Antagonistic Pythium against pathogenic Pythium on cucumber roots1

Antagonistic microorganisms introduced into the growing medium, the seed or the root, have been shown to reduce the attack of many soil-borne plant pathogenic fungi. In 1983, three experiments with Pythium oligandrum were carried out using cucumber seedlings artificially inoculated with Pythium splendens. The seedlings cv. Ideal Nova were grown in shallow boxes containing granulated rockwool fibre (Grodania). The plants were grown at a minimum temperature of 20°C in a glasshouse. Plants were treated with the antagonist either by coating the seeds, or by applying a disc of a PDA-culture to 9-day old seedlings. The pathogen was introduced 9 or 5 days later, respectively, by adding PDA culture discs to the rockwool; 15 and 38 days later, the area with attacked roots was calculated as a percentage of the box-bottom area. The experiments showed that it is possible to control P. splendens by either method of application. P. oligandrum was able to colonize the seed and rhizosphere and suppress P. splendens for at least 38 days. If future experiments are similarly positive, the methods for use in commercial horticulture should be worked out as soon as possible.     

Effect of 6-azauracil on infection with tobacco mosaic virus

6-Azauracil caused a marked reduction in the number and size of local lesions on excisedNicotiana glutinosa leaves or leaf discs inoculated with tobacco mosaic virus or its nucleic acid. The amount of infectivity recovered from tobacco (N. tabacum White Burley) leaf discs floated on the pyrimidine analogue solution and subsequently ground and assayed 24, 48, 72, and 96 h after inoculation with intact virus was reduced in the 24, 48, and 72 h series but reached the same level as that of the water-treated control discs in the 96 h series.By contrast, the amount of infectivity in stripped epidermal tissue ofN. glutinosa leaves inoculated with nucleic acid was not reduced in strips floated on the analogue for 24, 50, and 70 h as compared with water-floated controls. The analogue had no effect on infectivity of the virus in vitro and did not act as an inhibitor of infection.Part of this paper was presented at the International Symposium on Plant Pathology, held in New Delhi from December 27, 1966 till January 1, 1967. A summary will be published in the symposium volume entitled Plant disease problems.     

The development of resistance in Cladosporium cucumerinum aganst 6-azauracil,a chemotherapeutant of cucumber scab,and its relation to biosynthesis of RNA-precursors

Growth of the fungusCladosporium cucumerinum is inhibited by 6-azauracil (AzU), 6-azauridine (AzUR) and 6-azauridine-5′-monophosphate (AzUMP) on artificial media as well as on the cucumber plant. In cell-free extracts of this fungus the decarboxylation of orotidine-5′-monophosphate (OMP) is inhibited by AzUMP, but not by AzU or AzUR. The fungus itself is able to convert AzU and AzUR to the toxic AzUMP, an example of “lethal synthesis”.By irradiation of conidia with UV two types of AzU-resistant strains were obtained. The first type (III) did not convert AzU to AzUR. Therefore no lethal synthesis occurred when AzU was administered to this strain. The strain was, however, sensitive to AzUR and AzUMP. The second type (R) appeared resistant not only to AzU, but also to AzUR and AzUMP, in spite of the fact that conversion of AzU to AzUR and AzUMP did occur. No indications were obtained that the resistance of the latter type might be due to insufficient permeability towards the inhibitors or to a higher OMP-decarboxylase activity. The presence of an isoenzyme, more resistant to AzUMP than the original OMP-decarboxylase, was ruled out.The tolerance of the cucumber plant to AzU was paralleled by an extremely high activity of the enzyme OMP-decarboxylase in comparison with that found in the fungus.     

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.     

