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FAIR-CT95-0363
Development of Pyrethre Cultivation in Mediterranean Europe, for Valorisation on Domestic Insecticide and Public Health Markets |
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Contract No: | FAIR-CT95-0363 |
| Date Prepared: | March 2002, July 2001, September 1999, May 1999 | |
| Source: | Final Report Third Progress Report Second Progress Report First Progress Report |
AGRONOMY
Assessment of the agronomic and biochemical
potentialities of selected clones
A comparative test on the 18
clones selected has been realised in September 1996. The behaviour in
first year of culture of each was studied.
An analysis of flower
productions, pyrethrins content and proportions of each constituent
permitted us to choose 6 clones that were put in isolation in pairs in
October 1997 in order to obtain hybrids which will be evaluated from 2001
using a diallel test will regroup hybrids collected in both 1998 and 1999.
A strong variation in the physical features at anyone time and the
biochemical characteristics has been observed.
Direct seed drilling
The direct seeding of
pyrethrum was the object of several tests in three regions The first
results show that this technique of culture implantation is difficult, but
realistic. A field of two hectares situated in the department of Ardennes
gave some encouraging results.
Weed control
Five active matters herbicides
gave us satisfactory results in weeding of the adult plantations at the
end of winter and fall.
During summer, when pyrethrum is dormant, the
young yearly weeds can be controlled by one or several successive
applications of diquat.
The yearly weeds belonging as pyrethrum to the
family of the Compositae: Seneçio, Sonchus, matricaria... are
resistant to the herbicides which can used on pyrethrum. New
experimentation will be conducted in 1998 in an attempt to solve this
difficulty.
Study of diseases
Attacks of Sclerotinia sclerotiorum
have been observed on all fields.
Six months after the plantation, 5
to 10% of plants were already sick and had been destroyed by the fungus.
Treatments with fungicides done on each field in April and in
September with an association of vinchlozoline + carbenzime allowed
extensive further contamination to be successfully curbed.
200 three
year old plants which has remained healthy were isolated and transplanted
from a field which was very overgrown by the fungus and from which only
20% of all plants survived, into a very contaminated zone in order to
begin studies of plants which are resistant or simply tolerant to Sclerotinia
sclerotiorum.
Harvesting techniques
A fully mechanised
harvesting technique was tested with success for the first time in 1997 on
the fields.
The plants were cut with a machine to cut the rape and
the alfalfa called "andaineuse". After natural desiccation on
the field, the flowers were harvested with a combine equipped of a pick up
to modified alfalfa.
A treatment with an antioxidant before
harvesting prevented the losses of pyrethrins during the drying.
Tests
conducted in Spain with a matricaria harvesting machine on the one hand
and an harvesting machine to snap beans on the other hand gave relatively
unsatisfactory results.
These techniques will probably be abandoned in
1998.
PRODUCTION OF SOMACLONAL VARIANTS WITH RESISTANCE TO PATHOGENS
Different studies were initiated, allowing the
obtaintion of the first steps of organogen callus, without any
regeneration.
Protoplasts have been isolated and were conserved
several days.
A biological marker of tolerance against pathogens
seems to be defined.
Peroxydases are ones of molecules which react
against environmental changes.
Total peroxidase activity was measured
at different times during different experiments.
It was shown that
peroxidase activity increased after application of pathogens filtrates in
comparison with the control.
New experiments will be conducted to
determine the correlation between enzymatic activity, pathogens
applications and tolerance of various clones of pyrethrum. A combination
of the capacity to regenerate from callus under the pressure of selection
and of peroxidase activity after application of filtrate will be used to
select those platelets which are less sensitive to pathogens and then to
clone these in a highly efficient way.
In vitro tests with
biochemical analysis will be applied during the regeneration process and
on the culture in greenhouses for selection of resistant plants.
