AXENIC CULTURE STUDIES OF A NEW MARINE PHYCOMYCETE POSSESSING AN UNUSUAL TYPE OF ASEXUAL REPRODUCTION

Investigations of a nonfilamentous fungus (Schizochytrium aggregatum gen. et sp. nov.) reveal that it proliferates zoosporangia by successive bipartitioning of the thallus. Thus, its vegetative reproduction differs from that of other fungi, but resembles that of some possibly primitive chlorosphaeracean algae. Organism development is described from growth on natural substrates and synthetic media. Inoculation experiments involving diverse algae and phanerogam materials suggest that S. aggregatum is largely a saprophyte of vascular plant detritus in the littoral zone—a conclusion consonant with the results of a survey of its apparent distribution in coastal waters. This obligately marine organism requires 2.5-3.0% NaCl for optimal growth and does not develop in media which differ radically from sea water in potassium content. Neither KCl nor CaCl₂ can replace NaCl, and increased levels of KC1 do not mitigate the inhibitory effects of high concentrations of NaCl. The fungus grows well at 12–25 C and gives maximal yields when provided with cellobiose or the cleavage products of starch as carbon sources.     

Identification and characterization of rhizosphere fungal strain MF‐91 antagonistic to rice blast and sheath blight pathogens

Aim

To examine the inhibition effects of rhizosphere fungal strain MF‐91 on the rice blast pathogen Magnaporthe grisea and sheath blight pathogen Rhizoctonia solani.

Methods and Results

Rhizosphere fungal strain MF‐91 and its metabolites suppressed the in vitro mycelial growth of R. solani. The inhibitory effect of the metabolites was affected by incubation temperature, lighting time, initial pH and incubation time of rhizosphere fungal strain MF‐91. The in vitro mycelial growth of M. grisea was insignificantly inhibited by rhizosphere fungal strain MF‐91 and its metabolites. The metabolites of rhizosphere fungal strain MF‐91 significantly inhibited the conidial germination and appressorium formation of M. grisea. Moreover, the metabolites reduced the disease index of rice sheath blight by 35·02% in a greenhouse and 57·81% in a field as well as reduced the disease index of rice blast by 66·07% in a field. Rhizosphere fungal strain MF‐91 was identified as Chaetomium aureum based on the morphological observation, the analysis of 18S ribosomal DNA internal transcribed spacer sequence and its physiological characteristics, such as the optimal medium, temperature and initial pH for mycelial growth and sporulation production.

Conclusions

Rhizosphere fungus C. aureum is effective in the biocontrolling of rice blast pathogen M. grisea and sheath blight pathogen R. solani both in in vitro and in vivo conditions.

Significance and Impact of the Study

This study is the first to show that rhizosphere fungus C. aureum is a potential fungicide against rice blast and sheath blight pathogens.     

Rhododendron aureum Georgi formed a special soil microbial community and competed with above‐ground plants on the tundra of the Changbai Mountain,China

Rhododendron aureum Georgi is a perennial evergreen dwarf shrub that grows at all elevations within the alpine tundra of northern China. Previous research has investigated the plant communities of R. aureum; however, little information is available regarding interspecific competition and underground soil microbial community composition. The objective of our study was to determine whether the presence of R. aureum creates a unique soil microbiome and to investigate the relationship between R. aureum and other plant species. Our study site ranged from 1,800 to 2,600 m above sea level on the northern slope of the Changbai Mountain. The results show that the soil from sites with an R. aureum community had a higher abundance of nitrogen‐fixing bacteria and a higher resistance to pathogens than soils from sites without R. aureum. We emphasize that R. aureum promotes a unique soil microbial community structure that is distinct from those associated with other plants. Elevation and microbial biomass were the main influencing factors for plant community structure. Analysis of interspecific relationships reveals that R. aureum is negatively associated with most other dominant shrubs and herbs, suggesting interspecific competition. It is necessary to focus on other dominant species if protection and restoration of the R. aureum competition is to occur. In the future, more is needed to prove whether R. aureum decreases species diversity in the tundra ecosystems of Changbai Mountain.     

B12 and B1 auxotrophy of lower marine phycomycetes

Summary A study of the lower marine Phycomycetes Thraustochytrium roseum, T. aureum, and Schizochytrium aggregatum revealed their requirement for exogenous B₁₂ and B₁. T. roseum grew well when provided with only the pyrimidine moiety of the latter whereas the other fungi were dependent on the presence of the intact molecule. The response of these organisms to minute concentrations of B₁ suggests their value for assaying sea water for the vitamin.     

