Occurrence and Characterization of Penicillium Species Isolated from Post-Harvest Apples in Lebanon.

The apple is one of the most important fruit tree crops in the Mediterranean region. Lebanon, in particular, is among the top apple producer countries in the Middle East; however, recently, several types of damage, particularly rot symptoms, have been detected on fruits in cold storage. This study aims to identify the causal agents of apple decay in Lebanese post-harvest facilities and characterize a set of 39 representative strains of the toxigenic fungus Penicillium. The results demonstrated that blue mould was the most frequent fungal disease associated with apples showing symptoms of decay after 3-4 months of storage at 0 °C, with an average frequency of 76.5% and 80.6% on cv. Red and cv. Golden Delicious apples, respectively. The morphological identification and phylogenetic analysis of benA gene showed that most Penicillium strains (87.2%) belong to P. expansum species whereas the remaining strains (12.8%) belong to P. solitum. Furthermore, 67.7% of P. expansum strains produced patulin when grown on apple puree for 14 days at 25 °C with values ranging from 10.7 mg kg-1 to 125.9 mg kg-1, whereas all P. solitum did not produce the mycotoxin. This study highlights the presence of Penicillium spp. and their related mycotoxin risk during apple storage and calls for the implementation of proper measures to decrease the risk of mycotoxin contamination of apple fruit products.

A novel qPCR based-method for detection and quantification of three recurrent species of Penicillium isolated from bioaerosols in mold-damaged homes.

Fungal contamination of indoor environments can cause respiratory diseases and induce damages to building materials. Among the fungal species found in mold-damaged homes, Penicillium brevicompactum, P. chrysogenum and P. crustosum can be considered as recurrent strains. In this study, we therefore propose a rapid and novel qPCR-based method in order to allow the monitoring of these three fungal species. The method developed allows the quantification of the target DNA of these three Penicillium species with a limit of quantification of 0.01 ng/µL without significant difference with spectrophotometry quantification assay for DNA concentrations between 5 and 100 ng/µL. This technique also enables the rapid detection of these three species in complex mixtures of DNA extracted from 15 bioaerosols collected in mold-damaged homes and previously cultured on agar plate. This new sensitive and specific qPCR technique can thus be easily integrated into bioaerosol studies.

Species distribution and antifungal susceptibilities of clinical isolates of Penicillium and Talaromyces species in China.

Morphologically identified Penicillium (n = 103) and Talaromyces marneffei (n = 8) isolates were collected from various clinical sources between 2016 and 2017 at a medical centre in Beijing, China. Identification to species level was confirmed by sequencing of the internal transcribed spacer (ITS) region, ß-tubulin gene (benA) and RNA polymerase II second largest subunit (RPB2) gene. Of the 111 isolates, 56 (50.5%) were identified as Penicillium spp. and 55 (49.5%) as Talaromyces spp. Eleven species of Penicillium were detected, of which Penicillium oxalicum was the commonest, accounting for 51.8% (29/56), followed by Penicillium rubens (10.7%; 6/56) and Penicillium citrinum (10.7%; 6/56). Among the 55 Talaromyces isolates, nine species were identified, with Talaromyces funiculosus (36.4%; 20/55), Talaromyces stollii (27.3%; 15/55) and Talaromyces marneffei (14.5%; 8/55) being the most common. Of note, 89.3% (50/56) of the Penicillium isolates and 98.2% (54/55) of the Talaromyces isolates exhibited growth at 37°C. The isolates were mainly recovered from patients with pulmonary disorders (56.8%; 63/111), autoimmune disease (12.6%; 14/111) and AIDS (5.4%; 6/111). The azoles and amphotericin B exhibited potent activity against T. marneffei, while various levels of activity were observed against Penicillium and other Talaromyces species The echinocandins had the lowest MECs (MEC90, ≤0.12 mg/L) against most Penicillium and Talaromyces species, with the exception of T. marneffei whose MEC90 (4 mg/L) was five or more dilutions higher than that of the other species tested. These data on the species distribution and antifungal susceptibility expand the current clinical knowledge of Penicillium and Talaromyces species.

