Significant maize losses, estimated at 400,000 tons, being 13.5% of total maize production and valued at more than US$72 million are registered annually in Kenya due to lepidopteran stem borer pests. Host plant resistance to stem borers developed using the Bt technology has been used in several countries since 1996. Introduction of the Bt technology in Kenya started with the introduction of leaf tissue in 2001 and continued with testing in the biosafety greenhouse complex in 2004.

A confined field trial was initiated in may 2005 to test the efficacy of nine Bt maize events carrying cry1Ab and cry1Ba Bt genes against major stem borers in Kenya including Chilo partellus, Eldana saccharina, Sesamia calamistis, and Busseola fusca. Tests using the leaf damage scores on plants after field infestations with C. partellus and from leaf bioassays with the three other pests in the biosafety greenhouse complex indicate that control was found for C. partellus E. saccharina and S. calamistis but adequate control was not found for B. fusca. Additional Bt genes or events will need to be sought and tested for effective stem borer control in all maize growing ecologies in Kenya.

Unfortunately, a regrettable and inadvertent application of a systemic insecticide will prevent observation of the effects of the Bt delta-endotoxins beyond the early season vegetative stage of the crop. The problem was identified on observation that newly emerging leaves were clean even on plants previously showing extensive leaf damage including the non-transgenic CML216 control referred to above. A second infestation was done two weeks later and the result of the scores from the second infestation showed reduced leaf damage. Plants that were previously showing leaf damage are currently showing neither dead hearts nor reduced vigor that are common with susceptible plants. This indicates that the larvae may have died after feeding on the leaves and may not have burrowed into the stems to cause tunneling. While this may be expected in resistant plant on rare occasions, it has not been observed in CML216. It was therefore suspected that an additional factor may have caused the death of larvae, thereby preventing extensive damage even on susceptible plants.

On close inquiry, it was found that the field technician, with perfectly good reasons to control white grubs Phyllophaga spp that were destroying plants thereby further reducing the plant stand, applied Furadan®, a systemic insecticide without prior consultations with the principal investigators. Furadan® remains in the plant for up to 60 days after uptake and re-infestation may not offer solution to this problem. Without stem damage expected carrying the trial to 14 weeks as planned may not be worthwhile. More frequent irrigation was effected to try to leach out the Furadan® from the soil. The NBC was briefed on this during their visit at the site on 18 July 2005 and they recommended that the trial be terminated preferably at the 7-8 weeks stage and a second planting be made to obtain results over the full season; an important activity considering the level and extent of interest and attention that these trials have received locally and internationally.

Two issues are worth noting regarding the two confined field trials at Kiboko. First is that no biosafety conditions have been breached from planning, planting, transportation of materials as well as management of the trial. All major activities had KEPHIS inspectors participating as in the appended schedule of activities. All other records for compliance are up to date and available for inspection at the site. Secondly, the germplasm conversion to Bt is running as planned and controlled pollination has commenced.

Lepidopteran stem borers are of economic significance in crop production. In Kenya, stem borers are a problem in sorghum and maize production. Significant maize losses estimated at 400,000 tons, being 14% of total maize production, and valued at more than US$72 million are registered in annually in Kenya (De Groote et al, 2004). Among the control strategies of stem borers are cultural, chemical, biological, and host plant resistance. Each method has its advantages and disadvantages related to efficacy, cost, ease of adoption by farmers, and ability to fit into an integrated pest management strategy. Host plant resistance, where the plant protects itself against damage by insect pests can be arrived at by developing insect resistant varieties using conventional or biotechnology methods. The final products are seeds, an age old method of delivering technologies to farmers, and which is relatively easy to adopt and use.

Bt technology that involves use of modified and truncated genes from the common soil dwelling bacterium (Bacillus thuringiensis) was the chosen biotechnology method. Maize thus transformed with the effective cry genes will express delta-endotoxins that will control susceptible species of stem borer species, in a very specific way without affecting humans, livestock and the environment. Bt maize has proved to be a safe and effective product. Having undergone rigorous testing for food and feed safety, it has provided environmentally friendly and effective control of targeted pests, and the resistance is still durable after years of deployment (James 2004). This technology has been effectively used in controlling stem borers in 14 million hectares of maize globally by 2004, and has witnessed very high adoption rates since 1996 in both developed and developing countries (James 2005).

