Total Soluble Protein Patterns Research Articles

Cadmium and zinc induced similar changes in protein and glycoprotein patterns in tobacco (nicotiana tabacum l.) seedlings and plants

The effects of 10 μmol L-1 and 15 μmol L-1 cadmium (Cd), a nonessential toxic element and 25 μmol L-1 and 50 μmol L-1 zinc (Zn), an essential micronutrient, on proteins and glycoproteins of Nicotiana tabacum L. seedlings and plants were investigated after exposure to each metal alone or to their combinations. Changes in only few polypeptides related to heavy metal treatments were observed in tobacco seedlings and leaves of adult plants, while the greatest change in total soluble protein pattern was observed in plant roots. Differences between control and treated tobacco tissues were more pronounced in the glycoprotein pattern, which was analysed by application of different lectins. The majority of the detected glycoproteins in leaves and roots of adult plants can be considered as a result of enhanced glycosylation due to heavy metal stress. The difference in glycoproteins between Cd and Zn application on tobacco seedlings and adult plants could not be determined since enhanced glycosylation was noticed after treatment with either metal alone or in combination. Therefore, it can be concluded that both metals induced N- and Oglycosylation as a result of changed environmental conditions.

Improvement of Ethanol Production by Electrochemical Redox Coupling of Zymomonas mobilis and Saccharomyces cerevisiae

Zymomonas mobilis was immobilized in a modified graphite felt cathode with neutral red (NR-graphite cathode) and Saccharomyces cerevisiae was cultivated on a platinum plate anode to electrochemically activate ethanol fermentation. Electrochemical redox reaction was induced by 3 approximately 4 volt of electric potential charged to a cathode and an anode. Z. mobilis produced 1.3 approximately 1.5 M of ethanol in the cathode compartment and S. cerevisiae did 1.7 approximately 1.9 M in the anode compartment for 96 hr. The ethanol production by Z. mobilis immobilized in the NR-graphite cathode and S. cerevisiae cultivated on the platinum plate was 1.5 approximately 1.6 times higher than those cultivated in the conventional condition. The electrochemical oxidation potential greatly inhibited ethanol fermentation of Z. mobilis but did not S. cerevisiae. Total soluble protein pattern of Z. mobilis cultivated in the electrochemical oxidation condition was getting simplified in proportion to potential intensity based on SDS-PAGE pattern; however the SDS-PAGE pattern of protein extracted from S. cerevisiae cultivated in both oxidation and reduction condition was not changed. When Z. mobilis culture incubated in the cathode compartment for 24 hr was transferred to S. cerevisiae culture in the anode compartment, 0.8 approximately 0.9 M of ethanol was additionally produced by S. cerevisiae for another 24 hr. Conclusively, total 2.0 approximately 2.1 M of ethanol was produced by the electrochemical redox coupling of Z. mobilis and S. cerevisiae for 48 hr.

First Report of Phytophthora palmivora on Grevillea spp. in Italy.

The genus Grevillea (family Proteaceae) comprises over 300 species and is a popular and widely cultivated group of Australian plants. In the last 3 years, numerous potted grevilleas with symptoms of decline associated with a rot of feeder roots were found in ornamental nurseries in Sicily. Aboveground symptoms were reduced growth, yellowing of foliage, wilt, dieback, and death of the entire plant. The disease was observed on many commercial cultivars and was especially severe on G. alpina (mountain grevillea), G. juniperina (juniper-leaf grevillea), G. lavandulacea (lavender grevillea), and G. rosmarinifolia (rosemary grevillea) as well as the hybrid cultivars Clearview David (G. lavandulacea × rosmarinifolia) and Poorinda Rondeau (G. baueri × lavandulacea), while G. lanigera (woolly grevillea) cv. Mount Tamboritha and G. thelemanniana subsp. obtusifolia appeared resistant. A species of Phytophthora was consistently isolated from rotted roots of symptomatic plants using a selective medium (4), and pure cultures were obtained by single-hypha transfers. The species was identified as P. palmivora (E.I. Butler) E.I. Butler on the basis of morphological and cultural characters. On solid media, all isolates produced elliptical to ovoid, papillate sporangia with a mean length/width ratio of 1.8. Sporangia were caducous with a short pedicel (5 μm) and a conspicuous basal plug. All isolates were heterothallic (mating type A1) and produced oogonia and oospores only when paired with A2 mating type reference isolates of P. nicotianae and P. palmivora. Antheridia were amphyginous. Identification was confirmed by electrophoresis of mycelial proteins in polyacrylamide slab gels (1). The electrophoretic patterns of total soluble proteins and six isozymes (alkaline phosphatase, esterase, fumarase, NAD-glucose dehydrogenase, malate dehydrogenase, and superoxide dismutase) of isolates from grevillea were identical to those of a reference isolate of P. palmivora from Coronilla valentina subsp. glauca (2) but distinct from those of reference strains of eight other papillate species of Phytophthora included in Waterhouse's taxonomic group VI. Koch's postulates were fulfilled using 6-month-old rosemary grevillea plants that were transplanted into pots filled with soil that was artificially infested with chlamydospores (50 per gram of soil) produced in submerged cultures (3) by grevillea isolate IMI 390579. Plants were maintained in a glasshouse at 20 to 28°C and watered to field capacity once a week. One month after transplanting, infected plants showed decline symptoms similar to those of naturally infected plants. Control plants grown in pots containing noninfested soil remained healthy. P. palmivora was reisolated from roots of symptomatic plants. It appears that P. palmivora has become a widespread root pathogen in commercial ornamental nurseries in Italy (2).

