Esterase Isozyme Patterns Research Articles

In vitro mutagenesis and cell selection for the induction of black rot resistance in cauliflower

SummaryMutagenized (EMS and gamma ray treated) calli of cauliflower (Brassica oleracea var. botrytis) were screened for resistance against the culture filtrate of black rot pathogen Xanthomonas campestris pv. campestris. Survival of calli decreased with the increase in the concentration of culture filtrate and calli surviving at 30% level of culture filtrate were selected. Plants were regenerated from selected and unselected calli and investigated for isozyme pattern of esterase, peroxidase, catalase and ascorbate oxidase. Esterase isozyme pattern showed significant differences between the plants derived from selected and unselected calli and between those derived from EMS treated selected calli and gamma ray treated selected calli. In addition a high level of correlation was observed between resistance of calli to the culture filtrate and the resistance of regenerated plants to the pathogen.

Total protein and isozyme characterization in the flax zygotic embryo during development

Flax zygotic embryogenesis was studied for isozyme patterns of acid phosphatase and esterase and for content of total proteins. For acid phosphatase, six multiple molecular forms or isozymes, were identified during flax zygotic embryo development. For esterase, six isozymes were expressed during zygotic flax embryogenesis. Some of the isozymes were expressed during entire embryogenesis, and some were only transiently expressed or appeared near maturation. The amount of total proteins was very low at early embryogenesis and proteins in a range from 26.6 kDa to 97.4 kDa were expressed. During further flax embryo development the protein spectra became more diverse and the quantity of total proteins increased. Proteins ranging from 3.4 kDa to 97.4 kDa were present, and proteins of 20 kDa to 36.5 kDa were expressed in the highest amount. The results are discussed with reference to selected information in the literature.

Differential esterase expression in developmental mutants of Aspergillus nidulans.

Esterase isozymes were used to detect substrate-preference polymorphism in five strains of Aspergillus nidulans, and to show differential gene expression in developmental mutants in response to 5-azacytidine treatment. The medusa mutants B116, SM23, and M25 were selected in the presence of 5-azacytidine (5AC); also the G839 bristle mutant obtained in the absence of 5AC as well as the UT196 master strain and the normal segregant SM24 were used for the esterase studies. The esterase isozyme patterns of the A. nidulans strains observed with 4-methylumbelliferyl esters and alpha- and beta-naphthyl esters indicated a total of 18 isoesterases. Substrate preference for either 4-methylumbelliferyl esters and alpha- or beta-naphthyl esters was observed. Similarity between the different A. nidulans genotypes was 84.4-100%. The genomic similarity of the B116, SM23, and M25 mutant strains (100%) supports previous observations that specific DNA sequences might be targets for 5AC action in this filamentous fungus, and the differential expression of the Est-4 isozyme in the medusa developmental mutant and the Est-2 isozyme specifically detected in the bristle mutant G839 seems to indicate esterase isozymes as possible markers of biochemical differences among different developmental mutants of A. nidulans.

Detection and analysis of inter- and intraspecific diversity of Pratylenchus spp. using isozyme markers

The root-lesion nematodes of the genus Pratylenchus are an economically important group of plant parasitic nematodes that show high similarity among sibling species. Isozyme patterns obtained by isoelectrofocusing (IEF) were used to differentiate and establish genetic relatedness among Pratylenchus species. A total of 40 populations comprising 9 Pratylenchus species and Radopholus similis from broad host and geographic origins was examined to compare isozyme patterns of esterase (EST), hexoquinase (HK), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), phosphoglucose isomerase (PGI), phosphoglucomutase (PGM) and superoxide dismutase (SOD). Of these systems, only EST, MDH, PGI and PGM were useful for differentiation of P. vulnus, P. goodeyi, P. penetrans, P. scribneri, P. thornei and R. similis populations. The greatest intraspecific diversity was found within P. coffeae based on the isozyme patterns for MDH, PGI and PGM. Intraspecific variability was also detected among R. similis populations, which showed two isozyme patterns in EST and PGI systems. Less intraspecific variation was found within the P. penetrans group. The P. goodeyi population from Cameroon differed from the other populations in this specific group in its MDH, PGI and PGM phenotypes. Highly similar banding patterns of EST, MDH and PGI activity were found among the P. scribneri populations and the one population of P. agilis. A cluster analysis of the 40 populations, generated from the four enzyme banding patterns, produced groupings that broadly matched the previous classification into specific groups, reflecting intraspecific variability in some cases. The results confirm the potential use of isozyme patterns as markers for these nematode species and their value for diagnostic application.

