Fusarin C biosynthesis in Fusarium moniliforme and Fusarium venenatum

Fragments of polyketide synthase (PKS) genes were amplified from complementary DNA (cDNA) of the fusarin C producing filamentous fungi Fusarium moniliforme and Fusarium venenatum by using degenerate oligonucleotides designed to select for fungal PKS C-methyltransferase (CMeT) domains. The PCR products, which were highly homologous to fragments of known fungal PKS CMeT domains, were used to probe cDNA and genomic DNA (gDNA) libraries of F. moniliforme and F. venenatum. A 4.0 kb cDNA clone from F. venenatum was isolated and used to prepare a hygromycin-resistance knockout cassette, which was used to produce a fusarin-deficient strain of F. venenatum (kb = 1000 bp). Similarly, a 26 kb genomic fragment, isolated on two overlapping clones from F. moniliforme, encoded a complete iterative Type I PKS fused to an unusual nonribosomal peptide synthase module. Once again, targeted gene disruption produced a fusarin-deficient strain, thereby proving that this synthase is responsible for the first steps of fusarin biosynthesis.     

Authentic heterologous expression of the tenellin iterative polyketide synthase nonribosomal peptide synthetase requires coexpression with an enoyl reductase

The tenS gene encoding tenellin synthetase (TENS), a 4239-residue polyketide synthase nonribosomal-peptide synthetase (PKS-NRPS) from Beauveria bassiana, was expressed in Aspergillus oryzae M-2-3. This led to the production of three new compounds, identified as acyl tetramic acids, and numerous minor metabolites. Consideration of the structures of these compounds indicates that the putative C-terminal thiolester reductase (R) domain does not act as a reductase, but appears to act as a Dieckmann cyclase (DKC). Expression of tenS in the absence of a trans-acting ER component encoded by orf3 led to errors in assembly of the polyketide component, giving clues to the mode of programming of highly reducing fungal PKS. Coexpression of tenS with orf3 from the linked gene cluster led to the production of a correctly elaborated polyketide. The NRPS adenylation domain possibly shows the first identified fungal signature sequences for tyrosine selectivity.     

New PCR Assays for the Identification of Fusarium verticillioides, Fusarium subglutinans, and Other Species of the Gibberella fujikuroi Complex

Fusarium verticillioides and Fusarium subglutinans are important fungal pathogens of maize and other cereals worldwide. In this study, we developed PCR-based protocols for the identification of these pathogens targeting the gaoB gene, which codes for galactose oxidase. The designed primers recognized isolates of F. verticillioides and F. subglutinans that were obtained from maize seeds from several producing regions of Brazil but did not recognize other Fusarium spp. or other fungal genera that were either obtained from fungal collections or isolated from maize seeds. A multiplex PCR protocol was established to simultaneously detect the genomic DNA from F. verticillioides and F. subglutinans. This protocol could detect the DNA from these fungi growing in artificially or naturally infected maize seeds. Another multiplex reaction with a pair of primers developed in this work combined with a pre-existing pair of primers has allowed identifying F. subglutinans, F. konzum, and F. thapsinum. In addition, the identification of F. nygamai was also possible using a combination of two PCR reactions described in this work, and another described in the literature.     

6种常用农药与球孢白僵菌和蜡蚧轮枝菌的相容性

测定了3种杀虫剂和3种杀菌剂在田间常规浓度下对球孢白僵菌和蜡蚧轮枝菌孢子萌发和菌丝生长的影响。供试杀虫剂对球孢白僵菌孢子萌发的影响很小,萌发率超过85％,对蜡蚧轮枝菌孢子萌发影响较大,抑制作用最小的吡虫啉,抑制率达到25％。3种杀菌剂几乎完全抑制了孢子的萌发。杀虫剂对球孢白僵菌的菌丝生长有一定抑制作用,抑制率为22．60％-38．39％;对蜡蚧轮枝菌菌丝生长的影响小于对球孢白僵菌的影响,抑制率为10．06％．17．61％。供试杀菌剂对球孢向僵菌和蜡蚧轮枝菌菌丝生长的抑制作用都较大,抑制率都在50％以上。     