Mechanism of resistance of Cladosporium cucumerinum against 6-azauracil and 6-azauridinemonophosphate

The mechanism of resistance of strain R of Cladosporium cucumerinum resistant to 6-azauracil (AzU), 6-azauridine (AzUR) and to 6-azauridinemonophosphate (AzUMP) has been studied. Conversion of AzU into AzUR proceeds in the wild strain N and the resistant strain N equally well. Both, strain N and strain R convert AzUR into AzUMP. However, the amount of AzUMP detected in the resistant strain was approximately, three times lower than in strain N. This may be due to a change in the enzyme uridine kinase. The poor ability to form AzUMP from AzUR resulted in a higher activity of the enzyme orotidine monophosphate decarboxylase (OMP-decarboxylase) in strain R in this way interfering less with the incorporation of orotic acid into RNA of strain R than in that of strain N.     

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 6-azauracil against apple powdery mildew and apple scab

In 1964 and 1965 trials were made with 6-azauracil (AzU) against powdery mildew and scab in an apple orchard at Kloetinge (Zeeland) and at Wageningen. The activity against scab was moderate, but against powdery mildew the results were better than those obtained with Karathane. The activity against mildew is ascribed to the systemic action of AzU, which protected shoots not hit by the spray, as well as those developing after the treatment. Sprays with AzU also reduced the overwintering of powdery mildew in the terminal buds. When concentrations of 100 and 50 ppm were used, the young shoots developing were often malformed, but even with lower concentrations, still active against mildew, the trees were not completely free of slight abnormalities.Samenvatting In 1964 en 1965 zijn in een boomgaard te Kloetinge (Zeeland) en te Wageningen enige proeven genomen met 6-azauracil (AzU) ter bestrijding van meeldauw en schurft op appel. De activitieit tegen schurft was matig (Tabel 3 en 5), maar die tegen meeldauw was beter dan de werking van Karathane (Tabel 1, 2 en 4). Dit laatste wordt toegeschreven aan de systemische werking van AzU, waardoor ook niet geraakte en op het moment van bespuiting nog niet uitgegroeide scheuten beschermd worden. Bespuitingen met 6-azauracil reduceerden ook de overwintering van meeldauw in de eindknoppen (Tabel 6). Bij concentraties AzU van 100 en 50 ppm ontwikkelden zich scheuten met misvormingen (Fig. 1), maar ook de bomen die met lagere concentraties behandeld waren, bleven niet geheel vrij van lichte afwijkingen.     

Effectiveness of Trichoderma spp. obtained from re-used soilless substrates against Pythium ultimum on cucumber seedlings

Journal of Plant Diseases and Protection - Thirty-nine Trichoderma strains isolated from effective re-used substrates used in soilless systems and two commercial formulations (Trichoderma viride...     

Genetic diversity, aggressiveness and metalaxyl sensitivity of Pythium aphanidermatum populations infecting cucumber in Oman

Seventy three isolates of Pythium aphanidermatum obtained from cucumber from four different regions of Oman and 16 isolates of muskmelon from the Batinah region in Oman were characterized for aggressiveness, sensitivity to metalaxyl and genetic diversity using AFLP fingerprinting. Twenty isolates of P. aphanidermatum from diverse hosts from different countries were also included in the study. Most isolates from Oman were found to be aggressive on cucumber seedlings and all were highly sensitive to metalaxyl (EC₅₀ < 0·80 µg mL^?1). Isolates from cucumber and muskmelon were as aggressive as each other on both hosts (P > 0·05), which implies a lack of host specialization in P. aphanidermatum on these two hosts in Oman. AFLP analysis of all isolates using four primer–pair combinations resolved 152 bands, of which 61 (~40%) were polymorphic. Isolates of P. aphanidermatum from Oman and other countries exhibited high genetic similarity (mean = 94·1%) and produced 59 different AFLP profiles. Analysis of molecular variance indicated that most AFLP variation among populations of P. aphanidermatum in Oman was associated with geographical regions (F_ST = 0·118; P < 0·0001), not hosts (F_ST = –0·004; P = 0·4323). These data were supported by the high rate of recovery (24%) of identical phenotypes between cucumber and muskmelon fields in the same region as compared to the low recovery (10%) across regions in Oman, which suggests more frequent movement of Pythium inoculum among muskmelon and cucumber fields in the same region compared to movement across geographically separated regions. However, recovering clones among regions and different countries may imply circulation of Pythium inoculum via common sources in Oman and also intercontinental spread of isolates.     

