Differential Effects of Lithium Chloride on In Vitro Growth of Clavibacter michiganense subsp. nebraskense Depending upon Inoculum Source

The bacterium Clavibacter michiganense subsp. nebraskense (Corynebacterium michiganense subsp. nebraskense) was grown in broth cultures and inoculated into corn plants. The plating efficiency of cells from broth cultures was essentially the same on nutrient broth-yeast extract and the semiselective medium for this bacterium, CNS. However, when cells were isolated from Goss bacterial wilt- and blight-infected corn, very few were recovered on CNS compared with the amount recovered on nutrient broth-yeast extract agar. When lithium chloride was omitted from the CNS, recoveries from infected corn were nearly the same as on nutrient broth-yeast extract agar. No other ingredient of CNS was inhibitory, nor did substitution of other salts for lithium chloride cause equal inhibition. The amount of inhibition was proportional to lithium chloride concentration. The inhibition by lithium chloride occurred with several strains of the bacterium isolated from one corn cultivar and with one of the strains recovered from three different cultivars of infected corn.     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon

Differences in the production of shikonin derivatives by callus and suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. were examined. When Linsmaier and Skoog medium was used in suspension cultures, cell growth was not accompanied by the production of shikonin compounds. Shikonin derivatives were produced, however, when this medium was used in callus cultures. Differences in shikonin production were examined in terms of the nutrient supply, the effect of the agar itself, and the oxygen supply. Shikonin derivatives could be produced without agar by keeping the cells exposed to air while providing an adequate supply of nutrients. In callus cultures, the production of shikonin compounds was reduced remarkedly when the oxygen concentration in the atmosphere was lowered, evidence that shikonin production during L. erythrorhizon cell growth on Linsmaier and Skoog agar medium is enhanced by an abundant supply of oxygen.     

The biology ofAzospirillum-sugarcane association II. Ultrastructure

Summary Tissue cultures of sugarcane support abundant growth ofAzospirillum brasilense (SP 7). Visible after 1–2 weeks as a white or pink slime, this growth reaches 2×10⁸ bacteria/mm² on the surface of callus. Growth of the bacterium is strictly extracellular in viable callus, and instances of intracellular growth result from rupture of the cell wall during senescence of callus tissue. A significant proportion of the bacterial population on callus is pleomorphic. Varying the nitrogen source in the nutrient medium caused no obvious effect on callus cell structure. The presence of the bacterium caused structural alterations in callus cells which did not inhibit overall growth of the bacterium. Growth of callus as tight groups of cells lacking intercellular spaces may be important for the establishment of a long-term association withAzospirillum. The interface of bacteria and live callus tissue is at the surface of tight cell groups. Browning of the surface cell layers of these groups in the presence ofAzospirillum is not of the rapid nature known for hypersensitivity reactions. Rather, this production of phenolics appears to be due to the accumulation of extracellular bacterial metabolites. The ultrastructure of this and other callus reactions is described. As evidenced by organogenesis, the associated cultures have remained viable for at least 18–20 months.Florida Agricultural Experiment Station Journal Series No. 1695.     

Structural and physiological diversity among cystlike resting cells of bacteria of the genus <Emphasis Type="Italic">Pseudomonas</Emphasis>

Cystlike resting cells (CRC) of non-spore-forming gram-negative bacteria of the genus Pseudomonas, P. aurantiaca and P. fluorescens, were obtained and characterized for the first time; their physiological and morphological diversity was demonstrated. The following properties were common for all the revealed types of CRC as dormant forms: (1) long-term (up to 6 months or longer) maintenance of viability in the absence of culture growth and cell respiration; (2) absence of an experimentally detectable level of metabolism; (3) higher resistance to damage and autolysis under the action of provoking factors than in metabolically active vegetative cells; and (4) specific features of ultrastructural organization absent in vegetative cells: thickened and lamellar envelopes, clumpy structure of the cytoplasm, and condensed DNA in nucleoid. The differences in various types of CRC concern the thickness and lamellar structure of cell envelopes, as well as the presence and thickness of the capsular layer. In particular, forms ultrastructurally similar to typical bacterial cysts were revealed in pseudomonad populations growing on soil agar. Physiological diversity was revealed in different levels of viability preservation and thermal resistance in various types of CRC and depended on the conditions of their formation. The optimal conditions and procedures for obtaining P. aurantiaca and P. fluorescens CRC that retain the ability to form colonies on standard nutrient media are as follows: (1) a twofold decrease of nitrogen content in the growth medium; (2) an increased level of anabiosis autoinducer (C₁₂-AHB, 10^?4 M) in stationary cultures; (3) transfer of the cells from stationary cultures to a starvation medium with silica; (4) cultivation in soil extract; and (5) development of cultures on soil agar. The CRC from the cultures grown in soil extract or starvation medium with silica proved to be resistant to heat treatment (60°C, 5 min). In the CRC formed in nitrogen-limited media, the degree of heat resistance increased at longer incubation (1.5 to 6 months). CRCs on soil agar surface were resistant to desiccation. The ultrastructure of the morphologically varied types of P. aurantiaca CRC formed under simulated natural conditions is described for the first time. The data on the intraspecies diversity of pseudomonad dormant forms contribute to the concept of plasticity of the life style and adaptive reactions that ensure survival of these bacteria in unfavorable environmental conditions.     

