Biological changes in the rhizosphere of wheat after foliar application of chlorocholinechloride,urea and 4-chloro-2-methylphenoxyacetic acid

In field experiments wheat in the phase of shooting was sprayed with solutions of chlorocholinechloride (CCC) and urea, CCC and ammonium salt MCPA (Aminex) or CCC, urea and Aminex. The effect of the treatment on dry weight of overground parts of wheat, number of bacteria, production of carbon dioxide, urease activity and content of ammonium in the rhizosphere soil was investigated. In all cases evolution of carbon dioxide in the rhizosphere soil was higher than that in the control soil. Highest numbers of bacteria were found in the rhizosphere soil of plants treated with urea, the herbicide and their mixtures. Content of ammonium was higher in the control soil than in the rhizosphere soils, the urease activity was highest in the rhizosphere soil of plants treated with the solution of the herbicide and with the combination of the herbicide with urea.     

Metabolism of 2,4-dichlorophenoxyacetic acid, 4-chloro-2-methylphenoxyacetic acid and 2-methylphenoxyacetic acid by Alcaligenes eutrophus JMP 134

Of eleven substituted phenoxyacetic acids tested, only three (2,4-dichloro-, 4-chloro-2-methyl- and 2-methylphenoxyacetic acid) served as growth substrates for Alcaligenes eutrophus JMP 134. Whereas only one enzyme seems to be responsible for the initial cleavage of the ether bond, there was evidence for the presence of three different phenol hydroxylases in this strain. 3,5-Dichlorocatechol and 5-chloro-3-methylcatechol, metabolites of the degradation of 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid, respectively, were exclusively metabolized via the ortho-cleavage pathway. 2-Methylphenoxyacetic acid-grown cells showed simultaneous induction of meta- and ortho-cleavage enzymes. Two catechol 1,2-dioxygenases responsible for ortho-cleavage of the intermediate catechols were partially purified and characterized. One of these enzymes converted 3,5-dichlorocatechol considerably faster than catechol or 3-chlorocatechol. A new enzyme for the cycloisomerisation of muconates was found, which exhibited high activity against the ring-cleavage products of 3,5-dichlorocatechol and 4-chlorocatechol, but low activities against 2-chloromuconate and muconate.Non-standard abbreviations MCPA 4-chloro-2-methylphenoxyacetic acid - 2MPA 2-methylphenoxyacetic acid - PA phenoxyacetic acid     

Rhizosphere microflora and colonization of wheat roots byGaeumannomyces graminis var.tritici after foliar application of urea and benomyl

The effect of foliar application of 2% urea and 0.6% benomyl on changes in colonization of the rhizosphere by microorganisms and of roots by the fungusGaeumannomyces graminis (Sacc.)Arx etOlivier var.tritici Walker was followed in vegetation glass-house experiments. Treatment with a urea solution resulted in increased counts of bacteria (82 %),Pseudomonas fluorescens (46 %),Agrobacterium sp. (31 %) and antagonistic bacteria with respect to the used fungus isolate and in a decreased occurrence of micromycetes (63 %). Treatment of wheat with a benomyl solution resulted in an increased count of bacteria (43 %) and a decreased occurrence ofP. fluorescens (16 %),Agrobacterium sp. (50 %) and fungi (67 %). After treatment with both compounds the infection of roots byG. graminis considerably decreased as compared with untreated plants. The results are discussed from the point of view of the effect of application of the studied compounds to upper parts of wheat on the microflora colonizing its roots.     

Changes of microflora of wheat roots after foliar application of urea

Upper parts of wheat grown in containers with brown soil were four-times repeatedly treated with a solution of urea. The foliar application of urea resulted in a considerable increase of the number of bacteria in the roots and a decrease of the number of fungi. Seventeen and 13 genera of fungi could be detected in the rhizosphere of the treated plants, 3 and 5 weeks, respectively, after the application, whereas 23 and 16 genera could be identified in the rhizoplane of the treated plants. Fungi of the generaFusarium andPenicillium predominated in the root region. The foliar application of urea resulted in an increased relative occurrence of the genusFusarium and decreased relative representation of the genusPenicillium. Changes in the relative occurrence of other 18 genera of fungi present in small quantities on wheat roots could also be observed. The foliar application of urea affected not only numbers of fungi determined by the dilution plate method but also the occurrence of active hyphae.     

