Phytophthora Erythroseptica Research Articles

Field Observations on Blackleg in England1

ABSTRACTInoculation of seed tubers with Erwinia carotnvora (Jones) Bergey et al. var. atroseptica (van Hall) Dye before planting resulted in early death (especially with sprouted seed), blackleg stems and decreased yields. Apical inoculations had most, and stolon inoculations the least, effect on growth and yield. Even plants without blackleg symptoms were more stunted and had fewer stems than plants from uninoculated seed. Rotting of seed tubers often stopped in dry weather and started again later, probably caused, or assisted by, insects breaching the wound barrier.Many other organisms can also rot seed tubers including Phytophthora erythroseptica Pethybridge (pink rot), Fusarium spp. (dry rot), Verticillium spp. (wilts) and Rhizoctonia spp. (black scurf).The bacteria have been shown to spread along rows underground and to different stocks within the same field, presumably in air, but season to season spread by the potato digger has not been demonstrated.

Seasonal Changes in Molecular Forms of Acid Phosphatase and Ribonuclease of Potato Tubers and the Effects of Infection by Phytophthora erythroseptica

Sequential changes in total activity and molecular forms of acid phosphatase and ribonuclease from potato tubers were studied by seasonal assays and Sephadex gel filtration. Ribonuclease and p -nitrophenyl phosphatase activity fluctuated during storage of tubers, while β-glycerophosphatase declined to a low level coincident with initiation of sprout growth. In recently-lifted tubers acid phosphatase activity occurred in a single high molecular weight peak. Two new forms of lower molecular weight appeared during ageing of stored tubers. The influence of infection by a tuber-rotting fungus, Phytophthoraerythroseptica , on these seasonal changes was variable. No consistent effects on total hydrolase activities were observed, while post-infectional changes in molecular forms included a pronounced shift in the major acid phosphatase peak. The possible significance of this molecular weight change in infected samples is discussed in the light of recent evidence concerning the sub-unit structure of acid phosphatase from potato tubers.

Immunochemical Identification of a New Molecular Form of Acid Phosphatase of Host Origin Arising during Infection of Potato Tubers by Phytophthora erythroseptica Pethybr.

Summary: Changes in multiple forms of acid phosphatase from potato tubers during infection by Phytophthora erythroseptica were detected by polyacrylamide gel electrophoresis. In recently lifted tubers these changes included the appearance of a new molecular form of low electrophoretic mobility. Seasonal studies established that a similar molecular form developed in healthy tubers during storage. An antiserum to acid phosphatase from potato tubers was shown to be specific for the host enzyme. Utilizing a sectional immunofocusing procedure the novel molecular form from infected tissues cross-reacted with the antiserum, suggesting that this component originated from the host.

Lipoamide dehydrogenase of the phytopathogenic fungi Pythium ultimum and Phytophthora erythroseptica: Differences in conformation and kinetics

1. 1. Lipoamide dehydrogenase (NADH: lipoamide oxidoreductase, EC 1.6.4.3) was isolated from two closely related phytopathogenic fungi, Pythium ultimum and Phytophthora erythroseptica. The enzymes of both microorganisms differ considerably in their catalytic properties, the lipoamide dehydrogenase of P. ultimum being much more active (catalytic centre activity estimated for the reduction of lipoate by NADH: 5900 min −1; for the reduction of NAD + by reduced lipoamide: 100 000 min −) than that of Ph. erythroseptica (corresponding values, 720 min −1 and 4200 min −1). The Michaelis constants for both reactions were determined. 2. 2. Under identical conditions the fluorescence intensity of the enzyme from Ph. erythroseptica at 20°C is about three times higher than that of lipoamide dehydrogenase from P. ultimum. Upon anaerobic reduction with NADH, the Phytophthora enzymes shows beyond 500 nm the typical spectrum of the two equivalent reduced enzyme as reported for the pig heart enzyme (cf. Massey, V., Gibson, Q. H. and Veeger, C. (1960) Biochem. J. 77, 341–351 (ref. 1)), the Pyhthium enzyme becoming more bleached in the flavin region without being fully reduced. 3. 3. At pH 7.6 in phosphate buffer, higher concentrations of NAD + inhibit the activity of the lipoamide dehydrogenase from both fungi when lipoate is reduced with NADH. At pH 5.9 (optimum), NAD + stimulates the reaction in the case of the P. ultimum enzyme, but inhibits the Ph. erythroseptica enzyme. The results indicate that both enzymes have two NAD + binding sites. 4. 4. The results underline the strikingly high genetic variability within the fungus subfamily Pythieae.

