Spinach Roots Research Articles

First Report of Meloidogyne incognita Infecting Spinach in Southern Spain.

Severe plant yellowing and decline were recently observed in two commercial fields of spinach (Spinacia oleracea cv. Polka) in Encinarejo (Córdoba), southern Spain. Disease surveys revealed severe infections of main and feeder roots and a large soil population of the southern root-knot nematode Meloidogyne incognita. The nematode population was extracted and quantified from soil and root samples according to Barker (1) and identified by female perineal pattern, phenotype esterases, and host-differential test (3,4). M. incognita was found in 100% of soil samples and 85.7% of root samples with nematode population densities ranging from 44 to 378 eggs and second-stage juveniles (J2s) per 100 cm3 of soil and 162 to 725 eggs and J2s per 5 g of fresh roots. Infected roots showed large, regular galls on root tips and also along the main root axis and secondary feeder roots. Galling of root tips prevents further root growth into deeper soil layers and induced proliferation of secondary roots. The severe infections in roots of spinach suggest that parasitism of spinach roots by the nematode must contribute to stunting, yellowing, and decline of spinach as previously reported (2). To our knowledge, this is the first report of M. incognita infecting spinach in Spain.

Interactions between Spinacia oleracea and Bradysia impatiens: a role for phytoecdysteroids.

Plant produced insect molting hormones, termed phytoecdysteroids (PEs), are thought to function as plant defenses against insects by acting as either feeding deterrents or through developmental disruption. In spinach (Spinacia oleracea), 20-hydroxyecdysone (20E) concentrations in the roots rapidly increase following root damage, root herbivory, or methyl jasmonate (MJ) applications. In this inducible system, we investigated the plant defense hypothesis by examining interactions of roots, 20E concentrations, and larvae of the dark-winged fungus gnat (Bradysia impatiens). Root herbivory by B. impatiens larvae resulted in a 4.0- to 6.6-fold increase in root 20E concentrations. In paired-choice tests, increases in dietary 20E stimulated B. impatiens feeding deterrency. B. impatiens larvae preferred control diets, low in 20E, to those constructed from induced roots and those amended with 20E (25 to 50 micro g/g wet mass). When confined to 20E-treated diets, concentrations as low as 5 micro g/g (wet mass) resulted in significantly reduced B. impatiens survivorship compared to controls. The induction of root 20E levels with MJ resulted in a 2.1-fold increase in 20E levels and a 50% reduction in B. impatiens larval establishment. In a paired-choice arena, untreated control roots were damaged significantly more by B. impatiens larvae than MJ-induced roots that contained 3-fold greater 20E levels. Based on dietary preference tests, the 20E concentrations present in the MJ-induced roots (28 micro g/g wet mass) were sufficient to explain this reduction in herbivory. Interactions between spinach roots and B. impatiens larvae demonstrate that PEs can act as inducible defenses and provide protection against insect herbivory.

Identification of a cis Element for Tissue-specific Alternative Splicing of Chloroplast Ascorbate Peroxidase Pre-mRNA in Higher Plants

Alternative splicing events in the 3'-terminal region of chloroplast ascorbate peroxidase (chlAPX) pre-mRNA in spinach and tobacco, which produced four types of mRNA variants, one form (tAPX-I) encoding thylakoid-bound APX (tAPX) and three forms (sAPX-I, -II, and -III) encoding stromal APX (sAPX), were regulated in a tissue-specific manner. The ratio of the level of sAPX mRNAs (sAPX-I, -II, and -III) to tAPX-I mRNA was close to 1 in leaf, whereas the ratio in root was greatly elevated due to an increase in sAPX-III and a decrease in tAPX-I resulting from the alternative excision of intron 11 and intron 12, respectively. A putative splicing regulatory cis element (SRE), which is highly conserved in the sequences of chlAPX genes of higher plants, was identified upstream of the acceptor site in intron 12. The deletion of the SRE sequence diminished the splicing efficiency of intron 12 in tobacco leaf in vivo. Gel-shift analysis showed that SRE interacts strongly with a nuclear protein from leaves but not those from the roots of spinach and tobacco. These results indicate that the tissue-specific alternative splicing of chlAPX pre-mRNA is regulated by the splicing enhancer SRE.

