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This paper is devoted to analyzing the regularity in time and polynomial decay of solutions for a class of fractional reaction–diffusion equations (FrRDEs) involving delays and nonlinear perturbations in a bounded domain of Rd$\operatorname{\mathbf {R}}^{d}$. By establishing some regularity estimates in both time and space variables of the resolvent operator, we present results on the Hölder and C1$C^{1}$‐regularity in time of solutions for both time‐delayed linear and semilinear FrRDEs. Based on the aforementioned results, we study the existence, uniqueness, and regularity of solutions to an identification problem subjected to the delay FrRDE and the additional observations given at final time. Furthermore, under quite reasonable assumptions on nonlinear perturbations and the technique of measure of noncompactness, the existence of decay solutions with polynomial rates for the problem under consideration is shown. [ABSTRACT FROM AUTHOR]

Destruction of citrus fruits by fungal pathogens during preharvest and postharvest stages can result in severe losses for the citrus industry. Antagonistic microorganisms used as biological agents to control citrus pathogens are considered alternatives to synthetic fungicides. In this study, we aimed to identify fungal pathogens causing dominant diseases on citrus fruits in a specialized citrus cultivation region of Vietnam and inspect soilborne Bacillus isolates with antifungal activity against these pathogens. Two fungal pathogens were characterized as Colletotrichum gloeosporioides and Penicillium digitatum based on morphological characteristics and ribosomal DNA internal transcribed spacer sequence analyses. Reinfection assays of orange fruits confirmed that C. gloeosporioides causes stem-end rot, and P. digitatum triggers green mold disease. By the heterologous expression of the green fluorescent protein (GFP) in C. gloeosporioides using Agrobacterium tumefaciens-mediated transformation, we could observe the fungal infection process of the citrus fruit stem-end rot caused by C. gloeosporioides for the first time. Furthermore, we isolated and selected two soilborne Bacillus strains with strong antagonistic activity for preventing the decay of citrus fruits by these pathogens. Molecular analyses of 16 S rRNA and gyrB genes showed that both isolates belong to B. velezensis. Antifungal activity assays indicated that bacterial culture suspensions could strongly inhibit C. gloeosporioides and P. digitatum, and shield orange fruits from the invasion of the pathogens. Our work provides a highly effective Bacillus-based preservative solution for combating the fungal pathogens C. gloeosporioides and P. digitatum to protect citrus fruits at the postharvest stages.

The medicinal mushroom Cordyceps militaris is widely exploited in traditional medicine and nutraceuticals in Asian countries. However, fruiting body production in C. militaris is facing degeneration through cultivation batches, and the molecular mechanism of this phenomenon remains unclear. This study showed that fruiting body formation in three different C. militaris strains, namely G12, B12, and HQ1, severely declined after three successive culturing generations using the spore isolation method. PCR analyses revealed that these strains exist as heterokaryons and possess both the mating-type loci, MAT1-1 and MAT1-2. Further, monokaryotic isolates carrying MAT1-1 or MAT1-2 were successfully separated from the fruiting bodies of all three heterokaryotic strains. A spore combination of the MAT1-1 monokaryotic isolate and the MAT1-2 monokaryotic isolate promoted fruiting body formation, while the single monokaryotic isolates could not do that themselves. Notably, we found that changes in ratios of the MAT1-2 spores strongly influenced fruiting body formation in these strains. When the ratios of the MAT1-2 spores increased to more than 15 times compared to the MAT1-1 spores, the fruiting body formation decreased sharply. In contrast, when MAT1-1 spores were increased proportionally, fruiting body formation was only slightly reduced. Our study also proposes a new solution to mitigate the degeneration in the heterokaryotic C. militaris strains caused by successive culturing generations.