Changes in Isozyme Patterns During in Vitro Regeneration from Cotyledon Explants of Brassica Species

High frequency shoot regeneration from cotyledons excised from 4-d-old seedlings of Brassica campestris L. cv. M 27 and Brassica juncea (L.) Czern. cv. Pusabold was achieved on Murashige and Skoog's (MS) medium supplemented with 1.0 mg dm-3 N6-benzyladenine (BA) and 3 % (m/v) saccharose. Rooting occurred simultaneously with shoot formation on the medium containing 1.0 mg dm-3 BA and 0.5 mg dm-3 1-naphthaleneacetic acid. Cultures of cotyledon, cotyledon derived non-differentiating calli and differentiated calli with shoots and/or roots were analysed at different intervals for isozyme patterns of esterase and peroxidase. With the BA-induced development of shoot and/or root from non-differentiating callus, some conspicuous isozymes appeared which indicates the involvement of these isozymes in root and shoot development rather than in induction of morphogenesis in callus.

A study of resources of Fagopyrum (Polygonaceae) native to China

Morphology, taxonomy, reproductive biology, esterase isozyme patterns and chromosome number were studied in 16 accessions of cultivated and wild buckwheat native to Tibet, Sichuan, Guizhou and Yunnan. Among these are four established species Fagopyrum esculentum, F. tataricum, F. pleioramosum and F. gracilipes . The first three species are diploid (2 n=16) and the last one is tetraploid (2 n=32). In addition to the above, three new species are described F. zuogongense, F. megaspartanium and F. pilus . There are clear differences in esterase isozyme patterns between these species. The zymographs of esterase isozyme of diploid F. megaspartanium (native to Sichuan) and F. pilus (native to Tibet) are similar to those of F. esculentum and F. tataricum respectively. F. gracilipes , in the small achene group of the genus, has a zymograph of esterase isozyme that is very different from the species in the large-achene group. The diploid accessions of F. pilus and F. megaspartanium may be ancestors of cultivated buckwheat.

A study of resources of Fagopyrum (Polygonaceae) native to China

Morphology, taxonomy, reproductive biology, esterase isozyme patterns and chromosome number were studied in 16 accessions of cultivated and wild buckwheat native to Tibet, Sichuan, Guizhou and Yunnan. Among these are four established species: Fagopyrum esculentum, F tataricum, F. pleioramosum and F gracilipes. The first three species are diploid (2n = 16) and the last one is tetraploid (2n = 32). In addition to the above, three new species are described: F. zuogongense, F. megaspartanium and E pilus. There are clear differences in esterase isozyme patterns between these species. The zymographs of esterase isozyme of diploid F. megaspartanium (native to Sichuan) and F. pilus (native to Tibet) are similar to those of F. esculentum and F tataricum respectively. F. gracilipes, in the small achene group of the genus, has a zymograph of esterase isozyme that is very different from the species in the large-achene group. The diploid accessions of F. pilus and F. megaspartanium may be ancestors of cultivated buckwheat.

Molecular Characterisation of Low ODAP Somaclones of Lathyrus sativus

Somaclones of Lathyrus sativus having low ODAP contents were characterised at molecular level with respect to soluble protein pattern, esterase and ADH isozyme pattern, Southern hybridisation of genomic and mitochondrial DNA with specific probes. In 10-day-old seedling esterase isozyme pattern showed the presence of an unique band of Rm 0.73 in Bio L-12 and 15-8-1 and the absence of a band of Rm 0.80 in 15-8-1, as compared to parent and other somaclones. Southern hybridisation of genomic DNA with cDNA clone 29 showed an extra band of 13.5 kb in L-56 and 15-8-1 only. Hybridisation of mitochondrial DNA with mitochondrial specific ati ATPase probe showed differences with the pattern from Bio R-15 being unique. Some of these differences observed will be useful as marker for their identification.

