Evaluation of Propionibacterium acnes Isolates Using Contour-clamped Homogeneous Electric Field Gel Electrophoresis

Contour-clamped homogeneous electric field electrophoresis was performed to compare strains ofPropionibacterium acnes isolated from patients with chronic postoperative endophthalmitis. Propionibacterium acnes isolates were obtained from the vitreous humor of nine patients with chronic postoperative endophthalmitis following cataract surgery. In two of the patients, P. acnes isolates were also obtained from the aqueous humor as well as from the vitreous humor. Bacterial DNA was digested using Not I and Spe I restriction endonucleases. The DNA fragments were then subjected to contour-clamped homogeneous electric field electrophoresis and the DNA banding patterns were analysed. Eight nonidentical banding patterns were identified among the nine vitreous isolates of P. acnes. In each of the two cases from which aqueous and vitreous isolates were recovered from the same eye, the banding patterns were identical. Contour-clamped homogeneous electric field electrophoresis is a powerful method to distinguish P. acnes isolates based on DNA banding patterns and could be used in the epidemiological study of clinical processes caused by this organism.     

Mycotoxin formation by different geographic isolates of Fusarium crookwellense

Eighteen Fusarium crookwellense isolates from the continents of Australia, Europe, and North America were compared for their ability to produce mycotoxins on corn at 25 °C after 2 weeks. Extracts from corn fermented with each Fusarium isolate were analyzed by thin-layer chromatography (TLC) and gas chromatography/mass spectroscopy (GS/MS) for mycotoxins. Toxins detected were zearalenone (13 isolates), fusarin C (11 isolates), nivalenol (4 isolates), and diacetoxyscirpenol (2 isolates). Zearalenone and fusarin C were produced by isolates from each continent, while nivalenol was detected in the Fusarium isolates originating from Australia and one isolate from the United States.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned     

Electrophoretic protein patterns of three species ofPhytophthora associated with black pod disease of cocoa (Theobroma cacao L.)

When electrophoretic profiles of native proteins from vegetative mycelia ofPhytophthora palmivora, Phytophthora capsici and Phytophthora citrophthora causing black pod disease of cocoa in India were compared on a single Polyacrylamide gel, the isolates of same species were readily distinguished both qualitatively by visual similarity in banding patterns and quantitatively by calculating similarity coefficients. Similarity coefficients were generally much higher between isolates within a species than between isolates of different species. The dendrograms obtained after unweighted pair grouping with arithmetic averaging cluster analysis, revealed that all the isolates ofPhytophthora capsici were highly homogenous and formed a single cluster. The isolates ofPhytophthora citrophthora were resolved into two electrophoretic types which were clustered into two distinct sub groups.Phytophthora palmivora formed a separate group. Thus, the results reveal that polyacrylamide gel electrophoresis can be used successfully in distinguishing species and sub groups within a species ofPhytophthora encountered on cocoa. CPCRl contribution No. 914.     

Restriction pattern analysis of genomic DNA ofFrankia isolates

Summary Total genomic DNAs ofFrankia isolates were subjected to restriction enzyme digestion and subsequent agarose gel electrophoresis. Restriction fragment banding patterns were unique for each isolate and may therefore be used as a method to distinguish between isolates which may be morphologically indistinguishable. This method might be useful for practical purposes such as tracing specificFrankia strains during field studies.     

Aggressiveness spectrum and genetic variability of Fusarium spp. on rice and wheat varieties

