Biochemical and Genetic Polymorphisms for Carboxylesterase and Acetylesterase in Grape Clones of Vitis vinifera L. (Vitaceae) Cultivars

Native polyacrylamide gel electrophoresis (PAGE) was employed to show the highest number of esterase loci and to detect alpha- and beta-esterase polymorphisms in leaf buds of Vitis vinifera cultivars. A total of 16 esterase isozymes were detected in leaf buds from 235 plants including Italia, Rubi, Benitaka, and Brasil cultivars. Biochemical characterization of the grape esterases using ester substrates revealed alpha-, beta-, and alpha/beta-esterases with inhibitor tests distinguishing both carboxylesterases (EST-2, EST-3, EST-5, EST-6, EST-7, EST-8, EST-9, EST-10, and EST-16 isozymes) and acetylesterases (EST-4, EST-11, EST-12, EST-13, EST-14, EST-15 isozymes). No allele variation for alpha-, beta-, and alpha/beta-esterases was detected; however, EST-3 alpha-carboxylesterase was absent in 61.7% of vines, and EST-4 alpha/beta-acetylesterase was absent in one vine of Rubi cv. Null EST-3 carboxylesterase phenotype (61.7%) cannot be explained in this article, but the high genetic polymorphism in four V. vinifera clones is a positive aspect for genetic selection and development of new clones with different characteristics.     

Genetic structure and population dynamics of Xanthomonas axonopodis pv. manihotis in Colombia from 1995 to 1999

Restriction fragment length polymorphisms (RFLPs) were used to study the population genetics and temporal dynamics of the cassava bacterial pathogen Xanthomonas axonopodis pv. manihotis. The population dynamics were addressed by comparing samples collected from 1995 to 1999 from six locations, spanning four different edaphoclimatic zones (ECZs). Forty-five different X. axonopodis pv. manihotis RFLP types or haplotypes were identified between 1995 and 1999. High genetic diversity of the X. axonopodis pv. manihotis strains was evident within most of the fields sampled. In all but one site, diversity decreased over time within fields. Haplotype frequencies significantly differed over the years in all but one location. Studies of the rate of change of X. axonopodis pv. manihotis populations during the cropping cycle in two sites showed significant changes in the haplotype frequencies but not composition. However, variations in pathotype composition were observed from one year to the next at a single site in ECZs 1 and 2 and new pathotypes were described after 1997 in these ECZs, thus revealing the dramatic change in the pathogen population structure of X. axonopodis pv. manihotis. Disease incidence was used to show the progress of cassava bacterial blight in Colombia during the 5-year period in different ecosystems. Low disease incidence values were correlated with low rainfall in 1997 in ECZ 1.     

AFLP assessment of genetic variability in cassava accessions (Manihot esculenta) resistant and susceptible to the cassava bacterial blight (CBB).

Cassava bacterial blight (CBB) is caused by Xanthomonas axonopodis pv. manihotis (Xam). Resistance is found in Manihot esculenta and, in addition, has been introgressed from a wild relative, M. glaziovii. The resistance is thought to be polygenic and additively inherited. Ninety-three varieties of M. esculenta (Crantz) were assessed by AFLPs for genetic diversity and for resistance to CBB. AFLP analysis was performed using two primer combinations and a 79.2% level of polymorphism was found. The phenogram obtained showed between 74% and 96% genetic similarity among all cassava accessions analysed. The analysis permitted the unique identification of each individual. Two Xam strains were used for resistance screening. Variation in the reaction of cassava varieties to Xam strains was observed for all plant accessions. The correlation of resistance to both strains, had a coefficient of 0.53, suggesting the independence of resistance to each strain. Multiple correspondence analysis showed a random distribution of the resistance/susceptibility response with respect to overall genetic diversity as measured by AFLP analysis. A total heterozygosity index was calculated to determine the diversity within clusters as well as among them. Our results demonstrate that resistance to CBB is broadly distributed in cassava germplasm and that AFLP analysis is an effective and efficient means of providing quantitative estimates of genetic similarities among cassava accessions.     

