1. Role of Biopesticides in Agricultural Systems
Dr Errol Hassan
Adjunct Associate Professor
School of Agriculture and Food Sciences
The University of Queensland Gatton
Queensland, Australia

2. Tiny insects…BIG impact!

3. Insect impacts Economic impact (positive) Biological control
Parasitic/parasitoid wasps
Predators
Waste disposal
Dung beetles (reducing waste, ecosystem engineering)
Weed control
E.g. Lantana beetle, Cactoblastis spp. (prickly pear control), Salvinia destroyed by weevil Cyrtobagous salviniae
Pollination!
Crawford, D., ND
CSIRO, ND
Greb, P., ND

4. Insect impacts Economic impact (negative)
Introduced insects between cost (Australia):
4,665,250 tonnes of lost horticultural/agricultural production
$747,125,000 in control costs (Clarke, 1996)
These costs do not include reduced yield or control costs in livestock (e.g. ticks, lice, flies etc)
Crop herbivores or predators
Plant disease vectors
Human disease:
Dengue fever ($2.7m p.a. to control in Qld alone; mosquito-borne; Canyon, 2008)
Ross River fever (mosquitoes)
Malaria (mosquitoes)
Lyme disease (tick-borne)
The Bubonic Plague (rat fleas) – killed 1/3 of British population
Westmead Hospital, ND

5. Insecticidal Properties of Plants
A large number of different plants contain natural allelo-chemicals that have insecticidal properties (Morallo-Rejesus, 1986; Chun et al., 1994; Dev & Koul, 1997; Rathi & Gopalakrishnan, 2005)
Insecticidal properties derived from plants are generally termed phytochemicals and/or botanicals.
The efficacy of plant crude extracts that contain complex bioactive materials are effective than individually isolated and purified chemicals therefore reducing the potential for insect resistance.
Botanicals break down into non-toxic substances within hours /days when exposed to sunlight.

6. Benefits
The benefits of using plant products as insecticides in comparison to chemical insecticides include the following:
They have little or no harmful effects on non-target organisms (Schmutterer, 1997; Hedin et al., 1997, Khrisnamoorthy et al., 1999).
Insect resistance has not been reported.
They can be integrated in IPM (mixed with conventional pesticides), to provide better insect pest control.

7. Screening Process Selection of promising plant materials
Proper collection of selected plants
Authentication of plant material
Drying of plant materials
Grinding of the dried plants
Packing, storage and preservation
Extraction and fractionation of bioactive materials
Methods of separation and purification
Methods of identification of isolated compounds
Structure elucidation e.g. Ultra Violet, Infra Red Spectroscopy, Mass Spectrometry, H-Nuclear Magnetic Resonance and C-Nuclear Magnetic Resonance.

8. 1. Selection of Promising Plant Materials
The choice of promising plant depends on the following:
A plant that has biological activity.
A plant used in folk medicine.
A plant which shows a particular toxicity.

9. 2. Selection of Promising Plant Materials
Wild Plants
Cultivated Plants
Disadvantages
Advantages
Scattered in a widespread area
Wide availability
Inaccessible areas
Easy access
The collector must be a highly skilled botanist
The collector does not need to be a skilled botanist
Plant deficiencies may occur because of over-collecting
Continuous supply of plants

10. 3. Plant Collection Prosedure
The following precautions need to be considering when plant collecting:
The proper time of day, time of year and plant maturity since the stage of collection is dependent on the nature and quantity of bioactive materials found in certain species (dependent on season and time of collection).
The plant should be free of contamination (ie. Other plant growth, insect activity).
Plants should be free of diseases (ie. Unaffected by viral, bacterial, and fungal infection).

11. 4. Correct Plant Identification
It is important to correctly identify plants by the following methods:
Establish the plant identity by a taxonomist.
Collect common species in their expected habitat by a field botanist.
Compare the collected plant with a specimen in herbarium.

12. 5. Drying Plant Materials
The aim of drying plant materials provides the following:
Ease of handling and transport.
Ease of grinding.
Inhibiting the growth of micro-organisms.
Preserving bioactive materials.
Drying is undertaken in:
Shade and/or sunlight (natural light)
Hot-air drying and/or freeze drying (artificial light).