Influence of phosphorus and formononetin on isozyme expression in the Zea mays‐Glomus intraradices symbiosis

Maize (Zea mays L. cv. Great Lakes 586) plants were either inoculated with the vesicular‐arbuscular mycorrhizal (VAM) fungus Glomus intraradices Schenck and Smith, or grown in the presence of the isoflavone formononetin or were provided with both G. intraradices and formononetin. All plants were grown in soil containing one of five levels of inorganic P (between 8 and 110 µg g−1 soil). By 3 weeks there were significant differences in a number of enzyme activities and in the pattern of isoenzymes in roots colonized by the VAM fungus or treated with formononetin. One NAD‐malate dehydrogenase (MDH) isozyme was expressed only in mycorrhizal roots, whether treated or not with formononetin. Despite differences in the soil P level, the expression of this isozyme was not observed in non‐mycorrhizal roots, indicating specific expression in the mycorrhizae. We suggest that MDH isozyme could serve as a specific, early indicator of the Zea‐Glomus symbiosis. Differences in the esterase (EST) isozyme pattern were not detectable between VAM and non‐VAM roots, suggesting that this enzyme system is not a good parameter for the evaluation of mycorrhizal colonization. As available P in the soil increased, total EST activity appeared to increase as well. Interestingly, total peroxidase (POX) activity increased along with P suggesting that as plant P nutrition improved, both cell wall ramification and the quantity of defense peroxidases increased as well. Total POX activity from mycorrhizal roots was inversely correlated with root colonization, indicating that there was suppression of POX activity by the host under low soil P. Most interestingly, formononetin further decreased POX activity regardless of the level of P or mycorrhizal status. This may suggest one mechanism by which formononetin enhances root VAM colonization. The presence of this isoflavone suppressed POX activity in mycorrhizal roots allowing a rapid penetration and spread of the fungus in the root cortex. The interplay between host root, soil P levels, secondary metabolites and endogenous host enzyme activities and a particular VAM fungus has a profound effect on the efficiency, duration and functioning of an endomycorrhizal symbiosis.

Redescription of the Root-Knot Nematode Meloidogyne Maritima Jepson, 1987 (Nematoda: Heteroderidae), a Parasite of Ammophila Arenaria (L.) Link

The root-knot nematode, Meloidogyne maritima, a parasite of marram grass (Ammophila arenaria), is redescribed from the type locality, a coastal dune near Perranporth, UK. This species is characterized by second-stage juvenile 471 μm long, hemizonid posterior to excretory pore, tail 71.5 μm long, hyaline tail terminus indistinct; male head tapering, not set off, labial disc elevated, rounded and fused with crescent shaped medial lips, lateral lips present, stylet 20.5 μm long, with relatively small rounded knobs, slightly sloping backwardly, lateral field areolated; female stylet curved dorsally, 14.2 μm long, with rounded to transversely ovoid shaped knobs, slightly sloping backwardly, excretory pore located near level of stylet knobs, perineal pattern relatively small, rounded to ovoid and dorsal arch low with lateral field distinct. A malate dehydrogenase pattern, N1a type, and a VS1-S1type of esterase isozyme pattern were detected. Meloidogyne duytsi Karssen et al., 1998, was found on Elymus furctus in the foredunes of the type locality.

Sclerotinia nivalis, sp. nov., the pathogen of snow mold of herbaceous dicots in northern Japan

A new Sclerotinia, previously reported as S. intermedia from Japan, is described as Sclerotinia nivalis on the morphological basis of the sclerotial anamorph and teleomorph produced in culture. The characters assigning this species to the genus Sclerotinia are the tuberoid sclerotia superficially produced on suscepts, the small sclerotia produced on aerial mycelium in culture, the interhyphal spaces in medullary tissue of sclerotia, and the globose cells constructing the ectal excipulum of apothecia. It is distinguishable from S. sclerotiorum, S. minor, and S. trifoliorum by the intermediate sized sclerotia in culture, binucleate ascospores, the molecular mass of major proteins of sclerotia, and the patterns of esterase isozymes in sclerotial extracts. Although S. nivalis causes snow mold of various dicots, it is a mesophile having an optimum temperature for mycelial growth of around 20° It attacks edible burdock (Arctium lappa), Chryhsanthemum morifolium, Ambrosia elatior, carrot (Daucus carota), Angelica acutiloba, Ajuga reptans, and Plantago lanceolata.

