Pathogenicity of four Pythium species to wheat, barley, peas and lentils

Pythium heterothallicum, P. irregulare, P. torulosum and P. ultimum var. sporangiiferum were compared for pathogenicity to seedlings of winter wheat, spring barley, lentils and peas in growth chambers at 5, 10, 15, 20 and 25 C. These four fungi are among the most commonly isolated Pythium species from wheat roots and wheat-field soils in eastern Washington and northern Idaho, USA, where wheat, spring barley, lentils and peas are grown in various rotations. Pathogenicity was determined in artificially infested soils (500 propagules per g) based on ability to cause pre-emergence death and post-emergence stunting of seedlings. P. ultimum var. sporangiiferum caused significant pre-emergence death of the wheat at 15–25 C, lentils at 10–25 C. and peas at 5 25 C. P. irregulare caused pre-emergence death only of peas and only at 5 C. With the possible exception of lentils at 25 C, P. heterothallcum and P. torulosum caused no pre-emergence death of any of the four plant species. None of the species caused pre-emergence death of spring barley. P. ultimum var. sporangiiferum caused the most post-emergence stunting of wheat, peas and lentils at 10 C and above. Pythium irregulare caused as much or more stunting than P. ultimum var. sporangiiferum on wheat, lentils and peas at 5 C, and was the most pathogenic species on barley at 10, 20 and 25 C. P. irregulare caused significantly more post-emergence stunting of wheat at 5 C with than without chaff (added as a food base for the pathogen); this was not offset by adding ammonium sulphate with the chaff.     

Cyclic Lipopeptide Surfactant Production by Pseudomonas fluorescens SS101 Is Not Required for Suppression of Complex Pythium spp. Populations

ABSTRACT Previously, the zoosporicidal activity and control of Pythium root rot of flower bulbs by Pseudomonas fluorescens SS101 was attributed, in part, to the production of the cyclic lipopeptide surfactant massetolide A. The capacity of strain SS101 and its surfactant-deficient massA mutant 10.24 to suppress populations and root infection by complex Pythium spp. communities resident in orchard soils was assessed on apple and wheat seedlings and on apple rootstocks. Both strains initially became established in soil and persisted in the rhizosphere at similar population densities; however, massA mutant 10.24 typically was detected at higher populations in the wheat rhizosphere and soil at the end of each experiment. Both strains effectively suppressed resident Pythium populations to an equivalent level in the presence or absence of plant roots, and ultimately suppressed Pythium root infection to the same degree on all host plants. When split-root plant assays were employed, neither strain suppressed Pythium spp. infection of the component of the root system physically separated from the bacterium, suggesting that induced systemic resistance did not play a role in Pythium control. Strain SS101 only marginally suppressed in vitro growth of Pythium spp. and growth was not inhibited in the presence of mutant 10.24. When incorporated into the growth medium, the cyclic lipopeptide massetolide A significantly slowed the rate of hyphal expansion for all Pythium spp. examined. Differences in sensitivity were observed among species, with Pythium heterothallicum, P. rostratum, and P. ultimum var. ultimum exhibiting significantly greater tolerance. Pythium spp. populations indigenous to the two soils employed were composed primarily of P. irregulare, P. sylvaticum, and P. ultimum var. ultimum. These Pythium spp. either do not or rarely produce zoospores, which could account for the observation that both SS101 and mutant 10.24 were equally effective in disease control. Collectively, the results showed that (i) Pseudomonas fluorescens SS101 is very effective in controlling diverse Pythium populations on different crops grown in different soils and (ii) production of the cyclic lipopeptide massetolide A does not play a significant role in disease suppression. Other, as yet undefined mechanisms appear to play a significant role in the interaction between P. fluorescens SS101 and soilborne Pythium spp. communities.     

