Ultimum Trow Research Articles

Inhibition of Pythium ultimum in molecular fractions from gel filtration of soil extracts.

Filtered samples of aqueous extracts from clay and sandy soils (pH 5 to 6, Ontario) were fractionated using Sephadex and Bio-Gel. Responses of extension growth to the fractions were bioassayed with mycelia of Pythium ultimum Trow. Of 66 samples, 48% contained 1, or often several fractions that fully or partly inhibited growth; 5% contained stimulatory fractions; and 9% contained fractions with both inhibitory and stimulatory action; in 38% all fractions were without significant effects (at p < 0.01). Of 12 samples that in toto failed to exhibit, 4 nevertheless contained inhibitory fractions; this suggested a counteraction of inhibition by either stimulatory or nonstimulatory substances. Counteraction in several extracts was demonstrated with 36 fractions that were bioassayed both in purified agar (all 36 inhibited) and in agar amended with 0.5% yeast extract and 5% sucrose (only 1 inhibited). Estimations of the approximate molecular weights of the inhibitors showed that there were many different inhibitors, most of these (74%) with molecular weights between 3000 and 55 000. The results are considered as direct evidence for non-volatile inhibitors contributing to mycostatic situations in soils.

Infection of seeds of Pisum sativum by Pythium ultimum

Susceptibility of pea seeds to Pythium ultimum Trow, was negatively correlated with the proportion of seedlings emerging in the field and was associated with the quantity of soluble carbohydrates and nitrogenous compounds leached into moist sand. Pathogenicity was enhanced when seeds were coated with leachate or glucose but not when nutrients were added to sand in which they were sown. There were fewer macroscopic injuries and microscopic cracks in the testas of the less susceptible than in the more susceptible seeds, in which the cracks allowed passage of the fungus to infect the cotyledons. Faults in the seed coat are regarded as important in enhancing susceptibility to the fungus.

Evidence of Heritable Resistance to Fusarium solani f. sp. pisi and Pythium ultimum in Peas1

Ninety pea (Pisum sativum L.) populations were produced by crossing, in all possible combinations, five lines reported to be resistant to pea root and five commercial lines. Results from pathological tests conducted in soil artificially infested with F. solani (Mart.) Appel & Wr. sp. pisi (F. R. Jones) Snyd & Hans or P. ultimum Trow. did not agree with test results from soil naturally inrested with these pathogens and indicated that other pathogens or factors may be important in field soil. General combining ability effects were important in naturally and artifically infested soil, indicating a fixable amount of genetic variance for resistance to these pathogens. A highly significant correlation (r = .60, df = 53) between disease indices in soil artificially infested with F. solani and P. ultimum indicated that resistance to the two pathogens may be conditioned by the same factors.

An agar medium for the isolation and macroscopic recognition of Phytophthora spp. from soil on dilution plates.

An agar medium containing corn oil and antimicrobial compounds was developed on which Phytophthora colonies can be isolated and recognized macroscopically after 4 days of incubation. The presence of Pythium ultimum Trow, did not interfere with the recognition of P. cactorum colonies.The medium is suitable also for the isolation from soil of Phytophthora nicotianae var. parasitica, P. cinnamomi, and P. drechsleri, but less satisfactory for the enumeration of P. cryptogea, P. cambivora, and P. megasperma var. sojae.

