Broth dilution and disc diffusion methods in the susceptibility testing of pathogenic Candida albicans against four antimycotics

Susceptibility of Candida albicans isolated from mouthrinse specimens during episodes of acute pseudomembranous candidiasis in patients with haematological malignancies was tested by the broth dilution and disc diffusion methods using 24 and 48 h of incubation. The time factor did not significantly affect the results with 5-fluorocytosine. With amphotericin B, prolonged incubation doubled the geometric mean MIC of C. albicans as well as the number of isolates with intermediate sensitivity. With the shorter incubation in the disc diffusion assay, few isolates showed lowered sensitivity to clotrimazole; at 48 h, however, this figure was as high as 54%. The yeasts were highly sensitive to ketoconazole at 24 h, whereas at 48 h the results were bizarre. At 24 h, correlations between disc diffusion and broth dilution methods were satisfactory, with clotrimazole and ketoconazole accounting for most of the discordancies. Accordingly, the disc diffusion results should be recorded at 24 h.     

Effect of Bacillus subtilis on Growth and Protection of Onion against White Rot

The effect of isolates of Bacillus subtilis applied as seed treatments on plant growth and white rot on three onion cultivars was studied for two seasons on muck soil of Fraser valley of British Columbia. The isolates of B. subtilis provided significant season-long protection against onion white rot on all three cultivars. The most effective isolate (BACT 2) protected the onion cultivar Autumn spice against onion white rot in both years. plant height, number of leaves per plant, and bulb weight were not affected by treating seeds with the Bacillus isolates. Bulb neck diameter of the cultivar Autumn Spice was significantly reduced by two isolates in both seasons.     

Study on Rosellinia necatrix Isolates Causing White Root Rot Disease in Taiwan

White root rot is a serious soil‐borne disease of several woods and crops. Recently, white root rot of tea shrubs and ornamental trees has increasingly been observed in Taiwan. Thirty‐six isolates of white root rot pathogen, showing pear‐shape swellings adjacent to the hyphal septa, had been isolated from samples of white root rot collected from Taiwan for about 4 years. The pathogen isolates produced Dematophora anamorph. Conidia of the pathogen were one‐celled, hyaline, subglobal, with truncate base, 2.9–5.8 × 1.9–3.5 μm . Ascospore dimensions were in the range of 37.0–55.0 × 5.4–7.9 μm with a short, longitudinal and straight germ slit, which complied with Rosellinia necatrix. Based on molecular studies, the pathogen isolates collected from Taiwan except R701 were identified as R. nectarix. Isolate R701, which was relatively polymorphic in internal transcribed spacer DNA sequence than other isolates, was temporarily considered as R. necatrix‐related pathogenic Rosellinia spp. All the tea cuttings (Camellia sinensis) inoculated with isolates developed typical white root rot symptoms. Pathogenicity tests demonstrated the presence of variation in virulence among the Rosellina isolates. Most of the R. necatrix isolates originating from Acer morrisonense were less virulent than those that originated from other hosts. The pathogenic Rosellinia spp., isolate R701, was also highly virulent to both cultivars of tea cuttings.     

Evaluation of Trichoderma spp. as biocontrol agents against avocado white root rot

Five isolates of Trichoderma atroviride and one isolate each of T. virens, T. harzianum and T. cerinum were tested for in vivo biological control of white root rot of avocado (Rosellinia necatrix). Five of these Trichoderma isolates were previously selected as possible biological control agents on the basis of their capacity to control the disease and high levels of colonization of the avocado rhizoplane. Combinations of the five selected isolates were evaluated on cellophane for compatibility with each other and T. virens CH 303 was eliminated because of a high incompatibility with other Trichoderma isolates. The four remaining isolates, all T. atroviride, were tested singly and in combination for their capacity to control avocado white root rot. Isolate CH 304.1 provided the highest levels of control when tested singly or in combination with isolate CH 101.     

