Diversity among Single Zoospore Isolates Derived from Single-zoosporangia of Phytophthora sojae Kauf. and Gerd

Phytophthora sojae Kauf. and Gerd, a host specific pathogen to soybean, causes pre and postemergence damping-off and root and stem rot on soybean. The pathogen evokes severe yield losses in most soybean growing areas worldwide. The objective of this study was to determine phenotypic and genotypic diversity among single zoospore isolates (SZIs) originating from two single zoosporangia (Ps411-1 and Ps411-2) derived from the same parental isolate of P . sojae Ps411. Results showed that colony morphology and growth rate of 32 SZIs derived from sporangium Ps411-1 and 35 SZIs released from sporangium Ps411-2 did not significantly differ from the parental isolate Ps411. Pathogenicity of the SZIs was tested on three resistant and three susceptible Chinese soybean cultivars. While the majority of SZIs derived from sporangium Ps411-1(59.4%) and sporangium Ps411-2(71.4%) retained the same virulence spectrum as the parental isolate, the other SZIs of both progenies demonstrated either a higher or a lower level of virulence compared to that of parental isolate. A low level genetic variability in the populations of both single zoospore progenies was also demonstrated using the sequence-related amplified polymorphism (SRAP) technique. Cluster analysis separated the SZIs from both zoosporangia, Ps411-1 and Ps411-2, into four and three SRAP groups, respectively. No close correlation among SRAP and virulence could be established among SZIs. The results of this study suggest that virulence variability may be regarded as part of the total genetic changes among the zoospore progenies derived from single-zoosporangia. The pathogenic variability during asexual reproduction may play a role in changing the virulence structure of P . sojae .     

Isolation of microsatellite loci from expressed sequence tag library of Puccinia striiformis f. sp. tritici

We described twenty polymorphic microsatellite loci derived from the expressed sequence tags of Puccinia striiformis f. sp. tritici, which causes yellow rust disease on wheat. The numbers of alleles range from two to six and eight microsatellite loci show significant similarities to known genes. Observed and expected heterozygosities ranged from 0.12 to 0.78 and from 0.24 to 0.87, respectively.     

Purification and characterization of a zearalenone degrading enzyme produced by<Emphasis Type="Italic">Gliocladium roseum</Emphasis>

Enzymatic inactivation of fungal toxins is an attractive strategy for the decontamination of food and feeding stuff. A constitutively expressed enzyme opening the lactone linkage within the macrocyclic ring system of zearalenone (ZON) was isolated fromGliocladium roseum. The enzyme has been shown to catalyze the transformation of the mycotoxin ZON and therefore has been named ZON degrading enzyme. The resulting products of the enzymatic reaction are less toxic because they have lost their estrogenic capacity. In this study, we used scanning electron microscopy to evaluate the possible mycoparasitism betweenFusarium graminearum andG. roseum. The ZON-degrading enzyme could be isolated fromG. roseum cultures and biochemically characterized. It has been found to be similar to superoxide-dismutases at its N-teminus.     

Isolation and Identification of Streptomyces fradiae SU-1 from Thailand and Protoplast Transformation with the Chitinase B Gene from Nocardiopsis prasina OPC-131

Thirty-two strains of actinomycetes obtained from soil samples of Thailand were selected. Actinomycete strain SU-1 is the most effective in terms of antagonism of Fusarium moniliforme. It produces antifungal substances on agar medium against F. moniliforme. On the basis of microscopical observations of its morphology and biochemical tests as well as analysis of cell wall and fatty acid pattern, this strain was identified as Streptomyces fradiae. The chitinase gene B (chiB337) from Nocardiopsis prasina OPC-131 was inserted into an integrating plasmid pFIS318, an Escherichia coli–Streptomyces shuttle vector. The new plasmid pFIS319-1 carrying the chitinase gene was used to transform protoplasts of S. fradiae strain SU-1. The obtained recombinant strain SU-1 pFIS319-1 exhibited higher chitinase activity than the wild-type in chitinase induction medium. Chitinase activity after renaturing protein from SDS-PAGE was detected rapidly by using 4-methylumbelliferyl β-D-N,N′’-diacetylchitobioside as the substrate. S. fradiae SU-1 secreted two chitinases with estimated molecular masses of 26 kDa and 43 kDa whereas the recombinant strain secreted three chitinases of about 26 kDa, 31.5 kDa (ChiB), and 43 kDa. The supernatant of the recombinant strain grown in chitinase induction medium inhibited the hyphal extension of F. moniliforme.     

Einfluß von Tricyclazol auf die Entwicklung und Melaningehalte der Sklerotien von Botrytis cinerea

Effect of tricyclazole on production and melanin contents of sclerotia of Botrytis cinerea Tricyclazole retarded production of sclerotia of Botrytis cinerea on agar medium more severely than mycelium growth. At a concentration range (50–200 mg/l) that did not control Botrytis on grape leaves, sclerotia production was significantly reduced. There was a negative relation between the bleaching duration of sclerotia and the tricyclazole concentrations in the medium on which they were formed. Light microscopical studies showed that sclerotia from tricyclazole-containing medium contained a significantly poorer developed outer melanin layer than that from the control medium. Ultrastructural studies with 5 days old untreated sclerotia revealed intense electron impermeable deposits in the intercellular spaces and a small electron dense layer in the outer cell walls, on the other hand treated sclerotia of the same age showed only sporadic small pigmented deposits between the cells and the pigmentation of the outer cell wall was absent.     

