Malt Extract Agar Research Articles

87 Portuguese Groceries: A Potential Indoor Environment to Be Used as Public Health Sentinel for Fungal and Mycotoxins Contamination

Abstract This study aimed to characterize microbial contamination in Portuguese grocery stores. The research was carried out in 15 grocery stores in Cascais, Portugal. Electrostatic dust cloths (EDC) and surface swabs were used on 3 sampling sites: checkout, fruits/vegetable, and warehouse/dispenser. Fungal contamination was characterized by culture-based methods, through the inoculation onto two different culture media: malt extract agar (MEA) supplemented with chloramphenicol (0.05 %) and dichloran-glycerol agar (DG18). Screening of azole resistance was performed by inoculation of EDC extracts´ in azole-supplemented Sabouraud dextrose agar (SDA) media, according to EUCAST guideline. A total of 39 samples were screened for mycotoxins contamination. The highest fungal contamination was obtained from fruits/vegetables in swabs (76 % MEA; 71 % DG18). The most prevalent genera in swabs was Cladosporium sp. (54 % MEA). Regarding EDC, besides Penicillium sp. (63 %) the most prevalent were Aspergillus sections Circumdati (25 %) and Aspergilli (35 %). Penicillium sp. was highly prevalent in the checkout (61 % SDA; 87 % voriconazole), and warehouse/dispenser sectors (73% voriconazole; 52% posaconazole). Circumdati and Fumigati were the most prevalent Aspergillus sections in fruits/vegetables (73 % and 64 % in voriconazole, respectively). Four mycotoxins (fumonisin B1, B2, B3) were detected simultaneously in most of the 36 samples. Identifying the most critical workplaces in groceries concerning fungal and mycotoxins contamination is of upmost importance to human health (workers and consumers) when using a One Health approach.

Neocucurbitaria chlamydospora sp. nov.: A Novel Species of the Family Cucurbitariaceae Isolated from a Stink Bug in Korea

The fungal strain KNUF-22-18B, belonging to Cucurbitariaceae, was discovered from a stink bug (Hygia lativentris) during the investigation of insect microbiota in Chungnam Province, South Korea. The colonies of the strain KNUF-22-18B were wooly floccose, white to brown in the center on oatmeal agar (OA), and the colonies were buff, margin even, and colorless, reverse white to yellowish toward the center on malt extract agar (MEA). The strain KNUF-22-18B produced pycnidia after 60 days of culturing on potato dextrose agar, but pycnidia were not observed on OA. On the contrary, N. keratinophila CBS 121759T abundantly formed superficial pycnidia on OA and MEA after a few days. The strain KNUF-22-18B produced chlamydospores subglobose to globose, mainly in the chain, with a small diameter of 4.4–8.8 μm. At the same time, N. keratinophila CBS 121759T displayed a globose terminal with a diameter of 8–10 μm. A multilocus phylogeny using the internal transcribed spacer regions, 28S rDNA large subunit, β-tubulin, and RNA polymerase II large subunit genes further validated the uniqueness of the strain. The detailed description and illustration of the proposed species as Neocucurbitaria chlamydospora sp. nov. from Korea was strongly supported by molecular phylogeny.

Characterization of Three Fusarium spp. Causing Wilt Disease of Cannabis sativa L. in Korea

In July 2021, wilting symptoms were observed in adult and seedling hemp (Cannabis sativa L. cv. Cherry Blossom) plants grown in a greenhouse. As the disease progressed, yellowing and wilting symptoms on the leaves developed, resulting in whole plant death. In seedling plants, typical damping-off symptoms were observed. To identify the pathogen, the roots of diseased plants were sampled, surface sterilized, and cultured on potato dextrose agar (PDA) media. From the culture, 4 different fungal isolates were recovered and purely cultured. Each fungal isolate showed distinct growth shapes and color development on malt extract agar, oatmeal agar, sabouraud dextrose agar, and PDA media. Microscopic observation and molecular identification using ribosomal DNA internal transcribed spacer sequencing identified them as 3 Fusarium spp. and 1 Thielaviopsis paradoxa. Additional sequencing of elongation factor 1-alpha and β-tubulin regions of 3 Fusarium spp. revealed that 2 of them are Fusarium solani, and the other one is Fusarium proliferatum. To examine which isolate can act as a causal agent of wilt disease of hemp, each isolate was tested for their pathogenicity. In the pathogenicity test, F. solani AMCF1 and AMCF2, and F. proliferatum AMCF3, but not T. paradoxa AMCF4, were able to cause wilting disease in hemp seedlings. Therefore, we report that F. solani AMCF1 and AMCF2, and F. proliferatum AMCF3 as causal agents of Fusarium wilt of hemp plants. To our knowledge, this is the first report of the wilt disease of C. sativa L. caused by Fusarium spp. in Korea.

First Report of Didymella sinensis Causing Leaf Blight on Italian Ryegrass in China.

