Isolation and Characterization of Anti-Inflammatory Sorbicillinoids from the Mangrove-Derived Fungus Penicillium sp. DM815

Marine derived fungus has gained increasing ground in the discovery of novel lead compounds with potent biological activities including anti-inflammation. Here, we first report the characterization of one new sorbicillinoid ( 1 ) and fourteen known compounds ( 2 – 15 ) from the ethyl acetate (AcOEt) extract of a cultured mangrove derived fungus Penicillium sp. DM815 by UV, IR, HR ESI-Q-TOF MS, and NMR spectra. We then evaluated the anti-inflammatory effects of eleven sorbicillinoids ( 1 – 11 ) using cultured macrophage RAW264.7 cells. The results show that compound 9 , and to a lesser degree compound 5 , significantly inhibited the Gram-negative bacteria lipopolysaccharide (LPS)-induced upregulation of the inducible nitric oxide synthase (iNOS). Consistently, compounds 5 and 9 significantly reduced the level of nitric oxide (NO), the product of iNOS, induced by LPS. We further show that these two compounds dose-dependently inhibited LPS-triggered iNOS expression and NO production, but had no effect on proliferation of RAW264.7 cells in the presence of LPS. In conclusion, our study identifies novel and known sorbicillinoids as potent anti-inflammatory agents, holding the promise of developing novel anti-inflammation treatment in the future.     

蛇足石杉内生真菌无柄盘菌Pezicula sp. JR14中抑制乙酰胆碱酯酶的次级代谢产物分离

采用形态学及ITS-rDNA序列分析法,对具有抑制乙酰胆碱酯酶活性的蛇足石杉内生菌株JR14进行鉴定,确定为无柄盘菌Pezicula sp.。开展了乙酰胆碱酯酶抑制活性化合物的分离,从Pezicula sp. JR14乙酸乙酯提取物中分离到4个化合物,利用核磁共振及质谱技术将其鉴定为behenic acid (1)、himeic acid B (2)、secalonic acid A (3)和palmitic acid (4)。采用改进的Ellman比色法对分离的化合物进行活性检测,结果表明,himeic acid B (2)具有较强的乙酰胆碱酯酶抑制活性,其IC₅₀为205μg/mL（0.64mmol/L）。     

海绵共生真菌产黄青霉LS16抗菌活性物质的分离及结构鉴定

海洋真菌能够产生大量活性独特的次级代谢产物。为了探明海绵共生真菌产黄青霉LS16发酵液中抗副溶血弧菌Vibrio parahemolyticus的活性物质,本实验对副溶血弧菌Vibrio parahemolyticus的抑菌活性进行跟踪,采用VLC（vacuum liquid chromatography）、Sephadex LH-20柱层析、薄层层析和高效液相色谱等技术,从海绵共生真菌LS16乙酸乙酯发酵液中分离纯化得到5个化合物。进一步实验证明,化合物2具有抗副溶血弧菌Vibrio parahemolyticus活性。根据该化合物的波谱数据（¹H NMR、¹³C NMR）对其化学结构进行鉴定,确定其分子式为C₁₅H₁₅NO₃,为生物碱类化合物。     

Impact of carbon sources on growth and oxalate synthesis by the phytopathogenic fungus <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis>

The impact of various supplemental carbon sources (oxalate, glyoxylate, glycolate, pyruvate, formate, malate, acetate, and succinate) on growth and oxalate formation (i.e., oxalogenesis) by Sclerotinia sclerotiorum was studied. With isolates D-E7, 105, W-B10, and Arg-L of S. sclerotiorum, growth in an undefined broth medium (0.1% soytone; pH 5) with 25 mM glucose and 25 mM supplemental carbon source was increased by the addition of malate and succinate. Oxalate accumulation occurred in the presence of glucose and a supplemental carbon source, with malate, acetate, and succinate supporting the most oxalate synthesis. With S. sclerotiorum Arg-L, oxalate-to-biomass ratios, an indicator of oxalogenic potential, were dissimilar when the organism was grown in the presence of different carbon sources. The highest oxalate-to-biomass ratios were observed with pyruvate, formate, malate, acetate, and succinate. Time-course studies with acetate-supplemented cultures revealed that acetate and glucose consumption by S. sclerotiorum D-E7 coincided with oxalogenesis and culture acidification. By day 5 of incubation, oxalogenesis was halted when cultures reached a pH of 3 and were devoid of acetate. In succinate-supplemented cultures, oxalogenesis essentially paralleled glucose and succinate utilization over the 9-day incubation period; during this time period, culture pH declined but never fell below 4. Overall, these results indicate that carbon sources can regulate the accumulation of oxalate, a key pathogenicity determinant for S. sclerotiorum.     

