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Cranberry fruit rot (CFR) pathogens are widely reported in the literature, but performing large-scale analysis of their presence inside fruit has always been challenging. In this study, a new molecular diagnostic tool, capable of identifying simultaneously 12 potential fungal species causing CFR, was used to better define the impact of CFR across cranberry fields in Québec. For this purpose, 126 fields and 7,825 fruits were sampled in three cranberry farms distributed throughout the province and subjected to comparative analyses of fungal presence and abundance according to cultural practices, sampling times, and cranberry cultivars. All 12 pathogens were detected throughout the study, but as a first major finding, the analyses revealed that four species, Godronia cassandrae, Colletotrichum fructivorum, Allantophomopsis cytisporea, and Coleophoma empetri, were consistently predominant regardless of the parameters studied. Comparison of conventional and organic productions showed a significant reduction in fungal richness and relative abundance. Interestingly, Monilinia oxycocci was found almost exclusively in organic productions, indicating that fungicides had a strong and persistent effect on its population. Surprisingly, there were no significant differences in fungal relative abundance or species richness between fruit sampled at harvest or in storage, suggesting that there may not exist a clear distinction between field and storage rot, as was previously thought. Comparative analysis of fungal species found on eight different cranberry cultivars indicated that they were all infected by the same fungi but could not rule out differences in genetic resistance. This large-scale analysis allows us to draw an exhaustive picture of CFR in Québec and provides new information with respect to its management.

The complex etiology of cranberry fruit rot (CFR) (Oudemans et al., 1998) has made it difficult to precisely identify the fungi involved in CFR and their relative importance in North America. To remedy this situation, a multiplex PCR approach targeting the 12 most commonly reported fungi in CFR was recently developed (Conti et al., 2019). However, in surveys conducted in Eastern Canada, the molecular tool revealed the presence of an unknown fungus in more than 30% of the collected samples. Analyses were thus undertaken to identify this species. From 117 rotten fruit collected at harvest in 2017, 34 samples of the unknown fungus, all morphologically similar, were isolated but not detected using the molecular tool. Their ITS ribosomal regions were sequenced using universal primers (Vilgalys and Hester, 1990; White et al., 1990) and searched against the GenBank database using the Blastn tool (Altschul et al., 1990). The top match was obtained with Godronia cassandrae (accession number: MH855281 (Vu et al., 2019), 98-100% of identity and an E-value of 0.0), even though some isolates had minor nucleotide differences, as presented in the tree. Sequences were deposited in GenBank as accession numbers MT599989 to MT600022. Since G. cassandrae had been reported, albeit rarely, on cranberry in Michigan (Olatinwo et al., 2003), it was supposed to amplify with the molecular tool developed from the strain DAOM C216021 (AAFC, Ottawa, ON) identified in 1993 on Vaccinium angustifolium as G. cassandrae. Analysis of the sequences used to build the specific primers from this strain confirmed the DAOM strain as being Neocucurbitaria juglandicola, which was never diagnosed in our cranberry samples. To confirm this revised diagnosis, a multi-sequence alignment (MSA) was performed on the ITS regions of the isolates from rotten cranberries and sequences available for the genus Godronia in the NCBI nucleotide database (NCBI txid269064). This MSA allowed us to find discriminant regions between Godronia spp. A pair of PCR primers specific to G. cassandrae found on cranberry fruit was then designed (the forward and reverse sequences are AAT CAG TGG CGG TGC CTG TC and TAC CGC TTC ACT CGC CGT TAC, respectively), generating 196 bp amplicons, with an annealing temperature of 65°C. The diagnosis of 7,835 fruit sampled at three time points (harvest, after three and after six weeks of storage) in 2018, from four cranberry farms located in Quebec (CA) and Nova Scotia (CA), detected G. cassandrae in 2350 samples (30%). To assess the pathogenicity of four specimens from 2017, Koch's postulates were completed on two healthy fruit per isolate. The fruit were wounded with a sterilized pick and individually inoculated; two fruit were used as control. Based on our observations, the fungi isolated from cranberry fruit displayed a pale lemon yellow mycelium and black pycnidia. Conidia are hyaline, cylindrical and divided by a single septum. These morphological characters are similar to the ones described in the literature for G. cassandrae (Polashock et al., 2017). Rot symptoms appear as a discoloration from the firm, red and healthy cranberry fruit to a yellowish-orange softer fruit. Molecular characterization of the re-isolated fungus confirmed the presence of G. cassandrae. We report Godronia cassandrae for the first time as a major cause of CFR in Eastern Canada. Its prevalence in cranberry fields of Quebec and Nova Scotia suggests that it supplants Physalospora as the main fungus involved in CFR in Eastern Canada.

