Zoospores Of Phytophthora Palmivora Research Articles

Enhanced germination and electrotactic behaviour of Phytophthora palmivora zoospores in weak electric fields

Soil-dwelling microorganisms use a variety of chemical and physical signals to navigate their environment. Plant roots produce endogenous electric fields which result in characteristic current profiles. Such electrical signatures are hypothesised to be used by pathogens and symbionts to track and colonise plant roots. The oomycete pathogen Phytophthora palmivora generates motile zoospores which swim towards the positive pole when exposed to an external electric field in vitro. Here, we provide a quantitative characterization of their electrotactic behaviour in 3D. We found that a weak electric field (0.7–1.0 V cm−1) is sufficient to induce an accumulation of zoospore at the positive pole, without affecting their encystment rate. We also show that the same external electric field increases the zoospore germination rate and orients the germ tube’s growth. We conclude that several early stages of the P. palmivora infection cycle are affected by external electric fields. Taken together, our results are compatible with the hypothesis that pathogens use plant endogenous electric fields for host targeting.

Resistance to Black Pod Disease in a Segregating Cacao Tree Population

Black pod disease caused by Phytophthora spp. is the most important cacao tree disease worldwide. Genetic resistance has received much attention for the reduction of economic and environmental costs. In the present work, leaf disks from 282 individuals [F1(TSH-1188xCCN-51)] were inoculated using suspensions of 3 × 105 zoospores of Phytophthora palmivora and Phytophthora citrophthora. Twenty disks of each genotype were distributed in four blocks with five disks per plot. After 5 days, each inoculated leaf disk was assessed for the infection level. It was verified statistically that the progeny's parents were intermediate between the control clones Catongo (susceptible) and Sca-6 (resistant). A highly positive linear relationship was observed between infection levels caused by both Phytophthora species in the F1 progeny (r = 0.9935**). However, the interaction was highly significant. It was also observed that the curve distribution is skewed towards higher resistance. This suggests that few genes are involved in controlling the resistance to both species, and that they maybe linked and species-specific. Although the parents did not differ statistically, the F1 progeny segregated for resistance to each Phytophthora species and it is therefore an adequate population for breeding for resistance to black pod.

Defence responses induced by potassium phosphonate in Phytophthora palmivora-challenged Arabidopsis thaliana

Potassium phosphonate, a systemically translocated chemical used to protect plants against oomycete pathogens, induces rapid, localised defence responses in normally susceptible plants that are similar to those observed in an incompatible interaction. We demonstrated that in phosphonate-treated Arabidopsis thaliana seedlings inoculated with zoospores of Phytophthora palmivora, challenged cells respond with a rapid increase in cytoplasmic activity, the development of cytoplasmic aggregates, the release of superoxide, localised cell death and enhanced accumulation of phenolic materials around the infected cell. Pathogen development is severely restricted, and hyphae that develop in phosphonate treated seedlings appear distorted and sporangial production is inhibited. These rapid host responses are absent or muted in untreated inoculated seedlings, and the pathogen rapidly colonises seedling tissues, developing abundant sporangia within 24 h of inoculation. The presence of Mn(III)-desferal, a treatment that quenches superoxide release, abolishes hypersensitive cell death and facilitates pathogen development in phosphonate-treated plants, indicating that pathogen inhibition is a consequence of superoxide release rather than direct inhibition due to phosphonate.

Defective zoospore encystment and suppressed cyst germination of Phytophthora palmivora caused by transient leaching treatments.

The behaviour of encysting zoospores of Phytophthora palmivora during leaching conditions was studied. Zoospores encysted and germinated successfully on polycarbonate membranes after mechanical agitation. Transient (10 min) leaching treatments with nutrient-free buffer underneath the membranes resulted in abnormal encystment and poor germination. The disruption was greatest when leaching was applied during the first minutes after start of encystment and not observed after 20 min. The early sensitivity of cells to leaching coincided with the period when alkali-resistant cell walls were formed (2-6 min after mechanical agitation). Effects of calcium and organic nutrients on encystment during leaching and germination after these treatments were studied. The disruption of encystment by early leaching treatments, but not the suppression of cyst germination, was overcome by adding calcium chloride during mechanical agitation of zoospores. Leaching with calcium containing buffer resulted in suppressed cyst germination as was the case with buffer alone. Leaching with 0.1% peptone containing buffer promoted consistently high encystment and germination.

