Functional characterization of a tyrosinase gene from the oomycete Saprolegnia parasitica by RNAi silencing

Here we describe the first application of transient gene silencing in Saprolegnia parasitica, a pathogenic oomycete that infects a wide range of fish, amphibians, and crustaceans. A gene encoding a putative tyrosinase from S. parasitica, SpTyr, was selected to investigate the suitability of RNA-interference (RNAi) to functionally characterize genes of this economically important pathogen. Tyrosinase is a mono-oxygenase enzyme that catalyses the O-hydroxylation of monophenols and subsequent oxidation of O-diphenols to quinines. These enzymes are widely distributed in nature, and are involved in the melanin biosynthesis. Gene silencing was obtained by delivering in vitro synthesized SpTyr dsRNA into protoplasts. Expression analysis, tyrosinase activity measurements, and melanin content analysis confirmed silencing in individual lines. Silencing of SpTyr resulted in a decrease of tyrosinase activity between 38 % and 60 %, dependent on the level of SpTyr-expression achieved. The SpTyr-silenced lines displayed less pigmentation in developing sporangia and occasionally an altered morphology. Moreover, developing sporangia from individual silenced lines possessed a less electron dense cell wall when compared to control lines, treated with GFP-dsRNA. In conclusion, the tyrosinase gene of S. parasitica is required for melanin formation and transient gene silencing can be used to functionally characterize genes in S. parasitica.     

Distribution and Expression of Elicitin‐like Protein Genes of the Biocontrol Agent Pythium oligandrum

Pythium oligandrum has the ability to induce plant defence reactions, and four elicitin‐like proteins (POD‐1, POD‐2, POS‐1 and oligandrin) that are produced by this oomycete have been identified as elicitor proteins. The first three are cell wall protein elicitors (CWPs), and the latter is an extracellular protein. Pythium oligandrum isolates have been previously divided into two groups based on the CWPs: the D‐type isolate containing POD‐1 and POD‐2, and the S‐type isolate containing POS‐1. We identified the genes encoding these elicitin‐like proteins and analyzed the distribution of these genes among 10 P. oligandrum isolates. A genomic fosmid library of the D‐type isolate MMR2 was constructed and genomic regions containing the elicitin‐like protein genes were identified. Southern blot analyses with probes derived from pod‐1 and an oligandrin gene indicated that the 10 P. oligandrum isolates could be divided into the same groups as those based on the CWPs. The D‐type isolates carried pod‐1, pod‐2 and two oligandrin genes, termed oli‐d1 and oli‐d2, while the S‐type isolates carried pos‐1 and one oligandrin gene termed oli‐s1. Phylogenetic analysis of POD‐1, POD‐2, POS‐1, Oli‐D1, Oli‐D2 and Oli‐S1 with the previously defined elicitins and elicitin‐like proteins of Phytophthora and Pythium species showed the specific clade. These genes occurred as single copies and were present in the P. oligandrum genomes but not in the other nine Pythium species (Pythium iwayamai, Pythium volutum, Pythium vanterpoolii, Pythium spinosum, Pythium torulosum, Pythium irregulare, Pythium ultimum, Pythium aphanidermutum and Pythium butleri). Furthermore, RT‐PCR analysis demonstrated that all of these genes were expressed during the colonization of tomato roots by P. oligandrum, supporting the idea that they encode potential elicitor proteins. To investigate the genetic relationships between the D‐type and the S‐type isolates, physical maps of the flanking regions around pod‐1, pod‐2, pos‐1 and the oligandrin genes were constructed. The maps suggest that the D‐type isolates may be derived from the S‐type isolates due to gene duplication and deletion events.     

Cytological Effects of Cellulases in the Parasitism of Phytophthora parasitica by Pythium oligandrum

The ubiquitous oomycete Pythium oligandrum is a potential biocontrol agent for use against a wide range of pathogenic fungi and an inducer of plant disease resistance. The ability of P. oligandrum to compete with root pathogens for saprophytic colonization of substrates may be critical for pathogen increase in soil, but other mechanisms, including antibiosis and enzyme production, also may play a role in the antagonistic process. We used transmission electron microscopy and gold cytochemistry to analyze the intercellular interaction between P. oligandrum and Phytophthora parasitica. Growth of P. oligandrum towards Phytophthora cells correlated with changes in the host, including retraction of the plasma membrane and cytoplasmic disorganization. These changes were associated with the deposition onto the inner host cell surface of a cellulose-enriched material. P. oligandrum hyphae could penetrate the thickened host cell wall and the cellulose-enriched material, suggesting that large amounts of cellulolytic enzymes were produced. Labeling of cellulose with gold-complexed exoglucanase showed that the integrity of the cellulose was greatly affected both along the channel of fungal penetration and also at a distance from it. We measured cellulolytic activity of P. oligandrum in substrate-free liquid medium. The enzymes present were almost as effective as those from Trichoderma viride in degrading both carboxymethyl cellulose and Phytophthora wall-bound cellulose. P. oligandrum and its cellulolytic enzymes may be useful for biological control of oomycete pathogens, including Phytophthora and Pythium spp., which are frequently encountered in field and greenhouse production.     

