Biological control of Pythium ultimum–induced damping-off by treating cress seed with the mycoparasite Pythium oligandrum

Incorporation of the aggressive mycoparasite Pythium oligandrum into a carboxymethyl cellulose-based seed coating decreased damping-off of cress seedlings by Pythium ultimum in both naturally infested soil and artificially infested sand. This effect is attributed to the germination of P. oligandrum oospores on the seed surface with subsequent generation of a protectant mycelium around the seedling.
P. oligandrum oospores survived storage periods of 10—20 weeks at zero relative humidity both on glass slides and within seed coatings. The mycoparasite also survived an 18–month burial in natural soil, probably in the form of oospores.
The implications of these observations for biocontrol of seedling diseases by P. oligandrum are discussed.     

Use of fungal antagonists for biocontrol of damping-off and sclerotinia diseases

Two mycoparasites, Pythium oligandrum and Coniothyrium minitans, have been tested for their ability to act as disease biocontrol agents. P. oligandrum oospores, grown in a cane molasses liquid medium and coated onto cress and sugar-beet seeds using commercial thin-film or pelleting techniques, gave significant control of damping-off in cress and sugar-beet caused by Pythium ultimum and Aphanomyces cochlioides respectively, in glasshouse pot trials. In some cases, the control was equivalent to fungicide drenches or standard fungicide seed treatments, but little control was achieved with any treatment when the pathogen inoculum potential in the soil was high. Pre-planting application of a solid substrate preparation of C. minitans gave reproducible control of sclerotinia disease in the glasshouse. The degree of control was equivalent to that achieved with regular foliar sprays of vinclozolin, when there was less than 40% disease in the control plots naturally infested with Sclerotinia sclerotiorum. At higher disease levels biocontrol was lost. Nevertheless, C. minitans survived in the soil for over one year and continued to degrade sclerotia and reduce apothecial production. The commercial potential of these biocontrol agents is discussed.     

Factors affecting the control of Pythium ultimum damping-off of sugar beet by Pythium oligandrum

Application of Pythium oligandrum to a soil-based compost as a mycelial suspension (5 × 10² CFU g⁻¹ of dry compost) and oospore alginate pellets (10⁵ oospores/g of dry compost) controlled pre- and postemergence damping-off of sugar beet caused by Pythium ultimum to a level similar to metalaxyl seed treatment. Oospore seed treatments and aqueous suspensions of oospores applied to compost failed to control disease. Problems in the use of P. oligandrum oospore inocula for the control of damping-off were highlighted. It was shown that treatment of oospores with cellulase (20 g L⁻¹) increased germination approximately three-fold in comparison to untreated spores. Untreated and cellulase pretreated oospores were subsequently evaluated as seed treatments for their ability to control damping-off of sugar beet. The highest rate of pretreated oospores (10⁴ oospores/seed) gave levels of emergence and establishment in infested compost that were not significantly different from the uninfested controls, whereas seed treatment with untreated oospores gave no significant reduction in disease. In a trial carried out in a controlled environment to assess the effect of pH (4.5–8.0), P. oligandrum (10⁴ cellulase pretreated oospores/seed) was shown to control pre- and postemergence damping-off of sugar beet at pH 7.0 and 7.5 only.     

Pelleting of seed with the antagonist Pythium oligandrum for biological control of damping-off

Pythium oligandrum oospores, incorporated in clay carrier, survived a commercial seed-pelleting process to give protection against damping-off in sugar beet and cress induced by Pythium ultimum , together with damping-off in carrot caused by Mycocentrospora acerina.
It is suggested that field scale evaluation of P. oligandrum as a biocontrol of fungally incited seedling diseases is now both feasible and desirable.     

