Pythium Hyphae Research Articles

Clinical Characteristics, Histopathology, and Treatment Outcomes of Pythium Keratitis: A Retrospective Cohort Study.

PurposeTo identify factors associated with the prognosis in Pythium keratitis.Patients and MethodsThe medical records of 25 patients (26 eyes) diagnosed with Pythium keratitis at a referral institution were reviewed. The demographic and clinical characteristics, treatment, microbiological diagnosis, histopathological features, and outcomes were recorded. The histopathological specimens were reviewed. The patients were divided into a globe removal group and a globe salvage group. Univariate analysis was used to identify factors associated with poor outcomes.ResultsFifteen eyes (57.7%) were removed. Patients in the globe removal group were on average 16.4 years older (95% CI 6.98 to 25.88) than those in the globe salvage group, received the first medication (either topical antifungals or antibiotics) later than one day after the onset of symptoms (RR = 2.75, 95% CI 1.18 to 6.42), and had a maximal diameter of the infiltration area ≥6 mm (RR = 3.14, 95% CI 1.17 to 8.45). The globe removal group showed satellite, multifocal, or total corneal infiltration patterns (RR = 2.82, 95% CI 1.03 to 7.74) and a hypopyon (RR = 3.43, 95% CI 1.26 to 9.35) as risk factors. The histopathological examination showed a higher density of Pythium in the globe removal group than the globe salvage group (median 376 (interquartile range 323, 620) versus 107 (interquartile range 16, 260) hyphae per high power field; P = 0.035).ConclusionThe risk of globe removal in patients with Pythium keratitis increased with age, delayed initial topical antifungal or antibiotic treatment, advanced disease at presentation, and dense Pythium hyphae infiltration of the cornea. Early recognition and treatment are critical to successfully eradicate the infection.

Pesticidal activity of metal oxide nanoparticles on plant pathogenic isolates of Pythium.

CuO and ZnO nanoparticles (NPs) have antimicrobial effects that could lead to formulations as pesticides for agriculture or medicine. The responses of two soil-borne plant pathogenic Pythium isolates to the NPs were studied to determine the potential of these metal oxide NPs as pesticides. Growth of the P. ultimum isolate was more sensitive to CuO NPs than the P. aphanidermatum isolate. Growth in liquid medium with CuO NPs eliminated culturability whereas exposure to ZnO NPs resulted in stasis with growth resuming on transfer to medium lacking NPs. The citrate in the medium used for the growth assays was involved in enhanced release of the toxic metals from the NPs. Both CuO and ZnO NPs affected processes involved in Fe uptake. The NPs reduced levels of Fe-chelating siderophore-like metabolites produced by Pythium hyphae. CuO NPs inhibited, but ZnO NPs increased, ferric reductase activity detected at the mycelial surface. These findings illustrate that the toxicity of the metal oxide NPs towards Pythium was influenced by the medium, especially by the presence of a metal chelator. Environmental factors are likely to alter the pesticide potential of the metal oxide NPs when formulated for agricultural use in soils.

Interaction of Fluorescent Pseudomonads with Pythium ultimum and Rhizoctonia solani in Cucumber Roots

The effects of thefluorescent pseudomonadisolate CW2 on hyphae ofPythium ultimum (Trow) andRhizoctoniasolani(Khn)and root colonization were studied in gnotobiotic systems under scanning electron microscopy (SEM). Results of SEM revealed that the frequency of bacterial colonization was higher in cucumber roots infested with the fungi than in healthy roots. Isolate CW2 caused irregular and abnormal fungal growth. Swellings and shrinkages ofP. ultimumand R. Solanihyphae were obvious when cucumber roots were drenched with CW2. SEM studies were also conducted to evaluate the effect of CW2 on hyphae of both fungi on PDA medium.The hyphae ofP. ultimumand R. solanishowed distinct morphological alterations and degradationcompared to untreated healthy control hyphae ofPythium orRhizoctoniawhich were slender and uniform in shape.The results provide direct evidence of bacterial attachment and colonization to f ungal hyphae of both tested spp.and show that fungal growth to be significantly reduced in the presence of isolate CW2.

Characterization of the fungicidal activity of Calothrix elenkinii using chemical methods and microscopy

An investigation was directed towards biochemical characterization of cyanobacterium Calothrix elenkinii and analysis of the chemical nature and mode of action of its fungicidal metabolite(s) against oomycete Pythium debaryanum. Biochemical characterization of the culture in terms of carbohydrate utilization revealed the facultative nature of C. elenkinii. Unique antibiotic markers were also found for this strain. 16S rDNA sequencing of the strain revealed 98% similarity with Calothrix sp. PCC7101. The fungicidal activity was tested by disc diffusion assay of different fractions of the culture filtrate. A minimum inhibitory concentration of 10 microl was recorded for ethyl acetate fraction of the 7-weeks old culture filtrates. HPLC, followed by NMR spectral analysis demonstrated the presence of a substituted benzoic acid in the ethyl acetate fraction. Microscopic examination revealed distinct granulation, followed by disintegration of the hyphae of Pythium sp., indicating the presence of an active metabolite in the culture filtrates of Calothrix sp. The fungicidal activity of C. elenkinii can be attributed to the presence of 3-acetyl-2-hydroxy-6-methoxy-4-methyl benzoic acid. This is the first report of a benzoic acid derivative having fungicidal activity in cyanobacteria.