STUDY OF THE COMPARATIVE ACTIVITY OF THE ACTIVE STRUCTURES OF PYRETHRINS IDENTIFIED IN THE PLANT AS WELL AS THE POSSIBILITY OF BIOLOGICAL SYNERGY
Pyrethrin esters were collected and submitted to chemical analysis (NMR, MS) and re-chromatographied to draw standard curves in order to evaluate the content of pyrethrins in the extracts. These extracts were used for biological evaluations. A program was developed to extract new synergists from plants originated from Vietnam and Niger. Data from various literature indicates that synergist activity is often related to presence of the methylendioxybenzene group (benzodioxole) in the structure of the molecules. The synthesis of a series of new synergists has been undertaken in order to set up structure-activity relationships directed towards the insecticide activity of pyrethrum selected clones. An investigation of the availability of European industrial by-products as starting materials for the synthesis of new low-cost synergist has been undertaken. We are in the process of applying for a patent for the synthesis of these compounds. For confidential reasons, details on this research will be given only in the final report. Studies are continuing to monitor the possible formation of geometrical Pyrethrin isomers during the processing of the industrial extract and to find new synergists from natural origins.
IMPROVEMENT IN THE PROCESSES OF EXTRACTION AND PURIFICATION OF NATURAL PYRETHRINS
Plant matter extraction method
Batch process With mechanic stirring
Trials were initially developed with methanol. It was proved that the results of extraction were improved with the use of n-hexane as the extracting solvent. The most effective ratio solid: liquid is 1/4 1/5 The optimum number of extraction steps is three. The addition of water improves the yield of each step of extraction. The use of an antioxidant BHT slows down the loss of pyrethrins during the different steps of extraction and until the crude extract is recovered.
Semiantinous, counter current process trial with mechanical stirring
Trials have been developed in three steps of extraction getting similar results to the ones obtained on the batch process.
Continuos counter current process where the plant matter has been kept in stationary stage
A greater number of steps are required to extract the plant matter to the maximum. A lower rate of efficiency on the extraction process is observed in comparison to the processes involving stirring.
Extraction on supercritical conditions
Trials were developed in a pilot plant scale. The pilot process trial was based on laboratory experiments of batch process extraction. The results, in terms of yield, were similar to those obtained on a laboratory scale.
Purification trials on the crude extract
Preliminary
experiments has been conducted with the following results Methanol was
observed to be a suitable first step extractor agent.
Isolation of the six pyrethrins
Up to now,
it has not been possible to get an agreement in the analytical results of
a commercial refined extract. A possible way to over come this could be to
isolate solutions of each pyrethrin in order to obtain a response factor
for each compound. Until now, we have had difficulties in obtaining
cinerins and jasmolins in sufficient quantities.
Stability study on the plant matter
Very
little loss on palletised flowers harvested on the year 1997 has been
observed: less than 5 % six month after harvesting. The use of BHT during
the grinding and palletising processes is probably the main reason for the
deceleration of the pyrethrin losses.
IMPROVING THE BIOCHEMICAL ACTIVITY OF PYRETHRINS
Summary
Preliminary research and background information on agronomy and genetics The work started with evaluation of germplasm in the field. An analysis of 15 wild populations of Chrysanthemum cinerariaefolium collected on the Dalmatian coast has shown a wide variability in expression of the both agronomic and biochemical characters. Screening of about 600 plants selected ferom the best of these wild populations enabled identification of 18 individuals characterised by good agronomic performance characteristics; high strength, good standability, high production of flowers with abundant florets, combined with high contents of pyrethrin I and pyrethrin II. These 18 plants will all be used within for subsequent genetic improvement work on this species.
The germination rate of achenes obtained from self-fertilizing plants was found to be less than 3% whereas the germination rate of achenes obtained from cross-fertilizing plants ranged from 20- 60%. The high autosterility should make it fairly easy to obtain between two or more clones obtained from the selected 18 plants with a high potential.
Optimisation of the techniques and processes in agronomy and genetics The 18 clones were multiplied by micropropagation of 500 units from each over 6 months, without any particular. This was achieved by building upon previous works documented in the literature. The evaluation of these clones was initiated, through a comparative test of the 18 clones selected, in September 1996. During 1997, it enabled further selection of the 6 clones to be intercrossed in a two-allele test in order to create improved varieties, through clone hybridisation.
