Characterization of the Microbial Community Involved in the Suppression of Pythium aphanidermatum in Cucumber Grown on Rockwool

ABSTRACT The root pathogen Pythium aphanidermatum induced lower levels of disease in cucumber (Cucumis sativus) plants on unsterilized, re-used rockwool slabs than on heat-sterilized, re-used rockwool. Several recolonization treatments of the sterilized rockwool enhanced the suppressiveness of the rockwool. Microbial community structures in the different rockwool treatments were investigated by plate counts on selective media. Disease suppressiveness in the different rockwool treatments showed the highest correlation with the culturable number of filamentous actinomycetes in both experiments (r = 0.79 and 0.94), whereas the numbers of Trichoderma spp. correlated with suppression only in the first experiment (0.86). The numbers of total culturable bacteria, fluorescent pseudomonads, Bacillus spores, and fungi all showed lower correlations with disease suppressiveness. The filamentous actinomycetes enumerated with the plate counts were mainly Streptomyces spp., of which 10% were antagonistic toward P. aphanidermatum in dual culture. The composition of the bacterial and actinomycete populations was studied with polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE). Multivariate analyses of these patterns with canonical correspondence analysis showed significant correlations between the microbial composition and the disease suppressiveness. However, none of the bands in PCR-DGGE patterns occurred exclusively in the treatments that had enhanced disease suppressiveness. Bands extracted from the actinomycete-specific DGGE gels showed closest similarity with members of several actinomycete genera, i.e., Streptomyces, Mycobacterium, Microbacterium, Rhodococcus, Curtobacterium, and Tsukamurella. The possible mechanism of disease suppressiveness in used rockwool slabs, based on the results obtained with culture-dependent and culture-independent detection methods, is discussed.     

Induced suppressiveness to<Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp.<Emphasis Type="Italic">radicis lycopersici</Emphasis> in rockwool substrate used in closed soilless systems

Tomatoes grown in soilless systems can be seriously damaged byFusarium oxysporum Schlect f.sp.radicis lycopersici (Forl) causing Fusarium crown and root rot (FCRR). FCRR suppression can be achieved through the use of chemicals, selected substrates, composts and artificially introduced antagonistic microorganisms. This study evaluated the natural capacity of a used rockwool to suppress FCRR infections. New and used rockwool, sampled from closed soilless systems, was either autoclaved or not, either artificially inoculated withForl or not and, finally, sown with tomato seeds cv. ‘Cuore di Bue’. The effects of autoclaved/non-autoclaved and used/new rockwool on FCRR incidence were assessed by evaluating the symptoms of crown rot on the root — shoot transition zone of tomato seedlings. Non-autoclaved and inoculated used rockwool significantly reduced FCRR incidence when compared with non-autoclaved and inoculated new rockwool. Autoclaved and inoculated used rockwool did not suppress FCRR, similarly to new and inoculated rockwool. These findings are in accordance with other research that, on a cucumber/Pythium host/pathogen complex in a closed rockwool soilless system, demonstrated the key role of resident microflora in suppressing the root rot disease. http://www.phytoparasitica.org posting Dec. 8, 2006.     

Biocontrol of soil-borne fungal plant diseases by 2,4-diacetylphloroglucinol-producing fluorescent pseudomonads with different restriction profiles of amplified 16S rDNA

