Phytopathogenic Fungus Botrytis Cinerea Research Articles

Microbiological characterization for a new wild strain of Paenibacillus polymyxa with antifungal activity against Botrytis cinerea

A new bacterial strain with powerful antifungal activity against the phytopathogenic fungus Botrytis cinerea was characterized and identified as Paenibacillus polymyxa SCHC33. The kinetics of bacterial growth was fitted to the Monod model, with μmax=0.218h−1, Ks=0.087g/L, and YX/S=0.159gdry biomass/gglucose. The antifungal activity of the bacterium was detected in vitro as inhibition halos in antagonism bioassays against B. cinerea, and such activity was unchanged in magnitude when the assays were done in the same culture media with or without glucose. The ability of the bacterium to protect grapevine leaves against B. cinerea attack was determined as the percentage of damage to the plant tissue, after inoculation with different ratios of conidia and bacteria. Approximately 88% of the leaf surface was protected by the bacteria when a ratio of conidia:bacteria=1:100 was used. No significant damage was observed in the tissue of the leaves inoculated only with bacteria, whereas the leaves that were treated with solely conidia, the necrotic damage occurred in approximately 100% of their surface. Therefore, the results indicate that P. polymyxa SCHC33 has great potential to become the active component of a new biofungicide for control of B. cinerea.

Molecular characterization of a novel endornavirus from the phytopathogenic fungus Botrytis cinerea

The complete sequence of a novel endornavirus (Botrytis cinerea endornavirus 1, BcEV1) from the phytopathogenic fungus Botrytis cinerea strain HBtom-372 was determined. The BcEV1 coding strand is 11,557 nucleotides long, possessing an open reading frame (ORF) that codes for a polyprotein of 3,787 amino acid residues and lacks a site-specific nick. The polyprotein contains viral methyltransferase (MTR) domain, a cysteine-rich region (CRR), two putative viral helicase (DEXDc-like and Hel-1) domains, and an RNA-dependent RNA polymerase_2 (RdRp_2) domain. In phylogenetic analysis, BcEV1 clustered with several fungal endornaviruses, forming an independent clade, and it was detected in 4.2% of B. cinerea strains collected from central China.

The botrydial biosynthetic gene cluster of Botrytis cinerea displays a bipartite genomic structure and is positively regulated by the putative Zn(II)2Cys6 transcription factor BcBot6

Botrydial (BOT) is a non-host specific phytotoxin produced by the polyphagous phytopathogenic fungus Botrytis cinerea. The genomic region of the BOT biosynthetic gene cluster was investigated and revealed two additional genes named Bcbot6 and Bcbot7. Analysis revealed that the G+C/A+T-equilibrated regions that contain the Bcbot genes alternate with A+T-rich regions made of relics of transposable elements that have undergone repeat-induced point mutations (RIP). Furthermore, BcBot6, a Zn(II)2Cys6 putative transcription factor was identified as a nuclear protein and the major positive regulator of BOT biosynthesis. In addition, the phenotype of the ΔBcbot6 mutant indicated that BcBot6 and therefore BOT are dispensable for the development, pathogenicity and response to abiotic stresses in the B. cinerea strain B05.10. Finally, our data revealed that B. pseudocinerea, that is also polyphagous and lives in sympatry with B. cinerea, lacks the ability to produce BOT. Identification of BcBot6 as the major regulator of BOT synthesis is the first step towards a comprehensive understanding of the complete regulation network of BOT synthesis and of its ecological role in the B. cinerea life cycle.

Crop management as a driving force of plant growth promoting rhizobacteria physiology.

Crop management systems influence plant productivity and nutrient use efficiency, as well as plant growth-promoting rhizobacteria (PGPR), which are known to influence the growth of plants via phytohormone production, phosphate solubilization, nitrogen (N) fixation and antimicrobial activity. The objective of this study was to compare the influence of two crop management system on microbial PGPR features. PGPR isolated from the rhizospheres of Carica papaya L. grown under two distinct management systems (conventional and organic) were identified and characterized. The 12 strains most efficient in solubilizing inorganic phosphate belonged to the genera Burkholderia, Klebsiella, and Leclercia. N fixation was observed in the strains B. vietnamiensis from the conventional farming system and B. vietnamiensis, B. cepacia and Leclercia sp. from the organic farming system. The B. vietnamiensis, B. cepacia, Klebsiella sp. and Klebsiella sp. isolates showed antifungal activity, while Leclercia sp. did not. The strains B. vietnamiensis and Enterobcter sp. (isolated from the conventional farming system) and Klebsiella sp. (isolated from the organic farming system) were efficient at solubilizing phosphate, producing phytohormones and siderophores, and inhibiting the mycelial growth of various phytopathogenic fungi (Botrytis cinerea, Pestalotia sp., Alternaria sp., Phoma sp., Fusarium culmorum, Geotrichum candidum). Physiological differences between the isolates from the two crop management regimes were distinguishable after 10 years of distinct management.