Several callus induction media and regeneration media have been optimised on the basis of numerous tests. These organogenesis media are being adapted for the induction and development of somatic embryos. Various combinations of hormones, mineral elements and temperatures have been tested, as has the physiological age of the zygotic embryos.
As a result of extensive damage to cultivations caused by sclerotinia, work will concentrate on an investigation of variants exhibiting tolerance to this disease. The investigation of Phytophtora-toterant variants seems less urgent. Early in vitro tests are being developed for measuring the resistance to phytophtora. They will be used as a model for the preparation of more complex scierotinia resistance tests.
Optimisation of the techniques and processes in cultivation Experimental plantations were grown in Spain in 9 plots, distributed in 7 different areas. These plantations were grown in February 1996 on 4 hectares and in February 1997 on 6 hectares. So far the best results have been obtained in Andalusia, in areas with an altitude in the range of 500-800 m. Experimental plantations were also made in France, set up in September 1994 in 4 plots in the West-Atlantic and the Loire Valley regions. In Spring 1997, the behaviour of the cultivations was satisfactory.
The plantations were all grown from "miniclods", using know-how acquired by a company specialised in the production of young plants. The plantations: in France were made with a planting machine especially equipped for such plantations. This proved to be quite satisfactory once a few modifications had been made. Hence, techniques for planting appropriate for the development of cultivations over large areas have been established.
Control of weed Experiments with herbicides have identified several herbicides that may be applied before and after planting-out. These are both effective against weeds and well suited to pyrethrum. End-of-winter weeding tests have been initiated. A combination of two products applied following the emergence of weeds seems particularly promising. In due course, once these results have been substantiated (1998), a complete complete chemical weed control strategy will be formulated for pyrethrum cultivations.
Control of diseases Several pathogens have been identified on the first pyrethrum plantations both in France and in Spain. At the moment, only sclerotinia has proven detrimental to cultivation in France. The results of the first fungicide treatment tests gave poor results and hence trials had to be continued with preventative treatments for cultivations being performed systematically. In parallel, sclerotinia-resistant genotypes will be investigated using biotechnology methods.
Preliminary research and background information on chemistry and biology Industrial pyrethrum extracts were analysed and methods developed to characterize and to separate the major components. Authentic samples of pyrethrin I and pyrethrin II were obtained for biological evaluations. Studies were also undertaken in order to monitor the possible formation of geometrical pyrethrin isomers during the processing of the industrial extract and to find new synergists from natural sources.
Compativeactivity of pyrethrum extracts and different pyrethrins The comparative activity of commercial extracts of isolated pyrethrin I and pyrethrum II was measured on 5 species of insects. These were American cockroach, German cockroach, African leafworm, house fly and mosquito. The five species tested showed differing susceptibility to these two pyrethrins. The lethal doses as well as the repulsive effect also varied from one insect to another, depending on the pyrethrin tested. The pyrethrum extract was more potent than either of the isolated pyrethrins, pyrethrin I being more potent than pyrethrin II.
Evaluation of the synergistic activity of a range of natural and synthetic synergists Various ratios of pyrethrins/synergists ranging from 1/0 to 1/20 were tested. The activity of both pyrethrins was always increased by the synergists. The activity of pyrethrin II was increased to a greater extent by the use of synergists than that of pyrethrin I. The efficiency of the synergists varied in relation to the insect species. Of the five synergists tested, sesamin was the most effective, followed by piperonil butoxide, MB 599, MGK 264 and apiol.
Preliminary research and background information on the extraction process The analytical process analytical procedures were optimised. Processes included the reverse-phase HPLC assay method developed in France for assaying pyrethrins I and 11 in pyrethrum flowers. This enabled the selection of the 18 genotypes used in the selection programme. The use of two chromatography columns allowed for a better separation of the peaks, making their integration easier. Various extraction solvents were tested. These included methanol, ethanol and acetonitrile. The best results were obtained with methanol. Such extractions and other tests could first be performed on flowers harvested in May 1997.
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