Studies on Endogenous Bacteria in Potato Tubers Infected by Phytophthora erythroseptica Pethybr

Infection of potato tubers by the soft-rotting fungi Phytophthora erythroseptica and Pythium debaryanum resulted in multiplication of endogenous tuber bacteria. This effect was not evident after infection by dryrotting fungi. Some of the bacteria isolated produced pectolytic and hemicellulolytic enzymes in vitro and were themselves capable under certain conditions of degrading tuber tissue.

Changes in hydrolase activity of Solanum tuber tissues during infection by Phytophthora erythroseptica

Enzyme extracts from healthy potato tuber tissues and tissue culture cells showed varying phosphohydrolytic activity towards a range of phosphate esters. Infection of tissues by Phytophthora erythroseptica Pethyhr. resulted in increased acid phosphatase activity towards some substrates and decreased activity towards others. Changes in several carboxylic esterase activities occurred following tissue infections. Disk electrophoresis of enzyme extracts on Polyacrylamide gel, using α -naphthyl esters as substrates, indicated the existence of multiple forms of acid phosphatase and carboxylic esterases in both healthy and infected tissues.

Release of Hydrolytic Enzymes from Cytoplasmic Particles of Solanum Tuber Tissues During Infection by Tuber-rotting Fungi

SUMMARY Histochemical evidence showed that infection of tuber tissues and tissue-culture cells of Solanum tuberosum by Phytophthora erythroseptica, Phytophthora infestans and Fusarium caeruleum caused swelling and disruption of host cytoplasmic particles containing acid phosphatase, esterases and proteases. Heavy diffuse cytoplasmic staining for acid phosphatase was a consistent feature of infection by all three fungi, but staining reactions for esterases and proteases showed much less diffuse staining and a lesser degree of particle swelling. Biochemical assays showed that acid phosphatase was liberated from the particulate fraction to the supernatant fluid fraction of infected callus cells; acid ribonuclease behaved similarly. The biochemical evidence suggested that esterase activity of infected cells remained associated with a sedimentable fraction from cell homogenates. Assay methods for proteolytic enzymes did not confirm the histochemical evidence of a particulate localization of these enzymes in healthy or infected callus tissues. An excess recovery of ribonuclease from tissues infected with P. infestans and F. caeruleum was found; the significance of this is discussed. Attempts to separate a particulate fraction rich in hydrolases from homogenates of healthy or infected potato tubers were unsuccessful.

The disruption of lysosome-like particles of Solanum tuberosum cells during infection by Phytophthora erythroseptica Pethybr.

SUMMARY: Histochemical methods showed that tuber tissue and tissue culture cells of Solanum tuberosum contained phase-dense particles which absorbed neutral red and fluorescent dye, and particles which contained acid phosphatase and a non-specific esterase. It is suggested that these structures may be comparable to the lysosomes of animal tissues and the lysosome-like structures of plant cells which possess similar and additional properties. During infection of Solanum tissues by Phytophthora erythroseptica there was swelling and disruption of these host cell particles, accompanied by the release of acid phosphatase and esterase. Biochemical assay for acid phosphatase confirmed that infection of host cells resulted in the liberation of acid phosphatase from a particulate to the supernatant fluid fraction of cell homogenates.

Some observations on the zoospores of Phytophthora erythroseptica

The whiplash flagellum of zoospores of Phytophthora erythroseptica is not hairless but bears fine lateral hairs which are absent from the thin end-piece. These hairs seem to be of uniform thickness, not exhibiting the fine terminal portion present on hairs on the tinsel flagellum. Expanded knob-like structures which occur at the tips of flagella just before a zoospore settles down appear to be accumulations of disintegrated material.