Identification and Quantification of Pathogenic Pythium spp. from Soils in Eastern Washington Using Real-Time Polymerase Chain Reaction

ABSTRACT Traditional methods of quantifying Pythium spp. rely on the use of selective media and dilution plating. However, high variability is inherent in this type of enumeration and counts may not be representative of the pathogenic population of Pythium spp. Variable regions of the internal transcribed spacer of the rDNA were used to design species-specific primers for detection and quantification of nine Pythium spp. from soils in eastern Washington. Primer pairs were designed for Pythium abappressorium, P. attrantheridium, P. heterothallicum, P. irregulare group I, P. irregulare group IV, P. paroecandrum, P. rostratifingens, P. sylvaticum, and P. ultimum and used with real-time polymerase chain reaction. Standard curves were generated for each of the species using SYBR Green I fluorescent dye for detection of amplification. Seventy-seven isolates of Pythium were screened to confirm specificity of each primer set. DNA was extracted from soil and standard curves were generated for P. irregulare group I, P. irregulare group IV, and P. ultimum to correlate populations of each species in the soil with quantities of DNA amplified from the same soil. Examination of raw field soils revealed results similar to those observed in previous studies. This new technique for the quantification of Pythium spp. is rapid and accurate, and will be a useful tool in the future study of these pathogenic Pythium spp.     

Larval Bradysia impatiens (Diptera: Sciaridae) potential for vectoring Pythium root rot pathogens

A series of laboratory experiments were conducted to investigate the capacity of Bradysia impatiens (Johannsen) larvae to ingest propagules from two strains each of Pythium aphanidermatum (Edson) Fitzp. and P. ultimum Trow and transmit the pathogens to healthy geranium seedlings on a filter-paper substrate in petri dishes. The capacity of fungus gnat larvae to transmit P. aphanidermatum to seedlings rooted in a commercial peat-based potting mix and germination of Pythium oospores and hyphal swellings before and after passage through the guts of larval fungus gnats were also examined. Assays revealed that Pythium spp. transmission by larval fungus gnats varied greatly with the assay substrate and also with the number and nature of ingested propagules. Transmission was highest (65%) in the petri dish assays testing larvae fed P. aphanidermatum K-13, a strain that produced abundant oospores. Transmission of strain K-13 was much lower (<6%) in plug cells with potting mix. Larvae were less efficient at vectoring P. ultimum strain PSN-1, which produced few oospores, and no transmission was observed with two non-oospore-producing strains: P. aphanidermatum Pa58 and P. ultimum P4. Passage of P. aphanidermatum K-13 through larval guts significantly increased oospore germination. However, decreased germination of hyphal swellings was observed following larval gut passage for strains of P. ultimum. These results expand previous studies suggesting that larval fungus gnats may vector Pythium spp.     

浙江省春玉米苗期致病腐霉的研究

作者于１９９０年４、５月间，从浙江省东阳及天台县的玉米病苗上，共分离鉴定到Ｐｙｔｈｉｕｍ ａｃａｎｔｈｏｐｈｏｒｏｍ，Ｐ ａｐｈａｎｉｄｅｒｍａｔｕｍ，Ｐ ｄｅｂａｒｙａｎｕｍ，Ｐ ｇｒａｍｉｎｉｃｏｌａ，Ｐ ｉｒｒｅｇｕｌａｒｅ，Ｐ ｓｐｉｎｏｓｕｍ Ｐ ｔａｒｄｉｃｒｅｓｃｅｎｓ Ｐ ｕｌｔｉｍｕｍ 等８个种的腐霉。接种试验表明，这８种腐霉都能引起玉米苗期根病，造成种子腐烂、根腐和死苗，其中Ｐ     

Prevalence and pathogenicity of spinach root pathogens of the genus Pythium in Sweden

