一株番茄表面着生醋酸菌的分离鉴定及产酸条件优化

醋酸菌是食醋酿造过程中的关键菌种,性能优良的菌种对于产品品质的提升意义重大。以分离自番茄表面的产醋酸菌为研究对象,通过生理生化指标鉴定、16S rRNA编码基因比对及系统发育树构建等方法对其种类进行鉴定,并通过单因素实验、正交实验对鉴定为醋酸菌的菌株进行培养条件优化。结果表明,所分离的3株醋酸生产菌中,BQ-1被鉴定为醋酸杆菌属(Acetobacteraceae),在以酵母粉为主要氮源,蔗糖为主要碳源的培养基中,其最高产酸量为1823 g·L-1。由于该菌株在番茄表面具有很强的生长能力,因此有望应用于番茄果醋的酿造。     

The Effects of l-Naphthylacetic Acid on the Growth of Excised Tomato Roots

I-naphthylacetic acid (NAA) enhances main axis extension andlateral initiation of excised tomato roots cultured in 0.5 percent sucrose medium. NAA has a qualitatively similar effectupon the growth of root tips cultured in glucose medium or transferredafter 2 days' previous growth in standard (1.5 per cent sucrose)medium to a glucose medium containing NAA.NAA does not raisethe growth-rate of roots cultured in glucose medium to thatoccurring in presence of sucrose as carbon source. The enhancedgrowth resulting from addition of NAA to glucose medium is notdependent upon enhanced glucose uptake.The unique role of sucrosein the growth of excised tomato roots is discussed in the lightof the above observations and of studies on the ability of glucoseto maintain the growth of excised roots previously establishedby a period of culture in sucrose medium.     

A secreted lipolytic enzyme from Xanthomonas campestris pv. vesicatoria is expressed in planta and contributes to its virulence

A recombinase-based in vivo expression technology (RIVET) approach with Xanthomonas campestris pv. vesicatoria (Xcv) revealed that lipA, annotated as putative secreted lipase, is expressed during the interaction between this pathogen and tomato. Here, the tnpR and uidA reporter genes were used to show that lipA is strongly induced in XVM2 minimal medium and during the early stages of tomato infection by Xcv. A mutant strain impaired in lipA was generated by insertional mutagenesis. This mutant grew in a similar manner to the wild-type in rich medium, but its growth was significantly compromised in a medium containing olive oil as a single carbon source. The lipolytic activity of the extracellular fraction of the lipA mutant was reduced significantly relative to that of the wild-type strain, thus confirming that lipA indeed encodes a functional secreted enzyme with lipolytic activity. A plasmid carrying a wild-type copy of lipA complemented the lipA mutant for extracellular lipolytic activity. Dip inoculation experiments with tomato lines Hawaii 7998 (H7998) and Micro Tom showed that the lipA mutant grew to a lesser extent than the wild-type in tomato leaves. Following leaf syringe infiltrations, the mutant strain induced disease symptoms that were less severe than those induced by the wild-type strain, supporting a significant role of lipA in the pathogenicity of Xcv.     

Growth, pigment production and protease activity of Monascus purpureus as affected by salt, sodium nitrite, polyphosphate and various sugars

The effect of different levels of salt, sodium nitrite, polyphosphate and various sugars on growth, pigment production, protease activity and culture pH caused by Monascus purpureus was studied in broth medium and ground meat. The addition of sodium chloride (> 50.0 g l(-1)) and polyphosphate (> 3.0g l(-1)) to broth medium decreased mycelial growth, pigment production and protease activity of M. purpureus, whereas low concentrations of sodium nitrite (< 0.2 g l(-1)) promoted mycelial growth and pigment production. When the basal medium and ground meat contained salt, 150.0 g l(-1), the mould growth was stopped. The medium with fructose as carbon source proved to be the most suitable for mycelium growth and pigment production, with maltose and glucose being the second most productive. When sucrose and lactose were used as carbon sources, mycelium growth and pigment production were inhibited but the protease activity increased significantly. The mould showed more tolerance to salt and polyphosphate in ground meat than in broth medium and used sucrose as a carbon source as well as glucose for growth and pigment production in the meat mixture.     

