Integration of Pythium nunn and Trichoderma harzianum isolate T-95 for the biological control of Pythium damping-off of cucumber

Two biological control agents, Pythium nunn and Trichoderma harzianum isolate T-95, were combined to reduce Pythium damping-off of cucumber in greenhouse experiments lasting 3–4 weeks. T. harzianum T-95, a rhizosphere competent mutant, was applied to seeds and P. nunn was applied to pasteurized and raw soils naturally and artificially infested with Pythium ultimum. Some treatments were also amended with bean leaves to enhance the activity of P. nunn. The biological control of Pythium damping-off was evaluated in a Colorado soil (Nunn sandy loam) and an Oregon soil mix, which were replanted twice after 2 and 3 months. Interactions between P. nunn and T-95 were detected in the Colorado but not the Oregon soil. No consistent evidence of antagonism between P. nunn and T. harzianum was seen, and significant interactions were detected in the Colorado, but not the Oregon soil. In the first planting of some treatments, the combination of P. nunn and T. harzianum gave greater control of damping-off than either applied alone. P. nunn was most effective in soils that were pasteurized or amended with bean leaves. T. harzianum controlled Pythium damping-off in the Colorado, but not the Oregon soil. In both soils, disease declined over time in treatments amended with bean leaves but without P. nunn or T. harzianum added. This suppression was greater in the Colorado soil, which contained an indigenous population of P. nunn. This work demonstrates that two compatible biological control agents can be combined to give additional control of a soil-borne plant pathogen.     

Use of Trichoderma harzianum cultured on ground mesocarp fibre of oil-palm as seed treatment to control Pythium aphanidermatum,a causal agent of damping-off of Chinese kale seedling

Trichoderma harzianum, isolate T 01-22, was cultured on either sorghum grains, ground mesocarp fibre of oil-palm or oil-palm shell, both amended with urea fertilizer (100:1, w/w). T. harzianum cultured on ground mesocarp fibre was then used to coat seeds of Chinese kale (Brassica alboglabra Bailey) to control damping-off of seedlings caused by Pythium aphanidermatum. Biomass of T. harzianum cultured on ground mesocarp fibre of oil-palm was more effective than Captan and Benomyl, but less effective than Metalaxyl, in controlling damping-off of Chinese kale seedlings. Viability of T. harzianum growing on sorghum grains was reduced significantly during 7 months storage, followed by that of T. harzianum cultured on ground mesocarp fibre and oil-palm shell, both amended with urea fertilizer (46-0-0) at 100:1 (w/w).     

Potential for integrated biological and chemical control of damping-off disease caused by Pythium ultimum in tomato

Pythium ultimum (Trow) is one of the main causes of damping-off disease in many parts of the world. Control of the disease depends mainly on application of chemical fungicides. However, soil treatments with fungicides are not always feasible due to economical and ecological reasons. Soil-borne, non-pathogenic bacteria of the genus Pseudomonas fluorescens with the ability to antagonise fungal phytopathogens, represent a realistic alternative to chemical fungicides and show great promise with respect to protect plant roots from fungal-induced diseases. In an attempt to find an integrated control system of damping-off disease in tomato, fungicides including azoxystrobin, metalaxyl-M and pyraclostrobin were applied alone and in combination with P. fluorescens isolate CW2. The fungicides were tested in in vitro for their antagonistic potential against P. ultimum and for compatibility with CW2. It was found that the fungicides were fungitoxic to P. ultimum, but did not inhibit the growth of the P. fluorescens. The efficacy of the fungicides alone and in combination with CW2 was also tested in greenhouse experiments against damping-off disease on tomato. Two concentrations (5 and 10?μg?ml^?1) were applied. Damping-off incidence of tomato seedlings in Humosoil^?:sand mixture infested with P. ultimum was reduced following seed treatment with the fungicides. However, the degree of control obtained varied significantly depending on the fungicide used. Combined seed treatment with P. fluorescens and the fungicides resulted in a significant improvement in disease control and improved plant growth as indicated by shoot and root dry weights. Metalaxyl-M treatment applied alone or in combination with P. fluorescens, significantly protected tomato seedlings against damping-off. Strobilurin fungicides stimulated plant growth compared to metalaxyl-M. Combined treatment of tomato seeds with strobilurin fungicides and CW2 showed a moderate to good disease control and an increase in shoot and root dry weights.     