Several pathogenic Pythium spp. were isolated from spinach roots in a 4-year disease survey in commercial spinach fields in southern Sweden. Heterothallic and zoospore-producing isolates belonging to the filamentous, non-inflated zoosporangia (F) group of van der Plaats-Niterink were most abundant, followed by P. sylvaticum, P. ultimum var. ultimum and P. heterothallicum. The heterothallic hyphal swelling (HS) group and P. tracheiphilum were found in a few plants. Two types of pathogenicity tests were performed, to measure the effects of seedling infection and of root infection of older plants. These tests showed P. ultimum var. ultimum to be the most severe spinach pathogen inducing pre- and post-emergence damping off as well as root rot of older plants. Also P. heterothallicum and P. tracheiphilum damaged both seedlings and older plants, Pythium sylvaticum, Pythium 'group HS' and Pythium 'group F'were pathogenic only to seedlings, P. ultimum var. ultimum and Pythium group F were isolated in significantly higher frequencies from spinach grown in the autumn season than in the spring. No clear relationships were found between Pythium prevalence and disease severity index of surveyed field plants, between Pythium prevalence and plant developmental stage, or between prevalence of Pythium and other pathogens isolated. This is the first report of P. heterothallicum and P. sylvaticum being pathogenic on spinach.     

Suppression of Seedling Damping-Off Caused by Pythium ultimum, P. irregulare, and Rhizoctonia solani in Container Media Amended with a Diverse Range of Pacific Northwest Compost Sources

ABSTRACT Suppression of seedling damping-off disease caused by Pythium spp. and Rhizoctonia solani is a potential benefit of formulating soilless container media with compost. Thirty-six compost samples from Pacific Northwest commercial composting facilities were analyzed for a number of physical, chemical, and biological properties, including suppression of damping-off caused by Pythium ultimum, P. irregulare, and R. solani. The samples were produced from diverse feedstocks and composting technol ogies; this was reflected in a large degree of variability in the measured properties. When mixed with sphagnum peat moss and inorganic aggregates, 67% of the compost samples significantly suppressed P. irregulare damping-off of cucumber, 64% suppressed P. ultimum damping-off of cucumber, and 17% suppressed damping-off of cabbage caused by R. solani. Suppression of Pythium damping-off was related to the potential of compost to support microbial activity and a qualitative index of ammonia volatilization. Suppression of Rhizoctonia damping-off was not related to any one compost factor. Currently available compost products potentially could provide commercially acceptable control of damping-off caused by Pythium spp., but it is necessary to fortify composts with microbial antagonists for the control of R. solani.     

Interactions Between Trichoderma harzianum Strain T22 and Maize Inbred Line Mo17 and Effects of These Interactions on Diseases Caused by Pythium ultimum and Colletotrichum graminicola

ABSTRACT Seed treatment with Trichoderma harzianum strain T22, which results in colonization of plant roots but little or no colonization of shoots or leaves, had substantial effects on growth of and disease expression in maize inbred line Mo17. Shoots and roots of 10-day-old seedlings grown in a sandy loam field soil were larger (roots were nearly twice as long) in the presence of T22 than in its absence. Both main and secondary roots were increased in size and area and the root hair area was greater with T22. However, root hair area per unit of root length was greater in control plants. Increased growth probably was due to direct stimulation of plant growth in addition to effects from biological control of deleterious microflora. Seedlings of Mo17 grown in autoclaved or mefenoxamtreated sandy loam field soil were larger than those produced in untreated soil. However, seedlings grown in the presence of T22, either in treated or untreated soil, were larger than those produced in its absence. Infestation of soil with Pythium ultimum had little effect upon growth of Mo17. The presence of T22 increased protein levels and activities of beta-1,3 glucanase, exochitinase, and endochitinase in both roots and shoots, even though T22 colonized roots well but colonized shoots hardly at all. With some enzymes, the combination of T22 plus P. ultimum gave the greatest activity. Plants grown from T22-treated seed had reduced symptoms of anthracnose following inoculation of leaves with Colletotrichum graminicola, which indicates that root colonization by T22 induces systemic resistance in maize.     

