Cell Surface Saccharides in Three Phytomonas Species Differing in Host Specificity

ABSTRACT. Cell surface carbohydrates of three phytoflagellates, Phytomonas francai. Phytomonas serpens and Phytomonas sp. from different hosts including cassava, coreid insect Phthia picta and the milkweed plant Euphorbia hyssopifolia, respectively, were analysed by agglutination assays employing a battery of highly purified lectins with affinity for receptor molecules containing N-acetylglucosamine (d-GlcNAc), N-acetylgalactosamine (D-GalNAc), galactose, mannose-like (D-Man-like) residues and fucose, and by binding assay using radiolabeled [¹²⁵I]-wheat germ agglutinin (WGA) and fluorescent WGA lectin, as well as glycosidases of known sugar specificity, Escherichia coli K with mannose-affinity fimbrial lectin was also used as an agglutination probe. In general, the presence of D-GlcNAc. D-GalNAc and D-Man-like residues was detected in the phytomonads' plasma membrane. These sugar moieties were confirmed in whole cell hydrolysates as assessed by gas-liquid chromatography (GLC) which in addition, also showed the presence of galactose and xylose. However, marked differences in cell surface carbohydrate structures were observed. Wheat germ agglutinin, which binds to sialic acid and/or d-GlcNAc-containing residues, shows selective agglutinin activities for P. francai and Phytomonas sp., while Bandeiraea simplicifolia II agglutinin (which recognizes d-GlcNAc units) specifically bound to Phytomonas sp. Helix pomatia agglutinin which binds to D-GalNAc-containing residues reacted preferentially with Phytomonas sp. and P. serpens. Con A, which recognizes D-Man-like receptors, agglutinates all the phytomonads; however, the higher interaction was observed with Phytomonas sp. P. francai was selectively agglutinated in the presence of E. coli fimbrial lectin. Fluorescence WGA binding was significantly decreased by N-acetylglucosaminidase activities and the cell agglutination was not altered by neuraminidase treatment, suggesting the presence of an exposed D-GlcNAc moiety on the P. francai and Phytomonas sp. surfaces. Binding studies with [¹²⁵I]-WGA essentially confirmed the fluorescence WGA binding and agglutination assays.     

Enzymes of Ornithine-Arginine Metabolism in Trypanosomatids of the Genus Phytomonas1

Trypanosomatids recently isolated from the plants Euphorbia pinea, E. characias, E. hyssopifolia, Manihoi esculenta (cassava) and Lycopersicon sp. (tomato) plus the McGhee-Postell isolate of Phytomonas davidi have been examined for the presence of enzymes of ornithine-arginine metabolism. Arginase (EC 3.5.3.1) was not detected in the flagellates examined whereas arginine deiminase (EC 3.5.3.6) and citrullinehydrolase (EC 3.5.1.20) were present in all organisms. Phytomonas davidi and the isolate from E. hyssopifolia, besides these enzymes, also had ornithine carbamoyltransferase (EC 2.1.3.3). The enzymic constitution of these flagellates is compared from a taxonomic standpoint to that of previously studied trypanosomatids.     

A PCR-based survey on Phytomonas (Euglenozoa: Trypanosomatidae) in phytophagous hemipterans of the Amazon region

ABSTRACT. We have surveyed 244 hemipterans from Western Brazilian Amazônia for the presence of trypanosomatids and identification of members of the genus Phytomonas. Examination by phase microscopy of squashes of insect salivary glands (SG) and digestive tubes (DT) revealed that 44% (108/244) of insects from seven families harbored trypanosomatids. Infections were 5 times more frequent in Coreidae than in all other families together. Smears of SG and DT of the dissected insects were fixed on glass slides with methanol and stained with Giemsa for morphological analysis. DNA was recovered from these preparations and submitted to a PCR assay that permitted amplification of all trypanosomatid genera using primers of conserved sequences flanking a segment of the spliced leader (SL) gene. Upon PCR amplification of the recovered DNA, amplicons were hybridized with an oligonucletide probe (SL3′) complementary to a SL intron sequence specific for flagellates of the genus Phytomonas. Among the trypanosomatid‐positive insects, 38.8% harbored Phytomonas spp., corresponding to an overall Phytomonas prevalence of 17.1% among phytophagous bugs, their putative vectors. Since many Phytomonas are pathogenic in plants, this high prevalence in their vectors emphasizes the permanent risk of exposure to disease by native and cultured plants of the Amazon region.     

