Isolation of Pythium oligandrum from Egyptian soil and its mycoparasitic effect on Pythium ultimum var. ultimum the damping-off organism of wheat

Pythium oligandrum Drechsler bearing spherical sporangia with complex subglobose elements was isolated for the first time in Egypt from agricultural field soil cultivated with alfalfa (Trifolium alexandrinum) in El-Minia, Egypt. This fungus was found to be an active bio-control agent against P. ultimum var. ultimum, the damping-off organism of wheat. In agar plates, P. oligandrum parasitized P. ultimum var. ultimum hyphae with the aid of thin haustorial branches or infection pegs, eventually leading to host destruction. Incorporation of P. oligandrum into carboxymethylcellulose seed coating successfully eliminated pre-emergence damping-off of wheat caused by P. ultimum var. ultimum, whereas Post-emergence damping-off was prevented by adding inocula of P. oligandrum to the soil. This revised version was published online in June 2006 with corrections to the Cover Date.     

寡雄腐霉施用时期对设施黄瓜霜霉病的防治试验

寡雄腐霉是一种对多种植物病害有很好防治效果的新型生物农药。本文通过田间试验明确了寡雄腐霉在黄瓜霜霉病不同发病时期使用时的防治效果。结果表明,寡雄腐霉对黄瓜霜霉病预防效果达到68.9%,发病初期防治效果达到61.9%,发病后期防治效果只有23.5%。因此,在使用寡雄腐霉防治黄瓜霜霉病时一定要在未发病或发病早期使用才能达到理想的防病效果。     

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.     

Antagonistic actions of Pythium oligandrum and Trichoderma harzianum against phytopathogenic fungi (Fusarium oxysporum and Pythium ultimum var. ultimum)

Abstract

The possible biological control of damping-off fungi, Fusarium oxysporum and Pythium ultimum by Pythium oligandrum or Trichoderma harzianum was in vitro investigated. Results of comparing the antagonistic activity of P. oligandrum and T. harzianum in dual plates against the tested phytopathogens indicated different degrees of antagonism. After 12 days of incubation colony of the phytopathogenic fungus was completely overgrown by the antagonist, except for the interaction between T. harzianum and F. oxysporum which showed no overgrowth or any hyphal penetration by the antagonist. However, growth and proliferation of F. oxysporum colony was repressed. T. harzianum and P. oligandrum produced chitinase and β-1,3-glucanase when they were grown on liquid culture medium supplemented with chitin or fungal dried mycelium as a sole carbon source, and enzyme production was higher by T. harzianum comparing with P. oligandrum under the same condition. Fungal dried mycelium of F. oxysporum was the most selective carbon source for enzyme production, on the other hand, chitinase production was significant locked when P. ultimum dried mycelium was used as a carbon source. Production of volatile compounds by P. oligandrum or T. harzianum against F. oxysporum and P. ultimum was examined using the inverted plates method. F. oxysporum was inhibited by the antagonist volatile compounds and it is inhibited 100% by increasing the amount of inoculum size. Production of potential biocontrol agents provided with economically features and working under field conditions are recommended.     

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.     

瓜果腐霉菌丝中除草活性物质的初步研究

将瓜果腐霉进行液体发酵,其菌丝用等体积乙酸乙酯和甲醇进行依次萃取,甲醇萃取液旋转蒸发去溶剂后进行硅胶柱层析,以乙酸乙酯和石油醚(V/V=3:1和V/V=2:1)的混合液进行梯度洗脱,每50 mL收集为一个馏分,共收集到40个馏分。生物测定结果表明,以乙酸乙酯和石油醚(V/V=2:1)洗脱得到的馏分21 24对供试杂草马唐表现出了较强的活性,其对马唐的生长抑制作用均为4级。合并馏分21 24,以乙酸乙酯和石油醚(V/V=2:1)的混合液为展开剂进行等度洗脱,每50 mL收集为一个馏分,共收集到20个馏分。生物测定结果表明,馏分3对马唐有较强的抑制活性。HPLC分析发现,该馏分主要含有3个组分,其保留时间分别为12.7、14.0和30.5 min。     

