Differential isolatoion of Pythium species from soil by means of selective media,temperature, and pH.

Pythium aphanidermatum, with an optimum temperature for growth at 35C, grew welland was readily isolated from soil on pimaricin-vancomycin medium (MPVM) when incubated for 24h at 38-40C. The pH of the medium affected recovery; maximum numbers developed above pH 6.0. Other Pythium spp. were recovered on MPVM at 20-25C, but were excluded by incubation at 38-40C. These Pythium spp. included P. ultimun, P. paroecandrum, P. irregular, P. mamillatum, and an unidentified Pythium sp. These species grew well and were readily siolated from soil on gallic acid medium (GAM) when incubated for 24-8h at 20 C.P. aphanidermatum and P. myriotylum grew from mycelium on GAM, but their oospores did not germinate nor could they be isolated from soilon this medium. P. myriotylum grew well on MPVM, but was only rarely isolated, evenfrom soils with known high potential for disease caused by P. myriotylum. Propagules of Pythium were enumerated by a plate-dilution frequency method or by a smearplateethod is valuable for studies on the ecology, survival, and inoculum potential in soils with mixed populations of P. aphanidermatum and other Pythium spp.     

Ecology of Hymexazol-Insensitive Pythium Species in Field Soils

Soils from 100 irrigated fields (95 under vegetables, 5 under citrus) in different geographical locations in the West Bank (Palestinian Autonomous Territory) were surveyed for hymexazol-insensitive (HIS) Pythium species using the surface soil dilution plate (SSDP) method with the VP3 medium amended with 50 mg/L hymexazol (HMI) (VP3H50), over a period of 12 months. HIS Pythium species were isolated from 37% of the soils surveyed, with mean population levels ranging from 4.3-1422 CFU g(-1) dry weight. Eight HIS Pythium taxa were recovered on the VP3H50 medium, the most abundant of which was P. vexans (found in 29% of field soils surveyed). Seasonal variations in population levels of HIS Pythium species were studied in four fields over a period of 12 months. Significant seasonal variations in HIS population levels were detected in the four fields, with the highest population levels of HIS Pythium spp. encountered in spring and the lowest population levels in winter in three of the fields surveyed. Effects of HMI on linear growth and colony morphology of 149 Pythium ssp. isolates were examined on CMA amended with HMI at five concentrations. Pythium vexans isolates responded differently from those of the other Pythium species. Isolates of this important pathogen were more insensitive to HMI at high concentrations than the other main species tested. A large proportion of the P. ultimum isolates was either insensitive or weakly sensitive to HMI. Furthermore, a few isolates of other Pythium species were insensitive to the fungicide at various concentrations. The colony morphology of P. vexans isolates was not affected by HMI, whereas colonies of the other species showed sparse growth on the HMI amended medium relative to the control. The pathogenicity of P. vexans and P. ultimum isolates to cucumber seedlings was examined in growth chambers. Insensitive isolates of both species were found to be more virulent damping-off pathogens than the sensitive isolates. The present study demonstrates that HMI can not be used effectively in controlling Pythium spp. in soil inhabited with high densities of HIS Pythium spp. pathogens.     

Effects of Nematicides on Cotton Root Mycobiota

Baseline information on the diversity and population densities of fungi collected from soil debris and cotton (Gossypium hirsutum L.) roots was determined. Samples were collected from Tifton, GA, and Starkville, MS containing cotton field soil treated with the nematicides 1,3-dichloroproprene (fumigant) and aldicarb (granules). A total of 10,550 and 13,450 fungal isolates were collected from these two study sites, respectively. Of this total, 34 genera of plant pathogenic or saprophytic species were identified. Pathogenic root fungi included Fusarium spp. (40% of all isolations), Macrophomina, Pythium, Rhizoctonia, and Sclerotium. Fusarium and Rhizoctonia were the most common fungal species identified and included F. oxysporum, F. verticillioides and F. solani, the three Fusarium species pathogenic on cotton plants. Population densities of Fusarium were not significantly different among locations or tissue types sampled. Macrophomina was isolated at greater numbers near the end of the growing seasons. Anastomosis groups of R. solani isolated from roots and soil debris included AG-3, -4, -7, 2-2, and -13 and anastomosis groups of binucleate Rhizoctonia included CAG-2, -3, and -5. Occurrences and frequency of isolations among sampling dates were not consistent. Fluctuations in the frequency of isolation of Rhizoctonia did not correspond with changes in frequency of isolation of the biological control fungus, Trichoderma. When individual or pooled frequencies of the mycobiota were compared to nematicide treatments, no specific trends occurred between treatments, application methods or rates. Results from this study show that use of 1,3-D and aldicarb in cotton fields does not significantly impact plant pathogenic fungi or saprophytic fungal populations. Thus cotton producers need not adjust seedling disease control measures when these two nematicides are used.     

