Whole-genome and time-course dual RNA-Seq analyses reveal chronic pathogenicity-related gene dynamics in the ginseng rusty root rot pathogen Ilyonectria robusta.

Ilyonectria robusta causes rusty root rot, the most devastating chronic disease of ginseng. Here, we for the first time report the high-quality genome of the I. robusta strain CD-56. Time-course (36 h, 72 h, and 144 h) dual RNA-Seq analysis of the infection process was performed, and many genes, including candidate effectors, were found to be associated with the progression and success of infection. The gene expression profile of CD-56 showed a trend of initial inhibition and then gradually returned to a profile similar to that of the control. Analyses of the gene expression patterns and functions of pathogenicity-related genes, especially candidate effector genes, indicated that the stress response changed to an adaptive response during the infection process. For ginseng, gene expression patterns were highly related to physiological conditions. Specifically, the results showed that ginseng defenses were activated by CD-56 infection and persisted for at least 144 h thereafter but that the mechanisms invoked were not effective in preventing CD-56 growth. Moreover, CD-56 did not appear to fully suppress plant defenses, even in late stages after infection. Our results provide new insight into the chronic pathogenesis of CD-56 and the comprehensive and complex inducible defense responses of ginseng root to I. robusta infection.

A simple and effective method to discern the true commercial Chinese cordyceps from counterfeits.

The Chinese cordyceps, a complex of the fungus Ophiocordyceps sinensis and its species-specific host insects, is also called "DongChongXiaCao" in Chinese. Habitat degradation in recent decades and excessive harvesting by humans has intensified its scarcity and increased the prices of natural populations. Some counterfeits are traded as natural Chinese cordyceps for profit, causing confusion in the marketplace. To promote the safe use of Chinese cordyceps and related products, a duplex PCR method for specifically identifying raw Chinese cordyceps and its primary products was successfully established. Chinese cordyceps could be precisely identified by detecting an internal transcribed spacer amplicon from O. sinensis and a cytochrome oxidase c subunit 1 amplicon from the host species, at a limit of detection as low as 32 pg. Eleven commercial samples were purchased and successfully tested to further verify that the developed duplex PCR method could be reliably used to identify Chinese cordyceps. It provides a new simple way to discern true commercial Chinese cordyceps from counterfeits in the marketplace. This is an important step toward achieving an authentication method for this Chinese medicine. The methodology and the developmental strategy can be used to authenticate other traditional Chinese medicinal materials.

Verticillium isaacii is a Pathogen and Endophyte of Potato and Sunflower in the Columbia Basin of Washington.

The objective of this research was to test the hypothesis that Verticillium isaacii causes diseases of sunflower and potato plants. Two sunflower genotypes and one potato cultivar were inoculated with five V. isaacii isolates and three pathogenic V. dahliae isolates. Biomass, disease expression, and stem colonization were quantified. Overt wilt symptoms were observed on both sunflower genotypes and potato plants inoculated with a subset of the V. isaacii isolates. Biomass of both sunflower genotypes was not affected by V. isaacii infection. Tuber yields either decreased in response to infection by one V. isaacii isolate or were not affected by infection. Stems of sunflower and potato plants were infected by at least four of the five V. isaacii isolates. A new disease of sunflower and potato is documented. Evidence that V. isaacii exhibits different lifestyles including pathogenicity and endophytism is presented. Finally, this research documents variation in fungal lifestyles that can exist in samples from a single field.

Chamomile Floricolous Downy Mildew Caused by Peronospora radii.