First Total Synthesis, Structure Revision, and Natural History of the Smallest Cytochalasin: (+)-Periconiasin G.

The total synthesis of the smallest cytochalasin isolated so far, periconiasin G, which bears a seven-membered ring in lieu of the usual macrocycle, has been performed from both enantiomers of citronellal, relying on an intramolecular Diels-Alder reaction in favor of the natural endo stereochemistry. We show that, among the four synthesized stereoisomers, including the exo isomers, the one matching the NMR data of the natural product was not that assigned in the original report, imposing structure revision. The natural product, previously isolated from a plant-mutualistic fungus, was biologically investigated taking into account its natural history, showing significant effects against the phytopathogenic fungus Botrytis cinerea and thus opening new opportunities in combating this pest.

LAÇASSE CATALYZED-SYNTHESIS OF 4,4'-BIPHENYLDIAMINE FROM P-CHLOROANILINE. EVALUATION OF ANTIFUNGAL AND ANTIOXIDANT ACTIVITIES

ABSTRACT Laccase is a copper-containing oxidase that catalyzes reduction of molecular oxygen to water and the oxidation of a phenolic compound. In this paper, laccase was utilized to synthesize 4,4’-Biphenyldiamine using p -chloroaniline as substrate by means of a coupling reaction. The synthesized compound, 4,4’-Biphenyldiamine, presented low antifungal activity against the phytopathogenic fungus Botrytis cinerea , however the antioxidant ability, measurement by ORAC-PGR method, was higher than substrate.This work corresponds to the first report of synthesis 4,4’-Biphenyldiamine, from p -chloroaniline in a lacasse-catalyzed reaction. Keywords: p -chloroaniline, 4,4’-Biphenyldiamine, laccase, antioxidant activity, antifungal activity. e-mail: leonora.mendoza@usach.cl, milena.cotoras@usach.cl 1. INTRODUCTION Laccases (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) are copper-containing oxidases that catalyze the single-electron oxidation of a wide range of phenolic compounds and reduction of molecular oxygen to water

Genetic and Molecular Basis of Botrydial Biosynthesis: Connecting Cytochrome P450-Encoding Genes to Biosynthetic Intermediates.

Over two hundred species of plants can be infected by the phytopathogenic fungus Botrytis cinerea under a range of different environmental conditions. In response to these, the fungus produces unique terpenoid and polyketide metabolites. Parts of the plants may be killed by the phytotoxin botrydial, enabling the fungus to feed on the dead cells. In this paper, we describe the genetic and molecular basis of botrydial biosynthesis and the function of the five genes of the genome of B. cinerea that together constitute the botrydial biosynthetic gene cluster. Genes BcBOT3 and BcBOT4, encoding two cytochrome P450 monooxygenases, were inactivated by homologous recombination and were shown to catalyze regio- and stereospecific hydroxylations at the carbons C-10 and C-4, respectively, of the presilphiperfolan-8β-ol skeleton. The null mutants, bcbot3Δ and bcbot4Δ, accumulated key intermediates in the botrydial biosynthesis enabling the complete genetic and molecular basis of the botrydial biosynthetic pathway to be established. Furthermore, the bcbot4Δ mutant overproduced a significant number of polyketides, which included, in addition to known botcinins, botrylactones and cinbotolide A, two new botrylactones and two new cinbotolides, cinbotolides B and C.

Synthesis, Characterization, and Antifungal Activity of Phenylpyrrole-Substituted Tetramic Acids Bearing Carbonates.