In vitro plant regeneration in the genus Cucumis

Plants were regenerated from cotyledonary and root explants of cucumber (Cucumis sativus L.) cultivars and breeding lines of diverse sex type, growth habit, and processing quality and from cotyledonary explants of muskmelon (C. melo L.). Somatic embryogenesis was induced on a medium consisting of Murashige and Skoog (MS) salts supplemented with 1.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D), 1.0 mg/l α-naphthaleneacetic acid and 0.5 mg/l 6-benzylaminopurine. Embryos matured on the same medium without 2,4-D, and developed into normal plants on a hormone-free MS medium. Cucumber plants were also regenerated from cotyledonary protoplasts using a modified tomato protocol.     

Colonization of cellulose membranes by species of Verticillium

Verticillium dahliae Kleb. (from Brussels sprouts), non-pathogenic to tomato, and V. tricorpus Issac, a virulent pathogen to tomato, differed in their ability to colonize samples of commercial Cellophane, their rate of colonization being dependent upon external sources of carbohydrate. Cellophane and filter paper were both utilized in culture as the sole carbon source and it appears that both fungi have identical cellulase enzyme systems. Prolonged growth of the isolates is liquid medium containing sucrose as the carbon source resulted in lysis of the mycelium, which coincided with the production of polysaccharide material in culture.     

Callus cultures of tomato mutants: I. Nutritional requirements

Callus from hypocotyl, stem, and fruit tissue of tomato mutants was grown on a complex pea extract medium. The genotypes responded differently to the levels of nutrients and stimulators or inhibitors in the medium. Hypocotyl callus of yellow (r) tomato required K(2) SO(4) for quick establishment and continued steady growth for several months; callus of this mutant could also grow with 0.5 % dimethyl sulfoxide in the medium, although growth was less than the control. The red ghost (r(+) gh) mutant is sensitive to a toxic component in the pea extract, and makes its best growth with the standard minerals and vitamins, but in 1/2 concentration pea extract plus 5 % coconut water. Tangerine (t), red lutescent stem (r(+) l(2) ), and r(+) gh are mutants which respond differently to thiourea: t grows about the same at all concentrations, r(+) gh grows best at low thiourea, and r(+) l(2) grows best at the specific level of 20 mg/l thiourea. The recent active t or r(+) l(1) and r(+) l(2) isolates require supplementary auxin to which the older, slow-growing isolates do not respond. However, there is variation in growth response of different isolates of the same mutant. The several red (r(+) ) cultures are similar in their slow growth, but somewhat different in responses to specific nutrients. The recent (+) isolate is one of the most active cultures, in comparison to the slow growth of t callus isolated in 1964. It is therefore concluded that growth is affected both by the specific requirements of the mutant and by the age and vigor of isolates.     

Kinetic studies on citric acid production by Aspergillus niger. II. The two-stage process.

A two-stage process of submerged citric acid fermentation with replacement of growth medium by fermentation medium has been worked out. The optimum composition of mineral nutrients and pH of the fermentation medium of the second stage of the process were determined. An addition of 0.5 g/l of NH4NO3 as nitrogen source and 0.1 g/l of MgSO4-7H2O as magnesium source ensured effective conversion of sucrose to citric acid. An addition to KH2PO4, on the other hand, was definitely unfavourable as it considerably reduced the product yield. The medium for the second stage of fermentation should be acidified to about pH 2.2, while the water used for washing the mycelium from the remains of the growth medium should have a pH of 2.5--3.5. Under these conditions, with an initial sucrose concentration of 100 g/l, after 132 hr fermentation at 26 degrees up to 90 g/l of citric acid was obtained, which corresponds to a productivity of over 16 g/l. day. The highest activity for citric acid formation was found in three- or four-day-old mycelium.     

