Antifungal Secondary Metabolites Research Articles

Species richness and distribution patterns of leaf-inhabiting endophytic fungi in a temperate forest canopy

In 2005, researchers at the Leipzig Canopy Crane Research Facility collected living leaves of four temperate tree species at heights of between 15 and 33 m above the ground. Following surface sterilisation of the leaves, leaf-fragments were cultured on malt extract agar which allowed the growth of endophytic fungi into the surrounding medium. Isolated cultures were identified by morphology and sequence analysis of the D1/D2 region of the large subunit rDNA. Phylogenetic analysis established the taxonomic positions of the fungi. A total of 49 different taxa were identified, representing 20 families and ten orders. With the exception of one basidiomycetous yeast, all taxa belonged to filamentous ascomycetes. Species richness was highest on Tilia cordata and lowest on Quercus robur. Species-accumulation curves showed that the sampling effort was not sufficient to cover the majority of the likely species at the investigation site. Most endophytes proved to be ubiquitous within the canopy of the investigation site, but habitat preferences in terms of different tree species, different light regimes and season (sampling times) were obvious for some abundant endophytes. Apiognomonia errabunda and Aspergillus niger occurred predominantly on Q. robur, Diplodina acerina on Acer pseudoplatanus, one species of Phoma significantly prefered shaded leaves from the lower canopy layer whereas Sordaria fimicola prefered sun-exposed leaves from the upper tree crowns. Seasonal patterns were observed, for example, for A. errabunda, which was abundant in young leaves in the spring and almost completely absent in aged autumn-leaves, thus suggesting the accumulation of antifungal secondary plant metabolites during the growing season.

Using Serratia plymuthica to control fungal pathogens of plants.

Abstract Interest in biological control of plant pathogens has increased in recent years fuelled by trends in agriculture towards greater sustainability and public concerns over the use of hazardous pesticides in the environment. Most studies on the biological control of fungal plant pathogens have tended to focus on the use of antagonistic rhizobacterial strains belonging to the genus Pseudomonas or Bacillus . However, the development of biocontrol products based on isolates belonging to the Gram-negative genus Serratia is now gaining momentum. Serratia plymuthica is a ubiquitous bacterium that has been preferentially recovered from rhizospheres all over the world, both as a free-living and endophytic organism. Specific strains of S. plymuthica produce a broad palette of antimicrobial compounds and might hold great potential as broad-spectrum biocontrol agents. This review surveys the advances of biocontrol research with respect to plant-associated S. plymuthica strains focusing on the principles and mechanisms of action of S. plymuthica and their use or potential use for the biological control of fungal plant diseases. A cursory overview of the taxonomy and ecology of S. plymuthica is also provided. We highlight recent progress in the identification of antifungal secondary metabolites produced by S. plymuthica and pay special attention to the regulatory mechanisms underpinning the production of the latter metabolites. Finally, we discuss several strategies that may provide a basis to improve the efficacy of S. plymuthica -mediated biocontrol.

Oxachelin, a Novel Iron Chelator and Antifungal Agent from Streptomyces sp. GW9/1258

During a screening campaign for new antimicrobial and antifungal secondary metabolites from several thousand actinomycetes, a novel compound, isolated by activity guided fractionation, was oxachelin (1) from the new Streptomyces sp. GW9/1258. Oxachelin shows strong antibiotic activities against several fungi and Gram(+) bacteria. Additionally, oxachelin is a strong complexing ligand for Fe3+ (siderophore), possibly making it useful e.g. for iron excess diseases.

Thiamine-auxotrophic mutants of Pseudomonas fluorescens CHA0 are defective in cell-cell signaling and biocontrol factor expression.