Genetic Diversity Among Iranian Isolates of Rhizoctonia solani Kühn Anastomosis Group1 Subgroups Based on Isozyme Analysis and Total Soluble Protein Pattern

Abstract Rhizoctonia solani is a destructive fungal pathogen with a wide host range. The R. solani complex species includes several divergent groups delimited by affinities for hyphal anastomosis. In this study, genetic variation among 20 isolates of R. solani anastomosis group 1 (AG1) subgroups (AG1‐IA and AG1‐IB) collected from Mâzandaran province, Iran, and standard isolates of these subgroups, was determined by isozyme analysis and total soluble protein profile. Mycelial protein pattern and isozyme analysis were studied using denaturing and non‐denaturing polyacrylamide gel electrophoresis, respectively. A total of 15 enzyme systems were tested, among which six enzymes including esterase, alkaline phosphatase, superoxide dismutase, octanol dehydrogenase, lactate dehydrogenase and mannitol dehydrogenase generated distinct and reproducible results. The soluble protein patterns were similar among the R. solani isolates examined; however, minor differences in banding pattern were observed between the two subgroups. In isozyme analysis, a total of 64 electrophoretic phenotypes were detected for all six enzymes used. Based on cluster analysis and similarity matrix, the fungal isolates were divided into two genetically distinct groups of I and II consistent with the previously reported AG1‐IA and AG1‐IB subgroups in AG1. Group I represented all isolates belonging to AG1‐IA subgroup, whereas group II represented all isolates belonging to AG1‐IB subgroup. Results from isozyme analysis suggest that the subgrouping concept within AGs is genetically based.

First Report of Root and Crown Rot of Sage Caused by Phytophthora cryptogea in Italy

Sages are cultivated as aromatic and ornamental plants in Italy and represent the common name of certain species of Salvia and Phlomis (family Lamiaceae). In Sicily (southern Italy) during the summer of 2001, ≈40% of 1,400 2-year-old landscape plants of S. leucantha Cav. (Mexican bush sage or velvet sage) showed symptoms of stunting, chlorosis, and gradual dieback or sudden wilt, which are associated with root and crown rot. Plants were supplied by a commercial nursery, transplanted from pots in the spring, and irrigated using a trickle system. Phytophthora was isolated consistently from roots and basal stems of symptomatic plants on a BNPRAH medium (2). The species was identified as P. cryptogea Pethybr. & Laff., primarily on the basis of morphological and cultural characteristics. Five representative single-hypha isolates were characterized. On potato dextrose agar, they formed colonies with a slight petaloid pattern. Cardinal temperatures for mycelium growth were 2°C, minimum; 25°C, optimum; and 30 to 35°C, maximum. Hyphal swellings were abundant in aqueous culture. Sporangia were obpyriform, persistent, nonpapillate, and proliferous (2). All isolates were the A1 mating type and formed oogonia, amphigynous antheridia, and oospores in dual cultures with reference isolates of the A2 mating type of P. cryptogea and P. drechsleri. Identification was confirmed by electrophoresis of mycelium proteins on a polyacrylamide slab gel (1). Electrophoretic patterns of total soluble proteins from the sage isolates were identical or very similar to those from 10 reference isolates of P. cryptogea from various hosts, including isolate IMI 180615 (ex-type isolate). Conversely, the electrophoretic pattern of the isolates of P. cryptogea from sage was clearly distinct from those from reference isolates of other species included in Waterhouse's taxonomic group VI. Esterase (EC 3.1.1.2.) zymograms of the sage isolates corresponded to those of isolates of P. cryptogea included in electrophoretic group 2 (1). The pathogenicity of a representative isolate of P. cryptogea from sage was tested in the greenhouse using 4-month-old plants of Mexican bush sage. Inoculum was produced on a mixture of vermiculite and autoclaved oat seeds (4) and mixed with steam-sterilized sandy loam soil at a concentration of 4% (vol/vol). Plants were transplanted in pots (12 cm diameter) filled with infested soil; control plants were grown in pots containing noninfested soil. After transplanting, all pots were placed in shallow trays filled with water for 24 h to saturate the soil. All plants grown in infested soil showed extensive root necrosis and dieback ≈30 days after transplanting, and P. cryptogea was reisolated from roots of symptomatic plants. Control plants did not develop symptoms. Root and crown rot of sage caused by P. cryptogea has been reported previously in California (3). To our knowledge, this is the first report of P. cryptogea on sage in Italy. Root rot caused by P. cryptogea may be a potential problem for commercial cultivation of sage as no serious disease of this plant has been reported in Italy so far.