Effect of Entomopathogenic Nematodes on Mesocriconema xenoplax Populations in Peach and Pecan

The effect of Steinernema riobrave and Heterorhabditis bacteriophora on population density of Mesocriconema xenoplax in peach was studied in the greenhouse. Twenty-one days after adding 112 M. xenoplax adults and juveniles/1,500 cm³ soil to the soil surface of each pot, 50 infective juveniles/cm² soil surface of either S. riobrave or H. bacteriophora were applied. Another entomopathogenic nematode application of the same density was administered 3 months later. The experiment was repeated once. Mesocriconema xenoplax populations were not suppressed (P ≤ 0.05) in the presence of either S. riobrave or H. bacteriophora 180 days following ring nematode inoculation. On pecan, 200 S. riobrave infective-stage juveniles/cm² were applied to the soil surface of 2-year-old established M. xenoplax populations in field microplots. Additional applications of S. riobrave were administered 2 and 4 months later. This study was terminated 150 days following the initial application of S. riobrave. Populations of M. xenoplax were not suppressed in the presence of S. riobrave.     

广西北部地区腐霉种类和地理分布研究

从广西北部地区作物根围采集土壤标样323个,分离纯化出腐霉菌株260个,共15种。其中7种为广西新记录,即棘腐霉Pythiumacanthicum,浮游腐霉P.aquatile,宽雄腐霉P.dissotocum,缺性腐霉P.elongatum,孤雌腐霉P.amasculinum,禾生腐霉P.graminicola,绚丽腐霉P.pulchrum。优势种为瓜果腐霉P.aphanidermatum,其余7种为刺腐霉P.spinosum,德里腐霉P.deliense,昆明腐霉P.kunmingense,南宁腐霉P.nanningense,刺器腐霉P.acanthophoron,终极腐霉P.ultimum,镰雄腐霉P.falciforme。同时对腐霉的地理分布情况进行了初步探讨,发现腐霉分布有着明显的地域性。     

Host Specificity of Pathogenic Pythium Species: Implications for Tree Species Diversity

In the Janzen–Connell hypothesis, host-specific natural enemies enhance species diversity and influence the structure of plant communities. This study tests the explicit assumption of host specificity for soil pathogens of the genus Pythium that cause damping-off disease of germinating seeds and seedlings. We isolated Pythium spp. from soil of a tropical forest in Panama. Then, in an inoculation experiment, we determined the pathogenicity of 75 tropical isolates of unknown pathogenicity and seven pathogenic temperate isolates of Pythium on seeds and/or seedlings of eight tropical tree species. Only three tropical isolates, one identified as P. ultimum and two as P. aphanidermatum , were pathogenic. Tropical pathogenic isolates were pathogenic on 4–6 of eight tree species. Temperate isolates were pathogenic on 0–4 of eight species, indicating that some tropical tree species are susceptible to novel isolates of Pythium . No tree species was susceptible to all isolates and two species were not susceptible to any isolate. Collectively, these results indicate that these Pythium isolates vary widely in their pathogenicity, causing differential mortality of potential host species; likewise, the tree species vary in their susceptibility to a given Pythium isolate. These differences in pathogenicity and susceptibility indicate some support for the Janzen–Connell assumption of host specificity. While they are not restricted to a single species, their intermediate level of specificity suggests that Pythium spp. have the potential to have some effect on forest community structure and diversity.     

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

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

Biological Control of the Phytoparasitic Nematode Mesocriconema xenoplax on Peach Trees

Seven fluorescent Pseudomonas spp. capable of inhibiting reproduction of Mesocriconema xenoplax have been isolated from soil sites that suppress both nematode multiplication and Peach Tree Short Life (PTSL). One of these seven strains, Pseudomonas sp. BG33R, inhibits M. xenoplax multiplication in vivo and egg hatch in vitro. Mesocriconema xenoplax populations on peach seedlings inoculated with BG33R and planted into soil-solarized field plots remained at or below the economic threshold for nematicide treatment in South Carolina for nearly 18 months. Soil solarization alone induced a shift toward a microbial community that was suppressive to nematode multiplication. Additionally, five Tn5 mutants of BG33R, lacking the ability to kill eggs, have been generated. The Tn5 insertion site in each mutant has been cloned and sequenced. DNA sequence analysis has revealed a high degree of homology to several genes of interest because of their potential involvement in the production of the egg-kill factor. These Tn5 egg-kill negative mutants also no longer produce protease or salicylic acid while producing nearly twice the amount of fluorescent siderophore as the wild type parent.     