In plant–pathogen interactions, expression and localization of effectors in the aqueous apoplastic region play a crucial role in the establishment or suppression of pathogen development. Silicon (Si) has been shown to protect plants in several host–pathogen interactions, but its mode of action remains a source of debate. Its deposition in the apoplastic area of plant cells suggests that it might interfere with receptor–effector recognition. In this study, soybean plants treated or not with Si were inoculated with Phytophthora sojae and differences in the ensuing infection process were assessed through different microscopy techniques, transcript analysis of effector and defense genes, and effector (Avr6) localization through immunolocalization and fluorescence labeling. In plants grown without Si, the results showed the rapid (4 d post-inoculation) host recognition by P. sojae through the development of haustorium-like bodies, followed by expression and release of effectors into the apoplastic region. In contrast, Si treatment resulted in limited pathogen development, and significantly lower expression and presence of Avr6 in the apoplastic region. Based on immunolocalization and quantification of Avr6 through fluorescence labeling, our results suggest that the presence of Si in the apoplast interferes with host recognition and/or limits receptor–effector interactions, which leads to an incompatible interaction.

Phytophthora root rot, caused by Phytophthora sojae, is one of the most damaging diseases of soybean and the introgression of Rps (Resistance to P. sojae) genes into elite soybean lines is arguably the best way to manage this disease. Current bioassays to phenotype the gene-for-gene relationship are hampered with respect to reproducibility and long-term stability of isolates, and do not accurately predict horizontal resistance individually. The aim of our study was to investigate a new way of phenotyping P. sojae isolates and vertical and horizontal resistance in soybean that relies on zoospores inoculated directly into a hydroponic system. Inoculation of P. sojae isolates against a set of eight differentials accurately and reproducibly identified pathotypes over a period of two years. When applied to test vertical resistance of soybean lines with known and unknown Rps genes, the bioassay relied on plant dry weight to correctly identify all genes. In addition, simultaneous inoculations of three P. sojae isolates, collectively carrying eight major virulence factors against 64 soybean lines with known and unknown levels of horizontal resistance, separated the plants into five distinct groups of root rot, allowing the discrimination of lines with various degrees of partial resistance. Based on those results, this bioassay offers several advantages in facilitating efforts in breeding soybean for P. sojae resistance and in identifying virulence factors in P. sojae.

Summary Tritrophic interactions involving a biocontrol agent, a pathogen and a plant have been analyzed predominantly from the perspective of the biocontrol agent. We have conducted the first comprehensive transcriptomic analysis of all three organisms in an effort to understand the elusive properties of Pseudozyma flocculosa in the context of its biocontrol activity against Blumeria graminis f.sp. hordei as it parasitizes Hordeum vulgare. After inoculation of P. flocculosa, the tripartite interaction was monitored over time and samples collected for scanning electron microscopy and RNA sequencing. Based on our observations, P. flocculosa indirectly parasitizes barley, albeit transiently, by diverting nutrients extracted by B. graminis from barley leaves through a process involving unique effectors. This brings novel evidence that such molecules can also influence fungal–fungal interactions. Their release is synchronized with a higher expression of powdery mildew haustorial effectors, a sharp decline in the photosynthetic machinery of barley and a developmental peak in P. flocculosa. The interaction culminates with a collapse of B. graminis haustoria, thereby stopping P. flocculosa growth, as barley plants show higher metabolic activity. To conclude, our study has uncovered a complex and intricate phenomenon, described here as hyperbiotrophy, only achievable through the conjugated action of the three protagonists.

Background The interaction between oomycete plant pathogen Phytophthora sojae and soybean is characterized by the presence of avirulence (Avr) genes in P. sojae, which encode for effectors that trigger immune responses and resistance in soybean via corresponding resistance genes (Rps). A recent survey highlighted a rapid diversification of P. sojae Avr genes in soybean fields and the need to deploy new Rps genes. However, the full genetic diversity of P. sojae isolates remains complex and dynamic and is mostly characterized on the basis of phenotypic associations with differential soybean lines. Results We sequenced the genomes of 31 isolates of P. sojae, representing a large spectrum of the pathotypes found in soybean fields, and compared all the genetic variations associated with seven Avr genes (1a, 1b, 1c, 1d, 1k, 3a, 6) and how the derived haplotypes matched reported phenotypes in 217 interactions. We discovered new variants, copy number variations and some discrepancies with the virulence of previously described isolates with Avr genes, notably with Avr1b and Avr1c. In addition, genomic signatures revealed 11.5% potentially erroneous phenotypes. When these interactions were re-phenotyped, and the Avr genes re-sequenced over time and analyzed for expression, our results showed that genomic signatures alone accurately predicted 99.5% of the interactions. Conclusions This comprehensive genomic analysis of seven Avr genes of P. sojae in a population of 31 isolates highlights that genomic signatures can be used as accurate predictors of phenotypes for compatibility with Rps genes in soybean. Our findings also show that spontaneous mutations, often speculated as a source of aberrant phenotypes, did not occur within the confines of our experiments and further suggest that epigenesis or gene silencing do not account alone for previous discordance between genotypes and phenotypes. Furthermore, on the basis of newly identified virulence patterns within Avr1c, our results offer an explanation why Rps1c has failed more rapidly in the field than the reported information on virulence pathotypes. Electronic supplementary material The online version of this article (10.1186/s12915-018-0549-9) contains supplementary material, which is available to authorized users.

