An optimum environment for the culturing of cytospora isolates from stone fruits. I. Temperature

Summary Four isolates ofCytospora cincta Fr. and 2 ofC. leucostoma Fr. were obtained from diseased Italian prune, President plum and Bing cherry trees.The minimum temperature for growth of these fungi was found to be 3° C. Temperatures of 45 °C. were lethal to all cultures. The optimum temperature for theC. cincta isolates on solid and liquid media was found to be 30° C.; for theC. leucostoma isolates, nearly 25° C. OneC. cincta isolate produced greatest radial growth on the solid medium at 35° C., but in the liquid medium produced maximum mycelium at 30° C.All factors considered, the conclusion was reached that the best single temperature for laboratory culture of the fungi was 30° C.Approved by the Director of the Idaho Agricultural Experiment Station as Research Paper No. 493.     

Immobilized, thermostable S- and F-forms of the extracellular invertase from Candida utilis can hydrolyse sucrose up to 100 °C

When grown on a sucrose-containing medium, Candida utilis synthesizes and secretes two invertases: one of molecular size of 280 kDa (the S-form – Slow-migrating) and a new form of Mr of 62 kDa (the F-form – Fast-migrating). Prior to immobilization, purification of S- and F-forms of invertase increased the immobilization yield to 89–100%, in comparison with that of crude invertase preparation (52%). The immobilized purified S- and F-form of invertase remained partially active after 15 min at 100 °C; the F-form retained almost 30% of its maximum activity. The immobilized S-form or F-form of invertase almost completely inverted (95% hydrolysis) 60% (w/v) sucrose over 5 h continuous reaction at 80 °C. Moreover, at 90 °C the immobilized F-form hydrolysed 70% of 60% (w/v) sucrose over 5 h, while the capability of the immobilized S-form of inverting sucrose over 5 h reaction decreased from 80% to 45%.     

Studies on Aspergilli XVI. Effect of pH,temperature, and carbon and nitrogen interaction

Summary The effect of pH, temperature, and carbon and nitrogen interaction on the growth and sporulation ofAspergillus nidulans (Eidam)Wint.,A. rugulosus Thom &Raper,A. variecolor (Berk. &Br.)Thom &Raper andA. quadrilineatus was studied. All the moulds could grow on a wide range of pH (2.0 to 12.0) but the growth was poor on too acid and too alkaline media. Best growth ofA. rugulosus, A. quadrilineatus, andA. violaceus was seen at pH 6.5 and that ofA. nidulans andA. variecolor at pH 7.0. In general maximum production of perithecia was recorded between pH 6.0 and 8.0.All the above species ofAspergillus under study could grow between a temperature range of 10° C–48° C, but the growth was poor at 10° C and 48° C. The present moulds showed good growth at 20° C, 25°C, and 30° C. At 40° CA. nidulans andA. rugulosus showed moderate growth while the rest of the Aspergilli attained good growth. Temperatures between 20° C–30° C favoured excellent perithecial production.In general, little improvement in growth was noted on media containing good carbon and nitrogen sources. Malic acid was found to be useless when supplied singly. But, poor growth was recorded when supplied in combination with amino acids, amide, and peptone. This was due to the fact that these N sources also supplied carbon for their metabolism.     

Multiple temperature optima in collectotrichum graminicola

Summary The dry weights of three isolates ofColletotrichum graminicola (Ces.)Wills., growing at 10°, 15°, 20°, 30°, and 35° C in yeast extract liquid medium were recorded. Two temperature growth optima and minima occurred at 20°C, 30°C and 10°C, 25°C respectively.Portion of a Ph. D. thesis, The Ohio State University, Columbus 10, Ohio, U.S.A. Department of Botany and Plant Pathology. Paper Number 657.     