Geographical Differentiation of the Population of Xanthomonas axonopodis pv. manihotis in Colombia

Analyses of DNA polymorphism and virulence variation were used to evaluate the population structure of Xanthomonas axonopodis pv. manihotis, the pathogen causing cassava bacterial blight in Colombia. We collected strains from the major cassava-growing regions which can be grouped into different edaphoclimatic zones (ECZs) according to environmental conditions, production constraints, and economic parameters. DNA polymorphism was assessed by a restriction fragment length polymorphism analysis, using an X. axonopodis pv. manihotis plasmid DNA sequence (pthB) as a probe to evaluate the genetic relatedness among 189 Colombian strains. The sampling intensity permitted the estimation of genetic differentiation within and among ECZs, sites, and fields and even within an individual plant. A multiple correspondence analysis indicated that the Colombian X. axonopodis pv. manihotis population showed a high degree of diversity relative to X. axonopodis pv. manihotis populations studied previously, and the entire collection was grouped into seven clusters. A general correlation was observed between the clusters and the geographical origin of the strains, as each cluster was largely composed of strains from the same ECZ. Representative strains, identified with pthB, were further characterized by ribotyping, hybridization to two repetitive genomic probes (pBS6 and pBS8), and restriction analysis of plasmid contents to evaluate the complementarity of these markers. Virulence variation was observed within the Colombian collection. Strains of different aggressiveness were found in all ecological zones, but no correlation between virulence variation and DNA polymorphism was observed. The genetic and virulence analyses contribute to understanding the X. axonopodis pv. manihotis population structure in Colombia.     

Variations in the esterase expression pattern with respect to different light regimes in Drosophila agumbensis and Drosophila nagarholensis

Circadian clock enables organism’s to adapt under fluctuating environmental conditions by coupling of behavioral, physiological and molecular processes in a wide variety of organisms including bacteria, fungi, plants, birds and mammals. The endogenous circadian system functions to organize behavior and physiology to adapt to and anticipate environment changes in light and temperature. The present study is an attempt to understand enzyme profiles (alpha- and beta-esterases) of Drosophila agumbensis and Drosophila nagarholensis under light/dark (LD), constant dark (DD) constant light (LL), conditions over twenty generations. A polyacrylamide gel electrophoresis (7.5% – Native gel) was used to study the esterase expression patterns in two species of the montium subgroup of Drosophila. Alpha- and beta-esterase expression was significantly decreased in LL when compared to LD and DD at both the generations and species. In all the light regimes, females were found to have significantly higher level of α- and β-esterase expression than males. Flies were maintained under different light regimes showed difference in their expression patterns with respect to alpha- and beta-esterases. The present study showed that constant light conditions affect the expression of esterases in D. agumbensis and D. nagarholensis.     

Pathological and Molecular Characterization of Xanthomonas campestris Strains Causing Diseases of Cassava (Manihot esculenta)

Fifty-one strains representing Xanthomonas campestris pv. manihotis and cassavae and different pathovars occurring on plants of the family Euphorbiaceae were characterized by ribotyping with a 16S+23S rRNA probe of Escherichia coli and by restriction fragment length polymorphism analysis with a plasmid probe from X. campestris pv. manihotis. Pathogenicity tests were performed on cassava (Manihot esculenta). Histological comparative studies were conducted on strains of two pathovars of X. campestris (vascular and mesophyllic) that attack cassava. Our results indicated that X. campestris pv. manihotis and cassavae have different modes of action in the host and supplemented the taxonomic data on restriction fragment length polymorphism that clearly separate the two pathovars. The plasmid probe could detect multiple restriction fragment length polymorphisms among strains of the pathovar studied. Ribotyping provides a useful tool for rapid identification of X. campestris pathovars on cassava.     

Preliminary Evidence for Phytoalexin Production by Eucalyptus urophylla Leaves as a Response to Inoculation with the Incompatible Pathogen Acidovorax avenae pv. avenae