Fluorescent pseudomonads producing the antimicrobial compound 2,4-diacetylphloroglucinol (Phl) are being studied extensively for use as biocontrol agents of soil-borne fungal diseases. Some of them can produce pyoluteorin (Plt) in addition to Phl, whereas others synthesise only Phl. Here, a collection of seven Phl⁺ Plt^- pseudomonads, seven Phl⁺ Plt⁺ pseudomonads and seven Phl^- biocontrol pseudomonads were compared for protection of plant roots against fungal pathogens. The seven Phl⁺ Plt⁺ pseudomonads were identical by restriction analysis of amplified spacer ribosomal DNA (spacer ARDRA), whereas the Phl⁺ Plt^- pseudomonads and especially the Phl^- biocontrol pseudomonads were quite diverse by spacer ARDRA. Collectively, the Phl⁺ Plt^- pseudomonads proved superior to the Phl⁺ Plt⁺ pseudomonads and the Phl^- biocontrol pseudomonads for protection of tomato against Fusarium crown and root rot (in rockwool microcosms) or cucumber against Pythium damping-off (in non-sterile soil microcosms). There was no correlation between protection in vivo and inhibition of the corresponding fungal pathogen on plates. However, there was a significant correlation between the amount of Phl produced on plates and protection of tomato against Fusarium crown and root rot, but not with protection of cucumber against Pythium damping-off. Interestingly, the minority of strains unable to produce HCN, an extracellular protease, or both, were among those unable to protect plants in both pathosystems. A seedling assay was developed to compare pseudomonads for suppression of Fusarium crown and root rot in vitro, and a significant correlation was found between disease severity in vitro and in vivo. Overall, results suggest that promising biocontrol pseudomonads may be identified based on the ability to produce Phl and/or specific ARDRA-based fingerprints.     

Pathogenicity ofPythium species on cucumber in peat-sand,rockwool and hydroponics

Pythium spp. that cause damping-off of seedlings also can cause root rot of older plants and lead to yield reductions. This can be especially severe in soilless cultures where the fungus can spread easily with the nutrient solution. 39Pythium isolates obtained from discolored roots were assayed for their ability to cause damping-off on cucumber seedlings in sand-peat and for their pathogenicity in soilless culture of cucumber in rockwool or hydroponic solution. Isolates ofPythium aphanidermatum, P. irregulare, P. sylvaticum andP. ultimum were highly pathogenic in sand-peat, but onlyP. aphanidermatum strains were pathogenic in soilless conditions and led to root decay, plant death in rockwool culture and growth reduction in hydroponic culture. One strain ofP. aphanidermatum significantly reduced the yield of cucumber grown in rockwool under conditions similar to those of commercial cultures.     

Protection of cucumber against Pythium root rot by fluorescent pseudomonads: predominant role of induced resistance over siderophores and antibiosis

Four Pseudomonas strains were evaluated for their intrinsic properties conferring their ability to protect long English cucumber against Pythium aphanidermatum in hydroponic culture. Two of the strains, BTP1 and its siderophore-negative mutant M3, increased plant yield as compared with the non-inoculated control plants. Strain BTP7 was intermediate in its biocontrol activity while strain ATCC 17400 failed to reduce disease development. The role of pyoverdines could not be confirmed since treatment with either BTP1 or its siderophore-negative mutant M3 provided similar suppression of Pythium disease. In addition, no siderophores were detected in the nutrient solution. BTP1 did not inhibit pathogen growth in vitro on several media, suggesting that antibiosis was not a mechanism of suppression. Quantification of root bacterial populations did not indicate differences among the strains. On the other hand, roots treated with either BTP1 or its sid⁻ mutant M3 contained more antifungal phenolics than roots from any other treatments including controls. These results suggest that antifungal compounds induced by inoculation of cucumber roots with the fluorescent Pseudomonas strains BTP1 and M3 participate actively in the protection of cucumber plants against P. aphanidermatum     

杀菌剂SYP-4288对几种常见土传病害的防治效果

采用室内盆栽试验测定了杀菌剂SYP-4288对立枯丝核菌引起的棉花立枯病、大豆疫霉根腐病及瓜果腐霉菌引起的黄瓜猝倒病的防效。结果表明,SYP-4288推荐剂量下对3种病害具有较好防效。最高推荐剂量下对棉花立枯病、黄瓜猝倒病和大豆根腐病防效分别为72.67%±10.59%、100%和82.62%±12.48%。SYP-4288对西瓜枯萎病田间药效试验表明,SYP-4288最高推荐剂量下对西瓜枯萎病防效为56.82%~66.51%,好于或相当于对照药剂。基于室内与田间药效试验结果,SYP-4288可以推荐用于防治瓜果蔬菜常见土传病害。     