Synergistic effect of carboxymethylcellulose and Cryptococcus laurentii on suppressing green mould of postharvest grapefruit and its mechanism.

The synergistic effects of carboxymethylcellulose (CMC) combined with Cryptococcus laurentii FRUC DJ1 were studied on controlling green mould resulting from Penicillium digitatum in grapefruit fruit. The results indicate that both C. laurentii and the CMC treatment suppressed P. digitatum conidia germination. In addition, C. laurentii growth in vitro was not affected by low CMC concentrations, nevertheless, the biofilm of C. laurentii was enhanced. Compared with the control fruit, the grapefruit had a lower green mould in all treatments. Significantly synergistic effects were caused by combining C. laurentii and CMC on minimum decay incidence and lesion diameter. Combined treatment induced defence enzyme activities, including chitinase, ß-1,3-glucanase, peroxidase, polyphenol oxidase, and phenylalanine ammonia-lyase, together with disease tolerance-associated total phenol. Also, this combination inhibited the pathogen growth by adhered to the hyphae and reduced its infection in fruit wounds. Moreover, the commercial quality parameters in the combined treatment of C. laurentii and CMC, including weight loss, total soluble solids, ascorbic acid, and titratable acidity, were superior to single treatment. The combination of C. laurentii and CMC can not only control postharvest decay but also maintain fruit qualities. Thus, it can be used in grapefruit for commercial purposes.

Control of postharvest diseases caused by Penicillium spp. with myrtle leaf phenolic extracts: in vitro and in vivo study on mandarin fruit during storage.

BACKGROUND: In the postharvest handling of horticultural commodities, plant extracts with fungicidal activity are a valid alternative to synthetic fungicides. The fungicidal activity of myrtle leaf extracts from eight cultivars was studied in vitro against Penicillium digitatum, Penicillium italicum, and Penicillium expansum and on artificially inoculated mandarins with green and blue molds during storage for 12 days at 20 °C and 90% RH. RESULTS: Hydroxybenzoic acids, hydrolysable tannins, and flavonols were identified by high-performance liquid chromatography (HPLC). Despite sharing the same phenolic profile, extracts of eight myrtle cultivars significantly differed in the concentration of phenolics. Hydrolysable tannins are the principal subclass representing nearly 44.9% of the total polyphenols, whereas myricitrin was the most abundant flavonol in all cultivars. Myrtle extracts strongly inhibited conidial germination of the pathogens tested, although the greatest efficacy was observed against P. digitatum. At a concentration of 20 g L-1 , all the extracts completely inhibited fungi growth; only 'Angela', 'Tonina' and 'Grazia' extracts were effective at lower concentrations (15 g L-1 ). On inoculated fruit, myrtle extracts significantly controlled rot development. As a preventive treatment, 'Ilaria' and 'Maria Rita' extracts significantly reduced the rate of fruit with green mold decay lesions. When applied as a curative treatment, all the exacts decreased the incidence of decay. Against P. italicum, all the extracts applied as preventive treatments controlled decay effectively, while as curative treatment some of the extracts were not effective. All the extracts reduced the size of the infected areas. CONCLUSION: The results propose myrtle extracts as a possible natural alternative to synthetic fungicides. © 2021 Society of Chemical Industry.

Diversity of fungi associated with macroalgae from an estuarine environment and description of Cladosporium rubrum sp. nov. and Hypoxylon aveirense sp. nov.

Fungal communities associated with macroalgae remain largely unexplored. To characterize algicolous fungal communities using culture dependent methods, macroalgae were collected from different sampling sites in the Ria de Aveiro estuary, Portugal. From a collection of 486 isolates that were obtained, 213 representative isolates were selected through microsatellite-primed PCR (MSP-PCR) fingerprinting analysis. The collection yielded 33 different genera, which were identified using the ITS region of the rDNA. The results revealed that the most abundant taxa in all collections were Acremonium-like species: Alternaria, Cladosporium, Leptobacillium and Penicillium. The fungal community composition varied with macroalgae species. Through multilocus phylogenetic analyses based on ITS, tub2, tef1-α and actA sequences, in addition to detailed morphological data, we propose Cladosporium rubrum sp. nov. (type strain=CMG 28=MUM 19.39) and Hypoxylon aveirense sp. nov. (type strain=CMG 29=MUM 19.40) as novel species.