Leaves of eucalyptus ( Eucalyptus urophylla ) were infiltrated with a cell suspension of the incompatible pathogen Acidovorax avenae pv. avenae and showed a typical hypersensitive response within 24h. Necrotic leaf areas were excised, vacuum infiltrated with 40% ethanol and left under continuous agitation at room temperature for 24h. The diffusate was concentrated, partitioned with ethyl-acetate, concentrated to dryness and resuspended in a small volume of methanol. The biological activity of extracts was evaluated by an agar diffusion method against noncompatible bacteria ( A. avenae pv. avenae and Xanthomonas axonopodis pv. manihotis ) and fungi ( Penicillium sp. and Aspergillus sp. ). Inhibition haloes, when present, were always larger in extracts from leaves infiltrated with the incompatible bacterium than the water control. Thin layer chromatography resolution of crude extracts from leaves infiltrated with both incompatible pathogen cell suspension and water, followed by bioautography with Thieleviopsis paradoxa, consistently rendered, in both situations, a large, diffuse inhibition halo near the origin, assumed to be due to preformed antimicrobial substances. However, extracts from leaves infiltrated with the living cells of the incompatible pathogen gave rise to a smaller, second inhibition halo, near the front, that was interpreted as being one or several phytoalexins.     

Biofilm formation, epiphytic fitness, and canker development in Xanthomonas axonopodis pv. citri

The phytopathogenic bacterium Xanthomonas axonopodis pv. citri is responsible for the canker disease affecting citrus plants throughout the world. Here, we have evaluated the role of bacterial attachment and biofilm formation in leaf colonization during canker development on lemon leaves. Crystal violet staining and confocal laser scanning microscopy analysis of X. axonopodis pv. citri strains expressing the green fluorescent protein were used to evaluate attachment and biofilm formation on abiotic and biotic (leaf) surfaces. Wild-type X. axonopodis pv. citri attached to and formed a complex, structured biofilm on glass in minimal medium containing glucose. Similar attachment and structured biofilm formation also were seen on lemon leaves. An X. axonopodis pv. citri gumB mutant strain, defective in production of the extracellular polysaccharide xanthan, did not form a structured biofilm on either abiotic or biotic surfaces. In addition, the X. axonopodis pv. citri gumB showed reduced growth and survival on leaf surfaces and reduced disease symptoms. These findings suggest an important role for formation of biofilms in the epiphytic survival of X. axonopodis pv. citri prior to development of canker disease.     

Genetic Structure and Population Dynamics of Xanthomonas axonopodis pv. manihotis in Colombia from 1995 to 1999

Restriction fragment length polymorphisms (RFLPs) were used to study the population genetics and temporal dynamics of the cassava bacterial pathogen Xanthomonas axonopodis pv. manihotis. The population dynamics were addressed by comparing samples collected from 1995 to 1999 from six locations, spanning four different edaphoclimatic zones (ECZs). Forty-five different X. axonopodis pv. manihotis RFLP types or haplotypes were identified between 1995 and 1999. High genetic diversity of the X. axonopodis pv. manihotis strains was evident within most of the fields sampled. In all but one site, diversity decreased over time within fields. Haplotype frequencies significantly differed over the years in all but one location. Studies of the rate of change of X. axonopodis pv. manihotis populations during the cropping cycle in two sites showed significant changes in the haplotype frequencies but not composition. However, variations in pathotype composition were observed from one year to the next at a single site in ECZs 1 and 2 and new pathotypes were described after 1997 in these ECZs, thus revealing the dramatic change in the pathogen population structure of X. axonopodis pv. manihotis. Disease incidence was used to show the progress of cassava bacterial blight in Colombia during the 5-year period in different ecosystems. Low disease incidence values were correlated with low rainfall in 1997 in ECZ 1.     

Effect of food plants on the volume of repellent secretion obtained in adult Zonocerus variegatus (Orthoptera: Pyrgomorphidae)

The volume of secretion obtained from adult Zonocerus variegatus (Orthoptera: Pyrgomorphidae) was influenced by the type of food plants. Insects fed on leaves of cassava Manihot esculenta, bitter leaves Vernonia amygdalina, and a mixture of M. esculenta and Acalypha wilkesiana gave a good volume of secretion while Chromolaena odorata, Elaeis guinensis, Aspilia africana and Citrus sinensis did not favour secretion production. No significant difference was recorded in the volume of secretion obtained from Z. variegatus from the two seasons irrespective of the food plant. Similarly, food plants gave no significant difference on the volume of secretion between the two seasons.     