In the biocontrol strain Pseudomonas fluorescens CHA0, the Gac/Rsm signal transduction pathway positively controls the synthesis of antifungal secondary metabolites and exoenzymes. In this way, the GacS/GacA two-component system determines the expression of three small regulatory RNAs (RsmX, RsmY, and RsmZ) in a process activated by the strain's own signal molecules, which are not related to N-acyl-homoserine lactones. Transposon Tn5 was used to isolate P. fluorescens CHA0 insertion mutants that expressed an rsmZ-gfp fusion at reduced levels. Five of these mutants were gacS negative, and in them the gacS mutation could be complemented for exoproduct and signal synthesis by the gacS wild-type allele. Furthermore, two thiamine-auxotrophic (thiC) mutants that exhibited decreased signal synthesis in the presence of 5 x 10(-8) M thiamine were found. Under these conditions, a thiC mutant grew normally but showed reduced expression of the three small RNAs, the exoprotease AprA, and the antibiotic 2,4-diacetylphloroglucinol. In a gnotobiotic system, a thiC mutant was impaired for biological control of Pythium ultimum on cress. Addition of excess exogenous thiamine restored all deficiencies of the mutant. Thus, thiamine appears to be an important factor in the expression of biological control by P. fluorescens.

Posttranscriptional repression of GacS/GacA-controlled genes by the RNA-binding protein RsmE acting together with RsmA in the biocontrol strain Pseudomonas fluorescens CHA0.

In the plant-beneficial soil bacterium Pseudomonas fluorescens CHA0, the production of biocontrol factors (antifungal secondary metabolites and exoenzymes) is controlled at a posttranscriptional level by the GacS/GacA signal transduction pathway involving RNA-binding protein RsmA as a key regulatory element. This protein is assumed to bind to the ribosome-binding site of target mRNAs and to block their translation. RsmA-mediated repression is relieved at the end of exponential growth by two GacS/GacA-controlled regulatory RNAs RsmY and RsmZ, which bind and sequester the RsmA protein. A gene (rsmE) encoding a 64-amino-acid RsmA homolog was identified and characterized in strain CHA0. Overexpression of rsmE strongly reduced the expression of target genes (hcnA, for a hydrogen cyanide synthase subunit; aprA, for the main exoprotease; and phlA, for a component of 2,4-diacetylphloroglucinol biosynthesis). Single null mutations in either rsmA or rsmE resulted in a slight increase in the expression of hcnA, aprA, and phlA. By contrast, an rsmA rsmE double mutation led to strongly increased and advanced expression of these target genes and completely suppressed a gacS mutation. Both the RsmE and RsmA levels increased with increasing cell population densities in strain CHA0; however, the amount of RsmA showed less variability during growth. Expression of rsmE was controlled positively by GacA and negatively by RsmA and RsmE. Mobility shift assays demonstrated specific binding of RsmE to RsmY and RsmZ RNAs. The transcription and stability of both regulatory RNAs were strongly reduced in the rsmA rsmE double mutant. In conclusion, RsmA and RsmE together account for maximal repression in the GacS/GacA cascade of strain CHA0.

RsmY, a small regulatory RNA, is required in concert with RsmZ for GacA-dependent expression of biocontrol traits in Pseudomonas fluorescens CHA0.

In the plant-beneficial soil bacterium and biocontrol model organism Pseudomonas fluorescens CHA0, the GacS/GacA two-component system upregulates the production of biocontrol factors, i.e. antifungal secondary metabolites and extracellular enzymes, under conditions of slow, non-exponential growth. When activated, the GacS/GacA system promotes the transcription of a small regulatory RNA (RsmZ), which sequesters the small RNA-binding protein RsmA, a translational regulator of genes involved in biocontrol. The gene for a second GacA-regulated small RNA (RsmY) was detected in silico in various pseudomonads, and was cloned from strain CHA0. RsmY, like RsmZ, contains several characteristic GGA motifs. The rsmY gene was expressed in strain CHA0 as a 118 nt transcript which was most abundant in stationary phase, as revealed by Northern blot and transcriptional fusion analysis. Transcription of rsmY was enhanced by the addition of the strain's own supernatant extract containing a quorum-sensing signal and was abolished in gacS or gacA mutants. An rsmA mutation led to reduced rsmY expression, via a gacA-independent mechanism. Overexpression of rsmY restored the expression of target genes (hcnA, aprA) to gacS or gacA mutants. Whereas mutants deleted for either the rsmY or the rsmZ structural gene were not significantly altered in the synthesis of extracellular products (hydrogen cyanide, 2,4-diacetylphloroglucinol, exoprotease), an rsmY rsmZ double mutant was strongly impaired in this production and in its biocontrol properties in a cucumber-Pythium ultimum microcosm. Mobility shift assays demonstrated that multiple molecules of RsmA bound specifically to RsmY and RsmZ RNAs. In conclusion, two small, untranslated RNAs, RsmY and RsmZ, are key factors that relieve RsmA-mediated regulation of secondary metabolism and biocontrol traits in the GacS/GacA cascade of strain CHA0.