Kinetoplast DNA from plant trypanosomatids responsible for the Hartrot disease in coconut trees

Summary— Phytomonas parasites were isolated from crude sap of coconut trees affected with Hartrot disease in French Guyana (Hart 1 and Hart 2) and Brazil (Hart 3) and cultured in vitro. Two Phytomonas isolates obtained from weeds belonging to the Euphorbiaceae family and growing in an infected coconut tree plantation were also cultured (E hys and E hir). The kinetoplast DNA (kDNA) was purified and incubated with topoisomerase II which decatenates the huge network into free minicircles of 1.6 kilobase (kb) pair for Hart 1, Hart 2 and Hart 3 and 1.3 kb for E hys and E hir. Restriction endonuclease analysis showed that more than 90% of Hart 1 and Hart 2 minicircle content was homogeneous in base sequence while minicircles from Hart 3, E hys and E hir were heterogeneous. Minicircles exhibited restriction cleavage patterns characteristic of each Phytomonas isolate allowing their identification, except for the major class of Hart I and Hart 2 minicircles whose restriction maps were identical. Cross-hybridization experiments were performed by Southern blot. A high sequence homology was found between minicircies from Hart 1, Hart 2 and Hart 3 on one hand and those from E hys and E hir on the other. In contrast, minicircles from the Hartrot Phytomonas and those from the two Euphorbiaceae Phytomonas present little sequence homology. These data showed that minicircles from Phytomonas infecting coconut trees displayed biochemical properties different from those of other Phytomonas. This could lead to the elaboration of new molecular tools aimed to help to epidemiological studies, to an early diagnosis and to a better control of the disease.     

Polyamine biosynthesis in Phytomonas: Biochemical characterisation of a very unstable ornithine decarboxylase

The metabolism of polyamines as well as their functions as growth regulators in plants have been extensively studied for many years. However, almost nothing is known about the biosynthesis and roles of these substances in Phytomonas spp., parasites of several plants. We have used HPLC and electrophoretic analyses to investigate the presence and metabolism of polyamines in Phytomonas Jma strain, detecting both putrescine and spermidine but not spermine. Experiments carried out by incubation of intact parasites with labelled ornithine or putrescine showed the formation of radioactive putrescine or spermidine, respectively. These results indicated that Phytomonas Jma can synthesise these polyamines through the action of ornithine decarboxylase (ODC) and spermidine synthase. On the other hand, we could not detect the conversion of arginine to agmatine, suggesting the absence of arginine decarboxylase (ADC) in Phytomonas. However, we cannot ensure the complete absence of this enzymatic activity in the parasite. Phytomonas ODC required pyridoxal 5′-phosphate for maximum activity and was specifically inhibited by α-difluoromethylornithine. The metabolic turnover of the enzyme was very high, with a half-life of 10-15 min, one of the shortest found among all ODC enzymes studied to date. The parasite proteasome seems to be involved in degradation of the enzyme, since Phytomonas ODC can be markedly stabilized by MG-132, a well known proteasome inhibitor. The addition of polyamines to Phytomonas cultures did not decrease ODC activity, strongly suggesting the possible absence of antizyme in this parasite.     

Use of Monoclonal Antibodies for Differentiation of Different Isolates of Phytomonas (Plant Trypanosomatids)

The Phytomonas genus was created arbitrarily to designate plant trypanosomes. A serological study with polyclonal and monoclonal antibodies was carried out to situate these trypanosomatids with respect to other trypanosomatids - Herpetomonas, Crithidia, Trypanosoma - and to compare different plant trypanosome strains with each other. The use of monoclonal antibodies directed against two different isolates makes it possible to distinguish plant trypanosomatids according to their geographical origin and to separate clearly the plant trypanosomatids from the South of France from other lower trypanosomatids, which seems to justify creating the Phytomonas genus.     

