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Interactions between the mycoparasite Pythium oligandrum and two types of sclerotia of plant-pathogenic fungi.

The interactions between Pythium oligandrum hyphae and two types of sclerotia, i.e. the plano-convexoid sclerotium of Botrytis cinerea and the tuberoid sclerotium of Sclerotinia minor, were investigated by ultrastructural and cytochemical experiments. In the mycoparasitism of P. oligandrum, some differences in relation to sclerotium anatomy and the role of the rind layer in preventing invasion are documented. Both types of sclerotia showed neither alterations of the heavily melanised rind walls, nor direct rind wall penetration by P. oligandrum. This oomycete successfully entered B. cinerea sclerotia only through breaches at the junction of rind cells and corresponding to gaps in melanin deposits. On the other hand, none of these breaches was observed in the small sclerotia of S. minor, and P. oligandrum ingress in the sclerotia stopped at the inner rind layer. After the penetration of B. cinerea sclerotia by the mycoparasite, it extensively colonised the cortical and medulla areas by intercellular growth. The invaded tissues displayed pronounced alterations as well as some disorganisation of tissue in places. Colonisation was associated with severe chitin degradations of all host walls, which occurred even at some distance from P. oligandrum hyphae. The observation of wall thickenings in some P. oligandrum-hyphae suggests that the sclerotial cells constitute a harsh environment unsuitable for survival of the mycoparasite. These wall thickenings could be interpreted as P. oligandrum defence-like reactions.

Antibiosis↗

The putative gymnosperm plant defensin polypeptide (SPI1) accumulates after seed germination, is not readily released, and the SPI1 levels are reduced in Pythium dimorphum-infected spruce roots.

The putative plant defensin SPI1 cDNA from the conifer Norway spruce (Picea abies) is the only known plant defensin-like sequence from a gymnosperm. The predicted translational product SPI1 was not detected in the embryo or other parts of the seed by means of antibodies, but it accumulated in the root cortex after germination. In roots of seedlings infected with the root pathogenic oomycete Pythium dimorphum and the blue stain fungus Ceratocystis polonica, variable levels of SPI1 was detected during the first day as a response to the infection, however a significant increase was seen as an initial response to the root-rot fungus Heterobasidion annosum. After the first day of infection, the amount of SPI1 polypeptide was dramatically reduced in response to either of the pathogens, but not in response to the ectomycorrhizal fungus Laccaria bicolor. During the same time of infection, extensive damage to cortical root cells resulted from the infecting pathogens, but not from the mycorrhiza. These results indicate that pathogens may reduce the level of SPI1 by suppressing its expression, but may also reduce the SPI1 level by invading and disrupting the root cortical cells or by a combination of these mechanisms.

Defensins↗

Influence of temperature on Pythium splendens--induced root disease on carambola, Averrhoa carambola.

A series of glasshouse and incubator studies were conducted to investigate the role played by Pythium splendens in a decline disorder of carambola, Averrhoa carambola. Plants, 4-6 months old, were grown in native calcareous soil either infested or not infested with the pathogen. Isolates recovered from atemoya, carambola and passion fruit grew optimally at 30 degrees C, and significantly (P < 0.05) increased root necrosis and reduced root, shoot and total biomass of carambola. Temperature had a profound impact on the latter relationships. Two or more times more necrosis developed at 10 and 15 degrees C than at 25 and 30 degrees C. Total biomass accumulations were over four times greater at 30 degrees C than at 10 degrees C, and were always lower in soil infested with P. splendens. When biomass totals from infested and noninfested soil were compared, relative values were lowest at 15 and 20 degrees C and were almost two times greater at 30 degrees C than at 20 degrees C. Root infection by P. splendens was greatest at 15 and 20 degrees C, far below the species' optimum for growth, and at 30 degrees C was over nine times lower than at 15 and 20 degrees C. This is the first detailed report of P. splendens as a pathogen of carambola.