Effectiveness of Four New Pyrazole-pyrimidines on Phytopathogens: Ultrastructural Evidences on Pythium ultimum

Four newly synthesized molecules derived from pyrazole-pyrimidine were assayed on Botrytis cinerea Micheli, Fusarium moniliforme Sheld and Pythium ultimum Trow. All proved effective in inhibiting the growth of the phytopathogens at all of the test concentrations (10, 20, 50, 100 μg/ml). The most effective compound was 1-(3)nitrophenyl - 6 - trifluoromethylpyrazolo[3,4 - d ]pyrimidine 4(5H)-thione (CF₃3). Ultrastructural studies on P. ultimum treated with CF₃3 revealed alterations in the normal hyphal shape and, at high concentration, plasmolysis and damage to the wall texture was observed. At 20 μg/ml different vesicles were seen in the cytoplasm: some appeared quite dense, and specific cytochemical reactions indicated that they were most likely peroxysomes; other vesicles seem to be vacuoles of varying content. In some cases there was disintegration of the nuclear envelope. The effects on membrane lipids and interference in protein synthesis are hypothesized as possible mechanism of action of the molecule.     

Influence of Sugar Beet Seed Treatment with Pseudomonas fluorescens and Low Fungicide Doses on Infection with Pythium and Plant Yield and Quality

The effect of sugar beet seed inoculation with the bacterium Pseudomonas fluorescens and treatment with the fungicides Thiram 42‐S and Dithane S‐60 with and without seed inoculation aiming to control the root decay agents Pythium ultimum and Pythium debarianum was studied during a 2‐year trial on two soil types (Mollic Gleysols and Eutric Cambisols). The influence of the treatments on parameters of sugar beet yield and quality such as root yield, sugar content, sugar in molasses, sugar yield as well as percentage of the infected and decayed plants as a consequence of parasitic oomycete infestation will be described.     

Proteins Related to the Biocontrol of Pythium Damping-off in Maize with Trichoderma harzianum Rifai

Induced resistance has been evidenced as one of mechanisms of Trichoderma to control plant diseases, however, no study showed the change of host proteomics in Trichoderma-induced resistance of maize against damping-off caused by Pythium ultimum Trow. The mechanism of Trichoderma harzianum Rifai for controlling maize seedling disease caused by Pythium ultimum Trow was investigated firstly by proteome technique and the result suggested that T. harzianum strain T22 was not only able to promote seedling growth but also protein accumulation. One-dimensional electrophoresis assay showed that more bands appeared on the gel with T22 or T22 combined with P. ultimum （T22 ＋ P. ultimum） treatment than with other treatments. Enzyme assay showed that two chitinases of the root sample were more activated in the treatments with T22 than in the other treatments without T22. Proteins in the seedling roots from the various treatments were separated through protein extraction and 2-D electrophoresis technique. In the seedlings produced from the T22-treated seeds, there were 104 up-regulated proteins and 164 down-regulated proteins relative to the control, and 97 and 150, respectively, aftel treatment with T22 ＋ P. ultimum; however, with P. ultimum alone the values were much lower than with the other two treatments. The correlation coefficient values were 0.72, 0.51 and 0.49 for the comparison of protein spot distribution on gel among control with T22, P. ultimum and T22 ＋ P. ultimum, respectively. So it seemed that P. ultimum infection was more effective than T22 in interfering with the host proteome profile. Furthermore, analysis with MALDITOF-MAS showed that some important proteins associated with defensive reactions were identified in T22 or T22 ＋ P. ultimum treatments, including endochitinase, pathogenesis-related protein PRMS （pathogenesis-related maize seed）, GTP-binding protein, isoflavone reductase and other proteins related to respiration. All those proteins are probably part of the network of resistance or development-related proteins. Interestingly, P. ultimum treatment resulted in elimination of pathogenesis-related protein PRMS on gel, and therefore damping-off could be in part attributed to inhibition of the expression of this protein by P. ultimum infection. Some unknown proteins are also related to the defensive reaction of the host.     