Metabolism of 4-chloro-2-methylphenoxyacetic Acid by soil bacteria

A microorganism capable of degrading 4-chloro-2-methylphenoxyacetic acid (MCPA) was isolated from soil and identified as Flavobacterium peregrinum. All of the chlorine of MCPA was released as chloride, and the carboxyl-carbon was converted to volatile products by growing cultures of the bacterium, but a phenol accumulated in the medium. The phenol was identified as 4-chloro-2-methylphenol on the basis of its gas chromatographic and infrared characteristics. Extracts of cells of F. peregrinum and of a phenoxyacetate-metabolizing Arthrobacter sp. dehalogenated MCPA and several catechols but not 4-chloro-2-methylanisole. The Arthrobacter sp. cell extract was fractionated, and an enzyme preparation was obtained which catalyzed the conversion of MCPA to 4-chloro-2-methylphenol. The latter compound was not metabolized unless reduced nicotinamide adenine dinucleotide phosphate was added to the fractionated extract. The phenol in turn was apparently oxidized to a catechol by components of the enzyme preparation.     

Respiration and microflora of the rhizosphere soil of wheat influenced by foliar application of urea

The numbers of micromycetes and bacteria were investigated with respect to oxygen consumption in the rhizosphere soil of wheat and in non-rhizosphere soil. Plants after foliar application of urea (2 % solution) and non-treated plants were cultivated in degraded chernozem and garden soil in a green-house. Changes in oxygen consumption by the suspensions of rhizosphere and non-rhizosphere soils corresponded to changes in the number of bacteria designated as the rhizosphere effect (R/S). Values of R/S depended on the presence of organic substrates. Changes in oxygen consumption by the soil suspension from the rhizosphere of wheat occurring due to foliar application of urea corresponded to changes in the amount of microflora. The results obtained are discussed with respect to a possible utilization of the data to follow metabolic activity of soils in a natural environment (in situ) determined according to oxygen consumption by a soil suspension, and to assess changes in the microflora of rhizosphere and non-rhizosphere soil.     

Degradation of 4-chloro-2-methylphenoxyacetic acid in top- and subsoil is quantitatively linked to the class III tfdA gene

The tfdA gene is known to be involved in the first step of the degradation of the phenoxy acid herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA) in several soil bacteria, but bacteria containing other tfdA-like genes have been isolated as well. A quantitative real-time PCR method was used to monitor the increase in the concentration of tfdA genes during degradation of MCPA in sandy topsoil and subsoil over a period of 115 days. Quantitative PCR revealed growth in the tfdA-containing bacterial community, from 500 genes g(-1) soil to approximately 3 x 10(4) genes g(-1) soil and to 7 x 10(5) genes g(-1) soil for topsoil initially added to 2.3 mg MCPA kg(-1) (dry weight) soil and 20 mg MCPA kg(-1) (dry weight) soil, respectively. We analyzed the diversity of the tfdA gene during the degradation experiment. Analyses of melting curves of real-time PCR amplification products showed that a shift in the dominant tfdA population structure occurred during the degradation period. Further denaturing gradient gel electrophoresis and sequence analysis revealed that the tfdA genes responsible for the degradation of MCPA belonged to the class III tfdA genes, while the tfdA genes present in the soil before the occurrence of degradation belonged to the class I tfdA genes. The implications of these results is that the initial assessment of functional genes in soils does not necessarily reflect the organisms or genes that would carry out the degradation of the compounds in question.     

Effect of leaf age on the translocation movement of 4-chloro-2-methylphenoxyacetic acid (MCPA) inHordeum distichum L.