Fruit↗

Bacillus isolates from the spermosphere of peas and dwarf French beans with antifungal activity against Botrytis cinerea and Pythium species.

A range of isolation procedures including washing, sonication and incubation in nutrient broth were used separately and in combination to obtain potential bacterial antagonists to Botrytis cinerea and Pythium mamillatum from the testae and cotyledons of peas and dwarf French beans. Heat treatment was also used to bias this selection towards spore-forming bacteria. Ninety-two bacterial isolates were obtained, 72 of which were provisionally characterized as species of Bacillus. Four of these Bacillus isolates (B3, C1, D4 and J7) displayed distinct antagonism in vitro against Botrytis cinerea and P. mamillatum when screened using dual culture analysis. Further characterization of these antagonists using API 50CHB biochemical profiling identified isolate D4 as Bacillus polymyxa and isolates B3, C1 and J7 as strains of B. subtilis. In vitro screening techniques, using cell-free and heat-killed extracts of liquid cultures against Botrytis cinerea, demonstrated the production of antifungal compounds by these four Bacillus antagonists. With each isolate the antifungal activity was found not to be either exclusively spore-bound nor released entirely into the medium but present in both fractions. The antifungal compounds produced by these isolates were shown to be heat-stable. Their identification, production and release require further study for exploitation as biocontrol systems.

Antibiosis↗

Jasmonate signaling mutants of Arabidopsis are susceptible to the soil fungus Pythium irregulare.

Jasmonic acid has properties of a plant hormone, including the induction of specific genes associated with plant defense. We previously described jar1-1, an Arabidopsis jasmonate response mutant that exhibits reduced sensitivity to methyl jasmonate. We have further characterized this mutant and two new alleles; jar1-2 from a gamma irradiated population, and jar1-4 from a T-DNA mutant population. Seedling root growth in jar1-1 was equally insensitive to methyl jasmonate and jasmonic acid, indicating that the defect was not in the conversion of methyl jasmonate to the acid. None of the jar1 mutants showed an altered sensitivity to auxin, cytokinin, or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid, indicating that the lesion does not affect the general uptake or transport of hormones. A soil fungus, Pythium irregulare, was found to blight jar1-1. Cultures of this organism caused the symptoms in all three jar1 mutants but not in wild type, indicating that increased susceptibility was due to the lesion in the JAR1 locus. A fatty acid desaturase triple mutant that is defective in the biosynthesis of jasmonic acid (J. Browse, Washington State University) was also susceptible, confirming that jasmonate is involved in resistance. The jar1-1 locus was mapped to the lower end of chromosome 2, about 11.4 cM from as1 and 1.6 cM from cer8. These results establish that jasmonate signaling plays an important role in resistance to soil micro-organisms in plants.

Alleles↗

Signalling by the fungus Pythium ultimum represses expression of two ribosomal RNA operons with key roles in the rhizosphere ecology of Pseudomonas fluorescens F113.

Pseudomonas fluorescens F113 produces antifungal metabolites that protect the roots of sugarbeet from the fungus Pythium ultimum. The phytopathogen, in turn, has the ability to downregulate the expression of genes fundamental to the rhizosphere competence of the bacterial strain. This paper describes the characterization of two of these genes, which were isolated by screening a mini-Tn5::lacZ mutant bank for differential expression of beta-galactosidase in the presence of P. ultimum. In order to identify the genes affected in reporter mutants SF3 and SF5, the transposons and flanking regions were cloned. Sequence analysis of the regions flanking the transposons in SF3 revealed that mini-Tn5::lacZ had inserted into a tRNA(Ile) gene, which maps within a ribosomal RNA (rrn) operon. In SF5, the transposon inserted between the promoter of a second rrn operon and a gene encoding a 16S rRNA. Southern blot analysis demonstrated that there are five rrn operons in P. fluorescens F113 and that the transposons in SF3 and SF5 had inserted into two different operons. Further characterization of these mutants suggests that their reduced rhizosphere competence is not the result of reduced viability in the short term but may be accounted for partly by reduced growth rates under conditions that support rapid growth. Analysis of lacZ expression in the reporter mutants indicate that the marked rrn operons are regulated differently, suggesting different physiological roles.