Evolutionary Imprint of Catalytic Domains in Fungal PKS–NRPS Hybrids

Fungal hybrid enzymes consisting of a polyketide synthase (PKS) and a nonribosomal peptide synthetase (NRPS) module are involved in the biosynthesis of a vast array of ecologically and medicinally relevant natural products. Whereas a dozen gene clusters could be assigned to the requisite PKS–NRPS pathways, the programming of the multifunctional enzymes is still enigmatic. Through engineering and heterologously expressing a chimera of PKS (lovastatin synthase, LovB) and NRPS (cytochalasin synthase, CheA) in Aspergillus terreus, we noted the potential incompatibility of a fungal highly reducing PKS (hrPKS) with the NRPS component of fungal PKS–NRPS hybrids. To rationalize the unexpected outcome of the gene fusion experiments, we conducted extensive bioinformatic analyses of fungal PKS–NRPS hybrids and LovB‐type PKS. From motif studies and the function of the engineered chimeras, a noncanonical function of C‐terminal condensation (C) domains in truncated PKS–NRPS homologues was inferred. More importantly, sequence alignments and phylogenetic trees revealed an evolutionary imprint of the PKS–NRPS domains, which reflect the evolutionary history of the entire megasynthase. Furthermore, a detailed investigation of C and adenylation (A) domains provides support for a scenario in which not only the A domain but also the C domain participates in amino acid selection. These findings shed new light on the complex code of this emerging class of multifunctional enzymes and will greatly facilitate future combinatorial biosynthesis and pathway engineering approaches towards natural product analogues.     

Bioactive substances from insect pathogenic fungi

Insect pathogenic fungi have opened up a relatively untapped area of natural product research which, unfortunately, has not received much attention to date. Found in wild abundance in wet tropical Thailand, the insect fungi are shown to contribute not only as controllers of insect populations but also as rich sources of structurally novel biologically active substances.     

感染性疾病RNA病毒性病原体生物信息学检测方法的建立

目的以艾滋病病毒(HIV)为研究对象,建立感染性疾病RNA病毒性病原体的生物信息学检测方法。方法应用DNase-非序列依赖的单引物扩增技术,将艾滋病患者血清过滤后,经DNase处理去除血清中的内源DNA,提取血清病毒RNA,反转录合成病毒RNA的双链cDNA,TaqⅠ酶切双链cDNA,加接头分子,并以接头分子为引物非特异扩增病原基因;PCR产物克隆后,酶切鉴定并测序,经GenBankBLAST软件进行序列分析。结果非序列依赖的单引物扩增HIV患者血清中外源基因得到多个DNA片段;重组克隆质粒经EcoRⅠ和HindⅢ双酶切,可见插入片段存在;将所有PCR产物测序,序列与已知病原基因序列一致。结论应用DNase处理及非序列依赖的单引物扩增方法可以检测RNA病毒性病原体。     

利用植物病原真菌除草的研究进展

随着病虫草害的发生,化学药剂大量使用,带来了一系列的环境和生态问题,如抗药杂草种群的增加、土壤板结和水体污染等,严重影响了农业生态的可持续发展.植物病原真菌是引起植物发生病害的一类微生物,但对杂草而言,却是一类可开发利用的潜在资源.植物病原真菌侵染杂草的同时,分泌毒素抑制杂草生长或直接导致杂草死亡.对利用植物病原真菌防除杂草的研究进行了综述,以期为其进一步研究作理论基础.     