Microbiology of secondary infections in Buruli ulcer lesions; implications for therapeutic interventions.

BACKGROUND: Buruli ulcer (BU) is a skin disease caused by Mycobacterium ulcerans and is the second most common mycobacterial disease after tuberculosis in Ghana and Côte d'Ivoire. M. ulcerans produces mycolactone, an immunosuppressant macrolide toxin, responsible for the characteristic painless nature of the infection. Secondary infection of ulcers before, during and after treatment has been associated with delayed wound healing and resistance to streptomycin and rifampicin. However, not much is known of the bacteria causing these infections as well as antimicrobial drugs for treating the secondary microorganism. This study sought to identify secondary microbial infections in BU lesions and to determine their levels of antibiotic resistance due to the prolonged antibiotic therapy required for Buruli ulcer. RESULTS: Swabs from fifty-one suspected BU cases were sampled in the Amansie Central District from St. Peters Hospital (Jacobu) and through an active case surveillance. Forty of the samples were M. ulcerans (BU) positive. Secondary bacteria were identified in all sampled lesions (N = 51). The predominant bacteria identified in both BU and Non-BU groups were Staphylococci spp and Bacilli spp. The most diverse secondary bacteria were detected among BU patients who were not yet on antibiotic treatment. Fungal species identified were Candida spp, Penicillium spp and Trichodema spp. Selected secondary bacteria isolates were all susceptible to clarithromycin and amikacin among both BU and Non-BU patients. Majority, however, had high resistance to streptomycin. CONCLUSIONS: Microorganisms other than M. ulcerans colonize and proliferate on BU lesions. Secondary microorganisms of BU wounds were mainly Staphylococcus spp, Bacillus spp and Pseudomonas spp. These secondary microorganisms were less predominant in BU patients under treatment compared to those without treatment. The delay in healing that are experienced by some BU patients could be as a result of these bacteria and fungi colonizing and proliferating in BU lesions. Clarithromycin and amikacin are likely suitable drugs for clearance of secondary infection of Buruli ulcer.

Development and optimization of a loop-mediated isothermal amplification (LAMP) assay for the species-specific detection of Penicillium expansum.

Penicillium expansum is the main cause of Blue Mold Decay, which is the economically most significant postharvest disease on fruits. It occurs especially on pomaceous fruits such as apples and pears but also on a wide range of other fruits such as grapes or strawberries. Besides its negative economic effects on the industry, the fungus is also of health concern as it produces patulin, a mycotoxin known to provoke harmful effects in humans. A specific and rapid detection of this fungus therefore is required. In the current study, a loop-mediated isothermal amplification (LAMP) assay was developed and optimized for the species-specific detection of P. expansum. The assay showed high specificity during tests with genomic DNA of 187 fungal strains. The detection limit of the developed assay was 25 pg genomic DNA of P. expansum per reaction. The assay was successfully applied for the detection of the fungus on artificially contaminated apples, grapes, apple juice, apple puree, and grape juice. The developed assay is a promising tool for rapid, sensitive, specific, and cost-efficient detection of P. expansum in quality control applications in the food and beverage industry.

Capability of Penicillium oxalicum y2 to release phosphate from different insoluble phosphorus sources and soil.