Background Silicon (Si) is known to protect against biotrophic and hemibiotrophic plant pathogens; however, the mechanisms by which it exerts its prophylactic role remain unknown. In an attempt to obtain unique insights into the mode of action of Si, we conducted a full comparative transcriptomic analysis of soybean (Glycine max) plants and Phytophthora sojae, a hemibiotroph that relies heavily on effectors for its virulence. Results Supplying Si to inoculated plants provided a strong protection against P. sojae over the course of the experiment (21 day). Our results showed that the response of Si-free (Si−) plants to inoculation was characterized early (4 dpi) by a high expression of defense-related genes, including plant receptors, which receded over time as the pathogen progressed into the roots. The infection was synchronized with a high expression of effectors by P. sojae, the nature of which changed over time. By contrast, the transcriptomic response of Si-fed (Si+) plants was remarkably unaffected by the presence of P. sojae, and the expression of effector-coding genes by the pathogen was significantly reduced. Conclusion Given that the apoplast is a key site of interaction between effectors and plant defenses and receptors in the soybean-P. sojae complex, as well as the site of amorphous-Si accumulation, our results indicate that Si likely interferes with the signaling network between P. sojae and the plant, preventing or decreasing the release of effectors reaching plant receptors, thus creating a form of incompatible interaction. Electronic supplementary material The online version of this article (10.1186/s12870-018-1312-7) contains supplementary material, which is available to authorized users.

Aquaporins (AQPs) are of vital importance in the cellular transport system of all living organisms. In this study, genome-wide identification, distribution, and characterization of AQPs were determined in Arabidopsis lyrata, Capsella grandiflora, C. rubella, Eutrema salsugineum, Brassica rapa, B. oleracea, and B. napus (canola). Classification and phylogeny of AQPs revealed the loss of XIPs and NIP-IIIs in all species. Characterization of distinctive AQP features showed a high level of conservation in spacing between NPA-domains, and selectivity filters. Interestingly, TIP3s were found to be highly expressed in developing seeds, suggesting their role in seed desiccation. Analysis of available RNA-seq data obtained under biotic and abiotic stresses led to the identification of AQPs involved in stress tolerance mechanisms in canola. In addition, analysis of the effect of ploidy level, and resulting gene dose effect performed with the different combinations of Brassica A and C genomes revealed that more than 70% of AQPs expression were dose-independent, thereby supporting their role in stress alleviation. This first in-depth characterization of Brassicaceae AQPs highlights transport mechanisms and related physiological processes that could be exploited in breeding programs of stress-tolerant cultivars.

Summary On absorption by plants, silicon (Si) offers protection against many fungal pathogens, including powdery mildews. The mechanisms by which Si exerts its prophylactic role remain enigmatic, although a prevailing hypothesis suggests that Si positively influences priming. Attempts to decipher Si properties have been limited to plants able to absorb Si, which excludes the model plant Arabidopsis because it lacks Si influx transporters. In this work, we were able to engineer Arabidopsis plants with an Si transporter from wheat (TaLsi1) and to exploit mutants (pad4 and sid2) deficient in salicylic acid (SA)-dependent defence responses to study their phenotypic response and changes in defence expression against Golovinomyces cichoracearum (Gc) following Si treatment. Our results showed that TaLsi1 plants contained significantly more Si and were significantly more resistant to Gc infection than control plants when treated with Si, the first such demonstration in a plant transformed with a heterologous Si transporter. The resistant plants accumulated higher levels of SA and expressed higher levels of transcripts encoding defence genes, thus suggesting a role for Si in the process. However, TaLsi1 pad4 and TaLsi1 sid2 plants were also more resistant to Gc than were pad4 and sid2 plants following Si treatment. Analysis of the resistant phenotypes revealed a significantly reduced production of SA and expression of defence genes comparable with susceptible controls. These results indicate that Si contributes to Arabidopsis defence priming following pathogen infection, but highlight that Si will confer protection even when priming is altered. We conclude that Si-mediated protection involves mechanisms other than SA-dependent defence responses.

Recently, a new disease was reported on greenhouse tomato plants in both Quebec, Canada and Maine, United States. Symptomatic plants bore brown lesions at graft points and pruning sites, resulting in expanding cankers with clearly delineated margins. Diseased plants eventually wilted and died within a few weeks following the appearance of the first symptoms. The symptoms are reminiscent of infection by Fusarium oxysporum f. sp. radicis-lycopersici, with the notable difference of a discoloration of the pith area rather than the vascular tissues. A homothallic Fusarium sp. was consistently recovered from these lesions. Sequencing of the internal transcribed spacer and the partial translation elongation factor 1-α gene identified the species as F. striatum. Pathogenicity tests with F. striatum isolates from diseased tissues reproduced disease symptoms in tomato similar to those observed on tomato plants in the greenhouses. Specific detection of F. striatum from mycelia and diseased and disease-free plant tissues was achieved by developing a polymerase chain reaction-based test. These results establish, for the first time, that the species F. striatum is the cause of crown and stem rot affecting tomato in North America. In addition F. striatum was detected from all sampled tissues of plants delivered by the nursery common to both growers, suggesting that the transplants would be the source of the inoculum.

