Investigations on the destruxin production of the entomopathogenic fungus Metarhizium anisopliae

The dynamics of cyclic peptide destruxins (dtxs) produced by Metarhizium anisopliae strains V245 and V275 were monitored both on solid and in liquid media. The results showed that both strains did not produce dtxs in large-scale fermenter cultures or solid Czapek Dox (CD) agar. Production of the major dtxs A and B could be determined in both strains when grown on rice for up to 10-30 days. The main dtxs A, B, E, and E diol were detected in CD liquid culture filtrate from both strains after three days post-inoculation on. Parallel decrease of dtx E and increase of E diol in the culture medium were found, indicating that the latter is the hydrolytic product from the former. Production of dtxs A and B was significantly positively correlated. A negative correlation was observed between the production of the metabolites and pH value of the medium. The influence of different nutrient sources on dtx production was evaluated by using media with different carbon and nitrogen ratios as well as with different insect homogenates. The findings showed that the amount of dtxs A, B, and E increased with the increasing content of peptone in the medium. When insect homogenate was used as single nutrient source or added to CD medium, no toxins were detected in the culture filtrate. The potential risk posed by the toxic metabolites during mass production is discussed.     

Variation in Metabolite Production by Septoria nodorum Isolates Adapted to Wheat or to Barley

The production of secondary metabolites by S. nodorum strains was examined in relation to their adaptation to wheat or to barley. Eleven strains of each type previously characterized were tested for the production in culture of (-)-(3R)-mellein (ochracin), (3R)-O-metbylmellein; (-)-(3R,4R)-4-hydroxyniellein, (-)-(3R,4S)-4-hydro-xymellein, mycophenolic acid, septorine, N-methoxy-septorine, N-methoxyseptorinol, and a new compound (=UN).
Mellein was produced by every strain. Both 4-hydro-xymellein isomers were yielded by all wheat-adapted strains and six barley-adapted strains. Most of the strains produced mycophenolic acid. On the other hand the pigmentation of culture filtrates of wheat-adapted strains was pale-yellow whereas the pigmentation of barley-adapted strains was grey-blue. The productions of O-methylmellein, septorine, N-metboxyseptorine and N-methoxyseptorinol were typical for wheat-adapted strains. The detection of UN was restricted to the filtrates of barley-adapted strains. If the pigmentation of filtrates and the production of septorines, UN and O-methyl-mellein are considered the strains fell into two groups. This classification is related to the host adaptation except for one barley-adapted strain that showed characteristics of wheat-adapted strains and one wheat-adapted strain which did not produce septorines.     

Enhancement of antibiotic and secondary metabolite detection from filamentous fungi by growth on nutritional arrays

Aims: We asked to what extent does the application of the OSMAC (one strain, many compounds) approach lead to enhanced detection of antibiotics and secondary metabolites in fungi? Protocols for bacterial microfermentations were adapted to grow fungi in nutritional arrays. Methods and Results: Protocols for microfermentations of non‐sporulating fungi were validated using known antifungal‐producing fungi. Detection of antifungal activity was often medium dependent. The effects of medium arrays and numbers of strains on detection of antifungal signals were modelled by interpolation of rarefaction curves derived from matrices of positive and negative extracts. Increasing the number of fermentation media for any given strain increased the probability of detection of growth inhibition of Candida albicans. Increasing biodiversity increased detection of antifungal phenotypes, however, nutritional arrays could partly compensate for lost antibiotic phenotypes when biodiversity was limiting. Conclusions: Growth and extraction in microtiter plates can enable a discovery strategy emphasizing low‐cost medium arrays that can better exploit the metabolic potential of strains. Significance and Impact of the Study: Increasing fermentation parameters raise the probability of detecting bioactive metabolites from strains. The protocols can be used to pre‐select strains and their growth conditions for scale up that will most likely yield antibiotics and secondary metabolites.     

Effect of the cyclopeptolide 90-215 on the production of destruxins and helvolic acid by Metarhizium anisopliae

The effect of the cyclopeptolide 90-215 on the production of destruxins and helvolic acid in various Metarhizium anisopliae strains was investigated. Addition of 10.0 mg L⁻¹ of the cyclopeptolide to the production media increased the production of destruxins by 1.3-fold to 12.5-fold, whereas the production of helvolic acid was decreased by 1.6- to 11.0-fold. Fifty liters were fermented in Erlenmeyer flasks with the strain 86-23766 grown in medium supplemented with cyclopeptolide 90-215. The procedure for isolation and purification of destruxins was simplified due to the higher yield of destruxins. Good quantities of destruxins were obtained from this fermentation. The results of our studies show that the addition of small quantities of a suitable compound can drastically alter the production and relative ratios of secondary metabolites. This can have a wide range of potential applications in the area of metabolite production. Received 09 December 1996/ Accepted in revised form 09 April 1997     