Italian ryegrass (Lolium multiflorum Lam.) is a high-yield, high-quality forage grass and is cultivated widely in southern China. In April 2021, small black spots were observed on leaves of Italian ryegrass in the field of about 300 ha located in DuShan county, Guizhou province, China (25.62056°N, 107.53139°E). Approximately 1 to 3% of plants were affected. For isolation, eleven tissue pieces (about 0.5 × 1 cm) from four symptomatic leaves were surface-disinfested in 75% ethanol solution for 40s, rinsed thrice in sterilized distilled water and air dried; then these tissues were plated on potato dextrose agar (PDA), and incubated at 25°C for 4 days in the dark. Nine fungal isolates with similar colony characteristics were obtained, and three representative isolates (LMDS1, LMDS2 and LMDS3) were selected for further study. Colonies on PDA were 47 to 57 mm diam after 5 days, margin regular, dark gray in the center surrounded by white to gray, with floccose aerial mycelia on the upper side, and dark brown to black on the reverse side. There was no fungal sporulation when these isolates were incubated under continuous ultraviolet light on PDA, oatmeal agar (OA), malt extract agar (MEA) and potato carrot agar (PCA). ITS-rDNA, LSU-rDNA, and two other protein-coding genes (RPB2 and TUB2) were amplified with primers described by Chen et al. (2017). Sequences were deposited in GenBank (ON692740 to ON692742 for ITS, ON692775 to ON692777 for LSU, ON704660 to ON704662 for RPB2, and ON704657 to ON704659 for TUB2). BLAST analysis of all these four segments showed >99.7% identity with those sequences of ex-type isolate CGMCC 3.18348 of D. sinensis (Chen et al. 2017; Hou et al. 2020). Maximum likelihood (RAxML) phylogenetic tree based on the combined ITS, LSU, RPB2 and TUB2 alignments also showed these three isolates and the other two reported D. sinensis isolates formed a subclade with 100% bootstrap support. Referring to our previous method (Xue et al. 2020), five 8-week-old healthy plants of Italian ryegrass were spray-inoculated separately with a mycelial suspension of about 1.5 × 104 CFU/ml. In addition, five plants considered as non-inoculated controls were sprayed with sterilized distilled water. All plants were individually covered with transparent polyethylene bags for 5 days to maintain high relative humidity and placed in a greenhouse at 23 to 26°C. The small black spots similar to those observed on infected plants in the field developed on leaves fifteen days after inoculation. The symptoms consisted of brown to dark brown spots when leaves were severely infected; however, symptoms were not observed on non-inoculated plants (controls). Pathogenicity tests were carried out three times. The same fungus was re-isolated from the lesions, and confirmed by morphological characterization and molecular technique as described above, thus fulfilling Koch's postulates. To the best of our knowledge, this is the first report of D. sinensis causing leaf blight on Italian ryegrass in China. The accurate identification of this pathogen would be useful for the prevention and control of leaf spot on Italian ryegrass in the future.

First report of leaf spot disease caused by Xenodidymella glycyrrhizicola on Glycyrrhiza glabra

Glycyrrhiza glabra (liquorice) is a perennial shrub belonging to the Fabaceae family. It is a native of Mediterranean areas and primarily cultivated for its extracts which are used in the pharmaceutical and food industries, and in the manufacture of food supplements (Hayashi & Sudo, 2009). Several foliar diseases, such as powdery mildew, rust and leaf spots, have been reported to endanger the long-term viability and production of liquorice (Farr & Rossman, 2023). In 2022, a leaf spot disease was observed on c. 40% of the two-year-old plants in a liquorice field in Ankara, Turkey. Leaf spots were brown-to-black in colour, sometimes with a light brown centre. Lesions coalesced to large areas of blighted tissue with age (Figure 1a). Branches and petioles also became covered in oval lesions (Figure 1b). Pieces (5 × 5 mm) from lesion margins were surface sterilised in 70% ethanol (30 seconds) and 1% NaOCl (1 minute) and rinsed three times with sterile water. Phoma-like colonies were isolated from surface-sterilised parts of diseased tissues on potato dextrose agar (PDA). One representative isolate (Xg_Gg01), purified using the hyphal tip method, was selected for molecular, morphological and pathogenic characterisation and transferred to plates containing fresh PDA, oatmeal agar (OA), and malt extract agar (MEA) for seven days at 25°C with 12 hr light and 12 hr dark cycles. Colonies on MEA reached about 45 mm in diameter, margin regular and white, brown towards the centre, which was brown and orange due to conidial discharge from pycnidia (Figure 2a). Colonies reaching about 50 mm in diameter on the OA were marginally regular and olivaceous, with a colour change to black due to pycnidia formed towards the centre (Figure 2b). Colonies on the PDA reached about 43 mm in diameter, and the margin was regular and pale olive; the middle and centre were black due to dense pycnidia, and light brown conidial discharge was observed in the centre (Figure 2c). Pycnidia were mostly superficial or immersed in agar, ostiolate, 230–470 × 190–365 μm, and globose or subglobose and brown, becoming irregular-shaped and black with age (Figure 3a). Conidia were aseptate, variable in size and shape, oblong, broadly ellipsoidal, hyaline, becoming pale brown with age and measured 6.1 ± 9.3 × 3.4 ± 6.4 μm (Figure 3b). The internal transcribed spacer (ITS) of ribosomal DNA, partial 𝛽-tubulin gene (TUB2) and RNA polymerase second largest subunit (RPB2) loci of isolate Xg_Gg01 were amplified and bidirectionally sequenced with the universal primer sets; ITS1/ITS4 (White et al., 1990), BT2a/ BT2b (Glass & Donaldson, 1995), and 5f2 (Reeb et al., 2004)/7cr (Liu et al., 1999), respectively. A BLAST search of the resultant sequences (GenBank Accession Nos. OQ568230 for ITS, OQ571627 for TUB2, and OQ571628 for RPB2) showed 99.61–100% nucleotide identity with the holotype strain CBS 684.97 of X. glycyrrhizicola from New Zealand. The combined maximum-likelihood tree of ITS, TUB2, and RPB2 regions with 1000 bootstrap replicates showed that the isolate from the current study was clustered with strains of X. glycyrrhizicola derived from GenBank with strong support (Figure 4). Koch's postulates were fulfilled by spraying 10 ml of a spore suspension (106 spores/ml prepared in a 0.1% Tween-20 solution) of isolate Xg_Gg01 on each of five two-month G. glycyrrhizicola plants in trial pots. The plants were covered with plastic bags for 24 hr post inoculation. After seven days, spots were observed on the inoculated plants’ leaves and petioles (Figure 1c). No symptoms were observed on control plants, sprayed with sterile distilled water only. A morphologically identical X. glycyrrhizicola was isolated from affected tissues. To the best of our knowledge, this is the first report of X. glycyrrhizicola associated with leaf spots on G. glabra (Farr & Rossman, 2023). The fungus was previously reported on Glycyrrhiza lepidota in Iran (Ahmadpour et al., 2022) and New Zealand (Hou et al., 2020). However, G. glabra constitutes a new host record for this pathogen in Turkey and worldwide.