Abstract

A total of 106 Fusarium spp. were isolated from infected roots and soil samples of wheat and rice. Of the 106 isolates, 32 from wheat, and 74 from rice, were isolated. Six Fusarium spp. (F. oxysporum, F. moniliforme, F. poae, F. graminearum, F. tricinctum and F. equiseti) were identified at specie level. In aggressiveness tests Fusarium spp. root rot causing fungi were screened out into different aggressiveness classes according to disease severity scales. The aggressiveness of Fusarium spp. was studied on wheat varieties (Inqalab-91 and chakwal-86) and on rice varieties (Basmati-385 and IRRI-6) under controlled conditions. The overall total number of aggressive isolates was higher in rice than in wheat. However, the percentage of severely aggressive isolates was high in wheat, whereas the percentage of moderately and slightly aggressiveness isolates was high in rice. In rice, five isolates were non-aggressive and on wheat 17 were non-aggressive. Random Amplified Polymorphism DNAs (RAPDs) were used to study the polymorphism and genetic variations within the population of Fusarium spp. that established to study correlation between taxonomical and genetical characters of fungi. Five random primers were used P1 (5′-AGGAGGACCC-3′), P2 (5′-ACGAGGGACT-3′), PE7 (5′-AGATGCAGCC-3′), P14 (5′-CCACAGCACG-3′) and PE20 (5′-AACGGTGACC-3′). Each of the 10-mer primers produced results based on the respective banding patterns they generated in present investigations. Primers distinguished the F. oxysporum, F. moniliforme, F. graminearum, F. tricinctum, F. poa and F. equiseti. All the tested primers yielded amplification products, and that were reproducible. Although there was some intraspecific variation with primers, some strains were similar and some were different in banding pattern. In F. oxysporum, F. moniliforme, F. graminearum, F. tricinctum, F. poa and F. equiseti were seen clustered close to one another but each primer separated them unambiguously. All primer (P1, P2, P14, PE7 and PE20) combination produced 62 bands. All primers have shown interspecific and intraspecific variations in banding patterns.     

Pathogenicity of fungi associated with wheat and barley seedling emergence and fungicide efficacy of seed treatment

Presented study focused on the influence of Cochliobolus sativus isolates origin on pathogenicity towards wheat and barley seedlings in comparison with pathogenicity of certain Fusarium species and Microdochium nivale. The efficacy of fungicide seed treatment against C. sativus was estimated. The C. sativus isolates were collected from different locations and were isolated from wheat, barley and sunflower seeds. The pathogenicity of C. sativus, Fusarium species and M. nivale towards germinating seedlings were expressed as germination (GA) retardation and coleoptile growth rate retardation (CGR). Of wheat only, the CGR was significantly influenced by the isolate origin. The C. sativus isolates obtained from sunflower seeds were the most aggressive. Of the barley seeds, the barley isolates were the most aggressive. Barley was significantly more susceptible to damage by C. sativus isolates than wheat. The pathogenicity of tested fungal species declined in the order: F. culmorum, F. graminearum, C. sativus, F. avenaceum, M. nivale, F. poae for both barley and wheat. The results highlighted high pathogenicity potential of C. sativus equal to that of F. avenaceum and M. nivale. The symptoms of C. sativus on coleoptile and roots were very similar or the same as the symptoms caused by Fusarium species and M. nivale, except of white, pink or red colours. Of wheat sprouts, the fungicide efficacy (FE) against C. sativus declined in the order: tebuconazole + thiram, carboxin + thiram, quazatine, difenoconazole, iprodione + triticonazole (in term of GA) and carboxin + thiram, iprodione + triticonazole, tebuconazole + thiram, difenoconazole, quazatine (in term of CGR). In barley, the FE declined in the order: carboxin + thiram, iprodione + triticonazole, tebuconazole + thiram, difenoconazole, quazatine (in term of GA) and carboxin + thiram, tebuconazole + thiram, difenoconazole, iprodione + triticonazole, quazatine (in term of CGR).     

Molecular characterization of pathogenic Fusarium species in cucurbit plants from Kermanshah province, Iran

Fusarium is one of the important phytopathogenic genera of microfungi causing serious losses on cucurbit plants in Kermanshah province, the largest area of cucurbits plantation in Iran. Therefore, the objectives in this study were to isolate and identify disease-causing Fusarium spp. from infected cucurbit plants, to ascertain their pathogenicity, and to determine their phylogenetic relationships. A total of 100 Fusarium isolates were obtained from diseased cucurbit plants collected from fields in different geographic regions in Kermanshah province, Iran. According to morphological characters, all isolates were identified as Fusarium oxysporum, Fusarium proliferatum, Fusarium equiseti, Fusarium semitectum and Fusarium solani. All isolates of the five Fusarium spp. were evaluated for their pathogenicity on healthy cucumber (Cucumis sativus) and honeydew melon (Cucumis melo) seedlings in the glasshouse. F. oxysporum caused damping-off in 20–35 days on both cucurbit seedlings tested. Typical stem rot symptoms were observed within 15 days after inoculation with F. solani on both seedlings. Based on the internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA) restriction fragment length polymorphism (RFLP) analysis, the five Fusarium species were divided into two major groups. In particular, isolates belonging to the F. solani species complex (FSSC) were separated into two RFLP types. Grouping among Fusarium strains derived from restriction analysis was in agreement with criteria used in morphological classification. Therefore, the PCR-ITS-RFLP method provides a simple and rapid procedure for the differentiation of Fusarium strains at species level. This is the first report on identification and pathogenicity of major plant pathogenic Fusarium spp. causing root and stem rot on cucurbits in Iran.     