Due to insufficient amount of soluble phosphate and poor persistence of traditional chemical phosphate fertilizers in agricultural soils, the eco-friendly and sustainable phosphorus sources for crops are urgently required. The efficient phosphate-releasing fungal strain designated y2 was isolated and identified by the internal transcribed spacer of rDNA as Penicillium oxalicum y2. When lecithin, Ca3(PO4)2, or ground phosphate rock were separately used as sole phosphorus source, different phosphate-releasing modes were observed. The strain y2 was able to release as high as 2090 mg/L soluble phosphate within 12 days of incubation with Ca3(PO4)2 as sole phosphorus source. In the culture solution, high concentration of oxalic, citric, and malic acids and high phosphatase activity were detected. The organic acids contributed to solubilizing inorganic phosphate sources, while phosphatase was in charge of the mineralization of organic phosphorus lecithin. Afterwards, the fungus culture was applied to the soil with rape growing. During 50 days of incubation, the soil's available phosphate concentration increased by three times compared with the control, the dry weight of rape increased by 78.73%, and the root length increased by 38.79%. The results illustrated that P. oxalicum y2 possessed both abilities of solubilizing inorganic phosphorus and mineralizing organic phosphorus, which have great potential application in providing biofertilizer for modern agriculture.

Label-free surface enhanced Raman scattering spectroscopy for discrimination and detection of dominant apple spoilage fungus.

Fungal infection is one of the main causes of apple corruption. The main dominant spoilage fungi in causing apple spoilage are storage mainly include Penicillium Paecilomyces paecilomyces (P. paecilomyces), penicillium chrysanthemum (P. chrysogenum), expanded Penicillium expansum (P. expansum), Aspergillus niger (Asp. niger) and Alternaria. In this study, surface-enhanced Raman spectroscopy (SERS) based on gold nanorod (AuNRs) substrate method was developed to collect and examine the Raman fingerprints of dominant apple spoilage fungus spores. Standard normal variable (SNV) was used to pretreat the obtained spectra to improve signal-to-noise ratio. Principal component analysis (PCA) was applied to extract useful spectral information. Linear discriminant analysis (LDA) and non-linear pattern recognition methods including K nearest neighbor (KNN), Support vector machine (SVM) and back propagation artificial neural networks (BPANN) were used to identify fungal species. As the comparison of modeling results shown, the BPANN model established based on the characteristic spectra variables have achieved the satisfactory result with discrimination accuracy of 98.23%; while the PCA-LDA model built using principal component variables achieved the best distinguish result with discrimination accuracy of 98.31%. It was concluded that SERS has the potential to be an inexpensive, rapid and effective method to detect and identify fungal species.

Black aspergilli in Brazilian onions: From field to market.

The occurrence of black aspergilli in onions has been reported as frequent, and this group of fungi harbors potentially toxigenic species. In addition, Aspergillus niger has been reported as the causative agent of black mold rot, an important postharvest disease that causes damage throughout the world. Brazil stands out as one of the world's largest onion producers. However, few studies have been conducted to investigate the mycobiota in Brazilian onions. For this reason, we investigated the mycobiota of 48 market (n = 25) and field (n = 23) onion bulb samples. Nineteen soil samples were collected from the same fields and evaluated. In field onions and soil samples, Penicillium spp. was the prevalent fungal group, whereas in market samples A. section Nigri was the most frequent group. Due to the taxonomic complexity of this group, species identification was supported by phylogenetic data (CaM gene). A. welwitschiae was the most prevalent species in market samples. Black aspergillus strains were evaluated for fumonisin B2 (FB2) and ochratoxin A (OTA) production. Overall, 53% and 2.2% of the strains produced FB2 and OTA, respectively. The occurrence of FB2 and OTA was also investigated in onion bulb samples but none showed contamination with these mycotoxins.

New insights into the evolution of host specificity of three Penicillium species and the pathogenicity of P. Italicum involving the infection of Valencia orange (Citrus sinensis).