Penicillium digitatum exists in nature as a causative agent of green mould disease in citrus fruits at the postharvest stages. Inspections of the molecular mechanism of the host invasion by P. digitatum usually require suitable selection markers for genetic manipulation. In this study, we recruited the phleomycin resistance gene as a selection marker for the genetic transformation of P. digitatum using Agrobacterium tumefaciens. The results showed that the growth of the wild strain P. digitatum PdVN1 from fungal spores is quite sensitive to phleomycin and is inhibited at a concentration of 50 µg/ml, whereas growth from fungal mycelium is more tolerant and completely suppressed at a concentration of 200 µg/ml. Under optimised conditions, the A. tumefaciens-mediated transformation (ATMT) efficiency of P. digitatum with the phleomycin resistance marker could reach over 1000 transformants per 106 spores. All the tested transformants presented the integration of T-DNA in their genomes and were mitotically stable for the phleomycin resistance. Furthermore, the results also revealed the success for heterologous expression of the DsRed fluorescent gene in the fungus, in which the strong red fluorescent signal could be observed over the whole fungal mycelium and spores. Our work demonstrates for the first time that the phleomycin resistance gene can serve as a reliable selection marker for A. tumefaciens-mediated transformation of the postharvest pathogen P. digitatum. This selection marker can be exploited for T-DNA insertional mutagenesis and for functional investigations of target genes involved in citrus decay by P. digitatum.

Background: To optimize the multiplex polymerase chain reaction (M-PCR) technique to diagnose microdeletions of azoospermia factors (AZF) on the Y chromosome and initially apply the technique to diagnose male patients with sperm density less than 5× 106 million sperm/mL was assigned to do a test to check for AZF microdeletions on the Y chromosome. Methods: Based on the positive control samples which belong to male subjects who have had 2 healthy children without any assisted reproductive technologies, the M-PCR method was developed to detect simultaneously and accurately AZF microdeletions on 32 male patients with sperm densities below 5× 106 million sperm/mL of semen at the Department of Biology and Medical Genetics – Vietnam Military Medical University. Results: Successful optimization of the M-PCR technique including 7 reactions arranged according to each AZFabc region using 24 STS/gene on the Y chromosome. Initial application to diagnose AZF deletion on 32 azoospermic and oligospermic men reveals that AZFa deletion accounts for 6.25% (2/32); deletion of all 3 regions AZFa,b,c with 18.75% (6/32 cases); The combined deletion rate of AZFb,c is highest, accounting for 56.24% (18/32 patients). Conclusion: Successfully optimized the M-PCR technique in identifying AZF microdeletions using 24 sequence tagged sites (STS)/gene for azoospermic and oligozoospermic men. The M-PCR technique has great potential in the application of AZF deletion diagnosis. [ABSTRACT FROM AUTHOR]

Medicinal plants play important roles in traditional medicine, and numerous compounds among them have been recognized for their antimicrobial activity. However, little is known about the potential of Vietnamese medicinal plants for antifungal activity. In this study, we examined the antagonistic activity of twelve medicinal plant species collected in Northern Vietnam against Penicillium digitatum, Aspergillus flavus, Aspergillus fumigatus, and Candida albicans. The results showed that the antifungal activities of the crude extracts from Mahonia bealei, Ficus semicordata, and Gnetum montanum were clearly detected with the citrus postharvest pathogen P. digitatum. These extracts could fully inhibit the growth of P. digitatum on the agar medium, and on the infected citrus fruits at concentrations of 300–1000 µg/mL. Meanwhile, the other tested fungi were less sensitive to the antagonistic activity of the plant extracts. In particular, we found that the ethanolic extract of M. bealei displayed a broad-spectrum antifungal activity against all four pathogenic fungi. Analysis of this crude extract by enrichment coupled with high-performance liquid chromatography revealed that berberine and palmatine are major metabolites. Additional inspections indicated berberine as the key compound responsible for the antifungal activity of the M. bealei ethanolic extract. Our study provides a better understanding of the potential of Vietnamese medicinal plant resources for combating fungal pathogens. This work also highlights that the citrus pathogen P. digitatum can be employed as a model fungus for screening the antifungal activity of botanicals.

The conserved fungal velvet family regulatory proteins link development and secondary metabolite production. The velvet domain for DNA binding and dimerization is similar to the structure of the Rel homology domain of the mammalian NF-κB transcription factor. A comprehensive study addressed the functions of all four homologs of velvet domain encoding genes in the fungal life cycle of the soil-borne plant pathogenic fungus Verticillium dahliae. Genetic, cell biological, proteomic and metabolomic analyses of Vel1, Vel2, Vel3 and Vos1 were combined with plant pathogenicity experiments. Different phases of fungal growth, development and pathogenicity require V. dahliae velvet proteins, including Vel1-Vel2, Vel2-Vos1 and Vel3-Vos1 heterodimers, which are already present during vegetative hyphal growth. The major novel finding of this study is that Vel1 is necessary for initial plant root colonization and together with Vel3 for propagation in planta by conidiation. Vel1 is needed for disease symptom induction in tomato. Vel1, Vel2, and Vel3 control the formation of microsclerotia in senescent plants. Vel1 is the most important among all four V. dahliae velvet proteins with a wide variety of functions during all phases of the fungal life cycle in as well as ex planta.