Biological control of damping-off of sugar beet by Pseudomonas putida applied to seed pellets

Pseudomonas putida 40RNF applied to seed pellets reduced the occurrence of Pythium damping-off of sugar beet. A density of 6 × 10⁷ 40RNF per pellet reduced Pythium damping-off from 70 to 26% when seeds were sown in artificially infested soil (250 propagules Pythium ultimum per g dry soil). The efficacy of 40RNF was dependent on its density in the seed pellet (in the range 2 × 10⁴–6 × 10⁸ per pellet) and on the number of propagules of Pythium in soil. 40RNF declined to or stabilized at approximately 1 × 10⁶ per pellet 3 days after planting, and this was independent of the inoculum density. This indicated that the crucial steps resulting in damping-off of sugar beet caused by Pythium ultimum must occur within 3–4 days of sowing. 40RNF reduced pericarp colonization by P. ultimum by 43% 48 h after planting and caused a 68% decrease in the number of sporangia of P. ultimum in the surrounding soil (0.0–5.0 mm). P. putida 40RNF also reduced pre and post-emergence damping-off (from 69.5 to 37.5%) caused by indigenous populations of Pythium species in an infested soil and this was as effective as the fungicide hymexazol (69.5 to 40%).     

Control of fungal pathogens — preharvest Use of coniothyrium minitans and pythium oligandrum as disease biocontrol agents

When disease levels are low,Coniothyrium minitans gives biological control of Sclerotinia disease in lettuce andPythium oligandrum control of damping-off in cress and sugar beet equivalent to standard fungicide treatments, but both fail as the inoculum potential of the pathogens increases. Possible approaches for improving the use and efficacy of these biocontrol agents are discussed.     

Rhizosphere Competence of Pythium oligandrum

ABSTRACT The associations of Pythium oligandrum with the root cortex, rhizoplane, and rhizosphere were measured with 11 crop species. This work was expedited by the use of a semiselective technique for isolation of P. oligandrum from soil and plant material. Cortical colonization of roots by P. oligandrum was not detected, and the rhizoplanes of the roots of most crops were free of the fungus. However, P. oligandrum was detected in large quantities with every crop tested when roots with adhering soil (rhizosphere soil) were assayed. Different crop species and cultivars of cantaloupe, cauliflower, and tomato varied in rhizosphere densities of P. oligandrum, but rhizosphere population densities of the fungus were consistently higher than in nonrhizosphere soils with plants grown in P. oligandrum-infested sterilized potting mix or an unsterilized mineral soil. After transplanting tomatoes into potting mix infested with P. oligandrum, increases in CFU occurred over time in the rhizosphere but not in the nonrhizosphere soil. In trials on delivery methods of inoculum of P. oligandrum, the rhizosphere populations of tomato plants grown in potting mix were about sixfold higher compared to seed-coat treatments when ground, alginate pelleted oospores were applied to seedlings growing in plug containers prior to transplanting or to pots containing potting mix before direct seeding.     

Biological control of damping-off of sugar beet by Pseudomonas putida applied to seed pellets

Pseudomonas putida 40RNF applied to seed pellets reduced the occurrence of Pythium damping-off of sugar beet. A density of 6 × 10⁷ 40RNF per pellet reduced Pythium damping-off from 70 to 26% when seeds were sown in artificially infested soil (250 propagules Pythium ultimum per g dry soil). The efficacy of 40RNF was dependent on its density in the seed pellet (in the range 2 × 10⁴–6 × 10⁸ per pellet) and on the number of propagules of Pythium in soil. 40RNF declined to or stabilized at approximately 1 × 10⁶ per pellet 3 days after planting, and this was independent of the inoculum density. This indicated that the crucial steps resulting in damping-off of sugar beet caused by Pythium ultimum must occur within 3–4 days of sowing. 40RNF reduced pericarp colonization by P. ultimum by 43% 48 h after planting and caused a 68% decrease in the number of sporangia of P. ultimum in the surrounding soil (0.0–5.0 mm). P. putida 40RNF also reduced pre and post-emergence damping-off (from 69.5 to 37.5%) caused by indigenous populations of Pythium species in an infested soil and this was as effective as the fungicide hymexazol (69.5 to 40%).     

Biological control of damping-off caused byPythium ultimum andRhizoctonia solani usingTrichoderma spp. applied as industrial film coatings on seeds

Conidia of sevenTrichoderma strains were applied on cucumber or radish seeds as a simple methyl cellulose coating or through an industrial film coating process. The seeds were sown in a peat-based soil artificially infested byR. solani orP. ultimum. Four strains controlled damping-off caused byR. solani when applied as a simple coating or as an industrial film-coating. Also, four strains significantly reduced damping-off caused byP. ultimum in cucumber. A correlation was found between production of volatile antibiotics in vitro and control ofP. ultimum. Survival during storage varied according to the strain. Better survival was observed for two strains, with a decrease in conidial viability of one order of magnitude after storage for three and five months at 15 ° C and 4 ° C, respectively. The results show the feasibility of biocontrol of seedling diseases by some antagonists applied onto seeds through an industrial film-coating process.     

