Structural Aspects of the Parasitism of Sclerotia of Sclerotinia sclerotiorum (Lib.) de Bary by Coniothyrium minitans Campb.

Sclerotia of Sclerotinia sclerotiorum inoculated with pycnidiospores of Coniothyrium minitans were studied by means of light microscopy and transmission and scanning electron microscopy. The hyperparasite penetrated the walls of the rind cells by means of physical pressure and destroyed the cell contents. Penetration of medullary hyphae was by enzymic lysis and physical pressure; there was evidence to suggest that the hyperparasite may coil around the host cells before inserting infection hyphae.     

Penetration of Hyphae of Sclerotinia sclerotiorum by Coniothyrium minitans Without the Formation of Appressoria

In a study using scanning electron microscopy (SEM), the mode of hyperparasitism of Coniothyrium minitans on its host Sclerotinia sclerotiorum was investigated. The SEM micrographs confirm previous reports, from light microscopic studies, that hyphal tips of C. minitans invade the host hypha by direct penetration, without developing appressoria, and that indentation of the host cell wall at the point of penetration is often evident. There is no functional distinction between amain branch and a side branch hypha of the hyperparasite and tips of either type of hyphae are capable of invading host hyphae by direct penetration.     

Characterization of some culture factors affecting oxalate degradation by the mycoparasite Coniothyrium minitans

Aims:  To find possible approaches to utilize the mechanism of oxalate degradation by Coniothyrium minitans (Cm) in controlling the plant pathogen Sclerotinia sclerotiorum (Ss).
Methods and Results:  Differences in oxalate degradation by different Cm strains and effects of the initial oxalate concentration, ambient pH and nutrient factors on mycelial growth and oxalate degradation by Cm were studied in shaken cultures. Results showed that two wild-type Cm strains, Chy-1 and ZS-1, did not differ in oxalate degradation in modified potato dextrose broth (mPDB) amended with oxalic acid (OA). Cm could grow in mPDB amended with sodium oxalate (SO-mPDB) at pH 6·5 or with ammonium oxalate (AO-PDB) at pH 6·2, but oxalate degradation was very low; oxalate degradation was greatly enhanced in SO- or AO-mPDB with pH being lowered to 2·8–2·9. Similarly, oxalate degradation was higher than 90% in OA-amended mPDB at pH 4·4 but was reduced to be <22% at pH 7·0. Five carbon sources and three nitrogen sources investigated and nutrients from mycelia and sclerotia of Ss were favorable for the growth of Cm and OA degradation by Cm.
Conclusions:  Cm can degrade oxalate under acidic pH. Exudates from mycelia or sclerotia of Ss may serve as nutrients for Cm mycelial growth and degradation of oxalate secreted by Ss.
Significance and Impact of the Study:  The finding of oxalate degradation laid a foundation for mining-related genes in Cm for engineering plant resistance against Ss. Elucidation of the importance of acidic pH and nutrients from Ss in oxalate degradation by Cm will help to understand the interaction between Cm and Ss.     

Mycoparasitism by Coniothyrium minitans on Sclerotinia sclerotiorum and its Effect on Sclerotial Germination

Scanning electron microscopy showed that hyphae of Coniothyrium minitans produced appressorium-like swellings when they came in contact with Sclerotinia sclerotiorum in dual culture on PDA. The parasitized hyphae gradually skrank and collapsed, and hyphae of the mycoparasite were found inside the host hyphae. The mycoparasite hyphae grew inter- and intracellularly within the sclerotia of S. sclerotiorum. In the later stages of parasitism, hyphae of the mycoparasite proliferated extensively within the sclerotia and formed pycnidia near the sclerotial surface. At this stage, the sclerotia became flattened, soft and disintegrated. Sclerotia parasitized by C. minitans failed to germinate either myceliogenically or carpogenically.     

