Esterase Bands Research Articles

Characterization of the host genotype and fungal diversity in Scots pine ectomycorrhiza from natural humus microcosms using isozyme and PCR‐RFLP analyses

SUMMARYThe early development of indigenous and introduced Scots pine (Pinus sylvestris L.) ectomycorrhiza in natural forest humus was examined using molecular fingerprinting techniques. Non‐mycorrhizal or mycorrhizal (Suillus bovinus (L. ex Fr.) O. Kuntze or Paxillus involutus (Batsch ex Fr.) Fr.) seedlings were placed in transparent two‐dimensional microcosms on a thin layer of sieved humus to allow observation of ectomycorrhizal development and fungal growth. Twelve ectomycorrhizal morphotypes, mainly based on colour, gross morphology and outer mantle structure, were identified over a 3‐month period. No successional trends in mycorrhiza formation were observed. For further characterization, individual ectomycorrhiza representing the different morphotypes were subjected to analyses of esterase (EST) isozyme profiles and RFLPs of the internal transcribed spacer (ITS) from fungal rRNA genes following amplification using PCR. All 10 morphotypes analysed displayed different esterase isozyme profiles, and the characteristic s. bovinus species diagnostic band (S. b. EST8) was detected in two white morphotypes. Ten Pink and Black ectomycorrhiza were all separated into one and three groups, respectively. A fast‐running plant‐specific polymorphic locus (Z) was also confirmed in most ectomycorrhizal morphotypes. Diagnostic species‐specific (S. bovinus and Pink) EST bands were detected in intact external mycelium colonizing soil. Successful amplification of the ITS from individual ectomycorrhiza, of eight different morphotypes, was found to be mainly influenced by the DNA template concentration. Different RFLPs (Hinf I, Mbo I and Hha I) of the ITS placed the white morphotypes into two groups, one corresponding to S. bovinus, in agreement with the esterase fingerprinting. By contrast, Pink and Beige morphotypes displayed different EST profiles but nearly identical RFLP fingerprints. The ITS amplification success rate of standardized DNA template concentrations from 10 individual Pink and Black ectomycorrhiza was 90 and 50%, respectively. In all successful amplifications, the black morphotype yielded the smallest ITS fragment, similar to earlier reports for Cenococcum geophilum, that gave identical RFLPs. The ecological significance of the observed mycorrhizal diversity and combined application of these two identification methods is discussed.

Biotypes of the castor bean whitefly Trialeurodes ricini (Misra) (Hom., Aleyrodidae) in Egypt: biochemical characterization and efficiency of geminivirus transmission

AbstractThe newly introduced castor bean whitefly (CBW) Trialeurodes ricini (Misra) has become an important pest which is widely spread all over Egypt. Biochemical studies were conducted to distinguish the different T. ricini biotypes and to determine the biotype economic threshold based on its ability to transmit plant viruses. Forty‐five samples of T. ricini adults were collected from castor bean (Ricinus communis) at different geographical locations representing the Egyptian Delta and Nile valley governorates. Esterase banding patterns were investigated to evaluate the extent of variation among the tested populations of the castor bean whitefly. Electrophoresis of homogenates of individual T. ricini females showed that there are five different biotypes (C1–C5). The predominant biotype (C1 type) was found in about 80% of the tested locations. In transmission studies, the polymerase chain reaction (PCR) was used to detect geminiviruses in nucleic acid extracted from the different castor bean whitefly biotypes. PCR revealed that the five biotypes of T. ricini are viruliferous. To evaluate the efficiency of the predominant T. ricini biotype for transmitting the tomato yellow leaf curl virus (TYLCV), acquisition and inoculation access periods were determined. PCR indicated that 50% of whitefly adults collected from castor bean, acquired the virus after 10 h exposure to TYLCV infected tomato. Experimentally, the rate of transmission increased as the inoculation access period was lengthened and reached a maximum after 4 h. The obtained symptomatological results were also confirmed by PCR.

Isozyme polymorphism of esterases in the genus Lanistes (Mollusca:Prosobranchiata) and genetic analysis of populations.