<Emphasis Type="Italic">Haliphthoros milfordensis</Emphasis> isolated from black tiger prawn larvae (<Emphasis Type="Italic">Penaeus monodon</Emphasis>) in Vietnam

 A marine fungus was isolated from the black tiger prawn Penaeus monodon at Nha Trang, Vietnam, on March 20, 2001 and named isolate NJM 0131. The fungus was identified as Haliphthoros milfordensis from the characteristics of asexual reproduction, and its physiological characteristics were investigated. Although the optimum temperature for growth of the isolate was 25°–30°C, the fungus grew at a wide range of temperatures (15°–40°C). H. milfordensis grew well in 50%–100% seawater, but poorly in PYG agar containing 1.0%–5.0% NaCl and KCl. The fungus grew at a wide range of pH (4.0–11.0) with the optimum pH value of 7.0–9.0. The isolate also showed pathogenicity to swimming crab larvae (Portunus trituberculatus) by artificial infection, but mortality was not high. This is the first report of disease in the black tiger prawn P. monodon in Vietnam caused by H. milfordensis. Received: July 22, 2002 / Accepted: January 21, 2003 Correspondence to:K. Hatai     

Mycelial growth of the snow mold fungus, Sclerotinia borealis, improved at low water potentials: an adaption to frozen environment

The snow mold fungus, Sclerotinia borealis, shows optimal growth at 4°C on potato dextrose agar (PDA) and can grow even at subzero temperature. Its mycelial growth was improved on frozen PDA at −1°C and on PDA containing potassium chloride (KCl) (water potential, −4.27 to −0.85 MPa) or d(−) sorbitol (−3.48 to −0.92 MPa). Its optimal growth temperature shifted from 4 to 10°C on PDA amended with KCl or sorbitol, indicating that inherent optimal growth occurs at high temperatures. These results suggest that S. borealis uses concentrated nutrients in the frozen environment and that such physiologic characteristics are critical for the fungus to prevail at subzero temperatures.     

Endogenous respiration and regulation in a primitive marine fungus

The relationship between the respiration and the presence and utilization of endogenous and exogenous substrates was studied in the non-filamentous obligately marine fungus Thraustochytrium aureum. Using isotopic and manometric methods, it was shown that almost all exogenous glucose is assimilated, whilst almost all the oxygen consumption in the presence of exogenous glucose was due to oxidation of endogenous reserves. In contrast, exogenous glutamate, which cannot serve as the sole carbon source for growth, inhibits respiration of endogenous materials, and is itself rapidly oxidized. The uncoupler 2,4-dinitrophenol stimulates the oxidation of endogenous reserves without affecting the uptake and use of exogenous glucose. These data strongly support the idea of physiologic compartmentation in this organism.     

Pea chloroplast topoisomerase I: purification,characterization, and role in replication

A DNA-relaxing enzyme was purified 5 000-fold to homogeneity from isolated chloroplasts of Pisum sativum. The enzyme consists of a single polypeptide of 112 kDa. The enzyme was able to relax negatively supercoiled DNA in the absence of ATP. It is resistant to nalidixic acid and novobiocin, and causes a unit change in the linkage number of supercoiled DNA. The enzyme shows optimum activity at 37°C with 50 mM KCl and 10 mM MgCl₂. From these properties, the enzyme can be classified as a prokaryotic type I topoisomerase.Using a partiall purified pea chloroplast DNA polymerase fraction devoid of topoisomerase I activity for in vitro replication on clones containing the pea chloroplast DNA origins of replication, a 2–6-fold stimulation of replication activity was obtained when the purified topoisomerase I was added to the reaction at 70–100 mM KCl. However, when the same reaction was carried out at 125 mM KCl, which does not affect DNA polymerase activity on calf thymus DNA but is completely inhibitory for topoisomerase I activity, a 4-fold drop in activity resulted. Novobiocin, an inhibitor of topoisomerase II, was not found to inhibit the in vitro replication of chloroplast DNA.     

不同生长光强下8种红树植物幼苗叶片的光响应特征

为探讨红树植物光适应的生理生态策略,对6种真红树植物[无瓣海桑(Sonneratia apetala)、秋茄(Kandelia candel)、木榄(Bruguiera gymnorrhiza)、桐花树(Aegiceras corniculatum)、老鼠簕(Acanthus ilicifolius)、卤蕨(Acrostichum aureum)]和2种半红树植物[银叶树(Heritiera littoralis)、黄槿(Hibiscus tiliaceus)]的1 a生幼苗在不同生长光强(自然光强的100%、45%、30%、10%)下的光合光响应特征进行了研究。结果表明,不同生长光强对红树植物光响应特征的影响因物种而异,遮荫显著提高了秋茄和木榄的最大净光合速率(Pmax),而对其他红树植物的Pmax没有显著影响;秋茄在45%光强下具有较高的Pmax,木榄的Pmax则在45%和30%光强下显著高于其他2个处理。随着生长光强的下降,秋茄幼苗叶片的光饱和点显著上升,木榄、老鼠簕和卤蕨的光补偿点呈下降趋势,木榄和卤蕨的表观量子效率升高的同时暗呼吸速率下降。木榄、老鼠簕和卤蕨具有较强的耐荫性,...     