Evaluation of alternative nitrogen and carbon sources for sugarbeet suspension culture platings in development of cell selection schemes

Summary Low molecular weight nitrogenous impurity compounds as well as raffinose are negative quality factors that interfere with efficient processing of sugarbeet (Beta vulgaris L.) for sucrose. In order to identify nutrient media for cell selection of biochemical mutants or transgenics that might have reduced levels of these processing impurities, the ability of 10 endogenous compounds to serve as sole nitrogen or carbon source for suspension plating and subculture callus growth was evaluated. The most productive concentrations of nitrate, ammonium, l-glutamine, l-glutamate, urea, and l-proline as sole nitrogen sources supported plating callus growth at 106, 159, 233, 167, 80, and 52%, respectively, as well as the historical 60 mM mix of nitrate and ammonium in Murashige-Skoog medium. Glycine betaine and choline did not support growth. d(+) Raffinose and d(+) galactose supported plating callus growth only 67 and 25%, respectively, as well as sucrose as sole carbohydrate source. No callus growth occurred on glutamine, glutamate, or glycine betaine as the sole carbon or carbon plus nitrogen source. Platings on either nitrate or ammonium as sole nitrogen source did not differ in sensitivity to the nitrate uptake inhibitor phenylglyoxal, suggesting that phenylglyoxal lacks the specificity for use in selection for mutants of nitrate uptake. The ability of raffinose to be used as the carbon source, and glutamine or glutamate as the nitrogen source, may preclude their use for selection of genetic variants accumulating less of these processing impurities. However, mutants or transgenics able to utilize either glutamine, glutamate, or glycine betaine might be selectable on media containing any one of these as carbon, nitrogen, or carbon plus nitrogen source, respectively, that is incapable of supporting wild-type cell growth.     

Purification and properties of an intracellular leucine aminopeptidase from the fungus,Penicillium citrinum strain IFO 6352

An intracellular leucine aminopeptidase (LAP) fromPenicillium citrinum (IFO 6352) was purified to homogeneity using three successive purification steps. The enzyme has a native molecular mass of 63 kDa using HPLC gel filtration analysis and a molecular mass of 65 kDa when using SDS-polyacrylamide gel electrophoresis. This monomeric aminopeptidase showed maximum enzyme activity at pH 8.5. An optimum temperature was 45–50°C whenl-Leu-p-nitroanilide (pNA) was the substrate, and enzyme activity drastically decreased above 60°C. The Michaelis-Menten constants forl-Leu-pNA andl-Met-pNA were 2.7 mM and 1.8 mM, respectively. When the enzyme reacted with biosynthetic methionyl human growth hormone, it showed high specificity for N-terminal methionine residue and recognized a stop sequence (Xaa-Pro). The aminopeptidase was inactivated by EDTA or 1,10-phenanthroline, indicating that it is a metallo-exoprotease. Enzyme activity was restored to 90% of maximal activity by addition of Co²⁺ ions. The activity of EDTA-treated enzyme was restored by addition of Zn²⁺, but reconstitution with Ca²⁺, Mg²⁺ or Mn²⁺ restored some enzyme activity. It is likely that Co²⁺ ions play an important role in the catalysis or stability of thePenicillium citrinum aminopeptidase, as zinc plays a similar function in other leucine aminopeptidases.     

Influence of temperature upon growth and sporulation in two species of Phytophthora

The paper deals with the influence of temperature on the growth and sporulation of two species ofPhytophthora, viz.,P. palmivora Butl. andP. parasitica Dast. var.macrospora Ashby, the causal agents of fruit rots ofAchras sapota L. andAnona squamosa L. respectively. Germination of sporangia at different temperatures were also undertaken. There was marked variation in growth and sporulation of these two organisms. Isolate C (Phytophthora palmivora) showed no growth at 5° and 35°C, scanty growth at 10° and 32.5° with an optimum temperature between 26–28°C. On the other hand, Isolate S (Phytophthora parasitica var.macroscora) showed no growth at 10°C, but slight growth even at 37°C. Eight days exposure at 37°C completely stopped the growth of this Isolate. It showed best growth at 30°C and hence this was its optimum temperature. In general, Isolate C sporulated abundantly at all temperatures tested but reached its maximum at 25°C. On the other hand Isolate S showed best growth but failed to sporulate at any of the temperatures in 98 hours growth, although it sporulated freely when the incubation period extended up to two weeks. On the basis of temperature toleration the twoPhytophthora isolates are distinguished from each other as two different species. This confirms the earlier observations and nomenclature criterion as emphasized and formulated byTucker (1931). In the germination studies, it was observed that the indirect germination with the formation of abundant zoospores started from 5° and continued even up to 35°C, reaching maximum at 20°C. High temperature was not favourable for indirect germination. As the temperature proceeded increasing, the percentage of direct germination by formation of germ tubes also increased. Direct germination was observed from 10° which continued up to 37°C, with a maximum reach at 30°C. This confirms the epidemic of fruit rots in nature during monsoon season which is prevalent with the persistence of high humidity and rainfall.Taken from a thesis submitted by the author for the degree of Master of Science in the Faculty of Agriculture, Poona University, India.     