Inderjit, Mallik, A.U. (eds). Chemical ecology of plants: allelopathy in aquatic and terrestrial ecosystems

Inderjit, Mallik, A.U. (eds). Chemical ecology of plants: allelopathy in aquatic and terrestrial ecosystems

Regulatory RNA as mediator in GacA/RsmA-dependent global control of exoproduct formation in Pseudomonas fluorescens CHA0.

In Pseudomonas fluorescens CHA0, an antagonist of root-pathogenic fungi, the GacS/GacA two-component system tightly controls the expression of antifungal secondary metabolites and exoenzymes at a posttranscriptional level, involving the RNA-binding protein and global regulator of secondary metabolism RsmA. This protein was purified from P. fluorescens, and RNA bound to it was converted to cDNA, which served as a probe to isolate the corresponding chromosomal locus, rsmZ. This gene encoded a regulatory RNA of 127 nucleotides and a truncated form lacking 35 nucleotides at the 3' end. Expression of rsmZ depended on GacA, increased with increasing population density, and was stimulated by the addition of a solvent-extractable extracellular signal produced by strain CHA0 at the end of exponential growth. This signal appeared to be unrelated to N-acyl-homoserine lactones. A conserved upstream element in the rsmZ promoter, but not the stress sigma factor RpoS, was involved in rsmZ expression. Overexpression of rsmZ effectively suppressed the negative effect of gacS and gacA mutations on target genes, i.e., hcnA (for hydrogen cyanide synthase) and aprA (for the major exoprotease). Mutational inactivation of rsmZ resulted in reduced expression of these target genes in the presence of added signal. Overexpression of rsmA had a similar, albeit stronger negative effect. These results support a model in which GacA upregulates the expression of regulatory RNAs, such as RsmZ of strain CHA0, in response to a bacterial signal. By a titration effect, RsmZ may then alleviate the repressing activity of RsmA on the expression of target mRNAs.

Antifungal leaf-surface metabolites correlate with fungal abundance in sagebrush populations.

A central component in understanding plant-enemy interactions is to determine whether plant enemies, such as herbivores and pathogens, mediate the evolution of plant secondary metabolites. Using 26 populations of a broadly distributed plant species, sagebrush (Artemisia tridentata), we examined whether sagebrush populations in habitats with a greater prevalence of fungi contained antifungal secondary metabolites on leaf surfaces that were more active and diverse than sagebrush populations in habitats less favorable to fungi. Because moisture and temperature play a key role in the epidemiology of most plant-pathogen interactions, we also examined the relationship between the antifungal activity of secondary metabolites and the climate of a site. We evaluated the antifungal activity of sagebrush secondary metabolites against two fungi, a wild Penicillium sp. and a laboratory yeast, Saccharomyces cerevisiae, using a filter-paper disk assay and bioautography. Comparing the 26 sagebrush populations, we found that fungal abundance was a good predictor of both the activity (r2 = 0.36 for Saccharomyces, r2 = 0.37 for Penicillium) and number (r2 = 0.34 for Saccharomyces) of antifungal secondary metabolites. This suggests that selection imposed by fungal pathogens has led to more effective antifungal secondary metabolites. We found that the antifungal activity of sagebrush secondary metabolites was negatively related to average vapor pressure deficit of the habitat (r2 = 0.60 for Saccharomyces, r2 = 0.61 for Penicillium). Differences in antifungal activity among populations were not due to the amount of secondary metabolites, but rather to qualitative differences in the composition of antifungal compounds. Although all populations in habitats with high fungal prevalence had secondary metabolites with high antifungal activity, different suites of compounds were responsible for this activity, suggesting independent outcomes of selection on plants by fungal pathogens. The location of antifungal secondary metabolites on the leaf surface is consistent with their putative defense role, and we found no evidence supporting other functions, such as protection from ultraviolet light or oxidation. That the antifungal activity of sagebrush secondary metabolites was similar for two different fungi provides support for broad antifungal defenses. The incidence and severity of fungal disease in the field (caused by Puccinia tanaceti) were similar in moist and dry habitats, possibly reflecting an equilibrium between plant defense and fungal attack, as sites with greater fungal abundance compensated with more effective secondary metabolites. The geographic correlation between fungal abundance and antifungal secondary metabolites of sagebrush, coupled with our other data showing heritable variation in these metabolites, suggests that pathogenic fungi have selected for antifungal secondary metabolites in sagebrush.