KARI and CIMMYT through the Insect Resistant maize for Africa (IRMA) project have tested Bt maize following national rules and regulations since 2001, when cut leaves were imported into Kenya and used for leaf bioassays (Mugo et al. 2004). Further testing was done in the biosafety greenhouse complex (BGHC) after seeds were introduced in 2004 (Mugo et al. 2003).

Field testing as a confined field trial was proposed in the “Application for Field Evaluation, Leaf Bioassays, Seed Increase and Backcrossing of Maize Containing the cry1Ab or cry1Ba (Bt) Genes Under Confinement in the Open Quarantine Site (OQS) at Kiboko”. The application was approved by the Kenya NBC verbally on 9 February 2005 and in a letter dated 14 April 2005. The Kenya Plant Health Inspectorate Services (KEPHIS) spelt out the phytosanitary approval conditions and requested for a compliance document in a letter dated 12 April 2005. The compliance document was developed and presented to KEPHIS on 19 April 2005. KEPHIS subsequently issued an authorization permit No. 1/KCFT/2005 indicating the conditions to be met during the conduct of the trial on 10 May 2005. The Director KARI signed a letter of commitment to KEPHIS on 20 May 2005. The trials were sowed at the KARI Kiboko OQS that had been previously inspected and approved by the Kenya Standing Technical committee (KSTCIE) on 27 May 2005. This paper is a mid-term report for one of the two trials of the Bt maize testing in a confined field trial i.e. confirm under field conditions the effectiveness against Chilo partellus of the cry (Bt) gene events tested in leaf bioassays and in whole plants in the BGHC.

Genetic materials:

Seeds from nine Bt maize events all from inbred line CML 216 and the non-transformed version of the inbred line were used for the evaluation trial as detailed below (Table 1).

The nine events carried variously cry1Ab or cry1Ba Bt genes driven either by the maize ubiquitin or the rice actin promoters. The seeds were harvested and stored during the January-April 2005 period from the seed increase through selfing of BC3S1 in the biosafety greenhouse complex.

Table 1: List of Bt maize events and the non-transformed check sowed in the confined field trial at               KARI Kiboko for evaluation of resistance to major Kenyan stem borers.

Packaging and seed transportation from the BGHC to the open quarantine site and sowing

The seeds were counted and packaged the day before transportation. Seeds were packed by field plot rows with each envelope containing 21 seeds. The seed envelopes were packaged in plastic zip-lock bags which were then packaged in a small metal urn. The urn was then carried in a locked metal box. The boxes were then transported by the PIs by road while escorted by KEPHIS plant inspectors to the OQS at KARI Kiboko. These seeds were planted on the same day on 27 May 2005. Due to shortage of seed (since seeds were harvested from small single ears of inbred lines under greenhouse conditions), only one seed was sown per hill. No remnants seeds were found but the seed envelopes, Ziploc bags and biohazards bags where emptied seed packaging were held were destroyed by burning and burying the ashes in the disposal trenches within the open quarantine site. However, the metal urns and boxes were washed thoroughly before being returned to the BGHC use in future. Five perimeter border rows were grown with a tall hybrid 513 which was managed in the same way as the test materials as detailed below.

Field experimental design:

The experimental trial had three replications with 2 rows of 5 m length for each plot. The row to row distance was 75cm while the hill to hill distance was 25 cm giving an ideal plant stand of 21 plants per row and 42 plants per plot.

Treatments and crop management:

The field was irrigated immediately and irrigation maintained in such a way that the crop has not experienced drought stress in the sandy soils at the site. Basal NPK fertilizer was applied at sowing and topdressing done on split pattern at the 4th leaf and at the 7th leaf stages to the equivalent of the recommended 40kg N and 40kg P2O5 at the area. Weed control was done by hand.

Insect infestations and leaf damage scoring:

At the four-leaf stage on 17 June 2005, every plant in the first row was infested with 40 black head eggs of the spotted stem borer (Chilo partellus Swinhoe) by placing the egg mass in the plant funnel. The second row was neither treated nor infested. However, the perimeter hybrid borders were treated with bulldock (Bayer, i.a. beta cyfluthrin)). Stem borer damage was assessed two weeks after infestation on 1 July 2005 from each plant in the infested row on a 1-9 scale (1 least damage to 9 most damage). When it was observed that newly emerged leaves even in the plants with damage were clean, a second infestation with 20 C. partellus black head eggs was done on 4 July 2005. All remnant insects and their wrapping during both infestations were destroyed as for the case of seeds. However, the Petri-dishes were washed with water and taken back to the insectary at KARI Katumani.