Zoospores use electric fields to target roots

Species of Phytophthora and Pythium are a group of water molds, pathogens that colonize the roots of higher plants, and cause the familiar root rots. These Oomycetes have flagellated zoospores that allow them to swim in water and can carry them long distances. Zoospores are produced in large numbers and are the cause of most new infections on a wide variety of plant hosts. As they move through the soil, they are attracted to the tips of plant roots where they lodge, encyst and germinate to produce germ tubes that penetrate the roots. The targeting mechanisms of swimming zoospores have long been thought to consist only of chemotaxis toward root exudates, including those secreted from wounds. So how does one explain the marked spatial differences that exist in the sites of accumulation of zoospores at root surfaces or why zoospores of some species are attracted to wound sites and others are not? Although a few chemicals that elicit chemotaxis have been identified, including two isoflavones of soybean (daidzein and genistein) that attract the zoospores of the soybean pathogen, Phytophthora sojae, few other chemicals have been identified.Now Pieter van West and colleagues [1xOomycete plant pathogens use electric fields to target roots. van West, P. et al. Mol. Plant–Microbe Interact. 2002; 15: 790–798Crossref | PubMedSee all References][1] have shown that electrical signals can augment or override chemotaxis in mediating short-range tactic responses of oomycete zoospores at root surfaces, the first demonstration that electrotaxis plays a major role in a plant–microbe interaction under natural conditions. The roots of perennial rye grass (Lolium perenne) and wheat (Triticum aestivum) have been shown to have outward flows of positive current around the root cap, meristematic and elongation zones, that is, the apex or distal parts of roots are anodic, whereas the root hair zone and wound sites are cathodic. Among the evidence presented, Van West et al. [1xOomycete plant pathogens use electric fields to target roots. van West, P. et al. Mol. Plant–Microbe Interact. 2002; 15: 790–798Crossref | PubMedSee all References][1] showed that the positive or negative electrotactic responses of zoospores provide the most plausible explanation for the marked asymmetries of accumulation of zoospores on the surfaces of roots. For example, zoospores of Pythium aphanidermatum (cathodotactic) accumulated mainly at the rear of rye grass roots (anodic) and at wound sites, whereas Phytophthora palmivora zoospores (anodotactic) accumulated in the apical (anodic) zones and were excluded from the cathodic wound sites. These results were essentially reversed by treating the roots with fusicoccin, which reverses the electric field around the root.‘…the positive or negative electrotactic responses of zoospores provide the most plausible explanation for the marked asymmetries of accumulation of zoospores on the surfaces of roots.’This, and much other evidence, suggested to van West et al. [1xOomycete plant pathogens use electric fields to target roots. van West, P. et al. Mol. Plant–Microbe Interact. 2002; 15: 790–798Crossref | PubMedSee all References][1] that electrotaxis augments other root-targeting mechanisms, overriding chemotaxis when the effector molecules are at concentrations normal to the rhizosphere, and that chemotaxis and electrotaxis participate in the homing responses of zoospores toward roots. Perhaps, as they also suggest, root surface charges, generated as they are by the metabolism of living roots, ensure that zoospores colonize living rather than dead roots.

Calcium-Dependent, Genus-Specific, Autoaggregation of Zoospores of Phytopathogenic Fungi

Reid, B., Morris, B. M., and Gow, N. A. R. 1995. Calcium-dependent, genus-specific, autoaggregation of zoospores of phytopathogenic fungi. Experimental Mycology , 19, 202-213. Dense populations of zoospores of Phytophthora palmivora , Pythium catenulatum, and Pythium dissotocum formed multicell clumps, or autoaggregates. Autoaggregation was the result of active taxis and was shown to be density-dependent, calcium-requiring, and influenced by pH. In addition, autoaggregation appeared to be species-specific, since aggregates of Ph. palmivora did not attract zoospores of three Pythium species and aggregates of Py. catenulatum did not attract Ph. palmivora zoospores. Aggregation centers generated a calcium ion gradient and induced chemotropic growth of germ tubes emerging from zoospore cysts. Autoaggregation also functions to enhance zoospore accumulation at plant root surfaces, thereby increasing inoculum potential for infection. In the absence of roots, autoaggregation may enhance zoospore population survival.