Base Sequence↗

Use of polymerase chain reaction to detect the soft rot pathogen, Pythium myriotylum, in infected ginger rhizomes.

AIMS: The aims are to establish a polymerase chain reaction (PCR)-based method for detecting Pythium myriotylum in the rhizome of ginger and diagnosing ginger soft rot and screening health seed ginger. METHODS AND RESULTS: A booster PCR method was established for detection of P. myriotylum using a specific primer selected from rDNA ITS1 region coupled with universal primer ITS2. It successfully applied to the detection of P. myriotylum in naturally infected ginger rhizomes but not from DNA of ginger rhizomes collected from field without target fungus. CONCLUSIONS: A specific method for detecting P. myriotylum was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: The new PCR method has allowed us to monitor ginger for the presence of P. myriotylum as a way of disease diagnosis or healthy seed ginger examination.

Zingiber officinale↗

Detection of the low-germination-rate resting oospores of Pythium myriotylum from soil by PCR.

AIMS: To establish a sensitive and specific polymerase chain reaction (PCR)-based method for detecting Pythium myriotylum in soils. METHODS AND RESULTS: Oospores of P. myriotylum were separated from large soil particles by flotation in sucrose solution. The thick-walled oospores were disrupted by vortex with sea sand and its DNA was extracted by the Cetyl trimethyl Ammonium Bromide (CTAB) method. The recovered DNA was verified by PCR amplification of a 150-bp target sequence of P. myriotylum. Samples of 10 g of soil were assayed; thus, the detection limit by PCR-based method was 10 oospores per gram soil. The method was successfully applied for the detection of P. myriotylum in soils collected in March, prior to planting of ginger crops. CONCLUSIONS: A PCR-based method for detecting P. myriotylum from soil was achieved. SIGNIFICANCE AND IMPACT OF THE STUDY: The PCR method has allowed us to monitor the presence of P. myriotylum in soil prior planting season as a way of reducing or eliminating disease.

DNA, Fungal↗

Pythium perplexum isolated from soil in France: morphology, molecular characterisation and biological control.

Pythium perplexum (F-926) was isolated from a soil sample taken in the Burgundy region in France. In 1907, it was mistakenly described by Bulter as P. vexans. Despite morphological resemblance, the comparison between the internal transcribed spacer 1 regions of the ribosomal DNA of the two fungi leaves no doubt of their different identities. P. perplexum was found to be highly pathogenic to cucumber seedlings. Damping-off disease of cucumber caused by P. perplexum can effectively be controlled by using the soil bacterium Serratia plymuthica (B-781). The details of the morphology and the molecular characterisation of P. perplexum and its biological control with S. plymuthica are described here.

Antibiosis↗

Nasal and retrobulbar mass in a cat caused by Pythium insidiosum.

Nasal and retrobulbar infection caused by the Oomycete Pythium insidiosum is described in a cat. The diagnosis was established on three criteria. The staining of broad, sparsely septate hyphal elements in biopsy tissue using a fluorescein-labelled antiglobulin specific for P. insidiosum, detection of antibodies to P. insidiosum by an immunodiffusion test, and isolation of the aetiological agent in pure culture from the biopsy tissue. Treatment with ketoconazole for 6 weeks resulted in clinical improvement, but proptosis of the left eye slowly appeared after the discontinuation of treatment. This case represents a new host for P. insidiosum, namely, a domestic, shorthaired cat, from North Carolina, U.S.A.

Animals↗

Immunology and immunotherapy of the infections caused by Pythium insidiosum.