Phytophthora palmivora a New Pathogen of Lavender in Italy.

Root rot caused by Phytophthora nicotianae is considered the most serious disease of lavender in commercial cultivations in Italy. In summer 2001, in the Gela area (Sicily), ≈60% of 34,000 2-year-old landscape shrubs of English lavender (L. angustifolia) grown in a clay loam soil showed symptoms of dieback associated with root rot. Plants had been transplanted from pots in May and watered using a trickle irrigation system. A species of Phytophthora was isolated consistently from roots of symptomatic plants using potato dextrose agar (PDA) containing benomyl, nystatin, pentachloronitrobenzene, rifampicin, ampicillin, and hymexazol. The species was identified as P. palmivora on the basis of morphological and cultural characters. Ten representative single-zoospore isolates were characterized. On agar media, the isolates produced elliptical to ovoid, papillate sporangia, with a mean length/breadth ratio of 1.8. Sporangia, produced on sporangiophores forming simple sympodia (as many as 20 sporangia per sympodium), were caducous with a short pedicel (mean pedicel length = 5 μm) and a conspicuous basal plug. In addition to typical sporangia, all isolates produced sporocysts, i.e., subglobose, nonpapillate sporangia (2). The minimum temperature for mycelium growth on PDA was 10°C, the optimum was 27°C, and the maximum was 35°C. All isolates were A1 mating type. Antheridia were amphyginous. The identification was confirmed by electrophoresis of mycelial proteins on a polyacrylamide slab gel. Electrophoretic banding patterns of total soluble proteins and eight isozymes of the isolates from lavender were identical to those of a reference isolate of P. palmivora from olive (1). Conversely, the electrophoretic phenotype of the isolates from lavender was distinct from those of reference isolates of other species, including P. cactorum, P. capsici, P. citrophthora, P. nicotianae, and P. tropicalis. The pathogenicity of a representative isolate of P. palmivora from lavender was tested in the greenhouse using 6-month-old plants of English lavender, Rosea, a commercial cultivar very susceptible to root rot caused by P. nicotianae (3). Inoculum was produced on a mixture of vermiculite and autoclaved oat seeds (4) and mixed with soil (sand/lime/peat 1:1:1) at a concentration of 4% (vol/vol). Plants were transplanted to pots filled with infested soil. Control plants were grown in pots containing noninfested soil. After transplanting, all pots were flooded for 24 h by plugging the drain hole. Three months after transplanting all plants grown in pots containing infested soil showed extensive root necrosis and dieback symptoms. Control plants remained healthy. P. palmivora was recovered from roots of symptomatic plants. To our knowledge, this is the first report from Italy of P. palmivora on lavender. Root rot caused by P. palmivora may be a potential problem for commercial cultivation of lavender.

Collar and Root Rot of Olive Trees Caused by Phytophthora megasperma in Sicily.