For the aim of discovering new fungicide, a series of phenylpyrrole-substituted tetramic acid derivatives bearing carbonates 6a–q were designed and synthesized via 4-(2,4-dioxopyrrolidin-3-ylidene)-4-(phenylamino)butanoic acids 4a–k and the cyclized products 1′,3,4,5′-tetrahydro-[2,3′-bipyrrolylidene]-2′,4′,5(1H)-triones 5a–k. The compounds were characterized using IR, 1H- and 13C-NMR spectroscopy, mass spectrometry (EI-MS), and elemental analysis. The structure of 6b was confirmed by X-ray diffraction crystallography. The title compounds 6a–q were bioassayed in vitro against the phytopathogenic fungi Fusarium graminearum, Botrytis cinerea and Rhizoctonia solani at a concentration of 100 μg/mL, respectively. Most compounds displayed good inhibitory activity.

Synthesis and antifungal activity of 2H-1,4-benzoxazin-3(4H)-one derivatives

ABSTRACTA series of 2-alkyl-2H-1,4-benzoxazin-3(4H)-ones (4a-l) was easily synthesized by two-step process involving O-alkylation of 2-nitrophenols with methyl 2-bromoalkanoates and next “green” catalytic reductive cyclization of the obtained 2-nitro ester intermediates (3a-l). Further, 6,7-dibromo (5a-c) and N-acetyl (6) derivatives were prepared by bromination and acetylation of unsubstituted 2-alkyl-2H-1,4-benzoxazin-3(4H)-ones (4a-c). The novel compounds (3a-l, 4d-l, 5a-c and 6) were fully characterized by spectroscopic methods (MS, 1H and 13C NMR). 2-Alkyl-2H-1,4-benzoxazin-3(4H)-ones (4a-l, 5a-c and 6) were screened for antifungal activity. Preliminary assays were performed using two methods: in vitro against seven phytopathogenic fungi—Botrytis cinerea, Phythophtora cactorum, Rhizoctonia solani, Phoma betae, Fusarium culmorum, Fusarium oxysporum and Alternaria alternata—and in vivo against barley powdery mildew Blumeria graminis. The tested compounds displayed moderate to good antifungal activity at high concentration (200 mg L−1). The most potent compounds were 2-ethyl-2H-1,4-benzoxazin-3(4H)-one (4a), 2-ethyl-7-fluoro-2H-1,4-benzoxazin-3(4H)-one (4g) and 4-acetyl-2-ethyl-2H-1,4-benzoxazin-3(4H)-one (6), which completely inhibited the mycelial growth of seven agricultural fungi at the concentration of 200 mg L−1 in the in vitro tests. Moreover, 2-ethyl-2H-1,4-benzoxazin-3(4H)-one (4a) and 4-acetyl-2-ethyl-2H-1,4-benzoxazin-3(4H)-one (6) were also screened for antifungal activity at concentrations of 100 mg L−1 and 20 mg L−1. In the concentration of 100 mg L−1, the N-acetyl derivative (6) completely inhibited the growth of three strains of fungi (F. culmorum, P. cactorum and R. solani), while 2-ethyl-2H-1,4-benzoxazin-3(4H)-one (4a) completely inhibited only R. solani strain. At the concentration of 20 mg L−1, compound 6 showed good activity only against P. cactorum strain (72%).

CHARACTERIZATION OF THE ANTIFUNGAL ACTIVITY AGAINST BOTRYTIS CINEREA OF SCLAREOL AND 13-EPI-SCLAREOL, TWO LABDANE-TYPE DITERPENOIDS

The antifungal activity of Sclareol and 13-epi-Sclareol, two labdane-type diterpenoids, on mycelial growth of the phytopathogenic fungus Botrytis cinerea was evaluated. Diterpenoid fungitoxicity was assessed using the radial growth test method. Both diterpenoids inhibited the mycelial growth of B. cinerea in solid medium; however the inhibitory activity of Sclareol was slightly higher than 13-epi-Sclareol with IC50 value of 237.6 μg/mL and 268.8 μg/mL, respectively. On the other hand, both labdane-type diterpenoids did not alter the plasmatic membrane integrity; however the oxygen consumption of B. cinerea germinating conidia was affected. The evidence suggests that action mechanism of these molecules would be related to the uncoupling of mitochondrial oxidative phosphorylation. Finally, biotransformation of Sclareol by B. cinerea was analysed and the main biotransformed metabolite was identified as 3s-hydroxysclareol.