The Oomycete <Emphasis Type="Italic">Pythium oligandrum</Emphasis> Can Suppress and Kill the Causative Agents of Dermatophytoses

Pythium oligandrum (Oomycota) is known for its strong mycoparasitism against more than 50 fungal and oomycete species. However, the ability of this oomycete to suppress and kill the causal agents of dermatophytoses is yet to be studied. We provide a complex study of the interactions between P. oligandrum and dermatophytes representing all species dominating in the developed countries. We assessed its biocidal potential by performing growth tests, on both solid and liquid cultivation media and by conducting a pilot clinical study. In addition, we studied the molecular background of mycoparasitism using expression profiles of genes responsible for the attack on the side of P. oligandrum and the stress response on the side of Microsporum canis. We showed that dermatophytes are efficiently suppressed or killed by P. oligandrum in the artificial conditions of cultivations media between 48 and 72 h after first contact. Significant intra- and interspecies variability was noted. Of the 69 patients included in the acute regimen study, symptoms were completely eliminated in 79% of the patients suffering from foot odour, hyperhidrosis disappeared in 67% of cases, clinical signs of dermatomycoses could no longer be observed in 83% of patients, and 15% of persons were relieved of symptoms of onychomycosis. Our investigations provide clear evidence that the oomycete is able to recognize and kill dermatophytes using recognition mechanisms that resemble those described in oomycetes attacking fungi infecting plants, albeit with some notable differences.     

Biocontrol of black scurf on potato by seed tuber treatment with Pythium oligandrum

The biological control activity of Pythium oligandrum against black scurf of potato caused by Rhizoctonia solani AG-3 was evaluated in field experiments after treatment of potato seed tubers with P. oligandrum. Seed tubers infected with black scurf sclerotia were dipped for a few seconds in a suspension of 10³, 10⁴ or 10⁵ mL^?1 P. oligandrum oospores and were then air-dried. Each level of P. oligandrum-treatment significantly reduced the disease rates of stolon at a level similar to that achieved by chemical control. When P. oligandrum populations adherent to the surface of seed tubers were determined, oospore counts on tubers treated with 10⁴ or 10⁵ oospores mL^?1 were about 540/cm² or about 22,000/cm² just after dipping and decreased to about 170/cm² or 2900/cm² after a 3-week incubation, respectively. Confocal laser scanning microscopic observation with an immuno-enzymatic staining procedure showed that P. oligandrum hyphae had colonized the sclerotia and established close contact by coiling around the R. solani hyphae present on the surface of seed tubers, in a manner similar to that observed in the dual-culture test. Quantification of R. solani DNA by PCR indicated that the R. solani population was reduced on the seed tubers treated with P. oligandrum compared to untreated tubers. Furthermore, the ability of P. oligandrum to induce resistance against black scurf was determined using a potato tuber disk assay. Treatment of tuber disks with the cell wall protein fraction of P. oligandrum enhanced the expression of defense-related genes such as 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase, lipoxygenase and basic PR-6 genes, and reduced disease severity upon challenge with R. solani compared with untreated controls. These results suggest that biocontrol mechanisms employed by P. oligandrum against black scurf involve both mycoparasitism and induced resistance.     

寡雄腐霉发酵液对番茄生长的影响及对灰霉病的防治作用

为了研发对番茄灰霉病高效、稳定、安全的生物农药,试验利用自主分离获得的寡雄腐霉菌株制备发酵液,采用盆栽试验研究寡雄腐霉发酵液对番茄生长的影响和对灰霉病的防治效果及机制,并在大田生产中验证其生防效果。结果表明,盆栽试验中,寡雄腐霉发酵液促进健康番茄植株生长,植株总生物量和根系生物量分别增加9.5%和15.4%,提高了植株叶绿素含量、根系活力及氮、磷、钾吸收量,并使带病番茄植株的发病率和病情指数分别降低57.2%和60.3%,相对防治效果达60.3%,施用寡雄腐霉发酵液对番茄叶片细胞膜具有保护性,降低丙二醛含量,提高病原性相关酶""超氧化物歧化酶、多酚氧化酶和苯丙氨酸解氨酶活性。后续田间试验中寡雄腐霉发酵液对番茄灰霉病的防治效果达71.2%。说明寡雄腐霉发酵液能有效防治番茄灰霉病,还具有促进番茄生长的作用,并且可诱导番茄植株对病原菌的防御作用,应用前景广泛。     

