within Amazonian rainforest soils to find novel strains fungi capable of efficiently degrading the agriculturally and environmentally ubiquitous herbicide, glyphosate. Of 50 fungal strains isolated (using culture media supplemented with glyphosate as the sole carbon-substrate), the majority were Penicillium strains (60%) and the others were Aspergillus and Trichoderma strains (26 and 8%, respectively). All 50 fungal isolates could use glyphosate as a phosphorous source. Eight of these isolates grew better on glyphosate-supplemented media than on regular Czapek Dox medium. LC-MS revealed that glyphosate degradation by Penicillium 4A21 resulted in sarcosine and aminomethylphosphonic acid.

IN VITRO ANTAGONISTIC ACTIVITY OF GLIOCLADIUM SP.

Objective. Get effective isolates of Gliocladium sp. by in vitro evaluation of the antagonistic activity against phytopathogenic species of fungi obtained from different substrates. Methods. Study of the efficiency of Gliocladium sp. Was conducted against Botrytis cinerea, Fusarium solani, F. oxysporum, F. poae, Cladosporium sp., Alternaria tenuissima and A. arborescens. Fungi were identified by anamorph morphology. The dual culture was grown on Czapek-Dox medium with glucose. Incubation was carried out for seven days in a thermostat at 22–25 °C. Results. Gliocladium sp. was isolated from the roots of winter wheat of Bohdana variety after seed sprouting in the soil for three days. During this period, the fungi colonized the roots, while the analysis of the seeds on potato-glucose agar did not detect them. Isolates of the studied genus had a significant growth rate. Study of the antagonistic activity of Gliocladium sp. against B. cinerea and F. solani 1 showed high efficiency against the former and very low against the latter. Inhibition of fungal growth was different. B. cinerea developed worse when grown with Gliocladium sp. than when grown alone. During separate cultivation of F. solani 1, its growth lag was registered for the first two days of recording, compared to dual culture. The study of five different isolates showed their different effects on the studied fungi. Isolate No. 4 showed the highest antagonistic activity. Conclusion. The minimum percentage of inhibition of the development of phytopathogenic fungi using isolates of Gliocladium sp. was 15.6 % for F. solani 1, the maximum was 81.3 % for F. poae. Isolate No. 2 had the lowest parameters of fungal growth inhibition, but it was the only one that effectively restrained the development of F. oxysporum. Isolate No. 6 inhibited the development of B. cinerea by 69.9 %. Study of the efficiency Gliocladium sp. isolates against the representatives of various species and genera demonstrated their better effect on melanized fungi. The greatest growth inhibition was reported for Alternarium species and Cladosporium sp.

Variability in metabolites produced by Talaromyces pinophilus SPJ22 cultured on different substrates

BackgroundSeveral metabolites released by fungal species are an essential source of biologically active natural substances. Gas chromatography high resolution time-of-flight mass spectrometry (GC-HRTOF-MS) is one of the techniques used in profiling the metabolites produced by microorganisms, including Talaromyces pinophilus. However, there is limited information regarding differential substrates’ impacts on this fungal strain’s metabolite profiling. This study examined the metabolite profile of T. pinophilus strain SPJ22 cultured on three different media, including solid czapek yeast extract agar (CYA), malt extract agar (MEA) and potato dextrose agar (PDA) using GC-HRTOF-MS. The mycelia including the media were plugged and dissolved in 5 different organic solvents with varying polarities viz.: acetonitrile, dichloromethane, hexane, 80% methanol and water, and extracts analysed on GC-HRTOF-MS.ResultsThe study revealed the presence of different classes of metabolites, such as fatty acids (2.13%), amides (4.26%), alkanes (34.04%), furan (2.13%), ketones (4.26%), alcohols (14.89%), aromatic compounds (6.38%), and other miscellaneous compounds (17.02%). Significant metabolites such as acetic acid, 9-octadecenamide, undecanoic acid methyl ester, hydrazine, hexadecane, nonadecane, eicosane, and other compounds reported in this study have been widely documented to have plant growth promoting, antimicrobial, anti-inflammatory, antioxidant, and biofuel properties. Furthermore, T. pinophilus grown on PDA and MEA produced more than twice as many compounds as that grown on CYA.ConclusionThus, our result showed that the production of essential metabolites from T. pinophilus is substrate dependent, with many of these metabolites known to have beneficial characteristics, and as such, this organism can be utilised as a sustainable and natural source for these useful organic molecules.