Inhibitors of Plant Copper Amineoxidases

Abstract

In this review, inhibitors of plant copper amine oxidases from Lens esculenta seedlings, Pisum sativum seedlings, and Euphorbia characias latex are described. Reversible competitive inhibitors and non-competitive inhibitors, irreversible active-site directed inhibitors and mechanism-based inactivators are reviewed in regard to their mechanisms of action.     

Diversity of Trypanosomatids in Cockroaches and the Description of Herpetomonas tarakana sp. n.

In this study, we surveyed six species of cockroaches, two synanthropic (i.e. ecologically associated with humans) and four wild, for intestinal trypanosomatid infections. Only the wild cockroach species were found to be infected, with flagellates of the genus Herpetomonas. Two distinct genotypes were documented, one of which was described as a new species, Herpetomonas tarakana sp. n. We also propose a revision of the genus Herpetomonas and creation of a new subfamily, Phytomonadinae, to include Herpetomonas, Phytomonas, and a newly described genus Lafontella n. gen. (type species Lafontella mariadeanei comb. n.), which can be distinguished from others by morphological and molecular traits.     

Electrophoretic Analysis of Isoenzymes in the Identification of Trypanosomatids of the Genus Phytomonas1

Five strains of trypanosomatids of the genus Phytomonas, isolated from different species of Euphorbia {Euphorbia heterophylla, E. characias, E. pinea, E. hyssopifolia) and from Manihot escutenta, were cultured and compared through the electrophoretic mobility of isoenzymes of six enzymes: aspartate aminotransferase (EC 2.6.1.1), alanine aminotransferase (EC 2.6.1.2), phosphoglucomutase (EC 2.7.5.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.49), glucosephosphate isomerase (EC 5.3.1.9), and malate dehydrogenase (EC 1.1.1.40). The strains could be distinguished from one another by their respective isoenzyme profiles.     

Ectomycorrhizal fungi of Pinus pinea L. in northeastern Spain

 Although Pinus pinea L. is an important forest species in the Mediterranean region, few reports exist on its ectomycorrhizal associates. Sixty isolates, obtained from fungal sporocarps collected in mixed forests of P. pinea in Catalonia (northeastern Spain), were tested for ectomycorrhiza formation on containerized P. pinea seedlings when applied as mycelial inoculum produced in peat-vermiculite. A total of 17 isolates, in 8 genera (Amanita, Hebeloma, Laccaria, Lactarius, Pisolithus, Rhizopogon, Scleroderma and Suillus), formed ectomycorrhizas and the percentages of mycorrhizal short roots varied among isolates and species from 13% to 89%. Some of these fungi are cited for the first time in association with P. pinea. The results indicate further fungal candidates for controlled inoculation of P. pinea seedlings in the nursery. Accepted: 29 October 1998     

Kinetoplast DNA permits characterization of pathogenic plant trypanosomes of economic importance

Summary— Twelve Phytomonas isolates were obtained from different plants originating from several countries and cultured in vitro in complex media. The kinetoplast DNA (kDNA) was purified and observed by electron microscopy. The structure of kDNA from all isolates appeared as a large network of interlocked minicircles with some maxicircles extruding from the network, as has often been shown for Trypanosomatidae. Topoisomerase II resolved the kDNA network into free minicircles which were then analyzed by electron microscopy and by electrophoresis in agarose gel. The minicircle sizes varied from 1.3 to 2.8 kilobase pairs according to the Phytomonas isolate. The analysis by restriction endonucleases revealed a base sequence heterogeneity in the minicircles of 10 of these Phytomonas isolates. By contrast, in 2 Phytomonas isolates, more than 90% of their minicircle content was found to be homogeneous. Most interestingly, the minicircle cleavage patterns were found to be different between Phytomonas isolates and thus could be used to distinguish them.     