哈茨木霉菌(Trichoderma harzianum)和终极腐霉菌(Pythium ultimum)对玉米蛋白质组的影响(I)

 哈茨木霉(Trichoderma harzianum) T22菌株已普遍用于防治包括由终极腐霉(Pythium ultimum)引起的苗病或根腐病在内的各种病害。玉米自交系Mo17种子经T22处理后播种在接种腐霉或未接种的田间土壤内,5 d后取幼苗的根系或幼茎提取蛋白。结果表明:在接种腐霉菌的土壤内,未进行T22处理的5 d龄幼苗长势明显比对照差,而经T22处理的幼苗长势明显比对照好。T22和腐霉菌复合处理及T22单独处理对幼苗生长影响基本相同。本研究建立了蛋白质提取和双向电泳分离技术。通过双向电泳及相应的分析软件(PDQuest^TM 2-D softw are)可将不同处理的幼苗自交系蛋白进行分离。T22菌株处理的根系产生104种上游调控蛋白和164种下游调控蛋白,T22与腐霉菌复合处理可产生97种上游调控蛋白和150种下游调控蛋白,而用腐霉菌单一处理诱导的上游或下游蛋白的数量明显少于上述2个处理。T22或腐霉菌单一或复合处理的根系蛋白质组图谱与空白对照相比差异显著,它们与对照的蛋白质组图谱相似系数分别为0.72、0.51和0.49;T22与腐霉菌分别处理的蛋白质组图谱间也相差明显,两者的相似系数仅为0.65。进一步研究发现,T22菌体蛋白质组图谱与上述各种处理的蛋白质组图谱相似系数均很低,说明各种处理诱导后的幼苗根系蛋白质组组分主要来自植物本身,其变化主要因T22或腐霉菌的诱导所致。腐霉菌的侵染对寄主根系蛋白组图谱影响明显高于T22的作用。蛋白质组中各种蛋白质的质谱分析(M ALDI-TOF)与鉴定将另文发表。     

Amplified Fragment Length Polymorphism Analysis and Internal Transcribed Spacer and coxII Sequences Reveal a Species Boundary Within Pythium irregulare

ABSTRACT Pythium irregulare is a plant-pathogenic oomycete that causes significant damage to a variety of crops, including ornamentals and vegetables. Morphological as well as molecular studies have reported high levels of genetic diversity within P. irregulare sensu lato which has raised the question as to whether it is a single species or is actually a complex of morphologically similar (cryptic) species. In this study, we used amplified fragment length polymorphism (AFLP) fingerprinting and DNA sequence analysis of the internal transcribed spacer (ITS) region of the ribosomal genes (ITS region) and a portion of the mitochondrial cytochrome oxidase II gene and the spacer region between coxI and coxII to characterize 68 isolates of P. irregulare from the United States. The ITS sequence of a P. irregulare neotype at the CBS collection as well as ITS and coxII sequences for P. irregulare, P. spinosum, and P. sylvaticum from previous studies were included in our analysis. Cluster analysis identified a 19-isolate group (IR-II) that separated itself from the rest of the sample (IR-I). Population structure and sequence analyses supported the distinction of IR-I and IR-II and identified IR-II as P. irregulare sensu stricto. IR-I was designated Pythium sp. clade IR-I. Two insertion/deletion mutations and nine nucleotide substitutions in the ITS region and three in the sequence of coxII and the adjacent spacer region separated the two species. Additionally, they differed significantly (P > 0.01) in the frequency of 182 (77%) AFLP alleles. Gene flow results suggested that P. irregulare sensu stricto and Pythium sp. clade IR-I are cryptic species capable of exchanging favorable alleles (Nm = 0.72).     

Inhibition of polyamine biosynthesis in Phytophthora infestans and Pythium ultimum

Mycelial growth, polyamine concentrations and the activities of enzymes of polyamine biosynthesis and catabolism were examined in Phytophthora infestans and Pythium ultimum grown in the presence of difluoromethylornithine (DFMO), an inhibitor of omithine decarboxylase (ODC), and difluoro-methylarginine (DFMA), an inhibitor of arginine decarboxylase (ADC). Growth of P. infestans was reduced by DFMO and a mixture of DFMO + DFMA, but was increased by DFMA at concentratiotis of 5 MM and greater, Polyamine concentrations and ODC activity were significantly reduced in P. infestans grown on all inhibitor treatments. In contrast, growth, polyamine concentrations and enzyme activities were not affected in P, ultimum exposed to the inhibitors.
It seems unlikely that P. infestans and P. uttimum possess ADC activity, as neither of the products of ADC activity (agmatine and putrescine) could be detected in ADC assays. Although ODC from P. infestans was sensitive to DFMO, ODC from P. uttimum was insensitive to the inhibitor. Moreover, uptake of DFMO by P. infestans was three times greater than that observed by P. ultimum.     

