Photodynamic Inactivation of Bacillus Spores, Mediated by Phenothiazinium Dyes

Spore formation is a sophisticated mechanism by which some bacteria survive conditions of stress and starvation by producing a multilayered protective capsule enclosing their condensed DNA. Spores are highly resistant to damage by heat, radiation, and commonly employed antibacterial agents. Previously, spores have also been shown to be resistant to photodynamic inactivation using dyes and light that easily destroy the corresponding vegetative bacteria. We have discovered that Bacillus spores are susceptible to photoinactivation by phenothiazinium dyes and low doses of red light. Dimethylmethylene blue, methylene blue, new methylene blue, and toluidine blue O are all effective, while alternative photosensitizers such as Rose Bengal, polylysine chlorin(e6) conjugate, a tricationic porphyrin, and a benzoporphyrin derivative, which easily kill vegetative cells, are ineffective. Spores of Bacillus cereus and B. thuringiensis are most susceptible, B. subtilis and B. atrophaeus are also killed, and B. megaterium is resistant. Photoinactivation is most effective when excess dye is washed from the spores, showing that the dye binds to the spores and that excess dye in solution can quench light delivery. The relatively mild conditions needed for spore killing could have applications for treating wounds contaminated by anthrax spores, for which conventional sporicides would have unacceptable tissue toxicity.     

In vitro response of strawberry cultivars and regenerants to Colletotrichum acutatum

Diseases affecting strawberry (Fragaria × ananassa Duch.) have been of major concern in recent years because of their widespread occurrence and potential for yield loss. Anthracnose, caused by the fungus Colletotrichum acutatum, is one of the most serious diseases of strawberry worldwide. Tissue-culture induced (somaclonal) variation provides one strategy for generating disease-resistant genotypes. As part of a program to generate strawberry germplasm resistant to anthracnose, an in vitro screening system was used to evaluate several commercial cultivars, Chandler, Delmarvel, Honeoye, Latestar, Pelican and Sweet Charlie propagated in vitro, and shoots regenerated from leaf explants of these cultivars for resistance to C.␣acutatum isolate Goff (highly virulent). Regenerants with increased levels of resistance were identified from all of the cultivars. The greatest increases in disease resistance were observed for regenerants from leaf explants of cultivars Pelican and Chandler that exhibited 17.5- and 6.2-fold increases in resistance, respectively. The highest levels of anthracnose resistance (2 to 6% leaf necrosis) were exhibited by regenerants from explants of cultivars Pelican and Sweet Charlie. These studies suggest that generating somaclonal variation may be a viable approach to obtaining strawberry plants with increased levels of anthracnose resistance.     

In Vitro Evaluation of Antagonism of Endophytic Colletotrichum gloeosporioides Against Potent Fungal Pathogens of Camellia sinensis

An endophytic fungus isolated from Camellia sinensis, Assam, Northeastern India was identified as Colletotrichum gloeosporioides on the basis of morphological characteristics and rDNA ITS analysis. This endophytic fungus was evaluated for growth inhibition against tea pathogens Pestalotiopsis theae and Colletotrichum camelliae. One isolate of C. gloeosporioides showed strong antagonistic activity against Pestalotiopsis theae (64 %) and moderate activity against C. camelliae (37 %). Fifty percent cell-free culture filtrate from 5-day-old cultures showed highest antagonistic activity against both the pathogens although the inhibition percent was less as compared to dual culture. In the experiment of volatile compounds none of the isolates of C. gloeosporioides strains showed visible inhibition against P. theae and C. camelliae. The activity of extracellular hydrolytic enzymes chitinase and protease was also high in this culture fluid and measured 10 and 4.3 IU/μl, respectively.     

Molecular and Phenotypic Analyses Reveal Association of Diverse Colletotrichum acutatum Groups and a Low Level of C. gloeosporioides with Olive Anthracnose