The role of the plasma membrane in differentiation of Phytophthora palmivora zoospores

Sealed plasma membrane vesicles of about 90% purity were isolated from zoospores and mycelium of Phytophthora palmivora by chemical lysis and mechanical disruption, followed by differential and sucrose density gradient centrifugation. Both ATPase activity and proton transport (acridine orange quenching) showed similar properties: sensitivity to vanadate and calcium, insensitivity to oligomycin and molybdate, pH optimum 6·5, Km 3·6–3·8 m m for the substrate Mg:ATP = 1:1. These are characteristic properties of fungal and plant E1E2-type H+-pumping ATPase, suggesting that plasma membrane ATPase is involved directly in active proton transport across the membrane. Zoospores loaded with the fluorescent compound BCECF showed that internal pH rose during encystment in high (1–5 m m ) but not low (0·1–0·5 m m ) external calcium concentrations; pectin enhanced a pH rise only at high external calcium concentrations. Plasma membrane ATPase may play a vital part in controlling internal pH and providing an electrochemical gradient for calcium uptake, but high external concentrations of calcium lead to an uncontrolled rise in internal pH, due in part to inhibition of the proton pump by high internal concentrations of calcium. However, in normal physiological concentrations of calcium, the main function of calcium in zoospore encystment is as a secondary messenger within the zoospore, rather than as an inhibitor of ATPase.

Role of phosphatide acid during differentiation of Phytophthora palmivora zoospores

Summary: Zoospores of the fungus Phytophthora palmivora undergoing synchronous differentiation showed a rapid increase (250%) in phosphatidic acid (PA) concentration within 20 s of the inducing stimulus. There were only small (< 100%) changes in cGMP and cAMP and inositol phosphates during the same period. There was no consistent change in the concentration of diacyl glycerols during the first five minutes of differentiation. The addition of exogenous PA (3 mUm) induced zoospore differentiation, with the optimum concentration dependent upon the Ca2+ concentration in the suspension medium. Other phospholipids were ineffective as inducers. Both PA production and spore differentiation were Ca2+-dependent, and the addition of the Ca2+ channel blocker verapamil, or the removal of exogenous Ca2+ by EGTA addition, both reduced PA accumulation and slowed differentiation. We suggest that PA production in this organism arises via a stimulus-activated phospholipase D, and may act as a second messenger. There is no evidence for any role for cyclic nucleotides or inositol phosphates as second messengers during the early events of zoospore differentiation in this species.

The effect of modified pectin, pectin fragments and cations on Phytophthora palmivora zoospores

Chemically modified citrus pectin and native pectin were compared as inducers of differentiation in zoospores of the plant pathogen Phytophthora palmivora. Periodate oxidation or reduction of pectin completely destroyed all capacity of the polymer to induce cell differentiation. Methylation destroyed the capacity of the pectin to induce germination, but the methylated polysaccharide induced zoospore rounding and at least partial encystment at low concentrations. Some oligomers prepared from a partial pectinase digest of citrus pectin also induced zoospore differentiation, with tetra-, and pentasaccharides proving the most effective. These results suggest that free galacturonate residues in pectin are critical to the induction of complete zoospore differentiation, although the process can be initiated, but not completed, by a wider range of compounds. The use of K + in lieu of Tris—pectin resulted in an extended lag before pectin-induced differentiation began. However, after 90 min, no differences were observed between cells stimulated by potassium pectate and those stimulated by Tris—pectate. Dialysis of the native pectin reduced its efficiency as an inducer, but addition of the ashed dialysate to the dialysed pectin partially restored its capacity to induce zoospore differentiation.

Ultrastructural organization of freeze-substituted zoospores of Phytophthora palmivora

The ultrastructural organization of the zoospores of Phytophthora palmivora was examined using freeze substitution as a potential means of improving upon the images obtained with zoospores preserved by conventional chemical fixation. Despite die technical difficulties encountered, freeze substitution was effective in preserving the overall shape of the zoospores and membranous structures, including the plasma membrane, peripheral cisternae, and the water-expulsion vacuole. Coated pits, not previously reported on the plasma membrane of zoospores of fungi, were found in P. palmivora. The nucleus and cytoskeleton of zoospores were not as well preserved as with conventional chemical fixation. Unique features of freeze-substituted zoospores were compared with structures observed in previous studies of zoospores.

Accelerated ion fluxes during differentiation in zoospores of Phytophthora palmivora

Zoospores of Phytophthora palmivora show increased fluxes of Na + and Ca 2+ 3–5 min after they have been stimulated to differentiate with pectin. Both spontaneous and pectin-induced encystment are reduced below pH 6 and accelerated above pH 7. The ionophores monensin and A23187 induce slow differentiation when added together, but not when added separately. Ethanol (0.5%) also induces slow differentiation. Amiloride and verapamil inhibit pectin-induced differentiation and also reduce the onset of the Na + and Ca 2+ flux. A requirement for Ca 2+ for differentiation is confirmed, but a requirement for Na + could not be demonstrated.