Identification and characterization of rhizobacteria isolated by enrichment culture on spinach roots

Rhizobacteria can be used for biological control and environmental restoration. In this study, we performed enrichment culture of rhizobacteria, identified isolates, and investigated the physiological properties of the bacterial isolates. Five bacteria differing in their colony morphology were isolated from spinach roots as enriched rhizobacteria. Four isolates were identified by sequencing of 16S rDNA as β and γ-Proteobacteria; 16S rDNA sequencing was not completed on one isolate. Based on microscopic observation, we determined that at least two types of bacteria differing in their morphology co-existed in this isolate, and that it may not be possible to culture the two types separately. Based on tests of substrate utilization, we could not find the characteristics that were common to the isolates. One of the five isolates was inoculated into non-sterile soil, and we examined its root-colonizing ability. The test strain which was not detected in the non-rhizosphere soil, accounted for about 20% of the total bacteria on the roots. These results suggested that enrichment culture might be useful for isolating bacteria with a high root-colonizing ability

Explants of Ri-transformed hairy roots of spinach can develop embryogenic calli in the absence of gibberellic acid, an essential growth regulator for induction of embryogenesis from non-transformed roots

Hairy roots of spinach ( Spinacia oleracea L.) were obtained by inoculation of cotyledon explants with wild-type Agrobacterium rhizogenes A13. Integration of the transfer DNA (T-DNA) of a root-inducing (Ri) plasmid of A. rhizogenes into the plant DNA was confirmed by the polymerase chain reaction. Somatic embryogenesis from explants of hairy roots were induced even when the callus-induction (CI) medium did not contain gibberellic acid (GA 3), which is considered to be a key growth regulator that is critical for induction of somatic embryogenesis in spinach. Proteins extracted from hairy root-derived calli and from non-transformed root-derived calli generated on medium that contained 10 μM naphthaleneacetic acid (NAA), without GA 3, were analyzed by polyacrylamide gel electrophoresis. The proteins from the former calli included appreciably higher levels of a 31-kDa basic protein (BP31) (p I=8.8) than proteins from the latter. This 31-kDa protein also accumulated in embryogenic calli derived from non-transformed roots. Somatic embryos from hairy root explants, grown on medium without growth regulators, yielded regenerated plantlets with actively growing root systems and shoots that did not bolt.

Accumulation of a 31‐kDa glycoprotein in association with the expression of embryogenic potential by spinach callus in culture

Calli grown from segments of spinach (Spinacia oleracea L.) root in the presence of gibberellic acid (GA3) plus auxin, differentiated to yield somatic embryos after transfer to a medium without growth regulators, while calli formed in the absence of GA3 failed to generate any embryos. We extracted proteins from the two types of callus and analysed them by polyacrylamide gel electrophoresis. Compared with the proteins from calli formed on medium that contained only naphthaleneacetic acid (NAA) as a growth regulator, the proteins from calli grown in the presence of GA3 included appreciably higher levels of a 31-kDa basic protein (pI = 8.8). The protein resembled type I ribosome-inactivating proteins (EC 3.2.2.22) in terms of molecular mass, isoelectric point, sequence of amino-terminal amino acids and extent of glycosylation. The 31-kDa protein was barely detectable in extracts of various tissues from seedlings. Thus, it is possible that an increase in the relative level of this protein might be associated with the expression of embryogenic potential expressed by spinach callus.

ＮａＣｌ養液添加によるホウレンソウ根の高温障害の抑制

This study examined the possibility of reducing the heat injury of spinach incurred by the roots. In the first experiment, spinach plants (Spinacia oleracea L. cv. Okame) were controlled or subjected to salt stress by adding 43mM NaCl to 30°C nutrient solution for 11 days. In the second experiment, plants were grown in three different concentration of salt (control, 43mM, 130mM) or PEG (29gl-1) treatments each for 2 days at 34°C. In the first experiment, at 30°C solution temperature, root weight of 43mM NaCl treatment was larger than that of control, while at 20°C solution temperature, the weight was almost same between the treatments. In the second experiment, 43 mM NaCl treatment increased shoot and root weights compared to those of control. However, 130mM treatment as well as PEG treatment did not show any positive effect on growth. The results indicate that addition of 43mM NaCl could reduce the heat injury of spinach root under high nutrient solution temperatures.