Anthracnose (Colletotrichum spp.) is an important disease causing major yield losses and poor oil quality in olives. The objectives were to determine the diversity and distribution pattern of Colletotrichum spp. populations prevalent in olives and their relatedness to anthracnose pathogens in other hosts, assess their pathogenic variability and host preference, and develop diagnostic tools. A total of 128 Colletotrichum spp. isolates representing all olive-growing areas in Portugal and a few isolates from other countries were characterized by molecular and phenotypic assays and compared with reference isolates. Arbitrarily primed PCR data, internal transcribed spacer of rRNA gene and β-tubulin 2 nucleotide sequences, colony characteristics, and benomyl sensitivity showed Colletotrichum acutatum to be dominant (>97%) with limited occurrence of Colletotrichum gloeosporioides (<3%). Among C. acutatum populations, five molecular groups, A2 to A6, were identified. A2 was widely prevalent (89%), coinciding with a high incidence of anthracnose and environmental conditions suitable to disease spread. A4 was dominant in a particular region, while other C. acutatum groups and C. gloeosporioides were sporadic in their occurrence, mostly related to marginal areas of olive cultivation. C. gloeosporioides, isolated from olive fruits with symptoms indistinguishable from those of C. acutatum, showed same virulence rating as the most virulent C. acutatum isolate from group A2. C. acutatum and C. gloeosporioides isolates tested in infected strawberry fruits and strawberry and lupin plants revealed their cross-infection potential. Diagnostic tools were developed from β-tubulin 2 sequences to enable rapid and reliable pathogen detection and differentiation of C. acutatum groups.     

Molecular and phenotypic analyses reveal association of diverse Colletotrichum acutatum groups and a low level of C. gloeosporioides with olive anthracnose

Anthracnose (Colletotrichum spp.) is an important disease causing major yield losses and poor oil quality in olives. The objectives were to determine the diversity and distribution pattern of Colletotrichum spp. populations prevalent in olives and their relatedness to anthracnose pathogens in other hosts, assess their pathogenic variability and host preference, and develop diagnostic tools. A total of 128 Colletotrichum spp. isolates representing all olive-growing areas in Portugal and a few isolates from other countries were characterized by molecular and phenotypic assays and compared with reference isolates. Arbitrarily primed PCR data, internal transcribed spacer of rRNA gene and beta-tubulin 2 nucleotide sequences, colony characteristics, and benomyl sensitivity showed Colletotrichum acutatum to be dominant (>97%) with limited occurrence of Colletotrichum gloeosporioides (<3%). Among C. acutatum populations, five molecular groups, A2 to A6, were identified. A2 was widely prevalent (89%), coinciding with a high incidence of anthracnose and environmental conditions suitable to disease spread. A4 was dominant in a particular region, while other C. acutatum groups and C. gloeosporioides were sporadic in their occurrence, mostly related to marginal areas of olive cultivation. C. gloeosporioides, isolated from olive fruits with symptoms indistinguishable from those of C. acutatum, showed same virulence rating as the most virulent C. acutatum isolate from group A2. C. acutatum and C. gloeosporioides isolates tested in infected strawberry fruits and strawberry and lupin plants revealed their cross-infection potential. Diagnostic tools were developed from beta-tubulin 2 sequences to enable rapid and reliable pathogen detection and differentiation of C. acutatum groups.     

In Vitro Comparison of Hypericin and 5-Aminolevulinic Acid-Derived Protoporphyrin IX for Photodynamic Inactivation of Medulloblastoma Cells

Background

Hypericin (HYP) is a naturally occurring photosensitizer. Cellular uptake and photodynamic inactivation after incubation with this photosensitizer have neither been examined in medulloblastoma cells in vitro, nor compared with 5-aminolevulinic acid-derived protoporphyrin IX (5-ALA-derived PpIX).

Methods

In 3 medulloblastoma cell lines (D283 Med, Daoy, and D341 Med) the time- and concentration-dependent intracellular accumulation of HYP and 5-ALA-derived PpIX was analyzed by fluorescence microscopy (FM) and FACS. Photocytotoxicity was measured after illumination at 595 nm (HYP) and 635 nm (5-ALA-derived PpIX) in D283 Med cells and compared to U373 MG glioma cells.

Results

All medulloblastoma cell lines exhibited concentration- and time-dependent uptake of HYP. Incubation with HYP up to 10 µM resulted in a rapid increase in fluorescence intensity, which peaked between 2 and 4 hours. 5-ALA-derived PpIX accumulation increased in D283 Med cells by 22% over baseline after 5-ALA incubation up to 1.2 mM. Photocytotoxicity of 5-ALA-derived PpIX was higher in D283 Med medulloblastoma compared to U373MG glioma. The [lethal dose (light dose that is required to reduce cell survival to 50% of control)] of 5-ALA-derived PpIX was 3.8 J/cm² in D283 Med cells versus 5.7 J/cm2 in U373MG glioma cells. Photocytotoxicity of HYP in D283 Med cells was determined at 2.5 µM after an incubation time of 2 h and an illumination wavelength of 595 nm. The value was 0.47 J/cm².