13. 6. Changes May Occur When Drying Plants
The changes are:
Size and weight
Colour
Shape and appearance
Taste and odour
For example, slow drying of vanilla pods leads to release of vanillin from glucovanillic alcohol.

14. 7. Drying and Grinding
In general, plant material should be dried at temperatures below 30°C to avoid decomposition of thermolabile compounds.
Likewise, plant material should be protected from sunlight because of the potential for chemical transformations resulting from exposure to ultraviolet radiation.
To prevent the build-up of heat and moisture, air circulation around the plant material is essential. Therefore, it should not be compacted, and it may be necessary to use a fan or other means to provide airflow around or through the drying sample (Jones & Kinghorn, 2005).

15. 8. Extraction and Fractionation of Bioactive Materials
There is no universal method for the extraction of plant materials.
The precise mode of extraction depends on:
The texture of the plant material.
The water content of the plant material.
The type of substances to be extracted or nature of active constituents.

16. Extraction Extraction:
The separation of chemically active portions of plants or animal tissues through the use of selective solvents and suitable methods of extraction.
Methods of solvent extraction can be classified as:
Continous (percolation and soxhlex extraction):
Solvent flows through the plant material continuously. As constituents diffuse from the plant material into the surrounding solvent, the solvent becomes increasingly saturated but because the solvents are continually flowing the saturated solvent is replaced with less-saturated solvent.

17. Extraction Discontinuous
The solvent is added and removed in batches. Hence, once equilibrium is reached between the concentration of solute inside the plant material and the concentration in the solvent, extraction essentially stops until the solvent is decanted and replaced with new solvent.

18. Extraction Percolation An efficient method of extraction.
Suitable for medium to large sample sizes.
Usually not practical for small amounts of plant material.
Soxhlet extraction
Convenient methods of extraction of small to moderate volumes of plant material.
Amount of solvent needed for this extraction is minimal.
Heat needed to drive the extraction will likely cause thermolabile constituents to decompose product.

19. Extraction Maceration
A common method for extraction of small amounts of plant material in the laboratory is undertaken because it can be carried out conveniently in Erlenmeyer flasks. The flasks can be covered with parafilm or aluminum foil to prevent evaporation of solvent.
The solvent should be decanted through a screen or filter and fresh solvent added to the flask.
The sample is then mixed with the fresh solvent by stirring or swirling, and left to macerate again.

20. Continuous Hot Extraction Techniques
(Soxhlet extraction process)

21. Steam Distillation Process to Extract Essential Oils

22. A. Calamus Extraction Process Using Rotary Evaporator
Powder extracted byusing 4 different solvents (24 hrs)
Solvent(s) evaporated using water bath
A: Rhizome
B: White Powder

23. Solvent Choice
Factors that should be considered when choosing a solvent:
solubility of the target constituents,
safety ease of working with the solvent,
potential for artifact formation,
the grade and purity of the solvent.
As a general rule:
Benzene, petroleum, ether and hexane dissolves non-polar compounds (fats and waxes).
Chloroform, methanol, ethanol and water dissolves polar compounds (alkaloid, steroids, quinone, salts and sugars).
The solute affinity can be increased by using a mixture of solvents.

24. Example
Solubility of an aliphatic carboxylic acid in ethanol and acetone.
A mixture of ethanol and acetone.

25. Serial Dilution Figure: Emulsions of Acorus and L.petersonii extracts
A = L. petersonii
B = Acorus in chloroform
C = Acorus in methanol
D = Acorus in hexane
E = Acorus in Benzene A B C D E

26. Making Dilutions To make dilutions follow these steps:
Use volumetric flask and pipette to measure volume
Use the equation M1V1 = M2V2
1 = Current concentrated volumes
2 = Result(s) of diluted conditions
M = Moles as concentration
V = Volume

27. Dilution Formula
Let us assume the crude extract has 100% M (moles as concentration), what is V (volume) if we dilute crude extract to 10% for 10ml for stock solution?
M1 = 100%,M2= 10% V2 = 10 ml
M1V1 = M2V2, M2V2/M1 = V1
V1 = (10% x 10ml) / (100%) = 1 ml

28. Developmental Effect of A.calamus on P.xylostella
Rhizome extracts of A.calamus were tested in the laboratory for their effects on feeding, development, and mortality of P. xylostella.
Extract of Acorus at a concentration range of 2% - 10% showed anti-feedant effects related to the dose. It also reduced the size, weight and growth inhibitory effect in the larval stage of the test insect.
The A. calamus showed developmental disruption on adult and pupal stage in the next generation.