Evaluation of the Endophyte Enterobacter cloacae SM10 Isolated from Spinach Roots for Biological Control against Fusarium Wilt of Spinach

Six hundred sixty-three isolates of microorganisms, including fungi and bacteria, were collected from surface-sterilized roots of spinach (Spinacia oleracea L.) growing in commercial greenhouses in Kyoto Prefecture. These isolates were screened for their ability to control Fusarium wilt of spinach caused by Fusarium oxysporum f. sp. spinaciae. In primary screening, spinach seeds were treated with the isolates, sown in pots containing sterilized soil, and then challenge-inoculated with the pathogen. Nine bacteria were effective in reducing disease incidence. Subsequently, spinach seeds were treated with the selected isolates, then sown in an infested field and grown from June to July 1998. Four bacteria reduced disease incidence. One of these four, designated as SM10, significantly suppressed the disease. Based on bacteriological properties, SM10 was identified as a strain of Enterobacter cloacae. SM10 was observed within xylem vessels of spinach roots using light and immunoelectron microscopy, indicating E. cloacae SM10 was an endophytic bacterium of spinach.

The third naturally occurring attractant toward zoospores of phytopathogenic Aphanomyces cochlioides from the Spinacia oleracea host plant.

A bioassay-guided survey of spinach leaf constituents resulted in 5,4'-dihydroxy-3,3'-dimethoxy-6,7-methylenedioxyflavone being identified as the third naturally-occurring attractant in the host plant toward the zoospores of its pathogen, Aphanomyces cochlioides. The isolate showed attracting activity around Chromosorb W AW particles (60-80 mesh) coated with a 10(-5) M solution in a zoospore suspension. However, this activity was 1/100-1/1000 less than that of cochliophilin A, an attractant in the roots of spinach. Bioassays with the present isolate and related compounds revealed that 5,3',4'-trihydroxy-3-methoxy-6,7-methylenedioxyflavone did not possess attractant activity, but rather weak antagonistic activity toward the former two attractants from spinach.

Screening for strongly regenerative genotypes of spinach in tissue culture using subcultured root explants

A system for subculture of spinach (Spinacia oleracea L.) roots was established, and differences in regeneration; namely, embryogenic competence, among individuals of the `Nippon' cultivar were examined. Root tissues, excised from seedlings, were grown on medium without growth regulators and subcultured on the same medium and then on medium that contained 10 μM naphthaleneacetic acid and 0.1 μM gibberellic acid to induce callus formation. Calli were transferred to medium without growth regulators. All explants formed calli. However, the frequency of embryo formation varied among lines. Higher concentrations of gibberellic acid in the callus-induction medium had limited effects on somatic embryogenesis from poorly embryogenic lines. These results indicate that inherent factors are important for somatic embryogenesis in spinach and that the root subculture system is useful for identifying strongly regenerative genotypes among individuals of a single cultivar.

Production of iron enriched vegetables: Effect of feeding time on the rate of increase in foliar iron content and foliar injury