Development of a Defined Medium for Pythium oligandrum Oospore Production

Complex and defined media have been previously proposed for production of oospores of Pythium oligandrum , a fungal mycoparasite of several disease-causing fungi. However, oospore production in synthetic media requires long periods of incubation and yields lower oospore numbers than in complex media. Moreover, although complex media produce high oospore yields, these yields are not reproducible because of the variability in the composition of complex nutrient sources. In the present study, the average composition of molasses reported in the literature was utilized as a base to develop a new defined medium for P. oligandrum oospore production. Forty-two substrates defined in nine stock solutions: carbohydrates, vitamins, sterols, non nitrogenous acids, amino acids, minerals, nucleic acid bases, CaCl 2 and MgSO 4 were tested at two levels (present or absent) in two fractional factorial designs. Each of these nine variables had a significant main effect on oospore production. Furthermore, the effect of each variable, except for vitamins, depended on the level of each other variable, expressed by two-variable interactions. The maximal predicted oospore production was calculated from the polynomial regressions associated with the two fractional factorial designs. Oospore productions were 1.3 and 2 ×10 6 oospores mL -1 from the first and second designs. In order to optimize the oospore production, the two levels of each variable were modified and all variables were experimented in a selection of a complete factorial design (Plackett and Burman design). A fitted first-order polynomial regression equation provided the combination of levels of variables for optimal oospore production. A defined medium, based on this combination of levels, was used for P. oligandrum growth. The optimized oospore production after 7 days growth was 4 ×106 oospores mL -1 as predicted by the polynomial regression. Oospore yields, biomass produced from oospores and oospore freeze-drying tolerance were similar when P. oligandrum was previously grown in molasses or in the new defined medium.     

Pythium species associated with cavity spot on carrots in Norway

Carrot roots with cavity spot lesions from eight different counties in Norway were sampled and Pythium species were isolated on selective medium. Pythium spp. were characterised morphologically and by species-specific PCR. Laboratory experiments with inoculations of carrot roots were performed. A total of 130 isolates out of 230 Pythium -like isolates tested with PCR were identified as pathogenic species of Pythium. These were P. intermedium (29%), P. sulcatum (23%), P. sylvaticum (16%), P. violae (15%) and a possible new Pythium species designated P . ' vipa ' (18%). There were some differences between geographical regions and ages of cavities regarding the frequency of the different species isolated. When rating sunken lesions in the laboratory inoculation experiments, P. ' vipa ' was the most aggressive and P. violae the least aggressive species. P. intermedium and P. ' vipa ' caused more discolouration of the infected carrot tissue than the other species. The importance of the different Pythium spp. as agents of cavity spot in Norway is discussed.     

Effect of Storage on the Survival and Biocontrol Activity of Pythium oligandrum in Pelleted Sugar Beet Seed

Seeds of sugar beet were pelleted with oospores of Pythium oligandrum and stored for 6 years at 8 20IC. Mycelium of P. oligandrum grew from pelleted seed when plated on cornmeal agar (CMA) within 48 h from 100% of seeds stored for 0, 2 and 4 years, and from 93% of seeds stored for 6 years. The germination of oospores removed from pelleted seed immediately after pelleting was 30% on CMA after 18 h of incubation, but storage gradually reduced germination to only 16% after 48 h of incubation for oospores removed from seed stored for 6 years. The biocontrol activity of P. oligandrum -pelleted seed was also tested after 6 years of storage in mixes of soil naturally infested with Pythium spp. and Aphanomyces cochlioides , and sand. P. oligandrum -pelleted seed had no effect in reducing damping-off due to the combined effect of Pythium spp. and A. cochlioides in 5 and 1% (v/v) soil-sand mixtures. However, in the 1% (v/v) soil-sand mixture, P. oligandrum significantly reduced Pythium spp.-induced damping-off from 33 to 26%.     