Suppressive effect of composts from residual biomass on Pythium ultimum

Journal of Plant Diseases and Protection - Two experiments were established in the laboratory of the Department of Organic Farming and Cropping Systems and the greenhouse of the University of...     

Evaluation of a method for quantification of Pythium aphanidermatum in cucumber roots at different temperatures and inoculum densities

Journal of Plant Diseases and Protection - A method for the quantification of Pythium aphanidermatum density in cucumber roots based on an indirect enzyme-linked immunosorbent assay (Elisa) was...     

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.     

Defense enzymes induced in cucumber roots by treatment with plant growth-promoting rhizobacteria (PGPR) and Pythium aphanidermatum

Root and crown rot of cucumber caused by Pythium aphanidermatum can be suppressed by various rhizobacteria or PGPR (plant growth-promoting rhizobacteria). When cucumber roots were treated with Pseudomonas corrugata 13 or Pseudomonas aureofaciens 63–28, phenylalanine ammonia-lyase (PAL) activity was stimulated in root tissues in 2 days and this activated accumulation lasted for 16 days after bacterization. Peroxidase (PO) and polyphenol oxidase (PPO) activities were increased in roots 2–5 days after bacterization with P. corrugata strain 13. After bacterized cucumber roots were challenged with P. aphanidermatum, the enzyme activities of PAL, PO and PPO increased as the disease developed on the roots. These accumulations peaked 4–6 days after pathogen inoculation. A split root system demonstrated that the three enzymes could be systemically induced by the Pseudomonas strains 63–28 and 13, as well as P. aphanidermatum. Furthermore, isoperoxidase native PAGE (polyacrylamide gel electrophoresis) analysis indicated that the peroxidase isomer forms in cucumber roots induced by rhizobacteria were different from that in roots infected with P. aphanidermatum. These results suggest that the plant defense enzymes could be stimulated in cucumber roots which have been colonized by non-pathogenic rhizobacteria or in a compatible interaction between cucumber and P. aphanidermatum. The mechanisms of PO activation by the rhizobacteria may be different from those of pathogen infection.     

芽孢杆菌对黄瓜霜霉病的防治效果研究

经黄瓜霜霉病孢子囊萌发抑制试验明确,芽孢杆菌菌株Z-X-3、Z-X-10对黄瓜霜霉病菌有较强拮抗作用。离体叶片法和温室防病试验表明,两菌株对黄瓜霜霉病有较好的防治效果,相对保护效果分别达46.42％、41.55％;治疗效果分别达46.30％、44.44％,高于农药克霜氰的31.81％、37.03％。     

Production of tobacco mosaic virus in and its infectivity from leaves of two Nicotiana species treated with 6-azauracil

A correlation between the amount of virus as determined by infectivity test and that determined by a serological-spectrophotometric method was established in leaf discs ofNicotiana tabacum White Burley which had floated on a solution of 6-azauracil (AzU) before and after inoculation with tobacco mosaic virus. More virus, determined in the two different ways, was present in the AzU-than in the water-treated leaf discs at 24 and 48 h after inoculation. Thereafter, the amount of virus was less than that in the control. The inhibitory effect of the pyrimidine analogue on the amount of infectivity in sap from AzU-treated discs proved to be dependent on the dilution of sap used. The higher the dilution the more inhibition occurred.In inoculated leaf discs ofNicotiana glutinosa the formation of local lesions in the AzU-treated series was strongly inhibited at 48 h after inoculation whereas at that time the amount of virus determined by the serological-spectrophotometric method was higher than that in the water-treated control ones. The latter mentioned stimulating effect disappeared later than 48 h after inoculation and gave way to inhibition.The infectivity of tobacco mosaic virus from AzU-treated leaf discs of White Burley tobacco was 23% of that from water-treated controls when undiluted clarified sap was used.     