Growth interactions between calli and explants of rose plants in vitro

Growth of explants or calli of two rose cultivars ‘Sonia’ and ‘Golden Times’, was extensively promoted when they were grown on agar together with calli of rose rootstocks Rosa indica or Rosa canina, while growth of callus of a miniature rose cultivar was either not affected or inhibited. The growth of R. indica callus was inhibited when accompanied by explants of ‘Sonia’ or ‘Golden Times’. Promotion or inhibition of explants or callus growth was also observed when the agar medium was supplemented with conditioned liquid medium from cell suspension cultures of cv. Sonia or R. indica. Autoclaved conditioned medium from R. indica lost its promoting effect, while that from Sonia lost its inhibiting effect after autoclaving. The possible interaction between the rootstock and scion tissues is discussed.     

Effects of common components on hardness of culture media prepared with gelrite™

Summary Tissue cultures on properly solidified Gelrite media generally showed superior shoot proliferation and rooting, as well as shoot and root vigor and callus development to those on TC agar. Vitrification, or hyperhydricity, was observed in both Gelrite and agar media and minimized by increasing the gel concentrations. Rigidity of Gelrite media depended on combined levels of MS macrosalts, basal nutrient formulations, sucrose concentration, pH, and Gelrite concentration. Most MS macrosalts increased hardness of Gelrite gels; NH₄NO₃ had a decreasing effect. Rigidity of TC agar gels increased with reductions of MS macrosalts. A slightly softer Gelrite medium resulted when sucrose was excluded. Both Gelrite and agar media were softer at lower pHs and harder at higher pHs. Activated charcoal and mannitol increased gel hardness, and more noticeably of agar gels. NaCl addenda reduced rigidity, with their effects being more pronounced on Gelrite than on agar gels. Gelrite is a trademark of Kelco Division of Merck &amp; Co. (San Diego, CA), manufacturer of the gellan gum. Phytagel is a trademark of Sigma Chemical Co. (St. Louis, MO) for repackaged Gelrite. TC agar used for comparisons in this investigation is plant tissue culture tested agar obtained from JRH Biosciences (Lenexa, KS).     

Maceration of Clover and Grass Leaves by Lachnospira multiparus

A strain of Lachnospira multiparus, a pectin-hydrolyzing bacterium from the rumen, was incubated in nutrient media in the presence of surface-disinfected clover leaflets. When the culture flasks containing the leaflets together with Lachnospira were shaken after overnight incubation, extensive maceration of the leaflets was seen, although uninoculated control leaflets remained intact during a similar treatment. Examination of inoculated leaflets by transmission electron microscopy showed extensive invasion of intercellular areas of the mesophyll tissue but only minor invasion of vascular tissue. Cutting the leaves before incubation greatly increased the ability of L. multiparus to colonize and macerate the leaflets. Similar experiments with grass leaves are also described, and the possible role of maceration in the digestion of plant material in the rumen is discussed. Although Lachnospira stains gram variable and often gram negative, the ultrastructure of the cell wall was that of a gram-positive bacterium.     