Gamma-linolenic acid production by Cunninghamella echinulata growing on complex organic nitrogen sources

Growth of two strains of Cunninghamella echinulata on various nitrogen containing raw materials (corn gluten, corn steep, whey concentrate, yeast extract and tomato waste hydrolysate) yielded important amounts of biomass containing various quantities of γ-linolenic acid (GLA) rich cellular lipids. Especially, growth on tomato waste hydrolysate (TWH) yielded 17.6 g/l of biomass containing 39.6% oil and significant quantities of GLA corresponding to 800 mg/l GLA. Mycelium-bounded proteolytic activity was detected during early growth stages on TWH and declined thereafter, increasing the concentration of assimilable nitrogen in the medium. However, addition of glucose in the medium during the stationary phase triggered the biosynthesis of reserve lipid, since an increase of the proportion of neutral lipids from 45% to 79% in total lipids was observed, while polar lipids decreased from 35% to 12% and from 20% to 9% for glycolipids plus sphingolipids and phospholipids, respectively.     

Selection of tomato tissue cultures able to grow on ribose as the sole carbon source

When tomato (Lycopersicon esculentum Mill.) callus or cell cultures were placed on media containing ribose as the sole carbon source, the tissues turned dark brown and ceased growth. However, after approximately 60 days bright green tissue able to grow on ribose emerged from 3 % of the brown necrotic callus tissue pieces plated. The selected tissue was highly organized, consisting of leafy primordia and associated meristematic tissues, sustained growth on ribose, and demonstrated the capacity to regenerate whole plants for at least 3 years. Cultures able to grow on ribose could not be selected from liquid suspension cultured tomato cells or from callus which had been mechanically macerated into cell aggregates containing less than approximately 100 cells. Plants regenerated from ribose adapted cultures were abnormal, having shortened internodes and thicker greener leaves. Regenerated plants were both male and female sterile.Abbreviations BAP N⁶-benzylaminopurine - CFM callusforming medium - IAA indole acetic acid - SDM shoot determination medium - RCM ribose containing medium     

Organic acids, sugars, and L-tryptophane in exudates of vegetables growing on stonewool and their effects on activities of rhizosphere bacteria

The influence of stonewool substrate on the exudation of the major soluble carbon nutrients and of the auxin precursor tryptophane for Pseudomonas biocontrol agents was studied. To this end, the composition of the organic acids and sugars, as well that of tryptophane, of axenically collected exudates of seed, seedlings, and roots of tomato, cucumber, and sweet pepper was determined. The major results were as follows. i) The total amount of organic acid is much higher than that of total sugar. ii) Exudation of both organic acids and sugars increases during plant growth. iii) Citric, succinic, and malic acids represent the major organic acids, whereas fructose and glucose are the major sugars. iv) Compared with glass beads as a neutral substrate, stonewool substantially stimulates exudation of organic acids and sugars. v) It appeared that enhanced root-tip-colonizing bacteria isolated previously from the rhizosphere of tomato and cucumber grow much better in minimal medium with citrate as the sole carbon source than other, randomly selected rhizobacteria do. This indicates that the procedure which selects for excellent root-tip colonizers enriches for strains which utilize the major exudate carbon source citrate. vi) The content of L-tryptophane, the direct precursor of auxin, is approximately 60-fold higher in seedling exudates of tomato and sweet pepper than in cucumber seedling exudates, indicating a higher possibility of plant growth stimulation after inoculation with auxin-producing rhizobacteria for tomato and sweet pepper crops than for cucumber. However, the biocontrol strain Pseudomonas fluorescens WCS365, which is able to convert tryptophane into auxin, did not stimulate growth of these three crops. In contrast, this strain did stimulate growth of roots of radish, a plant which exudes nine times more tryptophane than tomato does.     

Plant Regeneration from Cotyledon Protoplasts of Tomato (Lycopersicon esculentum L.)

otyledon protoplasts of tomato (Lycopersicon esculentum L.) isolated from 2–3 week grown seedlings were cultured in MS liquid medium (2,4-D 1, 6-BA 0.1 mg/l) and fresh medium added subsequently. After 6 weeks culture, the cell clusters were transferred to semisolid medium (the additive same as in liquid medium, agar 0.3%). When the calli grew to 0.5 cm in diameter, transfer them to MS medium (6-BA 2, IAA 0.2 mg/l) for differentiation. The regenerated plants were obtained. After comparing different culture methods, tomato protoplasts grew better in double layers than in agar plate and hanging drops.     

