Novel EIicitin from Pythium oligandrum Confers Disease Resistance against Phytophthora capsici in Solanaceae Plants.

The mycoparasite Pythium oligandrum is a nonpathogenic oomycete that can boost plant immune responses. Elicitins are microbe-associated molecular patterns (MAMPs) specifically produced by oomycetes that activate plant defense. Here, we identified a novel elicitin, PoEli8, from P. oligandrum that exhibits immunity-inducing activity in plants. In vitro-purified PoEli8 induced strong innate immune responses and enhanced resistance to the oomycete pathogen Phytophthora capsici in Solanaceae plants, including Nicotiana benthamiana, tomato, and pepper. Cell death and reactive oxygen species (ROS) accumulation triggered by the PoEli8 protein were dependent on the plant coreceptors receptor-like kinases (RLKs) BAK1 and SOBIR1. Furthermore, REli from N. benthamiana, a cell surface receptor-like protein (RLP) was implicated in the perception of PoEli8 in N. benthamiana. These results indicate the potential value of PoEli8 as a bioactive formula to protect Solanaceae plants against Phytophthora.

Vascular pythiosis caused by Pythium aphanidermatum: the first case report in Asia.

BACKGROUND: Pythium, soil-borne plant pathogens, are in the class Oomycetes. They are not true fungi, but are related to diatom and algae. There are two human pathogens including P. insidiosum and P. aphanidermatum. To date, only one case of pythiosis caused by P. aphanidermatum has been reported. We present herein the first case of P. aphanidermatum vascular pythiosis in Asia. CASE PRESENTATION: A 47-year-old Thai woman, living in North Thailand, with ß thalassemia/hemoglobin E presented with acute recurrent arterial insufficiency of both legs. Emergent embolectomy with clot removal was performed. The pathology of the clot exhibited noncaseous granulomatous inflammation with many fungal hyphal elements. PCR identified P. aphanidermatum with 100% identity. Final diagnosis is vascular pythiosis. Unfortunately, the patient eventually expired after treatment with itraconazole, terbinafine, azithromycin, and doxycycline. CONCLUSIONS: To date, only one case of pythiosis caused by P. aphanidermatum has been reported. We present herein the first case of P. aphanidermatum vascular pythiosis in Asia.

Inhibition of Rhizoctonia solani RhCh-14 and Pythium ultimum PyFr-14 by Paenibacillus polymyxa NMA1017 and Burkholderia cenocepacia CACua-24: A proposal for biocontrol of phytopathogenic fungi.

Biocontrol has emerged in recent years as an alternative to pesticides. Given the importance of environmental preservation using biocontrol, in this study two antagonistic bacteria against phytopathogenic fungi were isolated and evaluated. These bacterial strains, identified as Paenibacillus polymyxa NMA1017 and Burkholderia cenocepacia CACua-24, inhibited (70 to 80%) the development of two phytopathogens of economic importance: the fungus Rhizoctonia solani RhCh-14, isolated from chili pepper, and the oomycete Pythium ultimum PyFr-14, isolated from tomato. The spectrum was not limited to the previous pathogens, but also to other phytopathogenic fungus, some bacteria and other oomycetes. Fungi-bacteria microcultures observed with optical and scanning electron microscopy revealed hyphae disintegration and pores formation. The antifungal activity was found also in the supernatant, suggesting a diffusible compound is present. Innocuous tests on tobacco leaves, blood agar, bean seed germination and in Galleria mellonella larvae showed that strain NMA1017 has the potential to be a biocontrol agent. Greenhouse experiments with bean plants inoculated with P. polymyxa exhibited the efficacy to inhibit the growth of R. solani and P. ultimum. Furthermore, P. polymyxa NMA1017 showed plant growth promotion activities, such as siderophore synthesis and nitrogen fixation which can contribute to the crop development.

Defective cytokine production from monocytes/macrophages of E-beta thalassemia patients in response to Pythium insidiosum infection.