Pseudozyma flocculosa (syn: Sporothrix flocculosa) was first discovered and described in 1987 as an epiphytic yeast on powdery mildew-infected clover leaves. It was subsequently found to be a powerful antagonist of powdery mildews which prompted its study and development as a biocontrol agent (BCA). Most BCAs exert their activity through the manifestation of one or more of the following modes of action: competition, parasitism, antibiosis and induced resistance. In the case of P. flocculosa, in vitro bioassays, electron microscopy studies, and chemical analyses all pointed to a single mode of action: antibiosis. This conclusion was reinforced by the characterization and purification of an active molecule, flocculosin, and by the demonstration of its powerful antimicrobial activity. This was further supported by the discovery of a complex gene cluster regulating the synthesis of flocculosin, a molecule nearly identical to ustilagic acid – a compound produced by U. maydis under the control of a sim...

Plants benefit greatly from silicon (Si) absorption provided that they contain Si transporters. The latter have recently been identified in the roots of some higher plants known to accumulate high concentrations of Si, and all share a high level of sequence identity. In this study, we searched for transporters in the primitive vascular plant Equisetum arvense (horsetail), which is a valuable but neglected model plant for the study of Si absorption, as it has one of the highest Si concentrations in the plant kingdom. Our initial attempts to identify Si transporters based on sequence homology with transporters from higher plants proved unsuccessful, suggesting a divergent structure or property in horsetail transporters. Subsequently, through sequencing of the horsetail root transcriptome and a search using amino acid sequences conserved in plant aquaporins, we were able to identify a multigene family of aquaporin Si transporters. Comparison of known functional domains and phylogenetic analysis of sequences revealed that the horsetail proteins belong to a different group than higher-plant Si transporters. In particular, the newly identified proteins contain a STAR pore as opposed to the GSGR pore common to all previously identified Si transporters. In order to determine its functionality, the proteins were heterologously expressed in both Xenopus oocytes and Arabidopsis, and the results showed that the horsetail proteins are extremely efficient a transporting Si. These findings offer new insights into the elusive properties of Si and its absorption by plants.

Flocculosin and ustilagic acid (UA), two highly similar antifungal cellobiose lipids, are respectively produced by Pseudozyma flocculosa , a biocontrol agent, and Ustilago maydis , a plant pathogen. Both glycolipids contain a short-chain fatty acid hydroxylated at the β position but differ in the long fatty acid, which is hydroxylated at the α position in UA and at the β position in flocculosin. In both organisms, the biosynthesis genes are arranged in large clusters. The functions of most genes have already been characterized, but those of the P. flocculosa fhd1 gene and its homolog from U. maydis , uhd1 , have remained undefined. The deduced amino acid sequences of these genes show homology to those of short-chain dehydrogenases and reductases (SDR). We disrupted the uhd1 gene in U. maydis and analyzed the secreted UA. uhd1 deletion strains produced UA lacking the β-hydroxyl group of the short-chain fatty acid. To analyze the function of P. flocculosa Fhd1, the corresponding gene was used to complement U. maydis Δ uhd1 mutants. Fhd1 was able to restore wild-type UA production, indicating that Fhd1 is responsible for β hydroxylation of the flocculosin short-chain fatty acid. We also investigated a P. flocculosa homolog of the U. maydis long-chain fatty-acid alpha hydroxylase Ahd1. The P. flocculosa ahd1 gene, which does not reside in the flocculosin gene cluster, was introduced into U. maydis Δ ahd1 mutant strains. P. flocculosa Ahd1 neither complemented the U. maydis Δ ahd1 phenotype nor resulted in the production of β-hydroxylated UA. This suggests that P. flocculosa Ahd1 is not involved in flocculosin hydroxylation.

Phenolic compounds are apparently important in the defence mechanisms of conifers. To test the hypothesis that phenolic compounds in resistant white spruce [Picea glauca (Moench) Voss (Pinaceae)] impart resistance against spruce budworm [Choristoneura fumiferana (Clemens) (Lepidoptera: Tortricidae)], we performed aqueous extractions of current-year shoots of white spruce that were tolerant of varying levels of budworm defoliation. High-performance liquid chromatographic profiles of water extracts of P. glauca needles differed between resistant and susceptible trees. Further nuclear magnetic resonance analyses identified two phenolic glucosides in susceptible white spruce, picein [3-(β-d-glucosyloxy)-hydroxy-acetophenone] and pungenin [3-(β-d-glucosyloxy)-4-hydroxy-acetophenone], and two phenolics in resistant white spruce, pungenol (3′,4′-hydroxy-acetophenone) and piceol (4′-hydroxyacetophenone). We focused on the performance of spruce budworm when piceol and pungenol were added to the diet. These two compounds significantly reduced larval survival, retarded development, and reduced pupal mass. Food consumption by sixth-instar spruce budworms was affected by a combination of the phenolic compounds. These results suggest that the two phenolic compounds reduce the pressure of spruce budworm herbivory on specific host tree phenotypes. Thus, the mechanism of defence in P. glauca apparently reflects a strategy of constitutive resistance.