Distribution of Diaminobenzidine Reaction Products in Zoospores of Phytophthora palmivora

Because microbodies in many filamentous fungi fail to stain with the 3,3′-diaminobenzidine (DAB) technique, the subcellular localization of DAB reaction products at two pHs was explored in an electron microscopic study of zoospores of Phytophthora palmivora. At pH 9.2, but not at pH 7.2, DAB reaction product was in the matrix of U-bodies and microbodies, indicating that both organelles contained catalasc. In contrast to a homogeneous distribution in microbodies, reaction product was limited to the core of U-bodies and absent from their halo and shell regions. At both pH 7.2 and 9.2, reaction product filled mitochondrial cristae, the site of cytochrome c oxidase. In addition to the organellar compartmentalization of reaction product, DAB reactions at pH 9.2 and 7.2 enhanced the prominence of an asymmetry in the trilamellar structure of the zoospore plasma membrane and the appearance of a cell coat covering the plasma membrane and flagellar sheath. This coat formed during zoosporogenesis, and DAB reactions intensified cell coat material that lined the inner surfaces of membranes involved in cytoplasmic cleavage. Since no reaction product formed in tissues incubated in reaction media to which sodium azide was added or DAB eliminated, reaction products were enzymatically produced. Because the DAB reaction highlights plasma membrane asymmetry and the presence of a cell coat, this technique should be useful in morphologically marking plasma membranes, enabling their recognition during zoosporogenesis or in isolated subcellular fractions of this fungus.

Distribution of Diaminobenzidine Reaction Products in Zoospores ofPhytophthora Palmivora

Because microbodies in many filamentous fungi fail to stain with the 3,3'-diaminobenzidine (DAB) technique, the subcellular localization of DAB reaction products at two pHs was explored in an electron microscopic study of zoospores of Phytophthora palmivora. At pH 9.2, but not at pH 7.2, DAB reaction product was in the matrix of U-bodies and microbodies, indicating that both organelles contained catalase. In contrast to a homogeneous distribution in microbodies, reaction product was limited to the core of U-bodies and absent from their halo and shell regions. At both pH 7.2 and 9.2, reaction product filled mitochondrial cristae, the site of cytochrome c oxidase. In addition to the organellar compartmentalization of reaction product, DAB reactions at pH 9.2 and 7.2 enhanced the prominence of an asymmetry in the trilamellar structure of the zoospore plasma membrane and the appearance of a cell coat covering the plasma membrane and flagellar sheath. This coat formed during zoosporogenesis, and DAB reactions intensified cell coat material that lined the inner surfaces of membranes involved in cytoplasmic cleavage. Since no reaction product formed in tissues incubated in reaction media to which sodium azide was added or DAB eliminated, reaction products were enzymatically produced. Because the DAB reaction highlights plasma membrane asymmetry and the presence of a cell coat, this technique should be useful in morphologically marking plasma membranes, enabling their recognition during zoosporogenesis or in isolated subcellular fractions of this fungus.

A model to explain ion-induced differentiation in zoospores of Phytophthora palmivora

Zoospores of Phytophthora palmivora undergo synchronous encystment followed by germination when exposed to sodium ions at 3–10 m M, calcium ions at 5–30 m M, and strontium ions at 0.3–10 m M. Lithium ions induce encystment at 3–10 m M but do not induce germination. Ferric, manganese, and barium ions.act as chaotropic agents, damaging the zoospore plasma membrane under both isoosmotic and hypoosmotic conditions, at concentrations as low as 30 μ M, ferric ions and 1 m M, barium ions. Cesium and ammonium ions are cytotoxic and induce lysis under hypoosmotic conditions. The capacity of cations to induce zoospore differentiation can be explained in terms of the following model. It is suggested that differentiation in P. palmivora requires the entry of sodium at one or more specific sites. Access to these sites is regulated by a calcium gated monovalent ion translocator with a high specificity for sodium. Calcium not only regulates sodium ion access but also regulates the cell's osmoregulatory system. Divalent cations act by competing for the calcium binding site, monovalent cations by restricting fodium ion access.