Demonstration of Activity of α‐Galactosidase Secreted by Cucumis sativus L. Cells

A simple, rapid and reproducible procedure for the identification of extracellular cucumber (Cucumis sativus L.) α‐galactosidase is described using callus cultures of seedlings from the tested plant, hairy roots of 2‐day‐old seedlings of cucumber germinating on agar plates as well as cell suspension cultures derived from callus cultures. For the determination of the intracellular and extracellular activities of α‐galactosidase, 6‐bromo‐2‐naphthyl‐α‐D‐galactopyranoside and p‐nitrophenyl‐α‐D‐galactopyranoside, respectively, were used as synthetic substrates. The extracellular α‐galactosidase activity was identified by evaluating the dye‐zones in agar medium. The enzyme from cucumber callus cultures and seedling roots, cultivated on agar plates supplemented with 6‐bromo‐2‐naphthyl‐α‐D‐galactopyranoside, hydrolyzed this substrate releasing 6‐bromo‐2‐naphthol. By simultaneous coupling with hexazonium p‐rosaniline the corresponding azodye was formed. Thus, the extracellular enzyme was detected by the presence of reddish‐brown zones on the agar plates around the plant material. The parallel extracellular and intracellular activities were determined in cell suspension cultures derived from callus cultures. The results show a 44.6% intracellular and 55.4% extracellular distribution of α‐galactosidase activity. The described agar plate method enables a rapid, simple and specific detection of plant producers of extracellular α‐galactosidase.     

Brevianamides A and B Are Formed Only After Conidiation Has Begun in Solid Cultures of Penicillium brevicompactum

In cultures of Penicillium brevicompactum grown on Czapek-Dox agar or on circles of dialysis membrane placed on Czapek-Dox agar, brevianamides A and B first appeared after conidiation had begun. The presence of these brevianamides imparted a yellow-green fluorescence to the penicilli and to the upper section of the conidiophores of actively conidiating cultures. Experiments with replacement cultures indicated that medium nutrient levels had little direct effect on brevianamide production. If P. brevicompactum was grown between two circles of dialysis membrane, no aerial hyphae were formed, and brevianamide production was not observed. Three brevianamide-minus mutants of P. brevicompactum were isolated.     

Induction of benzoquinone formation by activated carbon in Lithospermum erythrorhizon cell suspension cultures

Cultured cells of Lithospermum erythrorhizon which were capable of producing red naphthoquinone (shikonin) derivatives on Linsmaier-Skoog's agar medium stopped synthesizing these compounds when grown in liquid medium without agar. However, when the liquid medium was supplemented with a small amount of activated carbon, the cells produced a new orange benzoquinone derivative, echinofuran B, which may be considered an abnormal metabolite of geranylquinol, the key intermediate in the biosynthesis of shikonin. A similar effect of activated carbon was also observed with a variant cell line incapable of producing shikonin derivatives even on the agar medium. By contrast, the callus cultures grown on the agar medium as well as the dried roots of the intact plant were found to contain a small amount of echinofuran C, another new benzoquinone related to echinofuran B, in addition to shikonin derivatives.     

A method to compare the effect of ionic iron and iron chelates in nutrient solution cultures

A method is described by which the effect of chelated Fe can be compared with the effect of ionic Fe in nutrient solution cultures over a prolonged period of time. Plants are grown in two solutions in succession: the one containing all nutrient elements except Fe, the other one containing the Fe compound together with Ca(NO₃)₂. In experiments with soybeans (Glycine maxima (L.) Merr.) and with corn (Zea mays L.) it was shown that a 7-day cycle with the ratio of 4 days nutrient solution to 3 days Fe solution resulted in growth and Fe nutrition similar to plants grown with a normal nutrient solution.     

Comparison of fatty acid patterns in plant parts and respective callus cultures of Cucumis melo

Root, hypocotyl, cotyledon, stem and leaf of Cucumis melo var. utilissimus seedlings were used for callus induction. Comparison was made between these parts, between callus tissues originating from all the parts and between each part and its callus, with respect to the fatty acid composition of total lipids. In all the parts there was a greater proportion of unsaturated fatty acids, the predominant fatty acid in root, stem and leaf being linolenic acid whilst in the cotyledon linoleic predominated. In the hypocotyl these two acids were present in equal amounts. In callus cultures the proportion of saturated acids was greater and the predominant fatty acid was palmitic. The major unsaturated fatty acid in callus cultures was linolenic. The analysis showed that callus tissue and its respective plant part had different fatty acid patterns and that all the callus cultures had very similar patterns irrespective of their origin.     