In this work, we sought to understand how glycolipid production and the availability of nutrients could explain the ecology of Pseudozyma flocculosa and its biocontrol activity. For this purpose, we compared the development of P. flocculosa to that of a close relative, the plant pathogen Ustilago maydis , under different environmental conditions. This approach was further supported by measuring the expression of cyp1 , a pivotal gene in the synthesis of unique antifungal cellobiose lipids of both fungi. On healthy cucumber and tomato plants, the expression of cyp1 remained unchanged over time in P. flocculosa and was undetected in U. maydis . At the same time, green fluorescent protein (GFP) strains of both fungi showed only limited green fluorescence on control leaves. On powdery mildew-infected cucumber leaves, P. flocculosa induced a complete collapse of the pathogen colonies, but glycolipid production, as studied by cyp1 expression, was still comparable to that of controls. In complete contrast, cyp1 was upregulated nine times when P. flocculosa was applied to Botrytis cinerea -infected leaves, but the biocontrol fungus did not develop very well on the pathogen. Analysis of the possible nutrients that could stimulate the growth of P. flocculosa on powdery mildew structures revealed that the complex Zn/Mn played a key role in the interaction. Other related fungi such as U. maydis do not appear to have the same nutritional requirements and hence lack the ability to colonize powdery mildews. Whether production of antifungal glycolipids contributes to the release of nutrients from powdery mildew colonies is unclear, but the specificity of the biocontrol activity of P. flocculosa toward Erysiphales does appear to be more complex than simple antibiosis.

Flocculosin is an antifungal glycolipid produced by the biocontrol fungus Pseudozyma flocculosa. It consists of cellobiose, O-glycosidically linked to 3,15,16-trihydroxypalmitic acid. The sugar moiety is acylated with 2-hydroxy-octanoic acid and acetylated at two positions. Here we describe a gene cluster comprising 11 genes that are necessary for the biosynthesis of flocculosin. We compared the cluster with the biosynthesis gene cluster for the highly similar glycolipid ustilagic acid (UA) produced by the phytopathogenic fungus Ustilago maydis. In contrast to the cluster of U. maydis, the flocculosin biosynthesis cluster contains an additional gene encoding an acetyl-transferase and is lacking a gene homologous to the α-hydroxylase Ahd1 necessary for UA hydroxylation. The functions of three acyl/acetyl-transferase genes (Fat1, Fat2 and Fat3) including the additional acetyl-transferase were studied by complementing the corresponding U. maydis mutants. While P. flocculosa Fat1 and Fat3 are homologous to Uat1 in U. maydis, Fat2 shares 64% identity to Uat2, a protein involved in UA biosynthesis but with so far unknown function. By genetic and mass spectrometric analysis, we show that Uat2 and Fat2 are necessary for acetylation of the corresponding glycolipid. These results bring unique insights into the biocontrol properties of P. flocculosa and opportunities for enhancing its activity.

Flocculosin is an unusual cellobiose lipid secreted by the yeast-like fungus Pseudozyma flocculosa as part of its biocontrol arsenal against other fungi. Recent observations have suggested that the fungus degrades flocculosin to use it as a nutrient source during periods of food limitation. In this work, we sought to identify the catabolic steps leading to the degradation of flocculosin and its subsequent use by P. flocculosa. To this end, we characterized the catabolism of flocculosin through identification of degradation intermediates in a deprived medium using mass spectrometry. As the pH of the medium increased, the molecule was quickly deacylated and lost its antimicrobial activity thereby explaining conflicting results concerning the antimicrobial activity of this class of glycolipid. Following removal of both acetyl groups and the short fatty acid chain under alkaline conditions, the molecule was quickly and completely metabolized by P. flocculosa. Protein purification of culture filtrates confirmed the presence of degradative enzymes produced by P. flocculosa. These enzymes were found to degrade 3,15-dihydroxy-hexadecyl cellobioside (DHC) but not the acylated molecule, thus confirming the protective role of these groups against catabolism. These results are the first evidence of glycolipid degradation by producing organism and suggest that flocculosin can be recycled by P. flocculosa as a nutrient in addition to protecting its ecological niche.

The basidiomycetous fungus Pseudozyma flocculosa represents a promising new host for the expression of complex recombinant proteins. Two novel heterologous promoter sequences, the Ustilago maydis glyceraldehyde-3-phosphate dehydrogenase (GPD) and Pseudozyma tsukubaensis alpha-glucosidase promoters, were tested for their ability to provide expression in P. flocculosa. In liquid medium, these two promoters produced lower levels of intracellular green fluorescent protein (GFP) as compared to the U. maydis hsp70 promoter. However, GPD and alpha-glucosidase sequences behaved as constitutive promoters whereas the hsp70 promoter appeared to be morphology-dependent. When using the hsp70 promoter, the expression of GFP increased proportionally to the concentration of hygromycin in the culture medium, indicating possible induction of the promoter by the antibiotic. Optimal solid-state culture conditions were designed for high throughput screening of hygromycin-resistant transformants with the hsp70 promoter in P. flocculosa.