Inoculation of cucumber roots with zoospores of mycoparasitic and plant pathogenic Pythium species: Differential zoospore accumulation, colonization ability and plant growth response

Hydroponically grown cucumber (Cucumis sativus) seedlings were inoculated with zoospores of 1 mycoparasitic (Pythium oligandrum) and 2 pathogenic (Pythium aphanidermatum and Pythium group F) Pythium spp. During the first 2 days after inoculation, all the Pythium spp. caused reduction in the root length. However, roots treated with Pythium oligandrum quickly reached the length of the control and on the 8th day, and for the rest of the experimental period, stimulation of root elongation was noted. Pythium oligandrum was not pathogenic on cucumber and no differences in the fresh weights of control and Pythium oligandrum inoculated plants were observed in the course of the experiment. Pythium group F and Pythium aphanidermatum were pathogenic on cucumber seedlings, but their pathogenicities differed. Thus, while Pythium group F had a constant, negative influence on root length and plant growth, measured as fresh weight, Pythium aphanidermatum caused generalized necroses of the root system, inhibiting consistently root elongation and plant growth and finally causing plant death. Moreover, the zoospores of 2 mycoparasitic species, Pythium oligandrum and Pythium periplocum, were not attracted to roots of cucumber and accumulated on the roots in very low numbers compared to those of the pathogenic species, Pythium aphanidermatum, which were strongly attracted and accumulated in large numbers. Finally, it was also found that Pythium oligandrum colonized the roots very poorly, while Pythium group F and Pythium aphanidermatum were significantly better root colonizers. The significance of these findings is discussed in relation to the ecology of Pythium species and biocontrol.     

Antagonismus in vivo von fluoreszierenden Pseudomonaden gegenüber Rhizoctonia solani und Pythium aphanidermatum an Gurken und Buschbohnen

Fluoreszierende Pseudomonaden, die sich als Antagonistenin vitro gegenüberRhizoctonia solani undPythium aphanidermatum erwiesen haben, wurden an Gurken und Bohnen auf Antagonismusin vivo untersucht. Die Effizienz der Antagonisten in Auflaufversuchen gegenüberR. solani war niedriger als beiP. aphanidermatum. Die besten Isolate konnten in mitR. solani verseuchter Erde Auflaufraten von 30–40% erzielen, gegenüberP. aphanidermatum dagegen 40–60%. Die Wirkung der Antagonisten zur Verhütung von Spätinfektionen der Pflanzen war deutlich besser. So konnten 16 Isolate eine Schädigung durchR. solani an Bohnen zwischen 90 und 100% reduzieren. An Gurken konnten 12 Isolate eine Schädigung durchP. aphanidermatum auf 70–80% reduzieren.Fluorescent pseudomonads, which showed antagonism in vitro against Rhizoctonia solani and Pythium aphanidermatum, were investigated for antagonism in vivo using bean and cucumber. The efficiency of the antagonists in germination tests against Rhizoctonia solani was lower than against Pythium aphanidermatum. In soil infected with Rhizoctonia solani the germination rates of the seeds remained between 30 and 40%, whereas against Phytium aphanidermatum some Pseudomonas-strains caused germination rates of 40–60%. The preventive effect of the antagonists against fungal infection after the germination was better. 16 isolates reduced the damage caused by Rhizoctonia solani to bean between 90 and 100%. Damage caused by Pythium aphanidermatum to cucumber was reduced by 12 antagonists between 70 and 80%.

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Control of root zone pH is not effective in preventing Pythium aphanidermatum disease in cucumber

Journal of Plant Diseases and Protection - Pythium aphanidermatum is the most wide-spread root pathogen in greenhouse cucumber. Although environmental condi-tions are known to affect the disease...     