Intracellular serine proteinase behaves as a heat-stress protein in nongrowing but as a cold-stress protein in growing populations of Bacillus megaterium

A temperature increase from 35° to 40–42°C enhances the rise of cytoplasmic serine proteinase (ISP1) activity in Bacillus megaterium incubated in a sporulation medium. A temperature shift from 27°C in the growth medium to 35°C in the sporulation medium has the same effect. Elevated temperature stimulates the increase of ISP1 level when applied immediately after the transfer of cells from the growth to the sporulation medium (at T₀) or at T₃, when sporulation becomes irreversible. The cytoplasmic PMSF-resistant activity or the proteolytic activity associated with the membrane fraction is stimulated only slightly or not at all. A temperature increase to 45–47°C suppresses the rise of proteolytic activities in all cell fractions. In addition to the elevation of the ISP1 activity by an upward temperature shift, the rise of this enzyme in nongrowing cells is also stimulated by osmotic stress. In growing populations, in contrast to the rise of the ISP1 activity caused by elevated temperature in nongrowing cells, this proteinase is induced by low temperatures (24–27°C). The ISP1 activity roughly correlates with the enzyme protein concentration determined by immunoblotting.     

Production of Penicillin G acylase from Bacillus sp.: Effect of medium components

A Bacillus sp. producing a high level of intracellular penicillin G acylase (PAC) was isolated. The PAC production in this strain was induced by phenylacetic acid. Various carbon and nitrogen sources were evaluated for their effect on growth and PAC production at 28 °C and pH 7.0. Cells grown in medium supplemented with sucrose as carbon source and tryptone as nitrogen source produced maximum activity of 6.45 and 8.92 U mg^–1, respectively. Maximum concentration of PAC (10.1 Umg^–1) was produced by the cells grown in the medium containing sucrose and tryptone, which was twofold higher than the production in basal medium.     

Arabitol accumulation in Geotrichum candidum

Culture conditions which lead to the intracellular accumulation of arabitol and mannitol in Geotrichum candidum were investigated. The accumulation of arabitol was dependent on the concentrations of metabolizable hexoses, the non-metabolizable disaccharide sucrose, NaCl and KCl in the growth medium. In media containing 2% (w/v) glucose, fructose or l-sorbose cultures contained only mannitol after 48 h or 72 h growth. In media containing 10% (w/v) to 30% (w/v) glucose, or 25% (w/v) fructose or l-sorbose there was an increase in the total concentration of intracellular polyol due to the accumulation of arabitol. This pentitol was also found to accumulate intracellularly when the organism was grown in medium containing 34% (w/v) sucrose, 0.7 M NaCl or 0.7 M KCl in addition to 2% (w/v) glucose. Under the conditions tested no change in the accumulation of mannitol or ethanol-soluble carbohydrate, believed to be primarily composed of trehalose, was evident.Intracellular polyol was released during incubation of arthrospores obtained from media containing 25% or 10% glucose, in distilled water at 25° C, but no polyol was released under these conditions from arthrospores obtained from growth in 2% glucose medium.     

Studies on the growth of a thermotolerant yeast strain,Kluyveromyces marxianus IMB3, on sucrose containing media

A thermotolerant alcohol-producing yeast strain, Kluyveromyces marxianus IMB3 was shown to grow on sucrose (10% [w/v]) containing media at 45 °C. Under such conditions the organism reached stationary phase within 20 hours and yielded ethanol concentrations in the region of 33g/L. During growth on sucrose containing media the organism was found to produce a cell- associated activity capable of hydrolysing sucrose. This activity was shown to have a Km of 5.0mM when sucrose was used as the substrate. In addition the enzyme was shown to have a pH optimum of 5.0 and a temperature optimum of 50–55 °C and under those conditions the enzyme was shown to be relatively thermostable.     