Lactate Dehydrogenases in Oomycetes

Lactate dehydrogenases [D(-)-lactate: NAD oxidoreductase, E.C. 1.1.1.28] have recently been detected in a number of lower fungi particularly among the Oomycetes (Gleason and Price, 1969; Warren and Mullins, 1969; LeJohn, 1971) and the Chytridiomycetes (Gleason and Price, 1969; LeJohn, 1971). The Oomycetes known to synthesize these enzymes are Apodachlya brachynema (Hildebrand) Pringsheim, Araiospora sp., Sapromyces elongatus (Cornu) Coker, Mindeniella spinospora Kanouse, Rhipidium sp., Aqualinderella fermentans Emerson & Weston, Achlya ambisexualis Raper, Aphanomyces laevis DeBary, Pythium ultimum Trow, and Phytophthora dreschleri Tucker (Gleason and Price, 1969; Warren and Mullins, 1969; LeJohn, 1971; Gleason, unpublished data). The purpose of this study was to compare certain physical and kinetic properties of the lactate dehydrogenases from several of the Oomycetes listed above. The fungi used for this investigation were kindly made available by the following: Pythium ultimum (67-1) by Dr. Joseph Hancock, Department of Plant Pathology, University of California, Berkeley; Aphanomyces laevis (107-52) by Dr. Torgny Unestam, Institute of Physiological Botany, University of Uppsala; and the members of the Leptomitales by Dr. Ralph Emerson, Department of Botany, University of California, Berkeley. The methods for growth of the fungi and for preparation of cellfree extracts were the same as those described by Gleason and Price (1969). The reaction rates were measured spectrophotometrically at room temperature by a Zeiss PMQII spectrophotometer equipped with a strip chart recorder. The standard assay mixture for the backward reaction contained sodium pyruvate (3.3 X 10-4 M), reduced nicotinamide-adenine dinucleotide (NADH) (1.4 x 10-4 M), potassium phosphate buffer (0.1 M, pH 7.0), and enzyme in a final volume of 3.0 ml. The standard assay mixture for the forward reaction contained D(-)lithium lactate (0.1 M), NAD (2.0 X 10-4 M), tris hydrochloride buffer (0.1 M, pH 8.5), and enzyme in a final volume of 3.0 ml. The rate of the reaction was determined by change in absorbance at 340 mu

A Comparative Study of Two Components of the Poinsettia Root Rot Complex

THE MOST IMPORTANT pathological problem encountered by commercial growers of poinsettias ( Euphorbia piilcheirima Willd.) is caused by a complex of fungi composed of Thielaviopsis basicola (Berk. & Br.) Ferr., Rhizoctonia solani Kuhn, and Pythium ultimum Trow. Collectively these three organisms cause a condition referred to as the poinsettia root rot complex. Since more research has been conducted on P. ultimum and R. solani than on T. basicola, the present research was originally designed to investigate some of the environmental factors affecting the growth of Thielaviopsis and the development of the root rot caused by it. However, another fungus, Chalaropsis thielavioides Peyronel, frequently was obtained in isolations from diseased greenhouse poinsettias. Since C. thielavioides had not been reported as being a part of the poinsettia root rot complex, the author decided to investigate its importance as a pathogen on poinsettias. Numerous similarities between C. thielavioides and T. basicola were evident. The object of this work was to compare the two fungi. Prior to undertaking such a study, the pathogenicity of Chalaropsis on poinsettias had to be established. A comparison of Chalaropsis and two isolates of Thielaviopsis was made to determine the effects of environment on the growth of the fungi and the ability of the two fungi to produce disease symptoms on poinsettias.

Characterization and classification of mycorrhizae of Douglas fir. II. Pseudotsuga menziesii + Rhizopogon vinicolor

A common tuberculate ectomycorrhiza of Douglas fir in the Pacific Northwest, described earlier by Trappe, is further examined and defined. Tubercles consist of an outer rind of aseptate, amber, thick-walled hyphae encasing tightly packed inner elements mantled with septate, hyaline, thin-walled hyphae. Reported as a Phycomycete and a Basidiomycete, respectively, the two hyphal forms actually belong to a single fungus, Rhizopogon vinicolor A. H. Smith; cultural characteristics of this fungus are described. Pure culture mycorrhiza syntheses with both mycorrhizal and sporocarpic isolates and Douglas-fir seedlings are reported. Antagonism tests revealed the following inhibition of root pathogens by R. vinicolor: strong—Phytophthora cinnamomi Rands, Pythium debaryanum Heese, and Pythium sylvaticum Campbell & Hendrix; moderate—Fomes annosus (Fr.) Cke. and Poria weirii Murr.; and weak or none— Fusarium oxysporum f. pini (Hartig) Snyd. & Hans., Pythium ultimum Trow, Rhizoctonia solani Kuehn, and Macrophomina phaseoli (Maubl.) Ashby.