29. Developmental Effects
Methanol
Hexane
Chloroform
Benzene

30. Developmental Effect of Leptospermum petersonii Oil on P.xylostella

31. Biopesticides: Definition
An active ingredient or formulation that is effective in controlling pests and is typically derived from biological or natural origins.
Production agriculture is a highly complex system where growers and farm managers control inputs and processes in an effort to maximize quality, efficiency, and economic results.
As an input category, plant protection and plant enhancement chemicals help define the success of a given system.

32. Microbial Pesticides
Ingestion of Bt’s crystal protein from treated leaves.
Feeding stops within minutes after crystals are solubilised in the gut and the gut cells are damaged.

33. Biopesticide Market Growth Rate
Time Period
Crop Protection Market % Annual Growth Rate in Real Terms
Biopesticides Market % Annual Growth Rate in real Terms
6.8
0.1
2.2
8.0
9.0
-2.3
10.0
Forecasted after 2005
0.75*
6.0**
* Market growth limited by market saturation, generic, and regulatory impact
** Market growth slower due to the lack of introduction of new products
Sources: Phillips McDougall; Agro Report 2004; Biopesticides, Biocontrol and Semio-chemical markets; Scientific Consultants 2002: Biopesticides 6th Edition; V.1-Markets V 4-Companies.

34. Biopesticide Market Growth Rate
The global trend toward biopesticides is increasing because:
Increased commitment to integrated pest management programs (IPM) by key influencers. Key influencers are typically classified as extension specialists, agronomists, and consultants who advise growers.
Advances in formulation technology have made biopesticides easier to use.
Growing consumer awareness about pesticide use. Science and information technology have advanced, so has consumer awareness regarding pesticides residues on food.

35. Biopesticide Market Growth Rate
This awareness has helped shape public policy, which has resulted in increased use of biopesticides.
Increased demand for organically grown food while biopesticides are critical component of traditional agricultural programs.
Plant protection programs in organic production are largely dependent on biopesticides.
Organic acreage has increased
by nearly 400% in the past 10 years in USA.
Source: BCC Research Report 2010

36. Biopesticide Benefits
Enhanced crop quality and shelf life
Resistance management
Maintaining beneficial populations
Residue management
Labour and harvest flexibility
Worker safety
Environmental safety

37. Benefits in Resistance Management
Insects, weeds, and disease population can develop resistance to pesticides quickly.
With a limited number of pest control products available, the loss of a single important product can be devastating.
Biopesticides have more complex modes of action than their traditional counterparts. This means that insects and plant pathogens have difficulty in developing resistance to biopesticide products.

38. Benefits in Residue Management
Increasing consumer awareness toward the nature and application of pesticides is one of the driving forces behind the growing biopesticides trend.
A key benefit of biopesticide products in their ability to help the grower deliver produce with the absence of pesticide residue, or residue levels within regulatory parameters.

39. Benefits through Worker Safety
Worker safety is a benefit of working with biopesticides applications since there is a lack of human toxicity. This is advantageous when workers are required to re-enter the field and where mechanical harvesting is not possible.
Similarly, biopesticides have become increasingly important in greenhouse operations, where pesticides applications take place in enclosed areas.

40. Benefits through Environmental Safety
Environmental stewardship and best management practices are at the forefront of commercial farming operations.
Biopesticide products deliver on both fronts by providing solutions with a low impact to the environment without sacrificing effectiveness.
In general, biopesticides have minimal toxicity to birds, fish, or bees, help to maintain beneficial insect populations and break down quickly.
Biopesticides can be integrated effectively with traditional pesticide application methods.