Floricolous downy mildews (Peronospora, oomycetes) are a small, monophyletic group of mostly inconspicuous plant pathogens that induce symptoms exclusively on flowers. Characterization of this group of pathogens, and information about their biology, is particularly sparse. The recurrent presence of a disease causing flower malformation which, in turn, leads to high production losses of the medicinal herb Matricaria chamomilla in Serbia has enabled continuous experiments focusing on the pathogen and its biology. Peronospora radii was identified as the causal agent of the disease, and morphologically and molecularly characterized. Diseased chamomile flowers showed severe malformations of the disc and ray florets, including phyllody and secondary inflorescence formation, followed by the onset of downy mildew. Phylogeny, based on internal transcribed spacer and cox2, indicates clustering of the Serbian P. radii with other P. radii from chamomile although, in cox2 analyses, they formed a separate subcluster. Evidence pointing to systemic infection was provided through histological and molecular analyses, with related experiments validating the impact of soilborne and blossom infections. This study provides new findings in the biology of P. radii on chamomile, thus enabling the reconstruction of this floricolous Peronospora species' life cycle.

Botrytis polyphyllae: A New Botrytis Species Causing Gray Mold on Paris polyphylla.

Paris polyphylla is an important perennial medicinal plant in China. A disease similar to gray mold on P. polyphylla occurred at the seedling stage in March 2016 and 2017 in Tengchong city, Yunnan Province of China. The disease resulted in up to 50% mortality in serious cases. Isolates from diseased plants grew 10.6 mm/day at 20°C on PDA. After 21 days, sclerotia were spherical to elliptical (0.4-2.5 × 0.3-1.8 mm). Conidia from diseased tissues were hyaline to pale brown, long, ovoid, unicellular, and measured 15.1-24.5 × 8.8-13.4 µm; conidiophores were 526-1,064 ×12-15 µm. Isolates did not form conidiophores or conidia on PDA or MYA. A phylogenetic analysis based on G3PDH, RPB2, and HSP60 sequence data supported assignment of three representative isolates as a new species of Botrytis. Based on morphological, phylogenetic characteristics and Koch's Postulates, the causal agent of gray mold on P. polyphylla was identified as a novel species, Botrytis polyphyllae.

Identification and Evaluation of Reference Genes for qRT-PCR Normalization in Sparassis latifolia (Agaricomycetes).

Real-time quantitative polymerase chain reaction (qRT-PCR) has emerged as a powerful and popular tool for quantitating differences in transcriptional gene expression levels between samples. Validation of the stability of reference genes is a fundamental step before initiating qRT-PCR assays. Sparassis latifolia is an edible and medicinal fungus containing a remarkably high concentration of ß-glucan, which has many biological and pharmacologic activities. S. latifolia may be a model species for studying fungal photobiology because its fruiting body formation requires more light than other fungi. However, suitable reference genes for qRT-PCR have not yet been determined. In the present study, 10 candidate reference genes in S. latifolia were evaluated and validated under different developmental stages and light conditions. To evaluate the suitability of candidate reference genes, three popular software programs (geNorm, NormFinder, and BestKeeper), along with the delta Ct method, were used to analyze these genes; the final ranking was determined using RefFinder. According to our results, Actin and GAPDH were expressed at the most stable levels under different developmental stages and light conditions.

Ammonium Acetate Supplement Strategy for Enhancement of Chaetominine Production in Liquid Culture of Marine-Derived Aspergillus fumigatus CY018.

Pharmacological research on (CHA), a marine-derived quinazolinone alkaloid with significant cytotoxic activity, is restricted by low yields and is a problem that needs to be settled urgently. In this work, the selection of additional nitrogen sources and the optimization of additional concentrations and longer fermentation times using ammonium acetate, were investigated. CHA production was optimized to 62.1 mg/l with the addition of 50 mM ammonium acetate at 120 h of the fermentation in the shaker flask. This feeding strategy significantly increased 3- deoxy-arabino-heptulosonate-7-phosphate synthase activity and transcript levels of critical genes (laeA, dahp and trpC) in the shikimate pathway compared with the non-treatment group. In addition, the selection of the feeding rate (0.01 and 0.03 g/l/h) was investigated in a 5-L bioreactor. As a result, CHA production was increased by 57.9 mg/l with a 0.01 g/l/h ammonium acetate feeding rate. This work shows that the strategy of ammonium acetate supplementation had an effective role in improving CHA production by Aspergillus fumigatus CY018. It also shows that this strategy could serve as an important example of large-scale fermentation of a marine fungus in submerged culture.