Esterases from the digestive gland of the snails Lanistes carinatus and Lanistes bolteni collected from Egyptian governorates were extracted and analyzed using starch gel electrophoresis and five substrates. Twelve esterase bands were detected in both Lanistes species. The esterase bands were distributed in three main zones, which could be classified as acetylesterases, carboxylesterases, and cholinesterases. Depending on the substrates specificity, inhibition properties, and relative mobility of esterase bands, the three zones of esterase activity could be traced to eight genetic loci. Locality-specific loci were found. Inter- and intrapopulation variations are discussed. There is an absence of equilibria at all esterase loci in all populations studied, and a high proportion of genetic diversity in different esterase loci. The absence of interspecific variations proves that Lanistes snails in Egypt belong to one species.

Isozyme Variation among Isolates of Mucor piriformis

Isozyme polymorphisms among 59 isolates of Mucor piriformis from pears and nectarines were examined by vertical polyacrylamide gel electropho- resis. Among the enzyme systems tested, six enzyme activities, catalase, cx-esterase, glucose-6-phosphate de- hydrogenase, lactate dehydrogenase, malate dehy- drogenase and superoxide dismutase were used for detailed analyses. The variability of isozyme patterns revealed seven electrophoretic phenotypes for all the enzymes. No distinguishing morphological features were associated with the different electrophoretic phenotypes. In contrast, there was a correlation be- tween esterase, glucose-6-phosphate dehydrogenase and malate dehydrogenase banding patterns and the mating potency of the isolates from pears; those with mating abilities (plus and minus isolates) were clearly different from those without mating abilities (neutral isolates). In fruit inoculation pathogenicity studies, the plus and minus isolates were equally pathogenic to the neutral isolates. The 30 isolates from nectar- ines proved to be very homogeneous genetically, they did not reveal any polymorphisms for the 6 enzymes investigated. The potential application of the isozyme analysis to provide biochemical markers for Mucor species is discussed.

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.

Electrophoretic Evaluation of Esterase Isozymes from Turfgrass Seed Blends and Mixtures

Turfgrass seeds are commonly blended to improve performance. However, once physically blended, no fast, reliable technique is available to identify the cultivars and the proportion of each cultivar in the blend. The objective of this study was to isolate specific species and cultivars in seed blends or mixtures through examination of esterase banding patterns on electrophoretic gels, and to compare band intensities unique to each cultivar or species when blended in increasing or decreasing proportions. Two cultivars of Kentucky bluegrass (Poa pratensis L.) were blended by weight in proportions ranging from 0 to 100%. Protein extracted, separated on isoelectric focusing gels (pH gradient 3 to 9) and stained for esterase activity. Mixtures of fine fescue (Festuca spp.) species only and mixtures of both fine fescue species and Kentucky bluegrass cultivars were prepared in the same manner. Gels were visually analyzed for the presence of characteristic bands unique to each cultivar or species and for intensities of characteristic bands when cultivar proportions were varied within a seed blend or mixture. The results indicated that visual discriminations could be made between cultivars and species in turfgrass blends or mixtures and that bands unique to a cultivar or species varied in intensity with their concentration in a blend or mixture. These findings have importance to seed companies and consumers interested in monitoring the composition of seed blends and mixtures subsequent to physical mixing.

Description of Three Isozynte Polyntorphisms Associated with Insecticide Resistance in Green bug (Homoptera: Aphididae) Populations

Esterases have been associated with insecticide resistance in several aphid species. This report documents existence of 3 est erase polymorphisms associated with insecticide resistance in the green bug, Schizaphis graminum (Rondani). Est erases were resolved using polyacrylamide gel electrophoresis and Rf values were calculated for the 6 est erase bands that resolved. The 3 est erase polymorphisms observed have been named no noticeable elevated est erase, pattern 1 and pattern 2. Est erase bands visible on the polyacrylamide gel appear to have no elevated activity in the no noticeable elevated est erase strain. A single est erase band appears to be elevated in pattern 1 individuals and 3 est erase bands appear to be elevated in pattern 2. After testing >;10,000 green bugs, no individual green bug has been detected with both pattern 1 and pattern 2 banding. It is unknown whether the 2 elevated est erase forms are allelozymes or isozymes. Surface-residue bioassay data are presented that support the hypothesis that these elevated est erases may be important in insecticide resistance.

