Aniquinazolines A–D,Four New Quinazolinone Alkaloids from Marine-Derived Endophytic Fungus Aspergillus nidulans

Four new quinazolinone alkaloids, namely, aniquinazolines A–D (1–4), were isolated and identified from the culture of Aspergillus nidulans MA-143, an endophytic fungus obtained from the leaves of marine mangrove plant Rhizophora stylosa. The structures of the new compounds were elucidated by spectroscopic analysis, and their absolute configurations were determined on the basis of chiral HPLC analysis of the acidic hydrolysates. The structure for 1 was confirmed by single-crystal X-ray diffraction analysis. All these compounds were examined for antibacterial and cytotoxic activity as well as brine shrimp (Artemia salina) lethality.     

Diaporthe neotheicola,a new threat for kiwifruit in Greece

Although kiwifruit is considered as a crop with few phytopathological problems, new diseases have been identified in the last few years. This study is the first report of a shoot blight and canker disease of kiwifruit in Greece caused by the fungus Diaporthe neotheicola. The fungal species was identified based on fungal morphology and the analyses of the nuclear rDNA internal transcribed spacer (ITS), and the translation elongation factor 1-alpha (TEF-1α) gene regions.The pathogen caused distinct cankers on shoots of kiwifruit, while pycnidia were observed on the blighted shoots. The rate of development of D. neotheicola in vitro was reduced as temperatures increased from 25 to 30 °C, decreased from 20 to 10 °C, and was totally inhibited at 35 and 2–4 °C. The rate of conidial germination and the length of germ tube in vitro were reduced as temperatures increased from 25 to 30 °C, decreased from 25 to 10 °C, and was totally inhibited at 35 and 2–4 °C. A preliminary study on the effectiveness of the fungicides thiophanate methyl, carbendazim and tebuconazole against the development and germination of conidia of D. neotheicola and the disease symptoms was conducted. All fungicides were effective against the fungus in vitro. In addition, no canker was observed on artificially inoculated shoots treated with the fungicides. In general, the disease could be a threat for kiwifruit in Greece and its management should be investigated in the field.     

Cytosolic streaming in vegetative mycelium and aerial structures of Aspergillus niger

Aspergillus niger forms aerial hyphae and conidiophores after a period of vegetative growth. The hyphae within the mycelium of A. niger are divided by septa. The central pore in these septa allows for cytoplasmic streaming. Here, we studied inter- and intra-compartmental streaming of the reporter protein GFP in A. niger. Expression of the gene encoding nuclear targeted GFP from the gpdA or glaA promoter resulted in strong fluorescence of nuclei within the vegetative hyphae and weak fluorescence in nuclei within the aerial structures. These data and nuclear run on experiments showed that gpdA and glaA are higher expressed in the vegetative mycelium when compared to aerial hyphae, conidiophores and conidia. Notably, gpdA or glaA driven expression of the gene encoding cytosolic GFP resulted in strongly fluorescent vegetative hyphae and aerial structures. Apparently, GFP streams from vegetative hyphae into aerial structures. This was confirmed by monitoring fluorescence of photo-activatable GFP (PA-GFP). In contrast, PA-GFP did not stream from aerial structures to vegetative hyphae. Streaming of PA-GFP within vegetative hyphae or within aerial structures of A. niger occurred at a rate of 10–15 μm s^-1. Taken together, these results not only show that GFP streams from the vegetative mycelium to aerial structures but it also indicates that its encoding RNA is not streaming. Absence of RNA streaming would explain why distinct RNA profiles were found in aerial structures and the vegetative mycelium by nuclear run on analysis and micro-array analysis.Key words: aerial hypha, Aspergillus, conidia, conidiophore, cytoplasmic streaming, development, fungus, vegetative mycelium     

Antibacterial Cyclic Tripeptides from Antarctica-Sponge-Derived Fungus Aspergillus insulicola HDN151418

Three new aspochracin-type cyclic tripeptides, sclerotiotides M–O (1–3), together with three known analogues, sclerotiotide L (4), sclerotiotide F (5), and sclerotiotide B (6), were obtained from the ethyl acetate extract of the fungus Aspergillus insulicola HDN151418, which was isolated from an unidentified Antarctica sponge. Spectroscopic and chemical approaches were used to elucidate their structures. The absolute configuration of the side chain in compound 4 was elucidated for the first time. Compounds 1 and 2 showed broad antimicrobial activity against a panel of pathogenic strains, including Bacillus cereus, Proteus species, Mycobacterium phlei, Bacillus subtilis, Vibrio parahemolyticus, Edwardsiella tarda, MRCNS, and MRSA, with MIC values ranging from 1.56 to 25.0 µM.     