Blue and green molds, the common phenotypes of post-harvest diseases in fruits, are mainly caused by Penicillium fungal species, including P. italicum, P. digitatum, and P. expansum. We sequenced and assembled the genome of a P. italicum strain, which contains 31,034,623 bp with 361 scaffolds and 627 contigs. The mechanisms underlying the evolution of host specificity among the analyzed Penicillium species were associated with the expansion of protein families, genome restructuring, horizontal gene transfer, and positive selection pressure. A dual-transcriptome analysis following the infection of Valencia orange (Citrus sinensis) by P. italicum resulted in the annotation of 9,307 P. italicum genes and 24,591 Valencia orange genes. The pathogenicity of P. italicum may be due to the activation of effectors, including 51 small secreted cysteine-rich proteins, 110 carbohydrate-active enzymes, and 12 G protein-coupled receptors. Additionally, 211 metabolites related to the interactions between P. italicum and Valencia orange were identified by gas chromatography-time of flight mass spectrography, three of which were further confirmed by ultra-high performance liquid chromatography triple quadrupole mass spectrometry. A metabolomics analysis indicated that P. italicum pathogenicity is associated with the sphingolipid and salicylic acid signaling pathways. Moreover, a correlation analysis between the metabolite contents and gene expression levels suggested that P. italicum induces carbohydrate metabolism in Valencia orange fruits as part of its infection strategy. This study provides useful information regarding the genomic determinants that drive the evolution of host specificity in Penicillium species and clarifies the host-plant specificity during the infection of Valencia orange by P. italicum. IMPORTANCE: P. italicum GL_Gan1, a local strain in Guangzhou, China, was sequenced. Comparison of the genome of P. italicum GL_Gan1 with other pathogenic Penicillium species, P. digitatum and P. expansum, revealed that the expansion of protein families, genome restructuring, HGT, and positive selection pressure were related to the host range expansion of the analyzed Penicillium species. Moreover, gene gains or losses might be associated with the speciation of these Penicillium species. In addition, the molecular basis of host-plant specificity during the infection of Valencia orange (Citrus sinensis) by P. italicum was also elucidated by transcriptomic and metabolomics analysis. The data presented herein may be useful for further elucidating the molecular basis of the evolution of host specificity of Penicillium species and for illustrating the host-plant specificity during the infection of Valencia orange by P. italicum.

Diverse bioactive metabolites from Penicillium sp. MMA derived from the red sea: structure identification and biological activity studies.

A soft coral-derived fungus Penicillium sp. among other isolates e high antibacterial, anti-yeast and cytotoxic activities. The fungus, Penicillium sp. MMA, isolated from Sarcphyton glaucoma, afforded nine diverse compounds (1-9). Their structures were identified by 1D and 2 D NMR and ESI-MS spectroscopic data as two alkaloids: veridicatol (1), aurantiomide C (2); one sesquiterpene, aspterric acid (3); two carboxylic acids, 3,4-dihydroxy-benzoic acid; (4) and linoleic acid (5); three steroids, ergosterol (6), ß-Sitosterol (7), ß-Sitosterol glucoside (8) along with the sphingolipid, cerebroside A (9). Biologically, the antimicrobial, antioxidant, in vitro cytotoxicity and antibiofilm activities were studied in comparison with the fungal extract. The in silico computational studies were implemented to predict drug and lead likeness properties for 1-4. The fungus was taxonomically characterized by morphological and molecular biology (18srRNA) approaches.

HPLC-MS/MS Method for the Detection of Selected Toxic Metabolites Produced by Penicillium spp. in Nuts.

Penicillium spp. are emerging as producers of mycotoxins and other toxic metabolites in nuts. A HPLC-MS/MS method was developed to detect 19 metabolites produced by Penicillium spp. on chestnuts, hazelnuts, walnuts and almonds. Two extraction methods were developed, one for chestnuts and one for the other three nuts. The recovery, LOD, LOQ and matrix effect were determined for each analyte and matrix. Correlation coefficients were always >99.99%. In walnuts, a strong signal suppression was observed for most analytes and patulin could not be detected. Six strains: Penicillium bialowiezense, P. brevicompactum, P. crustosum, P. expansum, P. glabrum and P. solitum, isolated from chestnuts, were inoculated on four nuts. Chestnuts favored the production of the largest number of Penicillium toxic metabolites. The method was used for the analysis of 41 commercial samples: 71% showed to be contaminated by Penicillium-toxins. Cyclopenin and cyclopenol were the most frequently detected metabolites, with an incidence of 32% and 68%, respectively. Due to the risk of contamination of nuts with Penicillium-toxins, future studies and legislation should consider a larger number of mycotoxins.