The Effect of the acetone extract of Arctotis arctotoides (Asteraceae) on the growth and ultrastructure of some opportunistic fungi associated with HIV/AIDS

In this study, the effect of the acetone extract of Arctotis arctotoides (L.f.) O. Hoffm. (Asteraceae) on the growth and ultrastructure of some opportunistic fungi associated with HIV/AIDS was analyzed by means of scanning electron microscope (SEM). Remarkable morphological alterations in the fungal mycelia which were attributed to the loss of cell wall strength ranged from loss of turgidity and uniformity, collapse of entire hyphae to evident destruction of the hyphae. The elements responsible for giving the fungi their characteristic virulence were detected and quantified by energy dispersive X-ray microanalysis techniques. X-ray microanalysis showed the specific spectra of sodium, potassium and sulfur as the principal intersection of the four pathogenic fungi studied. Since these ions have the potential of fostering fungal invasion by altering the permeability of hosts' membranes, their presence was considered inherent to the pathogenicity of the opportunistic fungi. Hence, these findings indicate the potential of the crude extract of A. arctotoides in preventing fungal invasion and subsequent infection of host's membranes.     

Triplex Real-Time PCR Approach for the Detection of Crucial Fungal Berry Pathogens—Botrytis spp., Colletotrichum spp. and Verticillium spp.

Phytopathogens cause undeniably serious damage in agriculture by harming fruit cultivations and lowering harvest yields, which as a consequence substantially reduces food production efficiency. Fungi of the Botrytis, Colletotrichum and Verticillium genera are a main concern in berry production. However, no rapid detection method for detecting all of these pathogens simultaneously has been developed to date. Therefore, in this study, a multiplex real-time PCR assay for this purpose was established. Universal fungal primers for the D2 region of the large subunit ribosomal DNA and three multiplexable fluorogenic probes specific for the chosen fungi were designed and deployed. The triplex approach for the molecular detection of these fungi, which was developed in this study, allows for the rapid and effective detection of crucial berry pathogens, which contributes to a more rapid implementation of protective measures in plantations and a significant reduction in losses caused by fungal diseases.     

LysM Receptor-Like Kinase LYK9 of Pisum Sativum L. May Regulate Plant Responses to Chitooligosaccharides Differing in Structure

This study focused on the interactions of pea (Pisum sativum L.) plants with phytopathogenic and beneficial fungi. Here, we examined whether the lysin-motif (LysM) receptor-like kinase PsLYK9 is directly involved in the perception of long- and short-chain chitooligosaccharides (COs) released after hydrolysis of the cell walls of phytopathogenic fungi and identified in arbuscular mycorrhizal (AM) fungal exudates. The identification and analysis of pea mutants impaired in the lyk9 gene confirmed the involvement of PsLYK9 in symbiosis development with AM fungi. Additionally, PsLYK9 regulated the immune response and resistance to phytopathogenic fungi, suggesting its bifunctional role. The existence of co-receptors may provide explanations for the potential dual role of PsLYK9 in the regulation of interactions with pathogenic and AM fungi. Co-immunoprecipitation assay revealed that PsLYK9 and two proposed co-receptors, PsLYR4 and PsLYR3, can form complexes. Analysis of binding capacity showed that PsLYK9 and PsLYR4, synthesized as extracellular domains in insect cells, were able to bind the deacetylated (DA) oligomers CO5-DA–CO8-DA. Our results suggest that the receptor complex consisting of PsLYK9 and PsLYR4 can trigger a signal pathway that stimulates the immune response in peas. However, PsLYR3 seems not to be involved in the perception of CO4-5, as a possible co-receptor of PsLYK9.     

A Highlight on the Inhibition of Fungal Carbonic Anhydrases as Drug Targets for the Antifungal Armamentarium

Carbon dioxide (CO₂), a vital molecule of the carbon cycle, is a critical component in living organisms’ metabolism, performing functions that lead to the building of compounds fundamental for the life cycle. In all living organisms, the CO₂/bicarbonate (HCO₃⁻) balancing is governed by a superfamily of enzymes, known as carbonic anhydrases (CAs, EC 4.2.1.1). CAs catalyze the pivotal physiological reaction, consisting of the reversible hydration of the CO₂ to HCO₃⁻ and protons. Opportunistic and pathogenic fungi can sense the environmental CO₂ levels, which influence their virulence or environmental subsistence traits. The fungal CO₂-sensing is directly stimulated by HCO₃⁻ produced in a CA-dependent manner, which directly activates adenylyl cyclase (AC) involved in the fungal spore formation. The interference with CA activity may impair fungal growth and virulence, making this approach interesting for designing antifungal drugs with a novel mechanism of action: the inhibition of CAs linked to the CO₂/HCO₃⁻/pH chemosensing and signaling. This review reports that sulfonamides and their bioisosteres as well as inorganic anions can inhibit in vitro the β- and α-CAs from the fungi, suggesting how CAs may be considered as a novel “pathogen protein” target of many opportunistic, pathogenic fungi.     