Utilization of pretreated bagasse for the sustainable bioproduction of cellulase by Aspergillus nidulans MTCC344 using response surface methodology

Response surface methodology (RSM) was used to optimize the conditions for the production of endo β-1,4 glucanase, a component of cellulase by Aspergillus nidulans MTCC344 under solid state fermentation, using bagasse as the chief substrate. A four-factor-five-level central composite design was employed for experimental design and analysis of the results. Maximum cellulase activity (CMCase was 28.96 U g⁻¹) can be attained at the optimum conditions, 16.8 mm bagasse bed height, 60% moisture content, pH 4.25 and temperature 40 °C in the solid state fermenter. These data were rather close to the experimental results obtained (CMCase was 28.84 U g⁻¹). A. nidulans MTCC344 was able to hydrolyze pretreated bagasse completely after 8 days of incubation with significant endo β-1,4 glucanase activities. The results of Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD) and scanning electron microscopy (SEM) of bagasse showed structural changes through pretreatment, in favor of enzymatic hydrolysis. Bagasse with alkali pretreatment using sodium hydroxide is a source of lignocelluloses able to improve the yield of endo β-1,4 glucanase by the strain of A. nidulans. The endo β-1,4 glucanase produced during the bioconversion of cellulose to glucose by A. nidulans MTCC344 is strongly dependent on the pretreatment given before hydrolysis.     

Multi-gene phylogenies and phenotypic characters distinguish two species within the Colletogloeopsis zuluensis complex associated with Eucalyptus stem cankers

Colletogloeopsis zuluensis, previously known as Coniothyrium zuluense, causes a serious stem canker disease on Eucalyptus spp. grown as non-natives in many tropical and sub-tropical countries. This stem canker disease was first reported from South Africa and it has subsequently been found on various species and hybrids of Eucalyptus in other African countries as well as in countries of South America and South-East Asia. In previous studies, phylogenetic analyses based on DNA sequence data of the ITS region suggested that all material of C. zuluensis was monophyletic. However, the occurrence of the fungus in a greater number of countries, and analyses of DNA sequences with additional isolates has challenged the notion that a single species is involved with Coniothyrium canker. The aim of this study was to consider the phylogenetic relationships amongst C. zuluensis isolates from all available locations and to support these analyses with phenotypic and morphological comparisons. Individual and combined phylogenies were constructed using DNA sequences from the ITS region, exons 3 through 6 of the β-tubulin gene, the intron of the translation elongation factor 1-α gene, and a partial sequence of the mitochondrial ATPase 6 gene. Both phylogenetic data and morphological characteristics showed clearly that isolates of C. zuluensis represent at least two taxa. One of these is C. zuluensis as it was originally described from South Africa, and we provide an epitype for it. The second species occurs in Argentina and Uruguay, and is newly described as C. gauchensis. Both fungi are serious pathogens resulting in identical symptoms. Recognising them as different species has important quarantine consequences.Taxonomic novelty: Colletogloeopsis gauchensis M.-N. Cortinas, Crous & M.J. Wingf. sp. nov.     

Interference Efficiency and Effects of Bacterium-mediated RNAi in the Fall Armyworm (Lepidoptera: Noctuidae)

RNAi is an effective tool for gene function analysis and a promising strategy to provide environmentally friendly control approaches for pathogens and pests. Recent studies support the utility of bacterium-mediated RNAi as a cost-effective method for gene function study and a suitable externally applied delivery mechanism for pest control. Here, we developed a bacterium-mediated RNAi system in Spodoptera frugiperda based on four target genes, specifically, Chitinase (Sf-CHI), Chitin synthase B (Sf-CHSB), Sugar transporter SWEET1 (Sf-ST), and Hemolin (Sf-HEM). RNAi conducted by feeding larvae with bacteria expressing dsRNAs of target genes or injecting pupae and adults with bacterially synthesized dsRNA induced silencing of target genes and resulted in significant negative effects on growth and survival of S. frugiperda. However, RNAi efficiency and effects were variable among different target genes and dsRNA delivery methods. Injection of pupae with dsCHI and dsCHSB induced a significant increase in wing malformation in adults, suggesting that precise regulation of chitin digestion and synthesis is crucial during wing formation. Injection of female moths with dsHEM resulted in lower mating, fecundity, and egg hatching, signifying a critical role of Sf-HEM in the process of egg production and/or embryo development. Our collective results demonstrate that bacterium-mediated RNAi presents an alternative technique for gene function study in S. frugiperda and a potentially effective strategy for control of this pest, and that Sf-CHI, Sf-CHSB, Sf-ST, and Sf-HEM encoding genes can be potent targets.     

Antifungal activity of Vitex agnus-castus extract against Pythium ultimum in tomato

Vitex agnus-castus methanolic extract showed strong antifungal activity against Pythium ultimum in tomato under both in vitro and in vivo conditions. The 0.2% extract delayed the mycelial growth of the fungus and showed significant antifungal activity against P. ultimum on tomato seedlings with an efficacy comparable to that of the synthetic fungicide. To determine the involvement both of plant extract and pathogenic fungus in PR gene induction, tomato plants were treated with V. agnus-castus extract and/or inoculated with P. ultimum. The expression of four PR genes (PR-1, PR-2, PR-5, PR-6) was monitored at five time points within 48 h of the extract treatment and fungal inoculation. The PR-1 and PR-4 genes were activated directly by V. agnus-castus extract up to 12 h after treatments; at 24 h, the direct activation by plant extract disappeared and a synergistic inducing effect of extract and pathogen applied simultaneously on the plant was observed. The PR-6 gene was not activated directly by the V. agnus-castus extract but only when applied together with the pathogen; activation of the PR-6 gene occurred 24 h after treatments and the gene expression increased at 48 h. There was no activation of PR-5 gene by the plant extract. The ability of V. agnus-castus extract to enhance plant defence responses upon pathogen inoculation might be further investigated. The activation of various PR genes suggests that induction of defence responses by V. agnus-castus extract in tomato may be regulated by more than one signalling pathway.     