Chemical modulation of the metabolism of an endophytic fungal strain of Cophinforma mamane using epigenetic modifiers and amino-acids

Endophytic fungi are capable of producing a great diversity of bioactive metabolites. However, the presence of silent and lowly expressed genes represents a main challenge for the discovery of novel secondary metabolites with different potential uses. Epigenetic modifiers have shown to perturb the production of fungal metabolites through the induction of silent biosynthetic pathways leading to an enhanced chemical diversity. Moreover, the addition of bioprecursors to the culture medium has been described as a useful strategy to induce specific biosynthetic pathways. The aim of this study was to assess the effects of different chemical modulators on the metabolic profiles of an endophytic fungal strain of Cophinforma mamane (Botryosphaeriaceae), known to produce 3 thiodiketopiperazine (TDKP) alkaloids (botryosulfuranols A-C), previously isolated and characterized by our team. Four epigenetic modifiers, 5-azacytidine (AZA), sodium butyrate (SB), nicotinamide (NIC), homoserine lactone (HSL) as well as 2 amino acids, l-phenylalanine and l-tryptophan, as bioprecursors of TDKPs, were used. The metabolic profiles were analysed by UHPLC-HRMS/MS under an untargeted metabolomics approach. Our results show that the addition of the two amino acids in C. mamane culture and the treatment with AZA significantly reduced the production of the TDKPs botryosulfuranols A, B and C. Interestingly, the treatment with HSL significantly induced the production of different classes of diketopiperazines (DKPs). The treatment with AZA resulted as the most effective epigenetic modifier for the alteration of the secondary metabolite profile of C. mamane by promoting the expression of cryptic genes.     

Production of infectious hepatitis C virus of various genotypes in cell cultures

A unique hepatitis C virus (HCV) strain JFH-1 has been shown to replicate efficiently in cell culture with production of infectious HCV. We previously developed a DNA expression system containing HCV cDNA flanked by two self-cleaving ribozymes to generate HCV particles in cell culture. In this study, we produced HCV particles of various genotypes, including 1a (H77), 1b (CG1b), and 2a (J6 and JFH-1), in the HCV-ribozyme system. The constructs also contain the secreted alkaline phosphatase gene to control for transfection efficiency and the effects of culture conditions. After transfection into the Huh7-derived cell line Huh7.5.1, continuous HCV replication and secretion were confirmed by the detection of HCV RNA and core antigen in the culture medium. HCV replication levels of strains H77, CG1b, and J6 were comparable, whereas the JFH-1 strain replicates at a substantially higher level than the other strains. To evaluate the infectivity in vitro, the culture medium of JFH-1-transfected cells was inoculated into naive Huh7.5.1 cells. HCV proteins were detected by immunofluorescence 3 days after inoculation. To evaluate the infectivity in vivo, the culture medium from HCV genotype 1b-transfected cells was inoculated into a chimpanzee and caused a typical course of HCV infection. The HCV 1b propagated in vitro and in vivo had sequences identical to those of the HCV genomic cDNA used for cell culture transfection. The development of culture systems for production of various HCV genotypes provides a valuable tool not only to study the replication and pathogenesis of HCV but also to screen for antivirals.     

Major secondary metabolites produced by two commercial Trichoderma strains active against different phytopathogens

AIMS: Trichoderma harzianum strains T22 and T39 are two micro-organisms used as active agents in a variety of commercial biopesticides and biofertilizers and widely applied amongst field and greenhouse crops. The production, isolation, biological and chemical characterization of the main secondary metabolites produced by these strains are investigated. METHODS AND RESULTS: Of the three major compounds produced by strain T22, one is a new azaphilone that shows marked in vitro inhibition of Rhizoctonia solani, Pythium ultimum and Gaeumannomyces graminis var. tritici. In turn, filtrates from strain T39 were demonstrated to contain two compounds previously isolated from other T. harzianum strains and a new butenolide. The production of the isolated metabolites was also monitored by liquid chromatography/mass spectrometry during in vitro interaction with R. solani. CONCLUSIONS: This paper reports the isolation and characterization of the main secondary metabolites obtained from culture filtrates of two T. harzianum strains and their production during antagonistic interaction with the pathogen R. solani. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first work on secondary metabolites produced by the commercially applied strains T22 and T39. Our results provide a better understanding of the metabolism of these fungi, which are both widely used as biopesticides and/or biofertilizers in biocontrol.     

The effects of the piroplasm Babesia bigemina on the survival and reproduction of the blue tick,Boophilus decoloratus

Seven strains of the insect pathogenic fungus Verticillium lecanii have been examined in the Calliphora erythrocephala bioassay for the production, in surface culture on Czapek Dox medium, of insecticidal secondary metabolites. One strain which had lost its pathogenicity on storage yielded no active compounds. The remainder yielded dipicolinic acid (pyridine-2,6-dicarboxylic acid) which was responsible for the insecticidal activity of acidic extracts. Neutral extracts from two strains contained novel insecticidal C₂₅ compounds.     