Screening for microbial trehalases: extracellular trehalases produced byFusarium species

Two hundred microorganisms comprising actinomycetes, bacteria, fungi and yeasts were screened for extracellular trehalases by their growth on trehalose in solid and liquid culture.Candida albicans, Gelasinospora retispora and four isolates belonging to the genusFusarium produced extracellular trehalases. The production of trehalase by theFusarium spp. was influenced by the nutrient composition of the medium and the carbon source; of the substrates examined starch produced the highest enzyme titre. Trehalase was an inducible enzyme and was repressed when theFusarium spp. were grown on glucose. The properties of the trehalases from two of theFusarium spp. (isolates MU-105 and TR-8) were typical of non-regulatory trehalases. Activity of several α-glucosidases, an amylase, an invertase and a cellobiase was also demonstrated when the two isolates were grown on trehalose.     

RAPD analysis of Fusarium Isolates Causing “Mango Malformation” Disease in Pakistan

Mango Malformation (MM) disease is a major constraint to mango production. A total of 20 Fusarium isolates from MM-affected mango plants were collected from 14 locations in Pakistan and assessed for genetic diversity using the random amplified polymorphic DNA (RAPD) technique. A total of 393 fragments were amplified after screening with 50 random primers. The amplifications with 45 primers identified scoreable polymorphisms among the isolates. A genetic similarity matrix based on Nei and Li’s index determined coefficients ranging from 46.46% to 92.51%. These coefficients were used to construct a dendrogram using the UPGMA algorithm. The isolates grouped into two main clusters, comprising 13 and 7 isolates respectively, at a genetic relatedness of 52%. Within the clusters, Fusarium isolates were not necessarily related either by geographic origin or by the mango cultivar from which they were isolated. RAPD proved a reproducible and tractable means of differentiating Fusarium isolates. These findings also suggest that some infections originate not from adjacent plants within an orchard but from geographically distant areas; indicating that most probably infection occurs in nurseries prior to plants being transported around the country for subsequent cultivation, and that improved plant hygiene could significantly curb MM infection and spread.     

Purification and characterization of fibrinolytic alkaline protease from <Emphasis Type="Italic">Fusarium</Emphasis> sp. BLB

Fusarium sp. BLB, which produces a strongly fibrinolytic enzyme, was isolated from plant leaf (Hibiscus). Fibrinolytic alkaline protease was purified from a culture filtrate of Fusarium sp. BLB by precipitation with (NH₄)₂SO₄ and column chromatography with CM-Toyopearl 650M and Superdex 75. The purified enzyme was homogeneous on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight was 27,000 by SDS-PAGE. Maximum activity of protease was observed at pH 9.5 and 50°C. Purified protease was active between pH 2.5 and 11.5 and was found to be stable up to 50°C. The enzyme derived from Fusarium sp. BLB is useful for thrombolytic therapy because this enzyme showed pH resistance. The activity was inhibited by diisopropylfluorophosphate and phenylmethylsulfonyl fluoride. The N-terminal amino acid sequence of the enzyme showed a similarity to those of proteases from Fusarium sp., Streptomyces griseus, Bos taurus bovine, Katsuwo pelamis digestive tract, and Lumbricus rubellus.     

Temperature and seedling age affect susceptibility of perennial ryegrass seedlings to pathogenic fungi

Summary Effects of temperature and seedling age on survival of perennial ryegrass (Lolium perenne L.) seedlings grown on sand-wheat wholemeal cultures of different isolates ofFusarium spp. (9 isolates),Pythium spp. (9 isolates), andChaetomium spp. (1 isolate) are reported. Some isolates were virulent over the whole range of temperatures tested (7.5–27.5°C). The virulence of others depended on temperature. Most isolates were less virulent at intermediate temperatures (12.5–22.5°C) than at higher or lower temperatures. At 25°C ryegrass seedlings were susceptible to fungal attack for only a limited period after germination commenced. This period differed for different fungi, but for most isolates tested, seedlings were resistant after 2–3 days.     