SummaryThe effect of feeding time on the rate of increase in foliar Fe content and foliar injury was investigated. The roots of spinach (Spinacia oleracea L., cv. Leed), leaf lettuce (Lactuca sativa L., cv. Redfire) and bunching onion (Allium fistulosum L., cv. Hakata Kuronegi) were fed ammonium ferric citrate (AFC; 100 mg l–1) for 3, 6, 9, 12 and 15 h or EDTA-Fe (50 mg l–1) for 1, 3, 6, 9 and 12 h in a climatron (temperature: 27°C; relative humidity: 45–55%; PPFD: 380 μmol m–2 s–1). The foliar Fe content showed a near linear increase with time in both the AFC- and EDTA-Fe treatments. In the AFC treatment, foliar injuries in spinach occurred after feeding for 12 h and in lettuce and bunching onion after 9 h. In the EDTA-Fe-treatment, foliar injury in all the plants occurred by feeding for 6 h, and the Fe content in these leaves was obviously lower than that in the AFC-treatment for treatments of the same duration. Thus, EDTA-Fe is unsuitable for this treatment from a practical viewpoint. The foliar Fe content (mg per 100 g fresh weight) in spinach (blades), which had no foliar injuries after feeding AFC for 9 h, was 6.00 ± 0.92 (initial value: 0.82 ± 0.06). In lettuce and bunching onion after feeding for 6 h, it was 3.05 ± 0.40 and 2.00 ± 0.32 (initial value: 0.82. ± 0.05 and 0.58. ± 0.04), respectively. From these results, the incremental rate of the foliar Fe content per hour and the treatment time at which the foliar injury appeared can be estimated in advance given certain treatment conditions. Thus leaf vegetables in which the foliar Fe content is known can be produced without foliar injury.

Exogenous Ethylene Enhances Formation of Embryogenic Callus and Inhibits Embryogenesis in Cultures of Explants of Spinach Roots

Effects of ethephon, AgNO3, and AVG on formation of embryogenic callus and somatic embryogenesis were examined in cultures of explants of spinach (Spinacia oleracea L. `Nippon') roots. At 10 μm, ethephon more than doubled the frequency of formation of embryogenic callus as compared to that in cultures without ethephon. Silver nitrate at 10 mm and AVG at 1 μm each inhibited formation of embryogenic callus but neither reduced the growth rate of established callus. When ethephon was applied at 10 μm during embryogenesis, it completely inhibited embryo development. By contrast, AgNO3 at 10 μm markedly increased the number of embryos. Results suggest that ethylene might be essential as a plant hormone for formation of embryogenic callus but inhibits somatic embryogenesis per se in cultures of explants of spinach roots. Chemical names used: 2-chloroethylphosphonic acid (ethephon), silver nitrate (AgNO3), aminoethoxyvinylglycine (AVG).

Application of DGGE analysis to the study of bacterial community structure in plant roots and in nonrhizosphere soil

To analyze the structure of bacterial communities in spinach roots and in the nonrhizosphere soil, we used PeR-amplified 16S rRNA gene fragments separated by denaturing gradient gel electrophoresis (DGGE). DGGE revealed a large number of band patterns, which were ascribed to various bacterial species composing each of the bacterial communities. The pattern from the roots was less complex than that from the soil. It is considered that DGGE analysis is suitable for studies of bacterial community structure in soil-plant ecosystems.

Effect of soil salinity on population structure of fluorescent pseudomonads in spinach rhizosphere

Use of PGPR (plant growth-promoting rhizobacteria) for stimulating plant growth and for the biological control of soil-borne diseases is necessary mainly in fields with intensive cropping. The intensive cultivation systems in horticulture have generally led to soil salinity due tp the high rates of fertilizer application. Therefore, the high concentration of inorganic salts accumulated in the plant rhizosphere is likely to be a factor of salinity stress affecting both roots and root-associated rhizobacteria. In a previous report (Matsuguchi and Sakai 1995), we observed that high salinity of soil had induced a significant decrease in the populations of fluorescent pseudomonads in spinach roots. Furthermore, the adverse effect of high salinity of soil was more pronounced on Pseudomonas fluorescens strains than on Pseudomonas putida strains, resulting in an alteration of the composition of fluorescent pseudomonads in the roots where vP. putida} predominated. The objective of the present study was to determine the effect of soil salinity on the population structure, at the strain level, of fluorescent pseudomonad species colonizing spinach roots.

