Quantification of the biocontrol agent Pseudomonas fluorescens Pf153 in soil using a quantitative competitive PCR assay unaffected by variability in cell lysis- and DNA-extraction efficiency

Although often neglected, variability in cell lysis efficiency and DNA extraction yield represents the major hurdles of any polymerase chain reaction (PCR)-based quantification protocol in soil and other natural environments. In this study we developed a technique that minimizes the effects of these constraints, providing at the same time a reliable internal control to distinguish between PCR-inhibition and negative results. We used Pseudomonas fluorescens Pf153, a root-colonizing bacterium that shows biocontrol activity against tobacco and cucumber black root rot, as the target organism for PCR quantification. Prior to DNA extraction, the genetically engineered, cognate reference strain P. fluorescens CHA0/c2 was inoculated in a reference soil. CHA0/c2 in the reference soil and Pf153 in the soil sample were lysed in parallel and afterward the lysates were mixed in known proportions. CHA0/c2 carries the plasmid pME6031-cmp2 that contains an allelic variant (competitor) of the Pf153 specific sequence Pf153_2. In a quantitative competitive PCR (QC-PCR) assay the competitor allows the quantification of the target strain down to 0.66 Pf153 CFU/mg soil. Processing the reference strain in the same way as Pf153 enables the exact quantification of the target strain in biocontrol assays performed in natural soil, overcoming differences in DNA extraction efficiency and PCR amplification from different soil environments. This technique is easily adaptable to other Pseudomonas strains simply by replacing the competitor used here with one derived from a SCAR-marker which is specific for the strain of choice.     

Pre-unfolding resonant oscillations of single green fluorescent protein molecules

Fluorescence spectroscopy of a green fluorescent protein mutant at single-molecule resolution has revealed a remarkable oscillatory behavior that can also be driven by applied fields. We show that immediately before unfolding, several periodic oscillations among the chemical substates of the protein chromophore occur. We also show that applied alternating electric or acoustic fields, when tuned to the protein characteristic frequencies, give rise to strong resonance effects.     

Effects of permanent magnetic fields on the proliferation of Phalaenopsis protocorm-like bodies using liquid medium

Magnetic fields (MFs) have been applied for the first time in orchid micropropagation. Protocorm-like bodies (PLBs) – approximately 3 mm in diameter – first derived from leaf segment culture of Phalaenopsis Gallant Beau ‘George Vazquez’, were subcultured every 2 months, and served as initial explants. The proliferation of Phalaenopsis PLBs in liquid medium in the Miracle Pack^® culture system was affected by the action of different intensities and polarities of MFs: 0.1, 0.15 and 0.2 Tesla (T) at North (N) and South (S) poles. The MF of 0.1 T – S resulted in the greatest fresh weight of regenerated PLBs. The average number of neo-PLBs formed per clusters in the PLB treated by MF: 0.1–0.2 T was decreased compared to the control exposed to natural MF (5 × 10⁻⁶ T). The proliferation of PLBs under 0.15 T – MF at both N and S poles for 2 and 7 weeks demonstrated that a longer duration of exposure to an MF of 0.15 T, regardless of the polarity, resulted in greater biomass of newly formed PLBs and smaller average number of newly formed PLBs. The S pole of MF had stronger effects on Phalaenopsis PLBs proliferation than the N pole did in all treatments.     

Horticultural performance of ‘Folha Murcha’ sweet orange onto twelve rootstocks

Despite its outstanding position, the Brazilian citriculture is established on a very limited pool of varieties that limits its expansion and restricts the fruit availability throughout the year. This situation determines the urgent necessity of developing alternative scion and rootstock cultivars, with good performance under local conditions. ‘Folha Murcha’ sweet orange (Citrus sinensis (L.) Osbeck) is a late-harvest cultivar, suitable both for the juice processing industry and the fresh fruit market, being described as tolerant to citrus canker (Xanthomonas citri subsp. citri Schaad et al.), and less affected by citrus variegated chlorosis (Xylella fastidiosa Wells et al.). A study was conducted in Bebedouro, São Paulo State, Brazil, to evaluate the horticultural performance of ‘Folha Murcha’ sweet orange budded onto 12 rootstocks: the citrandarin ‘Changsha’ mandarin (Citrus reticulata Blanco) × Poncirus trifoliata ‘English Small’; the hybrid ‘Rangpur’ lime (Citrus limonia Osbeck) × ‘Swingle’ citrumelo (P. trifoliata (L.) Raf. × Citrus paradisi Macfad.); the trifoliates (P. trifoliata (L.) Raf.) ‘Rubidoux’, ‘FCAV’, and ‘Flying Dragon’ (P. trifoliata var. monstrosa); the ‘Sun Chu Sha Kat’ mandarin (C. reticulata Blanco); the ‘Sunki’ mandarin (Citrus sunki (Hayata) Hort. ex. Tanaka); the ‘Rangpur’ limes (C. limonia Osbeck) ‘Cravo Limeira’ and ‘Cravo FCAV’; ‘Carrizo’ citrange (C. sinensis × P. trifoliata), ‘Swingle’ citrumelo (P. trifoliata × C. paradisi), and ‘Orlando’ tangelo (C. paradisi × Citrus tangerina cv. ‘Dancy’). The experimental grove was planted in 2001, using a 7 m × 4 m spacing, in a randomized block design, with five replications and two plants per plot. No supplementary irrigation was applied. Fruit yield, canopy volume, tree tolerance to drought and to citrus variegated chlorosis, and fruit quality were assessed for each rootstock. Trees grafted onto the ‘Flying Dragon’ trifoliate were smaller in size, but had largest yield efficiency when compared to those grafted onto other rootstocks. Lower alternate bearing index was observed on trees budded onto ‘Cravo FCAV’ ‘Rangpur’ lime. Both ‘Rangpur’ lime rootstocks and the ‘Sunki’ mandarin induced higher tree tolerance to drought. The ‘Flying Dragon’ trifoliate induced better fruit quality and higher tolerance to citrus variegated chlorosis (CVC) to ‘Folha Murcha’ trees. A cluster multivariate analysis identified three groups of rootstocks with similar effects on ‘Folha Murcha’ tree performance. Among the 12 evaluated rootstocks, the ‘Flying Dragon’ trifoliate has a unique effect on plant growth, tolerance to drought and CVC, fruit yield and fruit quality of ‘Folha Murcha’ trees, and may be better suited for high-density plantings.     