Mutation of rpiA in Enterobacter cloacae decreases seed and root colonization and biocontrol of damping-off caused by Pythium ultimum on cucumber

Strains of Enterobacter cloacae show promise as biocontrol agents for Pythium ultimum-induced damping-off on cucumber and other crops. E. cloacae A145 is a mini-Tn5 Km transposon mutant of strain 501R3 that was significantly reduced in suppression of damping-off on cucumber caused by P. ultimum. Strain A145 was deficient in colonization of cucumber, sunflower, and wheat seeds and significantly reduced in colonization of corn and cowpea seeds relative to strain 501R3. Populations of strain A145 were also significantly lower than those of strain 501R3 at all sampling times in cucumber, wheat, and sunflower rhizosphere. Populations of strain A145 were not detectable in any rhizosphere after 42 days, while populations of strain 501R3 remained at substantial levels throughout all experiments. Molecular characterization of strain A145 indicated mini-Tn5 Km was inserted in a region of the E. cloacae genome with a high degree of DNA and amino acid sequence similarity to rpiA, which encodes ribose-5-phosphate isomerase. In Escherichia coli, RpiA catalyzes the interconversion of ribose-5-phosphate and ribulose-5-phosphate and is a key enzyme in the pentose phosphate pathway. Ribose-5-phosphate isomerase activity in cell lysates from strain A145 was approximately 3.5% of that from strain 501R3. In addition, strain A145 was a ribose auxotroph, as expected for an rpiA mutant. Introduction of a 1.0-kb DNA fragment containing only the rpiA homologue into strain A145 restored ribose phosphate isomerase activity, prototrophy, seedling colonization, and disease suppression to levels similar to those associated with strain 501R3. Experiments reported here indicate a key role for rpiA and possibly the pentose phosphate pathway in suppression of damping-off and colonization of subterranean portions of plants by E. cloacae.     

Association of Peronospora parasitica with Albugo candida on Brassica juncea Leaves

Associated infections between Peronospora parasitica and Albugo candida were observed on Brassica juncea leaves. A. Candida appeared first and was followed by P. parasitica. A. candida predisposes the host tissues towards susceptibility to P. parasitica.     

Isolation,identification, carbon utilization profile and control of Pythium graminicola,the causal agent of chilli damping-off

Postemergence damping-off of chilli caused by Pythium spp. is a common and serious problem in large chilli growing areas of India under the moist conditions that generally prevails during the sowing period. Therefore, in order to better understand this disease, an isolate belonging to the genus Pythium (Pythiales) was isolated from the infected chilli (Capsicum annuum L.) plant root parts collected from the fields of Chandauli district, Uttar Pradesh, India. Based on the congruence of cultural, morphological, cardinal growth rate and the sequence data analysis, the isolate was identified as Pythium graminicola. The molecular phylogenetic analysis based on ITS-rDNA sequences clustered the isolate with representative sequences for P. graminicola from GenBank in the Pythium clade. The isolate carbon utilization profiles were characterized using Biolog FF MicroPlate method. The results revealed that the isolate used a wide range of carbon sources, mainly carbohydrates, but also amino acids, suggesting the use of metabolic routes that include glycolysis/gluconeogenesis. Moreover, an in vitro colony growth inhibition assay was performed to determine the influence of chemical (fungicides) and biological (bacteria and fungi) antagonists over the pathogen using the poison plate and dual culture method, respectively. Overall, the results revealed that the presence of aggressive broad range biocontrol agents can be used as an effective environmentally friendly approach for management and control of damping-off in production systems. The antagonist can serve as a bio-efficient and eco-friendly alternative to synthetic fungicides for the development of an effective integrated pest management (IPM) system and obtaining higher yields.     