Pythium species associated with cavity spot on carrots in Norway

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

Effect of biocontrol strains of Trichoderma on plant growth, Pythium ultimum polulations, soil microbial communities and soil enzyme activities

Five strains of Trichoderma with known biocontrol activities were assessed for their effect upon pea growth and their antagonistic activity against large Pythium ultimum inocula. The effect of Trichoderma inocula upon the indigenous soil microflora and soil enzyme activities in the presence and absence of Pythium is assessed. In the absence of Pythium, Trichoderma strain N47 significantly increased the wet shoot weight by 15% but did not significantly affect the dry weight, whilst strains T4 and N47 significantly increased the root weights by 22% and 80%) respectively. Strains TH1 and N47 resulted in significantly greater root lengths. Pythium inoculation significantly reduced the root length and the number of lateral roots and nodules, and significantly increased the root and rhizosphere soil fungal populations. Pythium inoculation significantly reduced the plant wet and dry shoot weights and significantly increased the wet and the dry shoot/root ratio. All the Trichoderma strains reduced the number of lesions caused by Pythium and increased the number of lateral roots. The effect of the Pythium on emergence and shoot growth was significantly reduced by all the Trichoderma strains except strain To10. Inoculation with Trichoderma strains TH1 and T4 resulted in significantly greater wet root weights (62% and 57%, respectively) in the presence of Pythium compared to the Pythium control. Strain N47 significantly increased the shoot/root ratio compared to the Pythium control. Inoculation with Trichoderma strains T4, T12 and N47 significantly reduced Pythium populations. Pythium increased the activity of C, N and P cycle enzymes, whilst four Trichoderma strains reduced this effect, indicating reduced plant damage and C leakage. Overall, strains T4 and N47 had the greatest beneficial characteristics, as both these strains improved plant growth in the absence of Pythium and reduced plant damage in the presence of Pythium. The dual properties of these strains improve the commercial application, giving them an advantage over single action inocula, especially in the absence of plant pathogens.     

Seasonal Fluctuations of Soil and Tissue Populations of Ditylenchus dipsaci and Aphelenchoides ritzemabosi in Alfalfa

Population dynamics of A. ritzemabosi and D. dipsaci were studied in two alfalfa fields in Wyoming. Symptomatic stem-bud tissue and root-zone soil from alfalfa plants exhibiting symptoms of D. dipsaci infection were collected at intervals of 3 to 4 weeks. Both nematodes were extracted from stem tissue with the Baermann funnel method and from soil with the sieving and Baermann funnel method. Soil moisture and soil temperature at 5 cm accounted for 64.8% and 61.0%, respectively, of the variability in numbers of both nematodes in soil at the Big Horn field. Also at the Big Horn field, A. ritzemabosi was found in soil on only three of the 14 collection dates, whereas D. dipsaci was found in soil on 12 dates. Aphelenchoides ritzemabosi was found in stem tissue samples on 9 of the 14 sampling dates whereas D. dipsaci was found on all dates. Populations of both nematodes in stem tissue peaked in October, and soil populations of both peaked in January, when soil moisture was greatest. Numbers of D. dipsaci in stem tissue were related to mean air temperature 3 weeks prior to tissue collection, while none of the climatic factors measured were associated with numbers of A. ritzemabosi. At the Dayton field, soil moisture plus soil temperature at 5 cm accounted for 98.2% and 91.4% of the variability in the soil populations of A. ritzemabosi and D. dipsaci, respectively. Aphelenchoides ritzemabosi was extracted from soil at two of the five collection dates, compared to extraction of D. dipsaci at three dates. Aphelenchoides ritzemabosi was collected from stem tissue at six of the seven sampling dates while D. dipsaci was found at all sampling dates. The only environmental factor that was associated with an increase in the numbers of both nematodes in alfalfa stem tissue was total precipitation 1 week prior to sampling, and this occurred only at the Dayton field. Numbers of A. ritzemabosi in stem tissue appeared to be not affected by any of the environmental factors studied, while numbers of D. dipsaci in stem tissue were associated with cumulative monthly precipitation, snow cover at time of sampling, and the mean weekly temperature 3 weeks prior to sampling. Harvesting alfalfa reduced the numbers of A. ritzemabosi at the Big Horn field and both nematodes at the Dayton field.     