41. Resistance Management for Sustainable Agriculture
Effective management of pest insect populations in agriculture, horticulture, public health and animal health is dependent on a variety of inputs. This includes the supply of safe and highly efficacious insecticides.
If insecticides share a similar mode of action to control insect pests, there is a likely chance that the insect pests will develop resistance to those insecticides.
Cross-resistance occurs when compounds within a specific chemical group usually share a common target site within the pest.

42. Resistance Monitoring Methods
Reliable data on resistance, rather than anecdotal reports or assumptions, are essential to successful insect pestc resistance management.
A key to this is the availability of sound baseline data on the susceptibility of the target to the toxicant.

43. Insecticide Resistance Defined
Resistance is defined as a “heritable decrease in sensitivity to an insecticide”.
Cross resistance occurs when resistance to one insecticide confers resistance to another insecticide, even where the insect has not been exposed to the latter product.
Since pest insect populations are usually large in size and they breed quickly, there is always a risk that insecticide resistance may evolve, especially when insecticides are mis-used or over-used.

44. Mechanisms of Resistance
Metabolic resistance: Insects use an enhanced level of metabolic enzymes that detoxify particular insecticides faster than susceptible insects.
Target site resistance: The target site where the insecticide acts in the insect may be genetically modified to prevent the insecticide binding or interacting at its site of action. This reduces and/or eliminates the pesticide’s efficacy.
Penetration resistance: The insecticide may penetrate slowly through the resistant insect’s cuticle compared to the susceptible insects.
Behavioural resistance: Resistant insects may detect or recognise the presence of the insecticide and avoid it.

45. Strategies to Prevent and Delay Pest Resistance
Pest Monitoring
Pest monitoring is one of the key activities that users of insecticidal product can implement as part of their insecticide resistance management strategy.
Farmers should follow the progress of insect population development in their fields to determine if and when control measures are warranted. They should monitor and consider natural enemies when making control decisions.
After treatment, farmers should continue monitoring to assess pest populations and the effectiveness of any control measures implemented.

46. Strategies to Prevent and Delay Pest Resistance
Economic Injury Levels
Insecticides should be used only if insects are numerous enough to cause economic losses that exceed the cost of the insecticide plus application, or where there is a threat to public health.
Farmers are always encouraged to consult their local advisors about economic thresholds of target pests in their areas.

47. Sprayed Decision

48. Functional Botanical Pesticides
Deformity
Starvation
Repel
Pests
Weak No
Moulting
Sterilize
Unfit
Adults
Oviposition
Ovicidal
Contact
Action
Systemic Action
Egg Mortality
Larval Mortality
No Plant
Damage
Predator
Killing
Pesticide
Knockdown
No Damage
No Egg Laying
Botanical Pesticides
Pupae & Adult Mortality

49. Example of Insect Pest Damage
A cotton boll infested by H. armigera
The insect pest, Helicoverpa armigera (Heliothis) (Cotton Bollworm) is a major global pest of cotton, tomato, sweet corn and many other crops.
It is resistant to many synthetic pesticides.
The economic crop loss and pesticide control costs to Australian agriculture is $225 million per annum.

50. Example of Insect Pest Damage
The insect pest, Helicoverpa armigera (Heliothis) (Corn Earworm) feeding on sweet corn.
It is resistant to many synthetic pesticides.
The economic crop loss and pesticide control costs to Australian agriculture is $350 million per annum.

51. Biopesticide Effects on Helicoverpa armigera
Pupal (above left) and larval (above right) abnormalities due to feeding on lemon myrtle oil treated leaf (500 μg/cm2).

52. Example of Treated Cabbage Cluster Caterpillar
Deformed Cabbage Cluster Caterpillar pupae (left and below)

53. Effects of Formulated Products
The bar graph depicts the efficacy of different effects of formulated components of essential oils on Cabbage Moth (Plutella xylostella).

54. The University of Queensland Campuses
The UQ St. Lucia Campus, Brisbane
The UQ Gatton Campus

55. Sydney, Australia
Sydney, Opera House
Sydney, Harbour Bridge

56. Australian Indigenous People (Aboriginal)

57. Australian Native Marsupials and Traditional Hunting
Koala bear
Grey kangaroo
Red kangaroo
Boomerang

58. Great Barrier Reef and Sea Turtle, Shark and Clown Fish