Evaluation of environmental factors affecting the genetic diversity,genetic structure,and the potential distribution of Rhododendron aureum Georgi under changing climate

Understanding genetic variation and structure, adaptive genetic variation, and its relationship with environmental factors is of great significance to understand how plants adapt to climate change and design effective conservation and management strategies. The objective of this study was to (I) investigate the genetic diversity and structure by AFLP markers in 36 populations of R. aureum from northeast China, (Ⅱ) reveal the relative contribution of geographical and environmental impacts on the distribution and genetic differentiation of R. aureum, (Ⅲ) identify outlier loci under selection and evaluate the association between outlier loci and environmental factors, and (Ⅳ) exactly calculate the development trend of population of R. aureum, as it is confronted with severe climate change and to provide information for designing effective conservation and management strategies. We found high genetic variation (I = 0.584) and differentiation among populations (Φ_ST  = 0.703) and moderate levels of genetic diversity within populations of R. aureum. A significant relationship between genetic distance and environmental distance was identified, which suggested that the differentiation of different populations was caused by environmental factors. Using BayeScan and Dfdist, 42 outlier loci are identified and most of the outlier loci are associated with climate or relief factors, suggesting that these loci are linked to genes that are involved in the adaptability of R. aureum to the environment. Species distribution models (SDMs) showed that climate warming will cause a significant reduction in suitable areas for R. aureum, especially under the RCP 85 scenario. Our results help to understand the potential response of R. aureum to climatic changes and provide new perspectives for R. aureum resource management and conservation strategies.     

Proton-Translocating Inorganic Pyrophosphatase in Red Beet (Beta vulgaris L.) Tonoplast Vesicles

The substrate and ionic requirements of ATP and inorganic pyrophosphate (PPi) hydrolysis by tonoplast vesicles isolated from storage tissue of red beet (Beta vulgaris L.) were compared with the requirements of ATP-and PPi-dependent proton translocation by the same material. Both ATP hydrolysis and ATP-dependent proton translocation are most stimulated by Cl⁻ and inhibited by NO₃⁻. NaCl and KCl support similar rates of ATP hydrolysis and ATP-dependent proton translocation while K₂SO₄ supports lesser rates for both. PPi hydrolysis and PPi-dependent proton translocation are most stimulated by K⁺. KCl and K₂SO₄ support similar rates of PPi hydrolysis and PPi-dependent proton translocation but NaCl has only a small stimulatory effect on both. Since PPi does not inhibit ATP hydrolysis and ATP does not interfere with PPi hydrolysis, it is inferred that the two phosphohydrolase and proton translocation activities are mediated by different tonoplast-associated enzymes. The results indicate the presence of an energy-conserving proton-translocating pyrophosphatase in the tonoplast of red beet.     

Three species of Lagenidiales isolated from the eggs and zoeae of the marine crabPortunus pelagicus

Three species of Lagenidiales were isolated from the eggs and zoeae of the marine crabPortunus pelagicus. One of them,Lagenidium callinectes, is reported for the first time in Japan, with fungal infection in the eggs.Haliphthoros milfordensis was discovered from a zoea of the crab.Atkinsiella okinawaensis was also isolated from a zoea of the crab and described as a new species. Growth temperature range and optimum temperature of the fungi were examined. All of the isolates grew at various concentrations of NaCl or KCl, and optimum growth was observed on PYGS agar containing seawater. Pathogenicity to zoeae of the swimming crabPortunus trituberculatus was demonstrated by using a zoospore suspension of each fungus at 25°C.     

Is KCl a reliable extractant of15NH 4 + added to coastal marine sediments?