Immunological Detection of Greenbug (Schizaphis graminum) Esterases Associated with Resistance to Organophosphate Insecticides

Organophosphate resistance in the greenbug,Schizaphis graminum,has been at least partially attributed to two distinct patterns of nonspecific esterase isozymes (Type I and II). Antiserum raised against a purified Type II esterase was specific for this enzyme and did not cross-react with the Type I esterase, indicating that the two greenbug esterases are immunologically distinct. The antiserum reacted identically with the Type II esterase in both resistant and susceptible insects based on both immunoblots and quantitative immunoelectrophoresis despite the much higher activity of this isozyme associated with the resistant strain. These results indicate that the isozyme is immunologically indistinguishable in resistant and susceptible insects and that the resistance appears to involve a modification of the enzyme rather than overexpression. These results contrast with those obtained for the Type I esterase, which gave a strong reaction with an antiserum against the E4 esterase associated with resistance in the green peach aphid,Myzus persicae.Western blot analysis indicated that the Type I esterase is present at much higher levels than in the susceptible insects and is likely to involve a mechanism resulting in overexpression of the enzyme.

Differential expression of esterase and MDH isozymes during in vitro culture in maize (Zea mays L.)

Isozyme patterns of esterase and malate dehydrogenase were analyzed at different stages of in vitro culture of immature embryos and glumes of Zea mays L. viz. explant, callus formation, root formation and shoot formation. Significant changes in isoenzyme patterns of esterase and MDH were observed besides the appearance of specific and new isozymes. Specific fast migrating isozymes were noted in differentiating calli of embryo and glume calli which were absent at other stages suggesting a possible association of these isozyme patterns with in vitro differentiation.

Proteins and esterase isozymes during developmental stages of organophosphate resistant strains in Anopheles stephensi liston

The proteins and esterase isozymes from Anopheles stephensi were analysed by polyacrylamide gel electrophoresis. Changes in proteins and esterase isozyme patterns during developmental stages were studied. A few specific protein bands were present especially in the third instar larval stage. In most strains, eggs showed only one esterase band. During the larval development many bands were gradually formed and most anodal bands were lost upon pupation. The adults showed differences in intensity and mobility exhibiting sexual dimorphism. The protein and esterase isozyme patterns of organophosphate (fenthion, methyl parathion and malathion) resistant and susceptible strains during developmental stages were compared. The zymogram and relative mobility showed variation in the number of intensity of proteins and esterase bands in the resistant strains as compared to the susceptible strains.

Geographical Distribution of Meloidogyne Species (Nematoda: Tylenchida) in Tobacco Fields of Japan

The distribution of root-knot nematodes was surveyed in tobacco fields of Japan. In 1992 and 1994, 219 populations of root-knot nematodes in tobacco fields covering the northern most area of Honshu to the Ryukyu Is. were identified on isozyme patterns of esterase and malate dehydrogenase. In this work, four Maloidogyne species, M. incognita, M. arenaria, M. hapla and M. javanica, were found. In M. arenaria, two distinct esterase phenotypes, one (A-1) and two (A-2) band types, were found. Meloidogyne incognita was widely found from southern Tohoku to the Shinetsu districts and southward to the Ryukyu Is. This species was recorded for the first time in Gifu, Nagano, Fukushima, Yamagata and Miyagi Prefs. Meloidogyne arenaria (A-2) was widely found from the Ryukyu Is. to Akita and Iwate Prefs., however, its distribution in Kyushu and the Ryukyu Is. was patchy. This species was found for the first time in Akita and Iwate Prefs. Meloidogyne arenaria (A-1) was mainly concentrated in Kyushu and the Ryukyu Is., and was found in one locality in Shikoku. Meloidogyne hapla was distributed from Shinetsu to the Kanto distinct northward to Aomori Pref. Meloidogyne javanica was found only in the Ryukyu Is. In areas where the distribution of these species overlapped, two or three species were often found in a single field.

イネ寄生およびマコモ寄生ニカメイガの交尾時刻の違いについて

The Rice Stem Borer, Chilo suppressalis (WALKER), is a serious pest of rice plants in Asia. In Japan, host plants of are mainly rice plants (Oryza sativa) and the water-oat (Zizania latifolia). Our previous study indicated significant differences in insecticide susceptibility and esterase isozyme patterns between the rice-feeding and water-oat-feeding strains. In the present study, the mating time of the two strains was investigated in the laboratory. The mating time was significantly different. The peak of mating time of the rice-feeder was 5h earlier than that of the water-oat-feeder, suggesting that the two strains are reproductively isolated.