CHARACTERISTIC OF STREPTOMYCES SPECIES WITH ANTIMICROBIAL ACTIVITY AGAINST SELECTED PHYTOPATHOGENIC BACTERIA AND FUNGI

The object of this study was to find and characterise streptomycete isolates with antimicrobial activity accomplished by the agar plug method against phytopathogenic species. The test-microorganisms were selected according to their importance in agriculture. All of them belong to phytopathogenic species which reduce yields of agriculturally important plants worldwide. A total number of four phytopathogenic bacteria (Xanthomonas campestris (CCM 22), Pseudomonas syringae (CCM 2868), Erwinia amylovora (CCM 1114), Clavibacter michiganensis subsp. sepedonicus (CCM 7014)) and four phytopathogenic fungi (Botrytis cinerea, Fusarium poae, Alternaria tenuissima, Alternaria arborescens) were used for this experiment. Overall twenty streptomycete isolates showed antimicrobial activity against at least two of the test-microorganisms. These active isolates were subsequently characterized. Streptomycete isolates were observed for morphological characteristics, such as morphology and colour of aerial and substrate mycelium, colour of diffusible pigments, production of melanoid pigments on peptone-yeast extract iron agar and sporophore and spore chain morphology following the International Streptomyces Project. Genes coding polyketide synthase (PKS-I) and nonribosomal peptide synthetase (NRPS), which are considered to responsible for the synthesis of large number of biologically active compounds, like antibiotics and antifungals, that are produced by Streptomyces species largely, were screened. The occurrence of these genes in the genome of our isolates was in accordance with results from antimicrobial activity analysis. Classification to genus Streptomyces were confirmed by DNA sequencing.

Action mechanism for 3β-hydroxykaurenoic acid and 4,4-dimethylanthracene-1,9,10(4H)-trione on Botrytis cinerea

The mechanism of action of the diterpenoid 3β-hydroxykaurenoic acid and the anthraquinone 4,4-dimethylanthracene-1,9,10(4H)-trione on the phytopathogenic fungus Botrytis cinerea was studied. The effect of both compounds on the respiratory process and on the membrane integrity of B. cinerea was evaluated. The results showed that 3β-hydroxykaurenoic acid inhibited the growth of this fungus by disrupting the plasmatic membrane. This compound also partially affected oxygen consumption of B. cinerea germinating conidia. Conversely, 4,4-dimethylanthracene-1,9,10(4H)-trione did not produce membrane disruption of B. cinerea. The effect of this compound on mycelial growth was notably increased by the presence of an inhibitor of the cyanide-resistant respiration pathway. It also was shown that the anthraquinone inhibited oxygen consumption by about 80%; therefore this compound would act as a potent inhibitor of the cytochrome pathway of the respiratory chain to exert its antifungal effect.

A pyrosequencing-based method to quantify genetic substitutions associated with resistance to succinate dehydrogenase inhibitor fungicides in Botrytis spp. populations

The genetic underlying resistance mechanisms in the population of the phytopathogenic fungus Botrytis cinerea are well documented. Specifically, several genetic substitutions associated with succinate dehydrogenase inhibitor (SDHI)-based fungicide resistance have been identified in the succinate dehydrogenase gene. The objective of the present work was to develop a molecular tool for accurate quantification of these genetic substitutions within Botrytis populations. A test using the PyroMark Q24 instrument was designed to detect and quantify five genetic substitutions associated with SDHI resistance. The technique is based on sequencing by synthesis, and it generated quantitative and accurate data with a limit of quantification of a minimum of 500 spores. There was a linear relationship between the known and estimated percentages of spores with the targeted genetic substitutions and wild-type strains at ratios of 0-100%, with a 20% increment. With the pyrosequencing assay developed in this study, a large number of Botrytis spp. individuals can be characterised in a timely fashion with greater accuracy than by commonly used methods. Hence, pyrosequencing-based methods will be useful for improving our understanding of fungicide resistance, detecting the arrival of new genetic substitutions, monitoring shifts in fungal populations and assessing the effectiveness of antiresistance strategies, and for routine monitoring of fungicide resistance.

Nor-Mevaldic acid surrogates as selective antifungal agent leads against Botrytis cinerea. Enantioselective preparation of 4-hydroxy-6-(1-phenylethoxy)tetrahydro-2H-pyran-2-one

Solvent-free desymmetrisation of meso-dialdehyde 1 with chiral 1-phenylethan-1-ol, led to preparation of 4-silyloxy-6-alkyloxytetrahydro-2H-pyran-2-one (+)-3a with a 96:4 dr Deprotected lactone (+)-19a and the related racemic lactones 16a–18a present a lactone moiety resembling the natural substrate of HMG-CoA reductase and their antifungal properties have been evaluated against the phytopathogenic fungi Botrytis cinerea and Colletotrichum gloeosporioides. These compounds were selectively active against B. cinerea, while inactive against C. gloeosporioides.

