AIR2-CT94-1343 AZTEC: Innovative Technologies to Produce Environmentally Sound Natural Pesticides from Neem tree (Azadirachta spp.)

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AIR Cluster X - Inputs for Non-Food Crops : Biotechnology : Crops for Biological Control Products : Crops for Flavours/Fragrances : Crops for Pharmaceuticals/Cosmetics : Essential Oil : Flavours/Fragrances : Integrated Crop Protection & Biological Control : Pharmaceuticals/Cosmetics : Plant Genetics : Process Engineering

	Proposal No:	AIR2-CT94-1343
	Date Prepared:	September 1999
	Source:	Progress Report October 1997

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Progress Report October 1997

Summary

Objectives The general objectives are:

• the production by biotechnological means of natural pesticides from Azadirachta spp.(azadirachtin, marrangin and novel compounds)
• their evaluation through bioassays and field tests, in order to establish their potential as alternative pesticides - either in agricultural or civil environments - that have a minimal impact on the environment and on human health
• to obtain a greater understanding of the various fundamental and technical issues (fermentation technology, metabolic pathways, chemical and analytical aspects, biological activity, and product formulation) that have to be addressed to develop the target compounds at pilot scale

Activities As one of the main objectives of the AZTEC project was to enhance azadirachtin A production in plant tissue culture further work was carried out with a cell line VsB of Azadirachta indica that appeared to perform better than the cell line VmB. This was fully characterised, not only in terms of growth capabilities, but also in terms of expression and production of target compounds, including aspects of elicitation. Results clearly show that both cell lines are capable of expressing azadirachtin A, azadirachtin B, azadirachtin I and azadirachtinin. The VsB cell line had a greater capacity for total biomass production but the expression of the desired target compounds was at a level 10 to 100 lower than that o f the VmB cell line. The increase in total biomass production of the VsB cell line was insufficient to compensate for the decrease in target compound expression so it was concluded that the VmB cell line was most suited for further studies using a bioreactor.

The elicitation studies indicated that chitosan stimulated azadirachtin A production while jasmonic acid inhibits its production. This suggests that different reaction pathways are activated depending on the nature of the elicitor applied. These results indicate that it is possible to manipulate expression rates of the target compounds within the cell cultures, both positively and negatively.

As far as the stability of the cell lines from Azadirachta indica is concerned, it was concluded that the ability to accumulate biomass did not appear to be impaired with the time, but the expression of azadirachtin A obtainable at levels approaching those in the whole plant is not stable.

The regeneration of whole (shoot and root) plantlets of Azadirachta excelsa was achieved through a vegetative organogenesis process.

The results of biotransformation studies suggested that various enzymes including peroxidases, squalene synthetase and cytochrome P-450 are involved in the production of azadirachtin. Data derived from stimulation and inhibition experiments on cytochrome-P450-NADPH reductases suggested that this class of enzymes as well as other related enzymes such as monooxygenases and epoxidases are involved in many biogenetic steps.

Several biotransformation experiments conducted mainly using salannin and niznbin, did not give the expected results.

Fermentation experiments at laboratory scale were carried out with the VmB cell line incubated in the presence of 6 % sucrose, as prior shake-flasks experiments had shown that these conditions favoured biomass accumulation and production of the target compounds. The most important findings of these fermentation experiments were that:

• the cell line VmB is not destroyed by the change from shake-flask conditions to bioreactor conditions
• growth occurs
• expression of some of the characteristic secondary metabolites of Azadirachta indica occurs

Hence, the feasibility of the fermentation process was confirmed at pilot scale.

A four-stage extraction process was developed. It can easily be scaled-up to pilot plant or commercial plant size. The main equipment required is a decanter and phase-separation vessels. The solvents used can be largely recycled for which a wiped film or falling film evaporator is appropriate. Depending on the desired composition of the product, different extraction steps may be omitted without loss of material. The potential for selective extraction of the different molecules has been demonstrated at pilot scale but further work is necessary to fully define the process.

Chemical studies have been directed towards the isolation of triterpenoids and other products in seed and cell culture and towards a better understanding of the stability of Neem compounds during isolation and in storage after isolation. One result was the development of the 'Biotage' procedure for larger samples, and the SPE method for small samples. The effect of various parameters (pH, temperature, solvents, UV) on the decomposition of azadirachtin were determined. The conclusion is that compared to other natural insecticides, such as pyrethrin and rotenone, azadirachtin is a rather stable substance and its relative stability, together with its high potency and systemic action give high hopes for its future widespread use.

The main goal of the synthesis work has been to prepare a number of precursors for use in biotransformation reactions. A biomimetic approach was adopted based on a previously proposed biosynthetic route. In order to explore the chemistry a number of model systems and three precursors were developed, and introduced into the biotranformation studies. In order to develop more advanced precursors for biotransformation, it was necessary to develop methodology for extension at C8 of the left-hand side of azadirachtin. This was achieved after extensive effort, and various compounds produced in which the left-hand side of azadirachtin to had been extended.

Many samples from the tissue culture work have been tested for bioactivity and in general it was found that most of the characteristic triterpenoids produced in the whole plant are also present in the tissue cultures from the same plant. This is a very important result as it forms the prerequisite to justify a plant tissue culture approach to the production of these useful metabolites. Furthermore, this approach enables detailed study of the nature and regulation of the various metabolic pathways that lead to the formation of these different compounds.

In the plant tissue cultures some compounds, that are very bioactive, are produced that are not detectable in the seeds. These findings need further investigation as this might indicate the presence of other unknown compounds which might be of great scientific and practical interest.

The results of treating an economically important fungus such as Spheroteca fuliginea showed that the cell extracts after appropriate extraction can efficiently be used for controlling this type of infection.

The efficacy of the formulated compound was very interesting (similar to the result obtained with a farm spraying programme) towards Pandemis cerasana (Lepidoptera - Tortricidae) in spite of the high pest pressure (10.5% of the untreated plants were damaged and 5.5% not marketable because of the pest damage). A study on Phytometra gamma L. (Lepidoptera - Noctuidae) indicated that azadirachtin works as a moulting inhibitor: after the ingestion of some treated leaf portion larvae -quickly stopped feeding and in a few days died when they tried to moult. However, when activity was assessed using the pest Aphis grossypii Glov. (Rhynchota - Aphididae) results were unsatisfactory. Azadirachtin seemed to slow down the increase of the colony, but in a few days this effect was no longer evident.