The influence of two herbicides on the antifungal activity of some fungicides against Pythium butleri and Rhizoctonia solani causing damping-off of cowpea

The herbicides fluchloralin and alachlor applied to soil altered the effectiveness of fungicide treatments to seed and soil for controlling cowpea damping-off. These herbicides also modified the in-vitro toxicity of the fungicides to the mycelial growth of Pythium butleri and Rhizoctonia solani in a nutrient medium. Both herbicides reduced the toxicity of 2-methoxyethylmercury chloride (MEMC) and propamocarb to the growth of P. butleri, and of carbendazim to the growth of R. solani, but enhanced the toxicity of captafol and quintozene to P. butleri and R. solani, respectively. In pot tests, quintozene gave better control of R. solani damping-off in soil treated with fluchloralin or alachlor than in untreated soil, whereas disease control by carbendazim was decreased in similarly treated soils. Both herbicides attenuated the effectiveness of MEMC and captafol to control the damping-off caused by P. butleri; the efficacy of propamocarb was increased by alachlor but was decreased by fluchloralin. The implications of herbicide-fungicide interactions are discussed in the context of fungicidal control of root diseases in herbicide-treated soil.     

The incidence of Pythium spp. and Aphanomyces cochlioides associated with the sugar-beet growing soils of Britain

In a survey of fungi causing seedling diseases of sugar beet using a soil bioassay, Aphanomyces cochlioides and Pythium spp. were found to occur in 39% and 31%, respectively, of 341 sugar-beet fields selected in a stratified random sample in England, The frequency of A. cochlioides-infested soils varied widely in the different sugar-beet growing areas of the country. Soil pH was the single factor most strongly associated with the distribution of the pathogen, but regression models applied to combinations of factors indicated that soil texture and the interval between sugar-beet crops were also relevant to its frequency. It was detected less often in soils of high pH (≥ 75). heavy texture and where the interval between sugar-beet crops exceeded 5 years. No significant associations were found between the proportion of soils with Pythium spp. and soil or cropping factors.     

Cotton Seedling Preemergence Damping-Off Incited by Rhizopus oryzae and Pythium spp. and Its Biological Control with Trichoderma spp

ABSTRACT Planting the cotton cv. Sure-Grow 747 in cotton seedling disease plots during the 2001 growing season resulted in high levels of preemergence damping-off among the seedlings. Four cotton pathogens, Pythium aphanidermatum, P. ultimum, an unidentified Pythium sp., and Rhizopus oryzae, were isolated from diseased seed embryos and seedlings. Disease incited by the Pythium spp. could be controlled by seed treatment with Metalaxyl, but disease incited by R. oryzae could not. Seed treatment with Metalaxyl in naturally infested field soil was only partially effective; therefore, symptoms in 47% of the diseased seedlings could be attributed to R. oryzae. Susceptibility to disease appeared to be related to release in the spermosphere, by the germinating seeds, of compounds that stimulate pathogen propagule germination, because exudates from seed of the suscept Sure-Grow 747 and extracts from wheat bran induced pathogen germination and growth, whereas exudates from resistant cv. Stoneville 213 did not. However, even Stoneville 213 became susceptible when infested soil was amended with wheat bran. Seed treatment with preparations of Trichoderma virens parent, mutant, and hybrid strains gave effective biological control of preemergence damping-off. Disease control was attributable to metabolism by the biocontrol agent of pathogen germination stimulants released by the seed, because amendment of pathogen-infested soil with the propagule germination stimulants in wheat bran negated the protective effect of the seed treatment.     