Resting forms of <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis>

The ability of the symbiotrophic rhizobium Sinorhizobium meliloti P221 to produce cells having all the properties of resting forms (RFs) during the development cycles of the culture or after addition of the threshold concentrations of anabiosis autoinducers was demonstrated. The numbers, properties, and ultra-structure of S. meliloti resting forms depended on the conditions of growth and poststationary-phase incubation. In the four-month poststationary-phase, cultures grown in media deficient in some nutrient elements and energy sources (nitrogen, phosphorus, or oxygen), numerous cells (24–76% of the number of CFUs in the stationary-phase cultures) exhibiting a high degree of heat resistance and reversibly inhibited metabolic activity (the absence of endogenous respiration) were detected. According to their ultrastructure, all the resting forms detected in starving cultures were divided into three groups: (1) cystlike resting cells (CRCs) with thick cell envelopes and compacted nucleoids, (2) CRCs containing numerous (up to three-quarters of their volumes) polyhydroxyalkanoate inclusions, and (3) RFs similar to Azotobacter cysts. The resting forms obtained in the culture grown at high concentrations (5 × 10⁻⁵ M) of C₁₂-AHB, a chemical analogue of microbial anabiosis autoregulators, were incapable of endogenous respiration and retained the colony-forming ability. The CFU number after plating of these resting forms was twice as high as in the control culture; the heat resistance of these cells (55°C, 10 min) was an order of magnitude higher. The bacterial cells obtained from the resting forms either had a mixed (Swa⁺Gri⁺) type of motility in semisolid agar, typical of the dominant phenotype of the parent cells, or switched to the Gri⁺ type. Emergence of different motility phenotypes depended on the conditions of RF formation. More severe stress conditions of RF formation induced the emergence of the Gri⁺ type of cell motility. The results obtained can be used for development of a new generation of bacterial preparations based on bacterial CRCs which are able to preserve their viability for a long time and are highly resistant to stress impacts.     

Heme Requirement of a Novel Yeast-lysing Bacterium

A novel yeast-lysing bacterium (YLM-1) grew on nutrient agar containing yeast cell-free extract, but not on nutrient agar. We searched for the growth factor for YLM-1, which turned out to be hemin or hemoproteins, catalase or horseradish peroxidase. Also, the effects of growth accelerators and inhibitors on H₂O₂ metabolism were discussed.     

The effects of several factors on the growth of pure and mixed cultures of Azotobacter chroococcum and Bacillus subtilis

We studied the effect of a clay mineral, palygorskite, on the physiological activity of Azotobacter chroococcum and the phosphate-mobilizing bacterium Bacillus subtilis, as well as their mixed cultures, under various oxygen supply conditions during the utilization of phosphorus from readily and poorly soluble compounds (K₂HPO₄ · 3H₂O) and (Ca₃(PO₄)₂), respectively. During cultivation of the bacteria in a nutrient medium with Ca₃(PO₄)₂, the number of microorganisms was higher than that observed in a medium with K₂HPO₄. An increase in oxygen mass transfer in the nutrient medium was followed by a rise in the number of Bacillus subtilis cells and an inhibition of Azotobacter chroococcum growth. An addition of palygorskite (5 g/l) into the nutrient medium stimulated the growth of both bacteria and stopped the decreasing growth of Azotobacter chroococcum at high values of oxygen mass transfer. The number of Bacillus and, particularly, Azotobacter cells was two to five times lower in a mixed culture than in a monoculture. These differences were less significant during the cultivation of mixed cultures in medium with palygorskite.     

Persistence and spread of mycoplasma in axenic callus tissue cultures of tobacco (Nicotiana glauca Grah.) in the presence of kinetin and IAA in nutrient medium

A technique is described for the establishment of infectious axenic callus cultures originated from stems of tobacco plants suffering from a mycoplasma disease, potato witches' broom. When stem pieces from a diseased tobacco plant (Nicotiana glauca Grah.) are culture on a modified nutrient medium according toMurashige andSkoog (1962) with added kinetin (0.64 mg or 2.56 mg 1 000 ml^?1) and IAA (2 mg or 4 mg 1 000 ml^?1), the pathogen persists and spreads in the newly formed callus tissue. The presence of the pathogen was proved by implantation of callus slices into stems of tomato plants which indicate the disease very reliably. Reconstituted tobacco plants too recovered the infectious agent of potato witches' broom; 27 plants from the 35 reconstituted plants were diseased. Similar results were obtained with Crimean yellows. Calli were cultured and subcultured from 1 to 18 monthsin vitro and some infectious cultures are maintained and their investigation goes on. We suppose that kinetin plays a very important role for infectivity preservation in callus cultures.     