Characterization of the Sclerotinia sclerotiorum cell wall proteome

We used a proteomic analysis to identify cell wall proteins released from Sclerotinia sclerotiorum hyphal and sclerotial cell walls via a trifluoromethanesulfonic acid (TFMS) digestion. Cell walls from hyphae grown in Vogel's glucose medium (a synthetic medium lacking plant materials), from hyphae grown in potato dextrose broth and from sclerotia produced on potato dextrose agar were used in the analysis. Under the conditions used, TFMS digests the glycosidic linkages in the cell walls to release intact cell wall proteins. The analysis identified 24 glycosylphosphatidylinositol (GPI)‐anchored cell wall proteins and 30 non‐GPI‐anchored cell wall proteins. We found that the cell walls contained an array of cell wall biosynthetic enzymes similar to those found in the cell walls of other fungi. When comparing the proteins in hyphal cell walls grown in potato dextrose broth with those in hyphal cell walls grown in the absence of plant material, it was found that a core group of cell wall biosynthetic proteins and some proteins associated with pathogenicity (secreted cellulases, pectin lyases, glucosidases and proteases) were expressed in both types of hyphae. The hyphae grown in potato dextrose broth contained a number of additional proteins (laccases, oxalate decarboxylase, peroxidase, polysaccharide deacetylase and several proteins unique to Sclerotinia and Botrytis) that might facilitate growth on a plant host. A comparison of the proteins in the sclerotial cell wall with the proteins in the hyphal cell wall demonstrated that sclerotia formation is not marked by a major shift in the composition of cell wall protein. We found that the S. sclerotiorum cell walls contained 11 cell wall proteins that were encoded only in Sclerotinia and Botrytis genomes.     

Improved approach for transferring and cultivating Methanosarcina acetivorans C2A (DSM 2834)

Aim:  A method for cultivating Methanosarcina acetivorans was further developed to handle these anaerobic archaea without special equipment such as an anaerobic chamber.
Methods and Results:  Medium was filtered and oxygen removed under a nitrogen gas-phase. A dithiothreitol-filled syringe was used to transfer cells from high density grown cultures to new medium. Growth time and cell mass were determined, as well as cell viability was proven by light microscopy.
Conclusion:  Cell transfer and growth was successful using this approach.
Significance and Impact of the Study:  This updated technique allows almost every laboratory the opportunity to grow these methanogenic organisms for further studies. The described method could be used for proteomic analysis and is also interesting for further protein structure determination.     

Antibacterial activities of zinc oxide nanoparticles against Escherichia coli O157:H7

Aims:  To investigate antibacterial activities of zinc oxide nanoparticles (ZnO NP) and their mode of action against an important foodborne pathogen, Escherichia coli O157:H7.
Methods and Results:  ZnO NP with sizes of 70 nm and concentrations of 0, 3, 6 and 12 mmol l⁻¹ and NP-free solutions were used in antimicrobial tests against E. coli O157:H7. ZnO NP showed increasing inhibitory effects on the growth of E. coli O157:H7 as the concentrations of ZnO NP increased. A complete inhibition of microbial growth was achieved at the concentration level of 12 mmol l⁻¹ or higher. Scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Raman spectroscopy were used to characterize the changes of morphology and cellular compositions of bacterial cells treated with ZnO NP and study the mode of action of ZnO NP against E. coli O157:H7. The intensity of lipid and protein bands in the Raman spectra of bacterial cells increased after exposure to ZnO NP, while no significant changes in nucleic acid bands were observed.
Conclusions:  ZnO NP were found to have antibacterial activity against E. coli O157:H7. The inhibitory effects increase as the concentration of ZnO NP increased. Results indicate that ZnO NP may distort and damage bacterial cell membrane, resulting in a leakage of intracellular contents and eventually the death of bacterial cells.
Significance and Impact of the Study:  These results suggest that ZnO NP could potentially be used as an effective antibacterial agent to protect agricultural and food safety.     

Co-operative action by endo- and exo-β-(1→3)-glucanases from parasitic fungi in the degradation of cell-wall glucans of Sclerotinia sclerotiorum (Lib.) de Bary