Although infections caused by the straminipilan pathogen Pythium insidiosum were described in 19th century, it has been only recently that its epidemiology, immunology, treatment and other important traits were extensively studied. These studies were of paramount importance to theorize about the ecological niche for this pathogen, its host-parasite relationships, the antigens used for diagnosis, and the management of the infection using immunotherapy. P. insidiosum triggers in the infected host a T helper 2 [Th2] subset with an inflammatory reaction composed mainly of eosinophils and mast cells. These cells degranulate around the hyphal elements of P. insidiosum where a Splendore-Hoeppli-like reaction develops. In horses this reaction is so intensive that firm concretions called 'kunkers' develop. These data indicated that this pathogen might have developed an evolutionary strategy to conceal important antigens from the host immune system. Immunotherapy, a treatment approach that relies on the injection of antigens of P. insidiosum from in vitro cultures, has been successfully used in humans and horses to manage this disease. A switch from a Th2 to Th1 response is postulated as the most likely explanation of the curative properties of this approach. This review provides details on the serological, immunological, and immunotherapeutic methodologies used to diagnose and treat the infections caused by this pathogen.

Animals↗

Pythium insidiosum pleuropericarditis complicating pneumonia in a child with leukemia.

We describe a 12-year-old boy with acute myeloid leukemia who developed pleuropericarditis while he was neutropenic and was receiving intravenously administered antibiotic and antifungal therapy for pneumonia. A KOH preparation of the purulent material from an extensive diagnostic and therapeutic pleuropericardial drainage procedure revealed multiple irregularly septate hyphae, and cultures yielded the organism Pythium insidiosum. After completing a 12-month course of intravenously administered liposomal amphotericin B (AmBisome; Fujisawa Healthcare) and itraconazole, the patient remained alive, in clinical remission, and symptom free.

Amphotericin B↗

Successful medical therapy for deeply invasive facial infection due to Pythium insidiosum in a child.

Pythiosis occurs in animals and humans who encounter aquatic habitats that harbor Pythium insidiosum. Drug therapy for deeply invasive infections with this organism has been ineffective in humans and animals; patients have been cured only by radical surgical debridement. A 2-year-old boy developed periorbital cellulitis unresponsive to antibiotic and antifungal therapy. The cellulitis extended to the nasopharynx, compromising the airway and necessitating a gastrostomy for feeding. P. insidiosum was isolated from surgical biopsy specimens of the affected tissue. On the basis of in vitro susceptibility studies of the isolate, the patient was treated with a combination of terbinafine and itraconazole. The infection resolved over a period of a few months. The patient remained well 1.5 years after completing a 1-year course of therapy. Cure of deep P. insidiosum infection is feasible with drug therapy.

Anti-Infective Agents↗

Use of an immunotherapeutic vaccine to treat a life-threatening human arteritic infection caused by Pythium insidiosum.

A 14-year-old Thai boy presented because of a history of headache, mandibular swelling, and facial nerve palsy. A microorganism identified as Pythium insidiosum was cultured from the mandibular abscesses. Despite treatment with amphotericin B, iodides, ketoconazole, and surgery, the infection progressed. Magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA) of the neck revealed an aneurysm in the external carotid artery. The aneurysm was removed. MRA performed later showed stenosis of the internal carotid artery. Immunotherapy was recommended as a last resort. One hundred microliters of the P. insidiosum vaccine was subcutaneously injected into the patient's left shoulder, and 14 days later a similar dose was administered. Four weeks following the first vaccination, the patient's headache had disappeared, the facial swellings had dramatically diminished, the cervical lymph node had shrunk, and the proximal left internal carotid artery stenosis had significantly improved. One year after the vaccinations, the boy was considered clinically cured.

Arteries↗

Meiotic instability of Pythium sylvaticum as demonstrated by inheritance of nuclear markers and karyotype analysis.