First Report of Laurel Wilt Caused by Harringtonia lauricola (previously Raffaelea lauricola) on Northern Spicebush in Kentucky and Tennessee.

Laurel wilt (LW) is a vascular disease caused by the fungus Harringtonia lauricola (previously Raffaelea lauricola) and transmitted by its primary vector, the redbay ambrosia beetle (Xyleborus glabratus, RAB), both of which were first detected in the United States (US) in 2002, likely introduced from their native range in Asia (Fraedrich et al. 2008; Harrington et al. 2008). LW has since spread across the southeastern US causing the death of millions of native redbay, sassafras, silk bay, swamp bay and other native Lauraceae species (Hughes et al. 2017). Detection of LW on the deciduous understory shrub northern spicebush (Lindera benzoin) was previously reported in South Carolina (Fraedrich et al. 2016) and Louisiana (Olatinwo et al. 2021) and is hereby confirmed in Kentucky and Tennessee. Spicebush plants displaying typical LW symptoms were observed in September 2020 on a property spanning the KY/TN border (Christian Co., KY and Montgomery Co., TN). Several dense stands of spicebush exhibited leaf wilt, early fall leaf coloration, dead leaves on branches, and black streaks of discolored xylem. LW was already confirmed on sassafras in the area (Loyd et al. 2020), and there were abundant dead sassafras nearby. Ambrosia beetle boring dust was observed and callow female RABs emerged from containerized bolts of spicebush collected from the site, indicating that the vector used spicebush as a brood host. Samples of spicebush sapwood tissue collected from two symptomatic plants were plated onto CSMA (cycloheximide-streptomycin malt extract agar) medium. The cultures were grown at room temperature in ambient light, and a fungus was recovered and further transferred onto MEA (malt extract agar) and PDA (potato dextrose agar) media. The morphology of the two fungal isolates, referred to as LW415 and LW416, matched the typical white mucoid growth and ovoid conidia of H. lauricola (Harrington et al. 2008). DNA was extracted from conidia harvested from two-week-old MEA cultures using a modified method of Dreaden et al. (2014). The identity of the fungus was confirmed by performing PCR with the H. lauricola-specific microsatellite primer sets IFW and CHK (Dreaden et al. 2014, Parra et al. 2020). A positive amplification product was obtained for LW415 and LW416 for both primer sets, validating their identification as H. lauricola. To confirm pathogenicity, four spicebush seedlings (mean height 22.5 cm; mean ground line diameter 3.3 mm) were inoculated: two with H. lauricola isolate LW415 grown on PDA for two weeks at room temperature in the dark, and two were mock-inoculated with sterile PDA as a control. A scalpel was used to nick the spicebush stem at a bud about 5 cm above groundline, and a 3 mm2 agar plug was placed in the wound and wrapped with parafilm. The spicebush seedlings were maintained in a growth chamber with an average temperature of 24°C and a 15 h photoperiod. Wilt symptoms were evident on inoculated seedlings after two weeks, while the control plants remained healthy. Four weeks post-inoculation, black staining of the vascular tissue was present in the symptomatic seedlings, and a fungus matching the morphology of H. lauricola was consistently recovered, while no fungus was isolated from the control plants. These results provide additional evidence that northern spicebush populations may be threatened by LW and could serve as a reservoir for the pathogen and vector (Gramling 2010). The spread of LW and RAB on spicebush may gain importance as preferred hosts (e.g., sassafras) are killed.