Cacti as low-cost substrates to produce L-asparaginase by endophytic fungi.

This study aimed to select endophytic fungi to produce L-asparaginase and partially optimising the production of the enzyme using cacti as substrate. Seventeen endophytes were assessed for intracellular enzymatic potential in modified Czapek Dox's medium using L-proline as an inducer. The best producer was evaluated for intracellular and extracellular enzymatic activity in modified Czapek Dox's medium using flours of Opuntia ficus-indica and Nopalea cochenillifera as substrate. The biomass and L-asparaginase production profile was analysed and the best conditions for enzyme production were verified using factorial design. Penicillium decaturense URM 7966, Diaporthe ueckerae URM 8321, and Colletotrichum annellatum URM 8538 produced 0.76 U g- 1, 0.87 U g- 1, and 0.74 U g- 1L-asparaginase, respectively. Diaporthe ueckerae URM 8321 produced only intracellular L-asparaginase, using flours of N. cochenillifera (0.72 U g- 1) and O. ficus-indica (0.90 U g- 1) and the last was selected for the next steps. The ideal time for biomass and L-asparaginase production was 120h. The best conditions for enzyme production (1.67 U g- 1) were initial pH 4.0, inoculum concentration 1% and cacti flour concentration 0.2%; where was observed an increase of 46.11% in compared to the initial production. Opuntia ficus-indica flour is indicated as an alternative low-cost substrate for the production of L-asparaginase by the endophytic fungus D. ueckerae URM 8321.

<i>Trichoderma spp.</i> strains: growth and interaction at different temperatures

The paper examines the growth of Trichoderma spp. strains and their interaction at different temperatures. Trichoderma spp. is a genus of microscopic ascomycetes; these fungi belong to different ecosystems and are found in soil (rhizosphere). The study aims to analyze and compare the characteristics pertaining to the growth of Trichoderma species and the interaction between them. The experiments used the following media: a Czapek medium and a medium simulating plant root exudates (Cucumis sativus). The growth rate of Trichoderma spp. strains was determined at different temperatures. The use of a medium containing plant exudate models provided a different growth rate as compared to that obtained when using a rich growth medium; some variations in the macromorphology of colonies were also observed. The growth of Trichoderma longibrachiatum F2124 and Trichoderma viride F2001 strains was detected at 9 °С on the Czapek medium, while only the Trichoderma longibrachiatum F2124 strain was observed to grow on the medium simulating exudates. A significant growth inhibition was noted at 40 °С for all strains, except for Trichoderma longibrachiatum species. No colony growth was observed at 50 °С. All the strains grew within the temperature range of 12 to 28 °С. It was assumed that Trichoderma species growing in various media may interact differently with each other. Interaction between the three strains of Trichoderma spp. was examined via the surface culture method under different temperature conditions. The study found no significant differences in the interaction between the Trichoderma species.

Preliminary screening of toxigenic fungi and mycotoxin contamination: A case of agricultural products in Ivory Coast

Toxigenic fungi are generally known to contaminate agricultural products; the causes, rate, and extent of its effects on common products have less emphasis, especially in sub-Saharan African countries. This lack of emphasis could be attributed to the state of research, storage, and accountability that could have intimated the public with necessary measures to curb the menace.For the purpose of the food safety control, this study investigates the fungal presence and mycotoxins in major food crops such as cereals (millet, rice, white and yellow maize), vegetables (chili, gnangnan, haricot, melon, and okra), legumes (haricot), and root products (attieke, cassava flakes) in Abidjan, Ivory Coast. Each sample is serially diluted and inoculated onto Czapek yeast agar (CYA), malt extract agar (MEA) and potato dextrose agar (PDA), to screen for fungal contamination. Colony color, colony edge, mycelia color, conidia shape, and microscopic features are used to identify the isolates. In addition, thin layer chromatography (TLC) was used to identify seven mycotoxins in food commodities. It is revealed that, Aspergillus species are found in 64 (92%) of the samples, Penicillium species in 29 (41%), and Fusarium species in 20 (29%) of the samples. A significant incidence rate of aflatoxin B1 (AFB1) is found in vegetables (gnangnan, haricot, and melon), roots products (cassava flakes), cereals (rice, and white maize). According to this investigation, each sample contains one or more fungus and mycotoxins.