Extraction and characterization of a natural rubber from Euphorbia characias latex

A natural rubber was identified and characterized for the first time in the latex of the perennial Mediterranean shrub Euphorbia characias. Four different methods, i.e., acetone, acetic acid, trichloroacetic acid, and Triton® X‐100, followed by successive treatments with cyclohexane/ethanol, were employed to extract the natural rubber. The rubber content was shown to be 14% (w/v) of the E. characias latex, a low content compared with that of Hevea brasiliensis (30–35%) but a similar content to other rubber producing plants. E. characias rubber showed a molecular weight of 93,000 with a M_w/M_n of 2.9. ¹H NMR, ¹³C NMR, and FTIR analysis revealed the characteristic of the cis‐1,4‐polyisoprene typical of natural rubber. These results provided novel insight into latex components and will ultimately benefit the broader understanding of E. characias latex composition. © 2012 Wiley Periodicals, Inc. Biopolymers 97: 589–594, 2012.     

Long-term consequences of the alteration of the seed dispersal process of Euphorbia characias due to the Argentine ant invasion

The alteration of the seed dispersal process due to the Argentine ant invasion and its consequences on emergence, recruitment, distribution, and survival of seedlings of Euphorbia characias were analyzed. The study was carried out in two zones of Mediterranean cork‐oak secondary forest, one invaded by L. humile and the other non‐invaded. Two cohorts of E. characias seedlings (those emerged in 2001 and those emerged in 2002) were studied in three study plots in each zone. The level of seed loss due to lack of viability, parasitizing, and vertebrate predation did not differ between the two zones. The mean seed sowing depth was lower in the invaded zone (13.5 mm) than in the non‐invaded zone (22.4 mm). This depth difference implies a longer time needed for seedlings to emerge in the non‐invaded zone but not a different emergence percent under laboratory conditions. In the field study plots seedling recruitment did not differ between the two zones, probably due to the trade‐off between the differences in the initial number of seeds released (higher in the non‐invaded zone) and the different emergence proportions (higher in the invaded zone). As for the spatial characteristics of emerged seedlings, no differences in the mean seedling distance to the nearest inflorescence or to the nearest seedling or in the pattern of seedling distribution were found between zones. Seedling survival was assessed once a month until they had reached maturity or until all of them had died. The median seedling survival time was similar between the invaded and the non‐invaded zones. Survival curves also did not differ between zones. The present study suggests a functional equivalence of the Argentine ant after the replacement of the native ant species. Despite the initial differences found, the final reproductive success of E. characias was not altered after the invasion. However, the case of E. characias seems unlikely to be the rule, and the seed dynamics of other species may be altered, i.e. increased or decreased, and thus positively or negatively affected by the invasion.     

The Immune Response of Hemocytes of the Insect Oncopeltus fasciatus against the Flagellate Phytomonas serpens

The genus Phytomonas includes parasites that are etiological agents of important plant diseases, especially in Central and South America. These parasites are transmitted to plants via the bite of an infected phytophagous hemipteran. Despite the economic impact of these parasites, many basic questions regarding the genus Phytomonas remain unanswered, such as the mechanism by which the parasites cope with the immune response of the insect vector. In this report, using a model of systemic infection, we describe the function of Oncopeltus fasciatus hemocytes in the immune response towards the tomato parasite Phytomonas serpens. Hemocytes respond to infection by trapping parasites in nodular structures and phagocytizing the parasites. In electron microscopy of hemocytes, parasites were located inside vacuoles, which appear fused with lysosomes. The parasites reached the O. fasciatus salivary glands at least six hours post-infection. After 72 hours post-infection, many parasites were attached to the salivary gland outer surface. Thus, the cellular responses did not kill all the parasites.     

Plant fecundity and pre-dispersal reproductive losses in a common and a rare Euphorbia species (Euphorbiaceae)

Comparative studies on the reproductive biology of closely related plant species have provided valuable information to understand the causes and consequences of common-rare differences with possible applications for the management of threatened populations. The magnitude and spatiotemporal variability of seed production and pre-dispersal reproductive losses were studied for 3 years in the rare endemic Euphorbia welwitschii and in its widespread congener E. characias. The factors responsible for a decrease in potential seed production in these species were the lack of a functional ovary in the cyathium, ovary and fruit abortion, seed predation by insects and seed abortion. In E. characias, the moth Acroclita subsequana was also responsible for minor reproductive losses. The proportion of male cyathia varied significantly between seasons, populations and species, being consistently higher in E. characias than in E. welwitschii. Reproductive losses that resulted in ovary, fruit and seed abortion affected mostly the endemic species and were heavier in the populations located near the sea due to local adverse climatic conditions. Seed predators inflicted higher reproductive losses to the endemic species than to its widespread congener and their impact was particularly heavy at Risco. The two Euphorbia species differed markedly in cyathia production, with E. welwitschii producing systematically a lower number of cyathia than its widespread congener and this, together with higher levels of ovary, fruit and seed abortion, seemed to be the main reasons for the low reproductive output of this rare species.     