The Effect of Pectin and Related Compounds on Encystment and Germination of Phytophthora palmivora Zoospores

SUMMARY: Citrus pectin and other uronic acids (polygalacturonate, alginate and galacturonate) accelerated encystment and germination in suspensions of Phytophthora palmivora zoospores. Other anions, polyanions and polysaccharides containing α(1·4) linkages were not effective in this respect. The uronic acids tested differed from one another both in the concentrations required for maximal rates of encystment and in the extent to which they stimulated germination as well as encystment. Pectin-accelerated encystment did not require free Ca2+in the suspending medium, but when the Ca2+concentration exceeded 100μM, uronate-stimulated encystment and germination was suppressed. This blocking of uronate-accelerated encystment was almost specific for Ca2+, although Sr2+and Ba2+showed some effect. It is proposed that the action of uronates in accelerating encystment of P. palmivora zoospores is analogous to agonists which induce stimulus-mediated secretion in many animal cell types.

Comparison of cuprous oxide and metalaxyl with mixtures of these fungicides for thecontrol of Phytophthora pod rot of cocoa

Experiments were conducted using small‐scale, detached pod evaluation methods to compare the likely effectiveness in controlling black pod of cuprous oxide‐metalaxyl mixtures with these fungicides alone at approximately equivalent cost doses. All fungicide treatments were about equally effective and persistent except when rain fell soon afterspraying. Treatments containing cuprous oxide then gave very poor control of artificialinoculations with Phytophthora palmivora zoospores. Further laboratory' scale tests confirmed that cuprous oxide was readily leached by washing before the deposit completely dried whereas metalaxyl was very rapidly adsorbed on to ihe pods. A black pod control strategy is proposed using metalaxyi during critical wet periods and cuprous oxide for the rest of the year to reduce the possibility of metalaxyl resistance becoming apractical problem.

Synthesis of noncellulosic cell-wall β-glucan by cell-free extracts from zoospores and cysts of Phytophthora palmivora

Zoospores of Phytophthora palmivora, induced to encyst by vigorous agitation, synthesize alkali-insoluble β-glucans at a linear rate for ca. 10 min. Cell-free extracts of zoospores and cysts of P. palmivora contained particulate enzymes capable of synthesizing alkali-insoluble, noncellulosic β-1,3-, β-1,6-glucans from uridine diphosphate glucose. Similar levels of activity were detected in zoospores and cysts. Enzymatic activity was stimulated by Mg 2+; Ca 3+ and Mn 2+ were less effective. Cellobiose and other β-linked glucose disaccharides were strong activators. Evidently, the swimming zoospore is equipped with the enzymatic ability to make insoluble, noncellulosic β-glucan—a major component of the cyst wall; hence, a mechanism must exist which prevents the operation of glucan synthetase(s) until the time of encystment. The cell-free extracts also had the ability to synthesize soluble β-1,3-glucan. Most of this material was characterized as neutral mycolaminaran and mycolaminaran phosphate.

Negative Chemotaxis of Zoospores of the Fungus Phytophthora palmivora

Summary: Phytophthora palmivora zoospores are repelled by many low molecular weight cations. This negative chemotactic response occurs at a different threshold concentration for each cation, that for the most effective ion, H+, being 150 μM. The effectiveness of different cations parallels their ionic conductivities, the most active cations having the highest conductivities. It is suggested that the close approach of cations to the cell surface reduces the negative charge at the surface and hence changes the transmembrane potential, altering flagellum activity in such a way as to cause turning and negative chemotaxis.

Fatty acids, aldehydes and alcohols as attractants for zoospores of Phytophthora palmivora

ZOOSPORES of the important plant pathogenic fungi Pythium and Phytophthora are attracted to plant roots and to exudates and extracts from roots1. Attraction (positive chemotaxis) to amino acids2,3 which occur in root exudates, and ethanol4, which will be produced by roots in waterlogged soils, has been demonstrated, but no substance has been found which by itself is as powerful an attractant as root exudate. The attractiveness of root exudates must therefore be explained by additive or synergistic effects between the known attractants, or by powerful attractants which have hitherto escaped detection, or both. There is evidence for additive and synergistic effects5, but the fractionation of root exudates has not so far yielded evidence of additional chemotactic factors. We report here the existence of further attractants for fungal zoospores, and consider the possibility that they may be of significance in chemotaxis to plant roots.

Freeze-fracture and Freeze-etching Study of Encystment of Phytophthora palmivora Zoospores

Summary: Zoospores of Phytophthora palmivora were induced to encyst synchronously and changes in morphology, particularly in surface structure, were examined by freeze-fracture and freeze-etching techniques. A uniform ‘fuzzy coat’, 20 to 25 nm thick, covers the plasma membrane. This hitherto unknown structure surrounds the entire zoospore body and flagella. No structural correspondence between cyst wall microfibrils and plasma membrane particles was found. The pattern of distribution of plasma membrane particles remains unchanged during encystment.