Ethylene insensitivity impairs resistance to soilborne pathogens in tobacco and Arabidopsis thaliana

Transgenic ethylene-insensitive tobacco (Tetr) plants spontaneously develop symptoms of wilting and stem necrosis when grown in nonautoclaved soil. Fusarium oxysporum, F. solani, Thielaviopsis basicola, Rhizopus stolonifer, and two Pythium spp. were isolated from these diseased Tetr plants and demonstrated to be causal agents of the disease symptoms. Pathogenicity of the two Pythium isolates and four additional Pythium spp. was tested on ethylene-insensitive tobacco and Arabidopsis seedlings. In both plant species, ethylene insensitivity enhanced susceptibility to the Pythium spp., as evidenced by both a higher disease index and a higher percentage of diseased plants. Based on the use of a DNA probe specific for Pythium spp., Tetr plants exhibited more pathogen growth in stem and leaf tissue than similarly diseased control plants. These results demonstrate that ethylene signaling is required for resistance to different root pathogens and contributes to limiting growth and systemic spread of the pathogen.     

Biocontrol of Pythium in the pea rhizosphere by antifungal metabolite producing and non-producing Pseudomonas strains

AIMS: Four well-described strains of Pseudomonas fluorescens were assessed for their effect on pea growth and their antagonistic activity against large Pythium ultimum inocula. Methods and RESULTS: The effect of Pseudomonas strains on the indigenous soil microflora, soil enzyme activities and plant growth in the presence and absence of Pythium was assessed. Pythium inoculation reduced the shoot and root weights, root length, and the number of lateral roots. The effect of Pythium was reduced by the Pseudomonas strains. Strains F113, SBW25 and CHAO increased shoot weights (by 20%, 22% and 35%, respectively); strains Q2-87, SBW25 and CHAO increased root weights (14%, 14% and 52%). Strains SBW25 and CHAO increased root lengths (19% and 69%) and increased the number of lateral roots (14% and 29%). All the Pseudomonas strains reduced the number of lesions and the root and soil Pythium populations, while SBW25 and CHAO increased the number of lateral roots. Pythium inoculation increased root and soil microbial populations but the magnitude of this effect was Pseudomonas strain-specific. Pythium increased the activity of C, N and P cycle enzymes, while the Pseudomonas strains reduced this effect, indicating reduced plant damage. CONCLUSION: Strains SBW25 and CHAO had the greatest beneficial characteristics, as these strains produced the greatest reductions in the side effects of Pythium infection (microbial populations and enzyme activities) and resulted in significantly improved plant growth. Strain SBW25 does not produce antifungal metabolites, and its biocontrol activity was related to a greater colonization ability in the rhizosphere. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first critical comparison of such important strains of Ps. fluorescens showing disease biocontrol potential.     

琅琊山虫生真菌物种多样性和季节变化

在皖东琅琊山自然保护区设置的20个样方中共采集672份虫生真菌标本,分属于4科8属20种.优势种依次为球孢白僵菌、粉拟青霉、环链拟青霉、布氏白僵菌和玫烟色拟青霉,其中球孢白僵菌占绝对优势,相对多度高达73.8%.选用物种多度、Shannon-Wiener指数H′和Pielou均匀度E对琅琊山虫生真菌物种多样性和季节分布进行研究发现,虫生真菌数量丰富但物种多样性指数较低;夏季虫生真菌菌株最为丰富,均匀度相对较低,随着气温及降雨量的下降,菌株数量急剧下降,均匀度相对较高;不同季节白僵菌属的数量波动大,但拟青霉属波动较小.     

Occurrence of Pythium spp. and Phytophthora spp. in Norwegian Greenhouses and their Pathogenicity on Cucumber Seedlings

Samples of tomato, lettuce and cucumber submitted for diagnosis to the Plant Protection Centre at the Norwegian Crop Research Institute and samples of soil, water and cucumber collected from greenhouses employing hydroponic cultures were examined for the occurrence of Pythium spp. and Phytophthora spp. Two species of Phytophthora and 16 species of Pythium were identified. Phytophthora cryptogea was found on tomato and lettuce. Phytophthora nicotianae was found on tomato fruit. Phytophthora was not found on cucumbers. Pythium irregulare and Pythium group F were the two most commonly found Pythium species in hydroponically cultivated cucumbers. A pathogenicity test with 56 isolates was performed on cucumber seedlings. The most aggressive species were Pythium aphanidermatum, P. irregulare, Pythium paroecandrum and Pythium ultimum.     