An allelopathic substance exuded from germinating watermelon seeds

When watermelon seeds were cultured in a Petri dish together with amaranth, barnyard grass, cockscomb, lettuce or tomato seeds, the shoot growth of amaranth and cockscomb was markedly promoted, whereas the shoot growth of lettuce and tomato was inhibited. The shoot growth of barnyard grass was not affected. These results suggest that plant-selective allelopathic substance(s) affecting the shoot growth of other plant seedlings were exuded from watermelon seeds. An allelopathic substance was isolated from the exudates of germinating watermelon seeds and identified as vanillic acid by its spectral analysis and Rf value on TLC. Vanillic acid promoted the shoot growth of cockscomb at the concentrations of 300 to 10 mg/l and that of amaranth at the concentrations of 30 to 3 mg/l, although the shoot growth of amaranth was inhibited by 300 mg/l of vanillic acid. The shoot growth of lettuce and tomato was inhibited at the concentrations higher than 30 mg/l by vanillic acid. However, the shoot growth of barnyard grass was not affected at the concentrations used. All these results suggest that vanillic acid may play as a major component of allelopathic substance(s), which shows plant-selective activity, in the exudates of germinating watermelon seeds.     

The influence of humic acids derived from earthworm-processed organic wastes on plant growth

Some effects of humic acids, formed during the breakdown of organic wastes by earthworms (vermicomposting), on plant growth were evaluated. In the first experiment, humic acids were extracted from pig manure vermicompost using the classic alkali/acid fractionation procedure and mixed with a soilless container medium (Metro-Mix 360), to provide a range of 0, 50, 100, 150, 200, 250, 500, 1,000, 2,000, and 4,000 mg of humate per kg of dry weight of container medium, and tomato seedlings were grown in the mixtures. In the second experiment, humates extracted from pig manure and food wastes vermicomposts were mixed with vermiculite to provide a range of 0, 50, 125, 250, 500, 1,000, and 4,000 mg of humate per kg of dry weight of the container medium, and cucumber seedlings were grown in the mixtures. Both tomato and cucumber seedlings were watered daily with a solution containing all nutrients required to ensure that any differences in growth responses were not nutrient-mediated. The incorporation of both types of vermicompost-derived humic acids, into either type of soilless plant growth media, increased the growth of tomato and cucumber plants significantly, in terms of plant heights, leaf areas, shoot and root dry weights. Plant growth increased with increasing concentrations of humic acids incorporated into the medium up to a certain proportion, but this differed according to the plant species, the source of the vermicompost, and the nature of the container medium. Plant growth tended to be increased by treatments of the plants with 50-500 mg/kg humic acids, but often decreased significantly when the concentrations of humic acids derived in the container medium exceeded 500-1,000 mg/kg. These growth responses were most probably due to hormone-like activity of humic acids from the vermicomposts or could have been due to plant growth hormones adsorbed onto the humates.     

Optimisation of tomato Micro-tom regeneration and selection on glufosinate/Basta and dependency of gene silencing on transgene copy number

Key message

An efficient protocol of transformation and selection of transgenic lines of Micro-tom, a widespread model cultivar for tomato, is reported. RNA interference silencing efficiency and stability have been investigated and correlated with the number of insertions.