P?i aplikaci kyseliny 4-chlor-2-metylfenoxyoctové na první t?i listy je?mene se podle formativního ú?inku na klas ukázalo, ?e nedospělé listy nejsou schopny zprost?edkovat transport MCPA k vzrostnému vrcholu. Tento vztah souvisí z?ejmě s metabolickou aktivitou list?.     

Comparison of 16S rRNA gene phylogeny and functional tfdA gene distribution in thirty-one different 2,4-dichlorophenoxyacetic acid and 4-chloro-2-methylphenoxyacetic acid degraders

31 different bacterial strains isolated using the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, were investigated for their ability to mineralize 2,4-D and the related herbicide 4-chloro-2-methylphenoxyacetic acid (MCPA). Most of the strains mineralize 2,4-D considerably faster than MCPA. Three novel primer sets were developed enabling amplification of full-length coding sequences (CDS) of the three known tfdA gene classes known to be involved in phenoxy acid degradation. 16S rRNA genes were also sequenced; and in order to investigate possible linkage between tfdA gene classes and bacterial species, tfdA and 16S rRNA gene phylogeny was compared. Three distinctly different classes of tfdA genes were observed, with class I tfdA sequences further partitioned into the two sub-classes I-a and I-b based on more subtle differences. Comparison of phylogenies derived from 16S rRNA gene sequences and tfdA gene sequences revealed that most class II tfdA genes were encoded by Burkholderia sp., while class I-a, I-b and III genes were found in a more diverse array of bacteria.     

Effect of foliar application of urea on wheat growth in relation to plant age,soil fertility and light intensity

Summary Treatment of leaves of ‘Kaspar’ wheat seedlings, growing in low inorganic (NPK) fertilized soil, with urea, resulted in reduced growth until 24 days after the first application. Stimulation of growth was observed 31 days after the urea leaf treatment. Addition of NPK to the soil did not obviously change the effect of urea leaf treatment on seedlings, while increase of light intensity from 16.000 lux to 42.000 lux restricted the period of growth reduction. Leaf treatment with urea did not influence growth of one-month-old wheat plants for 14 days after the first application.     

Process intensification of immobilized lipase catalysis by microwave irradiation in the synthesis of 4-chloro-2-methylphenoxyacetic acid (MCPA) esters

4-Chloro-2-methylphenoxyacetic acid (MCPA) is a selective systemic herbicide which is absorbed by leaves and roots. MCPA esters are preferred due to their low water solubility and environmental friendliness. Esterification of MCPA with n-butanol was investigated as a model reaction using immobilized enzymes under the influence of microwave irradiation. Different immobilized enzymes such as Novozym 435, Lipozyme TL IM, Lipozyme RM IM and Lipase AYS Amano were studied under microwave irradiation amongst which Novozym 435 (immobilized Candida antarctica lipase B) was the best catalyst. Effects of various parameters were systematically studied on rates and conversion. Under microwave irradiation, the initial rates were observed to increase up to 2-fold. Under optimized conditions of 0.1 mmol MCPA and 0.3 mmol n-butanol in 15 mL 1,4-dioxane as solvent, Novozym 435 showed a conversion of 83% at 60 °C in 6 h. Based on initial rate and progress curve data, the reaction was shown to follow the Ping Pong bi–bi mechanism with inhibition by MCPA and n-butanol. Esterification of MCPA was also studied with different alcohols such as isopropyl alcohol, n-pentanol, n-hexanol, benzyl alcohol and 2-ethyl-1-hexanol.     