1. Two fungi, Coniothyrium minitans Campbell and Trichoderma viride Pers. ex Fr., were grown on autoclaved crushed sclerotia of the species Sclerotinia sclerotiorum, which they parasitize. 2. In vitro the crude culture filtrates would lyse walls isolated from hyphal cells or the inner pseudoparenchymatous cells of the sclerotia, in which a branched beta-(1-->3)-beta-(1-->6)-glucan, sclerotan, is a major constituent. 3. Chromatographic fractionation of the enzymes in each culture filtrate revealed the presence of several laminarinases, the most active being an exo-beta-(1-->3)-glucanase, known from previous studies to attack sclerotan. Acting alone this brought about a limited degradation of the glucan, but the addition of fractions containing an endo-beta-(1-->3)-glucanase led to almost complete breakdown. A similar synergism between the two enzymes was found in their lytic action on cell walls. 4. When acting alone the endo-beta-(1-->3)-glucanase had a restricted action, the products including a trisaccharide, tentatively identified as 6(2)-beta-glucosyl-laminaribiose. 5. These results are discussed in relation to the structure of the cell walls and of their glucan constituents.     

Changes during development in the permeability of sclerotia ofSclerotinia minor to an apoplastic tracer

Summary Sclerotia ofSclerotinia minor produced in culture are permeable to the apoplastic tracer sulphorhodamine G (SR) in early stages of their development, but become impermeable as the rind differentiates at the onset of maturation. Reduction in permeability corresponds with deposition of the dark brown pigment in the rind cell walls rather than initiation of the rind as a distinct surface layer. Fluorochrome permeation into cut sclerotia indicates that, while the rind is the primary barrier, the walls and extracellular matrix of the cortex and medulla of mature sclerotia also impede SR movement. Some cells take up fluorochrome into the protoplast. This indicates enhanced proton pumping activity at the cell surface, which suppresses ionisation of the fluorochrome, allowing it to cross the plasma membrane and accumulate in the hyphae. In intact sclerotia such hyphae are very rare and were detected only at one stage of development. However, in cut sclerotia at the two earliest stages of development most of the hyphae near the cut surface accumulated SR and it is possible that this is due to proton pumping activity induced by wounding.Abbreviations ECM extracellular matrix - SR sulphorhodamine G     

Histopathological studies of sclerotia of Rhizoctonia solani parasitized by the EGFP transformant of Trichoderma virens

Aims:  The aim of the study was to investigate the antagonistic interactions of Trichoderma species against Rhizoctonia solani sclerotia by enhanced green fluorescence protein (EGFP)-tagged transformant of Trichoderma virens TY009.
Methods and Results:  An EGFP was used as a report gene for transforming T. virens strain, and a stable EGFP transformant GF5 was obtained with the mycoparasitic activity against sclerotia of R. solani . Observation of parasitized sclerotia by fluorescence microscopy showed hyphae of transformant GF5 was able to invade into sclerotia and its colonization was mainly intercellar with uniformly distributed mycelium in sclerotia. The host cells were colonized, penetrated, and then the whole cells were replaced by transformant GF5 hyphae. Chlamydospores were seen after 10 days but mature ones after 20 days. Sclerotia became soft and decayed after 40 days but a few cells seemed not to be colonized completely.
Conclusions:  Trichoderma virens was able to parasitize sclerotia to make sclerotia soft and decayed, and its colonization was mainly intercellar in sclerotial tissues.
Significance and Impact of the Study:  This is first report of parasitism of sclerotia of R. solani by EGFP-tagged transformant, providing useful information for using T. virens as effective biocontrol agent.     

Characterization of lactosporin, a novel antimicrobial protein produced by Bacillus coagulans ATCC 7050

Aims:  To characterize the antimicrobial protein produced by Bacillus coagulans used in the probiotic dietary supplement (Lactospore^® Probiotic, Sabinsa Corp., Piscataway, NJ, USA).
Methods and Results:  Bacillus coagulans ATCC 7050 was grown at 37°C for 18 h. The cell free supernatant was concentrated 10-fold (lactosporin preparation, LP). The antimicrobial activity of LP was confirmed against Micrococcus luteus ATCC 10420 in a well diffusion assay. The proteinaceous nature of LP was determined following exposure to different enzymes. The activity of LP was pH-dependent but stable to heat. The isoelectric point of LP was determined to be 3·5–4·0. PCR analyses showed no similarity between lactosporin and known antimicrobial proteins produced by the Bacillus spp.
Conclusions:  Lactosporin is a novel antimicrobial protein. Initial characterization indicates that it may fall outside of the conventional classification of class I and II bacteriocins. Loss of activity after exposure to a number of proteolytic enzymes and lipase suggest that lactosporin may posses a lipid moiety which contributes to its inhibitory activity.
Significance and Impact of the Study:  The unique characteristics of lactosporin, including its antimicrobial activity against pathogenic micro-organisms, indicate that it may have potential for application in foods and personal care products.     