Olive (Olea europea L.) is grown on about 154,000 ha in Sicily (southern Italy). In the summer of 1999, a few 3-year-old olive trees with decline symptoms were observed in a recently planted commercial orchard in the Enna province (Sicily). The trees were propagated on wild olive (O. europea L. var. sylvestris Brot.) rootstock. Aerial symptoms, consisting of leaf chlorosis, wilting, defoliation, and twig dieback followed in most cases by plant death, were associated with root rot and basal stem cankers. A Phytophthora sp. was consistently isolated from rotted rootlets and trunk cankers using the BNPRAH (benomyl, nystatin, pentachloronitrobenzene, rifampicin, ampicillin, and hymexazol) selective medium. Pure cultures were obtained by single-hypha transfers. The species isolated from symptomatic olive trees was identified as P. megasperma Drechsler on the basis of morphological and cultural characteristics. All isolates were homothallic, with paragynous antheridia. The diameter of oospores varied from 28 to 42 μm (mean ± SE = 36.3 ± 0.4) when they were produced on potato-dextrose agar (PDA) and from 30 to 43 μm (mean ± SE = 37.8 ± 0.4) when they were produced in saline solution. Sporangia were non-papillate. Optimum and maximum temperatures for radial growth of the colonies on PDA were 25 and 30°C, respectively. At 25°C, radial growth rate was about 6 mm per day. The identification was confirmed by the electrophoresis of mycelial proteins on a polyacrylamide slab gel. The electrophoretic banding patterns of total soluble proteins and three isozymes (esterase, fumarase, and malate dehydrogenase) of the isolate from olive were identical to those of two isolates of P. megasperma obtained from cherry and from carrot in Italy and characterized previously (1). Conversely, they were clearly distinct from the electrophoretic patterns of four isolates of P. megasperma var. sojae Hildebr. from soybean (= P. sojae Kauf. & Ger.), from those of three isolates from asparagus tentatively identified as P. megasperma sensu lato (1) and from those of reference isolates of various species producing non-papillate sporangia, including P. cambivora (Petri) Buisman, P. cinnamomi Rands, P. cryptogea Pethybr. & Laff., P. drechsleri Tucker, and P. erythroseptica Pethybr. Pathogenicity of the isolate from olive was tested in the greenhouse at 18 to 25°C using 18-month-old rooted cuttings of olive cv. Biancolilla. Cuttings were inoculated on the lower stem by inserting a 3-mm plug taken from actively growing colonies on PDA into an incision made with a sterile scalpel. The wound was sealed with waterproof tape. Agar plugs with no mycelium were placed into the stem of cuttings used as a control. The bark was stripped and lesion areas were traced and measured 60 days after inoculation. The isolate from olive produced a brown necrotic lesion (mean size = 500 mm2) around the inoculation wound and was reisolated from the lesion. Conversely, the wound healed up on control plants. P. megasperma has previously been recognized as a pathogen of olive in Greece and Spain (3). However, this is the first report of P. megasperma causing root and collar rot of olive in Italy.

First Report of Root Rot Caused by Phytophthora cinnamomi on Avocado in Italy.

Root rot caused by Phytophthora cinnamomi Rands is generally recognized to be the most important disease of avocado (Persea americana Miller) wherever this tropical fruit tree is grown (3). The disease was first found in Italy in the spring of 1998. Eight-year-old trees, with symptoms ranging from initial to severe, were observed in an experimental field near Rocca di Caprileone, in Sicily. Few trees showed symptoms of both root rot and collar rot. Infected trees were of 13 commercial cultivars. Trees were grafted on two different rootstocks: Hass seedlings and G6 seedlings. G6 is a selection reported to have some field resistance to P. cinnamomi infections (2). However, no correlation was observed between symptom severity and rootstock. P. cinnamomi was isolated on BNPRAH selective medium (4) from trunk bark, feeder roots, and rhizosphere soil of diseased trees, and from roots of symptomless trees. The isolates, identified primarily on the basis of morphological and cultural characteristics, formed rosaceous colonies on potato dextrose agar (PDA) and on corn meal agar (CMA) coralloid-type mycelium, with abundant hyphal swellings, which were typically spherical and in clusters. Chlamydospores were either terminal or intercalary, and often occurred in characteristic grapelike clusters. Sporangia, which were produced in saline solution (1), were broadly ellipsoidal or ovoid, persistent, non-papillate and proliferous. The identification was confirmed by the electrophoresis of mycelial proteins on polyacrylamide slab gel. The electrophoretic patterns of total soluble proteins and eight isozymes (AKP [alkaline phosphatase], EST [esterase], FUM [fumarase], GLC [NAD-glucose dehydrogenase], G6PD [glucose-6-phosphate dehydrogenase], LDH [lactate dehydrogenase], MDH [malate dehydrogenase], and SOD [superoxide dismutase]) of the isolates from avocado were identical to those of two strains of P. cinnamomi, used as reference (isolate 70473 from International Mycological Institute, U.K., and an isolate from myrtle from the Institute of Plant Pathology, University of Catania, Italy). Conversely, the electrophoretic phenotype of the P. cinnamomi isolates from avocado was clearly distinct from those of reference strains of eight other species included in Waterhouse's taxonomic group VI. Pairings with isolates of a known mating type of P. cinnamomi, P. cryptogea, and P. drechsleri revealed that all the isolates from avocado were A2 mating type. It is possible that P. cinnamomi had been introduced into the experimental field on infected symptomless nursery trees. In Italy, root rot caused by P. cinnamomi could have a significant impact on commercial avocado plantings extending over about 20 ha. Moreover, this polyphagous pathogen may be a threat to other crops as well as to forest trees.