Currutaca: A Pythium Soft Rot of Xanthosoma and Colocasia spp. in Puerto Rico

The fungus Pythium ultimum Trow, was found the causal agent of a soft rot disease of taniers [Xanthosoma sagittaefolium (L.) Schott.] and taros [Colocasia esculenta (L.) Schott.]. A yellow pigmented bacterium occasionally associated with the rot turned out to be a saprophyte. Roots of diseased plants became mucilaginous and decayed rapidly. The infected rhizomes were soft and cheesy. The base of infected petioles were water-soaked and the leaves collapsed. The fungus was isolated in pure culture and grew well in most culture agar media, producing abundant sporangia at 20-30° C., but failed to grow at temperatures below 12° C. or above 36° C. Varieties of taniers and taro inoculated with P. ultimum showed differential degrees of susceptibility to infection when kept in an environment of 90-percent relative humidity and a temperature of 20° C. Definitive measures to control the disease effectively are not known. Use of clean "seed", planting in well drained soils, and crop rotation practices, however, have been recommended.

Interaction of nutrients and four antifungal antibiotics in their effects on Pythium species in vitro and in soil

Griseofulvin, streptomycin, aureomycin, and actidione, with and without nutrients, were tested againstPythium ultimum Trow (two strains),P. irregulare Buis., andP. rostratum Butler. Streptomycin was tolerated in the highest concentration, and actidione and aureomycin in the lowest.P. rostratum, a saprophytic species, was more resistant to the antibiotics than the pathogenic species, which were inhibited at low concentrations. The two strains ofP. ultimum behaved differently, the oogonial strain being more tolerant than the sporangial strain. Yeast extract was usually superior to sucrose or asparagine in counteracting the toxicity of antibiotics.

The lipids of Pythium ultimum

The lipids of the oömycete, Pythium ultimum Trow, grown under controlled environmental conditions, were identified and quantitatively determined. Lipid composition data are presented on the fungus grown at both 20° and 30° for a period of 14 days. The fungus contained from 3 to 48% total lipid depending upon its stage of growth. The triglycerides were most abundant, with lesser amounts of free fatty acids, phosphatides and monoglycerides. The major phosphatides were phosphatidyl choline, phosphatidyl ethanolamine, lysophosphatidyl ethanolamine and phosphatidyl glycerol. There were smaller amounts of diphosphatidyl glycerol, phosphatidyl inositol, phosphatidic acid and lysophosphatidyl choline. The major fatty acids were palmitic, oleic, linoleic, docosenoic, and docosadienoic acid. The fatty acid composition of the individual lipid classes was also examined.

The influence of nitrogenous compounds on growth of Pythium species.

The utilization of N compounds by P. ultimum Trow (strain I and II), P. rostratum Butler, and P. irregulare Buisman was examined in a chemically denned medium under controlled conditions in surface culture. All species were able to metabolize nitrate, ammonium, and organic nitrogen, and the amount of growth varied with the nitrogen source. In general, yeast extract, peptone, glycine, serine, histidine, cysteine, asparagine, aspartic acid, and glutamic acid supported favorable growth, whereas γ-aminobutyric acid, threonine, and alanine supported poor growth of these fungi. The addition of succinic acid at 0.02 M concentration to ammonium compounds further increased growth of four isolates.Preferential utilization of amino acids from a given mixture was recorded using paper chromatographic techniques. All four isolates gave more vegetative growth on mixtures of amino acids than when they were supplied singly.

