Development of a Simple Hydroponic Assay to Study Vertical and Horizontal Resistance of Soybean and Pathotypes of Phytophthora sojae.

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.

A comprehensive transcriptomic analysis of the effect of silicon on wheat plants under control and pathogen stress conditions.

The supply of soluble silicon (Si) to plants has been associated with many benefits that remain poorly explained and often contested. In this work, the effect of Si was studied on wheat plants under both control and pathogen stress (Blumeria graminis f. sp. tritici) conditions by conducting a large transcriptomic analysis (55,000 unigenes) aimed at comparing the differential response of plants under four treatments. The response to the supply of Si on control (uninfected) plants was limited to 47 genes of diverse functions providing little evidence of regulation of a specific metabolic process. Plants reacted to inoculation with B. graminis f. sp. tritici by an upregulation of many genes linked to stress and metabolic processes and a downregulation of genes linked to photosynthesis. Supplying Si to inoculated plants largely prevented disease development, a phenotypic response that translated into a nearly perfect reversal of genes regulated by the effect of B. graminis f. sp. tritici alone. These results suggest that Si plays a limited role on a plant's transcriptome in the absence of stress, even in the case of a high-Si-accumulating monocot such as wheat. On the other hand, the benefits of Si in the form of biotic stress alleviation were remarkably aligned with a counter-response to transcriptomic changes induced by the pathogen B. graminis f. sp. tritici.

Identification of genes potentially involved in the biocontrol activity of Pseudozyma flocculosa.

Flocculosin is an antifungal cellobiose lipid linked to the biocontrol activity of Pseudozyma flocculosa and whose structure is very similar to that of ustilagic acid produced by Ustilago maydis. In this work, homologs of the U. maydis cyp1 gene, involved in the biosynthesis of ustilagic acid, were isolated and sequenced from P. flocculosa and P. fusiformata, the latter species being also known to produce ustilagic acid. Interestingly, no homologs were found in four other closely related Pseudozyma spp. from which no evidence of ustilagic acid production has ever been obtained, thus supporting the specificity of cyp1 with ustilagic acid synthesis. In addition, a homolog of the U. maydis uat1 gene involved in the acetylation of the molecule and located next to the cyp1 gene was partially sequenced from P. flocculosa. All three newly sequenced genes showed strong sequence similarity to their counterparts in U. maydis. Cyp1 expression was monitored in conditions that were either conducive or repressive to flocculosin production. Expression increased markedly (>100x) when P. flocculosa was inoculated in a growth medium conducive to flocculosin production but was rapidly downregulated in a repressive medium (in vitro) or on powdery mildew-infected cucumber leaves (in vivo). This suggests that the molecule was preferentially synthesized early in the process of searching for a growth substrate. This study provides the first identification of genes involved in the production of flocculosin, a molecule potentially associated with the biocontrol properties of P. flocculosa.

In vitro antibacterial activity and antifungal mode of action of flocculosin, a membrane-active cellobiose lipid.

AIMS: To investigate the in vitro antibacterial activity and antifungal mode of action of flocculosin, a cellobiose lipid produced by Pseudozyma flocculosa. METHODS AND RESULTS: When tested against clinical bacterial isolates, the compound was particularly active against Gram-positive bacteria and its effect was not mitigated against isolates known as resistant to other antibiotics. The antifungal activity of flocculosin was found to be rapid and concentration-dependent. At lethal concentrations against Candida albicans, flocculosin caused a rapid leakage of intracellular potassium and inhibited acidification of the medium by plasma membrane ATPases suggesting a physical rather than a biochemical effect. TEM observations of cells exposed 6 h to flocculosin revealed disrupted membranes and disorganized mitochondria. CONCLUSIONS: Data obtained in this study confirm that flocculosin acts by disrupting the membrane surface of sensitive micro-organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The elucidation of an antifungal mode of action of flocculosin can be exploited in furthering its antimicrobial potential against fungi and bacteria whose cell membranes are particularly sensitive to the action of the molecule.

Alternative methods for genetic transformation of Pseudozyma antarctica, a basidiomycetous yeast-like fungus.