Penicillium brasilianum as a novel pathogen of onion (Allium cepa L.) and other fungi predominant on market onion in Korea

Onion (Allium cepa L.) is an important vegetable crop in Korea, but its production is severely affected by fungal pathogens during plant growth and bulb storage. We investigated the occurrence of fungi on market onion bulbs; identified the predominant fungal species based on the internal transcribed spacer region, β-tubulin region, and elongation factor 1-α gene sequences; and tested the pathogenicity of each predominant fungal species in onion bulbs. The genera Aspergillus (63.9%), Penicillium (15.5%), Fusarium (6.4%), Rhizopus (5.2%), and others (9.0%) were detected in the samples. Among these genera, Aspergillus awamori, Fusarium oxysporum, Penicillium brasilianum, and Rhizopus oryzae were identified as the predominant species. All of the fungi tested could infect both the inner layers and outer surfaces of onion bulbs and be re-isolated from the infected tissues. To our knowledge, this is the first report that P. brasilianum is a fungal pathogen of onion bulbs.     

HOTAIR Induces the Downregulation of miR-200 Family Members in Gastric Cancer Cell Lines

Background:Gastric cancer is the fourth most common human malignancy and the second reason for cancer morbidity worldwide. LncRNA HOTAIR has recently emerged as a promoter of metastasis in various cancer types, including GC, through the EMT process. However, the exact mechanism of HOTAIR in promoting EMT is unknown. Aberrant expression of the miR-200 family has been linked to the occurrence and development of various types of malignant tumors. This study investigates the correlation between the HOTAIR and miR-200 family gene expression patterns in GC cell lines. We investigated the miR-200 and HOTAIR due to their common molecular features in the EMT process. Methods:AGS and MKN45 cell lines were transfected with si-HOTAIR, along with a negative control. The effect of HOTAIR knockdown was also analyzed on cell viability and also on the expression of miR-200 family members, including miR-200a, -200b, and -200c, in cell lines using qRT-PCR. Statistical analysis was performed to find the potential correlation between the expression level of HOTAIR and miRs. Results:Our results showed significant increased miR-200 family expression level in transfected AGS and MKN45 GC cells (fold changes > 2; p < 0.001). Moreover, a negative correlation was observed between HOTAIR and miR-200 expression levels in GC cell lines (p < 0.05). Conclusion:Our findings showed a significant association between miR-200 family and HOTAIR expression levels in GC cell lines. Taken together, the HOTAIR-miR-200 axis seems to play a vital role in human GC, suggesting a potential therapeutic target in future GC treatment. Key Words: Gene expression, Long noncoding RNA, HOTAIR, MicroRNAs     

Cladosporium leaf-blotch and stem rot of Paeonia spp. caused by Dichocladosporium chlorocephalum gen. nov.

Cladosporium chlorocephalum (= C. paeoniae) is a common, widespread leaf-spotting hyphomycete of peony (Paeonia spp.), characterised by having dimorphic conidiophores. During the season, one stage of this fungus causes distinct, necrotic leaf-blotch symptoms on living leaves of Paeonia spp. In late autumn, winter or after overwintering, a second morphologically distinct conidiophore type occurs on dead, blackish, rotting stems. Conspecificity of the two morphs, previously proposed on the basis of observations in culture, was supported by DNA sequence data from the ITS and LSU gene regions, using cultures obtained from leaf-blotch symptoms on living leaves, as well as from dead stems of Paeonia spp. Sequence data were identical, indicating a single species with two morphs. On account of its distinct conidiogenous loci and conidial hila, as well as its sequence-based phylogenetic position separate from the Davidiella/Cladosporium clade, the peony fungus has to be excluded from Cladosporium s. str., but still belongs to the Davidiellaceae (Capnodiales). The leaf-blotching (cladosporioid) morph of this fungus morphologically resembles species of Fusicladium, but differs in having dimorphic fruiting, and is phylogenetically distant from the Venturiaceae. The macronematous (periconioid) morph resembles Metulocladosporiella (Chaetothyriales), but lacks rhizoid conidiophore hyphae, and has 0-5-septate conidia. Hence, C. chlorocephalum is assigned to the new genus Dichocladosporium.Taxonomic novelties: Dichocladosporium K. Schub., U. Braun& Crous, gen. nov., Dichocladosporium chlorocephalum (Fresen.) K. Schub., U. Braun & Crous, comb. nov.