The amino acid content of potato plant varieties and their resistance to attack by Phytophthora species

AbstractNutritional experiments were carried out to determine the suitability of 22 amino acids as sole sources of nitrogen for the culture of Phytophthora infestans and Phytophthora erythroseptica. The results showed that whilst P. erythroseptica was able to utilise all amino acids supplied reasonably well with the exception of DL lysine, P. infestans was more exacting in its requirements producing reasonable growth on less than half the number supplied. Examination of the free amino acid pool in the tissues of 14 varieties of potato tuber and 8 varieties of leaf showed that approximately 15 readily utilisable amino acids were present as the basic complement in the tuber tissues; the leaf tissues contained a smaller number of amino acids. Additions of water‐soluble tuber extracts to a nitrogen‐limited medium improved the growth of both fungi but the growth response of P. infestans was more varied than that of P. erythroseptica. There was no apparent correlation between the amino acid content of the potato tissues and the amino acid requirements of the parasite in relation to differential varietal resistance.

Asexual reproduction in Phytophthora erythroseptica

Sporangia are readily produced by Phytophthora erythroseptica Pethybr. when mycelial mats are grown in pea broth, washed and transferred to shallow layers of water or Petri's solution. Sporulation occurs between 8 and 26 ° C., (opt. c . 18–22 ° C.). Aeration of water is not necessary for sporangial production. Light stimulates more abundant sporulation but prolonged exposure to light is unnecessary. Sporangia are produced within a pH range of 4·0–8·5 (opt. c . pH 7·0–7·5). In colonies grown entirely in a synthetic liquid medium the addition of various carbon sources or thiamin did not induce normal development of sporangia. Direct germination was stimulated by fairly high temperatures while indirect germination was induced by cooling the cultures for a short period followed by a return to normal room temperatures. Sporangia in cultures frequently burst discharging undifferentiated protoplasm. A high percentage of bursting occurs in young cultures, especially when they are exposed for relatively long periods to low temperatures. Possible relationships between the fine structure of sporangia and the germination processes are discussed.

The fine structure of sporangia of Phytophthora erythroseptica Pethyb.

SUMMARY Young non-germinating sporangia of Phytophthora erythroseptica contain nuclei and cytoplasmic organelles, similar in appearance to those in other fungi, and a conspicuous central and other vacuoles. The sporangial wall consists of an outer homogeneous layer and an inner vesicular layer, prominent in the apical region. Peripheral cytoplasmic vacuoles are frequent, particularly where the vesicular layer is well developed. In old sporangia, which lose the ability to germinate indirectly (i.e. to produce zoospores), there is no vesicular layer and there is a change in the nature of the storage material of the vacuoles. When young sporangia are stimulated to indirect germination by low-temperature treatment the central storage vacuole disappears and there is a marked elongation of mitochondrial cristae, consistent with an increased rate of respiration. The sporangial wall becomes three-layered with the development of an inner homogeneous layer which separates the plasma membrane from an increasingly prominent vesicular layer. In the region of the papilla the vesicular layer shows a striking increase in thickness; it is suggested that this layer and the inner homogeneous layer play an important part in zoospore discharge.