High-speed counter-current chromatography (HSCCC) methods were developed for the study of induced defense metabolites in wheat (Triticum aestivum) against powdery mildew (Blumeria graminis f. sp. tritici). A single HSCCC purification step afforded extraction of mg-quantities of an induced compound with antifungal activity. Subsequent LC-MS and NMR analyses have led to the characterization of 5,6-O-methyl trans-aconitic acid, the first such report of this compound in a plant species. The inducible nature of aconitic acid was evidenced by comparing the metabolite profiles of leaf extracts from plants treated or not with soluble silicon and infected or not with powdery mildew. In a second step, dual-mode HSCCC was used to enhance the separation of other forms of aconitic acid in wheat. Based on these results, it was concluded that 5,6-O-methyl trans-aconitic acid plays an important role as a defense molecule in wheat plants and that HSCCC is a powerful separation method for purifying such compounds from complex plant-pathogen interactions.

Although Basidiomycetes represent the most evolved class of fungi, they have been neglected with regard to recombinant gene expression. In this work, basidiomycetous yeasts belonging to Pseudozyma spp. were studied with respect to their amenability to heterologous protein production. Single plasmid or cotransformation experiments routinely afforded 100 to 200 independent transformants for the two tested species of Pseudozyma. Green fluorescent protein (GFP) was expressed in the correctly folded conformation, as demonstrated by fluorescence microscopy, and hen egg white lysozyme (HEWL) was expressed in its active form, as revealed by its lytic activity on Micrococcus lysodeikticus cells. Protease analysis established that Pseudozyma spp. contained equivalent or less extracellular protease activity than yeasts and far less protease activity than ascomycetous filamentous fungi in similar culture conditions. This proteolytic activity was inhibited by over 97% with a combination of PMSF and Pepstatin A. N-glycosylation patterns of native Pseudozyma flocculosa secreted proteins were comprised of one or a few short glycan chains that possess a classic eukaryotic structure typical of higher fungi and animal cells. This is the first report of a Basidiomycete that possesses multiple intrinsic characteristics necessary for use as a heterologous gene expression system.

In this study, cucumber plants (Cucumis sativus) expressing induced resistance against powdery mildew (caused by Podosphaera xanthii) were infiltrated with inhibitors of cinnamate 4-hydroxylase, 4-coumarate:CoA ligase (4CL), and chalcone synthase (CHS) to evaluate the role of flavonoid phytoalexin production in induced disease resistance. Light and transmission electron microscopy demonstrated ultrastructural changes in inhibited plants, and biochemical analyses determined levels of CHS and β-glucosidase enzyme activity and 4CL protein accumulation. Our results showed that elicited plants displayed a high level of induced resistance. In contrast, down regulation of CHS, a key enzyme of the flavonoid pathway, resulted in nearly complete suppression of induced resistance, and microscopy confirmed the development of healthy fungal haustoria within these plants. Inhibition of 4CL ligase, an enzyme largely responsible for channeling phenylpropanoid metabolites into the lignin pathway, had little effect on induced disease resistance. Biochemical analyses revealed similar levels of 4CL protein accumulation for all treatments, suggesting no alterations of nontargeted functions within inhibited plants. Collectively, the results of this study support the idea that induced resistance in cucumber is largely correlated with rapid de novo biosynthesis of flavonoid phytoalexin compounds.

Leaf tissue harvested from cucumber plants (Cucumis sativus L.) expressing induced resistance against the powdery mildew fungus Podosphaera xanthii (syn. Sphaerotheca fuliginea, Castagne; Braun and Shishkoff) was extracted and analyzed for phytoalexin compounds. Fluorescence microscopy was then used to observe the production of these compounds in planta, and laser scanning confocal microscopy observations were made to locate the subcellular sites of phytoalexin accumulation. Phytochemical analyses and fluorescence microscopy observations revealed the production of autofluorescent C-glycosyl flavonoid phytoalexins within the epidermal tissues of disease-resistant plants undergoing fungal ingress. Phytoalexin production was triggered by the combination of an eliciting/inoculation treatment, and tissue autofluorescence of color characteristic of the phytoalexins reached a maximum 48 h after elicitation prior to subsiding following the collapse of the pathogen. After a second eliciting treatment, disease-resistant plants produced phytoalexins more rapidly in response to fungal challenge. At the cellular level, autofluorescent C-glycosyl flavonoid phytoalexins were observed associated with the plasma membrane of infected epidermal cells immediately following elicitation. In the hours that preceded the collapse of conidial chains, phytoalexins accumulated inside the haustorial complexes of the pathogen within the epidermal cells of disease-resistant plants. Taken together, the results of this study show the timely synthesis of C-glycosyl flavonoid phytoalexins at precise subcellular locations as a key defense reaction used by cucumber to create incompatible interactions with powdery mildew.