Culture media influence on vegetative growth and mycelia weight of Mycosphaerella fijiensis: implication for inoculum production

Six (6) culture media (Potato Dextrose Agar, Malt Extract Agar, AFPA Base, Czapek Dox Agar, Nutrient Agar and Yeast Extract), were tested for their effect on colony growth and mycelia weight of Mycosphaerella fijiensis. The isolate of M. fijiensis (Kaw10) produced vegetative mycelial growth on all six-culture media. Culture media effect on the level of vegetative mycelial colonization and mycelia weight was significant. The highest and lowest vegetative mycelia colonization was recorded on malt extract (23.5 mm) and AFPA Base (4.2 mm), respectively. Similarly, culture media effect on mycelia weight was significant. Malt extract agar produced more mycelia weight (0.34g) than the control, PDA (0.11g). Collectively, our data identify malt extract agar as a good medium for improving growth of Mycosphaerella fijiensis.

Identification and Management of Ascochyta Blight of Chickpea (Cicer arietinum L.) Prevalent in Bundelkhand Region of Uttar Pradesh, India

Field investigations were carried out during rabi (October–March), 2020–21 and 2021–22 in major chickpea-growing areas of Banda, Mahoba, Hamirpur and Chitrakoot districts of Bundelkhand region of Uttar Pradesh, India to find out a suitable management strategy for ascochyta blight disease in standing crop conditions. A survey study was carried out during January–February, 2022 to find out the occurrence of ascochyta blight. The overall severity of ascochyta blight observed in four surveyed districts ranged between 15.82–17.50%. The pathogen exhibited floccose and white to pale olive colored colony with irregular white periphery on potato dextrose agar media after 14 days of incubation. Maximum growth (46.2 mm) of mycelia was recorded on oatmeal agar media followed by the growth of 43.2 and 40.4 mm obtained in malt extract agar media and potato dextrose agar media, respectively. In standing crop conditions, two sprays of combination of Tebuconazole 50%+Trifloxystrobin 25% WG @ 0.1% gave the maximum reduction in disease incidence (47.05%) and disease severity (63.86%) along with the highest enhancement in grain yield (54.81%), number of pods plant-1 (55.56%) and 100 grain weight (62.28%). Results revealed that ascochyta blight of chickpea was prevalent in Bundelkhand region of Uttar Pradesh. Oatmeal agar medium can be used for cultivation of Ascochyta rabiei. In field, ascochyta blight can be managed by two sprays of combination of Tebuconazole 50% + Trifloxystrobin 25% WG @ 500 g ha-1 at symptoms initiation and after 15 days of first spray.

Taxonomy of Botryotrichum luteum sp. nov. based on Morphology and Phylogeny Isolated from Soil in Korea

In this study, a fungal strain KNUF-22-025 belonging to the genus Botryotrichum was isolated from the soil in Korea. The cultural and morphological characteristics of this strain differed from those of closely related species. On malt extract agar, strain KNUF-22-025 showed slower growth than most of the related species, except B. domesticum. The conidia size (9.6–21.1 × 9.9–18.4 µm) of strain KNUF-22-025 was larger than those of B. piluliferum, B. domesticum, and B. peruvianum but smaller than those of B. atrogriseum and B. iranicum. Conidiophores in strain KNUF-22-025 (137 µm) were longer than those in other closely related species but shorter than those in B. atrogriseum. Multi-locus analysis of molecular markers, such as ITS, 28S ribosomal DNA, RBP2, and TUB2 revealed that strain KNUF-22-025 was distinct from other Botryotrichum species. Thus, this strain is proposed as a novel species based on morphological characteristics along with molecular phylogeny and named Botryotrichum luteum sp. nov.

Isolation and Identification of some Yeasts from some Plants

The present study included isolating 71 local isolates of yeasts during three months, starting from 1/10/2021 to 1/1/2022 from different plant sources, as the fruits were obtained from local markets in Mosul, Kirkuk, Erbil and Dohuk and from home gardens. The isolates were diagnosed after culturing on solid nutrient medium MEA (Malt Extract Agar Medium) based on phenotypic tests including colony color, shape, diameter, nature of its edges, height, texture, brightness, surface shape, culture and microscopy at 40X powers to observe the shape of yeast cells and measure their sizes Using (E 10X) ocular and biochemical including Diazonium Blue B (DBB) Color test, growth in 25˚C and 37˚C, assessing ability to utilize nitrate as a sole nitrogen source, assessing preservative resistance of glacial acetic acid, growth at reduced water activities in high carbohydrate levels, growth at reduced water activities and high level of sodium chloride and ability mycelium formation test. The results showed that it belongs to 17 different species of yeasts Debaromyces, Rhodotorula, Pichia, Candida, Kluyveromyces, Geotrichum, Kloeckera, Saccharomyces, Cryptococcus, Zygosaccharomyces and Trichosporon.

Multilayer perceptron-genetic algorithm as a promising tool for modeling cultivation substrate of Auricularia cornea Native to Iran.