Chitinase and β-1,3-glucanase enzyme production by the mycoparasiteClonostachys roseaf.catenulataagainst fungal plant pathogens

Clonostachys rosea f. catenulata (syn. Gliocladium catenulatum) is an effective fungal biological agent against Fusarium root and stem rot and Pythium damping-off diseases on cucumber plants. Both chitinase and beta-1,3-glucanase enzymes were produced when C. rosea was grown on a synthetic medium containing chitin or laminarin as a sole carbon source, respectively. Chitinase production was also induced by Fusarium cell walls, while beta-1,3-glucanase activity was induced by both Fusarium and Pythium cell walls, as well as by growth on homogenized cucumber roots and on low-carbon media. Mycelial growth of Fusarium and Pythium, when exposed to C. rosea culture filtrates that contain glucanase activity, was significantly reduced compared with the controls, and cell walls of both pathogens were degraded. On excised cucumber roots, hyphae of C. rosea formed appressorium-like structures and coiled around hyphae of Pythium. In culture, C. rosea caused localized degradation of Fusarium hyphae. Cucumber root tissues colonized by C. rosea showed higher levels of beta-1,3-glucanase activity at 7 days post-application compared with untreated controls. To determine if this activity was derived from C. rosea, glucanase isoforms were separated on activity gels. Fungal culture filtrates and root extracts contained the same predominant 20 kDa isoform. Reverse-transcription polymerase chain reaction (RT-PCR) using primers designed to amplify a beta-1,3-glucanase gene in C. rosea confirmed glucanase expression on roots. These results show that C. rosea produces beta-1,3-glucanase in situ, which can degrade hyphae of Fusarium and Pythium and contribute to biological control efficacy.

First Report of Pythium Root Dysfunction of Creeping Bentgrass Caused by Pythium volutum in North Carolina.

In July and August of 2002 and 2003, a disease of unknown etiology was observed in Charlotte, NC on 'A-1' creeping bentgrass (CRB; Agrostis stolonifera L.) putting greens that were constructed in 2000. Symptoms appeared in irregular patches ranging from 15 to 30 cm in diameter. Grass in the affected areas was initially wilted and chlorotic, but later exhibited a yellow-to-orange foliar decline. Similar symptoms were observed in Durham, NC in July and August of 2003 on CRB greens established in 2001 with a 1:1 blend of 'A-1' and 'A-4'. The disease was initially diagnosed as take-all patch, but attempts to isolate Gaeumannomyces graminis var. avenae and other ectotrophic root pathogens were unsuccessful. Symptoms of the disease reappeared during periods of warm, dry weather in the fall of 2003 and spring of 2004. At that time, examination of affected root tissue revealed bulbous root tips, loose cortical structure, absence of root hairs, and abundant Pythium oospores and hyphae. These signs and symptoms are typical of Pythium root dysfunction (PRD) as described by Hodges and Coleman (2) in 1985 and Feng and Dernoeden (3) in 1999. Isolation of Pythium spp. was performed by plating directly on V8 agar (4) or baiting with 'A-4' CRB seedlings. Eleven Pythium isolates were obtained from Charlotte (seven via baiting) and 10 were obtained from Durham (all via baiting). All isolates were transferred to grass leaf-blade cultures (4) to induce development of sporangia, oospores, and antheridia for identification using the keys and descriptions of Dick (1). All isolates produced lobate sporangia, large oospores (27 to 33 ± 2.8 μm), and three to nine diclinous antheridia typical of Pythium volutum. Cone-Tainers (3.8 × 20 cm) containing sand meeting USGA specifications were seeded with 'A-1' CRB and grown for 6 weeks in the greenhouse. Each Cone-Tainer was inoculated by cutting the root system at a 5 cm depth, placing five to seven infested grass blades onto the surface of fresh sand, and then replacing the turf. Cone-Tainers inoculated with one of three P. volutum isolates and an uninoculated control (six reps each) were placed in a growth chamber with 12 h of light/dark periods at 24/16°C for 4 weeks to allow pathogen infection and disease development. After 4 weeks, the chamber temperature was raised to 32/26°C to induce symptom development. Two weeks after raising the temperature, all P. volutum isolates caused significant (P = <0.0001) foliar chlorosis and dieback (70 to 100% disease) and reduced root depth and mass by 25 to 65% compared with the uninoculated control. Roots of inoculated plants were colonized with Pythium hyphae, contained numerous oospores, and consistently yielded P. volutum in isolations. To our knowledge, this is the first reported occurrence of PRD in North Carolina and provides further support for the importance of P. volutum as a pathogen of creeping bentgrass. On the basis of our observations, the majority of pathogen activity and disease development occurs in the fall and spring, with foliar symptoms being induced by heat or other stresses.