Characterization of a Mutant from Lactobacillus amylovorus JCM 1126T with Improved Utilization of Sucrose

A strain YF43, which can grow on sucrose as rapidly as glucose, was isolated by mutation from Lactobacillus amylovorus JCM 1126, the type strain defective in sucrose utilization. Exogenous sucrose stimulated the production of invertase by strains YF43 and JCM 1126 simultaneously. In a medium containing fructooligosaccharide as the sole carbon source, the cells of strain YF43 showed high invertase activity in spite of poor growth. The two invertases produced in the cells grown on sucrose and fructooligosaccharide were an identical β-fructofuranosidase, as judged from properties of partially purified enzymes. These observations indicated that strain YF43 is a mutant improved for permeation of sucrose and not derepressed for the synthesis of invertase. Received: 23 May 2000 / Accepted: 26 June 2000     

The production of labeled betaine by incubation of osmolyte-freePseudomonas aeruginosa with radioactive choline

A simple, efficient, and economical method is presented for the preparation of radioactive betaine. It involves the incubation of radioactive choline with osmolyte-freePseudomonas aeruginosa previously grown in hyperosmolar medium with choline as an osmoprotectant. The summarized procedure was as follows: (i) bacteria were grown in high P_i basal salt medium (HP_i-BSM) with 20mm succinate, 18.7mm NH₄Cl, 0.8m NaCl, and 1mm nonradioactive choline. After the bacterial pellet was obtained, it was suspended in deionized water to release osmolytes accumulated during growth; (ii) suspension of the pellet, free of osmolytes, in hyperosmolar HP_i-BSM with [methyl-¹⁴C]-choline (55 nCi/nmol) without the carbon and nitrogen sources. Incubation of the mixture at 37°C for 8–30 h. When only 10% of the initial radioactivity remained in the supernatant, it was withdrawn after centrifugation and the pellet suspended in deionized water. This step released the accumulated betaine plus some contaminants. Purification of betaine contained in the aqueous supernatant was carried out after rotoevaporation to dryness and solubilization of the residue in methanol. The methanolic extract was rotoevaporated to dryness, the residue solubilized in 10% acetic acid and transferred to a Dowex 50-X8 column. After the column was washed with water and 2m NH₄OH, betaine was eluted by the addition of 4m NH₄OH. The total procedure for obtaining pure radioactive betaine resulted in a yield of 80%. The product obtained was chemically and radiochemically pure, with a specific radioactivity of 54±1 nCi/nmol.     

A preliminary evaluation of the use of microbial culture filtrates for the control of contaminants in plant tissue culture systems

The partitioning of carbon between reserve polysaccharide and alkaloid secondary products was investigated in batch cultures of transformed roots of Datura stramonium grown in media in which the carbon substrate concentration was held constant and the level of mineral nutrients was varied. The growth and accumulation of starch and hyoscyamine was examined in roots grown at temperatures of 20°C, 25°C or 30°C in media containing 5% sucrose and levels of mineral nutrients varying from 1/4 to twice the standard level of Gamborg's B5 salts. The dry matter content was highest (up to 15% w/w) in roots grown at either 20°C or 25°C in medium of the lowest ionic strenth (1/4 B5 salts) and decreased as the ionic strength was raised (down to 7% w/w with 2 B5 salts). Up to half of this decrease could be accounted for by loss of starch from the roots. At 20°C and 25°C, the starch content of the roots grown in medium of the lowest ionic strength (1/4 B5) was 40 mg g^-1 and 22 mg g^-1 fresh weight respectively but decreased to less than 1 mg g^-1 weight at either temperature when the ionic strength of the medium was raised to 2 B5. At 30°C, starch accumulation was severely inhibited in all media. In contrast, varying either the temperature or the ionic strength of the medium had only a small effect on hyoscyamine accumulation which remained at between 0.4–0.6 mg g^-1 fresh weight. Although increases in the level of mineral salts had little effect on the hyoscyamine content of the roots, total yields however, increased due to stimulation of growth. Time course experiments showed that cultures grown at either 20°C or 25°C continued to accumulate both starch and hyoscyamine into late stationary phase.     

Purification and characterization of a Bacillus sp. SAM1606 thermostable α-glucosidase with transglucosylation activity

We purified a novel -glucosidase to homogeneity from an Escherichia coli recombinant transformed with the -glucosidase gene from thermophilic Bacillus sp. SAM1606. The enzyme existed as mono- and multimeric forms of a promoter protein with a relative molecular weight of 64,000 and isoelectric point of 4.6. We isolated a monomeric form of the enzyme and characterized it. The enzyme was unique among the known -glucosidases in both broad substrate specificity and high thermostability. The enzyme hydrolysed a variety of O--d-glucopyranosides such as nigerose, maltose, isomaltose, sucrose, and trehalose efficiently. The molecular activity (k _O) and the Michaelis constant (K _m) values at 55°C and pH 6.0 for sucrose were 54.6 s^–1 and 5.3 mm, respectively. The optimum pH and temperature for hydrolysis were pH 5.5 and 75°C, respectively. The enzyme exhibited a high transglucosylation activity: it reacted with 1.8 m sucrose at 60°C for 70 h to yield oligosaccharides containing theanderose in a maximum yield of 35% (w/w). High thermostability of the enzyme (stable up to 65°C at pH 7.2 for 10 min) permits the transglucosylation reaction at high temperatures, which would be beneficial for continuous production of oligosaccharides from sucrose.     