The release of NH ₄ ⁺ and¹⁵N-labelled NH ₄ ⁺ by one-step KCl extraction was assessed in different types of coastal marine sediments. KCl was efficient to extract NH ₄ ⁺ from sandy sediments and less efficient in silt sediments, where an extended extraction period was required for obtaining a maximum NH ₄ ⁺ yield. Extraction at 0 or 20 °C had only a little effect on the efficiency of KCl. KCl gave always non complete recovery of¹⁵NH ₄ ⁺ in silt sediments. However, the added label could be fully recovered by addition of 80 mol·cm^–3 exogenous NH ₄ ⁺ prior to KCl, or when NaCl or ASW replaced KCl.¹⁵NH ₄ ⁺ was added to non-biological silt sediment, which was incubated at 0 °C up to 16 hours, to see the effect of physical processes on the partition of¹⁵NH₄ among porewater (29–49%) exchangeable (9–30%) and non-extractable, organic bound pools (24–42%). Total¹⁵N recovery was approximately 100%. KCl failed to remove¹⁵NH₄ which entered to unknown, bound pools in sediment. Only shortly after addition of¹⁵N (0.1 h), the extraction period resulted in significantly different¹⁵N recoveries (P < 0.05) in KCl extractable NH ₄ ⁺ , 17% versus 9% of label was recovered after 1 min or 60 min extraction of sediment, respectively. Two hours of incubation time were required for complete equilibrium of¹⁵NH ₄ ⁺ among porewater, exchangeable and organic bound pools. Sediments (silt) to which¹⁵NH ₄ ⁺ has been added in order to measure NH ₄ ⁺ turn-over and KCl is used as extractant, should be incubated for at least 2 hours, before taking a zero-time sample.     

Penicillium roqueforti conidia induced by L-amino acids can germinate without detectable swelling

Penicillium roqueforti is used for the production of blue-veined cheeses but is a spoilage fungus as well. It reproduces asexually by forming conidia. Germination of these spores can start the spoilage process of food. Germination is typically characterized by the processes of activation, swelling and germ tube formation. Here, we studied nutrient requirements for germination of P. roqueforti conidia. To this end,?>?300 conidia per condition were monitored in time using an oCelloScope imager and an asymmetric model was used to describe the germination process. Spores were incubated for 72 h in NaNO₃, Na₂HPO₄/NaH₂PO₄, MgSO₄ and KCl with 10 mM glucose or 10 mM of 1 out of the 20 proteogenic amino acids. In the case of glucose, the maximum number of spores (P_max) that had formed germ tubes was 12.7%, while time needed to reach 0.5 P_max (τ) was about 14 h. Arginine and alanine were the most inducing amino acids with a P_max of germ tube formation of 21% and 13%, respectively, and a τ of up to 33.5 h. Contrary to the typical stages of germination of fungal conidia, data show that P. roqueforti conidia can start forming germ tubes without a detectable swelling stage.

     

Precession-forced changes in South West African vegetation during Marine Isotope Stages 101–100 (2.56–2.51 Ma)

The intensification of Northern Hemisphere Glaciation (iNHG) is one of the critical climate thresholds in the Cenozoic. This study focuses on marine sediments recovered from Marine Isotope Stages 101/100 at the Ocean Drilling Program Site 1083 to assesses the impact of the iNHG on continental southern African vegetation through n-alkane (straight-chain hydrocarbon) abundance and δ¹³C values. The n-alkane abundance data yield a convoluted signal due to the number of controlling factors such as the source area, transportation routes and vegetation type. The C₃₁ n-alkane δ¹³C values, however, exhibit a cyclic pattern with a periodicity of c. 20 ka, and are not correlated to the abundance data. It is inferred that the signal does not represent a change in the geographical source of n-alkanes. Instead, we suggest that the variations are caused by water-stress-induced changes in either carbon isotope fractionation during C₃ photosynthesis or subtle changes in the proportion of C₃ and C₄ plants. These changes, unlike variations in oceanographic proxies, closely track precessional forcing factors and are independent of the prevailing obliquity-forced glacial/interglacial cycles. We conclude that the varying monsoon strength, rather than pCO₂ or temperature change, forced changes in southern African vegetation during this period.     

Fluorescein-Conjugated Folate as an Indicator of Specific Folate Binding to Paramecium1

Normal Paramecium tetraurelia cells stained with fluorescein-conjugated folate show intense fluorescence that can be reduced to near background autofluorescence with excess K₂-folate, but not with excess KCl. Mutant d4–534, which is not attracted to folate and does not specifically bind ³H-folate, shows reduced fluorescence when stained. This method of monitoring specific folate binding to cells can be adapted to a microscale for rapid screening of clones since cells are routinely fixed and stained in microtiter wells.     