BACKGROUND: Pythium insidiosum has been mainly reported to cause morbidity and mortality in thalassemia patients. P. insidiosum zoospores can germinate to be hyphae within a few hours; therefore, it is difficult to study the initial immune response that P. insidiosum zoospores induce. The present study aims to compare immune responses against P. insidiosum zoospore infection by comparing monocytes/macrophages from thalassemia patients with those from non-thalassemia controls. METHODS: In order to keepP. insidiosum in the zoospore stage in vitro for inoculation, the P. insidiosum zoospores were preserved without germination by treatment with inorganic hypochlorite solution. CD14+ cells were isolated from peripheral blood mononuclear cells of thalassemia and non-thalassemia donors and then left to transition to macrophages. Monocytes/macrophage culture was infected with P. insidiosum zoospores and culture supernatants were subjected to Th1/Th2 multiplex cytokine detection. RESULTS: Our study of cytokine production revealed that the basal level of GM-CSF produced by thalassemia monocytes/macrophages was lower than that observed in monocytes/macrophages of non-thalassemia individuals. Higher GM-CSF and IFN-γ response was also found when cells from non-thalassemia people were stimulated with P. insidiosum zoospores compared to thalassemia cells. It was also found that TNF-α, GM-CSF and IFN-γ productions from monocytes/macrophages of thalassemia patients who received iron chelator treatment were significantly higher than those produced from thalassemia patients without iron chelator treatment. CONCLUSION: For the first time, the present study demonstrates defective immune responses in monocytes/macrophages derived from thalassemia patients in response toP. insidiosum zoospore infection. The results also show an inverse correlation between iron overload and cytokine production in monocytes/macrophages of thalassemia patients. This finding could explain why thalassemia patients are susceptible to P. insidiosum infection.

Pulmonary pythiosis in a canine patient.

A Staffordshire terrier presented for evaluation of a chronic, nonproductive cough that was unresponsive to antibiotic therapy. A large mass identified in the pulmonary hilum was most consistent with tracheobronchial lymphadenopathy on radiographic and computed tomography (CT) images. Bronchoscopy confirmed a mass compressing the dorsal portion of the intrathoracic trachea. Bronchoscopic biopsies of the tracheal mass revealed necrosuppurative and eosinophilic inflammation with intralesional Pythium insidiousum hyphae. Pythiosis should be included as a differential diagnosis for tracheobronchial lymphadenopathy and bronchopneumopathy in dogs, especially when the patient is from or has visited a region endemic for Pythium insidiosum.

Pythium insidiosum: inhibitory effects of propolis and geopropolis on hyphal growth

Abstract Propolis and geopropolis are resinous products of bees showing antimicrobial effects. There is no data concerning their action against Pythium insidiosum - the causative agent of pythiosis, a pyogranulomatous disease of the subcutaneous tissue that affects mostly horses, dogs and humans. Fragments of 15 isolates of P. insidiodum were incubated with propolis and geopropolis extracts and evaluated for up to seven days to detect the minimal fungicidal concentration (MFC). Propolis inhibited three isolates at 1.0 mg mL-1 after 24 h and all other isolates at 3.4 mg mL-1. Geopropolis led to more variable results, exerting predominantly a fungistatic action than a fungicidal one. Propolis was more efficient than geopropolis in inhibiting P. insidiosum since lower concentrations led to no growth after 24 h. This effect may be due to propolis chemical composition, which has more active compounds than geopropolis. Propolis seemed to be a good candidate for in vivo studies, since treatment with conventional antifungal compounds is difficult in most of the cases, requiring extensive surgical debridement.

The role of strigolactones and ethylene in disease caused by Pythium irregulare.

Plant hormones play key roles in defence against pathogen attack. Recent work has begun to extend this role to encompass not just the traditional disease/stress hormones, such as ethylene, but also growth-promoting hormones. Strigolactones (SLs) are the most recently defined group of plant hormones with important roles in plant-microbe interactions, as well as aspects of plant growth and development, although the knowledge of their role in plant-pathogen interactions is extremely limited. The oomycete Pythium irregulare is a poorly controlled pathogen of many crops. Previous work has indicated an important role for ethylene in defence against this oomycete. We examined the role of ethylene and SLs in response to this pathogen in pea (Pisum sativum L.) at the molecular and whole-plant levels using a set of well-characterized hormone mutants, including an ethylene-insensitive ein2 mutant and SL-deficient and insensitive mutants. We identified a key role for ethylene signalling in specific cell types that reduces pathogen invasion, extending the work carried out in other species. However, we found no evidence that SL biosynthesis or response influences the interaction of pea with P. irregulare or that synthetic SL influences the growth or hyphal branching of the oomycete in vitro. Future work should seek to extend our understanding of the role of SLs in other plant interactions, including with other fungal, bacterial and viral pathogens, nematodes and insect pests.

