Refugium Area as an Important Tool for B.t. Cotton to Delay Resistance and Improve Host/Prey Availability for Natural Enemies Specially in the Monocultures Agro-ecosystem

Sobhy Temerak
Research Entomologist
Head of bio-control Unit
College of Agriculture
Assiut University, Egypt

INTRODUCTION There is a continuing need to increase food production as the world population is expected to be more than 6 billion by 2050. The cost for achieving production has become very high because of the need to control insect pests that cause an estimated loss of 8- 10$ billion annually. The difficulties experienced in controlling insect pests over the past 45 years have been largely due to the wide, unwise use of pesticides. Indiscriminate use of insecticides has resulted in the development of resistance in insects and the significant decrease of natural enemies.

Insect resistance to insecticides is one of the vexing problems in recent years due to the indiscriminate use of insecticides. About 400 species of insects and Acarina are known to have developed resistance to inorganic compounds, chlorinated hydrocarbons, organophosphate insecticides and carbamates (Brown, 1978 cited from B. Fakrudin et al 2004).

In Egypt, cotton lost 50% of its cotton yield due to the cotton leaf worm ( CLW ) Spodoptera littoralis resistance to Toxaphene countrywide in 1961. The product was applied 4-5 times on the same pest in the same season without any rotation program .For that reason, Egypt is considered one of the first countries applied rotation program on cotton since 1979( Temerak 2002 ).In 1971, 3000 of baflo chronic toxicity were appeared in Koutour KaferSheik Governorate due to the airplane application of Leptophos (Phosvil )on cotton. In 1975/76; MOA imported Gusathion methyl which arrived Egypt as gusathion ethyl. The last showed severe toxicity to cotton labors. Also. the same was happened with Gelecron to kill CLW eggmasses ( Temerak 2006 under publication ).

In India, farmers even apply 36 to 40 rounds of pesticides to the cotton crop of a duration of 150-180 days in a single season i.e., one spray for every five days (Banerjee et al., 2000). However , Fakrudin et al 2003 reported one spray every 2-3 days interval although the recommended spray is 8-10 sprays in India. The last representing a very high environmental pollution. Applying different class of chemicals that has different mode of actions delay resistance, decrease number of sprays and better efficacy (Temerak 2003a).

Bio-pesticides such as Bt (Bacillus thuringiensis) products are widely regarded as being the least harmful to natural enemies. Because of its selectivity and environmental safety, usage of Bt is increasing, particularly in IPM programs. Foliar application of Bt breaks down quickly under field conditions due to UV sensitivity and rainfall. (Gopalaswamy et al 2003) Also, it does not have stable results from year to another. Plant incorporated protectants (PIP) utilizing B.t. offered the best solution to elongate the bio-residual activity of this group in the field.

The primary target pest of this technology in India and several other countries is the cotton bollworm (CBW), Helicoverpa armigera (Hubner) which causes economic losses up to about 250 billion in India (cited from Fakrudin et al 2003).

The introduction of Bt transgenic crops is an important addition to the existing components of Integrated Pest Management. There is a considerable increase in global area under transgenic crops from 1.7 million hectares in 1996 to 52.6 million hectares in 2001, in which the share of Bt crops was 15% of the total area (James 2001). The economic advantage gained during 1999 by Bt cotton alone has been estimated to be $213 million in the USA. Cultivation of transgenic crops has led to a reduction in pesticide use and significant increase in yield (Cannon 2000). Of the $8.1 billion (US dollars) spent annually on insecticides worldwide, it was estimated that nearly $2.7 billion could be substituted with Bt biotechnology applications (Krattiger 1997.cited from Gopalaswamy et al 2003)

The technology is perceived to be effective and eco-friendly for reducing the considerable amount used from conventional insecticides. When resistance to insecticides had become a major problem, many hoped that use of Bt would not follow the same pattern. However insect resistance to Bt toxin has been reported in several populations of cotton bollworm in Australia, USA, and China since the inception of Bt cotton cultivation (Gould et al., 1997; Guhan et al., 2001 cited from B. Fakrudin etal 2004). Furthermore, the problems of pest management in B.t. cotton have been accompanied by the development of resistance to key CBW & Cotton leaf worm (CLW) pests (H. armigera, Kranthi et al. 2000; Pectinophora gossypiella Tabashnik et al. 2000a,b; S. exigua Moar et al. 1995; S. littorals, Muller-Cohn et al. 1996).