Control of microorganisms in the rhizosphere of wheat by inoculation of seeds withPseudomonas putida and by foliar application of urea

After inoculation of wheat seeds with various bacterial strains germination of plants was usually inhibited at first but growth was stimulated later. After inoculation withPseudomonas putida K 11 producing physiologically active compounds the total number of bacteria increased together with the bacteria: fungi ratio in the rhizosphere. These characteristic were further increased after foliar application of urea due to increased root exudation. Dry mass of upper wheat parts was about 15 — 80 % higher in green-house experiments, in which the plants were treated in the two above ways. More reliable results, were usually obtained by bacterization ofP. putida and foliar application of urea as compared with the situation when the seeds were inoculated without the foliar application or, on the contrary, after foliar application without inoculation of the seeds. Only when urea was applied early and in a soil contaminated with the fungusGaeumannomyces graminis var.tritici (causing “take-all” of the wheat) no favourable results could be detected. In these cases the foliar application without inoculation of the seeds was more successful. Symptoms of the disease of wheat roots caused byG. graminis were less frequently observed after the inoculation of the seeds with the strainP. putida K 11 and after the foliar application of urea.     

Effect of chlorocholine chloride and gibberellic acid on the anthocyanin synthesis in radish seedlings

Anthocyanin synthesis in radish ( Raphanus sativus L. cv. Scarlet Globe) seedlings after treatment with chlorocholine chloride (CCC) and gibberellic acid (GA) has been investigated. CCC promotes and GA₃ inhibits the synthesis. When both substances are given together, CCC reverses the inhibition caused by GA₃. Simultaneous external feeding of anthocyanin precursors (sucrose and phenylalanine) reverses the GA₃ inhibition. A higher amount of total free amino acids, in particular phenylalanine, was present in CCC-treated seedlings compared to controls grown on distilled water. The amount of phenylalanine was lower in seedlings treated with both CCC and GA₃ as compared to seedlings treated with CCC alone, and total free sugars (reducing plus non-reducing) was lower in CCC treated seedlings than in controls grown on distilled water. We conclude that CCC and GA3 control the anthocyanin synthesis at the level of precursors.     

Novel Insight into the Genetic Context of the cadAB Genes from a 4-chloro-2-methylphenoxyacetic Acid-Degrading Sphingomonas

The 2-methyl-4-chlorophenoxyacetic (MCPA) acid-degrader Sphingomonas sp. ERG5 has recently been isolated from MCPA-degrading bacterial communities. Using Illumina-sequencing, the 5.7 Mb genome of this isolate was sequenced in this study, revealing the 138 kbp plasmid pCADAB1 harboring the 32.5 kbp composite transposon Tn6228 which contains genes encoding proteins for the removal of 2,4-dichlorophenoxyacetic acid (2,4-D) and MCPA, as well as the regulation of this pathway. Transposon Tn6228 was confirmed by PCR to be situated on the plasmid and also exist in a circular intermediate state - typical of IS3 elements. The canonical tfdAα-gene of group III 2,4-D degraders, encoding the first step in degradation of 2,4-D and related compounds, was not present in the chromosomal contigs. However, the alternative cadAB genes, also providing the initial degradation step, were found in Tn6228, along with the 2,4-D-degradation-associated genes tfdBCDEFKR and cadR. Putative reductase and ferredoxin genes cadCD of Rieske non-heme iron oxygenases were also present in close proximity to cadAB, suggesting that these might have an unknown role in the initial degradation reaction. Parts of the composite transposon contain sequence displaying high similarity to previously analyzed 2,4-D degradation genes, suggesting rapid dissemination and high conservation of the chlorinated-phenoxyacetic acid (PAA)-degradation genotype among the sphingomonads.     

The effect of foliar application of urea on the root fungi of wheat growing in soil artificially contaminated withFusarium spp.

Repeated treatment of overground parts of wheat with a solution of urea was reflected in an increased count of bacteria and a decreased count of fungi on the roots of plants grown in soil contaminated with the fungusFusarium spp., which is known to be noxious to plants. The occurrence of some fungal genera on the roots of plants growing in contaminated and uncontaminated soil after foliar application of urea was also changed. The roots of treated planted were found to contain more bacteria with a lytic effect and retarding the growth of the fungus used for the contamination. The favourable effect of foliar application of urea on wheat was reflected in an increased dry weight of plants even if growing in a soil contaminated withFusarium.