A Taxonomic Study of Pythium Irregulare and Related Species

Positive identification of many Pythium species is a difficult task. When numerous isolates are examined, continuous series resulting from indistinct diagnostic characters and intermediate forms frequently can be recognized. Pythium spinosumt Sawada, P. irregulare Buisman, P. debaryanum Hesse, P. ultimum Trow, and P. pareocandrum Drechsler exemplify such a series. These species have spherical sporangia of approximately the same size. Morphological differences in the oospore, oogonium, and antheridium provide the diagnostic criteria separating these species. By definition P. spinosum with its plerotic oospore is readily distinguished from P. irregulare with its aplerotic oospore. Both species are characterized by an echinulate oogonium and a single monoclinous antheridium (5). Buisman (1) separated P. irregulare from P. debaryanum by its irregular oogonium which often has protuberances. Middleton (5) emphasized the presence of protuberances in P. irregulare as the principal difference between these 2 species. Conversely, Drechsler (4) reported a tendency for the development of protuberances in some cultures of P. debaryanum. Middleton (5) described P. debaryanum, P. ultimum, and P. pareocandrum as having an aplerotic oospore and a smooth oogonium. He rejected van Luijk's contention of synonymity based on the antheridial character of P. debaryanum and P. ultimum and distinguished P. debaryanum by its plurality of antheridia that originate some distance from the oogonium. Trow (7) characterized P. ultimum by its thickened oospore wall and one monoclinous antheridium originating adjacent to the oogonium. He sometimes observed 2 diclinous antheridia per oogonium in luxuriant cultures. Drechsler (2, 3) differentiated P. pareocandrum from P. ultimum by its non-thickened oospore wall, plurality of antheridia, and oospore suggestive of P. debaryanum.

ROOT EXUDATES FROM RED PINE SEEDLINGS AND THEIR EFFECTS ON PYTHIUM ULTIMUM

The role of root exudates in interaction between pine roots (Pinus resinosa Ait.) and Pythium ultimum Trow was studied. By paper chromatographic methods three sugars and 13 amino acids were definitely identified in the root exudate. Gamma amino butyric acid was most abundant, although asparagine, glycine, serine, and alanine were also present in large amounts. Valine, leucine, glutamine, aspartic acid, glutamic acid and proline were detected in smaller quantities. Among the sugars, both glucose and fructose were abundant while traces of arabinose were detected. When supplied singly, all amino acids and sugars except serine, leucine and arabinose, stimulated growth of the pathogen and germination of sporangia. Mixtures of sugars or mixtures of three or four amino acids were very effective in stimulating mycelial growth and development of germ tubes. The kind of nutrient influenced the number of germ tubes per sporangium. Favorable nutrient combinations, viz. mixtures of sugars, mixtures of three or four amino acids or root exudate solution, produced two to nine germ tubes, while unfavorable ones, viz., glycine, arabinose and serine, etc., produced one or two germ tubes.

New Pythium Species from South Australia

In a survey of the Pythium flora of several tree nurseries in South Australia (Vaartaja, 1964), the following species were most commonly found: P. irregidare Buisman, P. mamillatum Meurs, P. ultimum Trow, P. oligandrum Drechsler, P. acanthicum Drechsler, P. iwayamai Ito, and P. rostratum Butler. Among less frequently occurring species, three were distinctly different from any previously described. Their descrip? tions, which follow, are based on observations of pure cultures, mainly on cornmeal agar and, partly, on a great variety of other media. The zoospore stages were usually observed in cultures kept at low tempera? tures (12-15 C) and frequently rinsed with sterile water or soil extract. The terms used and the taxonomic approach to the genus are the same as those of Middleton (1943). The type cultures have been sent to the Centraalbureau voor Schimmelcultures, Baarn, Holland.

The growth of seedlings of some pioneer plantsd and the micro-organisms associated with their roots

Aseptic seedlings of Buddleja davidii French and Senecio squalidus L. on agar slopes with low nitrogen content grew well when inoculated with Moniliopsis sp., Mortierella alpina Peyron., M. renispora Dixon-Stewart, Pythium ultimum Trow or Rhizoctonia sp., while uninoculated seedlings made no growth and gradually died. The effect may have been due to some heat-labile hormone-like compound produced by the fungus, or to some physiological effect. The fungi penetrated the seedling roots, though after 6 months or less typical mycorrhizal structures were not formed. The course of development of natural endotrophic mycorrhiza on these and other similar pioneer plants is described with special reference to two species of Endogone.