Auricularia cornea Ehrenb (syn. A. polytricha) is a wood-decaying fungi known as black ear mushroom. Earlike gelatinous fruiting body distinguishes them from other fungi. Industrial wastes have the potential to be used as the basic substrate to produce mushrooms. Therefore, 16 substrate formulations were prepared from different ratios of beech (BS) and hornbeam sawdust (HS) supplemented with wheat (WB) and rice brans (RB). The pH and initial moisture content of substrate mixtures were adjusted to 6.5 and 70%, respectively. The comparison of in vitro growth characteristics of the fungal mycelia under the different temperatures (25, 28, and 30°C), and culture media [yeast extract agar (YEA), potato extract agar (PEA), malt extract agar (MEA), and also HS and BS extract agar media supplemented with maltose, dextrose, and fructose revealed that the highest mycelial growth rate (MGR; 7.5 mm/day) belonged to HS and BS extract agar media supplemented with three mentioned sugar at 28°C. In A. cornea spawn study, the substrate combination of BS (70%) + WB (30%) at 28°C and moisture contents of 75% displayed the highest mean MGR (9.3 mm/day) and lowest spawn run period (9.0 days). In the bag test, "BS (70%) + WB (30%)" was the best substrate displaying the shortest spawn run period (19.7 days), and the highest fresh sporophore yield (131.7 g/bag), biological efficiency (53.1%) and number of basidiocarp (9.0/bag) of A. cornea. Also, A. cornea cultivation was processed to model yield, biological efficiency (BE), spawn run period (SRP), days for pinhead formation (DPHF), days for the first harvest (DFFH), and total cultivation period (TCP) by multilayer perceptron-genetic algorithm (MLP-GA). MLP-GA (0.81-0.99) exhibited a higher predictive ability than stepwise regression (0.06-0.58). The forecasted values of the output variables were in good accordance with their observed ones corroborating the good competency of established MLP-GA models. MLP-GA modeling exhibited a powerful tool for forecasting and thus selecting the optimal substrate for maximum A. cornea production.

Salmon oyster mushroom: growth characteristics and protease production on a laboratory scale

Pleurotus have an easy cultivation and are excellent sources of proteolytic enzymes. The aimed was evaluate the mycelial growth and extracellular protease activity of Pleurotus ostreatoroseus. The mushroom was grown on potato dextrose agar (BDA) with yeast extract (YE) 0.5% (w/v). The radial mycelial growth velocity (RCV) and mycelial density occurred on different media containing 0.5% (w/v) YE: Potato Dextrose agar (BDA), GYP agar (glucose, yeast extract and peptone), malt extract agar (MEA), purple yam extract agar (EIR), torn yam extract agar (CEIN), aria extract agar (EA). Proteolytic activity was determined by the gelose block method. In mycelial growth (VCM) is used: cupuaçu exocarp (CC), açaí (Sac), pineapple (CsAb) and sawdust (SER), supplemented with rice bran (FA) or wheat bran (FT) were used. Proteases were extracted in sterile distilled water under 180 rpm agitation at 30 °C. Significant RCV was observed in MEA+YE (12.32 ± 0.10 mm/day). However, GYP agar showed strong mycelial density. Significant substrate mixture with strong mycelial density occurred on Sac+FA (0.61 ± 0.09 cm/day). The tested media were efficient in the production of extracellular proteases by this mushroom.

A Talaromyces Fungal Species with Strong Antimicrobial Activity from Deception Island, Antarctica

Deception Island is well-known for harboring highly diverse microbial communities due to its unique volcanic environment in Antarctica. Most studies focused on bacteria, and relatively little was known about the fungal species on this island. The present study was aimed to determine the antimicrobial production and nutrient utilization profiles of a soil fungus from Deception Island, designated as Im33. Our findings showed that the strain had maximum mycelial growth and sporulation on malt-extract agar (MEA) medium, but it demonstrated the strongest antimicrobial activity in yeast extract-malt extract broth (YMB) medium. Phylogenetic analysis of the internal transcribed spacer 1 and 2 regions showed that it is a species belonging to the genus Talaromyces. It was resistant to cycloheximide concentrations up to 1,000 mg/L and exhibited broad-spectrum antimicrobial activity against Gram-positive and Gram-negative test pathogens, as well as being able to utilize a variety of carbon sources. This is the first report of a Talaromyces species from Deception Island. The capability of the strain to produce broad-spectrum antimicrobial compounds and various enzymes indicated that Antarctic fungi, like their bacterial counterparts, have adopted various adaptation strategies to compete and survive in the extreme environment.

First Report of Postharvest Stem End Rot of mango Fruit (Mangifera indica) Caused by Diaporthe pseudomangiferae in China.