Development of Candida albicans Biofilms Is Diminished by Paeonia lactiflora via Obstruction of Cell Adhesion and Cell Lysis.

Candida albicans infections are often problematic to treat owing to antifungal resistance, as such infections are mostly associated with biofilms. The ability of C. albicans to switch from a budding yeast to filamentous hyphae and to adhere to host cells or various surfaces supports biofilm formation. Previously, the ethanol extract from Paeonia lactiflora was reported to inhibit cell wall synthesis and cause depolarization and permeabilization of the cell membrane in C. albicans. In this study, the P. lactiflora extract was found to significantly reduce the initial stage of C. albicans biofilms from 12 clinical isolates by 38.4%. Thus, to assess the action mechanism, the effect of the P. lactiflora extract on the adhesion of C. albicans cells to polystyrene and germ tube formation was investigated using a microscopic analysis. The density of the adherent cells was diminished following incubation with the P. lactiflora extract in an acidic medium. Additionally, the P. lactiflora-treated C. albicans cells were mostly composed of less virulent pseudohyphae, and ruptured debris was found in the serum-containing medium. A quantitative real-time PCR analysis indicated that P. lactiflora downregulated the expression of C. albicans hypha-specific genes: ALS3 by 65% (p = 0.004), ECE1 by 34.9% (p = 0.001), HWP1 by 29.2% (p = 0.002), and SAP1 by 37.5% (p = 0.001), matching the microscopic analysis of the P. lactiflora action on biofilm formation. Therefore, the current findings demonstrate that the P. lactiflora ethanol extract is effective in inhibiting C. albicans biofilms in vitro, suggesting its therapeutic potential for the treatment of biofilm-associated infections.

Structural insights into impact of Y134F mutation and discovery of novel fungicidal compounds against CYP51 in Puccinia triticina.

Sterol 14α-Demethylase Cytochrome P450 (CYP51) protein involved in ergosterol biosynthesis pathways turn out to be a crucial target for the fungicidal compound. However, the recognition mechanism and dynamic behavior of CYP51 in wheat leaf rust pathogen, Puccinia triticina, is still obscure. Previously, a mutation at position 134 (Y134F) was reported in five European isolates of P. triticina, conversely, structural basis of this mutation remains unclear. To address this problem, three-dimensional structure of CYP51 protein from P. triticina was successfully built using homology modeling approach. To assess the protein structure stability, wild and mutant-type CYP51 proteins bound with azole fungicide was subjected to 50 ns molecular dynamics (MD) simulations run. Observably, the comparative protein-ligand interaction analysis and binding free energy results revealed that impact of the mutation on the thermodynamics and conformational stability of the CYP51 protein was negligible. In addition, we carried out structure-based virtual screening and identified potent novel fungicidal compounds from four different databases and libraries. Consequently, through MD simulation and thermodynamic integration, four novel compounds such as CoCoCo54211 (CoCoCo database), ZINC04089470 (ZINC database), Allyl pyrocatechol 3,4 diacetate (Natural compound library), and 9-octadecenoic acid (Traditional Chinese Medicine database) has been predicted as potent fungicidal compound against CYP51 with XPGlide docking score of -11.41, -13.64, -7.40, and -6.55 kcal/mol, respectively. These compounds were found to form hydrogen bonds with heme group of CYP51, subsequently disturbing the stability and survival of fungus and can be used to control leaf rust in wheat.

Identification and Mode of Action of a Plant Natural Product Targeting Human Fungal Pathogens.