Moreover, the situation of beneficial arthropods is being disturbed because they are density dependant and Bt cotton areas do not harbour enough availability of host/prey for beneficials. No considerable work was done to quantify the effect of B.t. cotton areas on the abundance of natural enemies or beneficial arthropods.

Management of refugia as areas percentages versus free-toxin cotton areas and safety type of insecticides, will be play a significant role to dilute or decrease the two problems of resistance and conservation of beneficial arthropods

A -Refugia importance for natural enemies or beneficials
A.1 -Positioning and size of refuge areas, and its effect on natural enemies
No considerable work was done to find out the best size area of refugia and its relation to the abundance of beneficial arthropods. Refuge area as 20% treated with conventional insecticides (according to Matten 2000) may be not enough to serve and conserve natural enemies from insects, mites, true spiders and wild arthropods.

Refuge area as more than 20% may be recommended. May be 4% untreated non-B.t is better than 20% treated with conventional insecticides. Also, may be 20% as refuge areas is enough when use new safe insecticides instead of conventional insecticides

Moreover, mass release of some additional benficials e.g. Trichogramma or coccinellids may be incorporated and needed especially in mono-crop systems that use only 20% refuge areas. No considerable published work was seen until now to study such effect. Furthermore, information on the effect of beneficial enemies (that currently presented or released in the agroecosystem) on B.t. resistant insects are urgently needed

A.2- The need of new molecules to be applied in the refuge areas:
Therefore, care must be taken to ensure that refuges, particularly those sprayed with insecticides, produce adequate numbers of susceptible insects. Models and experimental data showed that separate but adjacent refuges might be superior to other strategies for insects, which can move between plants in their larval stage (Shelton et al. 2002).

The non-Bt.cotton areas must use highly safe insecticides in rotation to conserve natural enemies and also, not to produce resistant from using them. This can be done through alternate different class of relatively safe chemicals that are different in their mode of actions through IPM program (Temerak 2002).

One of the most important strategies suggested by several entomologists across the globe to manage and delay insecticide resistance is use of new molecules having novel modes of action. Recently some of the new molecules have entered the pesticide market, which can play a major role in resistant pest management (Viiaykumer et al 2004a )

A.2.1 - Using safe new insecticides to control sucking in non- B.t. cotton.
With the introduction of novel eco-friendly insecticides in the past 4-5 years, cotton pest management now appears to be very promising. The chloronicotinyls (imidacloprid, acetamiprid and thiomethoxam) and the insect growth regulator diafenthiuron are selectively more effective on the sucking pests and less toxic to beneficial insects as compared to all the conventional insecticides . (Kranthi et al 2004).

A.2.2- Using safe new insecticides to control CBW/CLW in non-Bt. cotton.
More interestingly, apart from the introduction of Cry toxins in the form of transgenic technology, chemicals such as spinosad, indoxacarb, emamectin benzoate, novaluron and lufenuron ensure effective control of H. armigera while being less toxic to beneficial insects in the cotton ecosystem. Kranthi et al 2004

A.3- Negative cross resistance of spinosad & non-Bt cotton of Spodoptera
Temerak 2003b indicated that spinosad was not being affected by the existing CLW resistance to conventional insecticides.New molecules with new mode of action are generally costly in short view , but the added value of decreasing or not being affected by the existing resistance mechanism to conventional may be considered not costly. From the economic point of view, intelligent farmers do not compare cost /ha based on single product cost but they are compared the whole package of the season . Such package may achieve less no. of sprays , better efficacy of resistant insects and good yield.