Nutritional study on <Emphasis Type="Italic">Embellisia astragali</Emphasis>, a fungal pathogen of milk vetch (<Emphasis Type="Italic">Astragalus adsurgens</Emphasis>)

Embellisia astragali is a strong, virulent pathogen that develops within milk vetch (Astragalus adsurgens). In order to determine nutrient requirements, the fungus was cultured on 9 carbon sources, 9 nitrogen sources, and 13 growth media in the dark at 25°C. Growth rates and sporulation capacity were measured after 4 and 12 weeks. All carbon sources supported growth, but only soluble starch, inulin, and dextrose supported sporulation. In general, better growth was obtained on disaccharides and polysaccharides than on monosaccharides. Compared with no growth on NH₄ ⁺-N and urea, the fungus grew little on all NO₃ ⁻-N, amino-N, and other organic-N such as peptone. There was no sporulation or very sparse conidia on almost all nitrogen sources with supplied dextrose or soluble starch as sole carbon source. The better growth and sporulation on most of the semidefined media than on defined media indicates that some components in plant or animal material may be vital to the fungus. Sporulation was positively correlated with growth rate in N source experiment at 12 weeks and in growth media experiment at 4 and 12 weeks. The fungus favors grow within agar with growth rate less than 1.18 mm day⁻¹.     

MICRURGICAL STUDIES IN CELL PHYSIOLOGY : V. THE ANTAGONISM OF CATIONS IN THEIR ACTIONS ON THE PROTOPLASM OF AM?BA DUBIA.

I. The Plasmalemma. 1. On the plasmalemma of amebæ CaCl₂ antagonizes the toxic action of LiCl better than it does NaCl, and still better than it does KCl. MgCl₂ antagonizes the toxic action of NaCl better than it does LiCl and still better than it does KCl. 2. CaCl₂ antagonizes the toxic action of LiCl and of KCl better than does MgCl₂: MgCl₂ antagonizes NaCl better than does CaCl₂. II. The Internal Protoplasm. 3. The antagonizing efficiency of CaCl₂ and of MgCl₂ are highest against the toxic action of KCl on the internal protoplasm, less against that of NaCl, and least against that of LiCl. 4. CaCl₂ antagonizes the toxic action of LiCl better than does MgCl₂: MgCl₂ antagonizes the toxic action of NaCl and of KCl better than does CaCl₂. 5. LiCl antagonizes the toxic action of MgCl₂ on the internal protoplasm more effectively than do NaCl or KCl, which have an equal antagonizing effect on the MgCl₂ action. III. The Nature of Antagonism. 6. When the concentration of an antagonizing salt is increased to a toxic value, it acts synergistically with a toxic salt. 7. No case was found in which a potentially antagonistic salt abolishes the toxic action of a salt unless it is present at the site (surface or interior) of toxic action. 8. Antagonistic actions of the salts used in these experiments are of differing effectiveness on the internal protoplasm and on the surface membrane.     

Increased biooxidation of lignosulfonates by Sporotrichum pulverulentum in the presence of polyacrylic acid

Summary The superiority of polyacrylic acid used as a buffer at 0.25% (w/v) during biodegradation of high molecular weight ¹⁴C-labeled lignosulfonates (LS) by the white rot fungus Sporotrichum pulverulentum is demonstrated. Compared to 2,2-dimethylsuccinate (DMS) the release of ¹⁴CO₂ from the LS occurs earlier, does not show the levelling-off symptom and reaches higher levels. Changes in pH values of the medium cannot be correlated with the stimulating effects of polyacrylic acid on the ligninolytic activity of the fungus. It seems that interaction between LS and also of dehydropolymers of coniferyl alcohol (DHP) with the polymeric buffer increases the accessibility to the fungus.     

Identification of sulphate-reducing ectosymbiotic bacteria from anaerobic ciliates using 16S rRNA binding oligonucleotide probes

The identity of ectosymbiotic bacteria of some marine, free-living anacrobic ciliates (Metopus contortus, Caenomorpha levanderi and Parablepharisma sp.) was studied using fluorescent-dye-conjugated oligonucleotides complementary to short sequence elements of 16S ribosomal RNA. The ectosymbiotic bacteria of all species hybridized with a eubacterial probe and those of the two former mentioned species hybridized with a general probe for sulphate-reducing bacteria, but not to a probe specific for Desulfobacter. The results support indirect evidence suggesting that ectosymbiotic bacteria of anaerobic ciliates are sulphate-reducers which depend on host metabolites for substrates.Abbreviations DMSO dimethyl sulfoxide - PBS phosphate-buffered saline, 137 mM NaCl, 2.7 mM KCl, 4.3 mM Na₂HPO₄, 1.4 mM KH₂PO₄, pH: 7.3 - TEAA triethylamonium acetate - 1 x SSC standard sodium citrate buffer, 150 mM NaCl, 15 mM Na₂ citrate, pH: 7.0 - 1 x Denh Denhardts solution, 0.02% ficoll, 0.02% bovine serum albumine, 0.02% polyvinol-pyralidone