Characterization of spontaneous gacS and gacA regulatory mutants of Pseudomonas fluorescens biocontrol strain CHAO.

In Pseudomonasfluorescens strain CHAO, the response regulator gene gacA controls expression of extracellular enzymes and antifungal secondary metabolites, which are important for this strain's biocontrol activity in the plant rhizosphere. Two Tn5 insertion mutants of strain CHA0 that had the same pleiotropic phenotype as gacA mutants were complemented by the gacS sensor kinase gene of P. syringae pv. syringae as well as that of P. fluorescens strain Pf-5, indicating that both transposon insertions had occurred in the gacS gene of strain CHA0. This conclusion was supported by Southern hybridisation using a gacS probe from strain Pf-5. Overexpression of the wild-type gacA gene partially compensated for the gacS mutation, however, the overexpressed gacA gene was not stably maintained, suggesting that this is deleterious to the bacterium. Strain CHA0 grown to stationary phase in nutrient-rich liquid media for several days accumulated spontaneous pleiotropic mutants to levels representing 1.25% of the population; all mutants lacked key antifungal metabolites and extracellular protease. Half of 44 spontaneous mutants tested were complemented by gacS, the other half were restored by gacA. Independent point and deletion mutations arose at different sites in the gacA gene. In competition experiments with mixtures of the wild type and a gacA mutant incubated in nutrient-rich broth, the mutant population temporarily increased as the wild type decreased. In conclusion, loss of gacA function can confer a selective advantage on strain CHA0 under laboratory conditions.

Pigment Diverse Mutants of Pseudomonas sp.: Inhibition of Fungal Growth and Stimulation of Growth of Cicer arietinum

A Pseudomonas strain MRS16 inhibited growth of different pathogenic fungi (Aspergillus sp., Fusarium oxysporum, Pythium aphanidermatum and Rhizoctonia solani) in vitro. Larger inhibition zones were obtained on nutrient agar and King's B media compared to potato dextrose agar and pigment production media. Mutants altered in production of fluorescent pigment were derived by nitrosoguanidine mutagenesis. The pigment overproducer mutant MRS16M-1 was more inhibitory whereas nonproducer mutant MRS16M-5 was less inhibitory than parent strain on nutrient agar medium. Addition of iron (100 µM FeCl3) in the medium decreased inhibition of fungal growth, suggesting the involvement of siderophores and other antifungal secondary metabolites. Seed bacterization of two cultivars of chickpea (Cicer arietinum cvs. H8618 and C235) differing in susceptibility to wilt caused initial root and shoot stunting at 5 d of growth followed by proliferation of secondary root growth at 10 d. Coinoculation of chickpea with Pseudomonas strain MRS16 or mutants and Rhizobium sp. Cicer strain Ca181 enhanced nodulation, nitrogen fixation and plant dry mass as compared to single inoculation with Rhizobium strain under sterile conditions.

Biotransformation of the Trichoderma metabolite 6- n-pentyl-2 H-pyran-2-one by cell suspension cultures of Pinus radiata

Cell suspension cultures of Pinus radiata metabolize the antifungal Trichoderma secondary metabolite 6- n-pentyl-2 H-pyran-2-one (6PAP) ( 1) via hydroxylation of the pentyl side chain. Examination of the culture medium following dosing studies with 1 revealed that 79–85% of this bioactive compound had been metabolised after 144 h. At that time, 34–40% of the metabolized dose was recovered as a series of monohydroxylated isomers of 1, the principal metabolite being 5-(2-pyron-6-yl)pentan-5-ol ( 7).