Rapid acclimation of root hydraulic conductivity to low temperature

Root hydraulic conductance of many species is substantially reduced by exposure to low temperatures. The objective of this research was to investigate the decrease and recovery of root hydraulic conductivity in spinach (Spinacia oleracea L.) root systems upon exposure to low temperature. Root hydraulic conductivity (Lp) was determined for detached whole root systems as the slope of the flux and an applied pressure gradient. Water flux (Jv), of root systems grown at 20°C, decreased immediately upon exposure to 5°C. After 2–5 h Jv recovered and reached a stable value after 12 h exposure to 5°C. In separate experiments, the root Lp of plants acclimated for 7 d at 5°C was 125% greater than that of isolated root systems acclimated for 12 h at 5°C. Lp of plants grown and measured at 5°C was about 50% of the Lp of plants grown and measured at 20°C. The rapid acclimation to low temperatures observed in detopped root systems was also indicated in intact plants at 20/5°C (shoot/root temperatures) using mass flow porometry. Acclimation of the root system after exposure to 5°C was apparent by recovery of stomatal opening. These results indicate that spinach root systems have the ability to acclimate rapidly to changes in temperature and to continue acclimating during prolonged exposure to low temperature.

Rapid acclimation of root hydraulic conductivity to low temperature

Abstract induced by increased resistance to water uptake or fluxin the roots at low temperature (Anderson andRoot hydraulic conductance of many species is sub-McNaughton, 1973; Babalola et al., 1968; Bohning andstantially reduced by exposure to low temperatures.Lusanandana, 1951; Kramer, 1942; McWilliam et al.,The objective of this research was to investigate thedecrease and recovery of root hydraulic conductivity 1982). This can be a problem in greenhouses when coldin spinach (Spinacia oleracea L.) root systems upon water is applied to rapidly transpiring plants. Leavesexposure to low temperature. Root hydraulic conduc- quickly wilt and can suVer injury. Root zone temperaturetivity (Lp) was determined for detached whole root is also important in field agriculture (Sharif Zia et al.,systems as the slope of the flux and an applied pres- 1994; Wraith and Ferguson, 1994). Low soil temperaturesure gradient. Water flux (Jv), of root systems grown limits spring transplanting and seeding and productivityat 20°C, decreased immediately upon exposure to of many plants. Although soil temperatures do not fluc-5°C. After 2–5 h Jv recovered and reached a stable tuate as rapidly as air temperatures, soil surface temper-value after 12 h exposure to 5°C. In separate experi- ature experiences diurnal temperature excursions, as wellments, the root Lp of plants acclimated for 7 d at 5°C as significant changes due to changes in season andwas 125% greater than that of isolated root systems weather. Leaf and bud acclimation to temperature is wellacclimated for 12 h at 5°C. Lp of plants grown and known, however, there is little information about themeasured at 5°C was about 50% of the Lp of plants ability of root systems to acclimate to temperature fluctu-grown and measured at 20°C. The rapid acclimation ations. The present study was undertaken to determineto low temperatures observed in detopped root sys- the ability and speed that spinach (Spinacia oleracea L.)tems was also indicated in intact plants at 20/5°C root systems can acclimate to low temperatures. Spinach(shoot/root temperatures) using mass flow porometry. was chosen for this study as it is frequently grown in theAcclimation of the root system after exposure to 5°C autumn, winter or early spring in diVerent regions of thewas apparent by recovery of stomatal opening. These country and may encounter a variety of soil and airresults indicate that spinach root systems have the temperatures.ability to acclimate rapidly to changes in temperatureand to continue acclimating during prolonged expo-sure to low temperature.

Chemotactic response to amino acids of fluorescent pseudomonads isolated from spinach roots grown in soils with different salinity levels

Chemotactic response to amino acids of fluorescent pseudomonads isolated from spinach roots grown in a low-salinity soil (LR-group) and its corresponding high-salinity soil (HR-group) was investigated. Furthermore, the amount and composition of amino acids in the root exudate under low- and high-salinity conditions were investigated. All the isolates examined showed a chemotactic response exclusively to the amino acid fraction, while most of them did not respond to the sugar and the organic acid fractions. Based on these results, the chemotactic response to 20 amino acids of randomly selected 48 isolates, of which 12 isolates each belongs to the LR- and HR-groups of both Pseudomonas putida and P. fluorescens, was investigated. The results showed that the spectrum of chemotaxis to 20 amino acids of the H-group of each species was markedly different from that of the corresponding L-group. Also, the amino acid composition of the root exudate in the high-salinity culture solution significantly differed from that in the low-salinity culture solution. The total amount of amino acids from the high-salinity solution was 2.83 μmol g-1 fresh roots, which was as small as 42% of that from the low-salinity culture solution. These results indicated that high salinity of soil may exert an effect on root colonization by fluorescent pseudomonads not only directly through the effect in the bacterial chemotaxis but also indirectly through the modification of the root exudation of amino acids qualitatively and quantitatively.