Extracellular Vesicles in the Fungi Kingdom

Extracellular vesicles (EVs) are membranous, rounded vesicles released by prokaryotic and eukaryotic cells in their normal and pathophysiological states. These vesicles form a network of intercellular communication as they can transfer cell- and function-specific information (lipids, proteins and nucleic acids) to different cells and thus alter their function. Fungi are not an exception; they also release EVs to the extracellular space. The vesicles can also be retained in the periplasm as periplasmic vesicles (PVs) and the cell wall. Such fungal vesicles play various specific roles in the lives of these organisms. They are involved in creating wall architecture and maintaining its integrity, supporting cell isolation and defence against the environment. In the case of pathogenic strains, they might take part in the interactions with the host and affect the infection outcomes. The economic importance of fungi in manufacturing high-quality nutritional and pharmaceutical products and in remediation is considerable. The analysis of fungal EVs opens new horizons for diagnosing fungal infections and developing vaccines against mycoses and novel applications of nanotherapy and sensors in industrial processes.     

黑曲霉实时荧光PCR检测方法的建立

目的建立黑曲霉实时荧光PCR检测方法。方法对6种主要病原曲霉(黑曲霉、烟曲霉、杂色曲霉、构巢曲霉、土曲霉及黄曲霉)的GAPDH基因序列进行比对分析,选择黑曲霉特异位点设计引物和探针,对黑曲霉进行实时荧光PCR扩增,并检测该方法的灵敏度及特异性。结果该方法可检出2.78×10-10μg/ml的黑曲霉基因组DNA;对亲源关系较近的11株不同种曲霉及4株其他属临床常见的病原真菌进行实时荧光PCR检测,未发现有交叉反应。结论已建立了灵敏度高、特异性好的快速检测黑曲霉的实时荧光PCR方法。     

Cloning and Characterization of the Ravidomycin and Chrysomycin Biosynthetic Gene Clusters

The gene clusters responsible for the biosynthesis of two antitumor antibiotics, ravidomycin and chrysomycin, have been cloned from Streptomyces ravidus and Streptomyces albaduncus, respectively. Sequencing of the 33.28 kb DNA region of the cosmid cosRav32 and the 34.65 kb DNA region of cosChry1‐1 and cosChryF2 revealed 36 and 35 open reading frames (ORFs), respectively, harboring tandem sets of type II polyketide synthase (PKS) genes, D ‐ravidosamine and D ‐virenose biosynthetic genes, post‐PKS tailoring genes, regulatory genes, and genes of unknown function. The isolated ravidomycin gene cluster was confirmed to be involved in ravidomycin biosynthesis through the production of a new analogue of ravidomycin along with anticipated pathway intermediates and biosynthetic shunt products upon heterologous expression of the cosmid, cosRav32, in Streptomyces lividans TK24. The identity of the cluster was further verified through cross complementation of gilvocarcin V (GV) mutants. Similarly, the chrysomycin gene cluster was demonstrated to be indirectly involved in chrysomycin biosynthesis through cross‐complementation of gilvocarcin mutants deficient in the oxygenases GilOII, GilOIII, and GilOIV with the respective chrysomycin monooxygenase homologues. The ravidomycin glycosyltransferase (RavGT) appears to be able to transfer both amino‐ and neutral sugars, exemplified through the structurally distinct 6‐membered D ‐ravidosamine and 5‐membered D ‐fucofuranose, to the coumarin‐based polyketide derived backbone. These results expand the library of biosynthetic genes involved in the biosyntheses of gilvocarcin class compounds that can be used to generate novel analogues through combinatorial biosynthesis.     