Mevalonate diphosphate decarboxylase (MVD; EC 4.1.1.33) is a key enzyme of the mevalonic acid (MVA) pathway. In fungi, the MVA pathway functions as upstream of ergosterol biosynthesis, and MVD is also known as Erg19. Previously, we have identified Aoerg19 in Aspergillus oryzae using bioinformatic analysis. In this study, we showed that AoErg19 function is conserved using phylogenetic analysis and yeast complementation assay. Quantitative reverse transcription–PCR (qRT-PCR) indicated that Aoerg19 expression changed in different growth stages and under different forms of abiotic stress. Subcellular localization analysis showed that AoErg19 was located in the vacuole. Overexpression of Aoerg19 decreased the ergosterol content in A. oryzae, which may due to the feedback-mediated downregulation of Aoerg8. Consistent with the decrease in ergosterol content, both Aoerg19 overexpression and RNAi strains of A. oryzae are sensitive to abiotic stressors, including ergosterol biosynthesis inhibitor, temperature, salt and ethanol. Thus, we have identified the function of AoErg19 in A. oryzae, which may assist in genetic modification of MVA and the ergosterol biosynthesis pathway.

Land consolidation is an effective solution for the hindrances in agricultural production and rural development caused by land fragmentation. In the Red River Delta of Vietnam, where land is still highly fragmented, the application of land consolidation is required. By using a bottom-up approach, the paper aims to clarify the effect of land consolidation on farm households in selected communities (as case studies) of two provinces (Hung Yen and Vinh Phuc) in the Red River Delta. With the primary structured and semi-structured interview method, 172 household questionnaires and 22 in-depth questionnaires (from local officials) were collected. The results indicated that land consolidation could either change the spatial structure or expand the area of land parcels, facilitate the conversion of crop structure, increase household incomes, accelerate mechanization in agricultural production, and create more job opportunities for agricultural laborers. However, we also found that the land consolidation process conducted in the case studies is inadequate and lacks integration with other related policies.

The filamentous fungus Aspergillus oryzae, also named as the koji mold, is a preferred host for enzyme production due to its prominent secretion ability into the culture medium. Fungal laccases are widely used in different industrial processes, especially in removing environmental pollutants. Up till now, little was known about the roles of some laccase genes from the black mold Aspergillus niger. The McoD laccase gene from A. niger includes three exons interrupted by two short introns. The respective coding sequence of the gene is computationally predicted to encode a secreted laccase of 563 amino acids. In this study, a binary vector carrying the A. niger McoD gene was successfully constructed for heterologous expression in the edible fungus A. oryzae. This vector was transformed into A. oryzae using the Agrobacterium tumefaciens-mediated transformation method. The transformation efficiency was relatively high in two different auxotrophic A. oryzae strains, including RIB40∆pyrG and VS1∆pyrG. All the tested transformants possess in their genomes the construct for expression of the McoD laccase gene under control of the strong A. oryzae amyB promoter. The selected transformants were examined for the laccase activity using the ABTS substrate. The results showed that in comparison to the wild-type fungal strains, the transgenic strains could oxidise ABTS to form the sea green colour, which can be seen directly on the agar plate.

The extraction of genomic DNA from microbial cells plays a significant role in PCR-based applications such as molecular diagnosis, microbial taxonomy, screening of genetically engineered microorganisms, and other such PCRbased applications. Currently, many methods for extraction of genomic DNA from microorganisms have been developed. However, these methods either require hazardous chemicals or consist of time-consuming steps for effective execution. In this study, we have established a simple and universal genomic DNA extraction method for different microorganisms including bacteria, yeasts, molds, and microalgae. Our method does not require harmful reagents such as phenol and chloroform for the extraction process to minimize the generation of hazardous wastes. The obtained genomic DNA products displayed high concentrations and represented a good purity level with the average 260 nm/280 nm absorbance ratios (A260/280) that range from 1.6 to 2.0. The DNA molecules further remained considerably intact when analyzed on agarose gels. More importantly, these DNA products were qualified through successful PCR amplifications of 16S rRNA gene, rDNA internal transcribed spacer (ITS), or 18S rRNA gene from genomes of bacteria, fungi, and microalgae respectively. Furthermore, with the extracted genomic DNA products, the processes of the identification of the haploid and diploid states of the Saccharomyces yeast strains or detection of putative strains of Aspergillus oryzae and Aspergillus flavus that have been isolated from infected food materials through PCR analyses are facilitated. The genomic DNA extraction method established in this study is easy to manage, time saving and costeffective, and environmentally friendly.