A distinct strain of lucerne Australian latent virus in white clover in New Zealand

A strain of lucerne Australian latent virus (LALV) was widespread in Trifolium repens (white clover) in Northland, New Zealand, occurring in 20–100% of plants tested on 23 of 26 farms. Infected plants showed chlorotic line patterns or were symptomless. In agar gel double diffusion serological tests, white clover isolates of LALV differed from lucerne isolates by two or three twofold dilution steps of antisera. In mechanical inoculation tests, white clover isolates did not infect lucerne and lucerne isolates did not systemically infect white clover. White clover and lucerne isolates also differed in their reactions in some other hosts. Six LALV isolates from white clover were indistinguishable in double diffusion serological tests and by enzyme-linked immunosorbent assay, although the isolates could be differentiated by some reactions in test plants. Purified LALV particles of lucerne and white clover isolates contained two nucleoprotein components and two RNA species of molecular weight 2.82 × 10⁶ (8150 nucleotides) and 2.46 × 10⁶ (7100 nucleotides) (estimated under denaturing conditions).     

Comparison of disc diffusion and epsilometer (E-test) testing techniques to determine antimicrobial susceptibiliy of Campylobacter isolates of food and human clinical origin

The antibiotic resistance profiles of 75 Campylobacter isolates of food and human clinical origin was determined by two agar diffusion susceptibility methods; disc diffusion and epsilometer-test (E-test). The most common therapeutic antimicrobials, erythromycin, ciprofloxacin and tetracycline were studied, along with chloramphenicol, ampicillin and naladixic acid. The resistance observed for each antimicrobial, as determined by both of methods, were statistically compared using Fisher two-tailed analysis.Of the six antimicrobials studied only two were shown to have statistically different patterns when resistance was compared by disc diffusion and E-test. The percentage of isolates resistant to clinically relevant antimicrobials using both techniques ranged from 6.6 to 21.3% for erythromycin, 25.3–26.6% for tetracycline and 33.3–36.0% for ciprofloxacin. The prevalence of multi-drug resistant (MDR) campylobacters (isolates resistant to 2 or more antimicrobials) for both disc diffusion and E-test was 44%. It can be concluded that, for four of the six antimicrobials assessed, antimicrobial resistance prevalences could be equally determined by either of the methods studied.     

Morpho‐pathological and Molecular Characterization of Bipolaris oryzae in Rice (Oryzae sativa)

Fifty isolates of Bipolaris oryzae from rice were characterized morpho‐pathologically and molecularly. Based on colony morphology and growth pattern on PDA, these isolates were grouped into four categories: black with suppressed growth (21 isolates), black with cottony growth (16 isolates), black with fluffy growth (12 isolates) and white with cottony growth (1 isolate). The frequency of the black and suppressed type was the highest (42%) with maximum aggressiveness (mean spore count of 1854/cm²), whereas the white and cottony growth isolate had lowest frequency (2%) and aggressiveness (548/cm²). Thirteen B. oryzae isolates (four isolates from Groups I, II and III and one isolate from Group IV) were further tested for their variability with random amplified polymorphic DNA (RAPD) primers. Twenty RAPD primers were screened, of which 10 gave amplification; however, only six primers gave reproducible results. Based on the molecular similarity of the RAPD profiles, the isolates were grouped in to three major clusters and maximum linkage distance between them was determined as 0.29 units. This study establishes the variability among B. oryzae isolates.     

ELECTROPHORETIC AND IMMUNOLOGICAL ANALYSES OF SEVEN CHLOROSARCINACEAN ALGAE1

Proteins from 7 species of chlorosarcinacean algae representing 4 genera were studied with starch and disc gel electrophoresis and immunology to determine biochemical relationships. Migration patterns of enzymes and serological reactions indicated that 4 species of Chlorosarcinopsis were closely related, whereas Friedmannia israeliensis, Fasciculochloris boldii, and Chlorosarcina longispinosa were more diverse. Malate dehydrogenase, glutamate dehydrogenase, nonspecific alpha esterases, and peroxidase (using pyrogallol as the hydrogen donor) were present in all 7 species. No peroxidase activity occurred with benzidine. Leucine aminopeptidase was obvious in only 2 species. A single distinct band of general protein in F. israeliensis and C. longispinosa suggested the presence of homogenous reserve protein. The 7 species exhibited serological affinity to 4 isolates of Protosiphon representing diverse morphology within the genus. The data are discussed relative to current knowledge of comparative morphology of chlorosarcinacean algae.     