The use of tissue culture method for the study of lichenized fungi (Lobariaceae Chevall., Parmeliaceae Zenker.) of the European part of Russia

Lichenized fungi as a multicomponent biological system are an interesting but difficult research object. In view of the complex interaction of the myco- and photobiont, the slow growth of natural thalli, their exceptional exactingness to environmental factors, the study of the biological characteristics of lichens is significantly difficult. This article discusses the problem of studying the anatomical and morphological structure of the myco- and photobiont of lichenized fungi using the tissue culture method on the example of rare species (Lobaria pulmonaria) and species with pharmaceutical potential (Usnea dasopoga, Cetraria islandica). The authors proposed a method for cultivating of myco- and photobionts of these species on synthetic nutrient media for the purpose of further research on the characteristics of lichenized fungi. Fragments of thalli, apothecia (for C. islandica), and soredia (for U. dasopoga and L. pulmonaria) were used as donor material. For the introduction of the photobiont under sterile conditions, a homogenate of the thallus region was prepared. Pure cultures of the myco- and photobiont of L. pulmonaria, U. dasopoga, and C. islandica were obtained on three types of hormone-free nutrient media, namely MS nutrient medium, modified MS medium with a reduced nitrogen content, Czapek medium. The verification of the research results was carried out taking into account the microscopy of the obtained cultures of myco- and photobionts. Using microscopy, the dimensional characteristics of the obtained mycobionts were determined. The diameters of the hyphae of L. pulmonaria, U. dasopoga, and C. islandica were 4.3–4.4 µm, 4.6 µm, and 4.1–4.3 µm, respectively, which corresponds to the size of the hyphae in natural samples. The use of the tissue culture method makes it possible to study and analyze the biological characteristics of lichenized fungi as a multicomponent biological system, as well as to contribute to the preservation of rare species and their components in the Red Data Book; while optimizing the method of cultivating myco- and photobiont on synthetic nutrient media, to reduce the anthropogenic load on natural populations of lichens when using them as medicinal raw materials.

SELECTION OF APPROPRIATE NUTRIENT MEDIUM FOR CULTIVATION OF PYRENOPHORA TERES DRECHSLER

Net blotch (Pyrenophora teres Drechsler) is the dominant pathogenic complex of barley leaf diseases worldwide. The study of various methods of controlling hemibiotrophic fungus requires careful preparation and development of a sufficient amount of pathogenic inoculum. The aim of our research was to select appropriate nutrient medium for obtaining P. teres inoculum with maximum aggressiveness parameters (colony growth rate, sporulation, short latency period) and ergonomics in preparation. Three nutrient media were used: carrot-beet agar (CBA), potato-glucose agar (PGA), Czapek's medium for standard methods and cultivation conditions. The selection consisted of 10 isolates for each nutrient medium and had statistically significant differences among the experimental variants. It was found that appropriate combination of parametres such as growth rate (5.3±0.5 mm/day), colony diameter (53.4±1.1 mm), latent period (4 days), sporulation intensity (4100 conidia in 1 ml) was observed in case of cultivation of P. teres isolates on carrot-beet agar on the 6th day. When cultivating the fungus on potato-glucose agar, rather high growth rates (5.2±0.8 mm/day), colony diameter (52.0±2.9 mm), sporulation intensity (3700 conidia per 1 ml) were revealed, the latent period was the longest among the studied variants, the first sporulation was detected on the 6th day. In case of usage of the standard Czapek medium, the minimum aggressiveness of P. teres isolates was revealed (growth rate - 3.2 ± 0.3 mm/day; colony diameter - 31.6 ± 1.9 mm; latent period - spores were revealed on the 4th day in 1 cup; the intensity of sporulation was 500 conidia per 1 ml). It was noted that there is a larger range of morphological types of colonies on carrot-beet agar, including morphotypes of isolates which grow on other media. Almost all experimental variants cultivated on CBA medium had a clear structured mycelium with a dark color, which indicates a sufficient availability of nutrients in the culture medium. Thus, it is reasonable to use carrot-beet agar in order to obtain P. teres inoculum with maximum aggressiveness parameters and low labor costs when using standard cultivation methods.