Pythium polare, a new heterothallic oomycete causing brown discolouration of Sanionia uncinata in the Arctic and Antarctic

Pythium polare sp. nov. is a new heterothallic oomycete species isolated from fresh water and moss from various locations in both the Arctic and Antarctic. This water mould is able to infect stems and leaves of Sanionia moss (Sanionia uncinata). Pythium polare causes brown discolouration in in vitro inoculation tests at 5 °C after 5 weeks of inoculation. It is characterized by globose sporangia with various lengths of discharge tubes releasing zoospores and aplerotic oospores with usually one to five antheridia. The sexual structures are only produced in a dual culture of antheridial and oogonial isolates. Phylogenetic analysis, based on ITS sequencing, places all isolated strains of P. polare in a unique new clade, hence it is considered a novel species. Pythium canariense and Pythium violae are the most closely related species of P. polare based both on morphology and the phylogenetic analysis.     

Influence of elevated CO(2) on the fungal community in a coastal scrub oak forest soil investigated with terminal-restriction fragment length polymorphism analysis

Sixteen open-top chambers (diameter, 3.66 m) were established in a scrub oak habitat in central Florida where vegetation was removed in a planned burn prior to chamber installation. Eight control chambers have been continuously exposed to ambient air and eight have been continuously exposed to elevated CO(2) at twice-ambient concentration (approximately 700 ppm) for 5 years. Soil cores were collected from each chamber to examine the influence of elevated atmospheric CO(2) on the fungal community in different soil fractions. Each soil sample was physically fractionated into bulk soil, rhizosphere soil, and roots for separate analyses. Changes in relative fungal biomass were estimated by the ergosterol technique. In the bulk soil and root fractions, a significantly increased level of ergosterol was detected in the elevated CO(2) treatments relative to ambient controls. Fungal community composition was determined by terminal-restriction fragment length polymorphism (T-RFLP) analysis of the internal transcribed spacer (ITS) region. The specificities of different ITS primer sets were evaluated against plant and fungal species isolated from the experimental site. Changes in community composition were assessed by principal component analyses of T-RFLP profiles resolved by capillary electrophoresis. Fungal species richness, defined by the total number of terminal restriction fragments, was not significantly affected by either CO(2) treatment or soil fraction.     

Fungal occurrence,disease incidence and severity,and yield of maize symptomatic for seedling disease in Mississippi

A study was conducted in Mississippi from 1995 to 1997 comparing soil rhizosphere fungi isolated from Pioneer 3167 hybrid maize (Zea mays L.) planted on Brooksville silty clay and Memphis silt loam soils. Maize seedlings were collected over four sampling dates from conventional and no-tillage plots. Eleven fungal genera consisting of nineteen species were isolated from these plants; Trichoderma spp. were most frequently isolated, followed by Fusarium spp. The highest disease incidence occurred in tilled plots of the latest planting date on Brooksville silty clay when samples were collected 17 days after planting. Root disease was most severe in 1996 from seedlings planted on the last planting date in tilled plots sampled 17 days after planting. Yields were significantly (P ≤ 0.05) higher on Brooksville silty clay soil than on Memphis silt loam in both 1995 and 1996. Yields were highest from no-tillage plots and from maize planted on the earliest date. There was a significant correlation between incidence of root infection and disease severity. There was no correlation between the incidence of root infection and yield or between disease severity and yield at either location. This revised version was published online in June 2006 with corrections to the Cover Date.     

The role of golf courses in maintaining genetic connectivity between common frog (Rana temporaria) populations in an urban setting

We studied population size, genetic diversity and differentiation of common frog (Rana temporaria) populations at urban golf courses and reference natural ponds in the greater Helsinki region, southern Finland. A total of 248 tadpoles from 12 locations (six golf courses, six reference sites) were genotyped with 13 polymorphic microsatellite markers. The most urban populations, situated in northern Helsinki, were the largest breeding sites having >120 (golf courses) and >200 (reference sites) spawn clumps at the time of sampling. On average, there was no difference in the number of spawns between the anthropogenic ponds at golf courses and the natural water bodies. Genetic variation within populations was substantial (H _O = 0.68) while genetic differentiation between populations was low (F _ST = 0.016; average distance = 17.6 km). The golf course populations did not differ from natural populations in terms of genetic variability or differentiation. Hence, our results suggest that golf courses contribute positively to urban amphibian populations by providing suitable water bodies for reproduction and green corridors for dispersal, thus preventing isolation and loss of genetic variability within populations.