Regulation of histidine biosynthetic enzymes in a mutant of Escherichia coli with an altered histidyl-tRNA synthetase

Summary A mutant of Escherichia coli was isolated that grew at a normal rate in minimal medium at 26°C, grew at a normal rate in minimal medium at 37°C only if exogenous histidine was supplied, and grew more slowly than normal at 42°C even in the presence of histidine. In very rich media the growth rate of the mutant was normal at 26°C and 30°C, but not at 37°C or 42°C. It may be described as a temperature-conditional histidine bradytroph with a decreased ceiling to its growth rate.The histidyl-tRNA synthetase of the mutant was found to be abnormal; in crude extracts the enzyme activity was less stable and had approximately a tenfold higher apparent K _Mfor histidine than normal.Under many growth conditions the histidine biosynthetic enzymes in the mutant were derepressed several hundred fold compared to the wild strain, even in the presence of exogenous genous histidine. In general, the degree of derepression in the mutant was proportional to the difference in growth rate between the mutant and normal strains; this relationship, however, did not hold below 30°C or above 37°C.The properties of the mutant could be related to the properties of its histidyl-tRNA synthetase by assuming that the enzyme participates both in protein synthesis and in histidine biosynthetic enzyme regulation and that at low temperature it functions relatively more effectively in protein synthesis than in repression, while at high temperature it functions relatively more effectively in repression.Abbreviations used tRNA transfer RNA - AICAR aminoimidazole carboxamide ribose-5-phosphate     

Immobilization studies of whole microbial cells on transition metal activated inorganic supports

Summary Whole cells of Saccharomyces bayanus, Saccharomyces cerevisiae and Zymomonas mobilis were immobilized by chelation/metal-link processes onto porous inorganic carriers. The immobilized yeast cells displayed much higher sucrose hydrolyzing activities (90–517 U/g) than the bacterial, Z. mobilis, cells (0.76–1.65 U/g). The yeast cells chelated on hydrous metal oxide derivative of pumice stone presented higher initial -d-fructofuranosidase (invertase, EC 3.2.1.26) activity (161–517 U/g) than on other derivatives (90–201 U/g). The introduction of an organic bridge between the cells and the metal activator led to a decrease of the initial activity of the immobilized cells, however S. cerevisiae cells immobilized on the carbonyl derivative of titanium (IV) activated pumice stone, by covalent linkage, displayed a very stable behaviour, which in continuous operation at 30° C show only a slightly decrease on invertase activity for a two month period (half-life=470 days). The continuous hydrolysis of a 2% w/v sucrose solution at 30° C in an immobilized S. cerevisiae packed bed reactor was described by a simple kinetic model developed by the authors (Cabral et al., 1984a), which can also be used to predict the enzyme activity of the immobilized cells from conversion degree data.     

Enhancement in activity of an invertase from the thermophilic fungus Thermomyces lanuginosus by exogenous proteins

A highly purified invertase from a thermophilic fungus Thermomyces lanuginosus showed enhanced activity when incubated with exogenous proteins. These proteins also stabilized the invertase when incubated at 50°C, 4°C and –20°C. However, none of these proteins stabilized the invertase at or above 55°C, the temperature of inactivation. This property was found to be specific for the thermophilic invertase, as no such activation was observed for the mesophilic invertases from yeasts.     