B-Refuge importance to reduce or delay resistance
B.1-Non-refuge
Mono-cultivation of Bt transgenic crops is likely to select intensely for resistance because pests will be exposed to Bt even when they are not causing economic damage (Mallet & Porter 1992).

B.2 - Examples of possible resistance
At the time, when resistance to insecticides has become a major problem, many hoped that use of Bt would not follow the same pattern. However insect resistance to Bt toxin has been reported in several populations of cotton bollworm in Australia, USA, and China since the inception of Bt cotton cultivation (Gould et al.,1997; Guhan et al.,2001 cited from B. Fakrudin et al 2004).

Helicoverpa armigera is capable of developing resistance to Cry1Ac in 7 to 8 generations (Kranthi et al. 2000). Highly mobile polyphagous pests such as Helicoverpa may develop resistance to Bt on one transgenic crop and then disperse, nullifying the effectiveness of a wide range of Bt transgenic crops expressing the same or similar Cry proteins. Pests with resistance to CryIA proteins in transgenic plants may also display significant resistance to Bt biopesticides.

Field collected pink bollworm , Pectinophora gossypiella quickly evolved resistance to Cry1Ac under laboratory selection (Patin et al. 1999, Simmons et al. 1998, Tabashnik et al. 2000b). Pectinophora gossypiella selected with Cry1Ac protoxin developed 300-fold resistance to Cry1Ac protoxin, and high levels of cross-resistance to Cry1Aa and Cry1Ab protoxin, and low levels of resistance for Cry1Bb protoxin (Tabashnik et al. 2000a). Three selections with Cry1Ac in artificial diet increased resistance of pink bollworm to >100-fold relative to a susceptible strain (Liu et al. 2001).

In general, Spodoptera spp. larvae are not very susceptible to the Cry toxins (Strizhov et al. 1996). However, Cry1C toxin had been reported to be toxic against S. exigua (Visser et al .1988) and Spodoptera littoralis (Van Rie et al. 1990). Selection to Cry1Ca caused 850-fold resistance to Cry1Ca and cross-resistance to Cry1Ab, Cry9C, and Cry2A, as well as to a recombinant Cry1E-Cry1C fusion protein in S. exigua (Moar et al. 1995), while in S. littoralis, 500-fold resistance to Cry1Ca and partial cross-resistance to Cry1D, Cry1E, and Cry1Ab has been recorded (Muller-Cohn et al. 1996).

This may undervalue the benefits of Bt in IPM approaches (Waage 1996), as it runs the risk of breakdown of resistance in the long-term.

B.3-Toxin-free areas ( refuge )
The primary strategy for delaying insect resistance to transgenic crops under large monocultures is to provide refuges of non-Bt crop plants that serve to maintain Bt-susceptible insects in the population. This potentially delays the development of insect resistance to Bt crops by providing susceptible insects for mating with resistant insects ( Liu et al. 1999). (Roush 1997b ; Vijaykumar et al 2004a ; Vijaykumar et al 2005a )

Currently suggested refuge strategy is very relevant and practicable under situations of monocropping in countries like US, Australia, China etc. But in countries like India, multiple cropping systems having a strong mix of several alternative hosts for bollworms such as chickpea, pigeonpea, sunflower, sorghum, maize and chillies which are grown both as sole crop and as mixed crop in the same area and in the same season as that of cotton which occupies only 5% of the total cultivable area and these alternative hosts are known to support high susceptible populations of the pest, thereby serving as natural refugia (Vijaykumar et al 2004 b)

B.4 - Refuge areas from non-cotton crops
On the other hand, farmers may be reluctant to sacrifice a large number of refuge plants to insects just to maintain susceptible alleles. In China, H. armigera naturally possesses a vast refuge as it can feed on corn, soybean, peanut, and many other crops. Studies that have monitored the sensitivity of H. armigera field populations to Bt insecticidal protein Cry1Ac from 1998 to 2000 indicated that H. armigera is still susceptible to Cry1Ac protein (Wu et al. 2002b).