Esterases and Esfenvalerate Resistance in AustralianHelicoverpa armigera(Hübner) Lepidoptera:Noctuidae

Larvae of pyrethroid-resistant AustralianHelicoverpa armigerahave enhanced esterase activity which is due to increased production of the enzymes. The most resistant individuals have approximately a 50-fold increase in esterase activity compared to susceptible populations. ResistantH. armigerahave additional esterase bands which are not detected in susceptible individuals. Increased esterase hydrolysis of 1-naphthyl acetate was correlated to the esfenvalerate resistance factor. Esterase bound readily to esfenvalerate. Homogenates of resistant insects hydrolyzed esfenvalerate and it is also likely that there was significant detoxification by sequestration. The prospect of biochemical assays for pyrethroid resistance and the possible genetic basis of the esterase resistance mechanism are discussed.

Biochemical and isoenzyme analyses of elephant grass, Pennisetum purpureum (Schum) varieties

This study characterized seven Pennisetum purpureum varieties, namely v. Anão, Bajra, Cameroon, Guaçu, Roxo, Taiwan A-144 and Uruckwami, through biochemical analyses, including protein, glucose and fructose contents, and polyacrylamide gel electrophoresis using the esterase system, by sampling 30, 60, 90, 120 and 150 day-old leaves. The number of nodes per stem and the percentage of bud emergence were also recorded. Variety Taiwan A-144 presented the highest number of nodes per stem and percentage of emerging buds. Protein concentration decreased gradually after 60 days for all varieties, except for Anão. Variety Guaçu presented the highest level of glucose in 90 day-old plants, whereas Cameroon presented the highest levels at 120 and 150 days. The esterase band patterns changed with plant age for all varieties, showing a tendency to increase the number of bands with time. The best age for discriminating between esterase bands of P. purpureum varieties was at 120 days, when most variation could be observed.

Characterization of General Esterases in Workers of the Eastern Subterranean Termite (Isoptera: Rhinotermitidae)

General esterases from eastern subterranean termite, Reticulitermes flavipes (Kollar), workers were examined for activity and were characterized by electrophoretic and biochemical methods. Greater activity was observed toward α-naphthyl substrates when compared Pared with ρ-nitrophenyl substrates. The specific activity of the esterases increased with a corresponding increase in α-naphthyl acetate concentration. Maximal activities were observed with a buffer pH of 7.6 and an incubation temperature of 45c. The calculated Michaelis-Menten constant (Km) was 72.53 µM and the Vmax was 33.77 µM/min/mg of protein. Nondenaturing polyacrylamide gel electrophoresis revealed the presence of seven esterase bands, which were named EI-E7. Bands EI-E4 (slowest gel mobility) were determined to be cholinesterase based on physostigmine inhibition. The remaining three bands were identified as carboxylesterases based on inhibition by paraoxon. The molecular weights of the seven bands ranged from 71.5 to 97.2 kD.

Discovery of a specific esterase band associated with photo- and thermo-sensitive genie male sterile rice

The discovery of germplasmic resources of PGMS and TGMS rices has opened a new avenue for the exploitation of rice heterosis by two-line method. Meanwhile, the two kinds of germplasmic resources have provided important materials for the studies of physiological genetics as well as developmental genetics. PGMS and TGMS lines possess the distinct trait of fertility transformation. However, up to now no convincing reports have been published with respect to the relationship between the morphological fertility

Studies on the strains of Trichogramma evanescens Westwood (Hym., Trichogrammatidae) collected from different hosts in Northern Maldova

AbstractBiological and morphological characteristics together with electrophoretic patterns were compared for five strains of Trichogramma evanescens W., collected from the eggs of Mamestra brassicae L., Ostrinia nubilalis Hb. and Agrotis segetum Schiff. in northern Maldova. Significant differences were observed in the biological characteristics including the proportion of female progeny, developmental period, longevity and fecundity. The strains also differed in the ratio of antennal hair length to the maximum width of flagellum in males. Patterns of esterase systems indicated that the strains differed in the mobility and intensity of esterase bands. All the strains had a strong preference for the host from which they were collected in the field. Crossing of the strains indicated that one strain was reproductively isolated. The results have application in selecting the strategy for establishing initial colony in mass rearing of parasitoids and for research on Trichogramma ecology and evolution.