Antifungal properties of native Trichoderma isolates against Sclerotium rolfsii and Pythium aphanidermatum infecting tobacco.

Isolates of Trichoderma native to tobacco rhizosphere, were evaluated for their antifungal properties against Sclerotium rolfsii and Pythium aphanidermatum, fungal pathogens causing collar rot and damping off diseases in tobacco. Isolate TvJt1 showed maximum HCN production (0.14 OD) followed by ThHt1 and minimum by isolates ThJnt and ThRt1 (0.08 and 0.09 OD). Indole acetic acid (IAA) production among the isolates varied from 6.48 to 14.82 pg ml(-1). Isolate ThJO1 produced maximum siderophore (13.48 m mol ml(-1), whereas isolate TvJt1 produced minimum content (6.24 m mol ml(-1)). Isolate ThJt1 showed maximum and significantly higher chitinase activity (62.12 p kat m;(-1)) as compared to other isolates, followed by isolate TvHt2. Isolate TvHt2 showed maximum and significantly higher ß-1, 3-glucanase activity (9.94 n kat ml(-1)) when compared to all other isolates. Isolate ThJt1 showed second highest activity of ß-1, 3-glucanase (6.75 n kat/ml). In dual culture, inhibition of mycelial growth of Pythium by isolate TvHt2 was maximum (72.45%) followed by ThJt1 whereas inhibition of mycelial growth of S. rolfsii by these two isolates was at a par (78.36% and 76.17%) and significantly higher than some isolates. Isolates TvHt2 and ThJt1 showed maximum and significantly higher inhibition of Pythium and Sclerotium growth (80.24% and 76%) by production of non-volatiles. Inhibition of Pythium and Sclerotium growth by production of volatile compounds by Trichoderma isolates ranged from 54.36 to 72.6% and 66.24 to 84.24% respectively. Hence, further investigation with potential isolates ThJt1 and TvHt2 in tobacco nursery may help to explore these bioagents for control of collar rot and damping off diseases in tobacco.

Antibiosis functions during interactions of Trichoderma afroharzianum and Trichoderma gamsii with plant pathogenic Rhizoctonia and Pythium.

Trichoderma afroharzianum is one of the best characterized Trichoderma species, and strains have been utilized as plant disease suppressive inoculants. In contrast, Trichoderma gamsii has only recently been described, and there is limited knowledge of its disease suppressive efficacies. Comparative studies of changes in gene expression during interactions of these species with their target plant pathogens will provide fundamental information on pathogen antibiosis functions. In the present study, we used complementary DNA amplified fragment length polymorphism (cDNA-AFLP) analysis to investigate changes in transcript profiling of T. afroharzianum strain LTR-2 and T. gamsii strain Tk7a during in vitro interactions with plant pathogenic Rhizoctonia solani and Pythium irregulare. Considerable differences were resolved in the overall expression profiles of strains LTR-2 and Tk7a when challenged with either plant pathogen. In strain LTR-2, previously reported mycoparasitism-related genes such as chitinase, polyketide synthase, and non-ribosomal peptide synthetase were found to be differentially expressed. This was not so for strain Tk7a, with the only previously reported antibiosis-associated genes being small secreted cysteine-rich proteins. Although only one differentially expressed gene was common to both strains LTR-2 and Tk7a, numerous genes reportedly associated with pathogen antibiosis processes were differentially expressed in both strains, including degradative enzymes and membrane transport proteins. A number of novel potential antibiosis-related transcripts were found from strains LTR-2 and Tk7a and remain to be identified. The expression kinetics of 20 Trichoderma (10 from strain LTR-2, 10 from strain Tk7a) transcript-derived fragments (TDFs) were quantified by quantitative reverse transcription PCR (RT-qPCR) at pre- and post-mycelia contact stages of Trichoderma-prey interactions, thereby confirming differential gene expression. Collectively, this research is providing information to elucidate the antibiosis mechanisms and disease suppressive activities of T. afroharzianum and T. gamsii against soilborne fungal and oomycete plant pathogens.

Complex interaction of deferasirox and Pythium insidiosum: iron-dependent attenuation of growth in vitro and immunotherapy-like enhancement of immune responses in vivo.

Pythium insidiosum iron acquisition mechanisms are unknown. We previously showed that the iron chelator deferasirox had weak activity in vitro and in rabbits with experimental pythiosis. Here we show that deferasirox causes damage to P. insidiosum hyphae in vitro, but that activity is diminished in the presence of exogenous iron. The tissue activity of the proinflammatory enzyme adenosine deaminase and the histological pattern observed in pythiosis lesions of rabbits treated with deferasirox were similar to the ones in animals treated with immunotherapy.