First Report of Dieback of Salix acmophylla Caused by Paecilomyces formosus in Iran.

Salix acmophylla Boss. (Brook willow), is native to Iran and mainly grows in natural riparian landscapes. It has excellent potential to use in erosion and phytoremediation projects. Since 2018, dieback has been observed on S. acmophylla at the Gilan-e Gharb forest (34°05'59.9"N, 46°00'52.6"E) in Kermanshah province, Iran. Affected trees showed wilting and yellowing of leaves, cankers, branch dieback, and decline (Fig. 1A, B). Small pieces (5 mm2) from the canker margins of 10 affected tree branches were surface sterilized with 75% ethanol for 30 s and 1% NaClO for 90 s. Subsequently, samples were rinsed three times with sterile H2O, plated on potato dextrose agar (PDA) supplemented with 100 mg/l streptomycin sulfate and 25 μg/mL chloramphenicole, and incubated at 25°C for 5 days. The same fungus was isolated from all samples. Pure cultures were obtained by monosporic isolation. A representative isolate, RU-PaMa-S1, was used for morphological and molecular characterization and deposited in the Fungal Reference Collection of the Ministry of Jihad-e Agriculture at the Iranian Research Institute of Plant Protection, Tehran, Iran. Radial growth of colonies on PDA and Czapek yeast extract agar (CYA) were 30 and 25 mm after seven days at 25 °C in the dark, respectively. Fungal colonies grew more rapidly on malt extract agar (MEA), and reaches a diameter of about 70 mm within 7 days at 25 °C in the dark. Color of colony on MEA, PDA, and CYA was light yellow to yellow-brown, which gets darker upon aging. In biochemical assay, our isolate grow and produced acid in culture on creatine sucrose agar medium. The conidiophore was irregularly branched out with one to seven phialides. The phialides were in whorls or solitary, flask shaped with cylindrical bases, and narrow apices. Conidia were one-celled, hyaline to yellow brown in mass, smooth, and ellipsoidal to cylindrical with truncate ends. Conidia were 3.0-9.6 (4.5) × 1.7-4.0 (2.6) µm (n = 50). Chlamydospores were present in two week old cultures. The morphology matched the description of Paecilomyces formosus (Sakag., May., Inoue and Tada) Houbraken and Samson (Samson et al., 2009). For an accurate identification, genomic DNA of RU-PaMa-S1 was extracted to amplify ITS regions, and β-tub gene with ITS1/ITS4 (White et al. 1990), and βt2a/βt2b (Glass and Donaldson 1995) primers, respectively. The sequences were deposited in GenBank (ITS: OL891502, β-tub OL944475). A BLAST search of GenBank showed that the ITS, and β-tub sequences of RU-PaMa-S1 were similar to those of Paecilomyces formosus. Baed on the multigene phylogeny and morphology, RU-PaMa-S1 isolate was identified as Paecilomyces formosus (Fig. 2). Molecular analyses based on sequencing of the ITS region, and parts of the β-tubulin and calmodulin genes, showed that P. formosus may be composed of three species, including P. maximus, P. lecythidis and P. formosus. However, the three species could be distinguished by only molecular data and not by microscopical examination (Samson et al. 2009). To fulfill Koch's postulates, 10 detached and 10 attached healthy branches from two 20-year-old S. acmophylla plants at the Gilan-e Gharb forest in Kermanshah province, Iran, were inoculated with 5-mm mycelial plugs of 5-day-old culture of RU-PaMa-S1 isolate (Fig. 1 C). Control branches were treated with agar plugs. The detached inoculated branches were stored in sterilized glass jar containing moist sponge and incubated at 25-30°C for 35 days until the appearance of the canker symptoms. After 35 days, inoculated branches were cut from the trees and transferred to the laboratory to check disease development and re-isolation of the pathogen. All inoculated points on detached and intact branches showed lesions similar to those observed on naturally infected trees, whereas controls were symptomless (Fig. 1 D, E). The same fungus was re-isolated from these lesions. P. formosus has been reported to infect a broad range of hosts, including Amygdalus scoparia, Malus domestica, Pistatia vera, and Querqus brantii (Sabbagh and Khosravi Moghaddam 2016; Sabernasab et al. 2019; Azizi et al. 2020). This is the first report of P. formosus on S. acmophylla in Iran and in the world. This finding provides crucial information on this high-risk disease to S. acmophylla and other forest trees and basis for identifying management strategies.