Isozyme Profile During Somatic Embryogenesis and In Vitro Flowering of Bambusa vulgaris

Peroxidase and esterase isozymes were investigated during plant regeneration via somatic embryogenesis in Bambusa vulgaris, The transition of non-embryogenic calli to embryogenic calli, somatic embryo development, germination and subsequent flowering of somatic embryo derived shoots were associated with selective expression or repression of isoforms of peroxidase and esterase. Non-embryogenic callus showed six peroxidase and four esterase bands. During somatic embryogenesis and germination of somatic embryos, some bands were suppressed and new isoforms of peroxidase and esterase appeared. During flowering, in addition to four peroxidase bands, a new unique esterase band ‘a’ appeared. Each developmental stage was thus associated with a definite isozyme profile.

Characterization of a spectrophotometric assay for juvenile hormone esterase

Two surrogate substrates, methyl 1-heptylthioacetothioate (HEPTAT) and methyl 1-hexylthioacetothioate (HEXTAT) were utilized to compare a new spectrophotometric assay with the standard radiochemical partition assay used to quantify juvenile hormone esterase (JHE) activity. The surrogate substrates were made with one common factor being a thiol ester moiety substituting for the ester moiety found in juvenile hormones (JHs) and a thioether replacing the 2,3-olefin of the JHs. As a result, nucleophilic attack by the serine residue of JHE at the carbonyl functional group results in a hydrolytic reaction and release of methanethiol. In the presence of Ellman's Reagent (DTNB) methanethiol will cleave the disulfide bond of DTNB resulting in a chromophore detectable at 405 nm. Methyl 1-hexylthioacetothioate and its oxygen ester analogue, methyl-l-hexylthioacetate, were compared for JHE activity. Statistical analysis of the slopes indicated a very small but significant difference between the hydrolytic rates for the thiol ester and oxygen ester. However, the data indicate that thiol esters can replace oxygen esters to quantify hydrolytic activity by the JHEs examined. Results gathered from different preparations of JHE including tissue culture media from a baculovirus expression system, affinity- and DEAE-purified enzyme, as well as insect hemolymph indicate an excellent correlation between the two assays. Isoelectric focusing of pure and crude JHE preparations resulted in coinciding peaks of hydrolytic activity when using the standard partition assay and the spectrophotometric assay, with no other peaks of activity found in the crude preparations with either substrate. Several esterase bands were found at different isoelectric points when gels were stained with a-naphthyl acetate. Substrates were further characterized by monitoring specific activity of JHE collected from hemolymph of larvae of Manduca sexta and Heliothis virescens