Isolation of Pythium Acanthicum,P. oligandrum,and P. periplocum from Soil and Evaluation of Their Mycoparasitic Activity and Biocontrol Efficacy Against Selected Phytopathogenic Pythium Species

Mycoparasitic Pythium species with spiny oogonia were surveyed in 50 Palestinian agricultural fields subject to different cropping practices using the Sclerotia Bait Technique (SBT) and the Surface-Soil-Dilution-Plate method (SSDP) with the selective VP3 medium. The mycoparasitic Pythium species were obtained from 21 (42%) soils using the SSDP method and from 37 (74%) soils using SBT. Pythium acanthicum and P. oligandrum were isolated by both methods, whereas P. periplocum was isolated only by the SBT. Using a newly modified dual plate culture method (MDPCM), the three mycoparasites showed varying antagonistic performance against several Pythium host species under a range of in vitro conditions. However, P. periplocum and P. oligandrum were found to be active biocontrol agents against P. ultimum, the damping-off organism of cucumber. This pathogen was antagonized, on thin films of water agar, by the three mycoparasites, and was moderately susceptible to P. periplocum while slightly susceptible to P. acanthicum and P. oligandrum. In direct application method in which antagonistic mycoparasites were incorporated into peat/sand mixture artificially infested with P. ultimum under growthroom conditions, Pythium oligandrum and P. periplocum (at 500 CFUg⁻¹) significantly improved seedling emergence and protected seedlings from damping-off. In the seed coating method, biocontrol by two types of seed dressing (homogenate- or oospore coated seeds), was comparable to that achieved by direct application. This revised version was published online in June 2006 with corrections to the Cover Date.     

Optimization of transgene-mediated silencing in Phytophthora infestans and its association with small-interfering RNAs

Methods for silencing genes in Phytophthora transformants have been demonstrated previously, but wide variation in effectiveness was reported in different studies. To optimize this important tool for functional genomics, we compared the abilities of sense, antisense, and hairpin transgenes introduced by protoplast, electroporation, and bombardment methods to silence the inf1 elicitin gene in Phytophthora infestans. A hairpin construct induced silencing three times more often than sense or antisense vectors, and protoplast transformation twice as much as electroporation. Using hairpins introduced into protoplasts, 61% of strains were silenced, and transgene copy number was positively correlated with silencing. The utility of bombardment was reduced by the occurrence of heterokaryons containing silenced and non-silenced nuclei, but silenced strains were obtainable from about 20% of primary transformants by single-nuclear purification. Most inf1-deficient strains were fully silenced, however some exhibited partial suppression. These produced inf1-derived RNAs of about 21-nt which correspond to both the sense and antisense strands of inf1, implicating an RNAi-like mechanism in silencing.     

Homothallic sexual reproduction of Pustula helianthicola and germination of oospores

Sunflower white blister rust has become an important disease in many countries with intensive cultivation of the important oil crop. The biology of the pathogen is still partly unclear, particular with respect to its sexual reproduction and primary mode of infection. Zoospores released from sporangia of Pustula helianthicola were isolated individually and used for the inoculation of sunflower in order to generate unithallic, genetically homogenous infections. Single zoospore inoculation of young seedlings resulted in mitotic sporulation within subepidermal blisters on cotyledons and true leaves after approximately 2 weeks. Three weeks postinoculation, the infected plants started forming oospores, hence indicating homothallic sexual reproduction of the pathogen. The development of oogonia and antheridia was studied using light and fluorescence microscopy. Oospores were isolated from infected plant tissue and used for infection and germination studies. Microscopic observation of isolated oospores showed germination that formed sessile vesicle-like structures, germ sporangia or only germ tubes. The rate of germination reached approximately 40 %. Germination was not dependant on a resting phase after oospore formation. Oospores applied to the above ground parts of sunflower seedlings lead to infections within a similar time frame as was achieved with mitotic sporangia. The results underline the importance of oospores for primary infection at the beginning of the season and for long-distance dispersal of the pathogen with sunflower seeds contaminated by oospores.     

Characterisation of Pythium paroecandrum and its antagonism towards Botrytis cinerea, the causative agent of grey mould disease of grape

Pythium paroecandrum (B-30), an oomycete, was isolated from soil samples taken from a wheat field in Genlis in the Burgundy region of France and was found to check the growth and development of Botrytis cinerea, a serious grapevine pathogen. The oomycete is a fast-growing organism, living on vegetable debris, and can be recognised by its catenulate hyphal swellings, catenulate oogonia, and monoclinous antheridia. When grown together with B. cinerea, the causal agent of the grey mould disease of the grapevine, P. paroecandrum shows a pronounced antagonism and suppresses its growth and its aptitude to provoke the grey mould symptoms. Morphological features of this oomycete, its antagonism to B. cinerea, the sequences of the internal transcribed spacer region of its nuclear ribosomal DNA, and its comparison with related species are discussed in this article.     