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     

Aggressiveness and production of cell-wall degrading enzymes by Pythium violae, Pythium sulcatum and Pythium ultimum, responsible for cavity spot on carrots

This study investigated the relationships between pathogenesis, types of symptoms and in vitro production of cell-wall degrading enzymes by P. violae, P. sulcatum and P. ultimum. The three pathogens, considered as the three Pythium species principally responsible for cavity spot on carrot roots, secreted only low levels of fatty acid esterases activity, suggesting they have limited ability to degrade suberin in the walls of the outer cell layers of carrot tissues. Among the enzymes that degrade cell-wall polysaccharides, only pectate lyases and cellulases were produced by P. violae, and these were produced late and in small amounts: the symptoms caused by P. violae were limited and typical of cavity spot. Conversely, P. ultimum caused maceration of tissues, and secreted polygalacturonases and -1,4-glucanases earlier and in larger amounts than P. violae. P. ultimum also produced a large diversity of proteins and cellulase isoenzymes. Although secreting all the monitored enzymes in higher quantity than the two previous species, P. sulcatum was responsible for only typical limited symptoms of cavity spot, with a brown colouring. The role of plant reactions induced in response to early pectinolytic enzyme production by P. sulcatum may account for this apparent inconsistency.Died on April 11, 1997     

黄瓜根分泌物中的7,8-苯并黄酮及其对枯萎病发生的影响

分别提取黄瓜枯萎病感病品种津研4号和抗病品种中农10号的根分泌物,感病品种根分泌物甲醇提取液能够刺激枯萎病菌的菌丝生长,而抗病品种根分泌物甲醇提取液则抑制病原菌生长。利用LC-MS/MS技术分别对感病品种和抗病品种根分泌物中的差异物质进行分析鉴定。与感病品种津研4号相比,抗病品种中农10号根分泌物中含有7,8-苯并黄酮(7,8-BF),含量为0.02μg/株。50μg/mL的7,8-BF能够显著抑制黄瓜枯萎病菌菌丝生长和孢子萌发,而100μg/mL的7,8-BF能显著抑制抗感品种侧根形成,但是抗病品种的耐受力高于感病品种。用7,8-BF处理黄瓜种子,枯萎病侵染程度显著降低。当7,8-苯并黄酮浓度为50μg/mL时,感病品种病情指数从71.7降为30.8,达到中抗水平。     

Species of Pythium in the Netherlands

A review is given on observations ofPythium spp. so far published. In personal investigations about 4000 isolates ofPythium species were obtained from soil, water, Daphnias and plants during 1966–1972. Special attention was paid to the progressive colonization of the newly reclaimed polder Zuidelijk Flevoland: the numbers of species per sample increased from 0 in 1966 to 13 in 1972. The percentage of samples which did not contain anyPythium decreased from 54 in 1968 to 0 in 1972. From Zuidelijk Flevoland altogether 21 species were isolated, from Oostelijk Flevoland 22, of which 19 occurred in both the polders. The soil depth down to 10 cm had little influence on the occurrence ofPythium species. From soils near Rhenen and Zutphen 12 and 19 species respectively were isolated, from flax and flaxfields 11, from forests 8, from roots of different plants 17 and from water 11. Besides species already previously recorded, 15 new records ofPythium species for the Netherlands are documented. Species with filamentous non-swollen sporangia preferentially occurred in water and wet soils while species with spherical sporangia or hyphal swellings dominated in cultivated soils, forests and in the roots of phanerogams. In 1968 species with filamentous non-swollen sporangia comprised 89% of the isolations from Zuidelijk Flevoland; this percentage decreased to 9 in 1972. The percentage of isolates with spherical sporangia or hyphal swellings from cultivated soils, forest soils or plant material was mostly over 90.P. rostratum Butler, though found in all kinds of soils, was often the only species which could be isolated from dry sandy soil samples.P. sylvaticum Campbell & Hendrix was the most common species in cultivated soils in the Netherlands (37% of the isolates in 1968–1972), followed byP. oligandrum Drechsler (22%) andP. paroecandrum Drechsler (11.5%).