Although development of H. armigera on Bt cotton was much slower than on common cotton, there was a high probability of mating between populations from Bt cotton and other sources due to scattered emergence pattern of H. armigera adults and overlap of second and third generations. Thus, in a cotton, soybean, and peanut mix system, non-cotton crops provided a natural refuge (Wu et al. 2002a). As indicated earlier in the diverse cropping systems of the tropics (Sharma et al. 2001), where the insects have several alternative and wild hosts, there may not be any need to grow the refuge crops (Gopalaswamy et al 2003).

B.5 -Positioning and size of refuge areas
Bt cotton is planted shall be fully surrounded by a belt of land called 'refuge' in which the same non-Bt cotton shall be grown." The size of the refuge belt should be such as to take at least five rows of non-Bt cotton or shall be 20% of the total sown area, whichever is more. The refuge strategy in time and space would serve to decrease selection pressure to the Bt toxin, as intercrossing between the bollworms from Bt cotton and non Bt refuge will dilute the resistance to Bt gene product (Vijaykumar et al 2004 b).

There is also a debate regarding the spatial design of the refuge system (separate/seed-mixture) to be adapted. Roush (1997a) pointed out that seed mixes can actually promote resistance development for insects that move from plant to plant.

Many researchers are concerned that cotton bollworm might become resistant to Bt toxins without a crop management strategy adapted to the farming systems. Considering the results obtained in resistance mechanism heritability as well as the role of natural and cultivated host plants in the dynamics of the bollworm populations, modelling is a tool already used to develop such a strategy in diverse situations . In the US, it led to the 'High Dose Refuge' strategy (HDR) combining a high level of toxin expression in the cotton plant associated with a toxin-free refuge inside the cotton area (Gould 1998).

The high dose strategy, combined with the use of refuges, is widely agreed to be the best technical approach for managing resistance, and evidence is accumulating that 'separate' refuges are more effective at conserving pest susceptibility than 'mixed' refuges (Cannon 2000).

Therefore, care must be taken to ensure that refuges, particularly those sprayed with insecticides, produce adequate numbers of susceptible insects. Models and experimental data showed that separate but adjacent refuges might be superior to other strategies for insects that can move between plants in their larval stage (Shelton et al. 2002).

Increasing the size of the refuge delays the development of resistance. Some workers have called for refuges as large as 50%, if farmers are allowed to spray them, which may present a dilemma and reduce farm profitability (Gould & Tabashnik 1998 cited from Gopalaswamy et al 2003 ).

Theoretical models suggest that pyramiding two dissimilar toxin genes in the same plant may require smaller refuges (Roush 1997a).The refuge fields must be within 0.8 km of their Bt fields (EPA/ USDA 1999).

B.6 - EPA opinion
The US Environmental Protection Agency (EPA), which regulates transgenic pesticidal crops, believes that scientifically sound long-term insect resistance management (IRM) strategies are essential to the protection of Bt microbial pesticides, transgenics, and reduction in the risks from the use of pesticides. The EPA has imposed mandatory IRM requirements for Bt cotton. Two structured refuge requirements have been imposed: 4% unsprayed or 20% sprayed crops (Matten 2000), and the refuge fields must be within 0.8 km of their Bt fields (EPA/ USDA 1999). Obviously, enforcing a similar system for small holding farmers will not be possible in most parts of Asia.

B.6 - Parallel development of Bt resistant insect & and the susceptible same insect from non Bt cotton
Although Bt cotton that produces Cry1Ac toxin has been effective against pink bollworm (Patin et al. 1999, Tabashnik et al. 2000b), the slower development of resistant larvae on Bt cotton as compared to susceptible larvae on non-Bt cotton could reduce the probability of mating between susceptible and resistant insects, and this asynchrony could reduce the expected benefits of the refuge strategy (Liu et al. 1999, Liu et al. 2001, Storer et al. 2001).