Wild manihot species do not possess C4 photosynthesis

Cultivated cassava (Manihot esculenta) has a higher rate of photosynthesis than is usual for C3 plants and photosynthesis is not light saturated. For these reasons it has been suggested that cultivated cassava could be derived from wild species possessing C4 photosynthesis. The natural abundance of 13C and activities of phosphoenolpyruvate carboxylase and phosphoglycolate phosphatase were measured in leaves of 20 wild cassava species to test this hypothesis. All the species studied, including M. flabellifolia the potential wild progenitor of cultivated cassava, clearly exhibited C3 not C4 characteristics.     

Sensitive and specific detection of Xanthomonas axonopodis pv. citri by PCR using pathovar specific primers based on hrpW gene sequences

A sensitive and specific assay was developed to detect citrus bacterial canker caused by Xanthomonas axonopodis pv. citri, in leaves and fruits of citrus. Primers XACF and XACR from hrpW homologous to pectate lyase, modifying the structure of pectin in plants, were used to amplify a 561 bp DNA fragment. PCR technique was applied to detect the pathogen in naturally or artificially infected leaves of citrus. The PCR product was only produced from X. axonopodis pv. citri among 26 isolates of Xanthomonas strains, Escherichia coli (O157:H7), Pectobacterium carotovorum subsp. carotovorum, and other reference microbes.     

Peptidotriazoles with antimicrobial activity against bacterial and fungal plant pathogens

We designed and prepared peptidotriazoles based on the antimicrobial peptide BP100 (LysLysLeuPheLysLysIleLeuLysTyrLeu-NH(2)) by introducing a triazole ring in the peptide backbone or onto the side chain of a selected residue. These compounds were screened for their in vitro growth inhibition of bacterial and fungal phytopathogens, and for their cytotoxic effects on eukaryotic cells and tobacco leaves. Their proteolytic susceptibility was also analyzed. The antibacterial activity and the hemolysis were influenced by the amino acid that was modified with the triazole as well as by the absence of presence of a substituent in this heterocyclic ring. We identified sequences active against the bacteria Xanthomonas axonopodis pv. vesicatoria, Erwinia amylovora, Pseudomonas syringae pv. syringae (MIC of 1.6-12.5 μM), and against the fungi Fusarium oxysporum (MIC<6.2-12.5 μM) with low hemolytic activity (0-23% at 50 μM), high stability to protease digestion and no phytotoxicity. These peptidotriazoles constitute good candidates to design new antimicrobial agents.     

Senescence-Inducible Expression of Isopentenyl Transferase Extends Leaf Life, Increases Drought Stress Resistance and Alters Cytokinin Metabolism in Cassava

Cassava (Manihot esculenta Crantz) sheds its leaves during growth, especially within the tropical dry season. With the production of SAG12-IPT transgenic cassava we want to test the level of leaf retention and altered cytokinin metabolism of transgenic plants via the autoregulatory senescence inhibition system. After confirmation of transgene expression by molecular analysis and phenotype examination in greenhouse plants, two transgenic plant lines, 529-28 and 529-48, were chosen for further investigation. Detached mature leaves of 529-28 plants retained high levels of chlorophyll compared with wild-type leaves after dark-induced senescence treatment. Line 529-28 showed significant drought tolerance as indicated by stay-green capacity after drought stress treatment. Field experiments proved that leaf senescence syndrome was significantly delayed in 529-28 plants in comparison with wild-type and 529-48 plants. Physiological and agronomical characterizations of these plants also revealed that the induced expression of IPT had effects on photosynthesis, sugar allocation and nitrogen partitioning. Importantly, the 529-28 plants accumulated a high level of trans-zeatin-type cytokinins particularly of corresponding storage O-glucosides to maintain cytokinin homeostasis. Our study proves the feasibility of prolonging the leaf life of woody cassava and also sheds light on the control of cytokinin homeostasis in cassava leaves.     

Effects of low temperature on wheat leaf proteins

Finnish wheat cultivars, winter wheat ‘Vakka’ and spring wheat ‘Apu’, grown for 9 days at 25° and then for 52 days at 2–4°, were called ‘hardened’. Proteins and esterases of young leaves were resolved by polyacrylamide gel electrophoresis (PAGE), porosity gradient PAGE (poroPAGE), isoelectric focusing (PAGIF) between pH 3 and 9, and by Na-dodecylsulfate (SDS)-PAGE. Hardened and unhardened leaves have different protein patterns after PAGE, PAGIF, poroPAGE and SDS-PAGE, respectively, as well as for esterases after PAGE and PAGIF. The PAGIF patterns of esterases show distinct changes of two bands especially for hardened leaves of winter wheat, one appearing, the other one disappearing.     