Two Bioactive Compounds, Uniformides A and B, Isolated from a Culture of Nocardia uniformis IFM0856T in the Presence of Animal Cells.

Two new peptides named uniformides A and B (1 and 2, respectively) were isolated from the cultured extracts of Nocardia uniformis IFM0856T in the presence of mouse macrophage-like cell line J774.1, in modified Czapek-Dox medium. These compounds were not produced in a culture containing only N. uniformis but in one that also included J774.1. Compounds 1 and 2 showed high cytotoxicity against J774.1 and suppressed the production of nitric oxide, IL-6, and IL-1β by inhibiting the NF-κB pathway.

First Report of Leaf Spot Caused by Colletotrichum fructicola on Myrica rubra in China.

Myrica rubra is an important fruit tree with high nutritional and economic value, which is widely cultivated in multiple regions of China. In January 2021, an unknown disease which caused leaf spot with approximately 20% (n=100 investigated plants) of incidence was discovered on the leaves of M.rubra in Jiujiang City of Jiangxi Province (29.71° N, 115.97° E). The initial symptoms were small pale brown spots (1 to 2 mm diameter) on the leaves, which gradually expanded into round or irregular dark brown spots with the occurrence of the disease, and the lesion developed necrotic tissues in the center at later stages, eventually leading the leaves to chlorotic and wilted. Ten diseased leaves with typical symptoms were collected and the leaf tissue (5 × 5 mm) at junction of diseased and healthy portion were cut. The surfaces were disinfected with 75% ethanol for 45 s, 1% sodium hypochlorite for 1 min, and rinsed in sterile water for 3 times then transferred to potato dextrose agar (PDA) at 28 ± 1 ℃ for 3 days. Five fungal single isolates with similar morphology were purified from single spores. On PDA medium, the colonies initially appeared white with numerous aerial hyphae, and the center of the colony turned gray at later stages, less sporulation. While on modified czapek-dox medium (Peptone 3g, K2HPO4 1g, MgSO4·7H2O 0.5g, KCl 0.5g, FeSO4 0.01g, Maltose 30g, Agar 15g, Distilled water 1000 mL, pH=7.0), the mycelia of the colony were sparse and produced a large number of small bright orange particles (conidial masses). Conidia were single-celled, transparent, smooth-walled, 1-2 oil globule, cylindrical with slightly blunt rounded ends, 14.45-18.44 × 5.54-6.98 μm (av=16.27 μm × 6.19 μm, n=50) in size. These morphological characteristics of the pathogen were similar to the descriptions of Colletotrichum fructicola (Ruan et al, 2017; Yang et al, 2021). To further confirm the identity of the pathogen, genomic DNA from a representative isolate was extracted with DNA Extraction Kit (Yeasen, Shanghai, China), and the internal transcribed spacer (ITS), glyceraldehyde-3-phosphatedehydrogenase (GAPDH), calmodulin gene (CAL), actin (ACT) and chitin synthase 1 (CHS 1) were amplified by using the primers ITS1/ITS4 (Gardes et al, 1993), GDF/GDR (Templeton et al, 1992), CL1C/CL2C (Weir et al, 2012), ACT-512F/ACT-783R and CHS-79F/CHS-345R (Carbone et al, 1999), respectively. The PCR amplified sequences were submitted to GenBank (GenBank Accession No. ITS, MW740334; GAPDH, MW759805; CAL, MW759804; ACT, MW812384; CHS-1, MW759803) and aligned with GenBank showed 100% identity with C. fructicola (GenBank Accession No. ITS, MT355821.1 (546/546 bp); GAPDH, MT374664.1 (255/255 bp); CAL, MK681354.1 (741/741 bp); ACT, MT364655.1 (262/262 bp); CHS, MT374618.1 (271/271 bp)). Phylogenetic tree using the maximum likelihood methods with Kimura 2-parameter model and combined ITS-ACT-GAPDH-CHS-CAL concatenated sequences, bootstrap nodal support for 1000 replicates in MEGA7.0, revealed that the isolate was assigned to C. fructicola strain (ICMP 18581 and CBS 125397) (Yang et al. 2021) with 98% bootstrap support. Pathogenicities of were tested on fifteen healthy M. rubra plants (five for wounded inoculation, five for nonwounded inoculation, and five for controls) in the orchard. Twenty leaves were marked from each plant, and disinfected the surface with 75% ethanol. Ten μL spore suspension (1.0 × 106 conidia/ml) of each isolate from 7-day-old culture were inoculated on the surface of 20 needle-wounded and 20 nonwounded leaves, respectively. Healthy leaves were inoculated with sterile water as controls by the same method. All inoculated leaves were sprayed with sterile water and covered with plastic film to remained humidification. After 5 days, all the wounded leaves which were inoculated with C. fructicola showed similar symptoms to those observed on the original leaves. Symptoms of nonwounded leaves were milder than the wounded inoculated leaves, while control leaves remained healthy. Finally, the C. fructicola was re-isolated from the inoculated leaves. C. fructicola has been reported on Juglans regia, Peucedanum praeruptorum, Paris polyphylla var. Chinensis in China (Wang et al, 2017; Ma et al, 2020; Zhou et al, 2020). As far as we know, this is the first report of C. fructicola causing leaf spot on M.rubra in China. This result contributes to better understand the pathogens causing diseases of M.rubra in this region of China and develop effective control strategies.