C --Additional ways to reduce resistance
C.1 - Other abiotic factors
Vijaykumar et al 2005b indicated that the efficacy of Bt cotton plants in killing H.armigera larvae is higher in irrigated condition compared to the Bt plants grown under rainfed conditions

C.2 - Pyramiding two dissimilar toxin genes in the same plant
Theoretical models suggest that pyramiding two dissimilar toxin genes in the same plant has the potential to delay the onset of resistance much more effectively than single-toxin plants released spatially or temporally, (Roush 1997b cited from Gopalaswamy et al 2003.

The basis for this strategy is sometimes referred to as "redundant killing" because insects adapted to one toxin may be susceptible to the second toxin. If the plants contain two Bt toxins at a high dose, insects that are able to survive on a plant with one high-dose toxin are rare, and insects that are able to survive on plants with two high-dose toxins will be very rare. (Roush 1997b and Adamczyk et al. 200).

Dual toxin (Cry1Ac and Cry2 Ac) Bt cottons will provide substantially better control of H. zea, S. frugiperda, and S. exigua compared with the existing single toxin (Cry1Ac) Bt cultivars, and may not require supplemental insecticidal applications (Stewart et al. 2001).

The strategy of "pyramiding," i.e., combining two toxins in a single transgenic plant will, at best, substantially reduce the size of the needed refuge and at worst, produce resistance to both toxins in the same amount of time as for a single toxin (Roush 1997b). Cross-resistance among toxins and the ability of insects to develop resistance to multiple toxins will limit the success of this approach (Roush 1998).

C.3 - Application of insecticides in B.t cotton when it is needed Vijaykumar et al 2005c in their Study on the pattern of cross-resistance of Cry 1Ac toxin selected (for seven generations) H.armigera to chemical insecticides (viz., cypermethrin, fenvalerate, endosulfan, quinalphos, chlorpyriphos, methomyl and spinosad) conducted under laboratory conditions using discriminating doses of insecticides revealed negative cross resistance as Cry1Ac toxin selected H. armigera individuals were more susceptible to all the chemical insecticides tested irrespective of the group, compared to the unselected larvae from non-Bt cotton fields. The study strengthens the concept of "using chemical insecticides" as one of the tools in Bt resistance management strategy to increase the life of Bt technology.

It is generally recommended that the conventional insecticides specially the new generation of PYs to be only used in B.t. cotton when it is needed and keep the new safe molecules for refuge areas. Such PYS have to be sprayed once per season. This differentiation will help to maintain considerable amount of beneficial in non-Bt plus may delay Bt resistance.

The new generation of PYs are Lambda cyhalothrin & Gamma cyhalothrin and Fenpropathrin . These groups are photo-stable pyrethroid insecticide for the control of both chewing and sucking insect pests in agriculture. It kills the insects by contact and stomach action. It offers knock down and residual control, and antifeeding and repellency properties extend the biological effect against both sucking and lepidopterous larvae.

SUMMARY The above is dealing with refuge size and positioning of old conventional and new molecules insecticides in the Bt cotton and free-toxin areas in relation to the conservation of beneficial arthropods

1-It is clear that refuge areas have to be accompanied Bt cotton specially in mono-crop system

2- Size of refuge area % will depend greatly on, single toxin or double toxin in the same plant, safe new insecticides or old conventional insecticides, diversity of crops or monoculture .For time being, it is generally assumed between 20-50%

2-It is generally assumed that using only safe new molecules in refuge areas will help in double ways the resistance and the availability of beneficial arthropods. Under this situation ,size of refuge should be 20%

4- It is generally assumed that using old conventional insecticides in rotation in refuge areas may help provided that refuge areas should not less that 50%

3- It is generally proposed that using only conventional insecticides specially the new generation of PYs in Bt areas when its needed, will help to combat CBW as well as sucking insects as well. It is also recommended to be used once per season.

There is a continuing need for a combined team of ecologists, geneticists, entomologist and plant breeders in determining system-wide impacts and devising optimal ways of deploying insect-resistant crops and reserve/conserve beneficial arthropods

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