Mango (Mangifera indica L.) is considered one of the most important tropical or subtropical fruit crops (Nelson et al.2008). China is the second-largest producer of mango (Kuhn et al. 2017). In June 2021, postharvest stem-end rot disease was observed on Narcissus mango (about 20% of the fruits showed similar symptoms of infections) in local agricultural market of Guangzhou, China. Black rot symptomatic lesions were observed on the fruit surface, which initially started from the stem end and progresses into decay, turning brown. To isolate and identify the pathogen, small pieces (3-5 mm2) were excised from the lesion margins of the fruits (n=54), which were surface sterilized by 1% NaOCl (1 min), 70% ethanol (30 s) and then washed twice with sterile distilled water. After sterilization, the tissues were cultured on potato dextrose agar (PDA). Three morphologically similar isolates (SXM-1/2/3) were obtained and the representative isolate SXM-1 was analyzed. Colonies surface initially had white-gray moderate aerial mycelia, in reverse umber with patches of pale luteous to luteous. On malt extract agar (MEA) surface dirty white, reverse greyish sepia with patches of sienna. Conidiomata pycnidia, black, erumpent to superficial on PDA, globose with neck, ostiole exuding cream conidial droplets; Conidiophores hyaline, smooth, 1-3-septate, branched, densely aggregated, cylindrical, straight to sinuous, 22-42 × 2.8-3.7 µm. Alpha conidia (n = 50) aseptate, hyaline, smooth, fusiform to somewhat short cylindrical, 3.2-11 ×1.3-3.5 µm. Beta conidia (n = 30) hyaline, smooth, curved or hamate 14.8-33.6 × 1.1-2.6 μm. According to morphological characterization, the representative isolate SXM-1 was similar to Diaporthe pseudomangiferae CBS 101339 (Gomes et al. 2013). For molecular identification, the internal transcribed spacer (ITS) region, histone H3 (HIS) and β-tubulin (TUB) genes (White et al. 1990; Carbone et al. 1999; Glass et al.1995) were amplified and sequenced, which were deposited in GenBank (ON243823, ON254656, ON254655). BLASTN analysis revealed that DNA sequences of the isolates (SXM-1/2/3) showed 99% identity with those of D. pseudomangiferae (MG576128.1, KC344149, MN329124.1), respectively. A phylogenetic tree analysis based on the concatenated sequences confirmed the isolates as D. pseudomangiferae. Pathogenicity tests were made with the representative isolate SXM-1. Healthy fruits were inoculated with 5 mm mycelial discs of the representative isolate SXM-1 after being wounded with a needle or non-wounded, control fruits were inoculated with sterilized PDA plugs. All inoculated and control fruits were incubated in the dark at 26°C for 7 days post-inoculation. Control fruits remained asymptomatic, whereas inoculated fruits were dark brown necrotic lesions with a roughly circular shape around the inoculation sites. Pathogenicity tests were performed in triplicate. The pathogenic isolates were successfully reisolated, thus confirming Koch's postulates. D. pseudomangiferae was associated with fruit peel of mango in Mexico and the Dominican Republic, and it has also been reported to cause inflorescence rot, rachis canker, and flower abortion in mango (Gomes et al. 2013; Serratodiaz et al. 2014). To our knowledge, this is the first report of D. pseudomangiferae causing postharvest stem-end rot of mango fruits in China. This finding suggests that D. pseudomangiferae is a potential problem for mango fruit production in China, and it is important to establish an adequate and effective control management of this disease.

Culture Media Options for Growth and Morphological Characterisation of Cercospora coffeicola Affecting Coffee in Zimbabwe

Cercospora leaf spot is fast turning into a critically important diseasein Zimbabwe. The disease is caused by Cercospora coffeicola whichsignificantly reduces productivity and quality of coffee. Disturbingly,optimum sporulation of Cercospora coffeicola in culture remains a limitingfactor for microbial analysis and quantitative studies of Cercosporaleaf spot. Faced with this challenge, an in-vitro study was conducted atCoffee Research Institute, Manicaland, Zimbabwe to examine growth ofCercospora coffeicola in different nutrient media and to determine the bestmedia for Cercospora coffeicola analysis. Six nutrient media were assessed(corn meal agar, oat meal agar, Czapek Dox agar, malt extract agar, yeastextract agar and potato dextrose agar) for the growth of Cercosporacoffeicola. The laboratory-based experiment was duplicated, laid out ina Completely Randomized Design, replicated three times and based onCercospora coffeicola nutrient inoculation. Data were collected on radialgrowth, colour and texture of mycelium at 3 and 6 days after inoculation.There were significant differences (p < 0.05) in the growth of Cercosporacoffeicola in media after 3 and 6 days. Malt extract agar had the greatestradial growth (34 mm and 32 mm) of Cercospora coffeicola for trials 1and 2 respectively, whilst the least growth was in the oat meal agar (14.2mm and 15.7 mm) for trials 1 and 2 respectively. There were variations incolour and texture of mycelium with malt extract agar, potato dextrose agarand oat meal agar associated with darker colours and rough texture whilesmooth white mycelia were found in corn meal agar. After considering allnutrient media, malt extract agar was found to be the best media for thegrowth of Cercospora coffeicola in-vitro. On the basis of our findings, theauthors recommend the use of malt extract agar as the primary media foridentification and characterisation of Cercospora coffeicola.

Chemical sensitivity phenome of an Armillaria mellea isolate determined using phenotype microarrays and potential use of caffeine-rich wastes for disease control

Armillaria fungi are frequently documented as economically and ecologically significant plant pathogens, recognised as the causal agents of Armillaria root rot disease. Armillaria mellea is one of the most aggressive pathogens in the Armillaria genus. None of the chemicals tested against Armillaria have been effective in fully eradicating an established A. mellea mycelium from an infection site and/or preventing plant mortality, which makes studies about the chemical sensitivity of A. mellea essential. In this work, the inhibitory effects of 120 different chemical agents on the growth rate of A. mellea were examined using the Biolog Phenotype MicroArray system of chemical sensitivity panels. Among the tested substances, aromatic and membrane function compounds showed the highest inhibitory activity against A. mellea. Interestingly, our results demonstrated promising potential for application of caffeine as an A. mellea-oriented fungicide. Further studies were conducted to explore the antifungal activity of a low-cost and locally available caffeine-rich waste, i.e. spent coffee grounds (SCG) against A. mellea. It is noteworthy that the hyphal growth of A. mellea was significantly inhibited when cultivated on malt extract agar supplemented with SCG. Current findings uncovered, for the first time, the potential use of caffeine-rich wastes for designing management strategies to practically control the spread of A. mellea.