A Penicillium rubens platform strain for secondary metabolite production.

We present a Penicillium rubens strain with an industrial background in which the four highly expressed biosynthetic gene clusters (BGC) required to produce penicillin, roquefortine, chrysogine and fungisporin were removed. This resulted in a minimal secondary metabolite background. Amino acid pools under steady-state growth conditions showed reduced levels of methionine and increased intracellular aromatic amino acids. Expression profiling of remaining BGC core genes and untargeted mass spectrometry did not identify products from uncharacterized BGCs. This platform strain was repurposed for expression of the recently identified polyketide calbistrin gene cluster and achieved high yields of decumbenone A, B and C. The penicillin BGC could be restored through in vivo assembly with eight DNA segments with short overlaps. Our study paves the way for fast combinatorial assembly and expression of biosynthetic pathways in a fungal strain with low endogenous secondary metabolite burden.

Microbiological quality, fungal diversity and aflatoxins contamination in carob flour (Prosopis flexuosa).

Carob flour is obtained from pods of some species of Prosopis, leguminous trees that abound in many desert habitats worldwide. Currently, this product is available in healthy food stores in several countries, including Argentina, as a nontraditional meal of growing interest with multiple applications for the preparation of puddings, biscuits and snacks, among others. The objective of the present study was to evaluate the microbial quality of carob flour on basis of the presence of deteriorative and pathogenic microorganisms. Fungal diversity of the mycobiota was also studied with a special interest in toxigenic fungi. Eighteen samples of carob flour (Prosopis flexuosa) were analysed. Standard plate count of aerobic mesophilic bacteria showed levels of contamination ranging from <102 (estimative) to 6.8 × 105 CFU/g; total coliforms from <102 (estimative) to 4.7 × 105 CFU/g; moulds and yeasts from 2.1 × 102 to 8.1 × 104. In all samples, the absence of Salmonella sp. was verified in 25 g and counts of Bacillus cereus less than 102 were observed. These results indicate that from the safety point of view the carob flour studied does not have a significant microbial load. Regarding to fungal contamination, Aspergillus and Penicillium were the genera more diverse in species and were present in all the samples. Some of the species identified were potential mycotoxins producers. Among the most frequently detected species in the studied mycobiota were the Aspergillus of the Flavi section, well recognized as potential aflatoxin producers. The A. flavus species was one of the most widely distributed, since it was detected in almost all samples. A. parasiticus and A. arachidicola were found more sporadically. Aflatoxins analysis demonstrated that a high proportion of the samples were contaminated with aflatoxins in concentrations relatively low, ranging from 1.26 to 20.33 µg/kg of total aflatoxins. Type G aflatoxins are much less frequent contaminants than type B aflatoxins, which is consistent with the fact that A. parasiticus and A. arachidicola (producers of type B and G aflatoxins) were detected sporadically, while A. flavus, which produces aflatoxins B1 and B2, was present in a high number of samples. Results of the present work indicate that carob flour is susceptible to Aspergillus section Flavi and aflatoxin contamination and should be subjected to aflatoxin monitoring prior to marketing as required for other traditional crops.

Complete Genome Sequence of Penicillium oxalicum Strain SGAir0226 Isolated from Outdoor Tropical Air in Singapore.

Penicillium oxalicum strain SGAir0226 was isolated from a tropical air sample collected in Singapore. The complete genome was assembled from long reads obtained from single-molecule real-time sequencing and was further polished and error corrected using short read sequencing data. The assembly comprises 20 contigs with a total length of 30.7 Mb. The genome was predicted to contain 8310 protein-coding genes, 237 tRNAs and 83 rRNAs.

Novel Penicillium species causing disseminated disease in a Labrador Retriever dog.