Colonization of Pythium oligandrum in the tomato rhizosphere for biological control of bacterial wilt disease analyzed by real-time PCR and confocal laser-scanning microscopy

It recently has been reported that the non-plant-pathogenic oomycete Pythium oligandrum suppresses bacterial wilt caused by Ralstonia solanacearum in tomato. As one approach to determine disease-suppressive mechanisms of action, we analyzed the colonization of P. oligandrum in rhizospheres of tomato using real-time polymerase chain reaction (PCR) and confocal laser-scanning microscopy. The real-time PCR specifically quantified P. oligandrum in the tomato rhizosphere that is reliable over a range of 0.1 pg to 1 ng of P. oligandrum DNA from 25 mg dry weight of soil. Rhizosphere populations of P. oligandrum from tomato grown for 3 weeks in both unsterilized and sterilized field soils similarly increased with the initial application of at least 5 x 10(5) oospores per plant. Confocal microscopic observation also showed that hyphal development was frequent on the root surface and some hyphae penetrated into root epidermis. However, rhizosphere population dynamics after transplanting into sterilized soil showed that the P. oligandrum population decreased with time after transplanting, particularly at the root tips, indicating that this biocontrol fungus is rhizosphere competent but does not actively spread along roots. Protection over the long term from root-infecting pathogens does not seem to involve direct competition. However, sparse rhizosphere colonization of P. oligandrum reduced the bacterial wilt as well as more extensive colonization, which did not reduce the rhizosphere population of R. solanacearum. These results suggest that competition for infection sites and nutrients in rhizosphere is not the primary biocontrol mechanism of bacterial wilt by P. oligandrum.     

Extraction of oospores of Peronospora viciae from soil

A method for the extraction of oospores of Peronospora viciae from soil is described. Approximately 75% of the oospores added to silty clay loam and loamy sand soil samples were recovered. Percentage recovery was independent of oospore density. This extraction method did not affect viability as determined by the tetrazolium bromide test and a germination assay and may therefore be used to study survival of oospores. Numbers of oospores extracted from soil samples taken from seven fields naturally infested with oospores of P. viciae f.sp. pisi ranged from 2 to 21 oospores per g soil. Oospore density was not significantly correlated with disease incidence as determined by a bioassay.     

Role of oospores as primary inoculum for epidemics of downy mildew caused by Peronospora arborescens in opium poppy crops in Spain

This study investigated the role of Peronospora arborescens oospores as primary inoculum for downy mildew of opium poppy and infection types that they may give rise to in Spain using an integrative experimental approach that combined pathogenicity tests in growth chambers and field microplots, together with molecular detection of P. arborescens infection by specific nested-PCR assays. The results demonstrated that oospores in infested soil or leaf debris were effective inoculum for ingress of the pathogen through underground plant tissues early in poppy seedling growth. This gave rise to systemic infections that reproduced the stunting, chlorotic syndrome frequently observed in affected plants in commercial fields. Additionally, infection of underground tissues of older plants by oospore inoculum could remain asymptomatic. Results also suggested that sporangia formed on infected plants are effective in producing secondary local infections that later may become systemic and either symptomatic or asymptomatic. Finally, and more importantly, those delayed symptomatic or asymptomatic systemic infections, as well as secondary local infections of capsules, can give rise to infected seeds. This research on the biology of P. arborescens on poppy plants and epidemiology of downy mildew may help to develop knowledge-based disease-management strategies of use in the protection of yields of opium poppy crops in Spain and elsewhere.     

Molecular characterization and pathogenicity of Pythium species associated with damping-off in greenhouse cucumber (Cucumis sativus) in Oman

A study was undertaken in 2004 and 2005 to characterize pathogens associated with damping-off of greenhouse-grown cucumber seedlings in 13 districts in Oman. Identification of Pythium to the species level was based on sequences of the internal transcribed spacer (ITS) of the ribosomal DNA. Of the 98 Pythium isolates collected during the survey, Pythium aphanidermatum , P. spinosum , P. splendens and P. oligandrum accounted for 76%, 22%, 1% and 1%, respectively. Pythium aphanidermatum was isolated from all of the districts, while P. spinosum was isolated from seven districts. Pathogenicity tests showed inter- and intraspecific variation in aggressiveness between Pythium species. Pythium aphanidermatum , P. spinosum and P. splendens were found to be highly aggressive at 25°C. However, the aggressiveness of P. spinosum decreased when the temperature was raised to 30°C, which was found to correspond to the lower frequency of isolation of P. spinosum in the warmer seasons, compared to the cooler time of the year. Pythium aphanidermatum exhibited limited intraspecific variation in the sequences of the ITS region of the rDNA and showed 100% similarity to the corresponding P. aphanidermatum sequences from GenBank. The ITS sequence data, as well as morphological characteristics of P. spinosum isolates, showed a high level of similarity within and between P. spinosum and P. kunmingense , and suggested that the two species were synonymous. This study represents the first report of P. spinosum, P. splendens and P. oligandrum in Oman.     