Abstract

Given its small size and ease of cultivation, the tomato (Solanum lycopersicon) cultivar Micro-tom is of widespread use as a model tomato plant. To create and screen transgenic plants, different selectable markers are commonly used. The bar marker carrying the resistance to the herbicide glufosinate/Basta, has many advantages, but it has been little utilised and with low efficiency for identification of tomato transgenic plants. Here we describe a procedure for accurate selection of transgenic Micro-tom both in vitro and in soil. Immunoblot, Southern blot and phenotypic analyses showed that 100 % of herbicide-resistant plants were transgenic. In addition, regeneration improvement has been obtained by using 2 mg/l Gibberellic acid in the shoot elongation medium; rooting optimisation on medium containing 1 mg/l IAA allowed up to 97 % of shoots developing strong and very healthy roots after only 10 days. Stable transformation frequency by infection of leaf explants with Agrobacterium reached 12 %. Shoots have been induced by combination of 1 mg/l zeatin-trans and 0.1 mg/l IAA. Somatic embryogenesis of cotyledon on medium containing 1 mg/l zeatin + 2 mg/l IAA is described in Micro-tom. The photosynthetic psbS gene has been used as reporter gene for RNA silencing studies. The efficiency of gene silencing has been found equivalent using three different target gene fragments of 519, 398 and 328 bp. Interestingly, silencing efficiency decreased from T0 to the T3 generation in plants containing multiple copies of the inserted T-DNA, while it was stable in plants containing a single insertion.     

Enhancement of population densities of fluorescent pseudomonads in the rhizosphere of tomato plants by addition of acibenzolar-S-methyl

Fluorescent pseudomonad isolates G309 and CW2, in combination with the resistance inducer acibenzolar-S-methyl (ASM), improved control of fungal and bacterial diseases on tomato plants. The interactions of the bacteria in the presence of ASM showed that in vitro growth of Pseudomonas fluorescens G309 and Pseudomonas sp. strain CW2 was not affected in King's B broth supplemented with 10 and 20 microM ASM. Also, the bacterial cells were not able to utilize ASM as a nutrient source. In vitro production of the two antimicrobial secondary metabolites phenazine-1-carboxylic acid and 2-OH-phenazine by the isolate CW2 was not affected within 3 days from incubation. In contrary, addition of ASM at a concentration of 20 microM to King's B liquid medium significantly increased production of salicylic acid by isolate G309. When roots of tomato plants were treated with G309 or CW2 cell suspensions containing 20 microM ASM, the number of bacterial cells recovered from the rhizosphere was significantly higher in the combined treatments than in the single applications 5, 10, and 15 days after inoculation. However, ASM at a higher concentration (50 microM) did not appreciably enhance the population sizes of either bacterial isolate in the rhizosphere. Enhanced bacterial cell densities in the rhizosphere of tomato plants were also determined following simultaneous treatments of tomato roots with 10 and 20 microM ASM in combination with the transformed isolate G309-384 (mini-Tn5gfp), which encodes the green fluorescent protein.     

The Role of Ethylene in Growth and Endopolygalacturonase Production by Sclerotium rolfsii

Growth of and endopolygalacturonase production by Sclerotium rolfsii was better on a defined mineral medium than on a medium containing segments of tomato leaf petioles. The effect of treatment with ethylene (10μl/l) upon endopolygalacturonase activity with investigated at various stages of growth, in a mineral defined medium. Addition of ethylene to a 10 days-old culture of S. rolfsii resulted in a decrease in activity by day 14, whereas the addition of ethylene to a 4, 6 and 8 days old cultures resulted in an increase in endopolygalacturonase activity. Ethylene seems to have little or no stimulating effect upon growth of S. rolfsii when applied after 8 days. However, inhibited fungal growth, after the addition of ethylene at earlier stages of growth, was obtained due to the depletion of oxygen from sealed culture flasks. Endopolygalacturonase was extracted and purified from control cultures after 14 days of growth. Fractionation of this enzyme protein on Sephadex G-100 gel filtration columns resulted in two peaks of activity measured by the release of reducing sugars from polygalacturonic acid (PGA).     