Biological control of cucumber and sugar beet damping-off caused by Pythium ultimum with bacterial and fungal antagonists

AIMS: Five bacterial strains belonging to Bacillus subtilis, Pseudomonas fluorescens and Ps. corrugata and two fungal strains belonging to Trichoderma viride and Gliocladium virens were evaluated for their efficacy in controlling sugar beet and cucumber damping-off caused by Pythium ultimum. METHODS AND RESULTS: The in vitro antagonistic activity of bacteria against various Pythium spp. was evaluated with dual cultures in various media. Pseudomonas strains inhibited the pathogen better than Bacillus strains. To identify potentially useful antagonist combinations, dual compatibility of antagonists was also evaluated, based on growth in two liquid media containing substrate previously used by other antagonists. Four pairs of bacteria were selected. Sugar beet damping-off biocontrol was attempted with bacterial seed treatments (individually and in pairs). Cucumber damping-off biocontrol was attempted with bacterial seed treatments and bacterial and fungal compost treatments. In sugar beet, satisfactory biocontrol was only achieved with Pseudomonas antagonists. Antagonist combinations did not show any superior biocontrol ability to individual antagonists and compatibility of bacteria in vitro did not correlate with compatibility in vivo. Bacterial seed treatments and fungal compost treatments failed to control cucumber damping-off. Better biocontrol in cucumber was achieved when bacterial antagonists were applied by drenching or by coating seed with bacteria in a peat carrier. CONCLUSIONS: Pseudomonas antagonists were superior to Bacillus antagonists in controlling damping-off in cucumber and sugar beet. Pseudomonas peat inocula maintained a good shelf-life 2 years after preparation. SIGNIFICANCE AND IMPACT OF THE STUDY: Pseudomonas peat formulations have the potential for development into commercial biopesticides.     

Effects of Temperature on Germination of Peronospora parasitica Conidia and Infection of Brassica oleracea

The effect of temperature on germination of a South African isolate of Peronospora parasitica , and infection of Brassica oleracea was studied. The optimum condition for germination was 20° C at 100% relative humidity. The percentage germination obtained was 80–98% and 70–80% between 15 and 25° C at 100% relative humidity, after a 12 and 6h incubation period, respectively. Optimum temperature for germ tube growth was also 20° C. The temperature range for maximum infection of seedlings of a highly susceptible cabbage cultivar and subsequent disease development in vitro was 15–25° C and 90–100% infection was achieved after 48 h of incubation. At<15°C and 26–30° C infection percentage was decreased to 40–50% and 35–40%, respectively. No disease incidence was recorded at temperatures above 35° C. A scanning electron microscope study of the infection process showed that penetration of cotyledons by germ tubes was mostly via stomata and occasionally directly through the cuticle. Results are discussed in relation to the need for future studies of P. parasitica in South Africa.     

大白菜霜霉病菌寄生霜霉孢子囊的保藏方法

通过离体子叶冷冻法、离体叶片冷冻法和10%二甲基亚砜+5%脱脂乳冷冻法等3种方法来保藏大白菜霜霉病菌寄生霜霉,并采用离体子叶接种法测定孢子囊的致病力,以筛选出一种较好的保藏方法。结果表明,保藏6个月后,离体子叶冷冻法和离体叶片冷冻法保藏的孢子囊的萌发率、发病率和病情指数均较高;而10%二甲基亚砜+5%脱脂乳冷冻法保藏6个月后仅有2.16%的孢子囊能够萌发,菌株致病力丧失,发病率和病情指数均为0。保藏12个月后,离体子叶冷冻法保藏效果最好,孢子囊萌发率达到62.22%,发病率为90%,病情指数为48.89;离体叶片冷冻法保藏的孢子囊的致病力较弱;10%二甲基亚砜+5%脱脂乳冷冻法保藏12个月的孢子囊全部失活。此外,采用离体子叶冷冻法保藏12个月的32株寄生霜霉复壮成功率达到了93.75%。离体子叶冷冻法适用于寄生霜霉的保藏,其孢子囊可长时间保持较高的萌发率。     