Comparative pathogenicity of Pythium species associated with poor seedling establishment of rice in Southern Australia

Isolates of Pythium attributable to thirteen named species and to species group F (sensu van der Plaats-Niterink) were isolated from roots of field-grown rice seedlings showing poor root development and lack of vigour. P. arrhenomanes and group F were the most commonly isolated species. The roots of 15-week-old rice plants yielded only P. flevoense, P. vanterpoolii, P. rostratum and Pythium group G of which the latter two were not obtained from seedling roots.
In pot tests with representative Pythium isolates, P. arrhenomanes was most pathogenic to rice seedlings, causing pre- and post-emergence death and average reductions in shoot and root growth in surviving seedlings of 48 and 70%, respectively. P. irregulare killed seedlings before but not after emergence, whilst the response of rice seedlings to P. myriotylum apparently depended on the length and timing of a cold shock treatment. P. pyrilobum did not reduce seedling number or shoot growth but reduced root growth. P. vanterpoolii and group F were not pathogenic in the tests. P. coloratum, P. echinulatum, P. flevoense, P. oedochilum, P. oligandrum, P. periilum, P. pyrilobum and P. rostratum were isolated infrequently and had no adverse effects on seedlings in pathogenicity tests, although P. myriotylum, P. oligandrum , and P. periilum were associated with significant increases in shoot growth. P. tracheiphilum was isolated from one site but its pathogenicity was not tested.
This is the first record of P. coloratum, P. echinulatum, P. flevoense, P. irregulare, P. oedochilum, P. oligandrum, P. periilum, P. pyrilobum and P. tracheiphilum from rice roots.     

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

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

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     

Transformants of Trichoderma longibrachiatum Overexpressing the beta-1,4-Endoglucanase Gene egl1 Show Enhanced Biocontrol of Pythium ultimum on Cucumber

ABSTRACT Nine transformants of Trichoderma longibrachiatum with extra copies of the egl1 gene were studied for mitotic stability, endoglucanase production, and biocontrol activity against Pythium ultimum on cucumber seedlings. The transformants showed a significantly higher level of expression of the egl1 gene in comparison to the wild type under both inducing and noninducing growth conditions. Transformants with the egl1 gene under the control of a constitutive promoter had the highest enzymatic activity. Both the endoglucanase activity and the transforming sequences were stable under nonselective conditions. When applied to cucumber seeds sown in P. ultimum-infested soil, T. longibrachiatum transformants with increased inducible or constitutive egl1 expression generally were more suppressive than the wild-type strain.     

Rhizosphere Competence of Pythium oligandrum

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

Genetic variation among populations of Pythium irregulare in southern Australia

Isolates of Pythium irregulare were sampled from seven cereal crops throughout South Australia to determine the extent of genetic diversity within this pathogen and the scale of genetic differentiation among populations. Data derived from 29 individual restriction fragment length polymorphism (RFLP) loci differentiated 54 DNA fingerprints among the 92 isolates analysed. Some isolates had two alleles at several RFLP loci and were scored as heterozygous. One such isolate was selfed in vitro and segregation ratios in the progeny were not significantly different from those expected for allelic variation in a diploid. These data provided evidence that outcrossing occurs within P. irregulare and may contribute to the high level of genetic variation within the species ( D _T = 0·502). Allelic frequencies were significantly different among all seven populations and G _ST values showed significant genetic differentiation between populations. The average genetic identity among populations was low and hierarchical cluster analysis provided no clear evidence that populations formed geographically related groups. These analyses indicate low levels of interpopulation gene flow within P. irregulare and imply that population differentiation results from genetic drift.