Variability in Fusarium oxysporum f. sp. ciceri causing vascular wilt in chickpea

The variability in cultural characteristics and the virulence among three isolates of Fusarium oxysporum f. sp. ciceri causing vascular wilt in chickpea was studied under laboratory conditions. The three isolates (Foc-1, Foc-2 and Foc-3) did not show any significant difference in their mycelial dry weight production at any temperature regimes, pH level or the growth media tested. The radial growth on PDA also did not differ significantly in the three isolates. However, some quantitative differences were noted in colony characters and septations in macroconidia of these isolates. The isolate Foc-1 exhibited dull white, thin and flat hairy growth, spreading out like thread, Foc-2 showed a white fluffy colony with irregular aerial margin, while Foc-3 exhibited a pinkish white, slightly fluffy colony with regular margin. Conidia also differed with regard to septation. Three to six septa were present in Foc-2, while there were 2–3 in isolates Foc-1 and Foc-2. These isolates differed significantly with regard to their virulence on test varieties. Isolate Foc-1 was more virulent that Foc-2 or Foc-3 and produced abundant spores.     

Detection and Molecular Diversity of Pantoea ananatis Associated with White Spot Disease in Maize,Sorghum and Crabgrass in Brazil

Bacteria of the genus Pantoea have become important plant pathogens worldwide in recent years. Pantoea ananatis was reported as the cause of maize white spot, a serious maize disease in Brazil, causing significant yield losses. However, very little information is available about how to detect this pathogen, its genetic variability and the putative alternative hosts in maize‐growing areas. To address these issues, we implemented a rapid and efficient PCR‐based method to identify P. ananatis isolated from leaves showing white spot symptoms and evaluated its genetic diversity in maize, sorghum and crabgrass. Of the 29 bacteria isolated from typical water‐soaked lesions of white spot disease that produced yellow colonies, 15 isolates were identified as P. ananatis by 16S rDNA sequencing and correctly detected by the PCR reaction, amplifying a specific fragment of the ice nucleation gene (ina). These P. ananatis isolates included 13 from maize, one from sorghum and one from crabgrass, while the other 14 yellow colony isolates were from other bacterial species, including two Pantoea species (Pantoea dispersa and Pantoea agglomerans) that were not amplified by the ina primers. These results indicate that the optimized PCR assay can be used to detect P. ananatis isolated from white spot lesions and could be used as a large‐scale and cost‐effective method of detecting this pathogen in leaf lesions on maize and other grasses. All isolates were evaluated for hypersensitive response (HR) on tobacco, revealing that some P. ananatis were able to induce HR. The high genetic variability revealed by rep‐PCR did not differentiated the P. ananatis isolates based on their hosts or HR reaction. The detection, characterization and diversity of P. ananatis from maize, sorghum and crabgrass in our study can be applied in understanding epidemiology and designing control strategies for maize white spot disease in Brazil.     

Specific and general effectiveness ofRhizobium trifolii populations from different agricultural locations

Summary The effectiveness ofRhizobium trifolii isolates from five locations in southern Britain representing contrasting soil types has been examined with five white clover varieties. The average effectiveness of Rhizobium isolates varied considerably as did the average productivity of plant varieties. The largest differences were, however, associated with Rhizobium population × plant variety interactions. These were often large enough to reverse relative yield differences between white clover varieties. The implications of these results for improving clover productivity in nitrogen fixation are discussed.     

Detection of Methicillin-Resistant Staphylococcus aureus Using mecA/nuc Genes and Antibiotic Susceptibility Profile of Malaysian Clinical Isolates