Fungal resistance to plant antibiotics as a mechanism of pathogenesis.

Many plants produce low-molecular-weight compounds which inhibit the growth of phytopathogenic fungi in vitro. These compounds may be preformed inhibitors that are present constitutively in healthy plants (also known as phytoanticipins), or they may be synthesized in response to pathogen attack (phytoalexins). Successful pathogens must be able to circumvent or overcome these antifungal defenses, and this review focuses on the significance of fungal resistance to plant antibiotics as a mechanism of pathogenesis. There is increasing evidence that resistance of fungal pathogens to plant antibiotics can be important for pathogenicity, at least for some fungus-plant interactions. This evidence has emerged largely from studies of fungal degradative enzymes and also from experiments in which plants with altered levels of antifungal secondary metabolites were generated. Whereas the emphasis to date has been on degradative mechanisms of resistance of phytopathogenic fungi to antifungal secondary metabolites, in the future we are likely to see a rapid expansion in our knowledge of alternative mechanisms of resistance. These may include membrane efflux systems of the kind associated with multidrug resistance and innate resistance due to insensitivity of the target site. The manipulation of plant biosynthetic pathways to give altered antibiotic profiles will also be valuable in telling us more about the significance of antifungal secondary metabolites for plant defense and clearly has great potential for enhancing disease resistance for commercial purposes.

Trichoderma /pathogen interactions: measurement of antagonistic chemicals produced at the antagonist/pathogen interface using a tubular bioassay

A tubular bioassay was used to measure analytically the local production and concentration of the antifungal Trichoderma secondary metabolite 6‐n‐pentyl‐2H‐pyran‐2‐one (6PAP) at the Trichoderma antagonist/pathogen interface. 6PAP levels significantly increased in the presence of the pathogen Botrytis cinerea, typically 300–700%, and were highest near the pathogen source. The level of response for a particular Trichoderma isolate was found to vary with the test organism used. Two products produced by biotransformation of 6PAP by B. cinerea in response to the interaction were also detected.

Combining proteolytic and phloroglucinol‐producing bacteria for improved biocontrol of Pythium‐mediated damping‐off of sugar beet

Pseudomonas fluorescens F113 and Stenotrophomonas maltophilia W81 protect sugar beet from Pythium‐mediated damping‐off through production of the antifungal secondary metabolite 2,4‐diacetylphloroglucinol and extracellular proteolytic activity, respectively. In this study, the two biocontrol strains were combined in a consortium, with the objective of improving upon the level of protection achieved when using each strain singly. Growth and in vitro production of 2,4‐diacetylphloroglucinol by F113 and extracellular lytic enzymes by W81 were not affected when inoculated in combination. The abilities of W81 and F113 to colonize the rhizosphere of sugar beet were essentially similar when the two strains were applied singly or coinoculated onto seeds in a 1 : 1 ratio, both in natural soil microcosms and under field conditions. Concomitantly, single inoculation with W81 or F113 effectively prevented colonization of sugar beet seeds by Pythium spp. in soil microcosms, without the necessity for combining both strains. However, this parity was not reflected in seed emergence where the combination of W81 and F113 significantly enhanced final sugar beet stands (to the level achieved with chemical pesticides) under microcosm conditions at 28 days after sowing. In a field experiment, the only inoculation treatment capable of conferring effective protection of sugar beet was that in which W81 and F113 were coinoculated, and this treatment proved equivalent to the use of chemical fungicides. In conclusion, when compared with single inoculations of either biocontrol strain, the combined use of a phloroglucinol‐producing P. fluorescens and a proteolytic S. maltophilia improved protection of sugar beet against Pythium‐mediated damping‐off.