Candida albicans is a major cause of fungal diseases in humans, and its resistance to available drugs is of concern. In an attempt to identify novel antifungal agents, we initiated a small-scale screening of a library of 199 natural plant compounds (i.e., natural products [NPs]). In vitro susceptibility profiling experiments identified 33 NPs with activity against C. albicans (MIC50s ≤ 32 µg/ml). Among the selected NPs, the sterol alkaloid tomatidine was further investigated. Tomatidine originates from the tomato (Solanum lycopersicum) and exhibited high levels of fungistatic activity against Candida species (MIC50s ≤ 1 µg/ml) but no cytotoxicity against mammalian cells. Genome-wide transcriptional analysis of tomatidine-treated C. albicans cells revealed a major alteration (upregulation) in the expression of ergosterol genes, suggesting that the ergosterol pathway is targeted by this NP. Consistent with this transcriptional response, analysis of the sterol content of tomatidine-treated cells showed not only inhibition of Erg6 (C-24 sterol methyltransferase) activity but also of Erg4 (C-24 sterol reductase) activity. A forward genetic approach in Saccharomyces cerevisiae coupled with whole-genome sequencing identified 2 nonsynonymous mutations in ERG6 (amino acids D249G and G132D) responsible for tomatidine resistance. Our results therefore unambiguously identified Erg6, a C-24 sterol methyltransferase absent in mammals, to be the main direct target of tomatidine. We tested the in vivo efficacy of tomatidine in a mouse model of C. albicans systemic infection. Treatment with a nanocrystal pharmacological formulation successfully decreased the fungal burden in infected kidneys compared to the fungal burden achieved by the use of placebo and thus confirmed the potential of tomatidine as a therapeutic agent.

Genotypic and Phenotypic Characterization of Streptomyces Species Causing Potato Common Scab in Uruguay.

Isolation and characterization of common scab (CS) pathogen Streptomyces spp. from Uruguayan potato tubers and soil samples were done in response to significant economic losses due to CS on potato in autumn 2010. Seventy of the 331 isolates were classified as pathogenic owing to their ability to induce necrosis on tuber disks and stunting of radish seedling. Streptomyces spp. causing CS on potato in Uruguay were found to represent a range of different species by virtue of their diverse morphological and physiological traits as well as rep-PCR, rpoB phylogenetic analysis, and multi-locus sequences analysis. We identified isolates primarily as Streptomyces scabiei, S. acidiscabies, and S. europaeiscabiei. However, some of the pathogenic isolates still remain to be identified at the species level. This highlights the need for improved methods for discrimination among pathogenic Streptomyces species. The presence of Streptomyces pathogenicity island (PAI) genes was analyzed, including genes encoding for thaxtomin synthetase (txtA, txtB), tomatinase (tomA), and a necrosis protein (nec1). Among the isolates that were pathogenic, 50% contained the four pathogenicity genes, 33% had an atypical composition of PAI marker genes, and 17% did not contain any genes. The absence of the genes reported to be involved in thaxtomin biosynthesis (txtA, txtB) was confirmed by whole-genome sequencing of two representative strains of this group. This finding suggests the participation of other virulence factors in plant pathogenicity.

Historic Late Blight Outbreaks Caused by a Widespread Dominant Lineage of Phytophthora infestans (Mont.) de Bary.

Phytophthora infestans (Mont.) de Bary, the causal agent of potato late blight, was responsible for the Irish potato famine of the 1840s. Initial disease outbreaks occurred in the US in 1843, two years prior to European outbreaks. We examined the evolutionary relationships and source of the 19th-century outbreaks using herbarium specimens of P. infestans from historic (1846-1970) and more recent isolates (1992-2014) of the pathogen. The same unique SSR multilocus genotype, named here as FAM-1, caused widespread outbreaks in both US and Europe. The FAM-1 lineage shared allelic diversity and grouped with the oldest specimens collected in Colombia and Central America. The FAM-1 lineage of P. infestans formed a genetic group that was distinct from more recent aggressive lineages found in the US. The US-1 lineage formed a second, mid-20th century group. Recent modern US lineages and the oldest Mexican lineages formed a genetic group with recent Mexican lineages, suggesting a Mexican origin of recent US lineages. A survey of mitochondrial haplotypes in a larger set of global herbarium specimens documented the more frequent occurrence of the HERB-1 (type Ia) mitochondrial haplotype in archival collections from 1866-75 and 1906-1915 and the rise of the Ib mitochondrial lineage (US-1) between 1946-1955. The FAM-1 SSR lineage survived for almost 100 years in the US, was geographically widespread, and was displaced first in the mid-20th century by the US-1 lineage and then by distinct new aggressive lineages that migrated from Mexico.