A novel milky disease organism from Australian scarabaeids: Ultrastructure

The unusual ultrastructure of a milky disease bacterium isolated from Aphodius tasmaniae is described and compared with a similar bacterium from Tipula paludosa. Both organisms possess an elongate sporangium with a small central spore, a multilayered exosporium, and longitudinally arranged extrasporal fibrils which arise from plates attached to the ends of the spore. The morphogenesis of the sporangium is described and illustrated for the organism from A. tasmaniae. It is suggested this organism and that from T. paludosa are closely related. Their taxonomic position remains obscure.     

A Comparative Study of CN-Resistant Respiration in Different Cultures of Tobacco Callus

The callus of Nicotiana rustica cv Gansu yellow flower and N. tabacum cv willow leaf were cultured on ordinary subculture medium (M-1) and on regeneration medium (M-2), respectively. No differentiation was observed in Gansu yellow flower tobacco callus cultures grown on both M-1 and M-2 medium. The respiration of both cultures was partially resistant to cyanide and markedly inhibited by m-chlorobenzhydroxamic acid. The relative contributions of alternative and cytochrome pathway were 31% and 47% of the total respiration, respectively, in M-1 callus cultures. The relative O₂ uptake of the two pathways was not changed significantly in M-2 callus cultures. In subcultured M-1 callus cultures of Willow leaf tobacco, the respiration mediated via alternative pathway was about 29 to 38% of the total respiration, and the cytochrome pathway still was the major respiratory pathway. In M-2 callus cultures in which differentiation occurred, the relative contribution of alternative pathway increased to 41 to 47% of the total respiration, and the cytochrome pathway decreased considerably. These results suggested that the change of respiratory electron transport pathway was probably related to the differentiation of tobacco callus cultures.     

Growth stimulation of Microcystis aeruginosa by a bacterium from hyper-eutrophic water (Taihu Lake, China)

Cyanobacteria are the causative organisms of the algal blooms that occur in Taihu Lake. Dissolved organic nitrogen (DON) comprises a significant composition of nitrogen (N) pool in the water and may increase the nutrient source of microalgae. In the present study, we investigated the relationship between Microcystis aeruginosa, Pseudomonas sp. A3CT isolated from Taihu, and DON compounds. Co-incubation (3 days) of the bacterium with six DON compounds (four free amino acids and two combined amino acids) was collected as six decomposed DON solutions. The decomposed DON solutions of six compounds were used to test the stimulatory effect of nutrient regeneration by the bacterium. The growth of M. aeruginosa was significantly enhanced by the six decomposed DON solutions. M. aeruginosa grew much better under the six decomposed DON solutions than the corresponding undigested DON forms. Especially, the decomposed l-lysine solution, not only avoided the inhibiting effect of lysine on M. aeruginosa, but significantly promoted the cyanobacterial growth. Further chemical tests indicated that A3CT transformed DON into NH₄ ⁺, which was utilized by M. aeruginosa. These results demonstrate that the bacterium plays an important role in decomposing unavailable DON forms into available NH₄ ⁺, which suggests that the bacterium contributes to the fast growth of M. aeruginosa. Moreover, this phenomenon, in conjunction with previous studies, indicates that the responsible and effective way of harmful blooms is reducing the N and P inputs (including DON and DOP).     

Purification, properties and physiological regulation of a putative outer-membrane porin for methanol in Methylophilus methylotrophus

A major outer-membrane protein was purified and partially characterised from the methylotrophic bacterium Methylophilus methylotrophus. The protein had a subunit M_r of 38 000 and was similar in terms of its biochemical properties to the recently characterised amide-urea porin (FmdC) from the same organism. Expression of the protein, as determined by SDS-PAGE and Western blotting of cells grown in continuous culture under various nutrient limitations, varied in a similar manner to that of methanol dehydrogenase and was maximal under methanol limitation. It was concluded that the protein is probably an outer-membrane porin for methanol.