Leaf bioassays on three stem borer species:

According to the application, and as it is not possible obtain meaningful data after infesting a maize with more than one stem borer species, responses to the other major stem borer species in Kenya were done using leaf bioassays. The species are: the African stem borer (Busseola fusca Fuller), the sugarcane borer (Eldana saccharina Walker), and Pink stem borer (Sesamia calamistis Hampson). One leaf was harvested from each of the representative plants second row (non-infested row) in on 29 June 2005. The leaves were packaged sealed in plastic bags, and placed in ice box with ice to remain fresh. These were transported by CIMMYT and KARI staff with the escort by a KEPHIS plant inspector to the BGHC in Nairobi. There, bioassays were performed with larvae from the three stem borer species.

The protocols followed in the bioassays were as described in Mugo et al. (2004). Briefly, a 1.5x1.5 inch leaf blade (but no mid-rib) was placed on a moistened filter paper in a Petri-dish. Ten neonate or first instars larvae of the particular stem borer species were introduced on the leaf, the Petri-dish sealed with parafilm, covered with dark plastic trays, and placed under room temperature for five days. Each of the five plants in the row was challenged with each of the three stem borer species. After the five days of incubation, the leaf area consumed as well as the number of larvae recovered alive and dead were counted. At the end of the bioassay, all materials (leaves, insects, paper, etc.) except the plastic Petri-dishes and boxes were autoclaved and later burnt to destroy all living material. The plastic Petri-dishes and boxes were sterilized by soaking in a bleach solution followed by extensive washing and rinsing.

As expected, due to shortage of seed and seedling pest damage, the crop stand was lower than ideal and this cut across transgenic and non-transgenic plots (data not shown). The test materials are all inbred lines and are therefore suitably enclosed by the vigorous hybrid 513 borders. The crop has gone through 54 days (about 6 weeks) and the plants look good, save for differences in plant types among entries. Events 127 and 216 showed rather poor plant types with narrowed and semi-chlorotic leaves, and shorter plants than the other events. A few plants have un-unidentified bacterial die back from the test materials as well in the hybrid borders.

Leaf damage score due to C. partellus damage from artificial infestation in the field

Leaf damage score taken 14 days after the first infestation with C. Partellus in the field showed significant differences among genotypes with the non-transgenic control showing average score of 3.5 on a 1-9 scale (Table 2 and Figure 1). Bt maize Event 6 had lower damage than the CML216 control but higher than all the other Bt maize events. Events 6, 93, 223, and 396 showed significantly higher damage scores than events 3, 10, 58, 127, and 216, which showed immune response (score of 1) to C. partellus. A significant observation was that though all plants in the CML216 control had stem borer damage on the leaves, only scattered plants had leaf damage among the Bt maize events 6, 93, 223, and 396, which showed damage (data not shown).

Leaf damage score taken 14 days after the second infestation with C. Partellus in the field as well showed significant differences among genotypes with the non-transgenic control showing average score of 2.0 (Table 2 and Figure 1). The level of damage was lower on both the non-transgenic CML216 as well as in the Bt maize events showing damage. However, there were no significant differences in leaf damage among the Bt maize events, though events 3, 10, 58, 127, and 216 still showed immune responses with no plant with any damage at all. As in the first infestation, leaf damage was spread in all plants in the CML216 control but only on some plants among the Bt maize events showing damage.

Table 2: Leaf damage scores 14 days after the first and 14 days after the second               infestation with Chilo partellus black head eggs at the Bt Maize confined field trial at KARI               Kiboko



Figure 1: Leaf damage scores 14 days after the first and 14 days after the second                infestation with Chilo partellus at the Bt Maize confined field trial at KARI CFT at                Kiboko

Leaf bioassays with B. fusca, E. saccharina, and S. calamistis from cut leaves in the BGHC.

After five days of incubation, the area consumed on the leaves by B. fusca on (37 mm2) was the largest when compared to the area consumed by E. saccharina (3 mm2) and by S. calamistis (7 mm2) stem borers (Table 3 and Figure 2). However, there were significant differences neither between transgenic events and the CML216 control nor among Bt maize events. The mean area consumed by E. saccharina showed significant differences with damage on Event 396 being comparable with the control. For S. calamistis, the control had much more damage than all the Bt maize events.