Prosystemin Overexpression in Tomato Enhances Resistance to Different Biotic Stresses by Activating Genes of Multiple Signaling Pathways.

Systemin is a signal peptide that promotes the response to wounding and herbivore attack in tomato. This 18-amino acid peptide is released from a larger precursor, prosystemin. To study the role of systemin as a modulator of defense signaling, we generated tomato (Solanum lycopersicum) transgenic plants that overexpress the prosystemin cDNA. We carried out a transcriptomic analysis comparing two different transgenic events with the untransformed control. The Gene Ontology categories of the 503 differentially expressed genes indicated that several biological functions were affected. Systemin promotes the expression of an array of defense genes that are dependent on different signaling pathways and it downregulates genes connected with carbon fixation and carbohydrate metabolism. These alterations present a degree of overlap with the response programs that are classically associated to pathogen defense or abiotic stress protection, implying that end products of the systemin signaling pathway may be more diverse than expected. We show also that the observed transcriptional modifications have a relevant functional outcome, since transgenic lines were more resistant against very different biotic stressors such as aphids (Macrosiphum euphorbiae), phytopathogenic fungi (Botrytis cinerea and Alternaria alternata) and phytophagous larvae (Spodoptera littoralis). Our work demonstrated that in tomato the modulation of a single gene is sufficient to provide a wide resistance against stress by boosting endogenous defense pathways. Overall, the data provided evidence that the systemin peptide might serve as DAMP signal in tomato, acting as a broad indicator of tissue integrity.Electronic supplementary materialThe online version of this article (doi:10.1007/s11105-014-0834-x) contains supplementary material, which is available to authorized users.

Novel aspinolide production by Trichoderma arundinaceum with a potential role in Botrytis cinerea antagonistic activity and plant defence priming.

Harzianum A (HA), a trichothecene produced by Trichoderma arundinaceum, has recently been described to have antagonistic activity against fungal plant pathogens and to induce plant defence genes. In the present work, we have shown that a tri5 gene-disrupted mutant that lacks HA production overproduces two polyketides, aspinolides B and C, which were not detected in the wild-type strain. Furthermore, four new aspinolides (D-G) were characterized. These compounds confirm that a terpene-polyketide cross-pathway exists in T. arundinaceum, and they may be responsible for the antifungal activity and the plant sensitization effect observed with the tri5-disrupted mutant. In addition, the molecular changes involving virulence factors in the phytopathogenic fungus Botrytis cinerea 98 (Bc98) during interaction with T. arundinaceum were investigated. The expression of genes involved in the production of botrydial by Bc98 was relatively repressed by HA, whereas other virulence genes of this pathogen were induced by the presence of T. arundinaceum, for example atrB and pg1 which encode for an ABC transporter and endopolygalacturonase 1 respectively. In addition, the interaction with Bc98 significantly repressed the production of HA by T. arundinaceum, indicating that a bidirectional transcriptional regulation is established between these two antagonistic fungi.

The High-Affinity Phosphodiesterase BcPde2 Has Impact on Growth, Differentiation and Virulence of the Phytopathogenic Ascomycete Botrytis cinerea

Components of the cAMP signaling pathway, such as the adenylate cyclase Bac and the protein kinase A (PKA) were shown to affect growth, morphogenesis and differentiation as well as virulence of the phytopathogenic fungus Botrytis cinerea. While loss of Bac caused drastically reduced intracellular cAMP levels, deletion of the PKA resulted in extremely increased cAMP concentrations. To regulate the intracellular level of the second messenger cAMP, a balance between its biosynthesis through adenylate cyclase activity and its hydrolysis by phosphodiesterases (PDEs) is crucial. Here, we report the functional characterization of the two PDEs in the ascomycete B. cinerea, BcPde1 and BcPde2. While deletion of bcpde2 resulted in severely affected vegetative growth, conidiation, germination and virulence, the bcpde1 deletion strain displayed a wild-type-like phenotype. However, the double bcpde1/2 deletion mutant exhibited an even stronger phenotype.Localization studies revealed that BcPde2 accumulates at the plasma membrane, but is also localized in the cytoplasm. BcPde1 was shown to be distributed in the cytoplasm as well, but also accumulates in so far unknown mobile vesicles. Overexpression of bcpde1 in the Δbcpde2 background rescued the deletion phenotype, and in addition an increased transcript level of bcpde1 in the Δbcpde2 strain was observed, indicating redundant functions of both PDEs and an interdependent gene expression.