Comparative Studies of the Mineral Nutrition of Three Species of Phytophthora

Summary: Nutritional experiments were carried out dealing with: (1) the utilization of sulphur compounds; (2) the utilization of phosphorus compounds; (3) the action of different mineral salts and their interaction. The results showed that Phytophthora erythroseptica grew well at 28° and pH 6·6 incubated for 20 days; P. parasitica grew well at 28° and pH 6·6 for 17 days; and P. infestans at 20° and pH 4·5 for 16 days. In the adjusted controlled medium pH changes were generally within one pH unit. The best carbon and nitrogen sources are stated. The only satisfactory sulphur sources for P. infestans were sodium sulphate and sodium thiosulphate. These compounds were also among the best S sources for P. erythroseptica and P. parasitica, but a number of other compounds also were utilized equally well, e.g. sodium metabisulphite, sodium sulphite, a-cysteine, sodium sulphide, sodium dithionite and methionine. For all organisms the best phosphorus sources were sodium dihydrogen orthophosphate, sodium metaphosphate and lecithin. Rate of utilization of the phosphorus was an important factor in mycelial yield. Factorial experiments were carried out in which P. erythroseptica and P. parasitica were incubated at 28° for 17 days. Statistical analysis of the results showed that under the given conditions optimal growth measured as mg. dry wt. was obtained in liquid media containing glucose, 25 g./l.; dl-asparagine, 4·0 g./l.; FeSO4.7H2O, 0·001 g./l.; thiamine, 0·8 mg./l.; ZnSO4.7H2O, 1 p.p.m.; H2MoO4, CuSO4. 5H2O, 0·02 p.p.m.; with varying amounts of K2HPO4, MgSO4.7H2O and CaSO4.2H2O for the different species specified. There was a balance between all combinations of K2HPO4, MgSO4.7H2O and CaSO4.2H2O for P. parasitica but not for P. erythroseptica, but there were significant interactions between the salts taken two at a time for both these fungi. There was interaction for all three salts together for both species. Phytophthora infestans and P. parasitica are more exacting in their nutritional requirements than P. erythroseptica.

A new variety of Phytophthora erythroseptica, which causes a soft rot of pea roots

The morphology and pathogenicity of a new variety of Phytophthora erythroseptica is described. The fungus was isolated from pea roots in the glasshouse although it was derived, almost certainly, from field soil. Infected roots were soft, sodden and faintly discoloured ; pea shoots wilted when root infection was severe. Peas were infected with the fungus, either by planting seed in soil inoculated with agar disks containing growing mycelium, or by immersing washed root systems in a suspension of zoospores. The zoospores encysted on the root tips, where they germinated and penetrated the host. Inter- and intracellular hyphae grew first along the stele and then into the cortex. Abundant sex organs usually formed in infected tissue and sporangia grew out from the roots. Infection was mild out-of-doors 0in the summer and was very severe in the glasshouse at 15°C.

Germination of Oospores of Phytophthora erythroseptica

GERMINATION of comparatively young oospores of Phytophthora erythroseptica has been obtained following passage through the digestive tract of the garden snail, Helix aspersa. These oospores were produced in oat agar cultures maintained at 20° C. for 4–6 weeks. Portions of the culture were fed to snails, and the excreta collected and kept in a moist atmosphere at room temperature. The old oogonial and antheridial walls appeared unaltered by this treatment, and within a few days germination of a number of oospores was observed.

Haustoria of Phytophthora erythroseptica in potato tubers affected with Pink Rot

An account is given of the occurrence of haustoria of Phytophthora erythroseptica Pethybr. in potato tubers affected with Pink Rot.

ANTAGONISM BY SPECIES OF ALTERNARIA AND THE ISOLATION OF A CRYSTALLINE SUBSTANCE FROM THE FILTRATE OF CULTURES OF A. RADICINA

Culture filtrates of five species of Alternaria were antagonistic to Phytophthora erythroseptica. Those of A. radicina exerted the greatest effect and they were followed in order of effectiveness by A. dianthicola, A. solani, A. brassicicola, and A. tenuis. The antagonistic principle in the filtrate of A. radicina was readily removed by activated carbon. By extracting this carbon with acetone, substantial yields were obtained of colorless, needlelike crystals, quite insoluble in water, but readily soluble in acetone, ethyl alcohol, ether, benzene, concentrated sulphuric acid, and 10% sodium hydroxide. The m.p. of the crystals, 204 °C. to 208 °C, and the deep pink color in solutions of sodium hydroxide distinguish the substance from alternaric acid. Water suspensions of the crystals were much less antagonistic to P. erythroseptica than the original filtrate and had little or no effect upon several species of bacteria.

Heterothallic Antibiosis in Mucor Racemosus

During a preliminary testing of stock laboratory cultures of molds for possible antibiotic properties, a culture of a phycomycete, Mucor racemosus Fres., indicated antibacterial effects. It was not known whether this culture represented a plus or a minus strain of the heterothallic fungus. Inasmuch as antibiosis has been reported to occur very infrequently in the phycomycetes, the antibiotic action of M. racemosus was investigated further. Wilkins and Harris (3) tested 19 phycomycetes for antibiotic action using Escherichia coli, Staphylococcus aureus, and Pseudomonas pyocyanea as test organisms. Negative results were obtained with all of these phycomycetes except for Phytophthora erythroseptica which exhibited antibacterial action against all three test organisms. Antibiotic tests also included heterothallic strains of nine different species of Mucoraceae. Among these were plus and minus strains of Mucor hiemalis, M. mucedo, and M. sexualis.