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.     

cDNA-AFLP display for the isolation of Peronospora parasitica genes expressed during infection in Arabidopsis thaliana

To identify genes from the obligatory biotrophic oomycete Peronospora parasitica that are expressed during infection in Arabidopsis thaliana we employed cDNA-amplified fragment length polymorphism (AFLP) display. cDNA-AFLP fragments from infected and non-infected leaves were separated in parallel by gel electrophoresis and displayed by autoradiography. Most differential gene fragments were derived from P. parasitica.     

Powder formulation of Burkholderia cepacia for control of rape seed damping-off caused by Rhizoctonia solani

Talc-based formulation of Burkholderia cepaci strain Bu1 was tested as seed and soil drenchs separately for its ability to control Rhizoctonia soloni the causal agent of rape seed damping-off in greenhouse and field trials. In general, the formulated bacteria was more effective to suppress the disease than the suspension of bacteria cells in carboxymethylcellulose solution (1% w/v), in both greenhouse and field trials. The formulation of strain Bul as soil and seed treatments had the greatest effect on reducing the rape seed damping-off in greenhouse and field trials (66.7, 53.3, 64.4 and 40% respectively). The formulation of strain Bu1 as soil and seed treatments were the most effective treatments to increase the root dry weights in the infected soil in greenhouse. The formulation of strain Bul as soil drench had the greatest effect on enhancement of the fresh weight of roots and stem fresh and dry weights. The formulation of strain Bu1 stored at 4 degrees C exhibited better shelf Life and efficacy in vitro than it's counterpart stored at 25 degrees C.     

Epicoccum Nigrum as biocontrol agent of Pythium damping-off and root-rot of cotton seedlings

Efficacy of Epicoccum nigrum and its exudate was tested in control the pre- and post-emergence damping-off and root-rot of Egyptian cotton (cv. Giza 83) in vitro and in vivo. Different isolates of Epicoccum nigrum reduced the radial growth of both Pythium debaryanum and P. ultimum significantly, by production considerable inhibition zones. In liquid cultures E. nigrum exudate showed a high fungicidal effect resulting in a significant reduction of the mycelial dry weight of the two investigated Pythium spp. Also E. nigrum exudate inhibited cellulase and pectinase activity by P. debaryanum and P. ultimum. Soaking of cotton seeds in E. nigrum exudate for different intervals resulted in significant reduction of root-rot severity of seedlings as well as the contamination of seeds and seedlings by fungi during and after germination. These treatments also stimulated germination of cotton seeds and enhanced the seedlings vigour significantly. In pot experiments, the use of E. nigrum as a soil mixture or seed dressing significantly alleviated the hazard effect of P. debaryanum. Pythium ultimum seemed to be weak or non pathogen to the used cotton cultivar (Giza 83). Application of E. nigrum or its exudate not only involved in protection of cotton seedlings against Pythium damping-off and root-rot but also enhanced their vigour and growth characteristics. The main conclusion of this study is that E. nigrum could be used successfully as environmentally safe and economic biological control agent to protect cotton (cv. Giza 83) from damping-off and root-rot diseases caused by P. debaryanum.     

寄生霜霉Peronospora parasitica(Pers.)Fr.孢子囊萌发方式的观察

<正>寄生霜霉Peronospora parasitica(Pers.)Fr.主要为害十字花科植物,引起许多蔬菜及油菜的霜霉病,常造成严重损失,寄主范围较广,常见寄主有油菜、甘蓝、花椰菜、白菜、芥菜、萝卜等,是霜霉属最重要的种之一(陆家云2001;张中义等1986)。侵染白菜引起白菜霜霉病。白菜小苗被害时,初在叶背     

Enhanced activity of introduced biocontrol agents in solarized soils and its implications on the integrated control of tomato damping-off caused by Pythium spp