Identification of causal agent of wilt of common sage (Salvia officinalis L.)

Summary Introduction: Common sage is cultivated in Europe and North America. It has strong antiviral, antibacterial and antifungal properties. This plant can be infected by different pathogenic fungi species, such as Alternaria alternata, Fusarium spp. (F. culmorum, F. equiseti, F. oxysporum), Phomopsis sclarea and Botrytis cinerea. Those species are the most frequently isolated fungi from sage stem base. Objective: The aim of this study was to identify the causal agent of common sage wilt disease. Methods: Studies were carried out in 2018–2020. 23 fungal isolates were identified based on their morphology and with use of PCR technique. Length and width of 100 conidia growing on SNA medium were measured after 7 days. Koch’s postulates were checked and the development of one fungus isolate (no. 13) was compared on seven media: the CMA, MEA, OA, PCA, SNA, PDA and Czapek medium. Sequences of the second largest subunit of RNA polymerase II (RPB2) were used to identify the pathogen. Results: The fungus formed 3 kinds of spores: thin-walled, hyaline, slightly folded at the base, mostly 4-cell macroconidia, oblong, hyaline one- or two-cell microconidia and oval thick-walled chlamydospores. The Koch’s postulates were fulfilled. The fungus formed the most abundant aerial mycelium on the Czapek medium, and the least on the CMA medium. On the SNA medium, the mycelium grew into the medium and the aerial mycelium was not formed. The obtained RPB2 nucleotide sequence was 100% similar to the Fusarium oxysporum sequence deposited in GenBank (NCBI). Conclusions: The results of this research can be used in further studies on the biological diversity of this species.