4种杀菌剂对瓜果腐霉病菌的室内毒力及田间防效测定

采用菌丝生长速率法测定了乙酸铜、噁霉灵、霜霉威、代森锰锌4种杀菌剂对瓜果腐霉病菌(Pythiumaphanidermatum)的毒力,各药剂的EC50值依次为:30.292 3、5.891 7、34.331 9、65.982 6μg/mL。可见噁霉灵30%水剂对瓜果腐霉病菌表现出较强的毒力。对4种杀菌剂还进行了田间黄瓜猝倒病的防治试验,其防效依次为83.65%、80.14%、80.91%、42.48%。根据室内毒力测定和田间药效试验结果,乙酸铜20%可湿性粉剂、噁霉灵30%水剂、霜霉威盐酸盐722克/升水剂对黄瓜猝倒病均具有较高的防效。     

Survival Dynamics of Aphanomyces cochlioides Oospores Exposed to Heat Stress

ABSTRACT To determine how exposure to heat effects their survival, oospores of Aphanomyces cochlioides isolate C22 were exposed in water to 35, 40, 45, or 50 degrees C for prescribed times and then examined for viability. The Weibull model was modified to represent the effects of temperature on survival of oospores. The final fitted model gave lethal doses for 50% of the oospores of 251, 49.8, 9.8, and 1.9 h at 35, 40, 45, and 50 degrees C, respectively. To determine if alternating high and low temperatures resulted in (i) recovery from heat damage during low temperature periods, (ii) increased susceptibility to heat damage, or (iii) if effects of heat damage were cumulative, oospores were examined after each of four 24-h cycles at 45 degrees C for 4 h and 21 degrees C for 20 h. Survival of oospores exposed to alternating high and low temperatures fit the cumulative effects model. Significant variability in heat tolerance among five isolates was observed (P< 0.001) but model parameters successfully accommodated this variability (R(2) = 0.96, P < 0.001). This research shows that under wet conditions, there are predictable patterns to mortality for A. cochlioides oospores exposed to continuous or fluctuating high temperatures.     

Interactions Between the Mycoparasite Pythium oligandrum and Sclerotia of the Plant Pathogen Sclerotinia sclerotiorum

Pythium oligandrum Drechsler is a mycoparasite which parasitizes hyphae of many fungal species. A detailed study of the interactions between P. oligandrum and the sclerotia of the plant pathogen Sclerotinia sclerotiorum (Lib.) de Bary is presented. Pythium oligandrum was present in Danish soils at concentrations between 4 and 26 cfu g^–1 soil. An increase in the natural population of P. oligandrum by addition of P. oligandrum zoospores to a soil reduced the ability of sclerotia of S. sclerotiorum to germinate myceliogenically and the sclerotia were colonized internally by P. oligandrum. This colonization and reduction of germination of sclerotia were also seen when sclerotia and P. oligandrum were incubated together in water. Small sclerotia were significantly more susceptible to parasitism by P. oligandrum than large sclerotia, and increasing the incubation time caused a further reduction in the germination ability of the sclerotia. P. oligandrum was able to pass through its entire life-cycle from zoospores to oogonia both with sclerotia as sole nutrient-source and in water containing exudates from the sclerotia. The cell wall degrading enzymes N-acetyl--D-glucosaminidase (NAGase), endo-chitinase, protease, -glucanase, -glucosidase and cellobiohydrolase were detected in culture filtrates of P. oligandrum cultivated with S. sclerotiorum. These findings suggest that P. oligandrum has a potential to reduce the survival of S. sclerotiorum sclerotia present naturally in soils, through mycoparasitic activity.