Based on previously reported techniques and methods to identify induced phenolic compounds, a shorter, more reliable methodology for identifying conjugated plant phenolics from cucumber was developed. During the initial analytical phase of this methodology, compounds are elicited, extracted, hydrolyzed, and fractionated. During the second semipreparative phase, scale-up chromatography is used to semipurify large amounts of extracted hydrolyzed induced compound, which are later purified with semipreparative high performance liquid chromatography. To demonstrate the merits of this methodology and the problematic nature of these defence molecules, comparisons are made with the methodologies of previous studies examining phytoalexin production in cucumber plants.Key words: Cucumis sativus, induced glycosylated phenolics, phenolic acids, C-flavonoid glycosides, phytoalexins, methodology.

Disease assessments and cytological investigations provided valuable information on the modes of action and efficacies of two prophylactic compounds, Milsana and benzothiadiazole (BTH), against powdery mildew development on long English cucumber. Milsana application significantly reduced disease incidence relative to inoculated controls through induction of localized resistance. Microscopic observations showed most haustoria had collapsed in the localized Milsana treatment and were encapsulated by an amorphous material impregnated by electron-opaque substances. The rapidity of haustorial collapse (within 4 days of treatment application) together with the encasement by electron-dense substances stained blue by toluidine blue O suggest that phenolics are possibly involved in the Milsana defense response. Cytochemical labeling of chitin with a wheat germ agglutinin/ovomucoid-gold complex showed that complete cellular disorganization of the fungus had occurred without disturbance to chitin in the walls of mycelia and haustoria. This may indicate that chitinolytic activity is not important in the Milsana-activated defense response. Application of high doses of BTH induced occasional cell wall thickening and accumulation of a compound that stained purple by toluidine blue O, but the defense response was weak, sporadic, and insufficient to reduce powdery mildew infection on cucumber. Responses to BTH could not be differentiated in terms of timing of the initial application or systemicity.

Pseudozyma flocculosa is related to the model plant pathogen Ustilago maydis yet is not a phytopathogen but rather a biocontrol agent of powdery mildews; this relationship makes it unique for the study of the evolution of plant pathogenicity factors. The P. flocculosa genome of ;23 Mb includes 6877 predicted protein coding genes. Genome features, including hallmarks of pathogenicity, are very similar in P. flocculosa and U. maydis, Sporisorium reilianum, and Ustilago hordei. Furthermore, P. flocculosa, a strict anamorph, revealed conserved and seemingly intact mating-type and meiosis loci typical of Ustilaginales. By contrast, we observed the loss of a specific subset of candidate secreted effector proteins reported to influence virulence in U. maydis as the singular divergence that could explain its nonpathogenic nature. These results suggest that P. flocculosa could have once been a virulent smut fungus that lost the specific effectors necessary for host compatibility. Interestingly, the biocontrol agent appears to have acquired genes encoding secreted proteins not found in the compared Ustilaginales, including necrosis-inducing-Phytophthora-protein- and Lysin-motif- containing proteins believed to have direct relevance to its lifestyle. The genome sequence should contribute to new insights into the subtle genetic differences that can lead to drastic changes in fungal pathogen lifestyles.

Silicon (Si) confers several benefits to many plant species when absorbed as silicic acid through nodulin 26-like intrinsic proteins (NIPs). The NIPs belong to major intrinsic protein (MIP) family, members of which form channels with high selectivity to control transport of water and different solutes. Here, comparative genomic analysis of the MIPs was performed to investigate the presence of Si transporter MIPs in soybean. Thorough analysis of phylogeny, gene organization, transcriptome profiling and protein modeling was performed to characterize MIPs in rice, Arabidopsis and soybean. Based on several attributes, two putative Si transporter genes, GmNIP2-1 and GmNIP2-2, were identified, characterized and cloned from soybean. Expression of both genes was detected in shoot and root tissues, and decreased as Si increased. The protein encoded by GmNIP2-2 showed functionality for Si transport when expressed in Xenopus oocytes, thus confirming the genetic capability of soybean to absorb the element. Comparative analysis of MIPs in plants provides opportunities to decipher gene evolution, functionality and selectivity of nutrient uptake mechanisms. Exploitation of this strategy has helped to uncover unique features of MIPs in soybean. The identification and functional characterization of Si transporters can be exploited to optimize the benefits that plants can derive from Si absorption.

Secretion of recombinant proteins aims to reproduce the correct posttranslational modifications of the expressed protein while simplifying its recovery. In this study, secretion signal sequences from an abundantly secreted 34-kDa protein (P34) from Pseudozyma flocculosa were cloned. The efficiency of these sequences in the secretion of recombinant green fluorescent protein (GFP) was investigated in two Pseudozyma species and compared with other secretion signal sequences, from S. cerevisiae and Pseudozyma spp. The results indicate that various secretion signal sequences were functional and that the P34 signal peptide was the most effective secretion signal sequence in both P. flocculosa and P. antarctica. The cells correctly processed the secretion signal sequences, including P34 signal peptide, and mature GFP was recovered from the culture medium. This is the first report of functional secretion signal sequences in P. flocculosa. These sequences can be used to test the secretion of other recombinant proteins and for studying the secretion pathway in P. flocculosa and P. antarctica.