Elicitin-like proteins Oli-D1 and Oli-D2 from Pythium oligandrum trigger hypersensitive response in Nicotiana benthamiana and induce resistance against Botrytis cinerea in tomato.

The biocontrol agent Pythium oligandrum and its elicitin-like proteins oligandrins have been shown to induce disease resistance in a range of plants. In the present study, the ability of two oligandrins, Oli-D1 and Oli-D2, to induce an immune response and the possible molecular mechanism regulating the defence responses in Nicotiana benthamiana and tomato were investigated. Infiltration of recombinant Oli-D1 and Oli-D2 proteins induced a typical immune response in N. benthamiana including the induction of a hypersensitive response (HR), accumulation of reactive oxygen species and production of autofluorescence. Agrobacterium-mediated transient expression assays revealed that full-length Oli-D1 and Oli-D2 were required for full HR-inducing activity in N. benthamiana, and virus-induced gene silencing-mediated knockdown of some of the signalling regulatory genes demonstrated that NbSGT1 and NbNPR1 were required for Oli-D1 and Oli-D2 to induce HR in N. benthamiana. Subcellular localization analyses indicated that both Oli-D1 and Oli-D2 were targeted to the plasma membrane of N. benthamiana. When infiltrated or transiently expressed in leaves, Oli-D1 and Oli-D2 induced resistance against Botrytis cinerea in tomato and activated the expression of a set of genes involved in the jasmonic acid/ethylene (JA/ET)-mediated signalling pathway. Our results demonstrate that Oli-D1 and Oli-D2 are effective elicitors capable of inducing immune responses in plants, probably through the JA/ET-mediated signalling pathway, and that both Oli-D1 and Oli-D2 have potential for the development of bioactive formulae for crop disease control in practice.

Prevalence of type III secretion system in effective biocontrol pseudomonads.

Functional type III secretion system (T3SS) genes are needed for effective biocontrol of Pythium damping-off of cucumber by Pseudomonas fluorescens KD, but whether biocontrol Pseudomonas strains with T3SS genes display overall a higher plant-protecting activity is unknown. The assessment of 198 biocontrol fluorescent pseudomonads originating from 60 soils worldwide indicated that 32% harbour the ATPase-encoding T3SS gene hrcN, which was most often found in tomato isolates. The hrcN(+) biocontrol strains (and especially those also producing 2,4-diacetylphloroglucinol and displaying 1-aminocyclopropane-1-carboxylate deaminase activity) displayed higher plant-protecting ability in comparison with hrcN(-) biocontrol strains, both in the Pythium/cucumber and Fusarium/cucumber pathosystems.

Insights into the molecular mechanism of tolerance to carboxylic acid amide (CAA) fungicides in Pythium aphanidermatum.

BACKGROUND: Tolerance to the oomycete-specific carboxylic acid amide (CAA) fungicides is a poorly understood mechanism in Pythium species. The root-rot and damping-off causative agent Pythium aphanidermatum and the CAA fungicide mandipropamid (MPD) were used to investigate the molecular basis of CAA tolerance. RESULTS: Five genes putatively involved in carbohydrate synthesis were identified and characterised: one chitin synthase gene, PaChs, and four cellulose synthase genes PaCesA1 to PaCesA4, of which PaCesA3 encodes the MPD target enzyme. These genes were differentially expressed throughout the life cycle of P. aphanidermatum. Mycelium treated with MPD concentrations slightly affecting mycelial growth did not cause a change in PaCesA3 expression nor a strong upregulation of PaCesA homologues. The high tolerance level of P. aphanidermatum and the lack of PaCesA upregulation imply that MPD tolerance is the result of a specific amino acid configuration in the cellulose synthase 3 (CesA3) target enzyme. Indeed, P. aphanidermatum displays the amino acid L1109 which is also associated with MPD resistance in artificial mutants of Phytophthora species. CONCLUSION: It is concluded that MPD tolerance in P. aphanidermatum is not caused by compensatory mechanisms but most likely by an inherent target-site configuration in PaCesA3 that hinders MPD binding to the enzyme pocket.

Implications of oligomeric forms of POD-1 and POD-2 proteins isolated from cell walls of the biocontrol agent Pythium oligandrum in relation to their ability to induce defense reactions in tomato.