Drop Method as a Quantitative Bioassay Method of Chemotaxis of Aphanomyces cochlioides Zoospore.

A drop method using fluorinated hydrocarbon (FC-72) was successfully applied as a quantitative assay to determine the threshold concentration of cochliophilin A, a potent Aphanomyces cochlioides zoospore attractant originally isolated from spinach roots. This method revealed that the number of zoospores attracted to droplets containing cochliophilin A increased with time, until approximately 60 to 90sec after injection of the droplet into a zoospore suspension. The threshold concentration, above which cochliophilin A was able to function as a zoospore attractant, was determined to be ca. 3.0×10-9M. Interestingly, zoospores attracted to droplets containing cochliophilin A at a concentration greater than 1.1×10-7M massed on the surface of the droplets, a behavior that was quite similar to that observed on the root surface of host plants. This result suggested that the drop method would be useful not only as a quantitative assay of chemotaxis, but also as a technique to investigate the mechanism of zoospore massing.

Plant regeneration from adventitious roots of spinach ( Spinacia oleracea L.) grown from protoplasts

An efficient system for plant regeneration from adventitious roots of spinach ( Spinacia oleracea L.) was established. The adventitious roots differentiated from calli that had grown from protoplasts. Protoplasts were obtained from the cotyledon of seedlings by partial digestion of cell walls, followed by mechanical maceration with forceps. The protoplasts formed colonies at a frequency of 1.6% in a Murashige and Skoog's (MS) medium containing 1 μM 2,4-dichlorophenoxyacetic acid (2,4-D) or 10 μM 1-naphthalene acetic acid (NAA) combined with 10 μM 6-(4-hydroxy-3-methyl-but- trans-2-enylamino) purine (zeatin). Calli derived from the colonies produced adventitious roots on MS medium containing 10 μM NAA and 0.1 μM gibberellic acid (GA 3). Root segments excised from these adventitious roots differentiated into many embryos when subcultured on MS medium supplemented with 30 mM fructose instead of sucrose. The embryos developed into flowering mature plants on a growth regulator-free medium.

Effect of calcium ion on spinach root colonization by fluorescent pseudomonads through chemotaxis

Abstract A non-motile mutant of Pseudomonas putida was selected by transposon mutagenesis of the motile wild-type strain isolated from spinach roots. The mutant was indistinguishable from the motile wild-type strain in terms of growth in laboratory media. When separately inoculated to spinach rhizo-sphere, no significant difference in the root colonization activity was observed between the mutant and the wild type. However, when a 1:1 cell mixture of the mutant and the wild-type strain was inoculated to the spinach rhizosphere, the rhizoplane population of the non-motile mutant was as low as one-sixtieth of that of the motile strain. This finding suggests that chemotactic movement is an essential trait for competitive colonization of the roots. In addition, chemotactic migration of the isolates from the root in a soil with low-salinity level (LR-isolates) was inhibited when the isolates were released to a soil with high-salinity level (H-soil). To reveal the factors inhibiting the chemotactic migration of the LR-isolates in the H-soil, effects of inorganic salts on chemotactic activities of the LR-isolates and HR-isolates (isolates from the root in the H-soil) were compared. The results showed that divalent cations were more inhibitory to the LR-isolates than monovalent cations, with the strongest inhibition stemming from Ca2+, and the chemotactic activities were completely inhibited in the presence of 50 mM CaCl2, though the HR-isolates were not inhibited. These results indicated that the high level of Ca2+ in the H-soil may account for the inhibition of the migration of the LR-isolates toward the root exudates.