Table 3: Means of leaf area consumed and mortality of larvae of three stem borer species after                feeding on maize leaves for five days.

As expected from the leaf area consumed, the mortality of B. fusca larvae (54%) was the lowest among the other stem borers where 66% was recorded for E. saccharina and 87% for S. calamistis (Table 3and Figure 3). Mortality was lowest for Event 6 and the control for E. saccharina larvae, while all Bt maize events recorded high mortality rates compared to the CML216 control.

Figure 2: Leaf area consumed by three stem borer species after feeding on maize leaves for five                days.

With the major trait of interest here being stem borer damage which is was scored and measured on per plant basis as is the practice, the effect of low plant stand is not as critical as would otherwise be were agronomic traits such as plant stature and grain yield been desirable to measure.

Control of C. partellus damage from artificial infestation in the field

The relative ranks of leaf damage scores observed in the field after infestation with C. partellus stem borer were similar across two infestations stages and stages of plant growth, although the absolute scores were lower during the second infestation. This indicates that events 3, 10, 58, 127 and 216 are very effective against C. partellus the most widely spread and damaging stem borer in Kenya. Further, the non-uniform leaf damage among plants in events 6, 93, 223, and 396 where immunity was not observed indicate some level of segregation within the ears representing these inbred lines in the trial.

Figure 3: Larvae mortality in three stem borer species after feeding on maize leaves for five days.

The germplasm tested was the BC3S2 generations with an expected level of inbreeding of more than 87.5%, hence the amount of segregation is that was observed as expected. The significance of these findings is that with further inbreeding in inbred lines (already on-going in the BGHC), all the Bt maize events, variously carrying cry1Ab and cry1Ba genes, and the corresponding delta-endotoxins will provide adequate control of C. partellus stem borer. These results are in agreement with those from Mugo et. al. (2003) and Mugo et al. (2004) who found complete control for C. partellus from cut leaves expressing cry1Ab and cry1Ba delta-endotoxins of maize grown in CIMMYT-Mexico. These results are also in agreement with observations made in the BGHC with Bt maize grown there (unpublished data).

Control of B. fusca, E. saccharina, and S. calamistis from bioassays in the BGHC.

Large leaf area consumed and the low larval mortality rated observed for B. fusca point to very low resistance among the Bt maize events against this pest. However, E. saccharina and S. calamistis were well controlled by both cry1Ab and cry1Ba delta-endotoxins from the Bt maize events tested here. These results also agree with those by Mugo et al. (2004) and Mugo et al. (2003) who found that Bt cry 1Ab, cry1Ba and other tested proteins were not effective in the control of B. fusca, and that S. calamistis was affected by cry1Ab, cry1Ba and other tested proteins. The results were, however, at variance with the findings of the same authors that E. saccharina was the least affected by cry1Ab, cry1Ba and other tested proteins. The results from field infestation with Chilo partellus and in bioassays with B. fusca, E. saccharina and S. Calamistis with cut leaves from the same plants when taken together indicate that we have Bt maize events to adequately control C. partellus, E. saccharina and S. Calamistis, but not for B. fusca. There is need to seek for additional cry genes or events of these same genes and test them since complete control of the major stem borer species is desirable for increased maize production in Kenya.

Set back in the field trial

While we stand firmly with these results in the field and BGHC as fully legitimate and adequate to make conclusions in one year field and lab testing, the field trial has encountered a set-back. This traces back to the observation that newly emerging leaves were clean even on plants previously showing extensive leaf damage including the non-transgenic CML216 control referred to above.

The second infestation was done and the result of the scores from the second infestation showed reduced leaf damage. Plants that were previously showing leaf damage are currently showing neither dead hearts nor reduced vigor that are common with susceptible plants. This indicates that the larvae may have died after feeding on the leaves and may not have burrowed into the stems to cause tunneling. While this may be expected in resistant plant on rare occasions, it has not been observed in CML216. It was therefore suspected that an additional factor may have caused the death of larvae, thereby preventing extensive damage even on susceptible plants.

On close inquiry, it was found that the field technician, with perfectly good reasons to control white grubs Phyllophaga spp that were destroying plants thereby further reducing the plant stand, applied Furadan® (insecticide-nematicide)1an, a systemic insecticide without prior consultations with the principal investigators (PIs). It is therefore suspected that Furadan® levels had not risen to critical levels by the time of first infestation and by the time of sampling leaves for bioassays that gave the results discussed above. Furadan® remains in the plant for up to 60 days after uptake and re-infestation may not offer solution to this problem.