Effect of cold cathode fluorescent lamps on growth of Gerbera jamesonii plantlets in vitro

The goal of this study was to evaluate the effect of cold cathode fluorescent lamps (CCFLs) on the growth of Gerbera jamesonii var. ‘Rui Kou’ plantlets in vitro in six different light quality ratios: 100% red CCFL (R), 80% R + 20% blue CCFL (B), 70% R + 30% B, 60% R + 40% B, 100% B and white CCFLs (W). Control radiation was provided by conventional heat-generating plant growth fluorescent lamps (PGFLs). Plantlets under CCFLs showed better plantlet height, SPAD value (i.e., leaf chlorophyll content) and root activity (as assessed by root dehydrogenase activity) than those growing under PGFLs while all other growth parameters were comparable with plants under conventional lighting systems.     

Determination of angiotensin I-converting enzyme activity in equine blood: lack of agreement between methods of analysis

Angiotensin-I converting enzyme (ACE) is a key regulator of blood pressure, electrolytes and fluid homeostasis through conversion of angiotensin I into angiotensin II. Recently, a genetic polymorphism of the ACE gene, which accounts for 47% of the variation of ACE activity in blood, has been advocated as a biomarker of athletic aptitude. Different methods of analysis and determination of ACE activity in plasma have been used in human and equine research without a consensus of a "gold standard" method. Different methods have often been used interchangeably or cited as being comparable in the existing literature; however, the actual agreement between assays has not been investigated. Therefore, in this study, we evaluated the level of agreement between three different assays using equine plasma obtained from 29 horses. Two spectrophotometric assays using Furylacryloyl-phenylalanyl-glycyl-glycine as substrate and one fluorimetric assay utilizing o-aminobenzoic acid-FRK-(Dnp)P-OH were employed. The results revealed that the measurements from the different assays were not in agreement, indicating that the methods should not be used interchangeably for measurement of equine ACE activity. Rather, a single method of analysis should be adopted to achieve comparable results and critical appraisal of the literature is needed when attempting to compare results obtained from different assays.     

Molecular and serological detection of Babesia spp. in neotropical and exotic carnivores in Brazilian zoos

Large and small piroplasms have been observed in the blood smears of various wild carnivores, but few studies utilizing molecular characterization have been done. The goal of this present study was to investigate the presence of Babesia sp. by molecular and serologic techniques in exotic and neotropical carnivores maintained in captivity at Brazilian zoos. Blood and sera samples were collected from 146 Brazilian wild felids, 21 exotic felids, 1 genet (Genetta tigrina), 3 European wolves (Canis lupus), and 94 Brazilian wild canids in Brazilian zoos in the S?o Paulo and Mato Grosso states and in the Federal District. A total of 53 wild felids (31.74%) and 10 wild canids (10.31%) were seropositive for Babesia canis by Indirect Fluorescent Antibody Test (IFAT). Antibodies were detected in ocelots, little-spotted cats, margays, pampas cats, jaguars, pumas, jaguarundis, crab-eating foxes, and bush dogs. Babesia sp. DNA, with high similarity to B. leo, was detected in one pampas cat and one genet.     

Penicilliosis in an African grey parrot (Psittacus erithacus)

Penicillium mycosis, or penicilliosis, is rare in animals. This report describes the clinical signs and pathological, immunohistochemical, and biomolecular findings in a case of avian penicilliosis in a Congo African grey parrot. On gross necropsy, pyogranulomatous changes were identified in the lung, liver, and kidney. Histopathology demonstrated periodic acid Schiff-(PAS) and Grocott-positive hyphae in these tissues. Immunohistochemistry confirmed the genus of the fungus and polymerase chain reaction (PCR) confirmed a high homology with Penicillium chrysogenum.     

Clavibacter michiganensis subsp. michiganesis: Tracking strains using their genetic differentiations by ISSR markers in Southern Turkey

There is no available data published related to the dissemination of bacterial canker caused by Clavibacter michiganensis subsp. michiganensis (Cmm) and its genetic diversity in Turkey. It is important to control new introduced inoculum sources by commercial seeds and seedlings. Pathogens were identified by morphological features and the identity was confirmed by PCR amplification using a specific primer PSA-4, PSA-R in addition to microbiological tests. ISSR markers showing high polymorphism ratios were selected and used to characterize Cmm strains. The collected strains were classified into different groups on the basis of ISSR-PCR fingerprints, which showed remarkable genetic specificity and diversity not previously identified in Cmm, suggesting that genetic differences are related to dissemination of the pathogen in the region. This is the first ever study carried out on the characterization of Cmm using ISSR. The selected ISSR primers to characterize Cmm can be used to determine genetic differences in further studies.