Electroporation and Agrobacterium tumefaciens-mediated transformation (ATMT) were adapted and optimized for genetic transformation of the basidiomycetous yeast-like fungus Pseudozyma antarctica as alternatives to the cumbersome PEG/CaCl(2)-mediated transformation of protoplasts. Electroporation yielded 100-200 transformants per mug of DNA per 10(8) cells after 3 days on selective medium. For its part, ATMT yielded 60-160 transformants per 10(6) input cfu after 5-10 days on a selective medium. Transformants obtained from both methods showed stable hygromycin resistance and strong expression of green fluorescent protein. Analysis of integration events revealed a limited number of predominantly tandem insertions in the genome of transformants, an improvement over PEG/CaCl(2)-mediated transformation. Both protocols relied on intact conidia of P. antarctica as starting material and thus eliminated the need for cell wall-degrading or weakening agents such as lytic enzymes or chemicals. Other advantages over protoplast transformation included higher yield of transformants and shorter recovery time of transformed colonies on selective medium.

The potential of Pseudozyma yeastlike epiphytes for the production of heterologous recombinant proteins.

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.

Cytological Evidence of an Active Role of Silicon in Wheat Resistance to Powdery Mildew (Blumeria graminis f. sp. tritici).

ABSTRACT Silicon (Si) amendments in the form of exogenously supplied nutrient solution or calcium silicate slag protect wheat plants from powdery mildew disease caused by the fungus Blumeria graminis f. sp. tritici. The most striking difference between Si- and Si+ plants challenged with B. graminis f. sp. tritici was the extent of epidermal cell infection and colonization by B. graminis f. sp. tritici. Histological and ultrastructural analyses revealed that epidermal cells of Si+ plants reacted to B. graminis f. sp. tritici attack with specific defense reactions including papilla formation, production of callose, and release of electron-dense osmiophilic material identified by cytochemical labeling as glycosilated phenolics. Phenolic material not only accumulated along the cell wall but also was associated with altered integrity of haustoria in a manner similar to localized phytoalexins as reported from other pathosystems. These results strongly suggest that Si mediates active localized cell defenses against B. graminis f. sp. tritici attack.

Selection of Antagonist Microorganisms Against Helminthosporium solani, Causal Agent of Potato Silver Scurf.

Silver scurf, caused by the fungus Helminthosporium solani, is an important disease affecting potato tubers. Control of the disease has been hampered by the development of H. solani strains resistant to thiabendazole, the only fungicide used in postharvest treatment. As a result, alternative control strategies are needed. In this study, 100 selected soil samples from the province of Québec were tested for their effect on silver scurf development on potato tubers. The results showed that 10 soils were able to decrease silver scurf development on tubers incubated at 10, 15, or 24°C. Many microorganisms were isolated from these soils and tested for their individual ability to reduce H. solani development using a whole-tuber assay. Several of them, including Alcaligenes piechaudii, Aquaspirillum autotrophicum, Arcanobacterium haemolyticum, Arthrobacter oxydans, Bacillus mycoides, Kocuria rosea, Streptomyces griseus, and a fungus of the class Zygomycetes displayed an ability to reduce the development of silver scurf on potato tubers at 10, 15, or 24°C. These results can find useful applications toward a biocontrol program of potato silver scurf as postharvest or seed tuber treatment.

Biological control in greenhouse systems.

The controlled environment of greenhouses, the high value of the crops, and the limited number of registered fungicides offer a unique niche for the biological control of plant diseases. During the past ten years, over 80 biocontrol products have been marketed worldwide. A large percentage of these have been developed for greenhouse crops. Products to control soilborne pathogens such as Sclerotinia, Pythium, Rhizoctonia and Fusarium include Coniothyrium minitans, species of Gliocladium, Trichoderma, Streptomyces, and Bacillus, and nonpathogenic Fusarium. Products containing Trichoderma, Ampelomyces quisqualis, Bacillus, and Ulocladium are being developed to control the primary foliar diseases, Botrytis and powdery mildew. The development of Pseudomonas for the control of Pythium diseases in hydroponics and Pseudozyma flocculosa for the control of powdery mildew by two Canadian research programs is presented. In the future, biological control of diseases in greenhouses could predominate over chemical pesticides, in the same way that biological control of greenhouse insects predominates in the United Kingdom. The limitations in formulation, registration, and commercialization are discussed, along with suggested future research priorities.