Enzyme Polymorphism in Umbilicaria cylindrica in Northwestern Greenland

Living Umbilicaria cylindrica (L.) Del. in the vicinity of Qaanaaq, NW Greenland is of special interest because it is found near sites where subfossils from the same population have recently emerged from beneath glacial ice. Electrophoretically-produced enzyme banding patterns of modern thalli were examined to determine whether isozymes indicated genetic variation within the population. Five enzymes were detected in each of six collection sites and the level of variability based on these was expressed as number of patterns per stand, Complement of the Simpson Index, and the Shannon Diversity Index. As is the case with other lichens examined previously, the U. cylindrica population exhibited appreciable enzyme polymorphism and, thus, potential for evolutionary change. Most enzymes were not detectable in glaciated fossil material, but two bands of esterase showed catalytic activity after approximately 1,350 yr. under ice. Umbilicaria cylindrica (L.) Del. is a variable circumpolar species (Llano 1950) that is common in the Arctic and occurs in a 5 km band of tundra adjacent to the icefield east of Qaanaaq Glacier (Fig. 1), Qaanaaq, NW Greenland (77*28'N, 69*2'W). At the receding margin of the ice, subfossil thalli of the same species were discovered in 1992 (Alstrup 1994) and, based on several samples, carbon dated to approximately 1,350 + 10 yr. This lichen in the vicinity of Qaanaaq is of special interest because it offers a rare opportunity to make phenotypic, ecological, and DNA comparisons between the past and present and to attempt to determine whether evolutionary changes have occurred during the last millennium. To this end, base sequences in mycobiont DNA are presently being investigated (Gargas et al. 1994), and results of a study on secondary chemistry of both recently-emerged and living thalli are now in press (Huneck et al. 1995). The evolutionary potential of any population depends in part on genetic variation, and enzyme polymorphism, which reflects innate variability, has been previously demonstrated in several lichens. The major objective of this study was to determine whether there was populational variation in electrophoretically-generated enzyme banding patterns and, thus, capacity for evolutionary change, in modem U. cylindrica from the Qaanaaq area. Possibilities for studying proteins of dead tissues are limited because of degradation in preserved specimens. Also, finds of fossils and subfossils are infrequent and quantities of available material small. Nev theless, functional enzymes could be of considerable systematic interest. No previous investigations had been made of proteins in deglaciated subfossil lichens, so a second objective was to test for enzyme activity in newly emerging thalli. MATERIALS AND METHODS Samples of Umbilicaria cylindrica were obtained from sites 6-14 (Fig. 1) in unglaciated moraines south of the icefield. In each of the latter sites at least 20 thalli were collected randomly within an area of no more than 3 x 3 m on 7-9 August 1993. When still moist, they were wrapped in ti sue, individually or in small groups, and sent with ice by a sequence of two air carriers to London, Ontario, Canada, arriving in the laboratory after 10 days. Specimens of extant thalli were placed in the University of Western Ontario lichen herbarium and material from three si es reserved for analysis of secondary products. From each of the six remaining sites, ten thalli, each approximately 1-1.5 cm diam., were set aside for DNA extraction in the laboratory of P. DePriest, Smithsonian Institution, U.S.A., and within 24 hr., larger thalli from the same six sites, each up to 2-3 cm diam., were prepared for protein analysis. To remove extracellular phenolics, thalli were washed in excess acetone, and the process was repeated four successive times. It was necessary to combine thalli in pairs to produce samples in the range of 0.38-0.45 g of ground material. From sites 10 and 12, ten samples were available, and from each of the sites 6, 9, 11, and 14, there 0007-2745/95/118-122$0.65/0 This content downloaded from 157.55.39.102 on Mon, 03 Oct 2016 04:50:17 UTC All use subject to http://about.jstor.org/terms 1995] FAHSELT ET AL.: ENZYMES OF UMBILICARIA FROM GREENLAND 119

In vitro inhibition of esterase activity in Liposcelis bostrychophila bad. and L. entomophila (end.) (Psocoptera: Liposcelididae)

Methods for assaying AChE and non-specific esterase activity from Liposcelis spp. were outlined. Liposcelis bostrychophila AChE was less sensitive to inhibition than L. entomophila to malaoxon and DDVP. Non-specific esterases of L. bostrychophila were more susceptible to inhibition than those of L. entomophila. Totals of 10 and 14 esterase bands were detected on PAGE for L. bostrychophila and L. entomophila, respectively. The link between esterase sensitivity to inhibition and the differential insecticide tolerance between the two Liposcelis spp. is examined.

The electrophoretic polymorphism of bacterial esterases

Abstract The specific activities of esterases and certain other molecular properties including immunospecificity indicate that the electrophoretic variations of these enzymes in bacterial populations are the result of allelic variations at specific gene loci. The esterase polymorphism of Enterobacteriaceae and some other species isolated from man or animals demonstrates that esterases can distinguish between bacteria at the species or subspecies level, both by their biochemical properties and by their electrophoretic differences. The esterase data complement DNA hybridization studies and agree with ribosomal DNA polymorphism, especially for delineating a phylogenetically distinct group of highly pathogenic strains in Escherichia coli. A two-dimensional electrophoretic profile obtained by establishing a direct correspondence between homologous esterase bands resolved by independent runs of isoelectric focusing and standard electrophoresis considerably improves the detection of allelic variations, whereas protein titration curves (electrophoresis in pH gradient) can be used to demonstrate the real electrophoretic homogeneity of allozymes or evalue their molecular relationship in terms of apparent amino acid substitutions. This overview establishes that esterases, by their significant electrophoretic polymorphism, are reliable molecular markers for systematics and epidemiology, and are suitable enzyme systems for studying population genetics and phylogeny.