Abundance of Pythium Species in Nursery Soils in South Australia

The abundance of Pythium propagules and frequency of 11 Pythium species were estimated in 16 soil plots in South Australian forest nurseries, in one ornamental nursery. and in one pine forest. A soil-plating technique Was utilized and a medium developed which was highly selective for these species and allowed their identification directly on the plates. The number of Pythium propagules in a unit volume of soil varied greatly. being lowest (24 per cubic centimetre) in the forest and highest (226 per cubic centimetre) in an alkaline loamy nursery soil. Loams had significantly more Pythium propagules than sandy soils. The immediately previous usage and soil amendments usually made no great difference to the total number of Pythium propagules. It is concluded that the abundance of pathogenic Pythium spp. in these nurseries is not the main factor governing the great variation found in the disease incidence of conifer seedlings. P. irregulare Buisman was frequent in all plots except the forest, and P. mamillatum Meurs in most sandy nurseries. The frequencies of P. ultimum Trow, P. paroecandrum Drechsler, P. rostratum Butler, P. iwayamai Ito, P. oligandrum Drechsler, P. acanthicum Drechsler, and P. echinulatum Matthews varied inconsistently between the plots. A new species, coded as P. col, was rare but occurred in eight plots. Another new species (P. dis) occurred only in one plot but frequently in this. This was a nursery freshly established on grassland pasture which differed from the others also in the frequencies of several other species.

Soil physical factors and the ecology of fungi: II. Behaviour of Pythium ultimum at small soil water suctions

With aluminium oxide grits as artificial soils and a technique which accurately controlled the moisture régime, neither suction, within the range 1–400 μ. water, nor pore size, in the range 2·7–100 μ radius of pore neck, taken as separate factors, had a major effect on the rate of hyphal growth of Pythium ultimum Trow. Sexual reproduction was unaffected by suction within the experimental range but was greatly reduced in intensity by a reduction in equivalent pore diam. from 45 to 15 μ. (diam. of oogonium c . 20μ). Growth from marginal disk agar inocula was little affected by the pores of the grit being air- or water-filled but growth occurred from submarginal disks only into drained grits. The frequent occurrence of fungi such as P. ultimum in wet environments is thus probably due as much to a toleration of conditions of poor gaseous exchange as to a positive need for abundant water. The ability to grow in waterlogged soils will, however, vary inter alia with the physiological status of the inoculum.

The Root Rot-Fusarium Wilt Complex of Peas

At least four fungal pathogens are involved in the root rot-Fusarium wilt complex of peas which is a serious problem following intensive cropping of peas in South Australia. The pathogens are Fusarium oxysporum f. pisi race 2 Snyder & Hansen, F. solani f. pisi Snyder & Hansen, Pythium ultimum Trow, and Ascochyta pinodella L. K. Jones. In susceptible pea cultivars there is a marked interaction between F. oxysporum and P. ultimum. P. ultimum alone causes initial stunting from which plants gradually recover; F. OX1Jsporum alone probably CRuses little damage; both fungi together CRuse initial stunting followed by severe wilt symptom about 6 weeks after sowing and death 2 weeks later. The importance ofF. solani and A. pinodella has not been fully determined, but they probably cause only minor damage.

Soil Disinfection

Summary The effect of dimethyl tetrahydrothiadiazine thione, sodium methyldithiocarbamate, and methylmercuric dicyandiamide on emergence of pea seeds was investigated in soil inoculated with Pythium ultimum Trow. With increased inoculum potential the first two materials proved superior to the third in giving increased emergence and growth of seedlings.

The Occurrence of Amino Acids in Four Species of Pythium

The use of chemical techniques as an aid in taxonomy has received considerable attention in the last few years. Paper chromatography has been used by many workers to determine quantitative or qualitative differences, or both, in fungi (1, 2, 3, 4). The object of this work was to determine if differences existed in the free amino acid content between isolates of Pythium aphanidermatum (Edson) Fitzpatrick, Pythium arrhenomanes Drechsler, Pythium debaryanum Hesse, and Pythium ultimum Trow.