The cell wall protein fraction (CWP) isolated from the biocontrol agent Pythium oligandrum induces defense reactions in tomato. CWP contains two novel elicitin-like proteins, POD-1 and POD-2, both with seven cysteines. To determine the essential structure in the defense-eliciting components of CWP, five fractions (F1, F2, F3, F4 and F5) were fractionated from CWP using cation chromatography and their components and disulfide bond compositions were analyzed. The expression levels of three defense-related genes (PR-6, LeCAS and PR-2b) were determined in tomato roots treated with each of the five fractions. Of the five fractions, F4 containing a heterohexamer of POD-1 and POD-2, and F5 containing a homohexamer of POD-1, both with disulfide bonds formed between all cysteine residues, induced the expression of three genes. F4 treatment also induced the accumulation of ethylene in tomato. The predicted three-dimensional structures of POD-1 and POD-2, and the results of SEC and MALDI-TOF MS analyses suggest that F4 consists of three POD-1 and POD-2 disulfide-bonded heterodimers that interleave into a hexameric ring through noncovalent association. These results suggest that this structure, which F5 also appears to form, is essential for stimulating defense responses in tomato.

Influence of cellular differentiation and elicitation on intermediate and late steps of terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

The invaluable antineoplastic bisindole alkaloids of Catharanthus roseus and their precursor, vindoline, are not produced in cell cultures. The intricacies involved in endogenous (cellular differentiation) and exogenous (elicitation) regulation of their biosynthesis need to be dissected out for favorable exploitation. This study aimed at elucidating the effect of Pythium aphanidermatum homogenate and methyl jasmonate (MeJa) on in vitro cultures (of cv. 'Dhawal') representing increasing level of differentiation (suspension < callus < shoots) in terms of alkaloid accumulation and transcript abundance of strictosidine beta-D: -glucosidase (SGD) and acetyl-CoA: 4-O-deacetylvindoline 4-O-acetyl-transferase (DAT) genes, representing intermediate and late steps, respectively, of terpenoid indole alkaloid biosynthesis. Elicitation of suspension cultures caused transcriptional upregulation of SGD and enhanced the accumulation of total alkaloids but did not produce vindoline as DAT transcripts were always found to be absent in suspension-cultured cells. Vindoline was also not detected in unelicited and MeJa-treated callus but appeared upon elicitation with fungal homogenate for 24 h that coincided with maximal DAT transcription. Transcript levels of both genes increased upon elicitation of callus but remained below levels present in the mature plant leaf. Elicitation caused appearance of vindoline in shoots and increased the transcript abundance of both genes beyond levels observed in the mature plant leaf. Differentiation was essential for expression of DAT but not SGD, and vindoline biosynthetic potential increased with it.

Use of cross-flow membrane filtration in a recirculating hydroponic system to suppress root disease in pepper caused by Pythium myriotylum.

Zoosporic pathogens in the genera Pythium and Phytophthora cause extensive root disease epiphytotics in recirculating hydroponic vegetable-production greenhouses. Zoospore cysts of Pythium myriotylum Drechsler were used to evaluate the effectiveness of cross-flow membrane filters to control pythiaceous pathogens in recirculating hydroponic systems. Four membrane filter brands (Honeycomb, Polypure, Polymate, and Absolife) were tested alone or in combination to determine which filters would effectively remove infective propagules of P. myriotylum from solutions and reduce disease incidence and severity. Zoospore cysts of P. myriotylum generally measured 8 to 10 microm, and it was hypothesized that filters with pore-sizes<5 microm would be effective at removing 100% of the infective propagules and protect pepper plants from root infection. Single-filter assays with Honeycomb and Polypure brands removed 85 to 95% of zoospore cysts when pore sizes were rated at 1, 5, 10, 20, or 30 microm. Single-filter assays of Polymate and Absolife brands were more effective, exhibiting apparently 100% removal of zoospore cysts from nutrient solutions on filters rated at 1 to 10 microm. However, plant bioassays with Honeycomb and Polymate single filters failed to give long-term protection of pepper plants. Double-filter assays with 1- and 0.5-microm Polymate filters significantly increased the protection of pepper plants grown in nutrient film technique systems but, eventually, root disease and plant wilt could be observed. Insect transmissions by shore flies were not factors in disease development. Scanning electron microscopy images of zoospore cysts entrapped on Polymate filters revealed zoospore cysts that were either fully encysted, partially encysted, or of unusually small size (3 microm in diameter). It was concluded that either the atypically small or pliable pleomorphic zoospore cysts were able to penetrate filter membranes that theoretically should have captured them.