Notes on the ultrastructure of the microconidium and stroma in Sclerotinia sclerotiorum

Summary An electron microscope study has been done on the structure of microconidia and stroma of Sclerotinia sclerotiorum. The microconidia showed the usual cell organelles already described for other species of this genus, e.g. a large nucleus and large lipid body, a few mitochondria and sparse endoplasmic reticulum. The stromatal ultrastructure presented hyphae with a rich content in lipid bodies and storage vacuoles as food reserve. Simple septa with plugged pores were occasionally seen. Although the existence of two kinds of hyphae, one with abundant food materials and the other type with a degenerated aspect, both hyphae showed a similar thickness in their cell walls approximately 0.2 , and the diameter of these hyphae was 1.5–3 .     

Cytological Study of Wheat Spike Infection by Bipolaris sorokiniana

The infection of wheat spikelets by Bipolaris sorokiniana , the causal agent of black point on grains and grain shrivelling, was examined by light and electron microscopy. Conidia of the pathogen germinated 6–12 h after inoculation on the surfaces of the different spike tissues. Extracellular sheaths were observed on germ tubes and appressoria attached to the surfaces of lemma, palea and seeds, but were only scarcely detected on the surface of conidia. Appressoria, frequently found over grooves, formed penetration hyphae invading the epidermal cell walls. Infection process was similar on the surface of the lemma, palea and glume. Growth of the fungus in the epidermal and parenchyma cells was found predominantly in the cell walls, and hyphae also extended intercellularly and intracellularly. Infection of seeds appeared to occur via two ways: (i) direct infection of the outer layers of the cell walls of the pericarp and (ii) through entering the stigma into the pericarp cells. Secretion of host cell wall hydrolytic enzymes at the apex of the penetrating hyphae may facilitate the spread of the fungus. In addition, toxins secreted by the fungus might explain the rapid death of host cells in contact with or distant to fungal cells. A host response to fungal infection involved the development of appositions between cell wall and plasma membrane in cells adjacent to fungal cells. Fungal hyphae were sometimes also surrounded by electron dense material.     

Effect of alternative treatments on seed-borne Didymella lycopersici in tomato

Aims:  To quantify the phytotoxicity and effect of alternative seed treatments based on acidified nitrite and elicitors of plant resistance (Tillekur and Chitosan) against seed-borne inocula of Didymella lycopersici .
Methods and Results:  Treatments tested were: nitrite [sodium nitrite in citric acid buffer (pH 2)] at 30, 100 and 300 mmol l⁻¹ and three exposure times (10, 20 and 30 min); Tillekur (in water) at 12·5, 25, 50, 100 and 200 mg ml⁻¹; Chitosan (in 0·05% acetic acid) at 2·5, 5, 10 and 50 mg ml⁻¹. Efficacy of treatments was determined in growth chamber experiments. Nitrite at 300 mmol l⁻¹ was completely effective, as was the fungicide, at controlling disease when applied for less than 20 min. Tillekur was as effective as the fungicide postemergence, but proved to be phytotoxic pre-emergence. Chitosan was significantly less effective than the other treatments.
Conclusions:  The high efficacy and low cost of acidified nitrite indicates that it is a suitable alternative to fungicides.
Significance and Impact of the Study:  There is currently a lack of effective seed treatments that can be used in organic and low-input crops. Treatments identified in this study can be considered as an effective alternative to chemical control against seed-borne fungal pathogens.     