This study sought to identify the factors and conditions that affected production of the antifungal glycolipid flocculosin by the biocontrol agent Pseudozyma flocculosa. For this purpose, different parameters known or reported to influence glycolipid release in fungi were tested. Concentration of the start-up inoculum was found to play an important role in flocculosin production, as the optimal level increased productivity by as much as tenfold. Carbon availability and nitrogen source (i.e., organic vs inorganic) both had a direct influence on the metabolism of P. flocculosa, leading to flocculosin synthesis. In general, if conditions were conducive for production of the glycolipid, carbon availability appeared to be the only limiting factor. On the other hand, if yeast extract was supplied as nitrogen source, fungal biomass was immediately stimulated to the detriment of flocculosin synthesis. Unlike other reports of glycolipid release by yeast-like fungi, inorganic nitrogen starvation did not trigger production of flocculosin. The relationship between the factors influencing flocculosin production in vitro and the conditions affecting the release of the molecule by P. flocculosa in its natural habitat appears to be linked to the availability of a suitable and plentiful food source for the biocontrol agent.

GFP technology was applied to the biocontrol agent (BCA) Pseudozyma flocculosa to study its development and interactions at the tritrophic level plant-powdery mildew-BCA. Transformation experiments with GFP led to the production of a strongly fluorescent strain, Act-4, that displayed biocontrol traits typical of P. flocculosa WT. Following inundative applications, growth of P. flocculosa Act-4 was closely and almost exclusively associated with the colonies of the pathogen regardless of the powdery mildew species or the host plant tested. Development of P. flocculosa Act-4 on control leaves alone was extremely limited 24 h after its application and was typical of the epiphytic growth characterizing this type of yeast-like fungus. Based on the strong correlation between the colonization pattern of the different powdery mildew species tested and the presence of P. flocculosa Act-4, as determined by its fluorescence, it seems that growth of the BCA is dependant on the presence of powdery mildews. These results demonstrate that the GFP technology can be used to study plant-pathogen-BCA interactions and fulfill a wide array of purposes ranging from fundamental observations of the biocontrol behavior of a BCA to very applied ones serving some of the requirements for the registration of BCA's such as defining their environmental fate.

Flocculosin, a glycolipid isolated from the yeast-like fungus Pseudozyma flocculosa , was investigated for in vitro antifungal activity. The compound displayed antifungal properties against several pathogenic yeasts. Synergistic activity was observed between flocculosin and amphotericin B, and no significant cytotoxicity was demonstrated when tested against human cell lines.

Extraction of cucumber leaf tissue expressing induced resistance against powdery mildew fungi revealed the presence of two new major C-glycosyl flavonoid products: vitexin-6-(4-hydroxy-1-ethylbenzene) (cucumerin A, 1) and isovitexin-8-(4-hydroxy-1-ethylbenzene) (cucumerin B, 2). In addition, the known C-glycosyl flavonoids apigenin-8-C-beta-D-glucopyranoside (vitexin, 3), apigenin-6-C-beta-D-glucopyranoside (isovitexin, 4), luteolin-8-C-beta-D-glucopyranoside (orientin, 5), and luteolin-6-C-beta-D-glucopyranoside (isoorientin, 6), as well as 4-hydroxycinnamic acid (p-coumaric acid, 7) and its methyl ester (p-came, 8), were found in higher quantities within resistant plants. The structures of 1-8 were elucidated using spectroscopic methods and unambiguously confirmed for 3-8 using co-chromatography experiments with authentic standards. On the basis of the results of this study and the reported biological activities of C-glycosyl flavonoids, these compounds would play a vital role in the defense strategy of this species by acting as phytoalexins.

Insertional mutagenesis was applied for the first time to a fungal biocontrol agent, Pseudozyma flocculosa , in an attempt to obtain mutants with altered antagonistic properties. Transformants were obtained via DNA-mediated transformation. Molecular analyses of the transformants revealed that multiple copies of the plasmid were integrated in tandem at one to many chromosomal loci. The transformants were screened for their biocontrol properties using standard bioassays, and the 160 tested transformants were classified into four groups: group I mutants (22 transformants) showed a stronger antagonistic effect than the wild type (WT) while those of group II (107 transformants) had a comparable antagonistic effect; group III mutants (17 transformants) had a decreased antagonistic effect relative to WT and group IV mutants (14 transformants) had lost their biocontrol properties. Culture extracts of the mutants (group IV) and WT were analyzed and compared for the presence of active metabolites which were then separated by solid-phase extraction and purified using conventional methods. Nuclear magnetic resonance experiments and analytical studies on a metabolite specifically produced by the WT revealed the presence of 2-(2′,4′-diacetoxy-5′-carboxy-pentanoyl) octadecyl cellobioside (flocculosin), a novel glycolipid with strong antifungal properties; the production of this compound would account for the biocontrol activity of P. flocculosa .