The use of mutant and engineered microbial agents for biological control of plant diseases caused by Pythium: Achievements versus challenges

Pythium species are devasting pathogens causing major crop losses, e.g., damping-off in sugar beet caused by Pythium ultimum and root-rot of tomato caused by Pythium aphanidermatum. The use of natural antagonistic microorganisms is a promising environment-friendly approach to control Pythium-caused plant diseases. There are several examples of biocontrol of diseases caused by Pythium species but the application of bioeffectors (biological control agents) is limited for various reasons, including the restricted amount of gene-modification based biotechnological progress. The regulations in many countries prevent genetically modified bioeffectors from being routinely deployed in field conditions. Our two connected aims in this review are (1) to compile and assess achievements in genetic modification of bioeffectors which have been tested for parasitism or antagonism towards a Pythium plant pathogen or biocontrol of a plant disease caused by a Pythium species, and (2) discuss how a better performing bioeffector could be engineered to improve biocontrol of Pythium-caused plant diseases. We focus on the role of seven key mechanisms: cellulases, carbon catabolite de-repression, glycosylation, reactive oxygen species, chitin re-modelling, proteases, and toxic secondary metabolites. Genetic modifications of bioeffectors include gene deletion and overexpression, as well as the replacement of promoter elements to tune the gene expression to the presence of the pathogen. Gene-modifications are limited to fungal and bacterial bioeffectors due to the difficulty of gene modification in oomycete bioeffectors such as Pythium oligandrum. We assess how previous gene modifications could be combined and what other gene modification techniques could be introduced to make improved bioeffectors for Pythium-caused plant diseases. The broad host-range of Pythium spp. suggests engineering improved antagonistic traits of a bioeffector could be more effective than engineering plant-mediated traits i.e., engineer a bioeffector to antagonise a plant pathogen in common with multiple plant hosts rather than prime each unique plant host.     

Implications of oligomeric forms of POD-1 and POD-2 proteins isolated from cell walls of the biocontrol agent Pythium oligandrum in relation to their ability to induce defense reactions in tomato

The cell wall protein fraction (CWP) isolated from the biocontrol agent Pythium oligandrum induces defense reactions in tomato. CWP contains two novel elicitin-like proteins, POD-1 and POD-2, both with seven cysteines. To determine the essential structure in the defense-eliciting components of CWP, five fractions (F1, F2, F3, F4 and F5) were fractionated from CWP using cation chromatography and their components and disulfide bond compositions were analyzed. The expression levels of three defense-related genes (PR-6, LeCAS and PR-2b) were determined in tomato roots treated with each of the five fractions. Of the five fractions, F4 containing a heterohexamer of POD-1 and POD-2, and F5 containing a homohexamer of POD-1, both with disulfide bonds formed between all cysteine residues, induced the expression of three genes. F4 treatment also induced the accumulation of ethylene in tomato. The predicted three-dimensional structures of POD-1 and POD-2, and the results of SEC and MALDI-TOF MS analyses suggest that F4 consists of three POD-1 and POD-2 disulfide-bonded heterodimers that interleave into a hexameric ring through noncovalent association. These results suggest that this structure, which F5 also appears to form, is essential for stimulating defense responses in tomato.     

Observations of in vitro gametangial copulation and oosporogenesis in Lagenidium giganteum

An in vitro liquid culture oospore production method yielding 5 × 10³ oospores/ml was used to follow the sequential events of gametangial copulation and oospore formation in Lagenidium giganteum. Observations were made with Nomarski differential interference microscopy and scanning electron microscopy. After septation and division of fungal thalli into a chain-like series of links, certain individual subthalli differentiated into gametaniga, oogonia, and antheridia. Antheridia issued a fertilization tube which made contact with, and fused to a single oogonium. Copulative behavior was relatively synchronous and necessitated physical contact between thalli. Sexual reproduction was manifested by the migration and condensation of gametes. Plasmogamy was achieved following the introduction of the male gamete into the oogonium. The fused gametes gave rise to a zygote. Small amounts of periplasm remained in the oogonium. Zygote maturation into a fully developed oospore was characterized by the deposition of a multilaminated oospore wall, the coalescence of lipids into a highly refractive central reserve globule surrounded by a layer of fine-grained cytoplasm.