Effect of salinity on pythium damping-off of cucumber and on the tolerance of Pythium aphanidermatum

A study was conducted to investigate the effect of salinity on pythium damping-off and tolerance to salinity among Pythium aphanidermatum isolates from different geographical origins, genetic backgrounds and saline/non-saline soils. Increasing irrigation-water salinity from 0·01 to 5 dS m⁻¹ significantly increased mortality in cucumber seedlings inoculated with P. aphanidermatum and reduced dry weight of non-inoculated seedlings. In vitro tests in culture media amended with different concentrations of NaCl showed that growth of P. aphanidermatum, P. spinosum and P. splendens isolates was stimulated or unaffected at salinity levels stressful for cucumber (electrical conductivity = 5 dS m⁻¹). Significant differences were found in tolerance to salinity among 47 P. aphanidermatum isolates obtained from different greenhouses. The concentration of NaCl required to reduce growth of P. aphanidermatum isolates by 50% varied from 23 to 62 dS m⁻¹, with an average of 46 dS m⁻¹, however, oospore production was more sensitive to salinity and no oospores were produced above 20 dS m⁻¹. Analysis of tolerance of P. aphanidermatum isolates to salinity showed no relationship between tolerance to salinity and geographical origins or genetic data. Isolates of P. aphanidermatum from greenhouses with no salinity problems were as tolerant to salinity as isolates obtained from salinity-affected greenhouses, suggesting lack of evidence for ecological adaptation within P. aphanidermatum in Oman. Increased mortality in cucumber seedlings at higher salinity levels may imply a synergistic interaction between salinity stress and salinity-tolerant Pythium species on cucumber seedlings, resulting in greater seedling losses.     

新疆加工番茄腐霉根腐病病原鉴定及其rDNA的ITS区段分析

为了明确新疆加工番茄根腐病的病原种类,从成苗期到果实成熟期对各主栽区的病原物进行分离,并进行形态学鉴定和ITS序列分析.结果表明,从162个根腐病样中获得120个分离物,其中腐霉菌93株,占总分离物的77.5%.腐霉菌分离物鉴定为3个种,瓜果腐霉Pythium aphani-dermatum(Edson)Fitzp,56株,占腐霉分离物的60.22%;简囊腐霉P.monospermum Pringsheim,6株,占6.45%;棘腐霉P.acanthicum Drechsler,2株,占2.15%;另有29个分离物因未诱发出雄器和藏卵器而无法鉴定到种.     

Effect of some organic amendments on onion bulb rot

The effect of soil amended with stalks of sunflower (Helianthus annuus L.), alfalfa (Medicago sativa L.) and Hungarian vetch (Vicia pannonica Crantz) on onion bulb rot caused byFusarium oxysporum Schlecht. f.sp.cepae (Hanz.) Snyder Hansen andAspergillus niger v. Tieghem was tested in a soil naturally infested with the pathogens under field conditions. All treatments significantly reduced bulb rot incidence. The fungal and antibacterial populations varied among all of the amended tested soils. Heterotrophic fungal populations were more effective than heterotrophic bacterial populations in the suppression of disease. The highest population of fungal microflora was found in plots amended with sunflower stalks. The carbon-to-nitrogen ratio and available moisture content of amended soils have an indirect suppressive role on disease, by increasing the heterotrophic fungal population. http://www.phytoparasitica.org posting June 18, 2002.     

Bacterial and fungal diseases in the major potato-growing areas of Tunisia

In a two year survey of Tunisian rustic potato stores, the losses were nearly equally distributed between dry rots induced by Fusarium solani and watery wound rot or leak caused by Pythium aphanidermatum and P. ultimum. Symptoms of leak were different from those normally induced by Pythium spp. Surveys of the growing potato crop confirmed the prevalence of these pathogens, which caused wilt and internal vascular stem necrosis. Most rotted progeny tubers also exhibited leak symptoms caused by P. aphanidermatum. Hardly any Erwinia diseases were found in Tunisia. The incidence of diseased plants was significantly higher in fields with successive cultivation of solanaceous crops, which may be attributed to a high rate of soil infestation by wilt-inducing pathogens such as Pythium spp., F. solani and Verticillium dahliae. The health of seed potatoes also played a significant role, affecting the incidence of rot in potato stores as well as in the soil before plant emergence. Therefore, Tunisian integrated management programmes for potato diseases should focus on soil disinfestation with appropriate crop rotation as well as seed quality and treatments.     