FUNGAL CELLWALL POLYSACCHARIDES ELICIT AN ANTIFUNGAL SECONDARY METABOLITE (PHYTOALEXIN) IN THE CYANOBACTERIUM SCYTONEMA OCELUTUM2

ABSTRACT The addition of fungal cell‐wall homogenates of Penicillium notatum and Cylindrocladium spathiphylli markedly stimulated the accumulation of tolytoxin, an antifungal secondary metabolite, in cultures of the cyanobacterium Scytonema ocellatum Lyngbye ex Bornet et Flahault. Evaluation of polysaccharides, proteins, and other polymers established that a limited range of polysaccharides, especially chitin and carboxymthylcellulose, selectively elicited enhanced tolytoxin accumulation in S. ocellatum. The elicitor activity of fungal cell‐wall preparations could be correlated with the chitin content of the preparation. Polymeric chitin was half‐maximally effective at a concentration (EC50) of 19 mg.L‐1, whereas chitin oligoments were more effective (EC50= 3.3 mg.L‐1) in eliciting enhanced tolytoxin accumulation. The elicitor activity of either purified chitin or an elicitor‐active fungal cell‐wall preparation could be destroyed by treatment with chitinase. The results suggest an ecological role for tolytoxin as an inducible chemical defense agent (phytoalexin) capable of protecting S. ocellatum against fungal invasion.

Isolation and Identification of Antifungal Metabolites Produced by Rice-Associated AntagonisticPseudomonasspp.

Antifungal secondary metabolites produced by six strains of rice-associated Pseudomonas spp. were determined. Five different media, pigment production medium (PPM), 1/5M523, potato-dextrose agar (PDA), yeast malt (YM), and King's medium B (KMB), were used to determine the best medium for plate assays of these strains against Rhizoctonia solani AG1, the rice sheath blight pathogen. Large inhibition zones were obtained on PPM, KMB, and YM using two strains of P. cepacia, In-b-6854 and In-b-6858, originating from the rice seed. The other four strains showed varying degrees of inhibition zones in the media tested. Their antifungal metabolites were isolated and compared with metabolites from reference strains and known compounds by regular and reversed-phase thin-layer chromatography. Pyrrolnitrin was present in the cell extracts from the two P. cepacia strains. Phenazine-1-carboxylic acid and pyocyanine were produced by two strains of P. aeruginosa, In-b-109 and In-b-784. A strain of P. putida, In-b-1821, produced 2,4-diacetylphloroglucinol. Several unidentified compounds were produced by a strain of P. fluorescens, In-b-7-14. Ten microliters of eluted spots from In-b-6854 and In-b-6858 inhibited the mycelial growth of R. solani 20 to 24 mm away.

Durchlässigkeit der Siebröhren für Druckströmungen.

Mollisia is a taxonomically neglected discomycete genus (Helotiales, Leotiomycetes) of commonly encountered saprotrophs on decaying plant tissues throughout temperate regions. The combination of indistinct morphological characters, more than 700 names in the literature, and lack of reference DNA sequences presents a major challenge when working with Mollisia. Unidentified endophytes, including strains that produced antifungal or antiinsectan secondary metabolites, were isolated from conifer needles in New Brunswick and placed with uncertainty in Phialocephala and Mollisia, necessitating a more comprehensive treatment of these genera. In this study, morphology and multigene phylogenetic analyses were used to explore the taxonomy of Mollisiaceae, including Mollisia, Phialocephala, and related genera, using new field collections, herbarium specimens, and accessioned cultures and sequences. The phylogeny of Mollisiaceae was reconstructed and compared using the nuc internal transcribed spacer rDNA (ITS) barcode and partial sequences of the 28S nuc rDNA (LSU) gene, largest subunit of RNA polymerase II (RPB1), DNA topoisomerase I (TOP1), and the hypothetical protein Lipin/Ned1/Smp2 (LNS2). The results show that endophytism is common throughout the Mollisiaceae lineage in a diverse range of hosts but is infrequently attributed to Mollisia because of a paucity of reference sequences. Generic boundaries within Mollisiaceae are poorly resolved and based on phylogenetic evidence the family included species placed in Acephala, Acidomelania, Barrenia, Bispora, Cheirospora, Cystodendron, Fuscosclera, Hysteronaevia, Loramyces, Mollisia, Neopyrenopeziza, Obtectodiscus, Ombrophila, Patellariopsis, Phialocephala, Pulvinata, Tapesia (=Mollisia), and Trimmatostroma. Taxonomic novelties included the description of five novel Mollisia species and five novel Phialocephala species and the synonymy of Fuscosclera with Phialocephala, Acidomelania with Mollisia, and Loramycetaceae with Mollisiaceae.