Ultrastructural and Cytochemical Studies on Cellulose, Xylan and Pectin Degradation in Wheat Spikes Infected by Fusarium culmorum

The cell wall components cellulose, xylan and pectin in different tissues of noninoculated healthy and Fusarium culmorum (W. G. Smith) Sacc-infected wheat spikes were localized by means of enzyme-gold and immuno-gold labelling techniques. The cell walls in the ovary, lemma and rachis of the healthy wheat spike showed labellings in different patterns and densities with cellulase-gold and xylanase-gold probes, as well as with the antipectin monoclonal antibody JIM7. The inter- and intracellular growth of the pathogen in the ovary, lemma and rachis of the infected wheat spike, not only caused pronounced alterations of cell walls and middle lamella matrices, but also led to marked modifications of cell wall components. The enzyme-gold and immuno-gold labellings in the infected host tissues revealed that the labelling densities for cellulose, xylan and pectin were significantly reduced in the cell walls of infected ovary, lemma and rachis as compared with corresponding healthy host tissues. The host cell walls in contact with or close to hyphae of the pathogen showed more marked morphological changes and much greater reduction of the labelling density than those in distance from the hyphae. These results provide evidence that F. culmorum may produce cell-wall-degrading enzymes such as cellulases, xylanases and pectinases during infection and colonization of wheat spikes tissues. Furthermore, at the early stage of infection (e.g. 3 days after inoculation), the degradation of pectin was greater than that of cellulose and xylan in the cell walls of the same infected host tissues, indirectly suggesting that the pectinases may be secreted earlier or exert higher activities than cellulases and xylanases.     

Micro-bioreactors for fed-batch fermentations with integrated online monitoring and microfluidic devices

In this study an array of micro-bioreactors based on the format of 48-well microtiter plates (MTP) is presented. The process parameters pH-value and biomass are monitored online by a combination of different sensors, the biolector measurement technology and conductance measurements. A microfluidic device dispenses two fluids individually into each well for controlling the pH-value of fermentations. The micro-bioreactor consists of four wells and two reservoirs. In each well a polyimide foil with platinum electrodes for conductance measurements is integrated. The microfluidic device is fabricated using softlithographic techniques and utilizes pneumatically actuated microvalves. The device is able to dispense volumes below 5nl. Finally, fermentations of Escherichia coli are carried out in the micro-bioreactor system. During the fermentation, the pH-value is measured optically and the biomass development is monitored by the scattered light signal. Meanwhile, the pH-value is controlled by dispensing sodium hydroxide and phosphoric acid. This micro-bioreactor demonstrates the possibility of online monitored and pH-controlled fermentations in micro-scale. The pH-value in the uncontrolled culture varies within the range of 6.46-8.83 whereas the pH-value in the controlled cultures can be kept within 6.85-7.07. This results in an increase in biomass in the pH-controlled culture compared to the nearly completely inhibited pH-uncontrolled culture.     

Inhibition of Rust Diseases of Cereals by Metabolic Products of Verticillium chlamydosporium

Verticillium chlamydosporium produced in submers culture several antifungal and/or phytotoxic compounds which were detected in a bioassay by using the pathogen-host system Puccinia coronata and oat seedlings. The antifungal compounds were also tested against P. recondita on wheat and P. sorghi on corn seedlings. The production of the active metabolic compounds highly depended on the nutrient solution (peptone-Czapek [PC] and malt extract [ME]) and on the fermentation times. Cell-free filtrates of PC-cultures of the fungus were highly phytotoxic; the fungitoxic and phytotoxic compounds were heat-labile and dialyzable. The ethyl acetate extracts of the PC-culture filtrates contained only the antifungal active substances. The antifungal compounds in ME-culture filtrates proved to be heat-stable, could be dialyzed and extracted with ethyl acetate. Ethyl acetate extracts of PC- and ME-culture filtrates at concentrations of 500 μg/ml reduced rust disease incidence by up to 80 % compared to the control treatment. Further studies with extracts of ME-culture filtrates displayed a distinct protective but no systemic activity. The extract interfered with the development of several infection structures of the rust fungi, mostly with the growth of germ tubes as well as with the formation of the aappressoria and haustorial mother cells. Three rust-active fractions were obtained by preparative layer chromatography on silica gel. One of these fractions exhibited phytotoxic activity. The most active antifungal fraction is identical with the macrolid antibiotic monorden which caused a desorientated spiral growth in P. coronata germlings on oat leaves.     

The behaviour of calves under four levels of lighting

The object of the present paper was to examine the influence of light intensity on the behaviour of calves. For this purpose, 20 calves of both sexes and of the breeds German Friesian, Simmental and their cross-breeds were kept in groups of 5 animals at 4 different levels of lighting: 2, 20, 100 or 130 lx. The behaviour of the animals was observed once a week by video. For each behaviour pattern and each light intensity, duration, frequency and daily rhythm were calculated. Furthermore, the behaviour sequences were examined.At 2 lx, the lowest level of lighting, the longest duration and most frequent phases of resting behaviour were noted. The daily rhythm of resting behaviour was more distinct in the better-lighted stable than in that with less lighting.Differences also occurred in feeding behaviour, which lasted longer at the higher light intensity.The influence of the light was clearest on social behaviour, and mainly on playing behaviour. The duration and frequency of play-fighting and solitary play-running increased with higher light intensity.Licking of objects was also significantly influenced by the level of lighting. It lasted longest at 130 lx and was shortest at 2 lx.Examination of the behaviour sequences showed that grooming and licking of objects were present in the majority of sequences. Apart from their functions, these activities are also of importance as displacement or redirected activities.The differences in the behaviour due to different lighting could be the result of worse visual ability at the lower light intensities.