Conclusion

By its 5-fold increase in fluorescence over autofluorescence levels HYP has excellent properties for tumor visualization in medulloblastomas. The high photocytotoxicity of HYP, compared to 5-ALA-derived PpIX, is convincingly demonstrated by its 8- to 13-fold lower . Therefore HYP might be a promising molecule for intraoperative visualization and photodynamic treatment of medulloblastomas.     

High-Frequency Ultrasound Assessment of Antimicrobial Photodynamic Therapy In Vitro

Ultrasound imaging is proving to be an important tool for medical diagnosis of dermatological disease. Backscatter spectral profiles using high-frequency ultrasound (HFUS, 10–100 MHz) are sensitive to subtle changes in eukaryotic cellular morphology and mechanical properties that are indicative of early apoptosis, the main type of cell death induced following photodynamic therapy (PDT). We performed experiments to study whether HFUS could also be used to discern changes in bacteria following PDT treatment. Pellets of planktonic Staphylococcus aureus were treated with different PDT protocols and subsequently interrogated with HFUS. Changes in ultrasound backscatter response were found to correlate with antimicrobial effect. Despite their small size, distinct changes in bacterial morphology that are indicative of cell damage or death are detectable by altered backscatter spectra from bacterial ensembles using HFUS. This highlights the potential for HFUS in rapidly and non-invasively assessing the structural changes related to antimicrobial response.     

Intraspecific Differentiation of Colletotrichum gloeosporioides sensu lato Based on In Silico Multilocus PCR-RFLP Fingerprinting

Colletotrichum gloeosporioides sensu lato is one of the most common and widely distributed plant pathogens in the world. Understanding fungal biodiversity is hinged on accurate identification and delimitation at the inter- and intraspecific levels. Sequences of the ITS1-5.8S-ITS2 region (ITS), β-tubulin (TUB), actin (ACT), and glyceraldehyde-3-phosphate dehydrogenase (GPDH) genes of 30 C. gloeosporioides sensu lato isolates, collected from anthracnose infected papaya fruits grown in the main production areas in Trinidad, were analyzed by in silico PCR-RFLP analysis with the aim of identifying which gene region(s) had the highest level of intraspecific polymorphism. Restriction site polymorphisms generated from 13 restriction enzymes enabled the identification of specific enzymes that were successful at intraspecific discrimination of the C. gloeosporioides isolates. Genetic distance values were reflective of the level of polymorphisms obtained for the four different gene regions. In both cases (calculated genetic distance and percentage of polymorphic loci from RFLP profiles), ACT and ITS gene regions had the highest level of restriction site polymorphisms and genetic diversity, GPDH and TUB had the lowest. Cluster analysis based on PCR-RFLP genetic distance data revealed sub-specific placement of the isolates which appeared to be gene-dependent. The implications of these findings are discussed relative to biodiversity monitoring and the need for multilocus, polyphasic investigations which must take into account the possibility of exaggerated estimates of genetic diversity.     

Detection and differentiation of Colletotrichum gloeosporioides isolates using PCR

An oligonucleotide primer (CgInt), synthesised from the variable internally transcribed spacer (ITS) 1 region of ribosomal DNA (rDNA) of Collectotrichum gloeosporioides was used for PCR with primer ITS4 (from a conserved sequence of the rDNA) to amplify a 450-bp fragment from the 25 C. gloeosporioides isolates tested. This specific fragment was amplified from as little as 10 fg of fungal DNA. A similar sized fragment was amplified from DNA extracted from C. gloeosporioides-infected tomato tissue. RAPD analysis divided 39 C. gloeosporioides isolates into more than 12 groups linked to host source and geographic origin. Based on the results obtained, the potential of PCR for detection and differentiation of C. gloeosporioides is discussed.     

Purification and characterization of an endochitinase produced by Colletotrichum gloeosporioides

The phytopathogenic fungus Colletotrichum gloeosporioides was analyzed for chitinase activity, the best production occurring at the fourth day. A 43 kDa endochitinase with specific activity of 413 U microg(-1) protein was purified corresponding to a 75% yield. The optima of temperature and pH for the enzyme were 50 degrees C and pH 7.0, respectively. The enzyme showed a high stability at 50 degrees C and pH 7.0. Values of pH from 5.0 up to 7.0 gave, at least, 50% of maximum activity, suggesting a biotechnological application. Further studies are in progress to determine the possible use of this endochitinase in biological control.     