Establishment of a gene transfer system for Pseudozyma flocculosa, an antagonistic fungus of powdery mildew fungi.

A reliable DNA-mediated transformation system has been developed for Pseudozyma flocculosa, a fungus that is antagonistic to powdery-mildew fungi. Plasmids harboring various selectable markers under the control of different promoters were tested. Molecular analyses demonstrated that successful transformation could be achieved using a plasmid that confers resistance to hygromycin B under the control of the Ustilago maydis hsp70 promoter and terminator sequences. On average, 1-40 (mean = 20) transformants were obtained per 10 microg of linearized DNA per 10(8) protoplasts. Southern analysis of the transformants revealed that, in each case, the vector had integrated in multiple tandem copies into the genome of P. flocculosa, and that integration events were random. Pulsed-field gel electrophoresis was employed to separate the genome of P. flocculosa into at least 11 chromosomes with sizes ranging from 0.55 Mb to 2.9 Mb. Hybridization with the plasmid indicated that integration of vector DNA had occurred in one to several chromosomes depending on the transformant examined.

Specificity and mode of action of the antifungal fatty acid cis-9-heptadecenoic acid produced by Pseudozyma flocculosa.

cis-9-Heptadecenoic acid (CHDA), an antifungal fatty acid produced by the biocontrol agent Pseudozyma flocculosa, was studied for its effects on growth and/or spore germination in fungi. Inhibition of growth and/or germination varied considerably and revealed CHDA sensitivity groups within tested fungi. Analysis of lipid composition in these fungi demonstrated that sensitivity was related primarily to a low intrinsic sterol content and that a high level of unsaturation of phospholipid fatty acids was not as involved as hypothesized previously. Our data indicate that CHDA does not act directly with membrane sterols, nor is it utilized or otherwise modified in fungi. A structural mechanism of CHDA, consistent with the other related antifungal fatty acids produced by P. flocculosa, is proposed in light of its activity and specificity. The probable molecular events implicated in the sensitivity of fungi to CHDA are (i) partitioning of CHDA into fungal membranes; (ii) a variable elevation in fluidity dependent on the buffering capability (sterol content) in fungi; and (iii) higher membrane disorder causing conformational changes in membrane proteins, increased membrane permeability and, eventually, cytoplasmic disintegration.

Molecular and Physiological Analysis of the Powdery Mildew Antagonist Pseudozyma flocculosa and Related Fungi.

ABSTRACT A number of phenotypic and genotypic characteristics were used to ascertain the identity and diversity of Pseudozyma flocculosa, a natural antagonist of powdery mildews that has received little attention in terms of taxonomy. To this end, several putative isolates of P. flocculosa as well as several closely related species were analyzed. Ribosomal DNA sequences distinguished P. flocculosa from other Pseudozyma spp. and identified two previously unknown Pseudozyma isolates as P. flocculosa. Random amplified microsatellites revealed three distinct P. flocculosa strains among the tested isolates. Biocontrol properties and antifungal metabolite production were limited to the P. flocculosa spp. Results produced useful molecular markers to (i) distinguish P. flocculosa from other related fungi, (ii) identify different strains within this species, and (iii) aid in the construction of isolate-specific molecular tools that will assist in research and development of P. flocculosa as a biocontrol agent of powdery mildew fungi.

Protoplast preparation and regeneration from spores of the biocontrol fungus Pseudozyma flocculosa.

This paper describes a specific protocol for yielding and regenerating protoplasts from spores of the recently described powdery mildew biocontrol agent Pseudozyma flocculosa. With this protocol, a large quantity of protoplasts was obtained from beta-mercaptoethanol-pretreated spores in 3-day-old cultures of P. flocculosa grown in YMPD. Enzymatic digestion was optimal with 0.5% Novozym 234 and 5% Glucanex prepared in 0.6 M KCl in 0.1 M citrate buffer. All liberated protoplasts fluoresced in the presence of fluorescein diacetate indicating that viability was nearly 100%. The regeneration rate was equally outstanding reaching 75% when 0.8 M sucrose was used as osmotic stabilizer in the regeneration medium. This protocol will find useful applications in genetic studies of this poorly characterized and understood biocontrol agent.