The plant-pathogenic fungus Verticillium longisporum is a causal agent of early senescence and ripening in cruciferous crops like Brassica napus. Verticillium wilts have become serious agricultural threats in recent decades. Verticillium species infect host plants through the roots and colonize xylem vessels of the host plant. The xylem fluid provides an environment with limited carbon sources and unbalanced amino acid supply, which requires V. longisporum to induce the cross-pathway control of amino acid biosynthesis. RNA-mediated gene silencing reduced the expression of the two CPC1 isogenes (VlCPC1-1 and VlCPC1-2) of the allodiploid V. longisporum up to 85%. VlCPC1 encodes the conserved transcription factor of the cross-pathway control. The silenced mutants were highly sensitive to amino-acid starvation, and the infected plants showed significantly fewer symptoms such as stunting or early senescence in oilseed rape plant infection assays. Consistently, deletion of single CPC1 of the haploid V. dahliae resulted in strains that are sensitive to amino-acid starvation and cause strongly reduced symptoms in the plant-host tomato (Solanum lycopersicum). The allodiploid V. longisporum and the haploid V. dahliae are the first phytopathogenic fungi that were shown to require CPC1 for infection and colonization of their respective host plants, oilseed rape and tomato.

The transcription factor Flo8/Som1 controls filamentous growth in Saccharomyces cerevisiae and virulence in the plant pathogen Magnaporthe oryzae. Flo8/Som1 includes a characteristic N-terminal LUG/LUH-Flo8-single-stranded DNA binding (LUFS) domain and is activated by the cAMP dependent protein kinase A signaling pathway. Heterologous SomA from Aspergillus fumigatus rescued in yeast flo8 mutant strains several phenotypes including adhesion or flocculation in haploids and pseudohyphal growth in diploids, respectively. A. fumigatus SomA acts similarly to yeast Flo8 on the promoter of FLO11 fused with reporter gene (LacZ) in S. cerevisiae. FLO11 expression in yeast requires an activator complex including Flo8 and Mfg1. Furthermore, SomA physically interacts with PtaB, which is related to yeast Mfg1. Loss of the somA gene in A. fumigatus resulted in a slow growth phenotype and a block in asexual development. Only aerial hyphae without further differentiation could be formed. The deletion phenotype was verified by a conditional expression of somA using the inducible Tet-on system. A adherence assay with the conditional somA expression strain indicated that SomA is required for biofilm formation. A ptaB deletion strain showed a similar phenotype supporting that the SomA/PtaB complex controls A. fumigatus biofilm formation. Transcriptional analysis showed that SomA regulates expression of genes for several transcription factors which control conidiation or adhesion of A. fumigatus. Infection assays with fertilized chicken eggs as well as with mice revealed that SomA is required for pathogenicity. These data corroborate a complex control function of SomA acting as a central factor of the transcriptional network, which connects adhesion, spore formation and virulence in the opportunistic human pathogen A. fumigatus.

In this paper, we analyze the regularity in time of solutions for nonlinear fractional reaction–subdiffusion equations (FrRSEs). By constituting regularity estimates of resolvent operator and employing fixed point theorems, we establish some results on the global existence and regularity in time of solutions to FrRSE in two different cases of nonlinear perturbations, namely, sublinear and superlinear cases. As an application of these results, several results on the existence and regularity of solutions in an inverse source problem governed by FrRSE with the additional measurements given at terminal time will be shown. [ABSTRACT FROM AUTHOR]

In this paper, we consider an inverse problem of determining a space-dependent source in the time fractional reaction-subdiffusion equation involving locally Lipschitz perturbations, where the additional measurements take place at the terminal time which are allowed to be nonlinearly dependent on the state. By providing regularity estimates on both time and space of resolvent operator and using local estimates on Hilbert scales, we establish some results on the existence and uniqueness of solutions and the Lipschitz type stability of solution map of the problem under consideration. In addition, when the input data take more regular values, we obtain results on regularity in time of solution for both the direct linear problem and the inverse problem above. [ABSTRACT FROM AUTHOR]