A novel protein elicitor (PaNie) from Pythium aphanidermatum induces multiple defense responses in carrot, Arabidopsis, and tobacco.

A novel protein elicitor (PaNie(234)) from Pythium aphanidermatum (Edson) Fitzp. was purified, microsequenced, and the corresponding cDNA was cloned. The deduced amino acid sequence contains a putative eukaryotic secretion signal with a proteinase cleavage site. The heterologously expressed elicitor protein without the secretion signal of 21 amino acids (PaNie(213)) triggered programmed cell death and de novo formation of 4-hydroxybenzoic acid in cultured cells of carrot (Daucus carota). Programmed cell death was determined using the tetrazolium assay and DNA laddering. Infiltration of PaNie(213) into the intercellular space of leaves of Arabidopsis (Columbia-0, wild type) resulted in necroses and deposition of callose on the cell walls of spongy parenchyma cells surrounding the necrotic mesophyll cells. Necroses were also formed in tobacco (Nicotiana tabacum cv Wisconsin W38, wild type) and tomato (Lycopersicon esculentum Mill.) but not in maize (Zea mays), oat (Avena sativa), and Tradescantia zebrina (Bosse), indicating that monocotyledonous plants are unable to perceive the signal. The reactions observed after treatment with the purified PaNie(213) were identical to responses measured after treatment with a crude elicitor preparation from the culture medium of P. aphanidermatum, described previously. The availability of the pure protein offers the possibility to isolate the corresponding receptor and its connection to downstream signaling-inducing defense reactions.

Behavior of pythium Torulosum zoospores during their interaction with tobacco roots and Bacillus cereus.

Bacillus cereus UW85 suppresses seedling damping-off diseases caused by Oomycetes and produces antibiotics that inhibit development of Oomycetes in culture. The goal of this study was to determine how UW85 and its antibiotics affected the behavior of an Oomycete, Pythium torulosum, in its interaction with plant roots. We studied tobacco seedlings inoculated with zoospores of P. torulosum and UW85 culture, culture filtrate, washed cells, antibiotics (zwittermicin A or kanosamine), purified from cultures of UW85, and UW030, a mutant of UW85 that does not suppress disease and does not produce the antibiotics. Microscopic observation revealed that all of the treatments inhibited zoospore activity around roots and encystment on roots. Treatment with UW85 culture, culture filtrate, zwittermicin A, or kanosamine delayed cyst germination and the elongation rate of germ tubes, whereas treatment with UW030 or washed UW85 cells did not. In an in vitro seedling bioassay of disease suppression, the antibiotics, zwittermicin A and kanosamine, suppressed disease singly or together, although UW85 culture suppressed disease more effectively than did the antibiotics. The results show that B. cereus cultures affect zoospore behavior in the presence of roots, and B. cereus-produced antibiotics, zwittermicin A and kanosamine, contribute to disease suppression and inhibition of germ tube elongation in the presence of the plant root.

The relationships between the toxicity and the primary and secondary structures of elicitinlike protein elicitors secreted by the phytopathogenic fungus Pythium vexans.

Elicitins are toxic and signaling proteins secreted by Phytophthora spp. responsible for the incompatible reaction and systemic hypersensitive-like necroses of diverse plant species leading to resistance against fungal or bacterial plant pathogens. Such proteins were observed in the culture filtrate of another species of the Oomycete genus, Pythium vexans. Two alpha elicitinlike proteins were purified and sequenced. One of these novel elicitins (Vex2) exhibited a 100-residue sequence instead of 98 while the other (Vex1) had an N-glycosylation site, effectively glycosylated (equivalent of 16 hexose residues). In addition to the point mutations already observed in Phytophthora species, we found several novel amino acid changes. Furthermore, circular dichroism revealed some differences in their structure in solution compared with the Phytophthora elicitins that were correlated with specific point mutations. These sequences permitted the establishment of a phylogenic tree, suggesting that Pythium vexans is a species close to the Phytophthora genus. The toxicity of the Pythium vexans elictins to tobacco leaves was investigated and correlated with the occurrence of the carbohydrate moiety of one of the two isoforms, observed for the first time in an elicitin.