Production of CoQ10 in fission yeast by expression of genes responsible for CoQ10 biosynthesis.

Coenzyme Q10 (CoQ10) is essential for energy production and has become a popular supplement in recent years. In this study, CoQ10 productivity was improved in the fission yeast Schizosaccharomyces pombe. Ten CoQ biosynthetic genes were cloned and overexpressed in S. pombe. Strains expressing individual CoQ biosynthetic genes did not produce higher than a 10% increase in CoQ10 production. In addition, simultaneous expression of all ten coq genes did not result in yield improvements. Genes responsible for the biosynthesis of p-hydroxybenzoate and decaprenyl diphosphate, both of which are CoQ biosynthesis precursors, were also overexpressed. CoQ10 production was increased by overexpression of Eco_ubiC (encoding chorismate lyase), Eco_aroF(FBR) (encoding 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase), or Sce_thmgr1 (encoding truncated HMG-CoA reductase). Furthermore, simultaneous expression of these precursor genes resulted in two fold increases in CoQ10 production.

The genome of the Tiger Milk mushroom, Lignosus rhinocerotis, provides insights into the genetic basis of its medicinal properties.

BACKGROUND: The sclerotium of Lignosus rhinocerotis (Cooke) Ryvarden or Tiger milk mushroom (Polyporales, Basidiomycota) is a valuable folk medicine for indigenous peoples in Southeast Asia. Despite the increasing interest in this ethnobotanical mushroom, very little is known about the molecular and genetic basis of its medicinal and nutraceutical properties. RESULTS: The de novo assembled 34.3 Mb L. rhinocerotis genome encodes 10,742 putative genes with 84.30% of them having detectable sequence similarities to others available in public databases. Phylogenetic analysis revealed a close evolutionary relationship of L. rhinocerotis to Ganoderma lucidum, Dichomitus squalens, and Trametes versicolor in the core polyporoid clade. The L. rhinocerotis genome encodes a repertoire of enzymes engaged in carbohydrate and glycoconjugate metabolism, along with cytochrome P450s, putative bioactive proteins (lectins and fungal immunomodulatory proteins) and laccases. Other genes annotated include those encoding key enzymes for secondary metabolite biosynthesis, including those from polyketide, nonribosomal peptide, and triterpenoid pathways. Among them, the L. rhinocerotis genome is particularly enriched with sesquiterpenoid biosynthesis genes. CONCLUSIONS: The genome content of L. rhinocerotis provides insights into the genetic basis of its reported medicinal properties as well as serving as a platform to further characterize putative bioactive proteins and secondary metabolite pathway enzymes and as a reference for comparative genomics of polyporoid fungi.

Highlights in pathogenic fungal biofilms / Aspectos sobresalientes en la formación de biopelículas por hongos patógenos

A wide variety of fungi have demonstrated the ability to colonize surfaces and form biofilms. Most studies on fungal biofilms have focused on Candida albicans and more recently, several authors have reported the involvement of other genera of yeasts and Candida species, as well as of filamentous fungi in the formation of biofilms, including: Cryptococcus neoformans, Cryptococcus gattii, Rhodotorula species, Aspergillus fumigatus, Malassezia pachydermatis, Histoplasma capsulatum, Paracoccidioides brasiliensis, Pneumocystis species, Coccidioides immitis, Fusarium species, Saccharomyces cerevisiae, Trichosporon asahii, Mucorales and Blastoschizomyces. There is a current interest in describing the particular characteristics of the biofilm formation by of these fungi. A major concern is the control of biofilms, requiring knowledge of the biofilm mechanisms. However, our knowledge of these microbial communities is limited, due to the complexity of these systems and metabolic interactions that remain unknown. This mini-review aims to highlight recently discovered fungal biofilms and to compare them with the current knowledge on biofilms. This manuscript is part of the series of works presented at the "V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi" (Oaxaca, Mexico, 2012) (AU)