Occurrence and variability of mycotoxigenicFusarium species associated to wheat and maize in the South West of Spain

The contamination of cereals with mycotoxins produced by species ofFusarium is an important risk to human and animal health. The toxigenic profile is different depending on theFusarium species considered and, in some species, differences can also be observed at intraspecific level. Information about the distribution and variability of the mycotoxigenicFusarium species allow prediction of the toxins that may occur and to devise control strategies. In this work, the occurrence of mycotoxigenicFusarium species associated to cereals was analysed in a wide sample of durum wheat fields (Triticum durum Desf.) and maize from the South West of Spain (Andalucía).F. equiseti, F. graminearum andF. culmorum were the most frequentFusarium species detected in wheat fields followed byF. sambucinum andF. avenaceum, whereas in the case of maize,F. verticillioides andF. proliferatum were the onlyFusarium species present. The relationships of the Spanish isolates from theF. equiseti, F. avenaceum andF. sambucinum species were analysed by nucleotide sequence comparison of a partial region of the Elongation Factor 1 alpha (EF-1α) with other sequences available in data bases. The results indicated thatF. avenaceum andF. equiseti showed high variability and that the SpanishF. equiseti isolates seemed to belong toF. equiseti type II. Presented at the EU-USA Bilateral Workshop on Toxigenic Fungi & Mycotoxins, New Orleans, USA, July 5–7, 2005 Financial support: MCYT (AGL2004/07549/C05/5). M. Jurado was supported by pre-doctoral fellowship by the MCYT     

Production of moniliformin by Canadian isolates of Fusarium

Twenty-eight Canadian isolates of Fusarium were tested for their ability to produce moniliformin in corn. Both F. moniliforme (2/6 isolates) and F. subglutinans (11/15 isolates) produced the mycotoxin, while F. graminearum did not. Field-corn inoculated with F. moniliforme M3783 was able to support production of both moniliformin and fusarin C.     

Production of zearalenone,moniliformin and trichothecenes in intact sugar beets under laboratory conditions

Five toxigenic isolates of Fusarium species were tested for the production of zearalenone, moniliformin and trichothecenes (deoxynivalenol, 15-acetyldeoxynivalenol, T-2, HT-2 and neosolaniol) when grown on solid sugar beet slices in the laboratory for thirty days. The isolates were also grown on a solid rice medium for comparison. High zearalenone and trichothecene-producing isolates originally obtained from corn and corn-based feedstuff were compared with isolates obtained from sugar beets. One moniliformin-producing isolate from wheat was included in the study. With the exception of moniliformin, all toxins were produced on both substrates; however, the rice medium yielded the greater concentrations except for HT-2 which was produced on sugar beets in equal or greater concentrations. Zearalenone production on rice reached 729–1943 gmg/g whereas on sugar beet it reached 72–193 gmg/g. The moniliformin-producing isolate grew well on both substrates; however, moniliformin was produced only on the rice substrate. This study demonstrates for the first time that Fusarium species can produce both zearalenone and the trichothecenes on a sugar beet substrate.     

Electrophoretic karyotypes of some related Mucor species

Contour clamped homogeneous electric field (CHEF) gel electrophoresis was used to obtain electrophoretic karyotypes from nine Mucorstrains representing five different species (M. bainieri, M. circinelloides, M. mucedo, M. plumbeus and M. racemosus). The chromosomal banding patterns revealed high variability among the isolates. The sizes of the DNA in the Mucor chromosomes were estimated to be between 2.5 and 8.7 Mb. The total genome sizes were calculated to be between 30.0 and 44.7 Mb. The applicability of these electrophoretic karyotypes for the investigation of genome structure, for strain identification and for species delimitation is considered.     