A fungal cell wall integrity-associated MAP kinase cascade in Coniothyrium minitans is required for conidiation and mycoparasitism

Coniothyrium minitans is an important biocontrol agent against Sclerotinia diseases. Previously, a conidiation-deficient mutant ZS-1T1000 was screened out from a T-DNA insertional library of C. minitans. CmBCK1, encoding MAP kinase kinase kinase and homologous to BCK1 of Saccharomyces cerevisiae, was disrupted by T-DNA insertion in this mutant. Targeted disruption of CmBCK1 led to the mutants undergoing autolysis and displaying hypersensitivity to the cell wall-degrading enzymes. The △CmBCK1 mutants lost the ability to produce pycnidia and conidia compared to the wild-type strain ZS-1. △CmBCK1 mutants could grow on the surface of sclerotia of Sclerotinia sclerotiorum but not form conidia, which resulted in much lower ability to reduce the viability of sclerotia of S. sclerotiorum. Furthermore, CmSlt2, a homolog of Slt2 encoding cell wall integrity-related MAP kinase and up-regulated by BCK1 in S. cerevisiae, was identified and targeted disrupted. The △CmSlt2 mutants had a similar phenotype to the △CmBCK1 mutants. The △CmSlt2 mutants also had autolytic aerial hyphae, hypersensitivity to cell wall-degrading enzymes, lack of conidiation and reduction of sclerotial mycoparasitism. Taken together, our results suggest that CmBCK1 and CmSlt2 are involved in conidiation and the hyperparasitic activities of C. minitans.     

Effect of physical, chemical and environmental characteristics on arbuscular mycorrhizal fungi in Brachiaria decumbens (Stapf) pastures

Aim:  To evaluate the effects of soil physical and chemical factors (pH, conductivity, humidity, available phosphorus and organic matter) and environmental factors (temperature, relative air humidity, altitude and atmospheric pressure) on arbuscular mycorrhizal fungi (AMF)– Brachiaria decumbens grass relationship. Furthermore to establish patterns of microbiological responses that allow to differentiate the study sites in two relief types.
Methods and Results:  Mycorrhizal characteristics (spore density, external hyphae and root colonizations by hyphae, vesicles and arbuscules), physical and chemical factors in soil and environmental factors were measured.
Conclusions:  The effect of physical, chemical and environmental factors on microbiological variables was related to the type of relief 'valley and hilly terrain'; the AMF behaviour was affected only over narrower ranges of evaluated variables. Similarly, the colonization of B. decumbens roots by AMF hyphae, vesicles and the mycorrhizal spore density follow different patterns according to the relief type.
Significance and Impact of the Study:  The type of relief is one of the factors to be taken into consideration to evaluate the AMF inoculum and root colonization of these pastures, because of the influence of slope – as physical property of soil – on AMF.     

Human embryonic stem cells secrete soluble factors that inhibit cancer cell growth

Objectives:  The aim of this study was to determine whether normal human embryonic stem cells (hESC) would secrete factors that arrest growth of human epithelial cancer cell lines.
Materials and methods:  Cell proliferation was examined using the MTT assay then haemocytometer cell counts. Staining with propidium iodide followed by flow cytometry was used to detect cell cycle stages. Heat denaturation and molecular fractionation experiments were also performed.
Results:  We found that hESC conditioned medium (hESC CM) inhibited SKOV-3 and HEY cell proliferation. Similar results were also obtained when we used breast and prostate cancer cell lines, whereas little or no inhibitory effect was observed when human fibroblasts were tested. Moreover, a co-culture model confirmed that inhibition of cancer cell proliferation is mediated by soluble factors produced by hESCs. We also determined that the proportion of cancer cells in G₁ phase was increased by hESC CM treatment, accompanied by decrease in cells in S and G₂/M phases, suggesting that the factors slow progression of cancer cells by cell cycle inhibition. Heat denaturation and molecular fractionation experiments indicated a low molecular weight thermostable factor was responsible for these properties.
Conclusions:  Our findings provide evidence that the human embryonic microenvironment contains soluble factor(s) that are capable of inhibiting growth of cancer cells, and that exposure to such factors may represent a new cancer treatment strategy.     