Protoporphyrinogen oxidase inhibitor herbicide effects on pythium root rot of sugarcane, pythium species, and the soil microbial community

ABSTRACT The effects of three protoporphyrinogen oxidase inhibitor herbicides, azafenidin, flumioxazin, and sulfentrazone, on Pythium root rot of sugarcane and the soil microbial community were evaluated in greenhouse experiments. Herbicides were applied as foliar and soil treatments. There were no consistent effects on plant growth or disease parameters. However, some herbicide treatments affected the relative frequency of isolation of Pythium spp. from roots and reduced colonization by the pathogenic species Pythium arrhenomanes. A comparison of sole carbon source utilization profiles indicated that soil-applied herbicides altered the functional diversity of the soil microbial community, with some variation depending on herbicide used. All three herbicides inhibited the in vitro mycelial growth of P. arrhenomanes, P. aphanidermatum, and P. ultimum. Active ingredients were less inhibitory than formulated product for azafenidin and flumioxazin but not for sulfentrazone.     

Foot Rot of Ulluco Caused by Pythium aphanidermatum

Severe rot of stem bases caused by Pythium aphanidermatum was found on ulluco (Ullucus tuberosus) grown in Kagawa Prefecture, Japan, in September 1999. The name “foot rot of ulluco” is proposed for this new disease. Received 14 November 2001/ Accepted in revised form 7 January 2002     

Conduciveness of different soilless growing media to <Emphasis Type="Italic">Pythium</Emphasis> root and crown rot of cucumber under near-commercial conditions

Substrates made from rockwool, coir dust, pumice and perlite were compared for conduciveness to Pythium root and crown rot in cucumber under near-commercial conditions. Rockwool slabs of 7 cm height were more conducive to the Pythium disease than coir dust slabs, pumice or perlite under these conditions. Temperature, oxygen concentration and water content were determined in the substrates to explain differences in conduciveness between the inorganic substrates rockwool, pumice and perlite by differences in the physical conditions. Temperature and oxygen concentration could not explain the differences but the higher disease level on rockwool was associated with a much higher water content of this substrate as compared to coir dust, pumice and perlite. Increasing the height of the substrate from 7 to 14 cm greatly decreased the percentage of diseased plants due to the Pythium disease on rockwool but had no effect on the level of disease on perlite when the substrate had been infested 4 cm below the planting hole. This difference in response in substrate height between rockwool and perlite could be explained by a much larger decrease in water content with substrate height in the rockwool than in the perlite substrate. Temperature in the substrates were above 30 °C for more than 6 h on sunny days in June and reached maximum values of 35 °C or more. These temperatures are highly favourable for the pathogen P. aphanidermatum but will have adverse effects on most biocontrol strains.     

新疆南疆枣树根腐病病原的分离与鉴定

<正>枣为鼠李科(Rhamnaceae)枣属(Ziziphus M ill.)植物,在我国拥有悠久的栽培历史。枣树的喜光、喜温、耐寒、耐旱等栽培特点,非常适合在新疆这种气候条件特殊的地区生长。近年来,随着新疆红枣产业的迅速发展,枣树病害问题也逐渐突显。2010和2011年,新疆南疆多地枣园出现枣树根腐病,该病害主要造成枣树实生苗和多年生枣树根部腐烂,叶片黄化,植株树势衰弱,最终导致植株     

Comparison of an in vitro and a damping-off assay to test soils for suppressiveness to Pythium aphanidermatum