Production of nisin-like bacteriocins by Lactococcus lactis strains isolated from vegetables.

Four bacteriocin producing lactic acid bacteria isolated from vegetables were identified as Lactococcus lactis strains on the basis of physiological and biochemical characteristics, carbohydrate fermentation patterns and analysis of total soluble protein pattern by SDS PAGE. The bacteriocins had a wide spectrum of activity as antagonism was detected not only towards a variety of lactic acid bacteria, but also to Staphylococcus aureus and Listeria monocytogenes. These bacteriocins were resistant to heating at 121 degree C for 15 minutes and showed highest activity at low pH (<5.0). They were inactivated by the proteolytic enzymes alpha-chymotrypsin and proteinase K, but not by lipase, alpha-amylase, catalase or lysozyme. These bacteriocinogenic Lactococcus strains were all immune to the bacteriocins produced as well as to commercial nisin. Bacteriocin producer culture supernatants showed a high degree (70 or 100%) of cross-reactivity in the nisin ELISA, suggesting similarity of the produced bacteriocins to nisin. The potential application of bacteriocin producing lactococci of vegetable origin for safety assurance of vegetable foods and controlling vegetable fermentations is discussed.

Classification of Pythium ‘group F’ based on mycelial protein and isozyme patterns

Taxonomic characteristics were compared among 10 isolates ofPythium ‘group F’ in tems of the electrophoretic patterns of their mycelial proteins and isozymes. These isolates were obtained from water of three ponds in different seasons and have an identical morphology of zoosporangia. Attempts to cross the isolates with each other themselves and with other isolates from the same group failed.Pythium ‘group F’ is the most dominant of the pythia in the aquatic ecosystem and is difficult to identify because of the lack of sexual reproductive structures. Isozyme analysis proved useful in this respect. Comparisons of banding patterns of total soluble proteins and isozymes revealed five subgroups inPythium ‘group F’. Two isolates obtained from water of different ponds in different seasons showed the same protein and isozyme patterns. Our findings indicate that the use of total soluble protein and isozyme patterns for determining the variation withinPythium ‘group F’ could become a valuable adjunct to the morphological and physiological criteria.

Plantlet Regeneration and Novel Protein Synthesis in a Long-term Cultured Callus of Rice in Response to Abscisic Acid.

Some physiological changes induced by abscisic acid (ABA) were investigated in a long-term cultured callus of rice, Oryza sativa, L. cultivar YUHKARA. The results were as follows : (1) The retardation of callus growth was evident, while dry matter content was apparently increased with the addition of abscisic acid (ABA). (2) Soluble protein content was increased both in fresh weight and dry matter bases. (3) The novel proteins (14, 18.5, 25, 45 kDa) were induced by ABA and disappeared after plantlet regeneration, while proteins of the same molecular weight existed in intact mature seed embryos and disappeared when the seeds were germinating. (4) Moreover, the electrophoretic patterns of total soluble proteins from callus being cultured with ABA, and from callus with regenerating plantlets, were very similar to those of total soluble proteins from mature seed embryos and germinating seed embryos in SDS polyacrylamide gel plates. (5) The practice that the callus precultured with addition of ABA, especially 10 mgL-1 ABA, followed by transferring to a regeneration medium gave us a high frequency of plantlet regeneration observed in the long-term culture of callus.

Stability of Markers Used for Identification of Two Rhizobium galegae Inoculant Strains after Five Years in the Field

The stability of identification markers was examined for two Rhizobium galegae inoculant strains after 5 years in the field. The two strains are genetically closely related, but differ in their lipopolysaccharides. Strain HAMBI 540 has lipopolysaccharide of the rough type, whereas that of strain HAMBI 1461 is of the smooth type. The properties that were examined for 10 field isolates of each inoculant type were symbiotic phenotype, phage type, intrinsic antibiotic resistance, maximum growth temperature, lipopolysaccharide and total soluble protein patterns, immunological properties, DNA restriction profiles, and DNA hybridization patterns, which were determined by using nifHDK and recA sequences as probes. Of these properties, all remained stable in soil, with the exception of some variation in intrinsic antibiotic resistance and the acquisition of an extra EcoRI restriction fragment by one of the isolates. Thus, both the rough and the smooth lipopolysaccharide phenotypes persisted equally well in soil.