Thermostability of l-phenylalanine aminotransferase from thermophilic bacteria

Summary Various mesophilic and thermophilic bacteria were screened for the presence of thermostable l-phenylalanine aminotransferases. With organisms from culture collections best results were obtained with Thermus aquaticus and Bacillus caldolyticus. Cell-free extracts of these bacteria contained enzymes which did not lose activity by heat treatment at 60°C for 25 min, although they became rapidly inactivated during incubation at 70°C. Bacillus species able to grow at 70–75°C in mineral medium with phenylalanine as the sole carbon- and energy source were subsequently isolated in pure culture. At 70°C Bacillus strain IS1 grew on phenylalanine with a doubling time of 35 min and synthesized a phenylalanine aminotransferase which only slowly lost activity when incubated at 70°C and was stable at 60°C for at least 7 h.During the purification of the phenylalanine aminotransferase from Bacillus IS1 only a single peak of activity was observed consistently. This enzyme showed activity with phenylalanine and tyrosine but not with aspartate. The apparent K _m values for phenylalanine and tyrosine were 0.95 and 0.77 mM, respectively. The enzyme had an optimum pH of 6.4 and a temperature optimum of 71.5°C for the deamination of phenylalanine. Similar levels of the enzyme were synthesized during growth of Bacillus IS1 on a variety of substrates, suggesting that it functions in phenylalanine (and tyrosine) biosynthesis rather than in phenylalanine catabolism.Dedicated to Prof. Dr. H. J. Rehm on the occasion of his 60th birthday     

Requirement for direct association of ammonia-excreting Anabaena variabilis mutant (SA-1) with roots for maximal growth and yield of wheat

Direct association between wheat roots and an ammonia-excreting mutant of the cyanobacterium Anabaena variabilis, strain SA-1, was required for maximal enhancement of growth of wheat plants in nitrogen (N)-free, hydroponic medium. Over 85% of the cyanobacterial mutant SA-1 inoculated to the roots were adsorbed under non-saturating conditions. The adsorption process of SA-1 to wheat roots was biphasic: an initial rapid adsorption was followed by a slow phase with about 10% of the initial adsorption rate. The maximal adsorption rate of filaments observed was 1.6 mg dry wt. SA-1 adsorbed·plant^–1·h^–1. Bypassing CO₂ fixation and sugar formation, the ¹⁴C label from [¹⁴C]sucrose was directly applied to leaf blades to study sugar translocation. The ¹⁴C label from this treatment appeared in the wheat culture medium within an hour. Nitrate-grown plants excreted about 30% of the ¹⁴C label into the medium, compared to only 10% excreted by wheat/Anabaena co-cultures. SA-1 assimilated 27% of all ¹⁴C translocated from [U-¹⁴C]sucrose applied to wheat leaves, and ¹⁴C label from this treatment was recovered from strain SA-1 after 30 min. Roots and cyanobacteria accounted for 51% of all radioactive label recovered in the plants co-cultured with SA-1 vs 20% for nitrate-grown plants. We studied the activity of -fructosidase (invertase) in wheat of variety Yecora rojo. Roots from nitrate-grown wheat plants produced high levels of invertase activity, which converted over 85% of 3 mm sucrose into glucose and fructose in 24 h. The rate of sucrose disappearance in the medium of co-cultures using A. variabilis SA-1, was 70% of that of nitrate-grown plants, but the levels of glucose and fructose in these cultures were always very low during sucrose conversion, suggesting hexose assimilation. To study the role of diffusible metabolites, a dialysis membrane was employed to separate the ammonia-excreting SA-1 from the wheat roots. Containing SA-1 in a dialysis bag away from direct root contact, severely limited leaf growth to less than one-third of the growth rate of nitrate control cultures. Ammonia produced by mutant SA-1 in dialysis bag cultures was excreted into the medium at 0.4 mm vs 1.2 mm in free-living cultures, but ammonia was not detectable in co-cultures with or without the dialysis bag containing the mutant. The nitrogenase activity derepressed in the mutant and responsible for ammonia excretion was always higher in the association co-cultures than in either free cells or in dialysis-bag cultures. The nitrogenase activity of strain SA-1 was highest (200 mol ethylene formed·mg^–1 Chl·h^–1) when the cyanobacterium was associated with the root tips. Dialysis membrane separation of plant and cyanobacterium severely inhibited growth of wheat during a complete growth cycle of 2 months. Total biomass and grain yield were very similar for control cultures without inorganic N or SA-1, and for diffusion cultures containing SA-1, kept in a dialysis bag around the roots. Total biomass of the association co-culture attained 75% of the biomass of the nitrate-grown control. It is proposed that wheat roots supplied fructose derived from sucrose for growth and nitrogen fixation of SA-1 in the light, and that ammonia excreted by SA-1 was utilized by the wheat plant for its own growth. Correspondence to: H. Spiller