Mutants of Escherichia coli producing pyrroloquinoline quinone.

In glucose minimal medium a PTS- strain of Escherichia coli [delta (ptsH ptsI crr)] could grow slowly (doubling time, d = 10 h). When the population reached 5 x 10(6) to 2 x 10(7) cells ml-1, mutants growing rapidly (d = 1.5 h) appeared and rapidly outgrew the initial population. These mutants (EF mutants) do not use a constitutive galactose permease for glucose translocation. They synthesize sufficient pyrroloquinoline quinone (PQQ) to yield a specific activity of glucose dehydrogenase (GDH) equivalent to that found in the parent strain grown in glucose minimal medium supplemented with 1 nM-PQQ. Membrane preparations containing an active GDH oxidized glucose to gluconic acid, which was also present in the culture supernatant of EF strains in glucose minimal medium. Glucose utilization is the only phenotypic trait distinguishing EF mutants from the parent strain. Glucose utilization by EF mutants was strictly aerobic as expected from a PQQ-dependent catabolism. The regulation of PQQ production by E. coli is discussed.     

Factors affecting the production of eremofortin C and PR toxin in Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Of 17 strains from the American Type Culture Collection that were studied for their ability to produce EC and PR toxin, 13 produced these metabolites. Toxin production by strains grown in solid media (10 cereals and 8 other agricultural products) was also investigated. Production of EC and PR toxin by fungi grown on cereals was greater than production of EC and PR toxin by fungi grown on legumes; fungi grown on corn produced the greatest amount of PR toxin. Addition of corn extracts to the culture medium greatly increased the production of EC and PR toxin in a coordinated manner, with no significant change in mycelial dry weight. The fungi produced the highest levels of EC and PR toxin at 20 to 24 degrees C depending on the strain. Toxin production was higher in stationary cultures than in cultures that were gently shaken at 120 rpm. The optimum pH for production of both EC and PR toxin was around pH 4.0. With regard to spore age, toxin levels did not change significantly when we used spores obtained from fungi that were grown at 24 degrees C for 3 up to 48 days.     

Factors affecting the production of eremofortin C and PR toxin in Penicillium roqueforti.

Eremofortin C (EC) and PR toxin are secondary metabolites of Penicillium roqueforti. Of 17 strains from the American Type Culture Collection that were studied for their ability to produce EC and PR toxin, 13 produced these metabolites. Toxin production by strains grown in solid media (10 cereals and 8 other agricultural products) was also investigated. Production of EC and PR toxin by fungi grown on cereals was greater than production of EC and PR toxin by fungi grown on legumes; fungi grown on corn produced the greatest amount of PR toxin. Addition of corn extracts to the culture medium greatly increased the production of EC and PR toxin in a coordinated manner, with no significant change in mycelial dry weight. The fungi produced the highest levels of EC and PR toxin at 20 to 24 degrees C depending on the strain. Toxin production was higher in stationary cultures than in cultures that were gently shaken at 120 rpm. The optimum pH for production of both EC and PR toxin was around pH 4.0. With regard to spore age, toxin levels did not change significantly when we used spores obtained from fungi that were grown at 24 degrees C for 3 up to 48 days.     

不同培养基对放线菌TRM10325抑制群感效应活性的影响

检测不同培养基条件下,放线菌TRM10325发酵液抑制群感效应的活性,初步了解其活性稳定性,并为筛选最优发酵培养基提供实验依据。选取17种合成培养基、26种天然培养基发酵放线菌TRM10325,采用微孔板半定量法检测其对紫色素杆菌群感效应以及表皮葡萄球菌生物膜形成的抑制作用。不同配方的培养基对放线菌10325抑制群感效应活性的影响也不相同,其中Am6培养基抑制群感效应效果最佳。成分不同的培养基,明显影响微生物不同次级代谢产物的产生。同一微生物在不同培养基中发酵,其次生代谢产物的种类和含量变化很大。最终选定Am6培养基为最适发酵培养基。     

The influence of medium composition on alkaloid biosynthesis by Penicillium citrinum

The fungus P. citrinum produces secondary metabolites, clavine ergot alkaloids (EA), and quinoline alkaloids quinocitrinines (QA) in medium with various carbon and nitrogen sources and in the presence of iron, copper, and zinc additives. Mannitol and sucrose are most favorable for EA biosynthesis and mannitol is most favorable for QA. Maximum alkaloid production is observed on urea. Iron and copper additives in the medium containing zinc ions stimulated fungal growth but inhibited alkaloid biosynthesis. The production of these secondary metabolites does not depend on the physiological state of culture, probably due to the constitutive nature of the enzymes involved in biosynthesis of these substances.     