Una amplia variedad de hongos poseen la capacidad para colonizar superficies y formar biopelículas (biofilms). La mayoría de los estudios efectuados sobre biopelículas de hongos han prestado atención a Candida albicans y, más recientemente, varios autores han descrito la implicación de otros géneros de levaduras y especies de Candida, al igual que de hongos filamentosos, en la formación de biopelículas, incluidos Cryptococcus neoformans, Cryptococcus gattii, especies de Rhodotorula, Aspergillus fumigatus, Malassezia pachydermatis, Histoplasma capsulatum, Paracoccidioides brasiliensis, especies de Pneumocystis, Coccidioides immitis, especies de Fusarium, Saccharomyces cerevisiae, Trichosporon asahii, mucorales y Blastoschizomyces. En la actualidad suscita interés la descripción de las características particulares de la formación de biopelículas de estos hongos. Una preocupación importante es el control de las biopelículas, que requiere una comprensión de los mecanismos de su formación. Sin embargo, nuestros conocimientos sobre estas comunidades microbianas son limitados debido a la complejidad de estos sistemas y a las interacciones metabólicas que aún no conocemos. Esta revisión tiene como objetivo poner de relieve las biopelículas fúngicas descubiertas recientemente y compararlas con los conocimientos actuales disponibles sobre ellas.Este artículo forma parte de una serie de estudios presentados en el «V International Workshop: Molecular genetic approaches to the study of human pathogenic fungi» (Oaxaca, México, 2012) (AU)

Characterization of the 5' flanking region of lipase gene from Penicillium expansum and its application in molecular breeding.

A major challenge for further promotion of lipase productivity in Penicillium expansum PE-12 is to find a suitable promoter that can function efficiently in this industrial strain. In this study, the 5' flanking region of P. expansum lipase (Ppel) containing a putative novel promoter sequence was characterized by fusing to ß-glucuronidase (GUS) and subsequently introducing into P. expansum. As a result, all the transformants showed blue color quickly after incubation in GUS detection buffer, suggesting a strong promoter activity of this fragment. Glucose repression was identified for the promoter, whereas olive oil acted as a positive regulator. Facilitated by this novel promoter, P. expansum PE-12 was genetically modified, with an improved lipase yield, via a recombinant plasmid with P. expansum lipase gene (PEL) under the control of Ppel promoter and TtrpC terminator. The highest lipase yield among the modified strains could attain 2,100 U/mL, which is more than twofold of the previous industrial strain (900 U/mL). The engineered strain through molecular breeding method as well as this new promoter has great value in lipase industry.

Assessing the microbial community and functional genes in a vertical soil profile with long-term arsenic contamination.

Arsenic (As) contamination in soil and groundwater has become a serious problem to public health. To examine how microbial communities and functional genes respond to long-term arsenic contamination in vertical soil profile, soil samples were collected from the surface to the depth of 4 m (with an interval of 1 m) after 16-year arsenic downward infiltration. Integrating BioLog and functional gene microarray (GeoChip 3.0) technologies, we showed that microbial metabolic potential and diversity substantially decreased, and community structure was markedly distinct along the depth. Variations in microbial community functional genes, including genes responsible for As resistance, carbon and nitrogen cycling, phosphorus utilization and cytochrome c oxidases were detected. In particular, changes in community structures and activities were correlated with the biogeochemical features along the vertical soil profile when using the rbcL and nifH genes as biomarkers, evident for a gradual transition from aerobic to anaerobic lifestyles. The C/N showed marginally significant correlations with arsenic resistance (p = 0.069) and carbon cycling genes (p = 0.073), and significant correlation with nitrogen fixation genes (p = 0.024). The combination of C/N, NO(3) (-) and P showed the highest correlation (r = 0.779, p = 0.062) with the microbial community structure. Contradict to our hypotheses, a long-term arsenic downward infiltration was not the primary factor, while the spatial isolation and nutrient availability were the key forces in shaping the community structure. This study provides new insights about the heterogeneity of microbial community metabolic potential and future biodiversity preservation for arsenic bioremediation management.

Does copper stress lead to spindle misposition-dependent cell cycle arrest?