Incidence of Filamentous fungi in some food commodities from Ivory Coast

This study surveyed important food crops consumed in Ivory Coast for fungi. To achieve this, the following local food items (attieke, cassava flakes, chili, gnangnan, haricot, melon, millet, okra, rice, white maize and yellow maize) were sampled from local markets (Adjame, Cocody and Youpougon) in Abidjan, Ivory Coast. These food crops were screened for fungal contaminants, and each sample was serially diluted to a concentration of 10 −5 . One hundred microliters of each mixture at 10 −3 10 −4 and 10 −5 were inoculated onto potato dextrose agar (PDA), Czapek yeast agar (CYA), and malt extract agar (MEA). The isolates were identified using morphological characters and confirmed by PCR with the internal transcribed spacer 1 and 4 primers (ITS1 and ITS4). A total of 227 isolates were morphologically identified and confirmed to be in the genera Aspergillus (54.9%), Penicillium (23.3%) and Fusarium (14.3%). Few isolated species were identified as Alternaria, Chaetomium , Cladosporium, Epicoccum, Emerica, Rhizopus and Tr ichoderma spp. The highest mean fungal load of 5.9 log 10 CFU/g was found in maize. Confirmed isolates were dominated by Aspergillus species which were frequent in cassava flakes, chili, gnangnan, haricot, rice and yellow maize. Penicillium species were found to be frequent in chili, haricot and rice, while Fusarium species highly prevalent in melon and millet. Isolates from food commodities in this study were grouping with known toxigenic fungal species.

Optimization of l-methioninase and l-arginase production by newly isolated marine yeast using response surface methodology

l-methioninase and l-arginase are important anticancer agents that are used extensively worldwide. In this study, fifteen marine yeast isolates were obtained from the marine environment of Red Sea and screened for l-methioninase and l-arginase production. A promising isolate, S5 was identified based on 18 S rRNA gene sequencing as Candida labiduridarumS5. Collectively, screening of different culture variables was achieved by Plakket-Burmn experimental design using Czapek Dox medium in order to determine the most significant parameters for l-methioninase and l-arginase production. With respect to the main effect of each variable, namely five independent factors, yeast extract, l-methionine, l-arginine, pH and incubation period had a significant effect on l-methioninase and l-arginase production by Candida labiduridarum. Subsequently, these significant parameters affecting enzymes production were optimized using the Box-Behnken and its representative four-factor response-surface method for each enzyme separately. These predicted conditions were verified by validation experiments and the optimal conditions for the highest l-methioninase production were l-methionine (3.4646 g/L), yeast extract (3.1414 g/L), pH (6.06) and for 38.6869 h. Meanwhile, the optimal conditions of l-arginase was l-arginine (3.5859 g/L), yeast extract (5.0808 g/L), pH (6.5758) and for 44.2805 h. The regression models demonstrated that the practical data could be suitably fitted into a first-order model with a significantly high (R2) value of more than 0.90 for all responses. The optimized process increased production of l-methioninase by 35.57 U/mg (2.2-fold) and l-arginase by 43.62 U/mg (4.9-fold), when compared to that obtained in the control medium. The total yield of the predicted l-methioninase and l-arginase was found to be near the practical result 32.8841 and 40.9934 U/mg respectively. Thus, this statistical approach enabled rapid identification and integration of key medium parameters for enhancing production of l-methioninase and l-arginase.

Endophytic community of Chaenomeles speciosa fruits: Screening for biodiversity and antifungal activity