Production of potato intraspecific somatic hybrids with improved tolerance to PVY and Pythium aphanidermatum

Somatic hybridization can be an interesting alternative for the selection of heterozygous and vigorous potato plants through combination of dihaploid genomes. The resulting hybrids can harbour interesting characters and thus can be used in agriculture if they are in agreement with agronomic criteria.

In this report, we used an intraspecific somatic hybridization technique for the production of tetraploid potato lines. Two parental combinations were used in protoplast electrofusion procedure: Aminca-Cardinal and Cardinal-Nicola. The selection of somatic hybrids was based on in vitro plant vigour. Therefore, among the 75 regenerated plants obtained from Aminca-Cardinal fusion, 3 putative hybrids were retained and 2 plant lines were selected among the 54 regenerated from the Cardinal-Nicola fusion. Heterosis was observed in the larger hybrid tuber size compared to the parents’. Our results also showed a precocity in the in vitro tuberization for the hybrids. Moreover, all of the regenerated putative hybrids were tetraploid (2n=4x=48 chromosomes). Isocitrate dehydrogenase and malate dehydrogenase isoenzyme analyses confirmed the hybrid nature of these lines. A molecular characterization performed by PCR amplification of simple sequence repeats and inter-simple sequence repeats confirmed that all these lines were somatic hybrids.

The effect of potato virus Y infection on these hybrid lines was tested by mechanical inoculation of plants cultivated in a greenhouse. The majority displayed a reduction of infection rate associated with a delayed appearance of symptoms compared to the parents. Moreover, complete resistance was noted for one hybrid line (CN2). All hybrids also showed improved tolerance to Pythium aphanidermatum infection during tuber storage or after plant inoculation.     

An annotated checklist of Pythium species from Iran

The genus Pythium, with slightly over 280 described species has been classified traditionally with other filamentous, coenocytic, sporangia-producing fungi as “Phycomycetes”. However, with recent advances in chemical, ultrastructural and molecular studies Pythium spp. are now considered as “fungus-like organisms” or “pseudo-fungi” and are placed in the kingdom Chromista or kingdom Straminopila, distinct from the true fungi or the kingdom Fungi. Little is known about the biodiversity of Pythium in Iran. This paper attempts to assess the position of the genus Pythium and provides details of the historical development of the study of Pythium in Iran. The survey list contains 33 species, 4 species groups and 1 unknown species of Pythium.     

Transmission of Pythium aphanidermatum to greenhouse cucumber by the fungus gnat Bradysia impatiens (Diptera: Sciaridae)

The fungus gnat (Bradysia impatiens) was examined for its ability to transmit Pythium aphanidermatum to cucumber plants. Larvae that had ingested oospores and mycelium, and then fed on the roots of young cucumber plants growing in rockwool readily introduced the fungus to them. Trans-stadial transmission of oospores from the larval to the adult stage of B. impatiens was demonstrated, although decreasing to a very low level (1.67%) in adults. However, external transmission of P. aphanidermatum on the surface of adults could not be shown. These results suggest that the larval stages of fungus gnats play a role in the dissemination of the fungus between cucumber plants but that adults probably play only a minor role.     

Pythium periplocum, an aggressive mycoparasite of Botrytis cinerea causing the gray mould disease of grape-vine

Pythium periplocum Dreschler has been found to be an aggressive mycoparasite of Botrytis cinerea, the causal agent of the gray mould disease of the grape-vine. When grown together, the former enters the latter's mycelium, branches freely within, coagulates its cytoplasm and finally tears its hyphae apart, bringing about widespread destruction of the grape-vine pathogen. Extensive coiling around the host, as reported in the case of other mycoparasites belonging to the genus Pythium, has not been observed here. The infected mycelium of B. cinerea fails to infect the grape-vine and does not induce the characteristic gray mould symptoms. Since P. periplocum is not a grape-vine parasite, it could be useful for the biological control of B. cinerea. A brief account of this mycoparasitism is discussed in this article.     