Because of its specific electrochemical properties, copper is an essential heavy metal for living organisms. As with other heavy metals, high levels can provoke damage. We examined gene expression under copper stress in wild-type fission yeast (Schizosaccharomyces pombe) through differential display. After the EC(50) concentration of CuSO(4) was determined as 50 µM, total RNA was isolated from cells treated or not with copper. The expression level of SPCC1682.13, ppk1, SPBC2F12.05c, and adg2 genes increased significantly under copper stress. Considering the functions of these genes are related to the cell cycle, cell division and chromosome dynamics, we hypothesize that retardation of the cell cycle under copper stress is relevant to the events that depend on the functions of these genes.

Gene fusions for the directed modification of the carotenoid biosynthesis pathway in Mucor circinelloides.

Several fungal species, particularly some included in the Mucorales, have been used to develop fermentation processes for the production of ß-carotene. Oxygenated derivatives of ß-carotene are more valuable products, and the preference by the market of carotenoids from biological sources has increased the research in different carotenoid-producing organisms. We currently use Mucor circinelloides as a model organism to develop strains able to produce new, more valuable, and with an increased content of carotenoids. In this chapter we describe part of our efforts to construct active gene fusions which could advance in the diversification of carotenoid production by this fungus. The main carotenoid accumulated by M. circinelloides is ß-carotene, although it has some hydroxylase activity and produces low amounts of zeaxanthin. Two enzymatic activities are required for the production of astaxanthin from ß-carotene: a hydroxylase and a ketolase. We used the ctrW gene of Paracoccus sp. N81106, encoding a bacterial ß-carotene ketolase, to construct gene fusions with two fungal genes essential for the modification of the pathway in M. circinelloides. First we fused it to the carRP gene of M. circinelloides, which is responsible for the phytoene synthase and lycopene cyclase activities in this fungus. The expected activity of this fusion gene would be the accumulation by M. circinelloides of canthaxanthin and probably some astaxanthin. A second construction was the fusion of the crtW gene of Paracoccus sp. to the crtS gene of Xanthophyllomyces dendrorhous, responsible for the synthesis of astaxanthin from ß-carotene in this fungus, but which was shown to have only hydroxylase activity in M. circinelloides. The expected result in M. circinelloides transformants was the accumulation of astaxanthin. Here we describe a detailed and empirically tested protocol for the construction of these gene fusions.

Characterization of CbCyp51 from field isolates of Cercospora beticola.

The hemibiotrophic fungus Cercospora beticola causes leaf spot of sugar beet. Leaf spot control measures include the application of sterol demethylation inhibitor (DMI) fungicides. However, reduced sensitivity to DMIs has been reported recently in the Red River Valley sugar beet-growing region of North Dakota and Minnesota. Here, we report the cloning and molecular characterization of CbCyp51, which encodes the DMI target enzyme sterol P450 14α-demethylase in C. beticola. CbCyp51 is a 1,632-bp intron-free gene with obvious homology to other fungal Cyp51 genes and is present as a single copy in the C. beticola genome. Five nucleotide haplotypes were identified which encoded three amino acid sequences. Protein variant 1 composed 79% of the sequenced isolates, followed by protein variant 2 that composed 18% of the sequences and a single isolate representative of protein variant 3. Because resistance to DMIs can be related to polymorphism in promoter or coding sequences, sequence diversity was assessed by sequencing >2,440 nucleotides encompassing CbCyp51 coding and flanking regions from isolates with varying EC(50) values (effective concentration to reduce growth by 50%) to DMI fungicides. However, no mutations or haplotypes were associated with DMI resistance or sensitivity. No evidence for alternative splicing or differential methylation of CbCyp51 was found that might explain reduced sensitivity to DMIs. However, CbCyp51 was overexpressed in isolates with high EC(50) values compared with isolates with low EC(50) values. After exposure to tetraconazole, isolates with high EC(50) values responded with further induction of CbCyp51, with a positive correlation of CbCyp51 expression and tetraconazole concentration up to 2.5 µg ml(-1).