Activity of NAD-dependent isocitrate dehydrogenase, isocitrate lyase, and malate dehydrogenase in Mucor circinelloides var. lusitanicus INMI under different modes of nitrogen supply

The growth and morphology as well as lipogenesis and activity of the enzymes of the tricarboxylic acid cycle and the glyoxylate cycle were studied in the fungus Mucor circinelloides var. lusitanicus INMI grown at various concentrations of urea (nitrogen source) added to the medium in different modes. It was shown that the maximum lipid content in the biomass was observed at a low (0.5 g/l) concentration of the nitrogen source, whereas the highest content of gamma-linolenic acid in the lipids was detected at high (up to 4.0 g/l) concentrations of the nitrogen source. It was found that, when the feed-batch mode of nitrogen supply was used, the amount of gamma-linolenic acid in total fatty acids was higher (up to 35%) than in the case of a single administration of the same amount of nitrogen source to the medium. The differences in the fatty acid composition and the unsaturation degree of the lipids from different subcellular fractions were demonstrated. The mycelium from the culture grown after a single administration of the nitrogen source was deformed to a great extent. The activities of the TCA cycle enzymes, NAD-dependent isocitrate dehydrogenase (IDH), and malate dehydrogenase (MDH) were lower than in the case of the feed-batch mode of urea addition, whereas the activity of isocitrate lyase (ICL), the key enzyme of the glyoxylate cycle, was higher. The coupling of the cell metabolism and the lipid composition of fungal cells and the process of cell differentiation in fungi depending on the conditions of nitrogen supply is discussed.     

The xylose isomerase gene from Thermoanaerobacterium thermosulfurogenes allows effective selection of transgenic plant cells using D-xylose as the selection agent

The xylose isomerase gene (xylA) from Thermoanaerobacterium thermosulfurogenes (formerly Clostridium thermosulfurogenes) has been expressed in three plant species (potato, tobacco, and tomato) and transgenic plants have been selected on xylose-containing medium. The xylose isomerase gene was transferred to the target plant by Agrobacterium-mediated transformation. The xylose isomerase gene was expressed using the enhanced cauliflower mosaic virus (CaMV) 35S promoter and the translation enhancer sequence from tobacco mosaic virus. Unoptimized selection studies showed that, in potato and tomato, the xylose isomerase selection was more efficient than the established kanamycin resistance selection, whereas in tobacco the opposite was observed. Efficiency may be increased by optimization. The xylose isomerase system enables the transgenic cells to utilize xylose as a carbohydrate source. It is an example of a positive selection system because transgenic cells proliferate while non-transgenic cells are starved but still survive. This contrasts to antibiotic or herbicide resistance where transgenic cells survive on a selective medium but non-transgenic cells are killed. The results give access to a new selection method which is devoid of the disadvantages of antibiotic or herbicide selection.     

Depression of sink activity precedes the inhibition of biomass production in tomato plants subjected to potassium deficiency stress

Tomato [Solanum lycopersicum (formerly Lycopersicon esculentum) L. cv. Momotarou] plants were grown hydroponically inside the greenhouse of Hiroshima University, Japan. The adverse effects of potassium (K) deficiency stress on the source-sink relationship during the early reproductive period was examined by withdrawing K from the rooting medium for a period of 21 d. Fruits and stem were the major sink organs for the carbon assimilates from the source. A simple non-destructive micro-morphometric technique was used to measure growth of these organs. The effect of K deficiency was studied on the apparent photosynthesis (source activity), leaf area, partitioning (13)C, sugar concentration, K content, and fruit and stem diameters of the plant. Compared with the control, K deficiency treatment severely decreased biomass of all organs. The treatment also depressed leaf photosynthesis and transport of (13)C assimilates, but the impact of stress on these activities became evident only after fruit and stem diameter expansions were down-regulated. These results suggested that K deficiency diminished sink activity in tomato plants prior to its effect on the source activity because of a direct effect on the water status of the former. The lack of demand in growth led to the accumulation of sugars in leaves and concomitant fall in photosynthetic activity. Since accumulation of K and sugars in the fruit was not affected, low K levels of the growing medium might not have affected the fruit quality. The micro-morphometric technique can be used as a reliable tool for monitoring K deficiency during fruiting of tomato. K deficiency directly hindered assimilate partitioning, and the symptoms were considered more detrimental compared with P deficiency.