In Vitro Fungicidal Photodynamic Effect of Hypericin on Trichophyton spp

Hypericin is a natural photosensitizer used in photodynamic therapy (PDT), which has shown in vitro antifungal effect against Candida spp. The aim of this study was to evaluate the in vitro fungicidal effect of hypericin-PDT on dermatophytes. Trichophyton rubrum and Trichophyton mentagrophytes strains were incubated with different concentrations of hypericin for different times and exposed to light-emitting diode lamp (602 ± 10 nm, 10.3 mW cm^?2, and fluence 37 J cm^?2). Using the optimal incubation time, 60 min, a 3-log fungicidal effect was achieved with hypericin concentration ranges of 10–20 μM for T. rubrum and 20–50 μM for T. mentagrophytes (p = 0.95). Confocal fluorescence microscopy showed the localization of hypericin inside the dermatophytes diffusely distributed in the cytoplasm of conidia and hyphae and outside the nucleus. In conclusion, hypericin-PDT has a fungicidal effect in vitro on dermatophytes. Hypericin seems to be a promising photosensitizer to treat localized dermatophytic infections such as tinea pedis and onychomycosis.     

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment.Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response¹. Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)².The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis.In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.     

Generation of Superoxide and In Vitro Inactivation of Viruses by Aldopentoses

Ren-1 renin is synthesized in the kidney of every mouse. Ren-2 renin has been observed in the submandibular gland (SMG) of male mice carrying two renin genes. However, it is not known if Ren-2 renin is in the kidney and blood of the two-renin gene mice. In this study, a direct ELISA for Ren-2 renin (SMG renin) was established by a sandwich method. This ELISA could measure the Ren-2 active renin in the range from 1 to 100ng and distinguish Ren-2 active renin from not only Ren-1 renin but also Ren-2 prorenin. By a combination of this assay system and conventional methods, the pro-form as well as the active form of Ren-2 renin was found in the kidney and plasma of male AKR mice carrying two-renin genes.     

In Planta Production of Indole-3-Acetic Acid by Colletotrichum gloeosporioides f. sp. aeschynomene

The plant pathogenic fungus Colletotrichum gloeosporioides f. sp. aeschynomene utilizes external tryptophan to produce indole-3-acetic acid (IAA) through the intermediate indole-3-acetamide (IAM). We studied the effects of tryptophan, IAA, and IAM on IAA biosynthesis in fungal axenic cultures and on in planta IAA production by the fungus. IAA biosynthesis was strictly dependent on external tryptophan and was enhanced by tryptophan and IAM. The fungus produced IAM and IAA in planta during the biotrophic and necrotrophic phases of infection. The amounts of IAA produced per fungal biomass were highest during the biotrophic phase. IAA production by this plant pathogen might be important during early stages of plant colonization.     

Chemical Control of Nematodes and Soil-borne Plant-Pathogenic Fungi on Cabbage Tranplants

Six general-purpose fumigants and one fungicide were applied by different methods and evaluated for control of nematode-fungus complexes on cabbage grown for transplant production. All chemicals reduced populations of nematodes and soil-borne fungi but varied greatly in effectiveness. Methyl bromide + chloropicrin (98% methyl bromide + 2% chloropicrin) (MBR-CP gas), DD + methyl isothiocyanate (DD-MENCS), methyl bromide + chloropicrin (67% methyl bromide + 31.75% cbloropicrin) (MBR-CP gel), and chloropicrin were more effective than sodium methyl dithiocarbamate (metham), pentachloronitrobenzene (PCNB), and potassium N-hydroxy-methyl-N-methyldithiocarbamate (Bunema) against Meloidogyne incognita. Populations of Pythium spp. and Fusarium spp. were reduced markedly by all treatments except PCNB. Plant growth, uniformity, and yield were greater when nematodes and fungi were controlled.     