The PIs take full responsibilities for these actions, but have made decisions to the effect that no treatment apart from weeding (by hand), irrigation, and fertilizer application will be effected without prior consultation with at least one of the PIs, and that no systemic pesticide will we used at the site. The technician has been efficient and effective and efficient and will continue managing the site. The immediate response was to effect frequent irrigation to leach out Furadan® from the soil.

Without stem damage expected carrying the trial to 14 weeks as planned may not be worthwhile. These findings and thoughts were presented to the members of the NBC during their visit to the trial site on 18 July 2005 and they recommended that this trial be terminated any time from that date and a second planting be made preferably at the same spot. This way, data could be generated for two seasons from this site. Seeds are available to sow this trial from the seed increases made in the BGHC.

Two issues are worth noting regarding the two trials at the Kiboko CFT. First is that no biosafety condition has been breached during planning, planting, transportation of materials as well as management of the trial. All major activities had KEPHIS inspectors participating who can collaborate with this view as in the appended schedule of activities. All other records for compliance are up to date and available for inspection at the site. Second is that the second trial where maize lines are being converted to Bt is running as planned and controlled pollination has commenced.

The Bt maize confined field trials have been run without any breach in biosafety and phytosanitary conditions and preliminary results on efficacy of the Bt maize events against stem borer through field infestations and through leaf bioassays have been obtained. The results indicate that tested Bt maize producing cry1Ab and cry1Ba delta endotoxins will control Chilo partellus E. saccharina and S. Calamistis but will not adequately control B. fusca and additional cry genes or events of these same genes needs to be sought and tested for effective stem borer control in all ecologies in Kenya.

However, a regrettable error made through inadvertently applying a systemic insecticide will prevent observation of the effects of the Bt delta-endotoxins beyond early season leaf damage. It is the recommendations of the NBC that this trial be terminated early enough to allow a second planting to obtain results over the full season. This is even more important considering the very high level and extensive attention that these trials have received locally and internationally.

Hugo De Groote, Charles Bett, James Ouma Okuro, Martins Odendo, Lawrence Mose, and Elizabeth Wekesa. 2004. Direct estimation of maize crop losses due to stem borers in Kenya, preliminary results from 2000 and 2001. In Friesen D.K. and A. F. E. Palmer (Eds.). Integrated Approaches to Higher Maize Productivity in the New Millennium. Proceedings of the 7th Eastern and Southern Africa Regional Maize Conference, Nairobi, Kenya, 11 - 15 February 2002. Mexico, D. F.: CIMMYT. Pp. 401-406 401.

James C. 2004. Global Review of Commercialized Transgenic Crops: 2002. Feature: Bt Maize. ISAAA Briefs No. 29 – 2003. International Service for the Acquisition of Agri-Biotech Applications (ISAAA). Web site http://www.isaaa.org/kc/bin/isasaabriefs/index.htm.

James C. 2005. Global Status of Commercialized Biotech/GM Crops: 2004. ISAAA Briefs No. 32-2004. International Service for the Acquisition of Agri-Biotech Applications (ISAAA). Web site http://www.isaaa.org/kc/CBTNews/press_release/briefs32/highlights/Highlights.pdf.

Mugo S., C. Taracha, S. McLean, J. Songa, B. Odhiambo, D. Bergvinson, and D. Hoisington. 2003. Screening cry proteins from straight and combination of events produced by Bt maize leaves for activity against Kenyan maize stem borers. A Report to the Kenya National Biosafety Committee (NBC) on the completion of the importation of Bt maize leaves

Mugo S., C. Taracha, D. Bergvinson, B. Odhiambo, J. Songa, D. Hoisington, S. McLean, I. Ngatia, and M. Gethi. 2004. Screening cry proteins produced by Bt maize leaves for activity against Kenyan maize stem borers. In: Friesen, D.K. and A. F. E. Palmer (eds.). Integrated Approaches to Higher Maize Productivity in the New Millennium. Proceedings of the 7th Eastern and Southern Africa Regional Maize Conference, Nairobi, Kenya, 11 - 15 February 2002. México, D. F.: CIMMYT, pp. 102-105.