具有抗哈维氏弧菌活性共生真菌HLZ-3菌株的筛选、鉴定及其培养条件

【背景】目前,海水养殖业中主要利用抗生素来防治哈维氏弧菌等病原菌,但抗生素的长期使用或滥用会对环境和人体健康带来危害,因此既环保又有效的生物防治方法具有广阔的应用前景。【目的】从海水产品共生微生物中筛选具有抗菌活性的菌株,对活性菌株进行鉴定并确定其合成抗菌活性物质的培养条件。【方法】利用沙氏和2216E培养基,以稀释涂布平板法从海水养殖动物中分离真菌和细菌;利用牛津杯法测定微生物发酵液抗水产病原哈维氏弧菌的活性;通过菌株的培养特征、形态特征和ITS序列分析对抗菌活性菌株进行鉴定;通过筛选发酵培养基的种类及盐度确定培养条件。【结果】从海蚌、白虾、海蛎子等9种样品中分离出微生物52株,其中真菌30株、细菌22株;筛选得到2株具有抗哈维氏弧菌活性的真菌菌株;其中一株活性菌株HLZ-3被鉴定为塔宾曲霉;菌株HLZ-3合成抗菌活性物质的培养条件为4%NaCl的大米培养基,28°C静置培养2周。【结论】实验结果为进一步分离纯化菌株HLZ-3所产抗菌活性次生代谢产物提供了基础。     

Biodegradation of α and β endosulfan in broth medium and soil microcosm by bacterial strain Bordetella sp. B9

Bacterial strains were isolated from endosulfan treated soil to study the microbial degradation of this pesticide in broth medium and soil microcosm. The isolates were grown in minimal medium and screened for endosulfan degradation. The strain, which utilized endosulfan and showed maximum growth, was selected for detail studies. Maximum degrading capability in shake flask culture was shown by Bordetella sp. B9 which degraded 80% of α endosulfan and 86% of β endosulfan in 18 days. Soil microcosm study was also carried out using this strain in six different treatments. Endosulfan ether and endosulfan lactone were the main metabolites in broth culture, while in soil microcosm endosulfan sulfate was also found along with endosulfan ether and endosulfan lactone. This bacterial strain has a potential to be used for bioremediation of the contaminated sites.     

Production of blastospores by three strains of metarhizium anisopliae (metch.) sorokin in submerged culture

Studies on blastospore production in different liquid media were conducted with three strains of Metarhizium anisopliae var. anisopliae (M. a.) derived from various countries (M. a. 43: Austria, M. a. 57: Brazil, M. a. 97: Philippines). Variation of six fermentation parameters (cornsteep products, carbohydrates, pH values, temperature, Tween 80, and polyethyleneglycol (PEG) 200) disclosed that the three strains of M. anisopliae differed in their growth pattern and physiology. In standard medium and in all tests, M. a. 57 produced the highest number of blastospores invariably amounting to > 10⁸ per ml, while mycelial pellets were never formed. The preferred carbohydrates were glucose and fructose. Blastospore production of M. a. 43 was increased by growth at 30°C, at pH 6.5 or by addition of 5% PEG 200. However, it was impaired by different concentrations of Tween 80 or higher concentrations of PEG 200 (10–15%). M. a. 97 produced most blastospores at 30°C, and the strain preferred basic (pH 8.0) as well as acid (pH 4.5) media. Blastospore production was increased by the addition of 5% PEG 200 or 0.4–1.2% Tween 80. Moreover, PEG 200 suppressed pellet formation effectively. Altogether, our results showed that for optimal blastospore production of Metarhizium anisopliae, suitable strain‐specific parameters have to be evaluated.     

Development and applications of destruxins: A review

The insecticidal and phytotoxic activities of destruxins (dtxs) have been well studied. The cyclodepsipeptides, which are dtxs mainly isolated from the fungus Metarhizium anisopliae and other fungi, have been well characterized in vitro and in vivo. A succession of important function, such as antitumoral, antiviral, insecticidal, cytotoxic, immunosuppressant, phytotoxic, and antiproliferative effects have been observed. To date, 39 dtxs derivatives have been identified. Dtxs possess a variety of biological activities, including acting as virulence factors for specific insects, a V-ATPase inhibitor that provides a basis for the development of new drug to against osteoporosis, cancer, or biological control agents, etc. Here, we focus on some of the research progress made on understanding dtxs during the last decade, introduce some of the newly identified dtx members, especially from M. anisopliae, and give an overview of the applications of dtxs. Using the dtxs to learn about and moderate biological events has advanced significantly during the past year. We believe that several ongoing dtx application fields may benefit from the reviewed information herein.