Cellular and Molecular Mechanisms Involved in the Interaction between Trichoderma harzianum and Pythium ultimum

The interaction between Trichoderma harzianum and the soilborne plant pathogen Pythium ultimum was studied by electron microscopy and further investigated by gold cytochemistry. Early contact between the two fungi was accompanied by the abnormal deposition of a cellulose-enriched material at sites of potential antagonist penetration. The antagonist displayed the ability to penetrate this barrier, indicating that cellulolytic enzymes were produced. However, the presence of cellulose in the walls of severely damaged Pythium hyphae indicated that cellulolytic enzymes were not the only critical traits involved in the antagonistic process. The marked alteration of the (beta)-1,3-glucan component of the Pythium cell wall suggested that (beta)-1,3-glucanases played a key role in the process.

Pre-inoculation of Ri T-DNA-transformed pea roots with Pseudomonas fluorescens inhibits colonization by Pythium ultimum Trow: an ultrastructural and cytochemical study

The influence exerted by Pseudomonas fluorescens, strain 63-28R, in stimulating plant defense reactions was investigated using an in-vitro system in which Ri T-DNA-transformed pea (Pisum sativum L.) roots were subsequently infected with Pythium ultimum. Cytological investigations of samples from P. fluorescens-inoculated roots revealed that the bacteria multiplied abundantly at the root surface and colonized a small number of epidermal and cortical cells. Penetration of the epidermis occurred through the openings made by the disruption of the fibrillar network at the junction of adjacent epidermal cell walls. Direct cell wall penetration was never observed and bacterial ingress into the root tissues proceeded via an intercellular route. Striking differences in the extent of fungal colonization were observed between bacterized and non-bacterized pea roots following inoculation with P. ultimum. In non-bacterized roots, the pathogen multiplied abundantly through most of the tissues while in bacterized roots, pathogen growth was restricted to the epidermis and the outer cortex. At the root surface, the bacteria interacted with the pathogen, in a way similar to that observed in dual culture tests. Most Pythium cells were severely damaged but fungal penetration by the bacteria was never observed. Droplets of the amorphous material formed upon interaction between the bacteria and the host root were frequently found at the fungal cell surface. Incubation of sections with a β-1,4-exoglucanase-gold complex revealed that the cell wall of markedly altered Pythium hyphae was structurally preserved. Successful penetration of the root epidermis was achieved by the few hyphae of P. ultimum that could escape the first defensive line in the rhizosphere. Most hyphae of the pathogen that penetrated the epidermis exhibited considerable changes. The unusual occurrence of polymorphic wall appositions along the host epidermal cells was an indication that the host plant was signalled to defend itself through the elaboration of physical barriers.

Induction of Differential Host Responses byPseudomonas fluorescensin Ri T DNA Transformed Pea Roots After Challenge withFusarium oxysporumf. sp.pisiandPythium ultimum

In the current study, the influence exerted by the plant growth-promoting bacterium Pseudomonas fluorescens strain 63-28 in stimulating plant defense reactions was investigated at the ultrastructural level by an in vitro system in which Ri T-DNA pea roots were either infected with the pea root rot fungus Fusarium oxysporum f. sp. pisi or with the soil-borne pathogen Pythium ultimum. Scanning electron microscopy (SEM) observations showed that bacteria abundantly colonized the root surface and established close contact with the outermost host cell layers through a thin mucilage. When bacterized pea roots were challenged with either P ultimum or F. oxysporum f. sp. pisi, strong differences in the extent of fungal damage were observed. Hyphae of P. ultimum were markedly collapsed, as illustrated by their wrinkled appearance, whereas Fusarium cells were apparently undamaged. In line with these SEM observations, cytological investigations of the root surface confirmed that the interaction between Pseudomonas fluorescens and F. oxysporum f. sp. pisi did not result in hyphal disturbances similar to those seen with Pythium hyphae. Restriction of Fusarium growth and development to the epidermis and outer root cortex and a marked decrease in pathogen viability were features observed only in bacterized pea roots. In pretreated roots, striking modifications of the epidermal and cortical cell walls as well as deposition of newly formed barriers were seen in response to Fusarium infection. Deposition onto the inner surface of the cell walls of callose-enriched wall appositions was associated with a lack of fungal ingress toward the vascular stele. Fungal cells in the vicinity of wall appositions frequently were surrounded by an aggregated material containing phenolic compounds, as shown by laccase-gold labeling. The labeling pattern obtained with this probe showed that phenolic compounds were widely distributed in Fusarium-challenged, bacterized roots. In addition to their infiltration at strategic sites of potential penetration, phenolic compounds were detected in the host cell walls. Use of the wheat germ agglutinin/ ovomucoid-gold complex provided evidence that the wall-bound chitin component in Fusarium hyphae colonizing bacterized roots was disrupted in places. However, chitin molecules still occurred over cell walls at a time when hyphae had undergone substantial degradation. The results reported in this paper show that pea root bacterization with Pseudomonas fluorescens strain 63-28 induces a set of plant defense reactions that culminates in the elaboration of physical barriers and the creation of a fungitoxic environment that adversely affects Fusarium growth and development.