Nutritional and biological activities of a sterile red fungus which promotes plant growth and suppresses take-all

The nutritional behaviour of a ‘Sterile Red Fungus’ (SRF) was studied with a view to the development of efficient field delivery systems for its use in plant growth promotion and disease protection. The vegetative cells of the fungus are binucleate with occasional presence of trinucleate terminal cells. Responses to 28 biochemical tests and 19 different carbon sources were determined to detect the enzymatic activities and preferred carbon source utilization for growth. Pectic enzyme patterns and manganese oxidation/reduction abilities of the SRF were also investigated. The SRF was found to have a wide range of enzymatic activity. It produced cellulase, esterase, ligninase, peroxidase, acid phosphatase, phenoloxidase and pectinase and required thiamine (0·6 g l −1 ) for growth. With the exception of chitin, lactose, dulcitol and l -arabinose it utilized all the carbon sources tested. It oxidized and reduced manganese and its isoenzyme pattern is characterized by distinct bands of esterase and polygalacturonase.

Geminivirus transmission and biological characterisation of Bemisia tabaci (Gennadius) biotypes from different geographic regions

SummaryEighteen populations of Bemisia tabaci, collected from different geographic locations (North & Central America, the Caribbean, Africa, the Middle East, Asia and Europe), were studied to identify and compare biological and genetic characteristics that can be used to differentiate biotypes. The morphology of the fourth instar/pupal stage and compound eye structures of adults were investigated using scanning electron microscopy and found to be typical of the species among all biotypes and populations studied. Setae and spines of B. tabaci larval scales from the same colony were highly variable depending on the host plant species or leaf surface characteristics. The location and the morphology of caudal setae, characteristic of all B. tabaci studied to date, were present in all colonies. However, differences in adult body lengths and in the ability to induce phy to toxic disorders in certain plant species were found between biotypes or populations. The recently identified “B” biotype, characterised by a diagnostic esterase banding pattern and by its ability to induce phytotoxic responses in squash, honeysuckle and nightshade was readily distinguished from non‐“B” biotype populations. None of the non‐“B” biotypes studied, were found to induce phytotoxic responses. Nine populations examined showed typical “B” biotype characteristics, regardless of country of origin. All tested populations, determined as “B” or “B”‐like biotypes successfully mated with other “B” biotype colonies from different geographic areas. Non‐“B” biotype colonies did not interbreed with other biotypes.The B. tabaci populations were tested for their ability to transmit 15 whitefly‐transmitted geminiviruses (WTGs) from different geographic areas with a wide range of symptom types. All WTGs were transmitted by the “B” biotype colonies and by most non‐“B” biotype colonies, with the exception of three viruses found in ornamental plants which were non‐transmissible by any colony. Some non‐“B” biotypes would not transmit certain geminiviruses and some geminiviruses were more efficiently transmitted than were others.

Genetic Analysis of Two Race 0 x Race 2 Crosses inCochliobolus carbonum

Progeny from two crosses of race 0× race 2 of Cochliobolus carbonum segregated in 1:1 ratios for loci controlling mating type, fungicide tolerance, and ability to sporulate on potato-dextrose agar. Segregations at single loci also were observed for three isozyme systems and for one of two bands in a fourth system. Progeny also segregated for four esterase bands, but the segregation patterns were complex. Pathogenicity to corn was inherited as two apparently complementary genes present in race 2 but lacking in race 0, which was avirulent on corn. Evidence was found for a new locus for production of pseudothecia and a new locus for production of asci and ascospores. These putative loci differ from the previously described single loci for pseudothecium and ascospore production in C. carbonum [...]