PHYTOPHTHORA ERYTHROSEPTICA PETHYBR. IN RELATION TO ITS ENVIRONMENT

Summary The present contribution, as part of a general study of pink rot of the potato, deals with Phytophthora erythroseptica Pethybr. in its relation to staling, light, acidity, moisture and temperature. In natural media the fungus is not, or but very slightly, affected by its own staling products, but, in general, is intensely intolerant to the presence of other fungi and bacteria. Ordinary daylight is not concerned in the growth or reproduction of the fungus. Growth occurs over a wide pH range (3·2 to circa 9·0–10·0); sexual reproduction occurs over this range except towards the limits of acidity and alkalinity; asexual reproduction was not investigated experimentally. It is considered that modification of the soil reaction, within practical limits, does not offer a means for control of the disease. The moisture factor controls directly the incidence of the disease in contaminated soil—with low moisture infection is inhibited; with normal moisture infection occurs via the dead or moribund stolon through the “heel” of the tuber; with high moisture the tuber is infected directly in the region of an “eye”. Sporangia are only formed under aquatic conditions and it is considered probable that “eye” infection is brought about through the agency of zoospores. The temperature limits of growth are 0–32°C. with an optimum at 25°C. Oospore production occurs only between the optimum and minimum growth temperatures (0–25°C), being inhibited above the growth optimum. Asexual reproduction seems to be similarly related to temperature.

ROOT ROT, SHOOT ROT AND SHANKING OF TULIP CAUSED BYPHYTOPHTHORA CRYPTOGEAPETHYBR. & LAFF. ANDP. ERYTHRO‐SEPTICAPETHYBR.

SUMMARYPhytophthora erythrosepticaandP. cryptogeahave been isolated on numerous occasions from diseased tulip plants on commercial nurseries, and the isolates, both in pure culture and in their effect on the host, have been compared with authentic cultures of these fungi. Both species have been proved to be, either separately or in conjunction, the cause of a serious root rot, shoot rot and shanking of forced tulips.Infection, which takes place from contaminated soil through the roots, begins as soon as the first roots are produced from the basal plate of the bulb. The parasite passes directly through the basal plate to the base of the flowering stem, leaving the old bulb scales practically un‐attacked. According to the time, or rapidity, of infection an affected bulb may produce no visible shoot, or it may give a shoot bearing a flower bud that remains blind because the flower stem is rotted at the base (shanking). Sometimes a bulb with some of its roots attacked may produce a marketable flower of poor quality.A very similar disease has been induced in outdoor tulips by contaminating the soil.The disease, which may result in the total loss of a forced crop, has not yet been reported outside Great Britain, where it seems to be widely distributed.There is no evidence that the disease is carried with the planted bulb in ordinary commercial practice, and the rate of spread in the glasshouse from one or more inoculated bulbs planted in the midst of healthy ones is comparatively slow.The susceptibility of varieties of tulips to the two species ofPhytophthorais briefly discussed.A contaminated water supply is a possibility, but appears to be relatively unimportant.Partial sterilization of the contaminated soil by steam, or formaldehyde, combined with hygiene, is an effective method of controlling the disease, but soil treatment with Cheshunt Compound is unsatisfactory.Certain strains ofPhytophthora cryptogeavar.Richardiaeand P.parasiticawere tested, but appear to be of little, or no, consequence as parasites of forced tulips.A large proportion of the success of the present investigations has been due to the stimulus, and ever‐ready helpfulness, of Dr Geo. H. Pethybridge and Mr W. C. Moore. The credit for identifying the fungi concerned lies almost entirely with Mr S. F. Ashby. The writer also wishes to express his thanks to his Laboratory Attendant, Mr A. Sherval, for taking the photographs.