In Vitro Morphogenesis of Arabidopsis to Search for Novel Endophytic Fungi Modulating Plant Growth

Fungal endophytes have shown to affect plant growth and to confer stress tolerance to the host; however, effects of endophytes isolated from water plants have been poorly investigated. In this study, fungi isolated from stems (stem-E) and roots (root-E) of Mentha aquatica L. (water mint) were identified, and their morphogenetic properties analysed on in vitro cultured Arabidopsis (L.) Heynh., 14 and 21 days after inoculation (DAI). Nineteen fungi were analysed and, based on ITS analysis, 17 isolates showed to be genetically distinct. The overall effect of water mint endophytes on Arabidopsis fresh (FW) and dry weight (DW) was neutral and positive, respectively, and the increased DW, mainly occurring 14 DAI, was possibly related to plant defence mechanism. Only three fungi increased both FW and DW of Arabidopsis at 14 and 21 DAI, thus behaving as plant growth promoting (PGP) fungi. E-treatment caused a reduction of root depth and primary root length in most cases and inhibition-to-promotion of root area and lateral root length, from 14 DAI. Only Phoma macrostoma, among the water mint PGP fungi, increased both root area and depth, 21 DAI. Root depth and area 14 DAI were shown to influence DWs, indicating that the extension of the root system, and thus nutrient uptake, was an important determinant of plant dry biomass. Reduction of Arabidopsis root depth occurred to a great extent when plants where treated with stem-E while root area decreased or increased under the effects of stem-E and root-E, respectively, pointing to an influence of the endophyte origin on root extension. M. aquatica and many other perennial hydrophytes have growing worldwide application in water pollution remediation. The present study provided a model for directed screening of endophytes able to modulate plant growth in the perspective of future field applications of these fungi.     

In planta production of indole-3-acetic acid by Colletotrichum gloeosporioides f. sp. aeschynomene

The plant pathogenic fungus Colletotrichum gloeosporioides f. sp. aeschynomene utilizes external tryptophan to produce indole-3-acetic acid (IAA) through the intermediate indole-3-acetamide (IAM). We studied the effects of tryptophan, IAA, and IAM on IAA biosynthesis in fungal axenic cultures and on in planta IAA production by the fungus. IAA biosynthesis was strictly dependent on external tryptophan and was enhanced by tryptophan and IAM. The fungus produced IAM and IAA in planta during the biotrophic and necrotrophic phases of infection. The amounts of IAA produced per fungal biomass were highest during the biotrophic phase. IAA production by this plant pathogen might be important during early stages of plant colonization.     

High Content Screening as High Quality Assay for Biological Evaluation of Photosensitizers In Vitro

A novel single step assay approach to screen a library of photdynamic therapy (PDT) compounds was developed. Utilizing high content analysis (HCA) technologies several robust cellular parameters were identified, which can be used to determine the phototoxic effects of porphyrin compounds which have been developed as potential anticancer agents directed against esophageal carcinoma. To demonstrate the proof of principle of this approach a small detailed study on five porphyrin based compounds was performed utilizing two relevant esophageal cancer cell lines (OE21 and SKGT-4). The measurable outputs from these early studies were then evaluated by performing a pilot screen using a set of 22 compounds. These data were evaluated and validated by performing comparative studies using a traditional colorimetric assay (MTT). The studies demonstrated that the HCS assay offers significant advantages over and above the currently used methods (directly related to the intracellular presence of the compounds by analysis of their integrated intensity and area within the cells). A high correlation was found between the high content screening (HCS) and MTT data. However, the HCS approach provides additional information that allows a better understanding of the behavior of these compounds when interacting at the cellular level. This is the first step towards an automated high-throughput screening of photosensitizer drug candidates and the beginnings of an integrated and comprehensive quantitative structure action relationship (QSAR) study for photosensitizer libraries.     

Conidium Germination and Appressorium Penetration of Colletotrichum gloeosporioides on Stylosanthes guianensis

Pre-penetration and early penetration phases of two pathogenic strains of Colletotrichum gloeosporioides (HM 502, HM 514) from Zaire were investigated quantitatively on four Stylosanthes guianensis genotypes (cv. Schofield, cv. Graham, ClAT 184 and CIAT 10136) of various levels of susceptibility. The results demonstrated control of the rates and type of conidial germination by the fungus, influenced to a lesser extent by the host genotype. Cell reactions were visible as papillae and cytoplasmic aggregations closely associated with the penetration peg. No fungal growth was observed when deposits of papillae occurred under appressoria-forming infection pegs, whereas the effect of the cytoplasmic aggregations varied according to the host genotype. In the interactions with strain HM 502, the highest rates of plant reaction were recorded on the resistant CIAT 184 genotype, while the highest level of penetration without reaction was observed on the most susceptible cv. Schofield. Cytoplasmic aggregations were also observed under a few ungerminated appressoria on this genotype. Differences were thus detected in the rates and types of conidial germination, in the rates and types of cell reactions under appressoria and in the appressorial penetration rates without cell reactions. However, these differences did not completely explain the range of macroscopic symptoms observed on the various genotypes.