The aim of this study is to compare methicillin-resistant Staphylococcus aureus (MRSA) detection methods and to generate antibiogram profile of S. aureus clinical isolates from two teaching hospitals in Malaysia including three reference isolates from American Type Culture Collection (ATCC). The mecA/nuc gene PCR amplification, spot inoculation test and oxacillin disc diffusion test were applied to compare its MRSA detection abilities. No disagreement between the three methods was observed. From 29 bacterial isolates (including the ATCC strains) tested, 19 isolates were confirmed as S. aureus with 14 isolates exhibiting multidrug-resistance. All isolates are still susceptible to vancomycin as indicated by the E-test result. Current biochemical tests are comparable with the molecular detection method for MRSA used in this study while multidrug-resistance traits are present in both MRSA and MSSA clinical isolates. Presently, mupirocin seems to be the best alternative for vancomycin against multidrug-resistant S. aureus infections in Malaysia. Susceptibility profile of 19 S. aureus isolates acquired from two teaching hospitals and ATCC towards 16 selected antibiotics was analyzed and an antibiogram was generated. Findings also indicated resistance against many of the available antibiotics and thus an urgent need to search for alternative antibiotics.     

Characterization of rhizospheric soil streptomycetes from Moroccan habitats and their antimicrobial activities

During an investigation on actinomycetes from rhizospheric soils from Moroccan habitats, 131 streptomycetes were recovered, morphologically characterized and assessed for their antimicrobial activity. Eleven isolates were characterized by the absence of an aerial mycelium. According to the colour of aerial mycelium, the rest were grouped into seven main classes, namely, grey, yellow, cream, white, green, red and polymorphic colours (pink, orange or violet). The grey colour class dominated (40%) and the red one was found only in rhizospheric soil of the Moroccan endemic plant Argania spinosa. About one third of the isolates (34%) produced soluble pigments of various colours and 14% produced melanoid pigments. Most of the isolates (83%) were active against one or more of the organisms tested (one gram-negative bacterium, three gram-positive bacteria, three yeasts and two filamentous fungi). Most antibiotic-producing isolates possess red and white colour. Strong antibiosis was exhibited against Streptomyces scabies, Staphylococcus aureus and Bacillus subtilis (75, 68 and 60% respectively), while only 14 and 8% of isolates displayed an activity against Escherichia coli and Verticillium dahliae respectively.     

Screening of terrestrial and freshwater halotolerant cyanobacteria for antifungal activities

Cyanobacterial cultures tolerating 200 mmol l⁻¹ sodium chloride isolated from terrestrial and freshwater habitats of North Maharashtra region of India were evaluated for antifungal activity. Aqueous, methanol, n-propanol, and petroleum ether extracts of 40 cyanobacterial isolates belonging to nine genera were examined for inhibitory activity against five fungal pathogens. Eighteen isolates belonging to genus Oscillatoria dominated the population of halotolerant cyanobacterial cultures. Four antifungal bioassays viz. double layer agar method, disc diffusion assay, silica gel method, and minimum inhibitory concentration (MIC) were used to screen the cultures for antifungal activity. Among the solvents used, methanol extracts showed 34.9% inhibition followed by n-propanol, petroleum ether, and water exhibiting 30.2%, 18.6% and 16.2% inhibition, respectively. The double agar layer method was found to be a suitable method in preliminary screening for handling large number of cultures without extraction of compounds. However, in later screening experiments, silica gel method was seen to be advantageous over MIC and agar disc diffusion methods.     

Genotypic characteristics in populations of Sclerotinia sclerotiorum from New York State,USA

White mould, caused by the fungus Sclerotinia sclerotiorum, is one of the most destructive diseases of beans globally. In New York State, USA, white mould causes substantial losses in soybean, snap, dry and succulent baby lima beans, which are grown successively in intensive crop rotations. Management strategies for white mould in these crops are reliant upon the prophylactic use of fungicides. No complementary information on the genetic structure of the populations of S. sclerotiorum in New York State, USA is available. Twenty isolates of S. sclerotiorum were collected from symptomatic bean plants within each of 10 fields across New York State, USA in 2014. Eight microsatellite (SSR) markers were used to characterise the genotypic diversity of the hyphal‐tipped isolates. Twenty‐four multilocus genotypes (MLGs) were detected within the population but one MLG was most prevalent. Although STRUCTURE analysis identified two subpopulations, these subpopulations were not associated with geographic location, suggesting no spatial structure to the population. In addition, the pathogen populations were predominantly clonal, with some evidence of infrequent outcrossing. These findings may assist in understanding the durability of management strategies for white mould and support the selection of representative isolates for host resistance screening for pathogen populations in the sampling area.     