Trichoderma species for biocontrol of soil-borne plant pathogens of pasture species

Soil-borne plant pathogens such as Rhizoctonia solani (Kuhn), Pythium ultimum (Trow) and Sclerotinia trifoliorum (Eriks) can reduce grass and forage legume establishment. The potential for biocontrol of these pathogens by Trichoderma fungi was evaluated. Following dual culture assays, nine Trichoderma isolates (five of Trichoderma atroviride and one each of Trichoderma hamatum, Trichoderma koningiopsis, Trichoderma viride and Trichoderma virens) were chosen for assessment in pot experiments. In the presence of R. solani, perennial ryegrass (Lolium perenne L.) emergence was increased by 60–150% by two isolates of T. atroviride and by 35–212% by the isolate of T. virens, with the increase depending on growing medium and amount of pathogen inoculum. Red clover (Trifolium pratense L.) emergence in the presence of S. trifoliorum was significantly increased by two T. atroviride isolates and the T. hamatum isolate. In the presence of P. ultimum, white clover (Trifolium repens L.) emergence was increased by 25–42% by one isolate of T. atroviride and the T. hamatum isolate. However, for all three pasture species, some Trichoderma isolates reduced seedling emergence. Seedling growth (shoot and root fresh weight/plant) of the three pasture species was significantly increased by one or more T. atroviride isolates. On the basis of these results for both disease reduction and growth promotion, four T. atroviride isolates were selected for field assessment as biocontrol agents of soil-borne pathogens of pasture species.     

Development of new formulations of Bacillus subtilis for management of tomato damping-off caused by Pythium aphanidermatum

Formulations of a strain of Bacillus subtilis AUBS-1 inhibitory to the growth of the damping-off pathogen, Pythium aphanidermatum, were developed for seed treatment. The formulations included a talc-based powder, lignite-based powder, lignite+fly ash-based powder, wettable powder, bentonite-paste, polyethylene glycol (PEG) paste and a water-dispersible tablet. Formulations were stored at room temperature for 2 years and frequently sampled to test their shelf life. Populations of bacteria in the formulations were stable for up to 2 years storage at room temperature (28°C). Viability of propagules in lignite, lignite+fly ash, bentonite paste, wettable powder and water dispersible tablet formulations was 100% for up to 1 year. However, the viability of propagules was significantly reduced in talc, wettable powder, PEG paste and tablet formulations beyond 1 year of storage. Seed treatment of tomato with these formulations resulted in effective control of damping-off caused by P. aphanidermatum, and also enhanced plant biomass under glasshouse and field conditions. Active rhizosphere colonization by the bacterium was observed on tomato plants grown from seeds treated with the above formulations.     

Suppression of Pythium ultimum by Biowaste Composts in Relation to Compost Microbial Biomass, Activity and Content of Phenolic Compounds

Seventeen composts from separately collected organic household waste plus one bark compost and one compost from grape marc were analysed for suppression of Pythium ultimum, phytotoxicity, microbial biomass and activity, substrate-induced respiration, extractible phenolic compounds and other physical and chemical parameters. Nine of the samples were mildly suppressive to P. ultimum, the others were conducive. The bark compost sample was strongly suppressive. Therefore of the examined composts, only the bark could be used to exert an economically relevant control of P. ultimum in horticultural media. A large part of the compost samples was slightly phytotoxic. Microbial biomass and SIR had only weak correlations with disease incidence. Microbial activity and content of extractible phenolics were positively correlated with disease incidence. None of the tested parameters were therefore suitable as a predictive test for suppression of P. ultimum with the compost samples used in this study.