Effects of milsana and benzothiadiazole on the ultrastructure of powdery mildew haustoria on cucumber.

ABSTRACT 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.

Silicon-mediated accumulation of flavonoid phytoalexins in cucumber.

ABSTRACT The controversial role of silicon in plant disease resistance, described mostly as a passive mechanical protection, has been addressed. Conclusive evidence is presented that silicon is involved in the increased resistance of cucumber to powdery mildew by enhancing the antifungal activity of infected leaves. This antifungal activity was attributable to the presence of low-molecular-weight metabolites. One of these metabolites, described here as a phytoalexin, was identified as a flavonol aglycone rhamnetin (3,5,3',4'-tetrahydroxy-7-O-methoxyflavone). This is the first report of a phytoalexin for this chemical group in the plant kingdom and of a flavonol phytoalexin in cucumber, a chemical defense long believed to be nonexistent in the family Cucurbitaceae. The antifungal activity of leaf extracts was better expressed after acid hydrolysis, extending to another plant species the concept that some phytoalexins are synthesized as glycosylated phytoalexins or their precursors.

Induction of systemic resistance to Pythium damping-off in cucumber plants by benzothiadiazole: ultrastructure and cytochemistry of the host response.

Benzo (1,2,3) thiadiazole-7-carbothioic acid S-methyl ester (BTH, CGA 245704), a non-toxic, synthetic chemical, was applied as a foliar spray to cucumber plants and evaluated for its potential to induce defense mechanisms in root tissues infected by the soilborne pathogen, Pythium ultimum Trow. In non-treated cucumber plants, fungal colonization was intense and paralleled marked host tissue damage, whereas in BTH-treated plants, pathogen ingress towards the vascular stele was apparently halted by the massive deposition of a phenolic-enriched material which occluded a large number of cortical and vascular parenchyma cells. This considerable increase in the accumulation of phenolics was accompanied by cytological disorders of the invading pathogen at a time when the wall-bound cellulose component was preserved. In addition to phenolic compounds, the occluding material contained large amounts of beta-glucoside residues. These residues gradually decreased in the areas neighboring fungal cells whereas phenolic deposition appeared to be more uniformly distributed throughout the occluded host cells. Pathogen penetration in non-occluded cucumber root cells coincided with other changes, mainly characterized by both the deposition onto the inner surface of the cell walls of some heterogeneous wall appositions and the coating of some intercellular spaces with an electron-opaque material. Evidence is provided in this study that BTH has the ability to induce SAR in cucumber. Exogenous, foliar applications of the chemical sensitize susceptible cucumber plants to react more rapidly and more efficiently to P. ultimum attack, mainly through the massive accumulation of phenolic compounds at sites of attempted pathogen penetration.

Novel butyrolactones with antifungal activity produced by Pseudomonas aureofaciens strain 63-28.

The bacterium Pseudomonas aureofaciens 63-28 is antagonistic to several plant pathogenic fungi, including Pythium spp. The bacterium produced at least four antifungal metabolites active against Pythium ultimum and Phytophthora cryptogea when tested in culture for antifungal activity. Two of these compounds were identified as the novel butyrolactones (Z)-4-hydroxy-4-methyl-2-(1-hexenyl)-2-butenolide and (Z)-4-hydroxymethyl-2-(1-hexenyl)-2-butenolide, by using NMR and GC-MS. All compounds were different from other antibiotics produced by Pseudomonas spp., including pyoluteorin, pyrrolnitrin, and 2,4-diacetylphloroglucinol, as determined by HPLC. This is the first report of butyrolactones with antifungal activity produced by a saprophytic Pseudomonas spp.

Interaction of Four Antagonistic Fungi with Botrytis aclada in Dead Onion Leaves: A Comparative Microscopic and Ultrastructural Study.