This report describes the phenotypic characteristics of a novel Penicillium species, Penicillium labradorum, isolated from a 3-year-old male, castrated, Labrador retriever with disseminated fungal disease. The dog's presenting clinical signs included lethargy, lymphadenopathy, tachypnea, moderate pitting edema, and nonweight bearing lameness associated with the right hind limb. Fine-needle aspirate biopsies from the sublumbar and prescapular lymph nodes were initially examined. The cytologic findings were consistent with pyogranulomatous inflammation with abundant extracellular and phagocytized fungal fragments and hyphae. Based on the morphology of the organisms and lack of endogenous pigment, hyalohyphomycosis was considered most likely, with Fusarium, Penicillium, and Paecilomyces species being considerations. Fungal isolates were obtained via culture of samples from the lymph nodes, and molecular identification testing originally identified an undescribed Penicillium species belonging to the Penicillium section Exilicaulis. BLAST searches and phylogenetic analyses performed approximately 1 year and 9 months after the isolation date revealed an isolate within the Penicillium parvum clade in the Penicillium section Exilicaulis but phylogenetically distant from the other species in the section, thus representing a new species, Penicillium labradorum. Antifungal susceptibility testing was also performed on the isolate and low minimum inhibitory concentrations were observed with terbinafine, voriconazole, and posaconazole, while in vitro resistance was observed with fluconazole. The dog had been previously treated with fluconazole, itraconazole, amphotericin B lipid complex, voriconazole, and terbinafine. Approximately 587 days after the initial diagnosis, the dog was euthanized due to worsening of clinical signs and concerns for quality of life.

Isolation and Identification of Endophytic Fungi in Kernels of Coix lachrymal-jobi L. Cultivars.

Coix lachrymal-jobi L. var. ma-yuen Stapf of Gramineae are annual or perennial herbs and an important food-medicine homologous plants of high value in nutrition, health protection, and comprehensive utilization. In recent years, the revival of researches on its roles in food and medicinal applications of this underutilized grass for food security and economic empowerment of rural communities has been seen . In this research, Coix kernel endophytic fungi were isolated and identified by fungal colony morphology observation combined with the PCR-amplified fungal internal transcribed spacer (ITS) sequence analyses. All together six isolates to five species of Coix endophytic fungi and two isolates to the genus level were identified from the kernels of six Coix cultivars: Penicillium expansum, Penicillium polonicum, Cladosporium cladosporioides, Alternaria alternata, Aspergillus flavus, and two genera of Aspergillus and Fusarium. Potential benefits and harms analyses showed that Penicillium expansum, Aspergillus oryzae, and Cladosporium cladosporioides can produce a variety of beneficial composite enzymes and have an extensive application in microbial chemistry, food science, and fermentation, whereas Penicillium, Aspergillus flavus, Alternaria alternate, and Fusarium can produce corresponding toxins harmful to plants, animals, and humans. These results not only provided a basis for the targeted prevention of contamination in the tissue culture of Coix kernels by the addition of specific antibiotics, but also enriched the endophytic fungi resource pool of Gramineae crops and suggested new ideas for the improvement, cultivation, post-harvest seeds/kernels storage, and the development of new natural drugs.

Challenging the charge balance hypothesis: reconsidering buffer effect and reuptake of previously excreted organic acids by Penicillium ochrochloron.

Penicillium ochrochloron was used in the past for the leaching of zinc from a zinc oxide containing filter dust via excreted organic acids. Organic acid excretion by P. ochrochloron was stimulated by the addition of an extracellular buffer (2-(N-Morpholino)ethanesulfonic acid, MES; or zinc oxide, ZnO: ZnO + 2 H+ → Zn2+ + H2O). It was tested if the buffer stimulated excretion of organic acid anions is due to the necessity of an anion efflux across the plasma membrane to maintain electroneutrality by balancing the excretion of protons by the H+-ATPase. This charge balance hypothesis was previously postulated for P. ochrochloron. Two strains of P. ochrochloron were studied, which differed in growth parameters and amount of excreted organic acids. From the results, it was concluded that charge balance at the plasma membrane is not the main reason for organic acid excretion in these two strains of P. ochrochloron. Furthermore, the phenomenon of reuptake of excreted organic acids in the presence of about 100 mM of glucose is confirmed. It is suggested that the equilibrium between extracellular and intracellular organic acid anions may be maintained passively by a facilitated diffusion transporter.