Leaf gas exchange of cassava as affected by quality of planting material and water stress

Field trial was conducted to study the effects of quality of planting material and prolonged water stress on leaf gas exchange of the cassava (Manihot esculenta Crantz) cultivar M Col 1684. Nutrient contents of planting material affected rootlet formation, but not leaf gas exchange. Net photosynthetic rate (PN), stomatal conductance (gs), and intercellular CO2 concentration (Ci) were significantly reduced by prolonged water stress. New leaves developed after recovery from water stress showed higher PN and gs, as compared to leaves of similar ages of unstressed plants. The higher PN was associated with higher leaf nutrient contents, indicating that photosynthetic capacity was enhanced in these leaves. These compensating characteristics may partly explain the small yield reduction often observed in stressed cassava.     

Leaf gas exchange of cassava as affected by quality of planting material and water stress

Field trial was conducted to study the effects of quality of planting material and prolonged water stress on leaf gas exchange of the cassava (Manihot esculenta Crantz) cultivar M Col 1684. Nutrient contents of planting material affected rootlet formation, but not leaf gas exchange. Net photosynthetic rate (PN), stomatal conductance (gs), and intercellular CO2 concentration (Ci) were significantly reduced by prolonged water stress. New leaves developed after recovery from water stress showed higher PN and gs, as compared to leaves of similar ages of unstressed plants. The higher PN was associated with higher leaf nutrient contents, indicating that photosynthetic capacity was enhanced in these leaves. These compensating characteristics may partly explain the small yield reduction often observed in stressed cassava.     

The β-esterase Gene Cluster of Drosophila melanogaster: is ψEst-6 a Pseudogene, a Functional Gene, or both?

Pseudogenes have been defined as non-functional sequences of genomic DNA that are originally derived from functional genes, but exhibit degenerative features such as premature stop codons and frameshifts that prevent their expression. However, there is increasing evidence that pseudogenes are often evolutionarily conserved and may have retained some functional role or acquired new ones. Pseudogenes may exhibit non-functional features as well as functional ones. We investigate, as a model case, the beta-esterase gene cluster of Drosophila melanogaster that includes the Est-6 gene and the psiEst-6 putative pseudogene. We study four samples derived from natural populations of east Africa (Zimbabwe), Europe (Spain), North America (California), and South America (Venezuela). The level of nucleotide diversity is higher in Africa than in the non-African populations. There is twice more nucleotide diversity in psiEst-6 than in Est-6. Linkage disequilibrium within the beta-esterase gene cluster is strong in non-African samples, but much lower in Africa. The population recombination rate is the same for psiEst-6 and Est-6 in Africa, but significantly different in non-African samples. Intragenic gene conversion events are detected within Est-6 and, with much higher incidence, within psiEst-6; intergenic gene conversion events are rare. The extensive intragenic gene conversion within psiEst-6 can be explained by the invasion of retrotransposons that promote a form of homology-dependent gene conversion upon excision. Tests of neutrality with recombination are significant for the beta-esterase gene cluster in the non-African populations but not in Africa. The Est-6 gene sequences exhibit a well-known allozyme dimorphic structure. The sequences of psiEst-6 are also dimorphic in North and South America, but they do not correspond at all (South America) or only imperfectly (North America) to the Est-6 allozyme dimorphism. Sequence dimorphism is less pronounced in the European and African samples. We suggest that demographic history (bottleneck and admixture of genetically differentiated populations) is the major factor shaping the nucleotide pattern in the beta-esterase gene cluster. However, there are some clear indications of positive selection shaping the distribution of nucleotide polymorphism within the cluster. Intergenic epistatic selection may play an important role in the evolution of the beta-esterase gene cluster, preserving psiEst-6 from degenerative destruction and reflecting its functional interaction with Est-6. The Est-6 gene cluster of D. melanogaster represents an example of a functionally interacting complex ('intergene') in which two components (Est-6 and psiEst-6) or more are required to perform the final function.