Antimikrobielle Dekontamination mit Syndets

Antimicrobial Decontamination Using Syndets The term ?decontamination”? was introduced, in order to include also those microorganisms (bacteria and fungi) which need not necessarily be removed by desinfection. They include germs which are often designated as contaminants, nosoparasites or not strictly pathogenic microorganisms. Several syndets have microbiostatic or even microbicide properties. Their high surface activity enables their spreading on bacteria and fungi, thus preventing germination. Owing to the soil removal properties of the syndets, the germs are depleted from the surface of the substrates and rinsed out. This sort of decontamination is necessary for the treatment of fungal infections of the skin, in order to prevent further growth and relapse. Comparative studies in vitro and in vivo showed Dermowas® to be best suited for the removal of pathogenic and not strictly pathogenic germs in the beginning of the therapy of dermatomycosis. The chances of cure are improved and the duration of treatment is reduced.     

海洋放线菌Y-0117农用活性代谢产物的研究

在农用抗生素筛选中发现代号为Y-0117的海洋放线茵菌株所产生的代谢产物对植物病原真菌具有良好的抑制作用。对Y-0117发酵液进行萃取、硅胶柱层析、制备薄层色谱、制备HPLC,分离纯化得到两个活性组分0117A和0117B。通过紫外光谱、质谱、核磁共振等手段,确定0117A的分子式为C35H58O8,分子量为604。结构与已知化合物Bafilomycin D相同;0117 B的分子式为C42H60O12．分子量为756,结构与近期发现具有抗癌活性的已知化合物Hygrobafilomycin相同。研究发现0117B对多种植物病原真菌具有良好的抑制活性,对稻瘟病菌的最小抑制浓度为15．63μg·mL^-1。     

运用SELEX技术筛选特异性高亲和IVB型纤毛结构蛋白RNA适配子

目的　获得特异性高亲和IVB型纤毛结构蛋白prepilS的RNA适配子 (aptamers) ,为开发预防、治疗伤寒杆菌肠热症的新型药物试剂奠定基础。方法　采用SELEX技术 (SystematicEvolutionofLigandsbyExponentialEn richment)筛选IVB型纤毛结构蛋白prepilS的特异性适配子 ,通过体外构建一个长度为 88nt含有 30个随机序列的单链DNA(ssDNA)文库 ,PCR扩增为双链DNA随机文库后 ,体外转录构建RNA随机文库 ,以Sepharose 4B为筛选介质 ,筛选具有特异性高亲和力prepilS蛋白的RNA适配子。结果　经 8轮筛选获得具有特异性高亲和prepilS蛋白的RNA适配子库。结论　成功地筛选出具有特异性高亲和力的RNA适配子库 ,该RNA适配子库可显著抑制伤寒杆菌侵入THP 1细胞     

Secondary Metabolites of the Rice Blast Fungus Pyricularia oryzae: Biosynthesis and Biological Function

Plant pathogenic fungi produce a wide variety of secondary metabolites with unique and complex structures. However, most fungal secondary metabolism genes are poorly expressed under laboratory conditions. Moreover, the relationship between pathogenicity and secondary metabolites remains unclear. To activate silent gene clusters in fungi, successful approaches such as epigenetic control, promoter exchange, and heterologous expression have been reported. Pyricularia oryzae, a well-characterized plant pathogenic fungus, is the causal pathogen of rice blast disease. P. oryzae is also rich in secondary metabolism genes. However, biosynthetic genes for only four groups of secondary metabolites have been well characterized in this fungus. Biosynthetic genes for two of the four groups of secondary metabolites have been identified by activating secondary metabolism. This review focuses on the biosynthesis and roles of the four groups of secondary metabolites produced by P. oryzae. These secondary metabolites include melanin, a polyketide compound required for rice infection; pyriculols, phytotoxic polyketide compounds; nectriapyrones, antibacterial polyketide compounds produced mainly by symbiotic fungi including endophytes and plant pathogens; and tenuazonic acid, a well-known mycotoxin produced by various plant pathogenic fungi and biosynthesized by a unique NRPS-PKS enzyme.