Thiol redox state and oxidative stress affect sclerotial differentiation of the phytopathogenic fungi Sclerotium rolfsii and Sclerotinia sclerotiorum

Aims:  To investigate the involvement of oxidative stress and thiol redox state (TRS) in sclerotial differentiation of Sclerotium rolfsii and Sclerotinia sclerotiorum.
Methods and results:  Oxidative stress in these fungi was assessed by lipid peroxidation, which was higher in comparison with their nonsclerotiogenic counterpart strains. TRS [measured as glutathione (GSH) and cysteine] was associated with oxidative stress and differentiation using the TRS modulator and antioxidant Ν -acetylcysteine (AcCSH) and the GSH biosynthesis inducer and inhibitor l -2-oxo-thiazolidine-4-carboxylate and l -buthionine- S , R -sulphoximine (BSO) respectively. Differentiation and oxidative stress was decreased by AcCSH in both fungi. The decrease of differentiation by BSO was not associated with oxidative stress in these fungi.
Conclusions:  Differentiation and oxidative stress in both fungi depends on the availability of antioxidant noncytotoxic –SH groups and is not depended on any direct antioxidant role of GSH and its precursor cysteine.
Significance and Impact of the Study:  This study helps to understand the mechanism(s) of sclerotial differentiation in these agriculturally important phytopathogenic fungi and proposes that AcCSH can be used as potent fungicide by (i) acting as growth inhibiting cytotoxic oxidant and (ii) sustaining these fungi in their undifferentiated hyphal stage where they are vulnerable to degradation by soil micro-organisms.     

Antifungal activity of thyme oil against Geotrichum citri-aurantii in vitro and in vivo

Aims:  To investigate antifungal effect of thyme oil on Geotrichum citri-aurantii arthroconidia germination and germ tube elongation, to reveal effects of thyme oil on morphological structures on fungal hyphae and arthroconidia and to assess potential bio-control capacities of thyme oil against disease suppression in vivo conditions.
Methods and Results:  Thyme oil controlled the growth of G. citri-aurantii effectively. Arthroconidia germination and germ tube elongation in potato dextrose broth was greatly inhibited by thyme oil. At 600 μl l⁻¹, it inhibited the germination of about 94% of the arthroconidia and the germ tube length was only 4·32 ± 0·28 μm. Observations using light microscope, scanning electron microscope and transmission electron microscope revealed ultrastructural modifications caused by thyme oil that included markedly shrivelled and crinkled hyphae and arthroconidia, plasma membrane disruption and mitochondrial disorganization. Thyme oil applied to 'Satsuma' mandarin oranges that had been artificially wounded and inoculated with G. citri-aurantii reduced sour rot from 78·1% among untreated control fruit to 14·1% after 5 days at 26°C. Thyme oil applied to intact fruits reduced the decay from 76% among untreated control fruit to 35% after 30 days at 20°C. Thyme oil treatment did not harm 'Satsuma' mandarin oranges when they were examined after treatment and storage at 20°C for 30 days.
Conclusions:  Thyme oil may provide an alternative means of controlling postharvest sour rot on citrus fruit.
Significance and Impact of the Study:  The use of such essential oil may constitute an important alternative to synthetic fungicides. They can be exploited in commercial production and applied under storage and greenhouse conditions.     

Transglutaminase down-regulates the dimerization of epidermal growth factor receptor in rat perivenous and periportal hepatocytes

Objective:  Recently, we found that transglutaminase 2 (TG2) might be involved in the difference in proliferative capacities between periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) through down-regulation of high-affinity epidermal growth factor receptor (EGFR). However, it is uncertain whether this high-affinity EGFR contributes to the hepatocyte growth signalling pathway. Here, we have investigated the influence of TG2 on EGF-induced EGFR dimerization and its phosphorylation, which are important steps in the hepatocyte proliferative/growth signalling pathway, in PPH and PVH.
Materials and methods:  PPH and PVH were isolated using the digitonin/collagenase perfusion technique. Amounts of TG2, EGFR dimerization and its phosphorylation were determined by Western blot analysis.
Results:  Pretreatment with monodansylcadaverine, an inhibitor of TG2, greatly increased EGF-induced EGFR dimerization and its phosphorylation in PVH compared with PPH. Conversely, treatment with retinoic acid, an inducer of TG2, significantly decreased EGF-induced EGFR dimerization and its phosphorylation with a significant increase in TG2 expression and its catalysed products, isopeptide bonds, in both subpopulations. It was found that EGFR served as a substrate for TG2.
Conclusion:  The present data showed good correlation with our previous data on EGF-induced DNA synthesis and EGFR-binding affinity to EGF. These results suggest that zonal difference in cell growth between PPH and PVH may be caused by down-regulation of EGFR dimerization and subsequent autophosphorylation through TG2-mediated cross-linking of EGFR.