First report of thousand cankers disease caused by the fungus Geosmithia morbida and its vector Pityophthorus juglandis on Juglans regia in France

Thousand cankers disease (TCD) of walnut trees is a disease complex involving the ascomycete fungus Geosmithia morbida and its insect vector, the walnut twig beetle Pityophthorus juglandis. The disease is named after the large number of cankers that appear on the tree, caused by the wounds made by P. juglandis when it feeds and builds galleries under the bark and the parasitic behaviour of the fungus growing in the tissue surrounding the galleries. Several Juglans species may be affected. High mortality of walnut trees caused by TCD has been observed in the USA since the mid-1990's, mainly on Juglans nigra planted in urban areas. In 2013, TCD was reported for the first time in Italy, in the region of Veneto, on a small number of Juglans nigra of different ages (Montecchio & Faccoli, 2014). As part of a national survey organised by the French plant protection organization, pheromone traps for P. juglandis have been installed in some urban areas where Juglans spp. are present. In August and September 2022, scolytids were trapped in parks in the cities of Bron and Lyon (region of Auvergne-Rhône-Alpes, 45°43'17.6“N 4°54'00.3”E, and 45°46'33.7“N 4°51'09.0”E respectively), and sent to the laboratory for identification. The morphological characters matched the features of Pityophthorus juglandis according to Bright (1981) (Fig. 1) and the identity was confirmed by sequence analysis of the mitochondrial cytochrome oxidase I gene. The barcode region was amplified using the primer cocktail from Germain et al. (2013). The 658-bp long amplicon was sequenced (GenBank Accession No. OP975730) and BLAST analysis showed 100% identity with P. juglandis (KF725084), confirming identification. Following this finding, an active survey was triggered on walnut trees around these traps, looking for symptoms of TCD. Foliar wilt, twig and branch diebacks, small brown bark cankers on the branch and trunk (Fig. 2), sometimes with a cracked appearance (Fig. 3), as well as exit holes were observed on several Juglans regia trees of different ages. Removal of the bark around the cankers disclosed a brown discolouration of the phloem and the presence of bark beetle galleries (Fig. 4) where some P. juglandis were collected. Sections of branch or bark showing cankers were sampled and sent to the laboratory for analysis. After surface sterilisation with 70% ethanol (v/v), pieces of diseased tissues around the necrotic margins of the cankers were cut and plated onto malt extract agar (MEA) medium supplemented with 100 ppm chloramphenicol, and incubated at 22°C in the dark. After five to seven days, whitish to beige and flat-growing colonies were observed. After subculturing on MEA in the same conditions, the culture was slow growing, lobate, plane, white to yellowish (Fig. 5). All the features observed matched the description of Geosmithia morbida in culture made by Kolařic et al. (2011), such as the roughened penicillate conidiophores bearing narrowly cylindrical to ellipsoid conidia, 4–5 μm long, sometimes organized in persistent chains. Total DNA was extracted from two cultures and tested positive in real-time PCR with the G. morbida-specific assay developed by Rizzo et al. (2020). In addition, the rDNA internal transcribed spacer region was amplified for the two cultures using ITS5 and ITS4 primers. The 584-bp long amplicon was sequenced (OP930845 and OP930846) and BLAST analysis showed 100% identity with G. morbida (FN434082). Since this first finding, the presence of both the insect and fungus was also confirmed on J. nigra in the same area. An intensive survey is underway to determine the extent of the disease. To our knowledge, this is the second report of P. juglandis and G. morbida in Europe and the first in France, where eradication measures have been put in place. Their potential spread may endanger the existence of walnut trees and the associated economy in the whole continent. The mycology research unit of the ANSES Plant Health Laboratory (LSV) is supported by a grant managed by the French National Research Agency (ANR) as part of the French government's “Investing for the Future” (PIA) programme (ANR-11-LABX-0002-01, Laboratory of Excellence-ARBRE). We would like to thank Massimo Faccoli (University of Padua) for exchanges on Pityphthorus juglandis identity and the situation of thousand cankers disease in Europe.

Cultivation and determination of nutrient contents of an edible wild Thai Hymenopellis

Hymenopellis species are taxonomically well-studied and widely distributed in tropical and temperate regions. It is well-documented in eastern and north America and can also be found in several Asian countries, including Thailand. In this study, we collected a wild Hymenopellis specimen in northern Thailand. The morphological and phylogenetic analyses inferred from a combination of the internal transcribed spacers (ITS) and the large subunit (nrLSU) of ribosomal DNA confirmed the taxonomic placement of the collected specimen to the genus Hymenopellis. Limited studies have documented the cultivation of Hymenopellis species. So far, only China commercially cultivates this mushroom. Pure mycelium was isolated from the internal tissues on malt extract agar (MEA). Sorghum grain was used in spawn preparation. Spawns were inoculated in bags of four different substrate formulas, and sawdust was used as the main substrate and other additives. The cultivation process was conducted for four months. Hymenopellis sp. (MFLU 19-1658) has an average yield ranging from 10.8±17.9 to 115.5±73.5 grams of basidiomata per substrate formula with an average biological efficiency ranging from 3.0±5.0 to 32.1±20.4% with the highest values exhibited by (A) sawdust (98%) + CaO (1%) + gypsum (1%), followed by (B) sawdust (30%) + corncobs (60%) + rice bran (8%) + CaO (1%) + gypsum (1%). The nutrient contents of the cultivated basidiomata were also determined in this study. The results indicated that the obtained basidiomata contained carbohydrate, crude fat, crude fiber, and protein at 49.2, 8.0, 16.5, and 17.8%, respectively. This study is the first to document the nutrient contents of the genus Hymenopellis.