Biosynthesis of gold nanoparticles by extracellular molecules produced by the phytopathogenic fungus Botrytis cinerea

The biosynthesis of gold nanoparticles (AuNPs) by extracellular components of the phytopathogenic fungus Botrytis cinerea was observed. Chloroauric acid (HAuCl4) reduction and the formation of AuNPs were evidenced by a color change in the free-mycelium culture filtrate, from a pale yellow to a dark purple, and through an absorbance peak of 540nm due to surface plasmon resonance of AuNPs. Characterization of the AuNPs showed triangular, hexagonal, spherical, decahedral, and pyramidal shapes of approximate sizes ranging from 1 to 100nm. The experimental data presented in this study demonstrates that extracellular putative NADH-dependent reductase activity participates in reduction of HAuCl4 and in the subsequent formation of AuNPs. However, this reductase is not the only active component secreted by the fungus in this process as the formation of AuNPs also occurs in conditions of high denaturation. The present study is the first to report on a rapid and eco-friendly method for the production of AuNPs by B. cinerea.

Application of Label-Free Shotgun nUPLC–MSE and 2-DE Approaches in the Study of Botrytis cinerea Mycelium

The phytopathogenic fungus Botrytis cinerea infects more than different 200 plant species and causes substantial losses in numerous crops. The B05.10 and T4 wild-type strain genomes have been recently sequenced, becoming a model system for necrotrophic pathogens, as well as opening up new alternatives in functional genomics, such as proteomics. We analyzed B. cinerea mycelium from these two wild-type strains, introducing label-free shotgun nUPLC-MS(E) methodology to complement the 2-DE-MS-based approach. We assessed the label-free nUPLC-MS(E) methodology for protein identification and quantification using five mycelium protein dilutions. A total of 225 and 170 protein species were identified by nUPLC-MS(E) in the B05.10 and T4 strains, respectively. Moreover, 129 protein species were quantified in both strains. Significant differences in protein abundance were found in 15 more abundant and 16 less abundant protein species in the B05.10 strain compared to the T4 strain. Twenty-nine qualitative and 15 significant quantitative differences were found using 2-DE. The label-free nUPLC-MS(E) was a reliable, reproducible and sensitive method for protein identification and quantification to study the B. cinerea mycelial proteome. Results obtained by gel-based and gel-free complementary approaches allow a deeper characterization of this fungus, as well as the identification of potential virulence factors.

A Functional Bikaverin Biosynthesis Gene Cluster in Rare Strains of Botrytis cinerea Is Positively Controlled by VELVET

The gene cluster responsible for the biosynthesis of the red polyketidic pigment bikaverin has only been characterized in Fusarium ssp. so far. Recently, a highly homologous but incomplete and nonfunctional bikaverin cluster has been found in the genome of the unrelated phytopathogenic fungus Botrytis cinerea. In this study, we provided evidence that rare B. cinerea strains such as 1750 have a complete and functional cluster comprising the six genes orthologous to Fusarium fujikuroi ffbik1-ffbik6 and do produce bikaverin. Phylogenetic analysis confirmed that the whole cluster was acquired from Fusarium through a horizontal gene transfer (HGT). In the bikaverin-nonproducing strain B05.10, the genes encoding bikaverin biosynthesis enzymes are nonfunctional due to deleterious mutations (bcbik2-3) or missing (bcbik1) but interestingly, the genes encoding the regulatory proteins BcBIK4 and BcBIK5 do not harbor deleterious mutations which suggests that they may still be functional. Heterologous complementation of the F. fujikuroi Δffbik4 mutant confirmed that bcbik4 of strain B05.10 is indeed fully functional. Deletion of bcvel1 in the pink strain 1750 resulted in loss of bikaverin and overproduction of melanin indicating that the VELVET protein BcVEL1 regulates the biosynthesis of the two pigments in an opposite manner. Although strain 1750 itself expresses a truncated BcVEL1 protein (100 instead of 575 aa) that is nonfunctional with regard to sclerotia formation, virulence and oxalic acid formation, it is sufficient to regulate pigment biosynthesis (bikaverin and melanin) and fenhexamid HydR2 type of resistance. Finally, a genetic cross between strain 1750 and a bikaverin-nonproducing strain sensitive to fenhexamid revealed that the functional bikaverin cluster is genetically linked to the HydR2 locus.