Progeny from a sexual outcross between opposite mating types of Pythium sylvaticum were analyzed for inheritance of RFLP and random amplified polymorphic DNA (RAPD) markers. Although most were inherited in expected Mendelian frequencies, several were not. Pulsed field gel electrophoresis was employed to examine these unexpected patterns of marker inheritance at a karyotypic level. Parental oogonial and antheridial isolates had different electrophoretic karyotypes and minimum number of chromosome-sized DNAs (13 and 12, respectively), however, summation of the sizes of all chromosomal bands for each isolate was similar at approximately 37 Mb. Progeny karyotypes differed significantly from each other and the parental isolates, ranging in estimated minimum number of chromosome-sized DNAs from 9 to 13 and the summation of band sizes within each isolate from 28.1 to 39.0 Mb. For the eight isolates most extensively analyzed, 80% of the progeny chromosome-sized DNAs were nonparental in size or hybridization grouping of cDNA clones and isolated RAPD markers. Based on the results of Southern analysis it appears that length mutations and perhaps aneuploidy and translocations have contributed to generation of karyotypic polymorphisms. Nineteen field isolates of P. sylvaticum collected from the same location also exhibited significantly different karyotypes, suggesting that the meiotic instability observed in the laboratory also is occurring in field populations.

Base Sequence↗

The effect of silicon on the infection by and spread of Pythium aphanidermatum in single roots of tomato and bitter gourd.

The effect of silicon (Si) supply on the infection and spread of Pythium aphanidermatum was studied in the roots of tomato [Lycopersicon esculentum (=Solanum lycopersicum), an Si excluder] and bitter gourd (Mormodica charantia, an Si intermediate accumulator). Individual roots were mounted into PVC compartmented boxes which allowed the application of Si and zoospores to defined root zones. Two days after inoculation, root growth was recorded, and P. aphanidermatum colonization of individual root sections was determined by ELISA. In tomato as well as in bitter gourd the root tip was the root section most sensitive to P. aphanidermatum infection. Application of Si did not affect severe root-growth inhibition by P. aphanidermatum in either species. However, continuous Si supply significantly inhibited the basipetal spread of the pathogen from the infected root apex in bitter gourd but not in tomato. Si application to the roots only during pretreatment or only during/after the infection of the roots failed to inhibit the spread of P. aphanidermatum. Determination and compartmentation of Si in the roots of bitter gourd revealed that apoplastic Si was not, but symplastic Si was, associated with the ability of the plant to reduce the spread of the fungus in roots. It is concluded that accumulation of Si in the root cell walls does not represent a physical barrier to the spread of P. aphanidermatum in bitter gourd and tomato roots. The maintenance of elevated symplastic Si contents is a prerequisite for Si-enhanced resistance against P. aphanidermatum.

Antiparasitic Agents↗

The type III secretion system of biocontrol Pseudomonas fluorescens KD targets the phytopathogenic Chromista Pythium ultimum and promotes cucumber protection.

The type III secretion system (TTSS) is used by Proteobacteria for pathogenic or symbiotic interaction with plant and animal hosts. Recently, TTSS genes thought to originate from the phytopathogen Pseudomonas syringae were evidenced in Pseudomonas fluorescens KD, which protects cucumber from the oomycete Pythium ultimum (kingdom Chromista/Stramenopila). However, it is not known whether the TTSS contributes to plant protection by the bacterium and, if so, whether it targets the plant or the phytopathogen. Inactivation of TTSS gene hrcV following the insertion of an omega cassette strongly reduced the biocontrol activity of the pseudomonad against P. ultimum on cucumber when compared with the wild type, but had no effect on its root-colonization ability. Analysis of a plasmid-based transcriptional hrpJ'-inaZ reporter fusion revealed that expression in strain KD of the operon containing hrcV was strongly stimulated in vitro and in situ by the oomycete and not by the plant. In vitro, both strain KD and its hrcV mutant reduced the activity level of the pectinase polygalacturonase (a key pathogenicity factor) from P. ultimum, but the reduction was much stronger with the wild type. Together, these results show that the target range of bacterial TTSS is not restricted to plants and animals but also can include members of Chromista/Stramenopila, and suggest that virulence genes acquired horizontally from phytopathogenic bacteria were functionally recycled in biocontrol saprophytic Pseudomonas spp., resulting in enhanced plant protection by the latter.

Artificial Gene Fusion↗