Fruit crops of the genus Chaenomeles Lindl are considered today as a superfood due to accumulation of biologically active compounds with antioxidant ability and known health-promoting properties. Successful introduction of this non-traditional culture in the steppe zone of Ukraine characterised by an unfavourable climate suggests the functioning of effective protective mechanisms in plants, including those that can be provided by the influence of endophytic microorganisms. However, there is little information about the endophytic community of Chaenomeles plants. Herein, the current study was aimed to isolate the endophytic fungi from the Ch. speciosa fruits and evaluate their biological activities against the phytopathogens. The study was carried out based on the collection of the Botanical Garden of Oles Honchar Dnipro National University (Dnipro city, Ukraine). Three media, namely PDA, MPA, and Gause’s medium were used for isolation of endophytic fungi. Colonies of isolates for identification were grown on PDA, Czapek's agar, and Czapek’s yeast autolysate media. Six fungal endophytic isolates derived from both peel and pulp of Ch. speciosa fruits have been morphologically identified using macroscopic and microscopic techniques, and assigned to the genus Penicillium (sections Chrysogena, Penicillium, Viridicata), and genus Talaromyces (section Talaromyces). Species P. expansum, P. viridicatum, and P. hirsutum were identified among the peel isolates, while P. chrysogenum, P. cyclopium, and P. purpurogenum were among the pulp isolates. Antagonistic ability of the endophytic isolates against phytopathogenic fungi was evaluated using the dual culture method. The results showed moderate to high antifungal capacity of the endophytic isolates against the phytopathogenic strains of the Fusarium genus. The growth inhibition of F. culmorum mycelium due to the influence of endophytic isolates was 51.5–81.3%, and the inhibition of the growth of F. oxysporum colonies was in the range of 68.4–86.6% as compared with control. There were no significant differences in the antagonistic ability between endophytic isolates derived from the peel and pulp of the fruit. Taken together, our findings indicated the great potential of the endophytic fungi from Ch. speciosa fruits as a source for the development of biocontrol agents and discovery of new bioactive compounds.

SCREENING OF DIFFERENT CULTURE MEDIA FOR GROWTH DEVELOPMENT, AND MORPHOLOGICAL CHARACTERISTICS OF Aspergillus niger

This research on the screening of different culture media for growth development, and morphological characteristics of Aspergillus niger was carried out between February and July 2021, to determine the best agar media for the growth of Aspergillus niger. Five different agar mediums are used to carry out the research. The agar mediums includes Potato Dextrose Agar mediums (PDA), Czapek's Yeast Extract Agar mediums (CYEA), Sabouraud's Dextrose Agar medium (SDA), Malt Extract Agar medium (MEA) and Potato carrot agar medium (PCA). A spoiled food samples was filled in a beaker with a normal saline and was allowed to dissolve for 15mins. The sample was checked very well every 4 hours and also filtered into a sterile tube. After that 1ml of the sample was collected using a sterile string and innoculate it in each of the already prepared agar mediums in a petri dishes and incubate it at 37oC for 48hours and count the observed fungal growth using the colony counter. PDA yielded a total growth count of 19.55x104 after 48hours of incubation at 37oC. CYEA yielded a total growth count of 10.0x104 after 48hours of incubation at 37oC. SDA yielded a total growth count of 5.00x104 after 48hours of incubation at 37oC. MEA yielded a total growth count of 1.20x104 after 48hours of incubation at 37oC and PCA yielded a total growth count of 7.5x104 after 48hours of incubation at 37oC. The study shows that Aspergillus niger grows mostly and was able to multiply rapidly on Potato dextrose agar medium (PDA) because these media contains the most nutritive components needed by the A. niger to carry out its metabolism and reproduce by its spore formation. And according to the morphological characteristics of Aspergillus niger they all have a black coloration when viewed under the light microscope.

Pathogenicity and Biological Characteristics of Septotinia populiperda Causing Leaf Blotch of Willow.

Salix babylonica is an important landscape tree in China and has been widely planted. In this study, the pathogenicity of Septotinia populiperda causing leaf blotch of Sa. babylonica to four willow species (Sa. matsudana, Sa. chaeomoloides, Sa. matsudana f. tortuosa, and Sa. suchowensis) and Populus tomentosa (Chinese white poplar) was determined. Its sexual stage and biological characteristics were studied. Leaves from four willow species and P. tomentosa were inoculated with mycelial plugs. Typical leaf blotches with sporodochia were produced on all inoculated leaves. Among the isolates studied, some developed conidia but sclerotia were rare. The sclerotia developed apothecia after induction at 4°C for 3 months in an incubator and 2 more months outdoors from January to March. The biological characteristics of S. populiperda showed that mycelium grew better on complete medium than on potato dextrose agar, Czapek's agar, and minimal medium. For mycelial growth, the optimal carbon source was dextrose and the optimal nitrogen source was yeast powder. Conidia germination rate was 59.4% at 24 h. The conidia germinated best in a 4% willow leaf extraction. The optimal temperature for conidia germination was 25°C, and the optimal pH was 4.