Characterization of General Esterases from Susceptible and Parathion-Resistant Strains of the Greenbug (Homoptera: Aphididae)

A susceptible and two parathion-resistant strains of the green bug, Schizaphis Graminum (Rondani), exhibit three different patterns of general esterase isozymes in native polyacrylamide electrophoresis gels. Characterization of general esterase activity using a-naphtholic esters as model substrates indicated that the three strains differed in isozyme composition. The type-II strain, which had the highest level of resistance, exhibited the highest levels of general esterase activity under all assay conditions, and the type-I strain had consistently higher levels than the susceptible strain. In all three strains, these esterases were more active toward a-naphtholic esters with side chains of six or fewer carbon atoms. a-Naphthyl propionate was the optimal substrate for both susceptible and type-I strains, and a-naphthyl butyrate for type II. Over 90% of esterase activity was localized in the cytosolic fraction of type-II greenbugs. In susceptible and type-I greenbugs, the activity was distributed equally between the cytosolic and microsomal fractions. Differences in kinetic properties of the general esterases from the three strains also were evident, further indicating differences in isozyme composition. Although the three strains differed in properties of the general esterase activities, the differences do not provide sufficient discrimination to distinguish reliably among the three strains using single aphid activity measurements.

A combination of molecular and morphological characters for delimitation of taxa in EuropeanParella

The merits of morphological characters, isozyme patterns and random amplified polymorphic DNA (R.A.P.D.) markers for bryophyte species delimitation were investigated using European species of Porella. Isozymes and R.A.P.D. markers allow rapid detection of genetic differences in morphologically highly-variable groups of organisms. Twenty-two clones of the European Porella species (eight species are currently recognised: P. arborisvitae, P. baueri, P. canariensis, P. cordaeana, P. obtusata, P. pinnata, P. platyphylla, P. platyphylloidea) were investigated together with three populations of Canadian P. platyphylloidea. Isozyme patterns of esterase, glutamate-oxaloacetate transaminase, peroxidase and malate enzyme were studied by electrophoresis. R.A.P.D. fingerprints were made using the polymerase chain reaction and six primers. Data were also obtained for 39 morphological characters scored for ten or more individuals of each population. Eight distinct isoenzyme patterns were obtained corresponding to the seven species and aberrant population 267. These results were supported by the R.A.P.D. analysis which showed population 267 to be close to P. platyphylla but also to possess affinities with P. platyphylloidea. The analysis allowed a reassessment of relationships between the European Porella species and the definition of improved morphological character sets for discriminating the taxa.

Interspecific somatic hybrids between Solanum khasianum and S. aculeatissimum produced by electrofusion

Mesophyll derived protoplasts of Solanum khasianum Clarke and S. aculeatissimum Jacq. were fused by electrofusion and resulted in interspecific hybrids. In total, 249 calluses were obtained from which more than 65% produced shoots. From the regenerated plantlets 45% could be identified as hybrids by chromosome counts (4x = 48) and esterase isozyme pattern. Most hybrids showing intermediate phenotypic characteristics, had a normal flower morphology with fertile pollen and produced fertile berries. More than 80% of the seeds germinated.

Carbon nutrition and hydrolytic and cellulolytic activities in the ectomycorrhizal fungus Pisolithus tinctorius

Four strains of an ectomycorrhizal fungus, Pisolithus tinctorius, were investigated for carbon nutrition, and for production of hydrolytic and cellulolytic enzymes. Glucose, mannose, and cellobiose supported rapid mycelial growth of all four strains. Fructose was utilized by two strains, SMF and S359. Of the 10 hydrolytic enzymes examined, acid phosphatase, acid α-galactosidase, acid esterase, and acid β-glucosidase were found in all four strains. β-Galactosidase was only observed in strain S359. α-Mannosidase, β-mannosidase, α-glucosidase, β-xylosidase, and proteinase were not detected in any of the four strains. Isozyme patterns of β-glucosidase and esterase in the four strains were compared by activity staining after native gradient gel electrophoresis. The isozyme pattern of β-glucosidase showed three major forms in all four strains. In addition, two more isoforms were found in strain S370. All strains shared two esterase bands, while strain S370 had three more isoforms. Study on strain SMF indicated that acid β-glucosidase was expressed constitutively, with increased activity in cellobiose-containing media. Under nitrogen-limiting conditions, a low level of endoglucanase and exoglucanase activity was observed in strains SMF and S359. Further study on S359 showed that high concentrations of nitrogen repressed the cellulolytic activity. When cellobiose served as carbon source, higher cellulolytic activity was observed. Cellulose did not induce higher activity.Key words: Pisolithus, ectomycorrhizal, β-glucosidase, hydrolytic enzymes, cellulolytic enzymes.