Fungal Pathogens: The Battle for Plant Infection

The attempted infection of a plant by a pathogen, such as a fungus or an Oomycete, may be regarded as a battle whose major weapons are proteins and smaller chemical compounds produced by both organisms. Indeed, plants produce an astonishing plethora of defense compounds that are still being discovered at a rapid pace. This pattern arose from a multi-million year, ping-pong?type co-evolution, in which plant and pathogen successively added new chemical weapons in this perpetual battle. As each defensive innovation was established in the host, new ways to circumvent it evolved in the pathogen. This complex co-evolution process probably explains not only the exquisite specificity observed between many pathogens and their hosts, but also the ineffectiveness or redundancy of some defensive genes which often encode enzymes with overlapping activities. Plants evolved a complex, multi-level series of structural and chemical barriers that are both constitutive or preformed and inducible. These defenses may involve strengthening of the cell wall, hypersensitive response (HR), oxidative burst, phytoalexins and pathogenesis-related (PR) proteins. The pathogen must successfully overcome these obstacles before it succeeds in causing disease. In some cases, it needs to modulate or modify plant cell metabolism to its own benefit and/or to abolish defense reactions. Central to the activation of plant responses is timely perception of the pathogen by the plant. A crucial role is played by elicitors which, depending on their mode of action, are broadly classified into nonspecific elicitors and highly specific elicitors or virulence effector/avirulence factors. A protein battle for penetration is then initiated, marking the pathogen attempted transition from extracellular to invasive growth before parasitism and disease can be established. Three major types of defense responses may be observed in plants: non-host resistance, host resistance, and host pathogenesis. Plant innate immunity may comprise a continuum from non-host resistance involving the detection of general elicitors to host-specific resistance involving detection of specific elicitors by R proteins. It was generally assumed that non-host resistance was based on passive mechanisms and that nonspecific rejection usually arose as a consequence of the non-host pathogen failure to breach the first lines of plant defense. However, recent evidence has blurred the clear-cut distinction among non-host resistance, host-specific resistance and disease. The same obstacles are also serious challenges for host pathogens, reducing their success rate significantly in causing disease. Indeed, even susceptible plants mount a (insufficient) defense response upon recognition of pathogen elicited molecular signals. Recent evidence suggests the occurrence of significant overlaps between the protein components and signalling pathways of these types of resistance, suggesting the existence of both shared and unique features for the three branches of plant innate immunity.     

Comparative analysis of the production of insecticidal and melanizing macromolecules by strains of Beauveria spp.: in vivo studies

Eleven strains of Beauveria bassiana, and a further five species of Beauveria sp., were tested by injection of 8x10(2) conidia into the haemocoel of the larvae of the lepidopteran Galleria mellonella with the aim of analysing their toxin producing activity in vivo. Although the virulent strains killed 100% of the insects at slightly different rates (4-6 days) there were significant differences in the pattern and intensity of host melanization caused by isolates. The majority of the isolates of Beauveria spp. induced a fast and intense melanization of the cuticle of the integument and of tracheal wall, which followed one of three patterns. Another small group of two B. bassiana strains, isolated from Ostrinia nubilalis, induced very weak or no melanization. Strains 618 and 101 of B. bassiana, were selected as models of "melanizing" and "non-melanizing" strains, respectively. Ultrastructural alterations of cells of hypodermal and tracheal epithelium and of haemocytes, assumed to be at least partially caused by fungal toxins, were revealed in larvae infected by both isolates. However, their effects on the fine structure of the hypodermis were different. Injection of sera obtained from haemolymph of insects infected with B. bassiana 618 showed that they have insecticidal, melanizing, and cytotoxic effects similar to those occurring during mycosis. Chromatographic studies and bioassays with fractions prepared from crude serum have allowed a partial identification of the toxic molecules secreted by the fungus in vivo. They are proteinaceous, as shown by protease treatments, thermolabile, negatively charged, and not glycosylated with alpha-d-mannose or alpha-d-glucose. If strain B. bassiana 618 produces melanizing macromolecules which are vivotoxins secreted during the mycosis, the mode of action of isolate 101 is different. Its capacity to kill the host depends on active mycelial development, and on the production of low molecular weight toxins.     