Soil solarization in combination with introduction of biocontrol agents (BCA) was evaluated as a potential disease management strategy for tomato damping-off caused by Pythium spp. A rifampicin resistant Pseudomonas fluorescens strain (PfT-8) and a carbendazim resistant Trichoderma harzianum strain (ThM-1) were introduced into soil following solarization. Tomato seeds were planted into treated field plots. The influence of soil solarization and application of biocontrol agents on damping-off incidence, plant biomass, rhizosphere population of introduced antagonists, and native Pythium spp. was assessed by two consecutive field trials. Damping-off incidence was significantly reduced in solarized plots compared to control. Soil inoculation of biocontrol agents into solarized plots resulted in the highest suppression of damping-off incidence (PfT-8 up to 92%; ThM-1 up to 83%), and increase in plant biomass (PfT-8 up to 66%; ThM-1 up to 48%) when compared to un-solarized control plots. Rhizosphere population of introduced biocontrol agents gradually increased (PfT-8 up to 102% and ThM-1 up to 84%) in solarized soils when compared to unsolarized control. The population of Pythium spp in rhizosphere soil was reduced up to 55% in solarized plots; whereas, application of BCA to solarized soils reduced the rhizosphere population of Pythium spp. by 86 and 82% in P. fluorescens and T. harzianum applied plots respectively.     

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.     

Light and Electron Microscopy of the Compatible Interaction Between Arabidopsis and the Downy Mildew Pathogen Peronospora parasitica

In this study, we focused on compatible interactions between Peronospora parasitica isolate Emoy‐2 and wild‐type (Oy‐0) and mutant (Ws‐eds1) Arabidopsis thaliana accessions by using light and transmission electron microscopy (TEM). Light microscopy of compatible interactions revealed that conidia germinated and penetrated through the anticlinal cell walls of two epidermal cells. Rapid spreading of the hyphal growth with formation of numerous haustoria within the mesophyll cells was subsequently followed by profuse sporulation in the absence of host cell necrosis on both wild‐type and mutant accessions. TEM observations revealed that coenocytic intercellular hyphae ramified and spread intercellularly throughout the host tissue forming several haustoria in host mesophyll cells. Intracellular haustoria were lobed with the diameter of 6–7 μm. Each haustorium was connected to intercellular hyphae in the absence of apparent haustorial neck. The cytoplasm of the haustorium included the organelles characteristic of the pathogen. Callose‐like deposits were frequently observed at sites of penetration around the proximal region of the haustorial neck. Apart from a few callose ensheatments, no obvious response was observed in host cells following formation of haustoria. Most of mesophyll cells contained normal haustoria and the host cytoplasm displayed a high degree of structural integrity. Absence of host cell wall alteration and cell death in penetrated host cell of both accessions suggest that the pathogen exerts considerable control over basic cellular processes and in this respect, response to this biotroph oomycete differs considerably from responses to other pathogens such as necrotrophs.     

A genetic interval and physical contig spanning the Peronospora parasitica (At) avirulence gene locus ATR1Nd

In Peronospora parasitica (At) (downy mildew), the genetic determinants of cultivar-specific recognition by Arabidopsis thaliana are the Arabidopsis thaliana-recognised (ATR) avirulence genes. We describe the identification of 10 amplified fragment length polymorphism (AFLP) markers that define a genetic mapping interval for the ATR1Nd avirulence allele, the presence of which is perceived by the RPP1Nd resistance gene. Furthermore, we have constructed a P. parasitica (At) bacterial artificial chromosome (BAC) library comprising over 630Mb of cloned DNA. We have isolated 16 overlapping clones from the BAC library that form a contig spanning the genetic interval. BAC sequence-derived markers and a total mapping population of 311 F(2) individuals were used to refine the ATR1Nd locus to a 1cM interval that is represented by four BAC clones and spans less than 250kb of DNA. This work demonstrates that map-based cloning techniques are feasible in this organism and provides the critical foundations for cloning ATR1Nd using such a strategy.     

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.