Screening of bacteria for the suppression of Botrytis cinerea and Rhizoctonia solani on lettuce (Lactuca sativa) using leaf disc bioassays

Two leaf disc bioassays were developed for screening bacteria as putative biological control agents of Botrytis cinerea and Rhizoctonia solani on lettuce. Aerobic spore and non‐spore forming bacteria were isolated from the phylloplane, rhizoplane and rhizosphere of symptom‐free lettuce plants grown in the presence and absence of chitin or composted bark soil amendments. Bacteria, previously isolated from other plants, were also included in the primary screen initially against B. cinerea. One hundred and twenty‐seven of 700 isolates reduced botrytis rotting of lettuce leaves by more than 50% in the primary screen. Following a secondary screen against B. cinerea, the lead 50 isolates were also tested for suppression of R. solani infection. Four isolates significantly reduced both botrytis and rhizoctonia leaf rotting. Eleven and five isolates gave control of botrytis and rhizoctonia, respectively, equal to that given by the standard fungicides Rovral WP (iprodione) and Basilex (tolclofos methyl). The two most effective isolates against B. cinerea and R. solani were both identified as Bacillus subtilis. Use of soil amendments did not increase the proportion of efficacious isolates recovered. Effective isolates were originally recovered from roots of oilseed rape and lettuce leaves. In general, it was found that bacteria which controlled one disease effectively did not control the second disease nearly as well. The bioassay protocols developed in this study were used successfully in screening a large number of bacterial isolates in a short time.     

Three Types of Sensitivity to Metalaxyl in Plasmopara viticola

The sensitivity of Plasmopara-isolates to metalaxyl was determined by a leaf disc test. For the first time this permitted the distinction and clear characterization of not only sensitive and resistant isolates but also of isolates belonging to a third type of sensitivity, termed the reduced sensitive type.     

Fluorescent Pseudomonas and cyclic lipopeptide diversity in the rhizosphere of cocoyam (Xanthosoma sagittifolium)

Cocoyam (Xanthosoma sagittifolium (L.)), an important tuber crop in the tropics, is severely affected by the cocoyam root rot disease (CRRD) caused by Pythium myriotylum. The white cocoyam genotype is very susceptible while the red cocoyam has some field tolerance to CRRD. Fluorescent Pseudomonas isolates obtained from the rhizosphere of healthy red and white cocoyams from three different fields in Cameroon were taxonomically characterized. The cocoyam rhizosphere was enriched with P. fluorescens complex and P. putida isolates independent of the plant genotype. LC–MS and NMR analyses revealed that 50% of the Pseudomonas isolates produced cyclic lipopeptides (CLPs) including entolysin, lokisin, WLIP, putisolvin and xantholysin together with eight novel CLPs. In general, CLP types were linked to specific taxonomic groups within the fluorescent pseudomonads. Representative CLP-producing bacteria showed effective control against CRRD while purified CLPs caused hyphal branching or hyphal leakage in P. myriotylum. The structure of cocoyamide A, a CLP which is predominantly produced by P. koreensis group isolates within the P. fluorescens complex is described. Compared with the white cocoyam, the red cocoyam rhizosphere appeared to support a more diverse CLP spectrum. It remains to be investigated whether this contributes to the field tolerance displayed by the red cocoyam.     

Oreocharis duyunensis (Gesneriaceae), a new species from Guizhou,China

Oreocharis duyunensis Z.Y. Li, X.G. Xiang et Z.Y. Guo (Gesneriaceae), endemic to Guizhou, China, is described as a new species. The new species is easily distinguished from other Oreocharis species by having a white, stellate ring‐like disc. Morphologically, the species is similar to Oreocharis chienii (Chun) Mich. Möller & A. Weber but differs by its densely white pilose leaves, mixed brown‐pilose, pubescent and glandular‐pubescent peduncle and pedicle, white stellate ring‐like disc, and linear‐lanceolate capsule. Based on phylogenetic reconstruction and interspecific genetic distances of nuclear ITS and chloroplast trnL‐F regions, O. duyunensis is a new species with uncertain phylogenetic position.