Testing of soil samples in greenhouse assays for suppressiveness to soilborne plant pathogens requires a considerable investment in time and effort as well as large numbers of soil samples. To make it possible to process large numbers of samples efficiently, we compared an in vitro growth assay with a damping-off assay using Pythium aphanidermatum as the test organism on tomato seedlings. The in vitro test compares the radial growth or relative growth of the fungus in soil to that in autoclaved soil and reflects suppressiveness of soils to the pathogen. We used soils from a field experiment that had been farmed either organically or conventionally and into which a cover crop (oats and vetch in mixture) had been incorporated 0, 10, 21, and 35 days previously. We obtained a significant, positive correlation between damping-off severities of tomato seedlings in damping-off assays and both relative and radial growth in vitro. In addition, radial and relative growth of P. aphanidermatum in the in vitro assay were positively correlated with several carbon and nitrogen variables measured for soil and incorporated debris. We did not find differences between the two farming systems for either growth measures of P. aphanidermatum or disease severities on tomato at different stages of cover crop decomposition. The in vitro assay shows potential for use with any fungus that exhibits rapid saprophytic growth, and is most suitable for routine application in suppressiveness testing.     

Role of Salicylic Acid in Systemic Resistance Induced by Pseudomonas spp. Against Pythium aphanidermatum in Cucumber Roots

Pseudomonas corrugata strain 13 and P. aureofaciens strain 63-28, applied to roots, induced systemic resistance against Pythium aphanidermatum in cucumber roots. Salicylic acid (SA) from bacterial culture or plant tissues was quantified by high performance liquid chromatography. Both strains produced SA in King's B broth and also induced cucumber root to accumulate endogenous SA one day after bacterial inoculation. Using a split root system, more SA accumulated in roots treated with bacteria than in distant roots on the opposite side of the root system in the first two days, but this difference disappeared after 3–4 days. SA levels were significantly higher in plants treated with bacteria compared to the split control, from one to five days after bacterization. SA did not inhibit mycelial growth of Pythium aphanidermatum at 100–200µgml^–1 in vitro, but higher levels inhibited mycelial growth. Zoospore germination increased at concentrations of 10–500µgml^–1, but decreased at 1000µgml^–1 compared to lower concentrations. Exogenously applied SA failed to induce local or systemic resistance against a challenge infection by the pathogen in planta. The results of this study show that exogenous applied SA does not induce systemic resistance to cucumber root rot caused by P. aphanidermatum, but endogenous SA accumulation in cucumber roots may be involved in induced systemic resistance.     

Evaluation of plant growth-promoting rhizobacteria for biological control of pythium root rot of cucumbers grown in rockwool and effects on yield

Three strains ofPseudomonas fluorescens (63-49, 63-28, and 15), one strain ofPseudomonas corrugata (13) and one strain ofSerratia plymuthica (R1GC4) were tested on rockwool-grown cucumbers for their ability to reduce Pythium root-rot caused byPythium aphanidermatum. These strains were previously selected for biocontrol ability from collections of >4000 bacteria. Strains 63-49 and 63-28 were tested on cucumber plants grown in rockwool in two replicatedPythium-inoculated trials conducted in British Columbia (B.C). Another inoculated, replicated trial was conducted in Quebec with all five strains. Cucumber yields (fruit number and weight) were measured over a ten-week harvest period. Strain 63-49 caused an early promotion of plant growth and increased cucumber yields at early harvests. No measurable effect ofPythium inoculation on disease development was observed in the Quebec trial, due to unfavourable cool weather. However, 63-49 significantly increased the total number of cucumbers (12%) and cucumber weight (18%), compared to the non-treated control. Strains 13, 15 and R1GC4 slightly increased the cumulative cucumber yields, but strain 63-28 had no effect. In the B.C. trial, inoculation withP. aphanidermatum reduced the number and weight of cucumbers by 27%. Treatments ofPythium-inoculated cucumbers with 63-49 significantly increased fruit number and weight by 18%, compared to thePythium-inoculated control. Strain 63-28 increased the cumulative number of cucumbers over time, compared to thePythium-inoculated control, but the increase was less than with 63-49. The use ofPseudomonas spp. in rockwool-grown cucumbers can increase yields, both in the presence and absence of Pythium root rot, and with variable seasonal conditions and disease pressures.