Axenic Cultivation and Ultrastructural Study of a Phytomonas sp. Isolated from the Milkweed Plant Euphorbia hyssopifolia1

ABSTRACT. A large number of trypanosomatids was seen in the latex of the milkweed plant Euphorbia hyssopifolia found in Rio de Janeiro, Brazil. The parasites, which belong to the genus Phytomonas, were isolated and axenically cultivated in a biphasic culture medium. The form and the structural organization of the parasites as found in the intact plant, in the isolated latex, and in the culture medium was studied by scanning and transmission electron microscopy.     

Ubiquity of Cysteine- and Metalloproteinase Activities in a Wide Range of Trypanosomatids

We have analysed the proteinase profiles of 11 species from 7 different genera of trypanosomatids by in situ detection of enzyme activities on SDS-PAGE gels containing co-polymerized gelatin as substrate, and the use of specific proteinase inhibitors. Our survey indicates that while cysteine- and metalloproteinases are distributed ubiquitously among trypanosomatids, there are marked differences between the enzyme profiles from the monogenetic (Crithidia, Herpetomonas, Leptomonas) and digenetic (Trypanosoma, Endotrypanum, Phytomonas, Leishmania) species. The detected metalloproteinase activities, ranging in size from 50–100 kDa, partitioned into the detergent-phase after Triton X-114 extraction, while most of cysteine proteinases, of three distinct molecular mass ranges (30–50 kDa, 80–100 kDa and 116–205 kDa), partitioned into the aqueous phase. Thus, within this group of organisms, the metalloproteinase activities seem to be predominantly membrane-associated proteins. We also show that the plant parasites of the genus Phytomonas exhibit a distinctive cysteine proteinase profile that might be exploited further as a criterion for taxonomy of the genus.     

Systematics of the Eimerian Parasites from North American Snakes of the Family Colubridae,and their Prevalence in the Colubrids of Iowa*

A survey of 117 Iowa snakes, representing 18 species within 12 genera, revealed the presence of 6 species of Eimeria, 5 of which are described as new and 1 of which (E. zamenis) is redescribed. Those species found, the average length-width dimensions of their oocysts ( in micrometers ), and the respective hosts from which they were isolated were as follows: E. attenuata sp. n., 22.2 × 12.6, from 1 of 25 red-sided garter snakes [Thamnophis sirtalis parietalis (Say)] and 1 of 14 northern water snakes [Natrix sipedon sipedon (Linnaeus)]; E. iowaensis sp. n., 17.8 × 14.5, from 1 of 25 redsided garter snakes; E. hydrophis sp. n., 15.4 × 10.9, from 5 of 14 northern water snakes and 1 of 1 diamond-backed water snake [N. rhombifera rhombifera (Hallowell)]; E. helmisophis sp. n., 13.8 × 10.6, from 1 of 5 western worm snakes [Carphophis amoenus vermis (Kennicott)]; E. collanuli sp. n., 33.1 × 18.3, from 1 of 14 prairie ring-neck snakes (Diadophis punctatus arnyi Kennicott), and E. zamenis, 31.0 × 17.0, from 1 of 6 eastern yellow-bellied racers (Coluber constrictor flaviventris Say) and 1 of 1 eastern milk snake [Lampropeltis triangulum triangulum (Lacépède)]. The overall infection rate for the 117 snakes examined was 9.2%; these data are tabulated. In addition, the possible synonymy of E. lampropeltis with E. zamenis is considered, and the probable status of E. attenuata, E. hydrophis, E. zamenis, and E. annea, as parasites of multiple host species, is reviewed with regard to the phylogenetic relationships of the respective hosts from which they have been reported.