Characterization of gibberellin producing strains of Fusarium moniliforme based on DNA polymorphism

The gibberellins are one of the major groups of growth promoting hormones and are secondary metabolites of the fungus Fusarium moniliforme (Perfect stage: Gibberella fujikuroi). Sixteen strains of Fusarium from different geographical regions and different hosts were analysed for their ability to produce gibberellins (GA) and for genetic relatedness by random amplified polymorphic DNA (RAPD). Range of gibberellin production varied between 28.9 to 600.0 mg g^-1 dry weight of mycelium in different strains of Fusarium. RAPD analysis showed completely different pattern between high, moderate and low producing strains. High producers formed nearly identical RAPD patterns, whereas the low and moderate producers gave heterologous amplification patterns. Since Fusarium pallidoroseum was in another group, it was possible to distinguish between different species of the genus Fusarium by RAPD. These investigations may find an application in the diagnosis of unknown Fusarium species and in distinguishing isolates of Gibberella fujikuroi within the section of Liseola. This revised version was published online in June 2006 with corrections to the Cover Date.     

Responses of alfalfa pollen and callus to filtrates from two isolates of Fusarium oxysporum

Summary For 25 Medicago sativa plants, measurements were made of in vitro pollen germination and growth and callus growth on mediums containing culture filtrate from two isolates of Fusarium oxysporum f. sp. medicaginis. In vivo resistance was determined by field inoculation with one isolate. There was no correlation between any in vitro measurement and in vivo resistance, and none of the in vitro measurements on control mediums were correlated. The only significant correlation for the same measurement between the two isolates was for pollen-tube length (r = 0.65). Percent of pollen germination was negatively correlated with callus growth for both isolates. Earlier work showed that callus growth in the presence of culture filtrate was linked to plant resistance, thus it appeared that percent pollen germination on culture filtrate had decreased for the more resistant plants. The haploid pollen may be used as a progeny test for identifying heterotic loci conferring resistance to Fusarium, but if the pollen of this plant species is used in direct selection by exposure to culture filtrate, the level of resistance to Fusarium may decrease.     

Fusarium root rot of coneflower seedlings and integrated control using <Emphasis Type="Italic">Trichoderma</Emphasis> and fungicides

Fusarium root rot (Fusarium spp.) is one of the most important seedling diseases of coneflower (Echinacea spp.) in Alberta greenhouses. Effects of microbial antagonists (Trichoderma spp.) and fungicides, including difenoconazole, fludioxonil, and a mixture of fludioxonil, metalaxyl and difenoconazole, on the management of this disease, were investigated in Alberta. Twenty Trichoderma isolates demonstrated antagonistic activity to Fusarium in agar plate bioassays, with inhibition rates ranging from 44 to 65%. Some Trichoderma isolates significantly ( p < 0.05) reduced disease incidence and severity on seedlings in greenhouse experiments. An in vitro bioassay indicated that difenoconazole and the mixture equally inhibited the growth of both Fusarium and Trichoderma, but, while fludioxonil strongly inhibited the growth of Fusarium, it had little effect on Trichoderma, according to the dose--response models developed ( p < 0.01, R²= 0.902-0.998). Two Trichoderma isolates, T1 and T13 were applied singly or in combination with a low rate of fludioxonil in greenhouse evaluations. The results suggested that fludioxonil and Trichoderma could be integrated into a disease management program for fusarium root rot in coneflower.     

Survey ofFusarium species in an arid environment of Bahrain

The prevalence and distribution of soil-inhabitingFusarium species in the soil of vegetable crops grown under custom-made plastic tunnels were studied at three geographical locations on the island of Bahrain. Six species ofFusarium representing a total of 1154 isolates were isolated on modified Komada medium. All species reported in this survey are recorded for the first time from the hot, arid desert of Bahrain.Fusarium solani andF. oxysporum were among the most frequently isolated fungi in all locations and crops.     

Occurrence and toxicity ofFusarium subglutinans from Peruvian maize

Twenty-five samples of maize kernels collected at harvest time from geographically different corn fields in Peru, were examined for the occurrence of toxigenicFusarium species. The most frequently recovered species wereF. subglutinans (48%),F. moniliforme (46%), andF. equiseti (5%). OtherFusarium species isolated (up to 1%) includedF. graminearum, F. acuminatum, F. solani, F. oxysporum, andF. culmorum. Assays ofFusarium culture extracts usingArtemia salina larvae, showedF. subglutinans as one of the most toxigenic species, and its toxicity was mostly correlated to the capability to produce beauvericin (BEA). All eight tested isolates ofF. subglutinans grown on autoclaved corn kernels produced BEA (from 50 to 250 mg/Kg) as well as moniliformin (M) (from 70 to 270 mg/Kg). This is the first report on BEA and M production by maize isolates ofF. subglutinans from South America.