First report of anthracnose on sugar beet (Beta vulgaris) caused by Colletotrichum incanum in Michigan, USA

In 2016, sugar beet leaf samples were received at the Michigan State University plant diagnostic laboratory with distinctive oblong lesions on the petiole (Fig. 1). The lesions contained acervuli with dark setae (Fig. 2). Lesions on the blade had light and dark rings that showed similar acervuli in a 90% humidity chamber. Pure cultures from single spore isolation were grown on malt extract agar (MEA). The resulting cultures produced acervuli-like structures with dark setae distributed throughout the conidiomata. Spores were curved and had a distinct gutule (Fig. 3). Based on these characteristics, isolates were identified as a Colletotrichum sp. A representative isolate, Co16-4, was grown in liquid V8 medium with shaking for seven days. Fungal tissue was lyophilised and DNA extracted using a DNeasy Plant Mini Kit (Qiagen, Germany). Partial sequences of the internal transcribed spacer (ITS), actin (ACT), and β-tubulin (TUB2) genes were amplified according to Damm et al. (2009) and submitted to the Research Technology Support Facility (East Lansing, USA) for Sanger sequencing. Sequences were compared to data in GenBank where ITS gave the closest match to C. spaethianum (>98%), while ACT and TUB2 gave the closest match to C. incanum (99 and 96%, respectively). Sequences were submitted to GenBank as Accession Nos. OP628186 (ITS), OP627081 (ACT) and OP627082 (TUB2). Due to the BLAST search ambiguity, sequences of 19 Colletotrichum species were retrieved from GenBank as identified in Yang et al. (2014). Sequences were concatenated and manually adjusted using Geneious Prime 2021.1.1 (Biomatters Ltd., Auckland, New Zealand). Alignment and a maximum-likelihood tree were generated in Mega version 11.0.13 (Tamura et al., 2021) using the Tamura-Nei substitution model with 1000 bootstrap replicates (Fig. 4). Based on this data, isolate Co16-4 was identified as Colletotrichum incanum. Koch's postulates were fulfilled by spraying sugar beet foliage (C869) at the 10- to 12-leaf growth stage with 10ml of a spore suspension (1×104/ml with 0.1% Tween 20). After seven days, an oblong lesion was observed on the petiole and irregular lesions developed on inoculated leaves. No lesions were observed on control plants. A morphologically identical Colletotrichum sp. was isolated from lesions. Colletotrichum incanum has been reported on soybean (Yang et al., 2014), but to our knowledge, no Colletotrichum spp. have been reported causing anthracnose on beet in the United States. The symptoms and morphology of the lesions on sugar beets are consistent with beet anthracnose reported in Japan and Canada which was attributed to C. dematium (Chiko et al., 1984; Gourley, 1966). In Japan, a radish isolate, originally identified as C. dematium, was recently identified as C. incanum (Gan et al., 2016). Leaves with similar lesions were found in commercial sugarbeet fields in 2021 and 2022, indicative of continuing disease presence, but yield effects are unknown.

Post-Harvest Fungi Associated with Cowpea (<i>Vigna unguiculata</i> L. Walp.) Seeds Produced in Burkina Faso

Cowpea is a very popular foodstuff among people in sub-Saharan Africa. In Burkina Faso, it is the main food legume, especially in rural areas. Its production is facing difficulties including post-harvest losses caused by fungi. Therefore, the objective of this study was to isolate and identify fungal strains associated with cowpea seeds produced in Burkina Faso. Thus, a total of 108 seed samples were collected in the three agro-ecological zones of Burkina Faso. The sanitary analysis of the seeds was carried out using the direct contact method. The isolation and purification of the isolates were performed on Potato Dextrose Agar medium while their identification was done through macroscopic and microscopic phenotypical characterization using different culture media (Potato Dextrose Agar (PDA), Malt Extract Agar (MEA) and Czapeck Dox Agar (CZA)) and different identification keys. A total of 10 fungal species were isolated, with predominance of Aspergillus flavus, Aspegillus niger, Macrophomina phaseolina, Fusarium oxysporium and Rhizopus sp. whose infection rates were 70.8% to 100% of seed samples. In addition to being present in all three zones, the infection rates of Aspergillus flavus (56.55%), Aspergillus niger (20.35%) and Rhizopus (32.80%) were higher in the Sahelian zone. In the Sudano-Sahelian zone, Macrophomina (50.66%) and Fusarium (18.88%) presented the highest infection rates, while Penicillium sp. showed the highest infection rate (2.84%) in the Sudanian zone. This finding demonstrated the necessity to improve post-harvest and conservation techniques of cowpea to limit crop losses and preserve the sanitary quality of this important foodstuff.