Biological Control of Pythium Damping-Off of Cauliflower by Trichoderma harzianum

Out of eight antagonists, one isolate of Trichoderma harzianum exhibited fastest growth rate and strong antagonism against Pythium apanidermaium in vitro. The prinicpal mechanism of antagonism was direct parasitization of Trichoderma by coiling around the hyphae of Pythium leading to digestion of protoplasmic contents and lysis. Occasional presence of Trichoderma in the lumen of Pythium hyphae was also recorded. Soil application of wheat bran saw dust (WBSD) preparation of Trichoderma gave 32.4 to 77.0 percent control of damping-off under glass house conditions. Seed coating with Trichoderma (1.6 × 10 10 spores/ml) resulted in 31% control of the disease. WBSD preparation of T. harzianum ; either live or killed, promoted the growth and vigour of cauliflower seedlings. Apron at 5 and Thiram at 100 μg/ml proved better and completely inhibited the radial growth of P. ahpanidermatum in vitro. None of the fungicides was found inhibitory to T. harzian am except Thiram. Seed treatement with Apron, Fytolan, SAN 506 F and Thiram gave 15.7 to 85.8% disease control. Integration of fungicidal seed treatment with soil application of T. harzianum resulted in 66.4% to 90% control of damping-off. Colony forming units of Trichoderma and Pythium monitored on selective media revealed that the population of Trichoderma remained almost stable where it was applied at higher rates but declined with time in lower rates of application. On the other hand, the population of Pythium declined in Trichoderma amended soil.

Chemotropism of germ-tubes from zoospore cysts of Pythium spp.

Zoospores of Pythium aphanidermatum and P. graminicola were attracted to malt-peptone agar (MPA), and their cysts germinated from the side nearest to MPA. Pre-encysted spores placed near MPA did not germinate preferentially towards the nutrient source, but the germ-tubes often changed direction and grew towards MPA. Vegetative hyphae of Pythium spp. did not show tropism to MPA, whereas those of Saprolegnia spp. did so. Zoospores of P. graminicola , but not P. aphanidermatum , were attracted to aerated zones and the cysts germinated towards the source of air. Pre-encysted spores germinated randomly with respect to air, but the germ-tube tips of P. graminicola (not P. aphanidermatum ) changed direction and grew towards aerated zones. Vegetative hyphae of both Pythium spp. grew towards air beneath coverslips on agar colonies. Zoospores seem to have a predetermined point of germ-tube outgrowth, and settle with this point nearest to a source of attractant. In all instances, factors that attracted zoospores also attracted germ-tube tips; but older vegetative hyphae behaved differently from germ-tubes in tropic responses.

Ultrastructural localization of acid phosphatase inPythium paroecandrum

Four cytologically distinct zones defined by the type of vacuoles and vesicles present can be distinguished in young hyphae ofPythium paroecandrum. Acid phosphatase is not associated with the vesicles and the fibrillar vacuoles present in the first and second zones from the hyphal apex but is localized in the vacuoles with electron translucent contents in the third and fourth zones. Microbodies and mitochondria do not contain acid phosphatase. The association of acid phosphatase with vacuoles in fungi of various taxonomic groups is discussed.

Nuclear structure and behaviour in the vegetative hyphae of Pythium aphanidermatum

Nuclear divisions taking place in the hyphae ofPythium aphanidermatum have been described. Typical prophase type of nuclei with elongate chromatin fibres have been observed. Achromatic structures resembling spindle fibres with well defined poles during anaphase configuration have been noticed. A mild pretreatment with acenaphthene vapours proved instrumental in arresting the progress of nuclear division. Stages similar to early and late anaphase and telophase with deeply staining chromosomal masses have been observed. A high concentration of acenaphthene treatment resulted in the formation of giant nuclei.