ABSTRACT The colonization of dead onion leaves by Botrytis aclada and the fungal antagonists Aureobasidium pullulans, Chaetomium globosum, Glio-cladium catenulatum, and Ulocladium atrum and the interactions between B. aclada and each of the four antagonists were studied at the microscopic and ultrastructural level. This approach was used in an attempt to understand the colonization pattern of these fungi and the nature of the biocontrol activity of the antagonists that have shown a potential to suppress spore production of Botrytis spp. on necrotic plant tissues. When applied alone, B. aclada and U. atrum were found throughout the leaf tissues in high densities after an incubation period of 6 days at 18 degrees C in a moist chamber. C. globosum and G. catenulatum colonized only the outer portions of the leaf, whereas A. pullulans appeared to be concentrated in the leaf stomata. When pathogen and antagonists were applied together, ultrastructural observations revealed that cells of B. aclada were plasmolyzed in the presence of G. catenulatum, suggesting a reaction to antifungal molecules. Antibiosis also seemed to be involved, albeit to a lesser extent, in the antagonistic interactions between B. aclada and A. pullulans or C. globosum. No evidence of direct parasitism was recorded. On the other hand, U. atrum appeared to completely exclude B. aclada from dead onion tissues when both fungi competed for the substrate. Ultrastructural observations of the in vitro interaction between the two fungi did not reveal parasitism or antibiosis by either fungus. Based on previous records of its biocontrol potential and observations of its colonizing properties, it appears that U. atrum can compete for and utilize necrotic tissues rapidly and extensively, thus, excluding competitors without any other antagonistic action.

Evidence of Phytoalexins in Cucumber Leaves Infected with Powdery Mildew following Treatment with Leaf Extracts of Reynoutria sachalinensis.

Phenolic compounds extracted from cucumber (Cucumis sativus L.) leaves were separated and analyzed for their differential presence and fungitoxicity in relation to a prophylactic treatment with Milsana (Compo, Munster, Germany) against powdery mildew (Sphaerotheca fuliginea). Based on our extraction and purification procedures, at least eight separate phenolic compounds with antifungal activity were identified as intrinsic components of cucumber plants. Of these compounds, six displayed a significant increase in concentration as a result of elicitation with Milsana, this being particularly evident when the plant was stressed by the pathogen. The combined amounts of these antifungal compounds in treated plants was nearly five times the level found in control plants. One week after Milsana application, some of the antifungal compounds obtained through hydrolysis of their glycosidic links were also detected in their free form, indicating that they are likely liberated from conjugated phenolics by enzymatic hydrolysis in planta. To our knowledge, these results provide the first direct evidence that cucumber plants produce elevated levels of phytoalexins in response to an eliciting treatment after infection.

Influence of a subinhibitory dose of antifungal fatty acids from Sporothrix flocculosa on cellular lipid composition in fungi.

Antifungal fatty acids produced by the biocontrol fungus Sporothrix flocculosa were studied on the basis of their effect on growth and cellular lipid composition of three fungi, Cladosporium cucumerinum, Fusarium oxysporum, and S. flocculosa, whose growth was decreased by 51, 33, and 5%, respectively, when exposed to 0.4 mg fatty acid per ml. The sensitivity to fatty acid antibiotics from S. flocculosa was related to a high degree of unsaturation of phospholipid fatty acids and a low proportion of sterols. The major responses of sensitive fungi to sublethal doses of antifungal fatty acids from liquid culture of S. flocculosa were: (i) a decrease in total lipid; (ii) an increase in the degree of fatty acid unsaturation (18:1 > 18:2 > 18:3); (iii) an increase in free fatty acids and phosphatidic acid and a decrease in total phospholipids; and (iv) an increase in sterol/phospholipid ratio. These modifications in lipid composition led to an increase in membrane fluidity in sensitive fungi as demonstrated by assessment of fluoresence anisotropy using liposomes and 1,6-diphenyl-1,3,5-hexatriene probe. This alteration in the physical state of lipids appears to be responsible for the previously demonstrated alteration of membrane structure and function in fungi confronted to S. flocculosa.