云南岩溶断陷盆地土地利用方式对丛枝菌根真菌群落结构的影响

【背景】丛枝菌根真菌(arbuscular mycorrhizal fungus,AMF)是一类能与绝大部分植物形成共生关系的真菌,在植被演替和生态恢复过程中发挥重要作用。【目的】了解云南岩溶断陷盆地不同土地利用方式对AMF群落结构的影响。【方法】采用Illumina HiSeq 2500高通量测序技术对云南岩溶断陷盆地林地、灌丛和草地等3种土地利用方式土壤样品的AMF群落结构进行分析。【结果】草地总磷(total phosphorus,TP)、速效钾(available potassium,AK)、交换性镁(exchangeable magnesium,E-Mg)、电导率(electrical conductivity,EC)和土壤温度(temperature,T)显著高于林地和灌丛,林地总氮(total nitrogen,TN)和有机碳(soil organic carbon,SOC)含量显著高于灌丛和草地。TN、SOC、AK和EC是AMF群落结构最主要的影响因子。草地样品的Chao1、ACE、Shannon和Observed species指数显著高于林地和灌丛样品,尽管Simpson指数差异不显著,但是也表现出了类似的变化趋势。9个土壤样品共产生953个AMF-OTU,鉴定出AMF 3个纲4个目9个科13个属。Glomeromycetes和Paraglomeromycetes为优势纲;Glomus和Paraglomus为优势属,其次为Claroideoglomus、Acaulospora和Diversispora。Glomus和Diversispora主要分布在草地和灌丛中,随着演替的进行其相对丰度有所下降,而Paraglomus的相对丰度逐渐上升。【结论】云南岩溶断陷盆地土地利用方式的AMF群落结构具有显著差异,土壤理化因子是重要的影响因素。     

细菌、真菌共代谢转化龙血树木质形成黄酮类化合物

利用龙血树产脂部位获得的真菌菌株和细菌菌株共同转化龙血树白木质,形成了木质成分中原来没有的黄酮类化合物,表明龙血树体内存在着一类能被转化成黄酮类化合物的前体物质,龙血树这种独特的抗性机制,可能与血竭形成有关。     

Honey bees can disseminate a microbial control agent to more than one inflorescence pest of oilseed rape

Pollen beetles (Meligethes aeneus) and cabbage seed weevils (Ceutorhynchus assimilis) are major pests of oilseed rape (Brassica napus) throughout Europe. In field cage experiments in both winter and spring rape, honey bees (Apis mellifera) effectively transported the entomopathogenic fungus Metarhizium anisopliae to the flowers, causing infection and mortality of both adult and larval pollen beetles, as well as of adult seed weevils. External conidiation was observed on many of the dead pest insects. Although some external conidiation also occurred on dead honey bees, reduction in honey bee colony size during the experiments appeared unrelated to the fungus. The potential of this technique for integration into pest management strategies for the crop, particularly in association with a trap crop, is discussed.     

丛枝菌根真菌对西瓜嫁接苗生长和根系防御性酶活性的影响

在温室盆栽条件下,研究丛枝菌根(AM)真菌地表球囊霉(Glomus versiforme)对连作土壤中西瓜自根苗和嫁接苗生长、根系膜透性、丙二醛(MDA)含量和防御性酶活性的影响.结果表明： 接种AM真菌能显著增加西瓜自根苗和嫁接苗的生物量,提高根系活力,降低根系膜透性和MDA含量.接种AM真菌的自根苗地上部鲜质量、地上部干质量和根系活力分别增加了57.6%、60.0%和142.1%,而接种AM真菌的嫁接苗分别增加了26.7%、28.0%和11.0%;自根苗（C）、嫁接苗（G）、接种AM真菌自根苗（C+M）和接种AM真菌嫁接苗（G+M）的根系细胞膜透性为C>G>C+M>G+M,根系MDA含量为C>G>G+M>C+M.接种AM真菌能提高西瓜自根苗和嫁接苗根系的苯丙氨酸解氨酶(PAL)、过氧化氢酶(CAT)、过氧化物酶(POD)、几丁质酶和β 1,3 葡聚糖酶活性,而且接种AM真菌的西瓜自根苗和嫁接苗根系POD、PAL和β-1,3-葡聚糖酶活性的峰值比不接种的提前2周出现.接种AM真菌能激活西瓜自根苗和嫁接苗与抗逆性有关的防御性酶反应,使根系对逆境产生快速反应,从而提高其抗连作障碍的能力.